ANALYTICAL STUDIES ON HONEY
A Thesis
submitted
to the University
in candidature
Doctor
for
of
Salford
the Degree of
of Philosophy
by
Ramchandra Parshotam
Department of Biological
Section,
Biochemistry
University
of Salford,
M5 4WT
Salford
Mistry
Sciences,
July
1987
ST
AVAILA
COPY
L
Variable print quality
To UMA -
for
her
inspiration.
-i-
DECLARATION
I hereby
for
a thesis
is
Salford
out
in
of
of Philosophy
under
the
Sciences,
supervision
of
that
and that
it
this
is not being
CANDIDATE:
SUPERVISOR:
SUPERVISOR:
work has not
the University
oe.
A"
cm
of
The work was carried
Biochemistry
of Dr.
been submitted
currently
as
now submitted
Section,
R. J. Washington
L. R. Croft.
degree.
DATE:
of Doctor
of Biological
Salford,
herein,
the work contained
of my own investigations.
the result
I certify
degree
that
the degree
the Department
University
and Dr.
declare
presented
for
for
any other
any other
ii
-
-
ACKNOWLEDGENENTS
like
I would
to express
for
R. J. Washington
to Dr.
throughout
his
investigation.
this
for
his
Dr.
D. A. Duddell
for
his
his
for
and interpretation
advice
are due to Mrs.
H. Thompson for
of
are due to
thanks
Thanks
analysis.
statistical
the automatic
operating
J. Pemberton
and to Dr.
on computation
the
L. R. Croft
due to Dr.
are also
A formal
guidance
and suggestions
guidance
My thanks
thanks
and sincere
gratitude
advice,
investigation.
in this
help
my profound
amino acid
analyser.
like
I would
to acknowledge
of honey samples
from
who participated
in this
project,
this
investigation.
able
to carry
Manley
Ratcliffe
and donation
I wish
typed
out
this
acknowledged
with
the members of
Limited,
sincere
Berinsfield,
Beekeepers
the British
without
the provision
gratitude
which
I could
I would
also
Oxford,
for
like
their
not
Association
have been
to thank
participation
of honey samples.
to recognise
thesis.
with
the care
The patience
deep appreciation.
and attention
with
and encouragement
which
Mrs.
of my family
M. Trotman
is
iii
-
-
C0NTENTS
Paste
DECLARATION
ACKNOWLEDGMENTS
CONTENTS
ABSTRACT
vii
CHAPTER1-INTRO.
The Amino Acid
-D
Composition
Processing
of Nectar
Extraction
and Processing
Honey Production
into
1
UCT10N-H0NEY
of Nectar
and Honeydew
3
Honey by the Honeybee
8
of Honey
9
11
and Trade
Honey Plants
14
Honey Composition
15
Honey Proteins
23
Honey Amino Acids
29
Honey Pollens
36
Mellissopalynology
40
Honey Adulteration
46
Honey Toxins
52
Aims
55
CHAPTER2
Equipment
-,
EXPERIMENTAL
and Materials
56
METHODS
Preparation
Subjective
The Isolation
57
of Honey Samples for
Analysis
Analysis
57
and Evaluation
and Concentration
60
of Proteins
in Honey
61
- iv -
Paize
in Residues
Quantitative
Estimation
of Proteins
Ultrafiltration
of Honey
The Determination
Determination
of Honey
of
of Proteins
of
Examination
and Identification
Substances by a Combination
Chromatography
64
Substances
Positive
72
the Free Ninhydrin
Quantitative
Microscopical
Positive
72
Positive
of the Ninhydrin
and
of Paper Electrophoresis
Analysis
of the Free Amino Acids
Amino Acid Analyser
Quantitative
Automatic
62
in Honey
the Free Ninhydrin
Separation
and Concentration
Substances of Honey
from
Analysis
Using
the
84
86
of Honey
91
Computation
93
Aýalysis
Statistical
RESULTS
CHAPTER3
-
The Colour
and Aroma of Honey
Analysis
Isolation,
Honey
99
101
of-Proteins
Concentration
and Estimation
Amino Acids in the United
Order of Pollen Types
in
124
of Analytical
Analysis
of Samples from the United
Analysis
of Samples from
of Analytical
101
and Pollens
Kingdom Samples Arranged
Amino Acids in the Foreign and Commercial
in Order of Stated Country Sources
Computer Processing
in
of Proteins
Positive
Analysis
Substances
of Ninhydrin
Present in Honey of Known Origin
Evaluation
Sources
79
the
Samples Arranged
184
209
Data
Foreign
Kingdom Survey
and Commercial
Honeys
Honeys
211
237
Data of Honey Samples from all
248
-v-
Page
CHAPTER4-DI
S' CUS.
S10N
PRELIMINARY SUBJECTIVE EXAMINATION OF HONEY SAMPLES
253
ASSESSMENTOF PRESENT WORK
255
Proteins
255
Enzymes
263
Ninhydrin
Positive
265
Substances
Pollens
280
PROCUREMENT
OF EXPERIMENTAL HIVE ON UNIVERSITY CAMPUS
283
Computation
284
and Statistical
Analysis
EVALUATION OF PRESENT WORK
290
Proteins
290
Enzymes
293
Ninhydrin
Positive
Substances
294
298
Pollens
Statistical
Analysis
298
ENVIRONMENTALAND PROCESSINGFACTORSAFFECTING HONEY
301
SIGNIFICANCE OF PRESENT WORK
312
FUTURE WORK
313
BIBL1
0G
RAPHY
APPEN
DI
CES
APPENDIX I
APPENDIX II
APPENDIX III
APPENDIX IV
LIST OF HONEY SAMPLES
QUESTIONNAIRE
DETERMINATION OF THE AMINO ACID
CONCENTRATIONIN A HONEY SAMPLE
COMPUTATION
318
330
339
340
343
- vi -
PaRe
APPENDIX VI
APPENDIX VII
APPENDIX VIII
APPENDIX IX
345
DISCRIMINANT ANALYSIS
APPENDIX V-A
DISCRIMINANT
HONEYS
-
-
ANALYSIS
OF THE U. K.
SURVEY
350
DISCRIMINANT ANALYSIS OF THE FOREIGN AND
COMMERCIALHONEYS
IDENTIFIED
POLLEN GRAINS
LIST OF PUBLICATIONS
356
359
364
- vii
-
ABSTRACT
The floral
and the geographical
by microscopic
examination
be applied
cannot
to highly
is a need for
there
This
samples.
Beekeepers
strained
and their
the help
with
Kingdom for
to such
the British
of
honey
members, who provided
the United
within
Thus,
can be applied
that
tedhnique
this
honeys.
free
or pollen
work was undertaken
Association
however
present,
technique
a chemical
present
of known origin
the pollen
of
of honey can be determined
origins
chemical
samples
evaluation
and comparison.
There were 192 samples
69 samples
and an additional
from various
proteins
by the
present
was abandoned
between
11 foreign
of
in the presence
phoresis
the proteins
The free
ninhydrin
were detected
obtained
of
honeys were examined
of polyacrylamide
sodium dodecyl
of
studies
the 261,
gel
This
sulphate.
indicated
that
origin
was not
the free
of
positive
substances
and then
concentrated.
by a combination
and 28 of
and chromatography
measurements
technique
and the geographical
chromatography
substances
origin
electroapproach
a correlation
possible.
and the
amino acids
were examined.
present
exchange
of foreign
those
and 5 English
preliminary
since
For 256 samples out
pollens
including
the U. K. survey
sources.
A selection
for
honey from
of English
these
the concentration
made on an automatic
of
the 13 amino acids
of
the 5 predominant
amino acid
of
of
Forty
of paper
ninhydrin
of
13 detectable
of
quantitative
amino acids
The average
analyser.
positive
electrophoresis
compounds were identified.
the 191 samples
pollens
by ion-
were separated
were
concentration
the U. K. survey
the U. K. have been given
and those
in Table
A.
- viii
-
TABLE A
Average
of thirteen
concentration
free
amino acids
Average concentration
(nMoles per g of honey)
SourcesfloralI
Amino
acids
123456
Lysine
123
170
122
78
107
133
90
167
170
124
146
136
Threonine
63
86
110
42
60
92
Serine
70
120
112
62
90
100
144
335
298
126
220
246
Aspartic
Glutamic
acid
acid
2,000
Proline
4,090
3,250
1,870
3,230
3,140
Glycine
44
76
100
46
66
67
Alanine
73
150
132
70
114
118
Valine
46
76
65
33
82
66
Isoleucine
43
107
62
30
70
67
Leucine
40
92
80
33
58
60
Tyrosine
55
238
100
120
75
120
170
312
180
110
218
208
44
29
17
8
8
191
Phenylalanine
Sample Number
1-
Brassica
2-
Trifolium
3-
Castanea
4-
CaUuna
5-
Myosotis
6-U.
repens
K. survey
- ix -
floral
The major
was found
It
the computer
to the
floral
concentration
source
large.
the detection
with
to categorise
used successfully
by correlation,
followed
using
measurements
The potential
to evaluate
the
role
sugar
unspecified
aspects
These aspects
to honeybees,
foreign
especially
with
processing
methods
the
honeys
and floral
with
of heating
effects
source
commercial
with
reference
region.
geographical
classifications
were indicated
honeyst
honeydew and nectar
were:
then
concentration
amino acid
of honey production
and/or
is
This
samples.
sources
can be
substances
the SPSS predictive
of using
of floral
pattern
a definite
SPSS to predict
the sub-group
provided
positive
in some cases,
the following
to be successful.
feeding
of ninhydrin
be correlated
could
The overall
(SPSS)
Sciences
Social
examined,
using
analysis
measurements
the samples
of
samples were sufficiently
coupled
discriminant
Package for
Statistical
package
the amino acid
sample.
by performing
that
by quantitative
honey were confirmed
of-each
of the original
analysis
pollen
that
sources
honey,
sugar
adulteration
products,
to plant
commercial
family.
INTR0DUCT10N
CHAPTER 1
1.
INTR0DUCT10N-H0NEY.
CHAPTER 1
The definition
is
the
of honey according
saccharine
flowers'.
issued
This
product
by the United
Inter-relationship
is
of
of Hymenoptera
is
there
me#ers
of
family
fZorea
and these
the Apia
are
and the well-known
(Bombus)
the bumblebee
be the Apia
Analytical
and also
of
species
dorsata,
of
the Apia
genus includes
genus or the
two genera
the
stingless
and these
are
and the MeZipona.
In the present
to will
The MeZipona
ovipositor
genii,
The Banbidea
meUifera.
species.
three
the Apia
cerena,
Within
membranous
their
genus has four
are commonly known consist
bees as they
the 1!rigona
Apia
with
by having
includes
The Apia
(1977).
bees and wasps are
The ants,
family
This
Bombidea and MeZipona.
honeybees
given
family.
insects
and are characterised
to form a sting.
modified
Apia,
this
the AcuZeata
products
and Daves
those
representing
Comninity.
on the honeybee
studies
was by Richards
of
regulations
Economic
and their
entomological
'Honey
is:
the nectar
those
with
of honeybees
species
and one such investigation
wings
in agreement
Kingdom and the European
There have been several
the order
by the bees from
gathered
definition
(1976)
to Pearson
data
one Trigona
in Table
1.0.
text
unless
stated
otherwise
the honeybee
referred
Apia
species
meUifera.
on the honey obtained
according
to various
The variation
in
from
the four
literature
the composition
sources
of
are
the honeys
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3.
by these
produced
but
genii
also
that
reported
lower
than
indica
from
the environmental
the amylase
those
locality.
activity
the Apia
of
Davies
(1975)
(1976)
and invertase
the Apia
those
meUifera
honey produced
by Apia
iridipennis
had a high
and glucose
oxidase
for
values
had the
but
Also
displayed
(1983)
content
with
=Uifera
and
reported
that
fZorea
and Trigona
reported
catalase
for
values
honey had the highest
and Txýona
and catalase
highest
melZifera-
caucasia
low diastase,
but
dorsata
Apia
Apia
same
cerena
by the Apia
meUifera
dorsata,
the
the enzymes amylase
of
produced
in comparison
invertase
diatase,
least
invertase
from
of the Apia
to that
the activities
Apia
cerena,
honeys were
the honey from Apia
that
Wakhle and Desai
content
honeys.
meZlifera
obtained
by the Apia
carnica.
(1968)
Vorwohl
honeys
in the honeys
collected
due to their
only
cerena
compared
that
observed
not
the Apia
meUifera
content
were higher
than
meZZifera
arises
conditions.
of
reported
had a low proline
Klemarewski
Apia
of honeybees
species
glucose
oxidase
content
iridipennis
activity
compared
to the others.
The Amino Acid
The raw materials
called
is
leaf
for
and honeydew.
are nectar
situated
Composition
in the region
'floral'
secreted
hoppers
the
'extrafloral'
and
by the plant-sucking
feeding
is
flowers
and Honeydew
honey by the honeybee
the production_of
Nectar
of
of Nectar
secreted
by the plant
or in other
nectaries,
insects
parts
respectively.
such as aphids,
on the phloem, or sap of plants
glands
of
the plant
Honeydew
coccoids,
and trees.
4.
The free
amino acids
of nectar
Mostowska (1965) reported
acids
in the nectars
of Brassica
TiZia
pseudbacacia,
acid,
phenylalanine,
that
napus var-
TrifoZium
cordata,
and CaUuna vulgaris.
aspartic
the presence of the following
PhaceZia tanacetifoZia
These amino acids were:
histidine,
glutamic
acid,
proline,
threonine
the amino acid content
Robinia
oleifera,
repens,
alanine,
leucine,
tyrosine
amino
arginine,
lysine,
ornithine,
He found
and valine.
ranged from 0.002 to 4.786 mg
of nectar
per 100g of dry matter.
Hanny and Elmore (1974) used the technique
layer
floral
nectar
were:
alanine,
asparagine,
acid,
a-amino
glutamine,
that
noted
serine,
the qualitative
composition
due to the amino acids
the cotton
the environmental
conditions
The percentage
was constant.
constituted
methionine,
of
composition
of
glutamic
These-researchers
and valine.
because
arginine,
acid,
lysine,
leucine,
amino acid
fluctuated
acid,
2,4-diaminobutyric
tyrosine
the extra-
These amino acids
y-aminobutyric
isoleucine,
glycine,
nectar
acid,
from
amino acids
hirsut=).
cysteine,
the quantitative
extrafloral
0.04% of
on average
nitrogen
the extrafloral
of cotton.
nectar
Baker
and Baker-(1975)
that
reported
serine
adipic
acid,
proline,
twenty
(Gossipium
of cotton
aspartic
ornithine,
but
and identified
chromatography
of gas- and thin-
alanine,
appeared
Baker,
asparagine,
Opler
the constitution
reviewed
aspartic
acid,
glutamic
to be the commonest amino acids
and Baker
cysteine,
lysine
(1978)
reported
and tyrosine
that
acid,
of nectar
glycine
and
of nectar.
the amino acids
were more frequently
and
5.
in the extrafloral
represented
only moderately
whereas these amino acids are
nectar
in the floral
represented
Inouye and Inouye (1980) reported
These amino acids were:
acid,
a- and y-aminobutyric
acid,
glutamic
acid,
methionine,
threonine,
Gilliam,
of citrus
per
tyrosine
100g of nectar
in
and that
its
(1981)
McCaughey and Moffett
in the floral
of amino acids
with
those
and Elmore
(1974).
the predominant
serine,
glutamic
Gilliam,
amino acids
the findings
of Baker,
was significantly
a thousand
amino acid
nectar
nectar
(1982)
species
composition
of
its
their
on the composition
(1978)
analysed
flowering
were aspartic
glutamic
Thus,
that
from
that
the constituents
plants
to have a taxonomic
that
reported
nectar
nectar.
and Baker
by Hanny
as reported
McCaughey and Moffett
whereas
hirsutum)
(Gossypiwn
of cotton
the floral
Gilliam,
nectar.
results
of cotton
in composition
different
Baker and Baker
over
Opler
in
level
in the extrafloral
amide were dominant
the nectar
that
had low level
Citma
most plants
and threonine;
acid
serine,
proline,
(1980) reported
compared
in
leucine,
7.5 to 79.1 mg of amino acids
unlike
the extrafloral
of
isoleucine,
and valine.
and high
pollen
aspartic
asparagine,
phosphoserine,
from
contained
extrafloral
a-amino adipic
histidine,
glycine,
McCaughey and Wintermute
cultivars
of proline
arginine,
phenylalanine,
tryptophan,
any other
a- and 0-alanine,
acid,
glutamine,
for
of
of HeZiantheUa
nectar
more than has been reported
quinquenervia
lysine,
the presence of a total
in the extrafloral
twenty four amino acids
nectar.
nectar.
acid,
and its
acid
confirming
nectar
extrafloral
floral
nectar.
of nectar
from
of
and reported
and phylogentic
that
the
significance
6.
and to be of benefit
to both the plants
and their
Freeman, Reid and Zaun (1983) reported
asparagine,
glutamine,
phosphoserine,
and taurine
in nectars
of four
to be present
in the detection
(1986) reported
that
(Orchidaceae)
and Epipactis
and the entantiomoric
composition
of the particular
differences
Indian
from
thirty
four
five
species
that
the nectar
of Hybanthus
high
amino acid
content
insect
plant
of Spathodea
nutrition.
characterised
tryptophan
representing
a-amino
and out
and isoleucine
acids.
nectars
indicate
amino acids
of
the
(Bignoniaceae)
The 142mera subuZata
the
were essential
a
valine,
The
two amino
for
was essential
of which
for
observed
nutrition.
had only
latter
eighty
exhibited
(Turneraceae)
to seven amino acids
and phenylalanine
insect
genera
workers
seven amino acids
for
of
that
reported
(Violaccae)
were essential
of which
three
eighty
These research
ennaespermus
campanuZata
by five
atropurpurea
and they
of angiosperms
acid
and glutamic
alanine
species
families
contained
of
and Swamy (1986) examined the nectars
one hundred
acids
of several
of both nectaries.
Bahadur, Chaturvedi
nectar
in nectars.
the amino acid content
Limodoxvm abortivum
leucine
These
species.
Pais et aZ.,
evolutionary
ornithine
species of Agave (Agavaceae).
also found variation
amino acids within
the presence of
phosphoethanolamine,
amino acids have as yet not been reported
These researchers
pollinators.
nectar
leucine,
insect
was
isoleucine,
nutrition.
7.
The free
of Honeydew
amino acids
(1953)
Mittler
that
reported
the number and amount of amino
in honeydew depended on the extent
acids
in the phloem
were present
has discussed
for
materials
Ewart
honeydew were exactly
(1956)
and Metcalf
in honeydew.
present
concentration
fluctuated
of honeydew and concluded
the origins
have reviewed
(1958)
Mittler
of amino acids
the
with
(1963)
Auclair
the
has given
that
reported
in
general
nectar.
on amino acids
the number and
sap and honeydew
the phloem
the host
of
the raw
for
the literature
development
seasonal
(1955)
that
same as that
and amides
amino acids
Morgenthaler
the plant.
sap of
these
to which
plant.
on the composition
references
of honeydew.
Srivastava
partition
and Vaishney
(1966)
identified
chromatography
in the honeydew exr-reted by Kerria
were:
a- and a-alanine,
glycine,
methionine,
proline,
researchers
found
were absent
from
insects
that
the plant
were reared.
They suggested
of proteolytic
Saleh and Salama, (1971)
honeydew came directly
of either
protein
from
breakdown
that
this
confirmed
that
ingested
as suggested
lysine,
These
and valine.
arginine
maerpphyUa
to tyrosine
on which
difference
in the
could
the amino acids
sap and were not
(1963)
the
presumably
insect
(1969).
by Salama and Rizk
by Auclair
glutamic
acid,
leucine,
enzymes present
reported
the
aspartic
from
amino acids
and
These amino acids
tyrosine
sap of Maghania
were later
These findings
Zacea.
of paper
amino acids
isoleucine,
threonine,
the eight
free
asparagine,
homoserine,
serine,
be due to the action
gut.
arginine,
histidine,
technique
seventeen
amides
acid,
the
using
in the
the product
or
8.
atmospheric
fixation
nitrogen
by Kennedy and Mittler
as suggested
(1953).
Noda,
Sogawa and
on distilled
water,
Hertel
traces
only
Lombard,
substances
observed
that
and small
in the amino acid
sap content
cordata
from
free
amino
the honeydew of
amino acids and two ninhydrin-
leaf
linden
seventeen
These researchers
extract.
fourteen
honeydew contained
compared
to that
the
of
honey.
in detail
by Maurizo
of nectar
liquid
part
by rapidly
nectar
to the warm dry
deposited
is
air
by the forager
is
then
into
bee to
and passively
actively
honey has been described
(1980a).
by the housebee
played
pumping out
its
of
for
reaches
the cells
to as unripened
from
proboscis,
the hive
in the nectar
into
Honey by the Honeybee
transferred
nectar
the end of
onto
sugars
into
of Nectar
and how the housebee
The active
referred
in
free
amounts of proline
engaged in the processing'of
then
were secreted.
detected
substance
the honey derived
the housebee
of
(1981)
in the TiZia
The way in which
the
reared
were
or the phloem
supply
but only fourteen
Processing
of
of water
and Buffa
tiZiae
reactive
nectar
insects
when
the variation
noted
and one ninhydrin-reactive
amino acids
that
the honeydew of AVzus persicae.
Rossetti
EucaUupterus
found
of amino acids
(1977)
in the changes
influenced
acids
Saito
and Kunkel
concentration
which
i
(1973)
honey.
of
involves
the honeystomach
thereby
the concentration
film
a thin
the nectar
exposing
evaporation.
When the concentration
approximately
60% (w/v)
the comb (Park
1933).
it
This-is
is
then
9.
The passive
of
the excess
to reach
the water
temperature
with
an airtight
water
from
the
to prevent
wax capping
surrounding
and thus
air
takes
filled,
of cells
humidity
and the relative
The housebee
It
the ripening
of
content
depends on the level
This
1927,1928,1933).
cells
the evaporation
in the combs.
unripened'honey
days for
movement,
(Park
the hive
this
20% or less.
the amount of air
of
from
water
involves
by the housebee
played
one to three
on average
honey
part
then
the
seals
the re-absorption
of
the risk
eliminating
of
fermentation.
is known that
It
oxidase
and glucose
1974),
(Rinaudo
and these
the honey to the amateur
and the United
for
example
Morse (1974),
(1980).
The description
moveable
frame hive.
W.B. C.,
Dadant,
plastic
traps
Although
on which
crown board,
(1980)
by these
authors
refer
Smith and Commercial
frames
boxes or supers
access
and roof.
of bees.
exist
All
popularity.
and Townsend
to the modern
of hives,
types
in Europe
by many authors;
Smith
a set of
floor
beekeeper
(1976),
several
of
and processing
semi-commercial
National,
namely
vary
which
consist
of
the
up to ten made of wood or
honey comb is constructed,
extension
to control
the extraction
use and regional
components:
and colony,
honey,
given
of Honey
have been described
Hooper
Langstroth,
in
size,
somewhat
following
and the
of America
States
elsewhere
for
available
introduction.
in the
and Processing
Extraction
The techniques
1973a and 1973b and Bergett
et aZ.,
be reviewed
will
invertase
adds the enzymes diastase,
the housebee
for
brood
the bulk
In addition
Some tropical
chamber for
of
there
honeys
queen
the combs and
are
sundry
are gathered
lo.
from either
or clay
wild
containers
which
The extraction
through
with
very
beekeeper
combined
Hooper
for
(Hicks,
(1976)
processing
fine
with
The
strainer.
strainers
mechanical
in
honey
or polishing
the honeys being
involves
have given
and storage
monohydrate
filtration.
or pressure
in detail
heating
controlled
the glucose
the various
honey by the amateur
packaging
0C
to 35
and then
filtering
can result
straining
and Dyce (1980)
the processing,
of
process
1969).
has described
extracted
Townsend (1980)
pressure
strained
straining
or a nylon
and to dissolve
present
is
The normal
process
the extracted
of
the yeast
high
presses
the removal
may have entered
that
material
to warm the honey
This
scale.
of pollen
to destroy
is
utilizes
filter
efficient
The processing
crystals,
once
the honey combs;
of
the honey
(54 mesh to 1 inch)
commercial
free
almost
logs
the uncapped honey combs often
process.
beekeeper
a cloth
semi-commercial
on a large
and extraneous
the extraction
used by the amateur
the uncapping
Then finally
of centrifuges.
the honey during
the combs involves
of honey from
to remove the wax bits
strain
the hives,
by the removal
the aid
with
destroyed
smashed and colonies
the honey from
of
the honey combs from
followed
of
are
hollowed
is complete.
harvesting
of
or from crude straw skips,
colonies
beekeeper.
extensive
used
methods
White
detailed
of honey for
(1978),
accounts
the commercial
producer.
Heather
state
protein
honey
from
upon standing
which
imparts
(caZluna
ling
due to
this
its
unusually
thioxotropic
forms
vuZgaris)
high
content
property
on it.
a gelatinous
of a thixotropic
Hence the
ii.
extraction
honey is different
The honey is extracted
mentioned above.
mechanical
of heather
and processing
inserted
probes or needles
either
into
cells
from that
by stirring
with
of the combs before
being spun out in the usual way or the honey is pressed out of the
combs with
the aid of a heather
be heated for
in plastic
comb honey'
outlets
to ensure perfect
to justify
from
sixty
the extra
Production
the world
of
1983 and 1984 were obtained
No. 21 and 22.
list
The reports
one countries
Australasia,
provided
sizes and sold as
there are sufficient
required
care and attention
combs.
figures
Estimated
convenient
containers
Honey
the years
in the usual
However, on the other hand combs containing
the heather honey can be cut out into
comm rcial
honey can then
two or three days at 400C before bottling
manner (Hooper, 1976).
'cut
The extracted
press.
Africa
honey production
from
the World
the actual
world-wide
and the
and Trade
tropical
Honey Crop Reports
production
or estimated
the Americas,
covering
for
and trade
Europe,
islands.
Production
In 1983 there
estimated
940,000
The estimate
was 934,100
tons
metric
metric
tons
of honey harvested
of honey production
of
in
1983 and 1984 have been shown in Table
of
a small
This
of
could
annual
increase
be attributed
the major
producers
of honey and in 1984 an
in world
to the
ten
2.0.
world-wide.
individual
The continued
honey production
increase
countries
trend
was reported.
in the number of colonies
such as the USSR, the
improved
yield
per
12.
TABLE 2
World
honey production
in 1983 and 1984 in
and trade
ten selected
countries
World
World honey
production*
Country
honey trade**
Imports
Exports
1983
1984
1983
1984
1983
1984
Argentina
28.0
33.0
29,874
29,227
-
-
Brazil
22.0
27.0
1+
-
-
Canada
34.8
44.1
9,752
9,522
239
284
Mexico
64.0
55.0
40,028
59,405
-
-
U. S. A.
93.0
95.0
3,781
3,384
41,715
49,823
German Fedral
Republic
15.0
18.0
12,960
9,409
75,830
66,385
190.0
198.0
16,000
19,890
6.5
7.5
100.0
115.0
Total
USSR
Japan
China***
United
+-
Kingdom
1.2
628
-
-
58,096
-
1,455
-
-
In thousand
metric
-
Preliminary
estimates
-
Based on imports
64,339
28,146
20,764
33,180
20,743
tons.
in metric
of major
tons.
importing
countries.
This value was quoted in the world honey crop report
No. 22 and referred
the
to as a misprint
regarding
in 1984.
actual
amount of honey exported by Brazil
13.
in countries
colony
conditions
in countries
proportion
of
and these
such as Argentina
the honey produced
countries
may not
A considerable
and Brazil.
was for
much surplus
increase
in exports
the country
within
consumption
export
weather
honey.
in honey
Trading
In 1983 a significant
in countries
by 42%, Mexico
such as Russia
an increase
Also,
and favourable
such as Canada and Brazil
such as the United
in
imports
States
have been shown in Table
the European
Economic
and the United
in countries
in 1983 and 1984 for
The largest
net
importers
Kingdom followed
by the United
States
ten countries
of honey were
such as the German Federal
countries
Preliminary
by 19% and Japan by 18%.
and imports
2.
by 90%.
by 48% and France
of honey was observed
of America
of honey exports
estimates
of honey was observed
Republic
of America
and
Japan.
(1980)
Crane
the world
production
Manufactured
Also
pharmaceutical
used directly
Willson
but
from
still
in honey.
that
use honey,
products
is
from mead, cake,
a wide
products
important
(1980).
users
(1980)
agent
see,
of honey
example,
examples
of honey and the whole
to legend
and will
aspect
have been covered
not be further
to health
into
Some honey
for
are given
bee products
cosmetics
candles,
for
figures
wax or other
inclusion
spread
Therein
to archaeology
by Crane
given
such as cough syrups.
as a therapeutic
and Crane
analysis
treatise
there
have also
and trading
are numerous and range
foods.
(1980)
and Willson
is also
Steyn
(1973),
of minority
of honey
in
mentioned.
the extensive
14.
Honey Plants
(1980)
Crane
economic
in Britain,
plants
Europe,
discussed
list
United
and discuss
States
their
such
authors
honey
the major
of America
contribute
the honey
of
Other
significances.
which
species
the characteristics
and Howes (1979)
(1978)
as White
of
and their
plant
She has also
scale.
and distribution,
characteristics
them,
important
the
of honey on a world
as a source
from
has listed
and the rest
the world.
75% of
honey produced.
the total
accounts
for
(CaUuna
sp. ) in the British
the greater
honey.
table
honey crop
is another
if
rearing
Oil
for
not
surplus
nectar
an equal,
honey
eastern
honey.
producers
Northern
United
terms
(Fagopyrum
United
States
Honey from citrus
Thyme and pine
sp. ) is also
forest
becoming
to
that
produced
are well
Russia,
of
to the beekeeper
brood
(Prunus)
by countries
Australia
New Zealand,
paeudoacacia)
produced
are rated
as
Buckwheat
the clover.
China
for
trees.
are produced
in large
a major
Mrunus/pyrus)
blossom
of America,
(Robinia
of America,
fruits
fast
Rumania and Yugoslavia
of quality,
as a unique
recognised
among the fruit
States
)
sp.
(Tilia
lime
(MaZus) and cherry
Apple
about
Honey from heather
sp. ) is
and is useful
such as Hungary,
in
is widely
Honey from acacia
and Tasmania.
in countries
Isles
of honey from clover
quantities
such as Canada,
the remainder.
1984). * Fruit
of nectar
source
honey from
While
(Brassica
(Sims,
in Britain
sp. ) constitutes
of
part
seed rape
are most valuable
Large
(TrifoZium
honey from clover
In Britain,
in North-
quantities
in
parts
and
of Europe.
known to Spain and California.
honeydew honey from Greece,
Also
Eucalyptus
15.
from Australia
Crane,
sixty
and mixed floral
(1984)
Day
and
Walker
to be a major
seven plants
For each plant
world.
information:
its
importance
flowering,
of
species
hundred
the following
and environmental
potential
hazards
in a country;
the
available
family;
conditions;
such as toxins;
time
of
honey yield
recommendations
in a country;
and
in the
of honey produced
have noted
and composition;
honey production
four
and comm n names and the plant
honeydew production;
composition;
increase
source
distribution
flow
nectar
from Mexico.
have identified
authors
and uses;
and honeybee
rating
these
the botanical
the form of plant,
economic
honeys
nectar
the year
of
and chemical
of planting
and the composition
to
and properties
the honey produced.
Moreover,
Europe,
the
Australia,
Canada are well
honey producing
countries,
is kept
at
on file
the United
of honey
sources
the
Kingdom and by national
the Uniýed
documented
although
International
in
of America,
States
the other
compared with
such documentation
Bee Research
organisations
that
exists
Association
with
elsewhere.
Honey Composition
In the following
topics
subject
only
the major
will
be briefly
to the present
and reviewed
study
in detail.
section
and other
literature
reviews
reviewed.
relevant
sections
of
However,
published
covering
importance
for
research
major
will
perpherial
to the particular
topics
of
importance
be described
16.
Factors
affecting
composition
(1980c)
White
has reviewed
of honey and suggests
the origin
influence
which
on the composition
is
the honey composition
that
Also,
the nectar.
of
the literature
the production
that
internal
the
of nectar
are
dependent
upon
factors
and external
important
contributory
to the honey composition.
factors
(1980a) has extensively
Maurizo
in detail
species,
are
some of
humidity,
and sunshine
the
outlined
internal
the
type-
and the wind,
the
or cultivated
variety
the
that
the age and maturity
surface,
the
reviewed
the
of
soil
time
of
of
size
internal
and external
fertilizers,
the
Shuel
that
soil
temperature
of
the day
(1982)
has also
the length
Similarly,
factors
belongs
such as the
the day and year,
important.
on the planto
factors
External
are also
and the
the plant
to which
and the use of
the production
the flower
the flower
of
races
factors.
influence
factors
and external
She has suggested
of nectar.
nectary
internal
how these
and discussed
the literature
influence
the
secretion
of nectar.
of beekeeping
The affects
techniques,
of honey either
by the beekeeper
the composition
of honey have been described
Morse
(1974),
respectively.
the commercial
processing
Townsend (1980).
and Ichimura
other
of
storage
Kushnir
and White
and Subers
(1978).
on
producer,
by Hooper
factors
of honey and the changes
by White,
(1974)
or by the commercial
have been described
The conditions
on the composition
been described
Also
and processing
extraction
(1976)
encountered
by Rodgers
and
during
(1980)
have a considerable
induced
by storage
(1964),
Echigo,
and
influence
have
Takenaka
17.
Physical
(1980d)
White
physical
has extensively
in water,
Moisture
the literature
reviewed
on the
Honey is a supersaturated
of honey.
properties
of carbohydrates
with
normally
solution
15% - 17% water.
only
content
White
of
of honey
properties
four
hundred
and ninety
showed that
samples
(Dyce,
and granulation
has been reviewed
1980).
the average
its
content
The amount
against
fermentation
of moisture
content
stability
The determination
(1969).
by White
extensively
moisture
of honey was 17.2%.
in honey determines
present
of water
(1962)
et aZ.,
Colour
Brice
syrup,
et aZ.,
caramel
the colour
Wootton,
six
Australian
Faraji-Haremi
honeys
accompanied
content.
Wootton,
not
in
an extensive
clearly
related
White
(1980d)
and Johnson
were stored
by appreciable
at
(1976)
are
of honey,
marple
similar.
These
physical
examination
reported
that
changes
in the
to
sugars
the extent
has reviewed
and free
total
(1976)
the effects
nitrogen
reported
amino acids
of darkening.
when
of honey colour
500C, darkening
Edwards and Faraji-Haremi
the amount of
on the colour
sugar products
out
carried
the colourants
of honey.
was not
changes
showed that
and other
solution
have also
researchers
of
(1956)
that
in honey were
-
of
storage
temperatures
of honey.
CrXstallisation
is basically
It
honey
to
its
the crystallisation
monohydrate
form.
of
The presence
the glucose
or absence
present
of nuclei
in
18.
influences
the onset
dependent
upon the glucose/water
problem
which
content
of
for
[glucose]/water
of honey.
ratio
as being
ratio
highly
as being
employed
the basis
of Austin's
values
of
would
on four
These values
hundred
acacia
and ninty
The subjects
(1977),
(1978),
that
predictably
samples
for
are
White
towards
tendency
regarded
workers
White
lead
(1962)
low in glucose
of honeys,
whereas
to rapid
granulation
carried
examined.
honey types
content
and high
of honey have been reviewed
(1980)
on
1.7 and lower
of
of honey they
non-crystallising
this
ratio
et al.,
values
non-granulation
of crystallisation
White
Thixotropic
which
the use of Austin's
were based on the measurements
There are exceptions
as tupelo,
(1924).
reported
2.1 and higher
tendency
who suggested
research
the
[glucose]-water/[fructose]
and Silsbee
ratio
and reviewed
the use of
as a measure of
to the
ideal
creates
the crystallisation
these
with
to a solid.
out
significant
associated
were generally
This
proposed
(1962)
et al.,
equivalent
by Jackson
(1958)
in moisture
increase
the
were studied
predicting
Furthermore,
crystallisation.
ratio
for
was White
It
crystallisation
on Austin
Later
The major
the honey.
to ferment
present
affecting
by Dyce (1931).
is
1980d).
phase above the crystals.
the yeast
The factors
ratio
and speed are
extent
(White,
from crystallisation
results
the liquid
conditions
its
but
of crystallisation
such
in fructose.
by Doner
and Landis
and Kevin
(1982).
thixotropy
in ling
(CaUuna
properties
Pryce-iones
(1944)
studied
honey
in that
the honey mobilised
solid
state.
Pryce-Jones
isolated
the
upon stirring
by alcohol
from
its
precipitation
sp. )
gelatinous
a material
19.
caused the
which
he demonstrated
thixotropic
by adding
that
were imparted
properties
this
to the clover.
to contain
1.86% of
contained
1.5% total
protein.
According
(1978)
to White
in Manuka (Leptospermium
(1957)
et aZ.,
honey of
India.
is
indeed
a protein
honey
whereas
other
the thixotropic
of molecular
weight
Deodikar
(Cmvi
Salford
over
agent
50,000.
samples
thixotropy
observed
in Karvi
work at
thixotropic
One sample of heather
also
thixotropy
that
Further,
honey of New Zealand.
project
1984 - 1987 has established
honey
Pryce-jones
similar
Subsequent
honey.
the protein,
scopatium)
reported
honey.
to clover
material
honey was found
about
in heather
property
caUoso)
the period
in heather
Hamilton
(1987)*
Chemical
of honey
composition
Honey contains
into
the following
minerals,
major
vitamins,
compounds which
carbohydrates,
esters
and volatile
on rare
and pollen.
can be arranged
amino acids,
hydroxy-
components,
occasions
proteins,
toxic
compounds
in low concentrations.
can be present
The amino acids,
be reviewed
groups:
acids,
lipids,
methylfurfural,
two hundred
nearly
later
proteins,
on in
the
and toxic
pollens
substances
will
the honey
solids.
introduction.
Carbohydrates
Glucose
The presence
nigerose,
and maltose
and fructose
of
kijibose,
sugars
constitute
such as erlose,
a, ý-treholose,
have been reported
(1959),
Hoban
and
85% of
about
isomaltose,
gentibiose,
by White
maltulose,
laminarebose,
and Maher (1953),
Watanabe and Aso (1960)
and Siddiqui
turanose,
sucrose
White
and Furgala
(1967).
20.
Siddiqui
identified
also
researchers
ten honey
and one pentasaccharide.
These
3-a-isomaltosylglucose,
melizitose,
isomaltotriose,
maltotriose,
of
isomaltopentose.
panose and
1-kestose,
of honey have been reviewed
The sugars
(1970),
(1977),
Doner
the presence
reported
one tetrasaccharide
trisaccharides,
Siddiqui
(1968)
and Furgala
by Thawley
extensively
White (19800
(1969),
(1982).
and by Prince
Minerals
The three
of
total
of minor
the amount of principal
ash,
the amount
and the
constituents
which
constituents,
metallic
of honey are:
the ash content
of
aspects
in extremely
occur
identities
minute
amounts.
and his
Schuette
the presence
by White
a lower
(1962)
mineral
than
the findings
summnrises
(1978).
those
(1975)
McLellan
of potassium
light
and his
reported
secretion
a relationship
was confirmed
classes
colour
honey
darker
of
Schuette
of
relationship
honey of
in honey than calcium,
more potassium
the rapidity
This
in that,
content
reported
in honey and suggested
elements
of pigmentation.
et aZ.,
by White
colleagues
of mineral
the degree
with
(1932,1937,1938,1939)
types.
there
Table
3
as presented
colleagues
that
has
was generally
magnesium or sodium due to
by the plant.
Vitamins
The following
thiamine,
acid,
vary
of
vitamins
riboflavin,
niacin,
folic
biotin,
to a large
different
been reviewed
have been reported
honey
and vitamin
acid
extent
between
types.
by White
ascorbic
acid,
K.
samples
pyridoxine,
in honey:
panthothenic
The amount of vitamins
of
The occurrence
(1978 and 198W).
to occur
similar
honey and also
of vitamins
in honey have
21.
TABLE 3
Mineral
content
of honey - from
(1932,1937,1938,1939)
Mineral
elements
the data
as given
Honey
colour
of
by White
Schuette
et aZ.,
(1978)
Parts
Number of
samples
Range
100-588
115-4733
per million
Average
Potassium(K)
Light
Dark
13
18
Sodium(Na)
Light
Dark
13
18
6-35
9-400
18
76
Calcium(Ca)
Light
Dark
14
21
23-68
5-266
49
51
Magnesium(Mg)
Light
Dark
14
21
11-56
7-126
19
35
Iron(Fe)
Light
Dark
10
6
1.20-4.80
0.70-33.50
2.40
9.40
Copper(Cu)
Light
Dark
10
6
0.14-0.70
0.35-1.04
0.29
0.56
Manganese(Mn)
Light
Dark
10
10
0.17-0.44
0.46-9.53
0.30
4.09
Chlorine(Cl)
Light
Dark
10
13
23-75
48-201
52
113
Phosphorous(P)
Light
Dark
14
21
23-50
27-58
35
47
Sulphur(S)
Light
Dark
10
13
36-108
56-126
58
100
Silica(S'02)
Light
Dark
10
10
7-12
5-28
205
1676
9
14
22.
Acids
of honey contribute
The acids
and its
micro-organisms
an enzyme catalysed
is
the predominant
reviewed
flavour.
reaction
of
(1969),
by Thawley
Gluconic
glucose
The acid
acid.
its
towards
the product
acid,
oxidase
of
of honey,
on glucose
of honey has been
composition
Stenikraus
against
stability
(1971)
et aZ.,
and White
(1978).
Volatiles
of honey which
The volatiles
flavour,
and
such as aroma
the
introduction
components
Ten-Hoopen
acetylaldehyde,
(1963)
quantities
in rape
reported
the presence
compounds they
and twenty
reported
and n-propanol,
be the products
of
ester
amino acids.
and unifloral
honey for
identified
and
technique
fifty
of gas liquid
and mass spectroscopy.
of a gas chromatography
of
than
in thyme.
in greater
separated
from honey.
hydrolysis
These researchers
3-methyl-
or may arise
from
(1983)
for
compounds responsible
two compounds.
these
and suggested
and Frattini
analysed
(1986)
used the
and a mass spectrometer
using
isolated
Multifloral
flavour
their
of gas liquid
Bonaga and Giumanini
could
corresponding
These researchers
combination
honey.
the one hundred
of
2-methyl-l-butanol,
upon storage
(1964)
Hadorn
in lavender
over half
and
formaldehyde,
and diacetyl
identified
Belliardo
Bicchi,
a
chromatography
anthranilate
in pentanol,
an increase
1-butanol
honey
of methyl
(1965)
Cremer and Riedmann
the presence
isobutyraldehyde
and clover
with
have been identified.
reported
acetone,
characteristics
However,
studied.
gas liquid
of
its
towards
has been little
the technique
of
number of volatile
contributes
chromatography
the combination
fifty
volatile
23.
from a unifloral
components
researchers
reported
main component
for
floral
that
ninteen
sativa)
honey.
components
had not
in honey.
They suggested
(3-aminoacetophenone)
the mixture
of
these
of
to be present
reported
previously
(Castanea
chestnut
These
been
that
the
may be specific
source.
Hydroxymethylfurfuralý
(HMF) results
5-Hydroxymethylfurfural
dehydration
and heat
Schade,
for
100g of honey.
indicative
(1980a)
reported
eight
of honey.
This
the
(1966)
subject
of
an average
samples
between
of commercial
was based on the data
and Chandler
0.06
et aZ.,
amongst
heat
that
mg of HIIF
during
treatment
amounts of HMF
White
and overheating.
for
seven hundred
one thousand
honey
to be 1.24 mg per
published
by Duisberg
(1974).
White
(1964)
and Subers
to 0.20
as excessive
HMF content
method
and conformational
Kushnir
reported
freshness
(1955)
the variability
White,
be limited
the loss
of
and twenty
Hadron
(1978)
White
of honey should
processing
are
honey contains
catalysed
in honey.
Winkler's
using
and heating,
storage
HMF formation.
fresh
showed that
per
of
influencing
factors
(1958)
the acid
present
HMF in honey demonstrated
the effect
of
of fructose
treatment
Marsh and Eckert
quantifying
honeys
from
(1980a)
100g
and
has reviewed
in detail.
Honey Proteins
The nitrogen
content
of proteins
and small
compounds.
A considerable
presence
of proteins
of'enzymes.
will
of honey
amounts
is
due mainly
of amino acids,
portion
The isolation,
now be reviewed.
to the presence
and other
of honey protein
characterisation
nitrogen
is due to the
and properties
24.
Proteins
The protein
honey from artificial
to solubility
have been described
groups
Paine
(1934)
et aL.,
from honey by the
technique
components
collodial
three
these
and 73,000
146,000
weights
molecular
White
(1962)
et al.,
to a protein
honey by the
techniques
Of
electrophoresis.
of approximate
third
detected
molecular
of
was a polysaccharide
9,000.
of about
weight
and the
(1953)
Helvey
boundary
two were proteins
components
separated
material
in buckwheat
and moving
of ultracentrifugation
(1978 and 1980c).
by White
of ultrafiltration.
present
or by assignment
tests
the colloidal
studied
of
solution,
immunological
or by alcohol,
acid
phosphotungstic
The assessment
by tannin
material
to distinguish
utilized
and blends.
admixtures
of proteinaceous
precipitates
three
of honey was initially
content
reported
content
of 0.25% for
and Kushnir
(1967b)
an average
four
value
hundred
corresponding
and ninety
samples
of honey.
White
by the
fractions
and ion-exchange
that
four
bee and two of
those
or greater
400,000
that
originated
molecular
results
appeared
weights
from
weight.
to seven protein
starch-gel
honeys
From these
had molecular
fractions
than
floral
fractions
protein
these
filtration,
four
These researchers
from eleven
to the bees.
by sugar-feeding
deduced
of gel
chromatography.
preparations
protein
While
technique
between
separated
electrophoresis
used the crude
and honey produced
the above authors
to originate
from
the
of 40,000
and 240,000.
the plant
were of
98,000
25.
distinct
five
(1975b)
and Diemar
Bergner
peaks by chromatography
that
reported
researchers
the bee and were identified
from
While
oxidase.
and glucose
on Sephadex G200 gel.
five
these
of
three
components,
two that
These
originated
invertase,
to be enzymes
the
into
the honey proteins
separated
diastase
from
originated
the plant
identified.
were not
(1978)
and Rudyj
White
determined
samples of honey using
and forty
These researchersIreported
seven hundred
for
the proteins
the Lowry photometric
a mean value
analysis.
169 mg of protein
of
per
100g of honey.
Bagdanov
to determine
honeys.
(1981)
the proteins
Croft,
Histry
and Washington
on the basis
bands.
and its
Richie
by the technique
for
band pattern
protein
four
the
noted
sensitivity
to resolve
and Guenther
technique
(1986)
in
gel
likely
of any of
of
as
the presence
to characterise
geographical
these
was useful
detection
enabled
foreign
honeys.
were unable
its
to
content
floral
were able
or absence
from bees fed on pollen
had been obtained
Reeder,
regard
of presence
They also
of adulteration
(1986)
method
was no significant
These researchers
sulphate.
honey sample with
any particular
there
reagent
and sixteen
bands on 10% polyacrylamide
protein
sodium dodecyl
that
ten honeydew and twenty
between
many as eleven
protein
an average
100g of honey and noted
differences
origin
reported
colour
Swiss honeys
of eighteen
These researchers
129 mg per
of
used the Bradford/Bio-rad
eleven
in detection
of honey
that
substitutes.
separated
isoelectric
of ultra-thin
layer
and intensity
were analysed
honey proteins
focussing
and scans
by a chemometrics
programme
26.
location
geographical
that
reported
to distinguish
the ability
revealed
of patterns
These researchers
computer.
on a mainframe
the
clustering
honey based on the
of collection.
Enzymes
The enzymes of honey have up to now been studied
from
the view
factors.
adverse
and other
invertase,
glucose
and systemmatic
and kinetic
properties
together
in honey but have as yet
will
the
reviewed
literature
(1980c) has also
have not
which
The origins
as being
chiefly
(1964),
Rinaudo
(1986).
Other
by
and
Vansell
ZaleWski
not
appropriate
(1978)
the presence
been studied
the
sources
and Freeborn
4.
Other
enzymes
have been shown to be present
and therefore
characterised
detailed
in honey.
and
White
of enzymes such as esterases
above have been identified
Ammon (1949),
(1973a
such
for
in detail.
honeybee
aZ.,
(1965).
in Table
has extensively
the enzymes listed
of
of enzyme activity
references
on the enzymes present
noted
et
been well
White
be reviewed.
not
with
phosphatases
and alkaline
type
name of enzymes,
enzymes have been tabulated
these
such as acid
in honey have been confirmed.
and catalase
oxidase
of enzymes by heat
of enzymes such as diastase,
The presence
The trivial
each of
and destruction
of quality
point
predominantly
and
1973b)
as pollen
(1929),
Bergner
Schepartz
and
Stadelmeier
and nectar
and
and
Sabir
have
been
(1977),
Subers
and
Bergner
suggested
Giri
(1938)
27.
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28.
Key to references
and notes
regarding
Table
4
Key to references
(1967a)
and Kushnir
1.
White
2.
Lampitt,
3.
Rina do et aZ.,
4.
White
5.
Rina do et aZ.,
6.
Takenaka
and Echigo
(1975)
7.
Takenaka
and Echigo
(1978)
Schepa,
9.
Schepartz
(1965a)
10.
Schepartz
(1966a)
11.
White
12.
Schepartz
13.
Stadelmeier
Hughes and Rooke (1930)
(1973b)
(1967a)
and Kushnir
(1973a)
(1964)
Subers
and
8.
and Subers
(1964)
(1966b)
and Bergner
(1986)
Notes
Name
Systematic
Enzyme No.
Trivial
3.2.1.2
a-1,4-glucan
hydrolyase
4-gluconoý
a-amylase
3.2.1.2
a-1,4-glucan
maltohydrolase
$-amylase
3.2.1.20
a-D-glucoside
1.1.3.4
O-D-glucose:
reductase
1.11.1.6
H202 :H202 oxido
Obtained
4+
glucohydrolase
02 oxido-
reductase
from Commission
a-glucosidase
-I
a-glucosidase
- II
(1)
Temps = Temperature
(2)
Refs
= References
I
on Enzymes,
Name
a-glucosidase
Glucose
oxidase
Catalase
1961.
Name allocated
to enzyme by researchers
1978.
Takenaka and Echigo,
0
29.
Honey Amino Acids
A relatively
is
have attempted
of honey
(1930),
Tillmans
were:
Schuette
of honey
content
in honey.
present
the amino acids
These workers
and Templin
Schuette
amino acids
to utilize
authenticity.
the nitrogen
of
portion
to the free
ascribed
its
small
I
Several
in order
workers
to determine
(1927),
and Kiesgen
(1944)
and Baldwin
and Vavruch
(1952).
Komamine (1960)
detected
the presence
and imported
acids
glutamic
cysteine,
tyrosine
honey also
the Finnish
imported
methionine
and the
asparagine
and glutamine.
Phadke (1962)
by Apia
produced
the honey graded
twelve
into
Apart
of
sets
reported
cerena
and valine.
samples
contained
the light
from
proline,
these
amino
ethanolamine
and
y-aminobutyric
contained
the presence
acid,
of proline
samples.
the dark
that
lysine,
arginine,
noted
in Finnish
a- and a-alanine,
were:
glycine,
contained
chromatography
amino acids
acid,
He also
in both
to be the highest
of paper
These amino acids
threonine,
serine,
technique
the following.
of
honeys.
acid,
aspartic
the
using
tryptophan
colour
classes
of honey
and tyrosine
whereas
colour
classes
did
not
contain
these
amino acids.
and Sohonie
Kalimi
Mahabaleshwar
were absent
in both
(India)
(1964)
honeys,
in that,
some variation
proline,
in the Gela and Harda honeys.
the Gela and Jambul
in any of
observed
the honeys.
honeys.
serine'and
Histidine
Isoleucine
in four
tyrosine
was absent
was not
present
30.
(1971)
Petrov
chromatography
in
acids
three
the presence
of
samples
of Australian
honeys;
stingy
(EucaZyptus
red gum
acid,
leucine,
tyrosine
did
not
whereas
isoleucine
honey.
In comparing
(Curti
lysine,
(1964)
and methionine
1966) Petrov
and Riganti,
most of
the amino acids
but not
arginine
that
to the differing
amino acids
amino acids
Petrov
varied
from
(1974)
using
2,000
or threonine,
those
the Italian
of
honey contained
Italian
honey
honeys.
This
in
different
the amounts'of
He suggested
collected
secretions,
the pattern
of
The content
of
this
widely,
nanomoles
amino acid
seventeen
researcher
amino acids
that
fed bees
sugar
due
an automatic
and one Trifolium
using
presumably
to 17,000
of
of amino acids
honey
in the Australian
detected
the
those
with
in the Australian
varied
similar.
the presence
species.
that
that
reported
of honeybee
content
was remarkably
quantitatively
results
and cysteine.
the amounts of amino acids
whilst
serine,
the Indian
honey with
found
clover
arginine,
phenylalanine
were present
(1972)
Hahn
and
Bergner
his
that
were not
the Australian
and white
proline,
In comparing
noted
(EucaZyptus
histidine,
'phenylalanine,
Petrov
amino
bark
alanine,
glycine,
hydroxyproline,
cysteine,
contain
acid,
and valine.
and Sohonie
of Kal-imi
glutamic
were:
and paper
sixteen
camaZduZensis)
These amino acids
cysteine,
hydroxyproline,
threonine,
ion-exchange
of
to detect
repens).
aspartic
technique
was able
macrorrhyncha),
(TrifoZium
the
using
variation
in the nectar,
the
per
observed
free
g of honey.
analyser
estimated
in six
amino acids
and also
total
Eucalyptus
the variation
between
different
was due to the amounts
those
secreted
by the bee
31.
formed
and those
as degradation
(1975)
Davies
acids
in ninty
honey
sugar
carried
eight
products
out an extensive
He was the
of amino acids
source
of a honey sample as well
that
by their
particularly
have a high
honey from four
in the ratios
sources
of
between
method of pollen
honeys
they
of
Thus,
sources
the
examined,
except
amino acids
also
increased
due to protein
by condensation
the honey obtained
observed
in
breakdown
reactions.
during
that
some honeys
the variation
that
of
more than balanced
between
the amino acid
the determination
the highly
subjective
work was required
use.
that
the
of all
the
reported
storage
from
the amount of
and they
amino
are variations
official
(1976)
decreased
sixty
further
that
in honey.
to characterise
method for
be recommended for
content
there
in place
the bees,
that
of
proposed
of honey,
generally
amino acids
same area,
Davies
recognised
analysis
while
He suggested
analysis.
amino acid
These researchers
of
by a selection
Edwards and Faraji-Haremi
Wootton,
free
suggested
to show that
samples
the method could
total
also
sources
Davies
honeys
since
be used as an instrumental
could
the geographical
before
areas
certain
of bees.
content
Davies
geographical
is much greater.
analysis
proline
that
be easily
could
of non-
the geographical
species
used the amino acid
He was able
ratios.
as the
were the major
Davies
to suggest
products
content.
proline
on in 1976,
Later
sugar
lower
and honeydew,
nectar
acid
could
non-honey
first
the amino
of
samples
be used to determine
ratios
reported
survey
of honey and eight
samples
products.
of pollen.
the
tree.
some individual
suggested
any loss
tea
that
changes
of amino acids
32.
The use of gas chromatography
amino acids
and protein
(1978).
Battaglini
and four
nectar
total
Gilbert
the United
from
These workers
analysed
of
samples
the Canadian
the Australian
acid
in order
Gilbert
of a honey
samples
Argentina
statistically
of honey
and Canada.
using
the canonical
to show good discrimination
occurred
and Canada.
between
They observed
and
by omitting
the free
that
suggested
in conjunction
with
identification
a reliable
between
the Argentinian
be discriminated
et aZ.,
to obtain
(1981)
Siddiqi
of paper
confirmed
the
She was able
samples
amino
another
of
of honey using
the
semi-quantitative
to demonstrate
amino acid
the presence
of mass spectroscopy.
such as citrulline,
analyser
assessment
a combination
Some samples were
and chromatography.
on the automatic
technique
as unusual
ninty
analysed
electrophoresis
analysed
by the
amino
five
Argentina
only
and could
group.
different
of
of origin.
country
also
results
method must be employed
analysis
technique
their
from Australia,
groups
free
honey.
Australia,
Kingdom,
Kingdom group
the United
the amounts of
spectrum
forty
analysed
They were able
analysis.
variates
groups
(1981)
et at.,
four
in the
differences
between
the amino acid
and
twenty
analysed
in
the proportions
and
that
the free
out by Bosi
and observed
not be used to characterise
sample could
obtained
workers
in the ratio
content,
They concluded
acids.
that
These research
amino acids,
and protein
of honey were carried
honeydew honeys
amino acid
to determine
technique
and this
used for
of pipecolic
Other
hydroxypipecolic
all
samples.
acid
amino acids
acid,
technique
in honey
grouped
kynurine
33.
were identified
and methyl-histidine
use of
be characterised
on the basis
proportions
relative
only
the presence
(1982)
Kanematsu et aZ.,
ratio
acid
honeys
vetch
(AstergaZus
from
detected
by the
her
technique
could
beekeepers
with
Davies
and Harris
analysis
between
(1982)
of
amino
British
found-that
the
was poor
compared
results
by plotting
to
the
identity
success
that
the
isoleucine/leucine
ratio
isoleucine/
the
honeys
acacia
by use of complex
that
it
was possible
honeys.
testing
of
from
were differentiated.
demonstrated
acids
the
originating
the
acid
could
content
Also
By plotting
ratio.
and Foreign
of
amino acid
honeys
of
by plotting
showed that
sinicus)
honey
and twelve
origins
the total
Rumania and Hungary
China,
classify
revealed
from various
the proline/glutamic
against
ratio
Japan,
statistical
that
not
be distinguished.
could
proline/phenylalanine
against
leucine
also
ratios
workers
Japan and China were distinguished
ratio
of paper
by plotting
that
against
and acacia
milk
but
one hundred
honeys
These research
proline/aspartic
Chinese
one another,
against
be distinguished.
the clover
and
to the fact
owing
trained
analysed
to demonstrate
and was able
amino acids
electrophoresis
pattern
by scientifically
and their
patterns
outlay.
capital
samples
methods
and
sample could
importance
on the
Furthermore,
analyser.
in the field
be applied
paper
amounts of amino acids
amino acid
automatic
little
trace
of
a honey
amino acid
a characteristic
provided
R
values
ARG
their
that
following
obtained
and chromatography
electrophoresis
not
its
of
Emphasis was placed
chromatography.
it
demonstrated
She also
standards.
from
These
by
the
canonical
to
correctly
researchers
ratio
variates
method
analysis
34.
for
the twenty five
results
could not be used more widely
further
data to test
for
Palma, Fontanarrosa
those
with
conformed
honeys
the Japanese honeys
to the
addition
did
not
(1971).
contain
such as season,
contained
honeys.
hydroxyproline,
and methionine
They also
noted
in
and tryptophan
identified
they
findings
their
These researchers
cysteine,
methionine
amino acids
sixteen
that
isoleucine
contained
in the Australian
present
were not
by Petrov
but
and threonine
phenylalanine
that
due to factors
(1982) reported
and Vigil
observed
the Indian
that
which
having collected
without
the variation
the
sampling and laboratory.
climate,
found
They suggested that
samples they used.
in the Australian
honeys.
(1982)
El-Sayed
of aspartic
amino acid
certain
acid.
and glutamic
acid
This
author
were
examined
high
suggested
identification
be used for
could
ratios
honeys
the clover
samples had a relatively
these
and that
representative,
that
reported
content
that
of country
of origin.
(1983)
Carter
of honey from a single
composition
of
that
is different
noted
Carter
also
individual
are negligible
However,
reported
researcher
of a unifloral
also
of
This
1982.
source
the variation
studied
in
terms of
in respect
considering
during
that
honey from a mixed
a mass of
overall
summer season
composition
during
content
that
than
the
season.
the contributions
in a jar
amino acid
1 to 2 Kg of pollen
floral
He
auguatifolium.
made by the pollens
its
the
amino acid
to the effect
to
amino acid
hive
in the amino acid
made calculations
amino acids
the free
namely Epilobium
source
the variation
its
in
of honey
composition.
which
is usually
35.
in a hive
present
the twenty
of
sixteen
honeys.
of Fiji
threonine
in the following
glutamic
Robinia
alanine,
leucine,
honeys
class
amounts
valine,
and cystine
honeys,
e. g. buckwheat
and Phacelia.
evident
in dark
class
colour
honeys
methionine,
and valine.
sanifoin
tanacetifoZia.
free
In contrast
Chepurnoi
of
colour
light
light
class
proline
in the
than
annuus),
amino acid
the dark
of
(Onobrychis
(HeZianthus
sunflower
to those
compared
lysine,
sp. ),
was an important
present
asparagine,
tyrosine
(Tilia
lime
pseudoacacia,
threonine
that
observed
colour
were:
(.Fagopyrum sp. ) and PhaceZia
buckwheat
amounts,
threonine,
serine,
honeys were:
These monofloral
tyrosine,
the main amino acids
histidine,
proline,
viciifoZia),
honeys
glutamine,
phenylalanine,
in highest
that
reported
monofloral
acid,
in varying
amounts.
(1983)
Chepurnoi
leucine,
that
noted
were found
in lowest
and cysteine
the presence
reported
seven common amino acids
They also
and glycine
the amino acid
honey produced.
Wimmer and Vakamoce (1983)
Poncini,
of
influence
most certainly
the final
of
composition
could
was more
colour
class
honeys.
(1983)
Campus, Madau and Solinas
honeys
of
amino acids
per
constituted
proline
that
and reported
cysteine,
that
on average
examined
on average
69% of
and tryptophan
methionine
honeys
these
100g of dry matter.
twenty
contained
They also
the
total
six
observed
amino acids
were present
Sardinian
73.42
mg
that
and
in very
small
amounts.
Bonafaccia
of
dubious
origin
et aZ.,
(1984)
contained
reported
less
total
that
free
commercial
amino acid
honey
and less
samples
36.
than
proline
composition
acid
derivatives
o-phthaladehyde
of
sixteen
fluorescence
acids
the following
and heath.
leatherwood
that
could
ratios,
amino acid
forty
for
five
French
individual
honey
free
of
amino
The
chromatography.
workers
Tasmanian
clover,
heath
that
reported
They also
amino acids.
proline/phenylalanine
be used to distinguish
hydroxyproline,
and gave low
acids
dandelion,
rape,
free
of
minutes
was because
This
the presence
These research
level
honey had the highest
researcher
twenty
within
of proline,
imino
ion-exchange
acacia,
This
and cystine.
examined
of
Chestnut,
were:
sources
the
sources
the technique
using
the presence
cysteine
(1986)
Speer and Montag
samples
were detected
with
to separate
in honey.
not be determined.
react
with
yields
from
amino
speed liquid
was able
of amino acids
could
does not
o-phthaldehyde
detection
however,
and cystine
of high
technique
amino acids
sample application,
cysteine
the
using
and fluorescence
chromatography
that
the free
samples although
was similar.
(1986)
Barnard
reported
honey
the genuine
reported
and proline/tyrosine,
and German heath
honeys.
Honey Pollens
is
Pollen
and adult
the chief
bees.
Without
source
1969).
has been discussed
in detail
The chemical
kinds
of
flowers
two different
The collection
composition
varies
plant
by Free
species
carried
of pollen
(1970)
of pollen
enormously.
both
the honeybee
the proteins
(Howells,
and grow
for
of proteins
cannot
develop
by the honeybee
and Butler
(1972).
from
different
obtained
Analysis
the larval
of pollen
from
out by Todd and Bretherick
thirty
(1942)
37.
that
revealed
pollen
5% fats,
about
oils
elements
magnesium and iron
were also
The presence
present.
acids,
organic
of other
sterols,
which
seven plants
However,
world.
are a major
only
acid
noted
in the pollen
of
the presence
six
examined.
was no species
difference
which
Lotti
determined
pollens
they
in
the
will
the presence
Virtanen
and pipecolic
plants.
reported
from
of
high
an extraordinarily
sixty
four
(1968)
in the amino acid
families
plant
that
reported
content
there
of eleven
grass
examined.
and Anelli
(1972) using an automatic
the free amino acid composition
belonging
and sixty
of hydroxyproline
and Jolliffe
Shellard
pollens
(1964)
in the pollen
amount of proline
they
yield
of pollens
reported
wind pollinated
and Musatova
Britikov
(1974).
in Zea mays pollen.
and hydroxyproline
acid
(1955)
and Kari
hundred
composition
(1955)
Grommer and Lunden
a-aminobutyric
four
grain,
of honey produced
source
the amino acid
and Linskens
the colour,
for
listed
and those
of Pollens
The Free Amino Acids
Nielsen,
shown to be
in detail.
here
be considered
such as potassium,
and pigments,
have listed
30% carbohydrates,
such as amino acids,
by Stanley
of pollens
composition
and chemical
acids
in detail
(1984)
Day
and
Walker
constituents
nucleic
above have been reviewed
Crane,
11% water,
and waxes and mineral
calcium,
phosphorous,
21% proteins,
contained
to fourteen
botanical
amino acid analyser
of twenty hand harvested
families.
These research
the free amino acid nitrogen
workers reported
that
fraction,
high
a
95%
to
of soluble
up
nitrogen
represented
content
of the pollens.
38.
id
were the predominant
ac
and glutamic
acid
from citrus
pollens
that
flowers.
cultivar
pollen
stored
flower
(1980)
McCaughey and Wintermute
Gilliam,,
also
than
of proline
amounts
aspartic
in the
amino acids
These researchers
higher
contained
that
reported
noted
the
pollen.
(1980)
Kauffeld
in the pollen
were present
acids
acid,
lysine,
arginine,
tryptophan,
tyrosine
and valine.
amino acids
glycine,
lysine,
the technique
of paper
by Apis
collected
that
reported
and serine;
proline
norvaline,
aminobutyric
lysine,
glutamine,
acid,
cysteine,
arginine,
serine,
proline,
of pollen
contained
(1982)
showed species
arginine
and tyrosine.
contained
the
that
showed
the monofloral
pollens
These research
workers
alanine,
lysine,
pollen
methionine,
hydroxyproline,
alanine,
contained
leucine,
and Jaglandaceae
and valine;
variation,
aminobutyric
hydroxyproline,
glycine,
by
ten amino acids
Ranunclaceae
observed
and valine
noted
and proline
hydroxyproline,
tryptophan
threonine,
Zhu and Jiang
serine
proline
phenylalanine,
contained
acid,
glutamic
arginine,
acid,
in
carpatica.
Omipositae pollen
leucine,
threonine,
researcher
identified
chromatography
meUifera
glutamic
a year.
(1981)
Ceausescu and-Mosarie
acid,
serine,
phenylalanine
throughout
variation
considerable
These amino
isoleucine,
This
amino acids
meUifera.
proline,
phenylalanine,
methionine,
ninteen
aspartic
hydroxyproline,
histidine,
glycine,
by Apis
collected
a- and 0-alanine,
were:
the following
showed that
leucine,
pollen
methionine,
and tyrosine.
that
the free
in that,
whereas
pollen
amino acid
pollen
of Pinus
of Ginko biZoba
content
eUiottii
contained
39.
Nadezhdin
(1983)
et aZ.,
constituted
approximately
researchers
noted
free
the total
Siberian
the
the pollen
(1984)
Sharma
and
Baruch
and type
that
than
amino acids
43% of
pollen
larch
observed
amino acid
contained
more
conifers.
in
wide variation
from
These
amino acid.
pollen
the other
of
in the pollen
of amino acids
the free
that
reported
ninteen
the number
they
plants
examined.
(1984)
Naumkin
methionine,
phenylalanine,
and valine
in the pollen
(Fagopyrum
buckwheat
were:
(Centaura
sp. ),
sp. ),
whereas
of
linden
(1985)
amounts
dmpacea
in both
compared
reported
the free
to that
proline
cornflower
(Sonchus
(Archium
reported
in amino acids
to be the poorest.
amino acid
of
content
rape pollen.
in large
was found
ZanceoZata
of Cunninghamia
the pollens
and ý-alanine
researcher
were found
that
sativue),
burdock
This
that
tyrosine
sow-thistle
to be the richest
found
was high
Feo et aZ.,
hirsut=),
sp. ).
lysine,
These plants
pr-atense),
and musk-thistle
(1985)
pollen
(Raphanus
was found
the buckwheat
Wang et aZ.,
ap. ),
(Carduus
pollen
raddish
plants.,
(EpiZobiwn
codlins-and-cream
leucine,
the following
raddish
asparagine,
arginine,
threonine,
serine,
(TrifoZium
red clover
sp. ) and musk-thistle
that
proline,
of
of alanine,
isoleucine,
histidine,
glycine,
acid,
glutamic
showed the presence
and Cephalotaxua
was found
in trace
amounts
(1985)
examined
the amino acid
in the former
pollen.
Rayner and Langridge
of bee-collected
Australian
pattern
plants.
for
pollen
pollen
from
ten
indigenous
These researchers
from both
these
and sixteen
noted
plant
that
sources
content
exotic
the amino acid
indicated
that,
4o.
the level
generally,
They also
requirements.
in
of amino acids
that
reported
whose low concentration
the pollens
were above bee
the amino acid
had been found
to be rate
tryptophan
limiting
for
the honeybee! s nutrition.
Mellissopalynology
The way in which
dust,
such as root,
pollen
spores
yeast,
of
analysis
'mellissopalynology'.
Typical
Maurizo
are Louveaux,
nology
found
elements
and other
microscopical
particles
constituents
(1980).
by Maurizo
the honey have been discussed
these
and other
grains
is
It
in honey which
enter
the microscopical
is known as
authorities
on the practice
and Vorwohl
(1970 and 1978)
of mellissopalyand Sawyer
(1975).
This
botanical
analysis
microscopical
origin
and geographical
adulteration
and the extraction
by LQuveaux,
Mmrizo
had been carried
1940.
a method for
pollen
and reviewed
in 1962.
for
of
up-dated
honey.
on
use
a mixture
(9 ml)
the pollen
during
to the
Briefly,
containing
sediment
include
for
Maurizo
the
obtained
acid
after
techniques
period
(1953)
Bee Botany
and this
'acetolysis'
published
(1970)
technique
established
was
method recommended
involved
(1 ml)
1930 -
method was revised
and Vorwohl
the procedure
sulphuric
of honeydew,
was demonstrated
analysis
of honey and this
Louveaux,
to
This
the
reveals
the presence
the pre-war
method of melissopalynology
(1978)
later
analysis
also
only
(1970 and 1978).
Commission
The International
the present
of
in Europe
out
but
techniques.
and Vorwohl
the development
Much of
of honey not
the addition
and acetic
centrifugation
anhydride
(2,500
revolutions
41.
of honey diluted
per minute)
and the resulting
and the
mixture
supernatant
the procedure
procedures
This
step.
acidic
plants
of acetolysis
step
structures
and Iverson,
and fifty
four
of pollen
most wide
of
recorded
the presence
in honey samples he examined.
(Rubus)
raspberry/blackberry
Chaubal
and Deodikar
bee plants
from
the western
determined
the
during
honey
of
the
An extensive
list
of
of
regions
seasonal
in
the pollen
hundred
the
source
six
species
that
the
Isles
(Prunuslpyrus),
fruit
(CaUuna).
an extensive
India.
minor
morphological
and accessory
These researchers
in the pollen
honey flows
grains
the British
the British
out
of
in
sixty
some major,
variation
successive
of
He reported
and heather
grains
from
on eight
repens),
(1965) carried
of pollen
characterisation
complex
matter,
to establish
sources
M-ifotium
clover
clarification
or lignin
analysis
author
in 1952 were the White
by a
of honey produced
samples
spread
archaeology,
acetolysis
extraneous
in order
honey
these
succeeded
of honey
important
a medium containing
1975).
out pollen
carried
supernatant
are many variations
or cellulose
survey
extensive
This
of honey.
also
the removal
(Faegri
Deans(1957)
Isles,
some cases
components
In the first
and this
Usually
in soil
in
was re-suspended
such as fossils,
science.
present
centifuged
to accommodate various
investigations
involves
at
There
chiefly
or in
0
70 C,
was mixed with
to mounting.
are preceeded
3: 5) to remove carbohydrates,
and detergent,
and forensic
allergy
pollen
sediment
sediment
of palynological
aspects
This
water
prior
glycerine/gelatin
in
was then
and the final
decanted
was
(w/v
water
incubated
removed.
of distilled
a mixture
with
composition
in the annual
which
were
identified
cycle.
has
42.
been presented.
also
(1969)
Howells
honeys
and reported
source
of pollen
in 18% of
15% of
in 64% of
samples.
important
most
difficult
identification
Prunuslpyrus
level
species
(1972)
Lieux
in fifty
four
commercial
easily
types,
this
Berchemia
Sawyer (1975)
twenty
eight
the major
individual
analysed
samples of
common pollen
grains
honey
that
samples
throughout
in
This
author
reported
of Louisiana
were
by pollen
geographically
honey plants
from
predominantly-TrifoZium
grains
of one hundred
in England.
and
He described
are characteristic
of
the
countries.
Adams, Smith and Townsend (1979)
of
types
pollen
and'SaZix.
the pollen
imported
an exact
and complicated
different
The major
scandens
as the
honey.
regions
family,
with
samples.
However,
honeys.
pollen
of honey pollens
filtering
eight
Louisiana
the Fabaceae
were of
Rubus,
repens,
fifty
in a few instances.
except
region
of
from one another
distinguished
ranked
the
level.
from different
the honeys collected
that
also
was more demanding
practice
identified
third
the classification
family
the plant
as a major
by Brassica
ranked
48% of
with
that
by the commercial
further
not
However,
source
occurred
followed
samples
Prunus/pyrus
reported
at
at
the
two Welsh
seventy
repens
While
secondary
James (1969)
was not
the TrifoZium
that
the samples.
the
from
the pollens
examined
of nectar
a season.
the honey samples
shaken from
the combs at
These researchers
collected
at
carried
out
frequent
identified
the end of
the
analysis
pollen
intervals
thirteen
season,
taxa
whereas
43.
total
a cumalative
In this
of
of
way they were able
the nectar
(1981)
Adams and Smith
pattern
of pollen
with
indicator
a sole
using
key index
has described
fifty
four
key for
the pollen
grains
species,
describing
their
Weber (1982)
electron
grains
belonging
orange
honeys
from
out
the other
the citrus.
orange
nectar.
of
eight
size,
the East
samples
these
source
and of
The
further
analysis
simple
as
shape,
coast
of
contained
samples
that
and also
a scanning
the presence
Quercus
Spain.
because
Only
three
large
iZex
the
tricolporate
of
group
as orange
of
plant
section.
more Quercus
contained
four
number and type,
aperture
identify
and
an identification
and thirty
microscope
in honey
two hundred
and the exine
to
method of
present
of
given
were considered
Weber concluded
blossom
the variations
the need for
grains
to the Fagaleae,
the eleven
season.
of
of pollen
of one hundred
in order
pollen
samples
indicated
He has also
used a light
microscope
picture
be beneficial.
a relatively
features
and structural
surface
would
of pollen
grains.
pollen
nectar
and microphotographs
cards
samples.
source.
identification
microscopical
active
a study
the reliability
of floral
Sawyer (1981)
that
therefrom
at present
to justify
investigations
its
over
season of a given
such information
of
the nectar
a much more complete
suggested
honey derived
the associated
lack
to obtain
used by a colony
sources
in
two were found
sixty
in eleven
honey
honeys
whereas
pollen
than
sweet
quantities
smell
of
of citrus
44.
(1982)
Porras
of
one hundred
This
Spain.
botanical
researcher
Erica
of one hundred
spectrum
samples
pollen
grains
were the most frequent
pollen
grains
were:
TrifoZium
The Rosaceae group
saZix
spp.;
spp.;
Apiaceae
able
to explain
and MipenduZa
the
The Brassicaceae
These
type.
hybridium,
and TrifoZium
Prunus,
MaZus, Sorbus
These researchers
uZwria.
in
variation
seasonal
the whole
representing
1977 and 1978.
is,
that
were
the-pollen
and abundant
repens
examined
content.
analysed
in the years
area of Finland
beekeeping
pollen
(1982)
and sixty
of Aracena,
to the EuCaZyptus,
The honeys
types.
and Koivulehta
Sierra
the
the most characteristic
by their
as evaluated
the sediments
of
from
belonged
survey
and Echium pollen
Helnuis
Varis,
that
reported
of honey
origin
of nectar
spectrum
samples of honey obtained
species
Cistus,
the pollen
studied
were
caused by weather
spectrum
conditions.
(1983)
Feller-Demalsy
examined
samples of honey from Quebec,
in
1977 and 1978.
most frequently
(hybridium
repens),
and/or
Moar (1985)
Canada,
author
represented
SoUdago,
pratense,
SaZix
in these
Vicia
sp.
(White
is
clover)
to bees in New Zealand
secondary
and minor
honey.
This
TrifoZium
sp.
on one hundred
He reported
nectar
occurred
researcher
species
TrifoZium
were:
analysis
the most important
pollen
the pollen
Rubus ep.,
and MeZiZotus
its
and
type.
honeys
four
and sixty
had been collected
that
cracca,
out pollen
carried
which
reported
samples of New Zealand
ninteen
repens
This
one hundred
that
and
Trifolium
source
available
as a dominant,
found
that
there
were
45.
generally
in floral
in honeydew honey than
by bees for
district
pollen
identification
careful
of flora
knowledge
of Pollens
Pollen
from different
size,
taken
into
that
grains
(the
(Sawyer,
if
the
those
(Fir)
of
in
considerably
have to be
The smallest
pollen
the AVosotis
arvensis
are only
6 Mm in
up to 145 pm in
occurring
due to their
Castanea
size
from the
the
small
size.
size,
after
sterile
grains
mean that
the
Moreover,
of AVosotis
arvensis,
and Mimosa pudia
officinale
Whereas,
are known
on the other
may be due to the bees deliberately
'under-representation'
(Howells;
pollen
was collected.
grains
in honeys.
to be 'over-represented'
almost
does not
two plants
pollen
Cynoglossum
sativa,
the pollen
two different
same number of
same amount of nectar
are
analysis.
in a honey sample this
to be found
pollen
vary
1981).
However,
off
upon
a sound background
with
These grains
are the Abies.
within
were dependent
These variations
in honey are
are found
be determined
could
plants
pollen
common forget-me-not).
The largest
are
during
from
of New Zealand.
shape and structure.
account
wind
in Honey
The Characteristics
their
honey originating
together
and vegetation
grains
was that
noted
such deductions
but
analysis
plants
and Coprosma were worked
that
of New Zealand
of pollen
Also
plantain
Moar observed
pollen.
a particular
from wind pollinated
honey.
such as grass,
plants
pollinated
limits
derived
more pollen-types
collecting
1969).
the nectar,
In some citrus
and hence only
ten
those
varieties
to twenty
hand
dusting
(Lime)
of Tilia
the anthers
percent
of
the
46.
citrus
Robinia
pseudoacacia,
1970 and 1978).
and Vorwohl,
(Weinmannia)
Maurizo
(Thymus vuZgaria,
thyme
in New Zealand
were under-represented
and kamahi
in New Zealand
were over-represented
EpZiZobium
(Louveaux,
from manuka (Liptospermum)
Whereas pollen
of
Curcurbitaceae,
from
The pollens
(Knightia)
and rewarewa
honey.
Banksia,
chamaencrion,
The pollens
sample.
are known to be under-represented
and Rosmarinus
citrus
in a honey
may be present
pollen
(Moar,
honey
1985).
Honey Adulteration
Honey being
essentially
in price
than other
higher
or fructose
leads
Under this
broad
heading
adverse
processing
conditions,
and finally
delibrate
essences.
sold
in
the four
in
lead
Various
the extent
developed
trades.
defects
the literature
the production
practices
important
The various
flavourl
to the lowering
of
of
were aimed at
these
or of
these
adulterated
deliberately
for
the catering
1.
with
Reports
aspects
of defects
been shown.
Usually
these
of honey.
the quality
practices.
the addition
glucose
to the various
have been developed
techniques
syrup,
associated
have been shown in Figure
of honey have also
and degree
fructose
aspects
temptation
one can group
of water,
honeys
a 'honey
pertaining
to the
to distinguish
the artificial
as such to provide
and confectionary
found
is
as honey from
made and sold
each of
It
now much
bad practice
addition
of high
and especially
syrups
artificial
solution
agents
misrepresentation,
by the beekeeper
sugar
sweetening
adulteration.
of possible
products
a flavoured
and utilized
At first
of acid
inverted
to determine
the technique
syrups.
The
47.
FIGURE 1
Defects
in
the production
of honey
Invert
Adulteration
[deliberate]
I
sugar
(A)
NN,
--ýHigh-fructose
corn
Excessive
storage
Processing
[adverse]
Total
heating
(C)
pollen
Blending
via
(B)
syrup
and prolonged
(D)
removal
machinery
Defects
Foreign honey as English
admixture
of both (E)
resentation
or an
*11
Addition
Excessive
of
(F)
flavours
sugar
feeding
(G)
Beekeeping
[bad management]
honey may not
A sample from Nigeria
found to be molasses
donated by a visiting
than honey.
rather
Represents
the appropriate
page.
on the following
reference
list
be unifloral
student
was
can be found
(I)
48.
List
of
references
for
Figure
1
A-
Wiley (1892)
Shannon (1916)
Sherwood (1924)
B=
Doner and White (1977)
White and Doner (1978)
(1979)
Doner, White and Philips
(1979)
Kushnir
White (1979)
White and Robinson (1983)
Kanematsu (1983)
C=
Schade, Marsh and Eckert (1958)
White, Kushnir and Subers (1964)
Hase et aZ., (1973)
White (1980a)
(1980)
White and Siciliano
D=
Hicks (1969) general commercial practice
for many products
In the case of honey
so as to enhance sales appeal.
by pollen
of identification
removes possibility
analysis.
E-
(1981)
Siddiqi
Sawyer (1975)
F=
Croft
G-
Siddiqi
(1980)
(1981)
49.
resorcinol
(Fiche)
have since
then been intensively
of
the levels
of
invert
at high
storage
of
the amount of
for
the purpose
reported
free
relatviely
corn
(Mermelstein,
enzymes
honeys.
in honey.
atomic
This
with
is because
by using
1975).
Instrumental
the presence
a highly
method utilizes
to detect
(1978)
two dimensional
A method has become predominant
This
that
the presence
(Horwitz,
12
13
mass
and
1980).
are
of honey have been
et aZ.,
and in bulk
to determine
mass spectrometer
of
syrup
that
filtered,
inconsistent
reported
paper
invert
or thin
sugar.
(HFCS) has become a major
honey adulterant.
cheaply
factors
of misrepresentation
during
by Louveauxt
the honey was naturally
and botanical
Maurikos
of honey
suggested
either
in honey adulterated
fructose
and developed
that
The measurement
the extraction
These researchers
amino acids
chromatography
of possible
1978).
the honey had been excessively
by Sawyer (1975).
High
1964 and White,
indicate
in the detection
layer
Marsh and Eckert,
have been reported
involved
patterns
the honey
analysis
The, apicultural
of
that
of pollen
or that
or sugar-fed.
during
or
(Schade,
heating
increase
during
sediment
in pollen
levels
of addition
was due to
This
obtained
(1970).
The measurements
as an indication
temperatures
and Subers,
(1911)
te'st
sediment
and Vorwohl
low amounts of
poor
and modified.
ambient
to excessive
Kushnir
White,
Maurizo
studied
to be inadequate.
have proven
has been subjected
1958,
(Feder)
the hydroxymethy1furfural
that
prolonged
and aniline
of hydroxymethy1furfural
syrup
the fact
(1908)
Test
source
HFCS can be produced
immobilised
isomerase
glucose
methods have been examined
of HFCS in adulterated
in the detection
specialised
of
stable
of HFCS
isotope
carbon
ratio
isotopes
50.
In order
of
isotopes
to explain
to briefly
review
three-carbon
obtained
from corn
(C ),
4
biochemical
current
the
the major
(C
plants
(maize)
In C3 plants
carboxylase
compound is
immediately
compound,
converted
cycle.
cells
oxaloacetate,
dioxide
above
from
then
(Prince,
(Prince,
the carbon
In this
1983).
bound
first
six-carbon
the
three-
taken
up by
of
is
case the Co2 is
dioxide
The immediate
cells.
This
cells
therein.
molecule
to reform-phosphoenolpyruvate
enters
up by
taken
1983).
dioxide
carbon
the mesophyll
to the chloroplasts
the
is
The resulting
compound oxaloacetate.
transferred
enzyme releases
(1983).
the assimilation
to two molecules
in the mesophyll
present
and thence
plant
first
by the enzyme phosphoenolpyruvate
bound to phosphoenolpyruvate
then
in
syrup
corn
by the enzyme ribulose-1,5-diphosphate
the Kartschak-Hatch-Slack
is
fructose
Here the CO is
2
cycle.
the atmospheric
the four-carbon
the honeybee
of
dioxide
carbon
3-phosphoglycerate
In C4 plants
is
is
in the chloroplasts.
present
carboxylase
of nectar
high
the
and
3)
the atmospheric
to ribulose-1,5-diphosphate
carbon
sources
as used by Edwards and Walker
Bassham-Benson-Calvin
the
is necessary
dioxide.
carbon
of atmospheric
it
theory.
in the C3 and C4 plants
The differences
can be measured,
which
is known to be a four-carbon
the terminology
using
in utilization
in a mass spectrophotometer,
expensively
is known that
It
are
12 and 13 by plants
of carbon
very
although
is a difference
there
that
Bassham-Benson-Calvin
product
compound oxaloacetate
to the bundle
sheath'
'A decarboxylating
bound to form
The released
cycle
the
carbon
as described
51.
12
is
the remainder
(0/oo)
contains
isotope
Walker,
enzyme ribulose-1,5-diphosphate
co
net
is
of -25.4
corn
0 /oo
with
has a6
syrup
-9.80/oo.
a6
13
fructose
Serra
of
The
13
C value
13
a6
C value
13
honey has a6
with
C value
and high
a range
fructose
to
of -9.5
method can be used to detect
corn
syrup.
Invert
and Walker,
These are:
and high
it
since
has
1983).
to detect
honey adulterated
layer
the thin
pressure
sugar
liquid
chromatography
chromatography
1983).
applied
of honey with
and sugar
enabled
methy1furfural
a level
1979)
and Gomez (1986)
adulteration
sugar with
been developed
syrup.
corn
(Kushnir,
et aZ.,,
These methods
at
(Edwards
of -11.00/oo
the
against
a6
be distinguished
cane can also
methods have also
(Kanematsu
detect
with
that
1987).
of -9.70/oo
fructose
gel
on silica
C value
high
C value
Other
with
13
known
with
to -27.40/oo
of -22.5
isotope
from'sugar
high
a range
Hence the carbon
honey adulterated
produced
is
per
carboxylase.
sugar
(1978 ) showed that
and Doner
White
abundance
discriminates
produce
1983 and Croft,
CO and
2
as parts
It
produce
the C4 plants
(Prince,
of -9.00/oo
1983).
13
isotope
the
C expressed
carboxylase
the C3 plants
that
whereas
of -28.30/oo
in
the enzyme phosphoenolpyruvate
than
more
2
effect
6
13
Since
1%
approximately
ratio
(Edwardsand
the C3 and C4 plants
enrichment.
The differences
CO
2*
by the
expressed
thousand
dioxide
carbon
atmospheric
13
between
in the phenomenon known as isotope
results
is
in the metabolism
difference
This
products
four
different
different
methods
sugar
the determination
were:
the detection
5% or more;
four
of
the adulterant
invert
sugar
to
products.
of hydroxy-
adulterant
common glucose
syrup
at a
53.
in honey from New Zealand
(1966),
and White
Karaka
Scott
II
in
of British
the province
honey
honey sample.
(1973)
has extensively
listed
has
and
the
to the
graynotoxin
of Grouse Mountain
These two compounds
was
which
literature
the
reviewed
the reported
honeys by animals
of
I (andromedotoxin)
on toxic
the consumption
symptoms after
and humans.
(1975)
Jachimowic, z and El-sherbiny
per
invert
that
reported
30 mg of hydroxymethylfurfural
containing
from
nectar
poisonous
in Canada.
Colombia
in this
of poisonous
toxic
was only
the region
to graynotoxin
related
are closely
White
that
the presence
reported
in a honey sample from
found
and Turner
and Clinch
reported
caevigata)
(1971)
et aZ.,
and III
not
(1968)
by Hodges
studied
bees.
worker
adult
Palmer-Jones
(Corynocarpus
tree
(1968)
and Clinch
Turner
(1968 and 1975).
have been extensively
sugar
100g of honey was
honeybee.
fed
the
to
when
and Corner
Neufeld
Majak,
of AstragaZus
v.
miser
serotinus
glycocide
a nitropropanol
which
(1980)
reported
was found
resulted
that
the nectar
to contain
in
a misertoxin,
the poisoning
of
the
honeybees.
(1981)
White
has reviewed
and identification
has described
experimental
of
these
toxic
of
the
toxic
literature
and human subjects.
compounds,
that
gelsemine,
on the occurrence
compounds in honey.
symptoms of poisoning
animals
anhydroandromedol,
given.
the
the
by these
This
compounds in
The structure
is,
acetylandromedol,
tutin
and hyenanchin
author
of
six
andromedol,
have also
been
54.,
(1982)
Atkins
of
the honeybees
(AescuZus
buckeye
death
sp. ),
MýZochin
western
aZbum),
henbane
(Eyoscyamus
With
increased
the
insects
deter
to have similar
liable
also
these
with
of crops
colony
insecticides
on the honeybee
Baird
Homan
and
and
Apart
honey
tasting.
source
honey
is also
individual
bitter
toxic
are
The spraying
honeybees
or
The effects
of
(1984)
by Arzone
these
compounds in
flavours
characteristics
and aroma which
(Ligustr-um
taste
are
due to
Isles
In the British
sp. ) has been described
is
low concentrations,
and aroma or bitter
by the honeybees.
(Senecio
flavour
of
of
collected
honey from privet
have a distinctly
of crops
must be carried
or minimised.
to unpleasant
The main origins
from ragwort
destruction
to
(1983).
subject
of nectar
the
hippocastanwn)
hybrids.
have been studied
from the presence
to have a strong
Also,
of
is negligible
the honeybee
chestnut
or pesticides
rate
the mortality
so that
(Veretum
(AescuZus
on the honeybee.
effects
insecticides
subnerticiUata),
and insecticides
use of pesticides
cause damage and/or
that
(AstragaZus
hellebore
sp. ) and its
(Rhododendron
and rhododendrons
horse
summer
arrowgrass
(AscZepias
caZifornicum),
niger),
sp. ),
locoweeds
seaside
milkweed
(Veretum
hellebore
(Cuscuta
racimifZora),
whorled
the california
(SoZanum
nigrum),
shade
night
(KaZmia ZatifdZia),
rwritima),
false
out
(CyriZZa
of poisoning
the plants
are:
dodder
venenosus),
laurel
mountain
black
caZifornica),
or leather-wood
titi
listed
These plants
to them.
carnas(Zygadenus
the effects
and has also
by pesticides
are poisonous
which
in detail
has discussed
by Howes (1979)
somewhat nauseous.
)
has been described
sp.
and was quoted
to be quite
to
uneatable.
55.
(1976)
Further,
it
flavours
and aroma are often
lost
when crystallisation
Howes (1979)
also
noted
was pointed
are completed.
to have caused
has been reported
limes
working
such as TiZia
flowering
orbicuZaris
annuus,
taste
by Crane,
(Crane,
1980)
and processing
tomentosa
In other
season.
honeys
lime
flavours,
plant
strong
sources
and TiZia
parts
from Agave,
sativa
and other
offensive
amongst honeybees
TiZia
and Castanea
to have unpleasant
been reported
a bitter
in the
these
honeydew from
fatalities
Australia,
jacobaea
Senecio
that
that
petioZaris,
late
Americas,
Europe,
world,
out by Hooper
of
the
Hezianthus
species
have
aroma and possess
have been listed
(1984).
Day
and
Walker
AIMS
1.
To determine
chemical
selected
show the variation
of
components
such compounds with
of honey and to
floral
or geographical
source.
2.
The determination
use of
information
this
in
highly
even
3.
The correlation
pollen
4.
analysis
To explore
detect
amino acids
to aid
strained
and with
the possible
of honey and the
identification
pollen-free
of chemical
adulteration,
of honey.
the free
of
samples.
methods
computer
as to source
with
the
traditional
predictions.
uses of chemical
misrepresentation
identification
and gross
to
mishandling
EXPERIMENTAL
CHAPTER 2
56.
EXPERIMENTAL
CHAPTER 2
Equipment
Amicon Standard
filter
to exclude
cell
No. 402,
material
below
tank,
Electrophoresis
10,000
daltons
PAGE and 'tent'
Instruments
Ltd.,
Olympus FHT microscope
fitted
Carnerley,
used to record
TSM1 Multisampler
Technicon
Instruments
from Technicon
Gel scanner
Ltd.,
sp8-500
PYE UNICAM Ltd.,
bottles
Plastic
(UK) Ltd.,
Unit
through
(London).
Gallenkamp
adaptors
Hailton
Nebraska,
analyser
Close,,
X10
eyepiece
and was
of X400.
recommended magnification
Spectrophotometer
York
head,
microscope
Model UA-5 was purchased
UV/VIS
chromatank
Surrey.
amino acid
Company, Lincoln,
Specialities
with
at the
grains
pollen
were
were purchased from
triocular
with
Olympus OM2 camera was fitted
'Shandon'
tanks,
X10 and X40, were purchased
and objectives
weight
molecular
400V
'Shandon'
the
power
pack,
output
vokam
and
Shandon Southern
and a UM10 membrane
unit
High Wycombe, Bucks.
from Amicon Ltd.,
purchased
filtration
was purchased
Hants.
Basingstoke,
from Instrumentation
Ti. S. A.
PYE Unicam was purchased
from
Cambridge.
Street,
(100 mls)
2, Broad Lane,
were purchased
Cottenham,
from Kartell
Plastics
Cambridge.
Materials
Normal
distilled
listing
laboratory
chemicals
of analytical
water were used as required.
of
chemicals
used.
These were:
reagent
quality
Given below is a partial
and
57.
Bovine
30 - 50 units
a-galactosidase-activity,
and Folin.
grade V free
Invertase
serum albumin,
's reagent
Ciocalteau.
of
per mg of
were purchased
catalase
Bakers
solid,
from
and
yeast
Sigma Chemical
Company, London.
NON'-methyl-bis-acrylamide,
Acrylamide,
Blue
Coomassie Brilliant
N, N', N", N"I-tetr;;.
Black
were purchased
Laboratory
from Hopkin
courtesy
and arrangements
who also
made a donation
Many of the honeys
received
donated
All
Appendix
towards
of
the cost
origin
Buckinghamshire.
and Basic
Health,
provided
of
the
fushin
Essex.
the
through
Beekeepers
Association
These survey
survey.
1 lb jars
were samples
by Manley
and provided
of various
or by retail
the honey samples
grade
Chadwell,
were samples
foreign
Other
according
analar
were
and provenance.
by named individuals
I.
Colubrook,
made by the British
as shipped
oxford.
Berinsfield,
Ltd.,
and Williams,
known
origin
of
were
samples
containers
and 0-mercaptoethanol
12B, propanr2-ol
Most of the honeys
bulk
Poole.
methylenediamine
purchased from Koch Light
Napthalene
Ltd.,
sulphate,
(Na)
50
Mech
Us
-
50-X8.20
and Dowex resin
from BDH Chemicals
were purchased
sodium dodecyl
received
to the
label
taken
Ratcliffe
from
the
Ltd.,
honeys were either
purchase.
were catalogued
or information
and listed
in
provided.
METHODS
Preparation
All
of Honey Samples for
the honey samples apart
which was stored
Honey samples
that
from the China Buckwheat, Code No. 209,
at 40C, were stored
were received
Analysis
at room temperature,
in pieces
of
0
18 - 22 C.
combs or containing
comb
58.
fragments
and wax were first
thorough
were solid
upon receiving
Preparation
required
honey when received
above all
most of it
had crystallised.
placed in an oven maintained
through. a layer
honey was then strained
on the sieve cloth
honey was left
The remaining
Both these portions
original
until
The
debri
Before
overnight.
to ensure that
all
This
the crystalline
on the sieve cloth
was discarded.
China buckwheat honey were pooled and
of strained
in ten plastic
containers
cleaned plastic
container.
collected
This
honey was
crystallised
temperature,
the honey was again stirred
honey had dissolved.
four hours.
to break up the crystals.
thoroughly
at this
was
container
plastic
twenty
and wax, and
of mesh and*bench cloth.
and the remaining
warmed at 550C and stirring
pound plastic
comb fragments
This
0
at 45 C, for
a fifty
in
bees and bee fragments,
container
straining
400C
with
at
warming
gentle
of the China buckwheat Honey (Code No. 209)
contained
semi-solid
or
the crystals.
The China buckwheat
debris
or creamed
had crystallised
Those samples that
stirring.
to dissolve
stirring
could be
any analysis
Also those samples which were soft-set
performed on them.
required
before
strained
(500 mls),
and the remainder
This honey was then stored
in its
40C
at
required.
Prep ration
of the United Kingdom survey honeys
These honey samples were received
Those samples
that
were received
of combs and wax were first
samples were strained
within
strained
through
beaker (100 mls) and then left
in plastic
pieces
before
a mesh cloth
containers
(100 mls).
of combs or with
being
placed
analysed.
over
in an oven maintained
fragments
These
the mouth of a
0C
for
40
at
an hour.
59.
Crushing the combs with
straining
Occassionally
process.
it
comb and honey on the mesh cloth.
remained on it
honey that
the residue
honey was then
This strained
(100 mls).
bottle
the
the crushed
The comb fragments with
Honey samples that were
as described
were treated
or crystalline
soft-set
to stir
was necessary
were discarded.
in a clean plastic
collected
facilitated
the aid of a glass rod often
being
above before
analysed.
honey was donated
This
its
during
which
conditions
A portion
received
mimicked
those
and thereafter
with
storage
gentle
heating
this
and this
that
ensured
with
manipulated
the honey was subjected
or no overall
to
on the honey's
effect
sample when received
minutes
of
following
after
the
was
the normal
one-third
followed
portion
and then
previously.
allowed
the honey was warmed at
from
the aid
to settle
0C
60
for
the hive
by the commercial
of honey after
to room temperature
Then the honey was strained
out with
These conditions
extraction
at 40C was equilibrated
as described
amount of honey
total
conditions.
by the beekeeperv
This
honey was carried
to three
hours.
bottling.
two hours.
strained
After
encountered
of
period
to the
processing
before
from
removal
was carefully
to one-third
equivalent
was subjected
producer
have little
would
who upon request
OC.
4
at
stored
combs it
and composition
consistituents
Croft
was done so that
This
heating.
minimal
by Dr.
from the
removal
Honey (Code No. 261)
Chorley
of English
Preparation
through
Vigorous
over
cheese cloth
stirring
of
the
about
two
of a spatula
for
for
of one hour.
a period
a period
of
twenty
four
a
60.
The remaining
the heat
treated
of identification
honey will
Chorley
Whereas the not heat
treated
of the honeys
of the Brassica
appearance
the China buckwheat.
or crystalline
soft-set
fragrance
of
of
flowers
to the strong
fermentation
some of
honeys
Nearly
with
by sensory
pale
samples
samples
blossom,
of
in
the
some of
ranged
black
of
were either
from a gentle
the light
sugar
pleasing
colour
honeys.
The sign
fed samples
and in
colour
smell
of hydroxymethy1furfural
characteristic
brown almost
were liquid.
dark
honeys by the distinct
the bulk
The
crystalline
white
especially
the
and foreign
examination.
received
a few that
only
unpleasant
the U. K. survey
of
to the dark
the
all
and fruit
was evident
Evaluation
from a soft
the aroma of the
In general,
honey,
ranged
Chorley
Code No. 261.
original
samples were determined
portion
one-third
of English
portion
and odour
treated
to by the Code No. 261a.
Analysis-and
appearances
and commercial
colours
the heat
two-third
Subjective
The colour,
was not
above was used as a control
be referred
to by its
be referred
honey will
described
just
honey that
Chorley
sample.
For the purpose
of English
the English
of
to the conditions
subjected
for
two-thirds
of alcohol.
was evident
in
An odour
some commercial
samples.
The honey samples were not
building
pipetting
especially
or tasting
Furthermore,
the different
designed
tasted
for
as the
radioactive
laboratory
analysis
was within
and any mouth
was forbidden.
considerable
experience
aromas and flavours.
was required
in recognising
a
61.
An example of the questionnaire
beekeepers
is
given
in Appendix
II
and their
and 6 have been given
No. 2,3,4,5
The Isolation
to the participating
sent
under
and Concentration.
to questions
replies
their
appropriate
sections.
in Honey
of Proteins
Ultrafiltration
The basic
passage
achieved
Amicon Standard
10,000
pressure
of proteins
present
protein
(Code No. 194),
Positive
on the
separation
above a
solution
membrane filter
by
in honey was achieved
nitrogen
pressure
Model 402 of 400 ml capacity,
honeys
from
whose molecular
content;
Canada (199),
the
in
an
fitted
with
weight
exclusion
a
Staffordshire
foreign
and commercial
samples
were
these
samples were:
Australia
Banksia.
Chile
(214),
Polyflora.
(222),
Mexico Yucatan (227) , New Zealand
pure
This
sizes.
daltons.
Representative
examined
pore membrane which
of molecular
using
cell,
UM10 semi-permeable
for
This
and macromolecules.
a positive
ultrafiltration
accelerated
from a
daltons
membrane.
The concentration
is
range
low
of soluble
10,000
than
micromolecules
of a defined
by applying
semi-permeable
limit
less
is based on the use of a controlled
separation
permits
both
the separation
those
components
containing
solution
is
principle
weight
molecular
involved
(229)
,
Hungary
Yellow
Box (246)
and English
(248).
Procedure
The procedure
a few modifications.
diluted
with
used by Bergner
and Diemar
A sample of honey
0.033M sodium phosphate
(25g)
buffer
(1975a)
was followed
was weighed
(50 mls)
with
out and then
at pH 6.5.
62.
This
honey mixture
pressure
square
of
four
inch)
of buffer
hundred
This
that
including
macromolecules
The solution
the reducing
of 10 mls.
This
of less
This
protein
and the weight
dried
10,000
daltons
of the
solid
iti
This
components
had been removed.
to a
from honey was then
product
in
vacuo
was noted.
the presence
of hygroscopicity
the process
filtrate
was concentrated
concentrate
was stored
residue
to prevent
pentoxide
soluble
cell
cell
solution.
and other
than
the
until
Benedict's
with
the Amicon-standard
within
freeze-dried
was repeated
sugars
(20 mls)
in the Amicon standard
solution
process
pounds per
Portions
cylinder.
A
cell.
(sixty
Pascals
a nitrogen
on testing
result
indicated
volume
using
were added when the
gave a negative
kilo
and four-teen
was applied
10 to 15 mls.
reached
to the Amicon standard
was transferred
of phosphorous
and microbial
contamination.
It
in
order
was found
to perform
necessary
to minimize
the
of bacterial
the risk
filtration
at 40C
process
contamination
the honey
of
solution.
Estimation
Quantitative
of Proteins
Ultrafiltration
of
in Residues
from
Honey
The Lowry Method
Proteins
presence
when reacted
of alkaline
tungstic
tungstate
mixed
acid
and/or
and involves
copper
(1951)
by Lowry et aZ.,
the
Folin-Ciocalteau's
with
that
solutions,
bring
about
by the loss
is,
a reduction
of up to three
molybdate
ions.
following
chemical
This
mixed
species:
reagent
under
of
the
given
conditions
the phosphomolybdic-
oxygen
acid
in
is
atoms from
the
final
the
chromagen
63.
10H
3H O-P
-13Wo3*5Moo3o
2
205
20
and
4Moo 10H
3H O-P
-14WO
2
205
3*
20
3'
is
The result
reduced
species
the production
have a characteristic
which
750 rm and Xmin 405 rm).
The copper
bonds of the proteins
the peptide
to the mixed
of electrons
of the amino acid
of
of one or more of several
the proteins.
acid
functional
Hence,
thereby
chromagen,
protein-serum
albumin
Solution
as standard
form
It
(X
745 max ,
chelates
with
facilitating
the transfer
particularly
in
is
alter
the sensitivity
in
for
concentration
to use a soluble
conventional
(Peterson,
the vicinity
1979).
and reagents
solutions
A consisted
O. lM sodium hydroxide
present
as the amino acids
each protein,.
of
colour
Thus increasing
groups.
so does the response.
Preparation
ions
blue
possible
2% (w/v)
of
bisodium
carbonate
dissolved
in
solution.
of, l% (W/V) copper sulphate
.
of 2% (w/v) sodium potassium
Solution
B1 consisted
Solution
B2 consisted
Reagent
B was obtained
by mixing
equal
Reagent
C was obtained
by mixing
solution
volumes
(CuSO 5H 0).
4'
2
tartarate.
of
B1 and
solution
B2.
the ratio
50: 1 (v/v)
Reagent
reagent with
and was freshly
D was obtained
by mixing
2 volumes of water.
A with
reagent
B by
made when required.
1 volume
of Folin-Ciocalteau's
64.
Procedure
(1951)
The method of Lowry et aZ.,
was used to"prepare
(5 mls)
water
5 mls.
and each 1 ml
(0.5
Aliquotes
the protein
reagent
reagent
D (0.5
stand
read at a wavelength
protein
were also
for
thirty
values
A standard
calibration
measurements
for
in
the diluted
the
standard
of each
-Furthermore,
and for
honey
500 M,
at wavelength
This
was
no protein,
was done to check
at the 500 nm measurements,
by plotting
standards
the
against
The concentration
ultrafiltration
their
of proteins
were obtained
from
of Proteins
in Honey
Electrophoresis
Proteins
groups
0.04
was obtained
after
but
concentration
to
allowed
curve.
The Detemination
Disc-Gel
reagents
each of the protein
honey residues
being
adding
a spectrophotometer,
values.
that
solutions,
the absorbance
of 750 =.
to
were mixed with
After
than
from
diluted
0
23 C, before
at
all
of less
curve
known concentration
appropriate
is,
determined
of concentration
accuracy
diluted
minutes,
that
at a wavelength
re-read
absorbance
for
at 20 ug per ml
absorbance
samples with
ten minutes
the range
in distilled
was further
solution
of 500 nm using
standards
and dissolved
immediately.
to a blank,
relative
solution
for
serum albumin
of the residues
and honey concentrates
and mixing
at room temperature
covering
of each of these
and left
ml)
standards
was weighed
of this
mls)
standards
C (5 mls)
Bovine
Portions
per ml.
(0.1g)
of honey
ultrafiltration
for
of protein
20 to 260 ug of protein
from
is,
a series
was used.
always
projecting
contain
amino acid
from the polypeptide
residues
structure
with
acidic
and these
and basic
polar
groups
65.
ionise
in aqueous solutions.
charge
on'the
on the
chains
side
The term relative
such as net
factors
free
formed
pore
sizes
that
concentration,
by varying
on these
and then
media.
blue
the presence
of
only
can be produced
is,
the degree
the degree
calibration.
of acrylamide
a co-monomer methylenebis-
occurs
is
added as an accelerator.
the
of polymerisation
referred
total
acrylamide
length
or chain
through
of cross-linking
by
or other
riboflavin
by varying
as a
acts
initiation
after
is
and bisacrylamide.
mixed polymers
changing
Electrophoresis
to as polyacrylamide
the
carried
gel
(PAGE).
electrophoresis
Protein
The effect
by the polymerisation
from ammonium persulphate,
of acrylamide
proportions
a suitable
by prior
N, N, N", N"'-tetramethylenediamine
compounds.
and also
formed
factors
including
cross-linking.
be determined
Polymerisation
cross-linking
Different
are
function
and gel
and relative
(CH2 w CO-NH-CH *NH-CO-CH - CH which
2
2)
agent.
radicals
size,
of migration
known as electrophoresis.
is
applied
a complex
can only
gels
(BIS)
acrylamide
is
CH-CONH in
2)
(CH
monomer
2n
is
molecular
Polyacrylamide
out
field
direction
their
electrodes,
mobility
charge,
these
of all
the
when an electric
mobility
the
solution,
The process
the protein.
of
surface
towards
of the protein
the
depend on the number and pKa of the various
will
protein
pH for
At any given
movement in
protein
destaining
stain
individual
dye can be measured
is
such as napthalene
by thorough
Thus the relative
gels
washing
of
detected
black
and after
or coomassie
each gel with
movement of the proteins
before
by staining
staining
with
blue
the appropriate
to the bromophenol
(Smith,
1976).
66.
of buffers
Preparation
Electrophoresis
(28.8g)
amine
buffer
dissolved
of Tris(hydroxymethyl)-methyl-
consisted
in distilled
to a final
water
of 100 mls
volume
at pH 8.5.
(0.38M
Gel buffer
in distilled
dissolved
pH 8.9)
tris-HC1,
(50 mls)
water
consisted
(4.5g)
tris
of
to pH 8.9 and then
and adjusted
100
to
mls.
up
made
Procedure
of Sargent
The procedure
dissolved
were
(0.1428g)
in
tetramethylene
and filtered.
Polymerisation
This
prepared
solution
gel
0.5
to within
7% (w/v)
vertical
Distilled
water
was then
and also
air
completed
when a distinct
division
gel below.
These tubes
buffer.
care
The water
to exclude
Electrical
air
connections
carefully
in
into
layered
a flat
appeared
between
of a
(1 ml).
placed
with
were so arranged
the upper
that
gel
so as to
The process
and gel
from the bottom
in a
serum caps.
above the
in the
tubes
electrophoresis
was removed and replaced
were then
was mixed
were maintained
surface.
minutes
bubbles
the
ends sealed
thirty
layer
solution
by the addition
These tubes
to obtain
was, normally,
N, N1, N", N"'-
of ammonium persulphate
lower
their
with
(99 mls).
pipetted
top.
a
with
and N, N'-methylene-bis-acrylamide
was catalysed
solution
cm from the
was followed
was added and the
was carefully
position
exclude
buffer
gel
(30 ul)
diamine
freshly
(6.28g)
Acrylamide
few modifications.
(1975)
and George
of gelling
formation
water
was evident
layer
by the
electrophoresis
tank
electrophoresis
of each of
the upper
the
and the
taking
tubes.
surface
of
the
67.
tube was the cathode.
(0.01g)
0.05% (w/v)
under
This
then
transferred
7% acetic
for
acid
Detection
for
gel
of
Honey
initially
the
increased
blue
tube by
the
of
gels
the
were
12B (w/v)
black
gel was carefully
acid
became clear.
the protein
bromophenol
of one hour.
10% acetic
in
acid
so as to
circumference
The stained
10% acetic
to allow
The cast
and the
The
The distance
solution.
length
were
oxidase
and
gel
in honey samples
was investigated
as described
of marker
with'napthalene
black
used as an indicator
about
on Polyacrylamide
of enzymes such as diastase,
electrophoresis
bring
was performed
a period
destained
containing
Enzyme Activity
the application
will
of
each gel.
The presence
invertase
and a drop
of napthalene
minutes.
bands,
for
round
solution
in
were stored
moved by the protein
measured
not
glycerol
and syringe.
and then
water
The regions
gels
water
needle
thirty
of this
were removed from
the, gels
forcing
and 100 jil
was then
per tube
of
(5 mls),
50 volts
at
The voltage
to a staining
distilled
in
washed
solution.
current
of 4 milliamps
of
with
Electrophoresis
solution.
the gel.
involved
water
one drop
with
blue
a hypodermic
using
cast
destained
in distilled
electrophoresis
gel
in
from ultrafiltration
of constant
a current
After
rimming.
The residue
bromophenol
conditions
maintain
(250 mls).
together
sample to enter
the
was filled
Was dissolved
was applied
solution
compartment
buffer
electrophoresis
honey
Each buffer
was not
for
carried
the presence
the reduction
glucose
by performing
that
above except
dye bromophenol
of
Gels
blue
out.
stage
and staining
Instead
or absence
the yellow
the
polyacrylamide
picric
of
the
acid
picric
of glucose.
acid
involving
gels
was
Glucose
to the
68.
orange
red picramic
glucose
During
acid.
is
glucose
oxidase,
resulting
from the catalytic
invertase
on their
Hence,
after
action
of
the enzyme
consumed where as glucose
is
a by-product
action
substrates
the enzymes diastase
of
starch
and
in either
0C
for a period
sucrose at 37
gels
1% solutions
of one hour.
of
commonly used in biochemistry
laboratories
incubated
in their
distilled
and washed with
honey sample.
carbohydrate
These gels were
as described
solution
carbohydrate
solution
two drops of 4M sodium hydroxide
was added.
at 600C for
to the
the gels and picric
this
period
solution.
acid and
of picric
The tubes
in a water bath
acid were warmed gently
After
twenty minutes.
above.
solution
water to remove excess carbohydrate
polution
or
sources
were also applied
These gels were then immersed in a dilute
containing
glucose
or other
the gels were removed from their
incubation
After
appropriate
starch,
Also samples of the enzymes
from microorganisms
own and also mixed with
the honey
containing
mentioned above were obtained
gels on their
and
respectively.
sucrose,
the replicate
electrophoresis
were incubated
proteins
the catalytic
of warming the tubes were
bath
from
the
water
and the changes in colour
removed
on the gels in
the form of bands were noted.
Sulphate-Polyacrylamide
Sodium Dodecyl
detergents
By including
during
fractionation,
proteins'that
gel
resolution
results
with
pH, in a 1% SDS solution
bind
are obtained
and most importantly
to molecular
are now proportional
electrophoresis
most proteins
such as sodium dodecyl
better
electrophoresis
Gel Electrophoresis
SDS will
which
be referred
was also
to SDS and dissociate,
(SDS)
sulphate
in
terms
the ability
of
of
The polyacrylamide
weight.
to as SDS-PAGE.
OAM with
disulphide
the
respect
At neutral
to mercaptoethanol,
linkages
are broken
69.
by the mercaptoethanol,
secondary
subunits
of protein
consisting
is
structure
lost
and the complexes
and SDS assume a random-coil
configuration.
Protein
in
treated
shape and an identical
SDS bound per unit
than
rather
weight
effective
molecular
sieving
is
charge
related
property
of
the separation
enabling
for
to molecular
of molecules
for
(Sargent
by the
SDS
Thus the
because
the
of
can be obtained
ranging
weight
10% T cross-linking
5% T cross-linking
SDS per g
the amount of
size
of molecular
the
residues.
weight
pore
of
determined
By varying
gel.
the desired
daltons
lo, 000 to 100,000
the
fact
in
because
1.4g
the amino acid
of
only
is
constant:
is
on the protein
and bisacrylamide
acrylamide
is
have a uniform
they
This
to mass ratio.
of protein
the intrinsic
mobility
daltons
charge
The charge
of protein.
way behave as though
this
to 20,000
from
to 350,000
1975 and Smith,
and George,
1976).
Preparation
of buffer
Gel buffer
and solutions
consisted
0.2% sodium dodecyl
sample buffer
sulphate
buffer
solution
N, N'-methylene-bis-acrylamide
water
5% acrylamide
solution
at pH 7 and
together
consisted
with
at pH 7
1% 0-mercaptoethanol.
gel buffer
of
buffer
diluted
1: 1 with
water.
Acrylamide
distilled
of 0.01M sodium phosphate
consisted
Electrophoresis
buffer
sulphate.
dodecyl
1%
sodium
and
distilled
of 0.2M sodium phosphate
1: 1 with
(100 mls)
solution
distilled
(10V consisted
(0.6g)
and stored
of acrylamide
both
these
in a dark
was prepared
water.
of
(22.2g)
were dissolved
brown bottle
by diluting
and
the
in
at 40C.
10% acrylamide
70.
Procedure
The procedure
for
(4 mls)
a period
ethanol
(0.05g)
for
and this
of two hours,
to ensure
the disulphide
was found
it
dissolved
two hours,
were also
and deareated.
(25 pl)
cast
followed
a constant
After
This
of honey
incubated
and then
gel
0C
37
at
(13.5
solution
(1.5
solution
and English
as described
mls)
freshly
mls)
and
was activated
Chorley
current
were then
of
forty
Then the current
of
6 milliamps
and the
electrophoresis
brilliant
was measured.
blue
applied
of honey
(1.25g)
blue
solution
to the
gels.
at a constant
minutes
honeys
above and also
from ultrafiltration
was performed
a period
moved by the marker
of coomassie
by $-mercapto-
the honey proteins.
in
a drop of 0.05% bromophenol
Electrophoresis
the gel.
at
described.
a drop of glycerol,
to enter
(I ml)
0C
37
by N, N', N", N""-tetramethylenediamine
thoroughly.
of raw honey and residues
for
cleavage
solution
of sample buffer
per tube
complete
sample
suitable.
261 and 261a were prepared
with
was incubated
1% ammonium persulphate
as previously
each mixed with
solution
acrylamide
and mixed
in
from ultrafiltration
residues
a few
with
dissolved
(15 m1s) was mixed with
was added,
Solutions
was followed
(2g)
bonds present
in sample buffer
Gel buffer
prepared
that
(1969)
raw honey
that
was suitable
of all
Moreover,
was found
It
modifications.
buffer
of Weber and Osborn
per tube
for
removal
of
The gels
dissolved
(100 Ul)
the
gels,
of
sample
four
hours.
the distance
were stained
in
2 milliamps
of
so as to maintain
a period
the
were
together
to allow
was increased
solutions
similar
current
in order
Code Nos.
a mixture
in a solution
of 50%
71.
methanol (454 mls) and glacial
acetic
This being a mixture
solution.
(50
methanol
mls) and water
(895 mls).
the regions
were made until
solution
of glacial
Then the length
became clear.
The resolution
of the gels.
bands were also measured from the top
of the stained
close together,
of 0.1 cm were clearly
visible.
the position
moved by the
leading
accuracy
±0.1
Gel
is,
that
separated
by a distance
and trailing
band.
edges of the protein
of distances
the distances
by averaging
was obtained
The
moved by the bands was on average
cm.
scanning
The destained
tubes
were
rubber
with
manual.
a speed
distilled
the
of
6.33
scanner,
The tubes
mm per
then
minute.
the
this
gel
were
at
the
then
sealed
gel
were
then
the
top
scanner
tubes
by
values
of
the
placed
gels
entering
to
warm
manufacturers
560 nm and
for
and
with
was allowed
a wavelength
The absorbance
the
of
tubes
bubbles
no air
tapping
made as described
scanned
the
sealing
were
the
with
usually
to
Before
by gently
These
tubes.
scanning
there
containing
adjustments
were
that
removed
tubes
Prior
necessary
The gels
were
water.
These
gel
water.
was ensured
bubbles
first.
placed-in
distilled
with
it
on the
scanner
up and
filled
stoppers.
horizontally
were
gels
stoppers
up with
topping
rubber
then
The air
present.
the
bands were sharp
protein
For broad bands, 0.5 cm and wider,
density
of measurement
the proteins
not containing
and the bands occuring
of greatest
Frequent changes of destaining
of each gel was measured and the
moved by the protein
distances
in destaining
(75 mls) and
acid
acetic
The
overnight.
water and destained
distilled
gels were then washed with
(46 mls),
acid
each
at
band
as
72.
displayed
by the absorbance
were usually
that
in
some honeys
thus
the destaining
weights
there
of nearly
60,000
from
examined ranged
were bands which
Determination
of
occurred
Although
the presence
by various
reported
complexes
have also
Hence in
the present
are considered
of
to be of major
importance.
of
top of the
the
near
proteins.
weight
in honey have been
of amines,
forming
(1975)
of
ninhydrin
positive(1981).
and Siddiqi
these,
the Free Ninhydrin
the
imino
amides,
be referred
compounds will
However,
and Concentration
Separation
Davies
these
study
in
Positive
amino acids
substances
been reported,
present
However,
molecular
the presence
and other
gels
Honey
substances.
positive
ninhydrin
free
of
researchers,
peptides
small
acids,
very
the Free Ninhydrin
Substances-of
on the
the proteins
all
to 20,000.
of high
the presence
suggesting
was completed.
process
bands obtained
the protein
of
the molecular
the honeys
gels
position
Gels
on the chartpaper.
were noted
scanned a week after
The relative
suggest
meter
to as
free
amino acids
Positive
Substances
Honey
Ion-exchange
chromatography
The principle
absorb
onto
or eluted
by changing
contains
ion-exchange
an ion-exchanger
An ion-exchanger
that
of
the
is
covalently
are electrostatically
reversibly
ionic
usually
linked
is
chromatography
so that
that
molecules
charged
molecules
can be bound
or pH environment,
a three
dimensional
network
or matrix
charged groups to which counter
bound using conditions
that
give stable
ions
and tight
73.
binding.
a different
Then using
are eluted
Cation
the ion-exchanger
off
exchangers
+
Na etc.,
Cl
and eluted
with
buffer.
Anion
ions
counter
and similarly,
acid buffers.
with
and eluted
of charge.
extent
ions such as H0
alkaline
basis
are retained
etc.,
their
and
counter
3H
Resin-quantanary
exchangers contain
to
ions
the
pH conditions
and/or
according.
Resin-SO
contain
are retained
such as OH
ionic
Procedure
for
The procedure
the separation
from a honey sample as described
determinations.
quantitative
trials
of preliminary
was then
procedure
check
for
acid
to solutions
or honey plus
in Table
are given
from the ion-exchange
modified
method
6.
in Table
B as given
weighed
in whole
propan-2-ol/0.5M
33% glucose
resin
and concentration
of amino
out by applying
dissolved
in
the
A and
solutions
known
10% (v/v)
either
solution
or a carbohydrate
to
these
A, B and S1 contained
amino acids
acid
of
of recovery
to the amino acid
These solutions
hydrochloric
in order
standards
The assessment
modified
known amounts of
containing
was carried
final
This
amino acid
or in part
thirteen
amounts of
5.
to ensure
were made as a result
experiments.
The composition
recovery.
solutions
and recovery
and
was adopted
were incorporated
The modifications
applied
standards
amino acid
(1981)
by Siddiqi
and improvements
modifications
several
of amino acids
and concentration
containing
and 33% fructose.
Once satisfactory
was then
applied
samples
of honey,
honey amino acid
recoveries
individually
in a like
concentrates
were obtained
to all
manner.
the
the modified
two hundred
Further,
were preliminarily
for
procedure
and fifty
six
a few randomly
checked
for
chosen
the presence
74.
TABLE 5
Concentration-of
Amino acids
amino acids.
in
solutions
A, B and Sl
Concentration
of, amino acids in solution
as prepared by weighing
(nMoles per ml)
(nMoles per 0.6 mls)
A*
B**
Sl***
(nMoles per ml
Bl****
394
394
1180
483
418
418
1250
513
Threonine
502
502
1510
616
Serine
380
380
1140
466
596
596
1790
730
Proline
4960
4960
14880
6080
Glycine
618
618
1850
757
Alanine
474
474
1420
580
Valine
324
324
970
397
Isoleucine
398
398
1190
488
Leucine
388
388
1160
476
Tyrosine
650
650
1950
797
Phenylalanine
814
814
2440
998
Lysine
Aspartic
Glutamic
acid
acid
Solution A was made up in 10% propan-2-ol/0.5M
acid solution.
hydrochloric
B was made up in 33% glucose plus 33% fructose solution.
Solution S1 was made up in 10% propan-2-ol/0.5M
hydrochloric
acid solution.
Solution Bl concentration
values given are those from the total
10.6 ml, that is, Solution B (10 mls) + Solution S1 (0.6 m1s).
Solution
75.
TABLE 6
Separation
and concentration
chromatography
No.
on solutions
of amino acids
Amino acid solutions
or honey samples
and honey samples
of amino acids
Amount
used
(mls or g)
by ion-exchange
Addition
of Sl*
(mls)
IxC
12
AAAA
1
Solution
A*
10.0
+
2
Solution
Al*
10.0
+
3
Solution
A2*
10.0
+
+
4
Solution
B*
10.0
+
+
5
Solution
Bl*
10.0
+
+
6
U. K. Survey Sample
Code No. 174
10.0
+
+
U. K. Survey Sample
Code No. 174
10.0
o. 6
+
+
China light
amber
Code No. 211
10.0
-
+
+
China light
amber
Code No. 211
10.0
0.6
+
+
Mexico Yucatan
Code No. 227
10.0
-
+
+
Mexico Yucatan
Code No. 227
10.0
o. 6
+
+
7
8
9
10
11
o. 6
For amino acid composition
and concentration
Al, A2, B, Bl and S1 refer
to Table 5.
in
solutions
The technique
of ion-exchange
chromatography
was not
(10 mls) was evaporated
but the solution
to dryness
in text using vacuum pump and liquid
nitrogen.
1
ion-exchange
-
IXC - modified
2
-
AAAA - Automatic
+
-
relevant
experimental
procedure
performed.
-
-
relevant
experimental
procedure
not
Amino Acid
chromatography
Analysis
procedure.
procedure.
performed.
A,
performed
as described
76.
of proteins
the Bio/Rad
using
of 1 ml of coomassie
acid
absorbance
amino acid
concentrates
A standard
reference
duplicate.
Furthermore,
composition
consistency.
Also,
thirteen
honey samples
to check
at 20 pg per ml
out
were carried
assays
step
of Resin
The viscous
cation
for
of honey required
exchange
the binding
(500 mls)
lon ger coloured
no longer
acidic.
resin.
acid
to protonate
portions
in honey.
in distilled
6M hydrochloric
with several
of the
of distilled
by the
and stored
ten minutes
Sl was added to
effects
of other
the use of a large
Dowex 50-X8 20 - 50 US mesh (Na).
resin,
compounds present
in portions
similar
honey
Binding
property
facilitated
similar
of similar
to ensure
of solution
and the
recoveries
the processing
amino acids
5 were checked
amino acids
for
of
during
on the added amino acids.
consistituents
Preparation
intervals
in Table
given
(10g)
for
5.
a de-proteinization
that
from solutions
recoveries
as those
settle
All
assayed.
at random periodical
of honey samples,
resin
solutions
obtained
A and B, Table
serum albumin,
indicated
The results
were also
required.
was not
size
bovine
similarly
was also
concentration
in
of amino acid
protein,.,
at 595 nm were obtained.
measurements
at 595 =
measurements
the addition
to 1 ml of honey amino
G-250 based reagent
Blue
involved
This
assay.
Then absorbances
concentrates.
Similarly,
micro
free
amino acids
This
resin
water
This
water
until
suspension
eluents
was then
(500 mls).
To this
was added and the mixture
the resin.
of distilled
The protonated
water
(100 mls)
This
and other
was initially
the
mesh
washed
were no
allowed
washed resin
was stirred
resin
until
to
for
was washed
the washings
were
77.
Ion-exchange
Chromatography
A prepared
(10.0g)
sample of honey as described
was weighed
(10 mls)
water
by gentle
Procedure
to a temperature
warming
was then
solution
could
of a glass
distilled
in
the beaker
a2
ml portion
of distilled
stirrer
magnetic
components
and other
pipette
pasteur
was washed with
and the
twice
and honey mixture
fifteen
for
no loss
distilled
water
by pipette.
the
and then
process
water
The pooled
vacuum oil
head fitted
the aid
of a
containing
the aid
The amino acid
stirring
This
process
for
of
(5 mls)
solution
This
ten minutes
was repeated
was added to the washed
was then
mixture
and all
supernatants
pump at 1=
a
bound resin
stirred
for
ten minutes
off and retained.
was carefully
pipetted
I
The resin was then washed twice
twice more.
(2 mls)
free
the
the carbohydrates
with
with
of
supernatant
was repeated
distilled
was
were discarded.
the supernatants
bound resin.
transfer
binding
effective
of resin.
(10 mls)
once more with
with
of honey was removed carefully
A 7M ammonium hydroxide
amino acid
for
The supernatant
to ensure
and transferred
quantitative
was stirred
minutes,
supernatant'removed
and all
was repeated
Thus,
water.
to the resin.
amino acids
This
rod.
and the
of honey were left
traces
no more greasy
or on the glass
The resin
ensured.
until
The beaker
(2 mls)
water
honey
This
no more honey
until
by means of droplets.
be transferred
by means of droplets
resin
was ensured
mixing
0
40 C.
exceeding
added to the. protonated
rod were then washed with
glass
not
distilled
with
Thorough
rod.
Honey
was used.
thoroughly
and mixed
the aid
with
solution
accurately
before
the
were then
of mercury
evaporated
pressure
to a B14 cone (Washington,
1966).
with
to dryness
the aid
Liquid
with
were pooled.
collected
supernatants
This
using
a
of a microsplash
nitrogen
was used
78.
in the vacuum pump traps
as a coolant
from contaminating
ammonia and water
of the extracted
contamination
This
was done by rinsing
(0.5
mls)
in
twice
the usual
to prevent
amino acids
in vacuo at a pressure
dried
to a minimum.
was reduced
head with
the microsplash
The final
The ammonia
the vacuum oil.
into
to remove any splashings
manner.
the evaporated
distilled
flask
the
and evaporated
was left
product
water
overnight
Sodium hydroxide
of 0.5 am of mercury.
pellets
were used as desiccant.
It
was found
removed from the
contact
Dissolution
2.5
in
solution
flask
was discharged
in
left
either
This
process
the
flask
was then
of 10% propan-2-ol/0.5M
3
The 2.5 cm drum vial
0.5M hydrochloric
the concentrated
Each of
were then
acid
stored
following
the
that
or on the
ml)
concentrated
in a refrigerator
until
inside
procedure
hydrochloric
and brought
of a pasteur
into
flask.
the
of
and transferred
to a clean
All
pipette.
solution
from
of
walls
flask
to the
pipette
the
traces
no more visible
were
the pipette.
by 0.2 mls
0.3 mls and finally
acid
mixture
be discharged
glass
the dried
containing
(1.5
could
by adding
hydrochloric
flask
wall
to ensure
be sucked up by the pasteur
could
honey amino acid
these
the aid
the vial
repeated
glass
warming
with
that
into
product
gentle
and any solution
was transferred
pipette
with
3
cm drum vial
the
In order
product
inside
the accessible
was ensured
pre-weighed
the
all
with
be effectively
not
1 ml of 10% propan-2-ol/0.5M
was added to the dried
solution
acid
could
manipulation.
the dried
of all
Initially,
was utilized.
product
by physical
flask
transfer
quantitative
the dried
that
and transferring.
10%
propan-2-ol/
-
product
was weighed
and-the
weight
of
determined.
honey amino acid
at 40C until
samples
required.
thus
prepared
After
use
79.
these
to the refrigerator
samples were returned
Examination
and Identification
of
by a Combination
Substances
required.
next
until
Positive
the Ninhydrin
of Paper Electrophoresis
and Chromatography
Paper Electrophoresis
Biological
for
groups
or gain
to the electrode
Originally
applied.
equally
to an inert
The rate
support
and charge
distribution
compounds,
referred
and ionic
of the medium,
for
power decreases
Amino acids
properties,
that
the pH of the
of
ampholtyes
the
the
solution.
they
both
size,
shape
by the pH
It
the
and conditions
acid
and can ionise
Thus the direction
phase.
of support.
PAGE > cellulose
display
applies
two running
influenced
same mixture
are ampholytes
are pH dependent
of
separation
power is
in the order
field
and a bound water
and by the nature
are compounds which
is,
the process
depends upon their
as resolving
has been shown experimentally
resolving
a mobile
molecules
in
can migrate
when an electric
were used but
and therefore
to here
strength
polarity
containing
these
of
to become the charged
molecules
of opposite
-COOH or -NH 21
groups,
in solution
H
solutions
of migration
polar
These charged
-COO and -NH3
solution
is
can loose
example
containing
molecules
and extent
acetate
> paper.
and base
according
to
of migration
and can be used to produce
the required
separation.
RRR
H N+-CH-COOH
332
pH
form
Ionic
Migration
Acidic
Cation
Towards Cathode
H N+--Clt--COO- ==;
Isoionic
point
Zwitterion
Stationary
H N-CH-COOBasic
Anion
Toward Anode
80.
By employing
acids
for
except
and therefore
reported
the oxidation
paper
product
the
towards
migrate
to be found
technique,
at pH 1.9
electrophoresis
in honey.
cysteic
is
acid
for
given
amino
as cations
exist
Cysteic
cathode.
The basis
chromatography,
the natural
all
has not
acid
the other
part, of
the paper
after
been
the
electrophoresis
procedure.
of buffer
Preparation
buffer
Electrophoresis
100%) formic
distilled
(104 mls)
acid
consisted
and glacial
of a mixture
acetic
was made up to two litres.
water
adjusted
so-called
of
(58 mls)
acid
(98% -
and with
The pH was checked
and
needed to pH 1.9 ± 0.1.
if
Procedure
20
T&atman
No.
chromatography
of
A sheet
to give
cut
the longer
rods
to avoid
stages
spot
size
The paper was then
or contact
contamination
or amino acid
drying
with
of 0.5
in between
buffer
The loaded
compartments.
tank
so that
the
longer
(250 mls)
paper
to become the positive
terminal
a pasteur
pipette
the paper
all
for
the honey
a constant
minimum
the sample application
was then
arranged
into
in
evenly
the electrical
with
electrophoresis
compartments.
was ensured
to the Vokam power pack.
electrophoresis
connections
the
the buffer
of sample application
when connected
the buffer
in each of
was placed
57 cm edges dipped
the origin
moistening
on glass
and 14 cm from the 57 cm edge.
The 57 cm edge nearest
After
so as to maintain
with
by a micropipette,
was applied
The origin
40
from
10
the
cm side
cm
was
supported
the bench whilst
with
(25 jil)
sample
cm diameter.
Electrophoresis
40 x 57 cm and arranged
the dimensions
with
edge on the right.
concentrate
in
a sheet
46 x 57 cm, was
paper,
buffer
were made.
applied
by
81.
Electrophoresis
was carried
four
hours.
tank
and then hung in a fume cupboard
The paper was carefully
dried
To this
substances
in honey were applied
not
arranged
1.5
of
positive
solutions
between
These standard
the paper.
and dried
groups
the sample origin
ninhydrin
so that
25 pl
were applied
positive
to occur
These
standards.
of different
RF values
positive
have been found
The solutions,
substances,
10 cm line
cm on the
ninhydrin
as reference
two applications
to their
according
of
a period
at room temperature.
thirty
literature
exceed 0.5 cm in diameter.
the ninhydrin
to dry
paper,
to the
in
were spotted
solutions
did
in
for
removed from the electrophoresis
electrophoresis
according
which
250 volts
out at
at
the
spot
of each of
of standards
intervals
of
57 cm edge
and the
substance
groups
were:
(a)
arginine,
a-alanine,
(b)
histidine,
gýycine,
(c)
lysine,
(d)
Cysteine,
(e)
asparagine,
serine,
tyrosine,
proline,
glutamic
aspartic
acid
valine
and phenylalanine;
methionine
and leucine;
and isoleucine;
tryptophan
acid,
and threonine;
hydroxyproline,
y-aminobýtyric
acid
and pipecolic
acid;
(f)
ornithine
(g)
glucosamine,
(h)
kynurine
and 0-alanine;
' citruline,
and a-aminobutyric
glutamine
and 0-aminobutyric
acid;
acid.
Paper Chromatograp
The technique
between
compound
phase.
of chromatography
a stationary
At any instant,
depends upon partition
phase which
an equilibrium
is
is
set
polar
of a given
and a mobile
up and then
covalent
displaced
82.
the tendency
until
due to its
the
of
phase and its
The initial
for
solvent
forces
on the cellulose.
the dry
responsible
in descending
movement and thereafter
(Smith
by gravity
aided
is
paper
and Seakins,
1963).
of solvent
Preparation
The butanol-acetic
acid-water
(1981).
to Siddiqi
This
(200 mls) and glacial
butan-l-ol
(75 mls)
water
groups
of
movement is
and Macek,
1976 and Hais
the major
of
and a weak ion-exchange
and other
2 cm of solvent
chromatography
according
bonding
forward
forces
attractive
On paper
support.
attraction
capillary
first
the
hydroxyl
involving
the components
by the various
solid
hydrogen
include
attraction
phase to carry
is balanced
solubility
stationary
mechanism
of the mobile
solvent
mixture
(BAW) was prepared
solvent
mixture
consisted
acetic
acid
of
(30 mls) and distilled
at pH 4.3.
Procedure
Descending
chromatography
to the origianl
angles
bottom
end of
short
chromatography
with
this
electrophoresis
(150 mls)
The BAW salvent
acid and ninhydrin
for
was allowed
in
into
related
the solvent.
and the
The
and development
hours.
The paper
was
overnight.
substance
derivatives
is a universal
compartment
chromatank
four
at right
a Shandon Chromatank.
a fume cupboard,
Positive
of colour
and similar
twenty
in a direction
the solvent
the paper was dipped
the Ninhydrin
The formation
amino acids
in
paper was then hung in the
solvent
of
using
was placed
hung
dry
to
then
and
removed
Detection
was performed
by the reaction
method for
compounds.
between
amino
detecting
the presence of
The reaction
mechanism of
83.
formation
the colour
proposing
has been disputed
different
pathways
(Hais
and Macek,
derivatives
The use of metal
and stability
formation
the
chelates
the
enables
of colour
research
the
of
1963 and Metzler,
the formation
of
workers
colour
1977).
enhancement
complexes
of sensitivity
(Haworth
and
1969).
Heathcote,
Preparation
of solutions
The ninhydrin
reagent
was prepared
to Haworth
according
and
(1969).
Heathcote
Cadmium acetate
acetate
for
by various
(2g)
(200 mls).
in
stock
acetic
glacial
Ninhydrin
in cadmium acetate
solution
reagent
This mixed reagent was stored
longer
not
case
any
(40 mls)
acid
and distilled
cadmium
water
by dissolving
was prepared
stock solution
by dissolving
was prepared
(2g)
ninhydrin
(24 mls) and propanone (200 mis).
in a refrigerator
until
and in
required
than seven days.
Procedure
Ninhydrin
it
by dipping
positive
in
the cadmium acetate-ninhydrin
fume
hung
in
then
a
cupboard
was
hours
some three
positive
of concentrated
became visible
was then
sulphuric
Each sheet
days and then
a week later
ninhydrin
acid
positive
the
complexes.
The chromatogram
concentration
cylinder
every
colour
This
After
of ninhydrin
The
were outlined.
to achieve
examined
to record
chromatogram
reagent.
sealed
(5 mls),
was then
on the
at. room temperature.
and these
in a dark
stored
environment.
resulting
to dry
due to the high
the spots
complexes
chromatogram
were detected
substances
with
a test
tube
an ammonia free
day for
the next
and position
chromatogram
of
three
the
was either
84.
the patterns
recognise
differed
Considerable
or traced.
photographed
of
the ninhydrin
in kind
and concentration.
Quantitative
Analysis
Amino Acid
identification
as these
.
Amino Acid
of ion-exchange
Using
Analyser
of individual
by measuring
the absorbance
be compared with
This
wavelength.
at a given
imino
the ninhydrin-amino
of
acid,
The absorbance
proline.
that
obtained
with
photoelectrically
acid
570 nm for
being
eluted
The quantitative
can be evaluated
amino acid
by the
individually
of
compounds can be automated.
positive
440 nm for
followed
chromatography
determination
and quantitative
determination
formed
complexes
Analysis
The technique
ninhydrin
positive
of the Free Amino Acids
the Automatic.
to
was required
experience
colour
complex
amino acids
and
can then
measurement
a corresponding
amino acid. standard
of known concentration.
is
It
on a capillary
spheres
is
in order
10-
6
m.
high
to maintain
an adequate
and constant
and sample especially
may be measured before
are approximately
a true
satisfied
the separation
and inversely
However,
flowrate.
as the
equilibrium
coloured
is
in most commercial
or resolution
to the
as the bead diameter
The other
sample application,
resin
proportional
needs to be increased
pressure
separation
ion-exchange
resolution
In general
to the column length
the applied
are repetitive
reagents
6x
with
after
and mixing
area and bead diameter.
cross-sectional
decreases,
filled
column
of diameter
proportional
diffusion
to prevent
essential
substantially
majorrequirements
mixing
consistent
products
reached.
of
of
the reaction
These conditions
instruments.
85.
Procedure
.
honey amino acid
To the concentrated
standard
buffer
mixture
to the analyser
by first
cartridge
ion-exchange
column system containing
the basic
amino acids while
buffer
Citrate
amino acids.
the other
of 600C by means of a-water
The samples
coil
was then
colour
the
passed
reactions
filters
interference
The output
to take
amino acids,
displacement
at constant
coil
This
place.
housed two flow
of different
from the colorimeter
maintained
cell
approximately
in
a mixing
This
reagent.
0C
95
for
with
the
passed
through
mounted
at 570 nm and at 440 rm.
was then recorded
on a chart
for
seventeen standard
recorder
each different
amino
amino acids at
each programmed cycle was completed in
two hours.
(nMoles) of each amino acid was then determined
The concentration
from the area under
known standards
at
assemblies
wavelengths
The normal run detecting
25 nMoles concentration,
temperature
was finally
solution
in the form of peaks which are characteristics
acids.
were pumped
pumps through
of 1% ninhydrin-hydrazine
a heating
through
amino acids and
the column were then mixed
off
quantitites
which
colorimeter
and neutral
and neutral
bath and a circulator.
once eluted
discrete
with
the basic
These columns were operated
these columns.
the acidic
separates
by means of two positive
on to a. two
One column separates
resin.
pH 5.25 for
pH 3.25 and pH 4.25 for the. acidic
at high pressure
being loaded onto an equilibrated
The samples were then eluted
resin.
containing
acid
samples (20 Ul each)
duplicate
From this
was added.
were applied
(25 nMoles) in O. lM hydrochloric
of norleucine
solution
(25 V1) an internal
sample
the
obtained
trace
response
on chart
curve
paper.
in
comparison
with
the
This was done by using the
86.
formulae
of
derived
as shown in Appendix
amino acids
per unit
sample weight
The mature
is
grain
pollen
source,
and differences
between
contours
and apertures
can lead
(1975),
and Iverson
Light
dyes,
found
acids
carboxylic
1974).
and Linskins,
to stain.
in relative
either
for
1971).
always
the
basic
sporopollenin
exines
of different
the extent
of pollen
even under
ideal
by the bee stored
in
conditions.
the hive
pressures
by honey ready
possible
(Howells,
1969).
pollen
in honey of
foreign
family
level
level
is
only
(James,
origin,
1969).
species
is
moieties,
source
Also
variable
the degree
of reactive
to the species
For pollen
identification
in some cases,
their
vary*in
or in
and subject
identification
(Stanley
of pollen
the masking
of
has shown
of hydroxy-
series
sporopollenin
of different
40OX, is
by. selective
Research
exine
generated
osmotic
features
grain
example,
and Sawyer (1981).
of about
fushin.
that
surface
See, for
the polymerised
suggests
identification
possible
been collected
in
shape,
to identification.
floral
its
of
as to size,
the pollen
proportions.
(Heslop-Harrison,
not
characteristic
grains
stains
This
cross-linking,
Moreover,
of
However,
or in
of
usually
example,
dye selectivity
response
of Honey
Moore and Webb (1978)
by enhancement
with
this
Analysis
at a medium magnification
microscopy
made easier
that
the concentration
Analysis
Microscopical
staining
Hence,
was calculated.
Microscopical
Quantitative
Faegri
III.
groups
level
that
is
has
to great
to genus
especially
was possible
to plant
87.
P-ent-atIliv-gs
The method for
to
for
grains
pollen
those
the preparation
the microscopical
described
techniques
the microscope
of
of honey,
examination
by Louveaux,
slides
Maurizo
of
was according
(1970)
and Vorwohl
and Sawyer (1981).
Preparation
of the
(7g)
Gelatin
on a hot
(0.5g)
phenol
(0.1g)
stains
in distilled
To this
gelatin
Two grades
solution
was dissolved
in
of
stained
glycerine
way.
gelatin
(1.5
was added and labelled
as the
glycerine
gelatin
labelled
and
added
was
Honey (10g)
This
water
in
the
was carefully
was transferred
as possible,
and spread
pipette.
over
coloured'
stain.
water
centrifuged
(20 mls)
using
on to a microscope
an area
at
off
of honey sugars.
a pasteur
slide,
with
to dry.
the pollen
carefully
in distilled
pipette.
as completely
15 x 15 mm by means of
slide
0
40 C,
by gentle
at 2500 revolutions
so as to remove traces
off
mls)
grains
was dispersed
The sediment
pipetted
solution
(3.0
solution
was decanted
The supernatant
The microscope
in an oven maintained
fuschin
the pollen
of
was then
of sediment.
The sediment
placed
and
stain.
in distilled
and re-centrifuged
The supernatant
a pasteur
and
basic
fuschin
coloured'
slides
was dissolved
ten minutes.
no loss
(10 mls)
(I ml)
were prepared
'Light
basic
'Dark
as the
honey solution
for
per minute
ensuring
(30 mls)
of the microscope
warming.
70% ethanol
(30 mls)
To one glycerine
Preparation
by warming
(50 mls)
glycerine
gelatin
following
The other
water
(160 mls).
water
mls)
(42 mls)
stirring.
was added with
fuschin
Basic
distilled
was dissolved
bath.
water
gelatin
glycerine
the tip
of
smear was then
88.
Staining
and mounting
After
basic
drying
fuschin
stained
a lot
of
intermediate
were
A coverslip
slide.
and the
slide
at
the
400C for
coverslip
with
with
grains
containing
for
Moreover,
both
of
were
light
the
the
and dark
used.
positioned
of
distribution
by placing
Excess
of
between
the
of
pollen
twizzers.
In
the
coverslip
between
stain
the slide
the
of
edge
a pair
become trapped
should
the
near
the help
with
the
a tissue
paper
and left
was then
sealed
along
gelatin
pressing
to set.
overnight
the edges with
stain
coverslip
in an oven maintained
was removed by gently
stain
a clear
from becoming
nail
infected
Grains
were identified
by using
thirty
containing
the identification
prepared
by Sawyer (1975).
The full
smears
pollen
the
of
pollen
was used.
a combination
drops
two
or fungus.
with
and standard
stain
the glycerine
grains
conjunction
For
stain.
coloured
ten minutes.
by Ray Williamson
two hundred
coloured
of Pollen
Pollen
pollen
little
was ensured
Identification
of
containing
A uniform
to prevent
bacteria
smears
carefully
The coverslip
varnish
Pollen
bubbles
way no air
and the
with
was carefully
smear and lowered
this
was stained
smears
pollen
stains
smear
dark
the
smear
pollen
pollen
light
the
pollen
coloured
the
stain.
with
the
of
Also
reference
list
and fifty
pollen
sample slides
of all
six
four
different
key,
index
by Sawyer (1981)
fresh
the pollen
samples
reference
slides
pollen
cards
prepared
grains,
in
and microphotographs
and diagrams
from known sources
of pollen
grains
were obtained
were prepared.
grains
that
were identified
of honey have been given
in
the
in Appendix
VIII.
89.
In
list
this
the identity
levels,
classification
then
the
of related
for
example
of
identifying
six
in
Brassica
a honey sample.
pollen
throughout
the present
the nearly
related.
identity
whose
classed
Counting
near
of
Pollen
Grains
grains
were counted
an exception
in
grains
to be present
elsewhere,
and were quoted
especially
as such
was made for
pollen
the
grains
foreign
family
even at plant
the pollen
Counting
at
smear with
frequency
was according
to Louveaux,
were then
classes
Maurizo
field
the visual
least
Between 200 - 300 total
the centre.
classes.
in
of the microscope.
eyepieces
and presented
frequency
discussed
whose distinctive
Pollens
Pollen
and others
However,
the
by Crane,
are likely
not be determined
into
identified
described
nearby
species
7 are
in Table
honey sources.
world
to genus only
TrifoZiwn
could
the edge of
or near
Listed
problems
honeys
level
were
as unidentified.
Pollen
objective
alone.
analysis.
identifiable.
were readily
pounded considerable
reason
were limited
identity
genus;
species
cultivated
For this
the identity
isi.
that
was possible,
as important
species
botanical
each pollen
In most cases
out of the nine
species
related
closely
related
for
name.
species
by genus and
followed
list
this
by pollen
species
(1984)
Day
and
Walker
from
plant
the Brassica
European
in
given
to three
was specified
family
plant
cormon English
equivalent
to a group
is
that
Also
species.
name the
of each pollen
for
as seen through
was carried
two counts
pollen
grains
each samples
and Vorwohl
distributed
according
The following
terms
(1970).
to their
out usually
at
minimim
were counted
of honey.
This
The pollen
percentages
were used for
the
expressing
grains
into
the
90.
TABLE 7
of common European
List
species
of
the
genus Brassica
Species
Plant
family
Genus
Cruciferae
Brassica
it
Botanical
name
English*
name
Campestris
Field
juncea
Chinese
napus var,
napobrasswa.
Shaljam
napus var**
oZeifera
Oilseed
nigra**
Black
Mustard
rapa
Winter
Rape
Cabbage
Mustard
Rape
There are other English names besides the common names,,
by Crane, Walker and Day, 1984.
these names have been listed
Adequate
honey yields
are obtained
from
these
plants.
91.
frequency
forty
classes.
five
percent
represented
pollen
'Important
Minor
three
and over
present
Pollen'
and fifteen
percent
percent
'Predominant
These were:
pollen
present,
between
sixteen
represented
which
'Secondary
percent,
between
present
Pollen'
represented
Pollen'
five
and forty
pollen
'Minor
and
percent
Pollen',
three
less
represent
than
present.
pollen
Computation
data
The experimental
fifty
six
This
for
social
and writes
out
to a file.
sciences
in
The data
such files
has
for
data
analysis
1975 and Hadlaihull
et aZ.,
for
analysis
statistical
in at
computer
using
from
a file
SPSS commands
the available
computer,
the
reads
and the
visual
display
1982 and 1984).
1982 and Eckersley,
to the prime
access
prime
information
were typed
(Noland,
(VDU) terminal
the
(Nie
statistical
(SPSS) version
Sciences
programs
on the Salford
The SPSS package
procedure
Social
and
1981).
and Nie,
units
for
750 computer
Prime
of statistical
comprises
package
and was written
contained
Package for
the Statistical
available
9.
The Salford
two hundred
the
each of
the use of a computer
samples warranted
and interpretation.
analysis
for
gathered
files,
editing
SPSS package
The
and carrying
have been listed
below:
the RETURN key a few times
By pressing
PLEASE' appears
password
the prime
are
on the
typed.
system
is
screen.
When the
then
the
The user
OK, appears
available.
ISALFORD PRIME
name followed
on the
screen
LOGIN
by the
access
to
92.
2.
By. typing
Then the information
to be opened.
can be typed
in detail
in Appendix
When all
After
in a pre-determined
of the data
arrangement
3.
the relevant
line,
last
the
the EDIT appears
typed,
4.
for
'SPOOL DATl-UK,
-AT,
PRINT the
errors
been made the
the
6.
filename
file
file,
opening
of a file
and the
series
the
point
LEAR' the computer
line
on the
for
printer
can then be located
in
displays
the
by typing
the contents
printing.
on the print-out.
can be made by
computer
of the
the contents
is
screen
transfer
will
once more
filename
can be obtained
file
of this
file
on the
commands TOP, BOTTOM, NEXT, LOCATE9
the editor
can be rectified.
When all
the corrections
as described
can be closed
the SPSS control
procedure
file
previously
of control
was TEST1.
statements,
and obeyed by the
was created
as described
for
have
except
This
SPSS control
each specifies
SPSS.
by following
DATl-UK.
has been given
of a SPSS controlfile
filename
recognised
file.
the
need not be typed.
Another
and format
into
Then OK, appears
to the Lear-Siegler
By using
the
of
have been. described
files
At this
screen.
DATl-UK.
ED DAT1-UK which
screen.
file
this
the RETURN key and then
by pressing
Ammendments to the DAT1-UK file
typing
The format
data have been entered
in the DATl-UK file
Any errors
into
to be entered
the data
of the contents
of DAT1-UK file
5.
in
screen
order.
a file
has enabled
IV.
on the
example,
A print-out
in
INPUT on the
ED and obtaining
The structure
in Appendix
file
the
contains
a command which
VI(b)
a
is
93.
7.
The TEST1 file
made by using
8.
be checked
can also
the SPOOL and EDIT c
analysis
were carried
out by typing
line
Lear-Siegler
by typing
log
will
Hence in
hundred
two samples
and ninty
in
arranged
The other
honeys
the numerical
of the
four
as given
the vast
fifty
in Appendix
considered
samples
appropriate
of statistics
sort
(1981)
et aZ.,
of Dr.
of honey,
data
the analytical
computer
the
foreign
of
one
honeys
Kingdom survey
in Appendix
I.
and commercial
to their
according
known
I.
biochemical
collected
reference
and Davies
Department
the
completed
one contained
for
information
the
the use and application
was not my field
Pembertont
to process
with
is
session
Analysis
understandable
amount of numerical
six
Gilbert
to obtain
at the
code number given
order
Statistical
In order
printed
system.
samples
in an alphabetical
of origin
and is
an OK, on the screen
the United
of
The
contains
were compiled,
order
the sixty
contained
arranged
country
files
way two'data
this
RESULTV.
which
terminal
from the prime
the user
TEST1
file
output
When the
printer.
previously.
file
SPSS control
the
analysis
the command LO after
out
in
as the
of the statistical
the results
and corrections
'SPSS TEST1, -LISTINGS
defined
RESULT1 is
errors
mands as described
detailed
The statistical
-LISTINGS
for
to Davies
and Harris
of expertise,
of Mathematics,
information
two hundred
of
and
statistics
was
(1975 and 1976),
(1982).
the
from
Since
experience
University
gathered.
the
subject
and advice
of Salford
was
94.
familiarisation
After
recommended by Dr.
between
VI(b))
as collected
type
and improvements
which
SPSS considerably
per
out
as described
as 'TESTV
labelled
files
SPSS control
groups
into
(Appendix
appropriate
The details
VI)
2.
carried
out
briefly
outlined
(Appendix
groups
(Appendix
modifications,
below.
classification
that
changes
of samples
and selection
in SPSS control
modifications
the
of
and improvements
concentration
measurements
less
ten samples
is,
than
were carried
subclasses
file
and the
were obtained
Relevant
VI(d))
of these
nine
Pemberton,
'DAT1-UK'
was made by replacement
and classification
processing
(Appendix
random selection
enabled
which
into
V(a).
labelled
1, filename
'COPYF filename
to Appendix
much needed
and multifloral
file
of the raw data
Copies
attempt
to require
of amino acid
of unifloral
in Appendix
(refer
honey samples
of Dr.
sample number groups,
and defining
group
my initial
These modifications
out.
transformation
of small
distance
the predictive
enhanced
were carried
root
square
of Mahalanobis
was considered
groups,
pollen
was
one modification
SPSS to classify
was
analysis
'DISCRIMINANT'
After
groups.
Upon the recommendations
improvements.
and removal
of Discriminant
calculation
with
the
specified
predominant
file
for
biochemistry
of both
aspects
A subprogramme
such as pollen
populations
which
were:
Pemberton.
on the SPSS package
raw data
using
with
the use and application
and statistics
available
the relevant
to each of
in a particular
(e),
improvements
have been detailed
the
command
the copy
commands
group
recoding
of
for
sample
of honey samples
and allocation
VI(d)(ii),
by using
of appropriate
VI(c)),
SPSS control
(f)
and Appendix
and changes
VII)'.
of command
in Appendix
V and
95.
Discriminant
As it
is
is
the process
boundaries
of
Canonical
functions
which
these
is
This
distance.
SPSS (Appendix
for
of
samples
combinations
are known
degrees
of
group
variate
of
variable
separation.
is
can be
which
the minimum distance
known as Mahalanobis
method developed
'MAHAL' which
all
containing
by Mahalanobis
and obeyed by
recognised
carrying
file
into
out
nine
grains
concentration
described
two samples
and detailed
the discriminant
and subsequent
in
analysis.
of
classification
in a honey sample were used for
predominant
classification
and ninety
and
earlier.
estimation
the pollen
the biochemical
the one hundred
as described
predicted
amino acid
linear
varying
each of
From the microscopical
The SPSS then
of amino
The SPSS control
The SPSS was initiated
allocating
of Fisher's
combinations
statistical
file
was used for
the distribution
variates
canonical
are linear
by the complex
data
information
VI(c)
One.
the determination
is
error
minimise
VI(a)).
of honey was required.
Appendix
will
to a group.
The maximisation
a stepwise
The U. K. survey
analytical
of
analysis
of group
calculation
on each canonical
by the term
described
and is
their
give
axis.
defined
is
groups
that
A number of these
on co-ordinate
between
is
analysis
each sample has a value
plotted
the
variates
variates,
as canonical
Thus,
enables
to statistical
Discriminant
space which
field
of this
section
concentrations.
acid
which
Honeys
computers
are accepted.
a multi-dimensional
discriminant
linear
definitions
of
a honey sample of unknown origin
allocating
analysis.
Kingdom Survey
the application
of computation
within
specialised
in
understood
following
the
problems
on the United
Analysis
frequency
class
of honey samples
measurements.
pollen
groups.
basis
the
on
of
96.
to improve
In order
pollen
major
performed
five
major
pollen
on the original
amino acid
groups,
those
evaluated.
.
pollen
Discriminant
analysis
each of the
representing
These three
major
to five
honey samples
major
sources
representing
analysis
on the honey samples
of honey in
sources
Brassica4
were:
was also
in
otherwise
and samples which
the years
analyses
were also
five
individual
pollen
the United
TrifoZium
repens
were heated
which
and sugar
analysis
feeding
or
Moreover,
or otherwise.
Similar
groups.
on the honey samples
on honey samples
performed
of heating
effects
samples
were sugar-fed
three
on the combined
performed
1981 and 1982;
harvested
the
unifloral
I
Discriminant
of
In the
values.
and CaZZuna.
these
of
and modifications
concentration
was performed
three
the n=ber
of'samples
from nine
groups were reduced
were also
Kingdom.
the classification
representative
was also
performed
on a sample
of
honey.
The details
of
the procedure
above have been described
Analysis
Discriminant
in Appendix
on the Foreign
Discriminant
analysis
foreign
and comercial
on the
data file
containing
each of the sixty
For the United
following
countries:
sometimes
China.
of the
all
four
analysis
statistical
listed
V(a).
Honeys
and Commercial
was performed
using
honey samples.
the biochemical
the
subprogram
The foreign
'DISCRIMINANT'
and commercial
information
and analytical
for
samples of honey was required.
Kingdom the major
Australia,
Discriminant
sources
of
imported
honey are
the
Canada,, Mexico and New Zealand and
analysis
was performed
on the
samples
of
97.
honey
from the
originating
honey samples.
VII(a)
The SPSS control
and the control
Discriminant
following
the
of
origin
they
was not
detailed
on the
was also
foreign
VII(b)
patterns
samples.
between
honeys
containing
samples whose country
a country
code from which
on the basis
that
and samples
were used
and commercial
family;
to have originated
in. Appendix
to distinguish
performed
same plant
English
with
and described
in Appendix
known but were allocated
and amino acid
together
and honeydew honeys;
nectar
were most likely
content
file
analysis
from the
originating
pollen
countries
given
analysis
groups:
four
statements
discriminant
to perform
first
of their
pollen
were or were not
processed.
The details
of the procedure
above have been described
Analysis
Discriminant
Survey
Foreign
and the
The data
file
was appended onto
the United
(APPEND)',
honey samples
were pooled
the
containing
the tail
In this
in at
end of
and the
in
together
foreign
a visual
the U. K. survey
display
analysis
statistical
of
in Appendix
required
were to change the number of cases
'IF'
Appendix
control
VI(c)
V(a)
unit
either
and VII(b)
statements.
could
and
a sequential
data
order.
honey samples
file
by typing
UK SURVEYDATA
terminal.
the
SPSS control
be used.
The only
files
modifications
to 256 and to replace
These control
and 54 to 59 in Appendix
foreign
and commercial
detailed
five
Kingdom
and Commercial
'COPYF
FOREIGN AND COMMERCIALDATA FILE,
the command
FILE
listed
analysis
V(b).
on the Combined Honeys of
honey samples
commercial
the statistical
in Appendix
Kingdom survey
The United
of
VII(c).
statements
the
60
in
56
and
were
98.
Discriminant
analysis
was performed
United Kingdom and the foreign
U. K. survey and six other
groups Brassica,
TrifoZiwn
to distinguish
between the
and commercial honeys; between the
country
groups;
and between the three pollen
repens and Castanea common to the combined
samples.
The details
of the procedure
above have been described
Note that
in
at a visual
July
1983.
and format
carried
of the statistical
However,
unit
terminal
some of these
due to the updating
out at regular
listed
in Appendix V(c).
the commandsused to initiate
display
analysis
intervals.
the SPSSwhich were typed
were correct
up to
commands have changed
and revision
of
the end of
in
the computer
context
systems
RESULTS
CHAPTER 3
99.
RESULTS
CHAPTER
The Colour
The colour
3
and Aroma of Honey
for
and aroma were assessed
the honey samples
that
were
examined.
In assessing
not
considered
honeys produced
white
colours
by the plant
genera
the Echium honey and then
'Viguieral
The light
honeys.
and Prunuslpyrus
of
colour
These were followed
Rubus and Robinia.
of
investigation
this
honeys.
Lotus
and
Helianthus
reddish
dark
brown
in colour.
Chile,
Hungary,
light
the extra
ambers to
spectrum.
light
water
7rifoZium
to
light
ambers of
repen4,
golden
the Impatiens
and the
light
to dark
Vicia
and also
amber colours.
colours
of
the
honeys were characteristically
those
in colour.
India,
determined
to medium ambers were distinctive
with
While
black
of Brassica,
amber were distinctive
The heather
honeys.
brown almost
China,
from
to dark
yellow
a broad
one has the nearly
The Castanea
the honeydew honey were associated
The golden
covered
by the white
the
was
the honeys were therefore
of
increasing
of
method as this
a spectrophotometric
and in
in the order
Arranged
honey samples numerical
the various
of
The colours
suitable.
inspection
by visual
via
obtained
were not
values
the colours
Mexico
of
dark
the Fagopy2ýum honeys
Furthermore,
some of
and Spain honeys,
the medium and then
were rather
the Australia,
the colours
to the dark
ranged
red brown
ambers.
Characterisation
experience
as considerable
However,
honeys
by sight
of
it
was not
and smell,
the aroma of honeys
was required
the purpose
although
of
proved
in judging
the present
identification
to define
difficult
the various
study
of
fragrances.
to assess
source
can be
100.
quickly
hundreds
could
to the
established
samples of
of
it
Nevertheless,
of
the darker
the Australia,
of
or
saw dust
the
Indian
was evident
in
sulphur
to destroy
performed.
Some of
aromas while
or
that
others
of burnt
produced
samples
sample which
Yet
of
and then
the honeys
were marred
wax.
and fir
did
had an aroma which
with
sulphur
was characteristic
suggesting
wet wood
One of
compounds such
the practice
the honey
collecting
fermentation
of
smell
and especially
processed
honeys
samples.
the aroma of hydroxymethylfurfural
was commercially
similar
not have any characieristic
by the distinct
in others
fragrance
trees
in some countries
colonies
Some of
the European
smell
is known that
of burning
It
fragrances
had a distinct
aroma associated
samples had a characteristic
smell
fruits.
and citrus
the U. K. survey
some of
honeys had
class
had distinct
some of
A distinct
as sulphur
still
in
and identified.
and characteristic
honeys
foreign
was evident
dioxide.
is
flowers
values
and the non-volatiles
colour
The aroma of pine
terpenes.
of
presence
strong
some honeys
garden
eucalyptol.
turpentine
light
seeing
numerical
been isolated
not
the
to the
China and other
Moreover,
the volatiles
that
Moreover,
of apples,
with
associated
the
all
not
compared
honeys.
those
that
since
was found
smell
pleasant
such as
to
at a time.
honey aroma have as yet
with
associated
sweet
a variety
be evaluated
not
show judge
of a honey
satisfactign
in
some commercially
and of a controversial
of engine
one
was evident.
oil
nature,
used for
lubricating
machinery.
These personal
examination
as collected
storage.
of
observations
samples
from
as received
the hive
or yet
were the
impressions
and may not
from
the
be true
sample after
on initial
for
the
long
sample
term
101.
ANALYSIS OF PROTEINS
Isolation,
Concentration
The presence
of
for
the standard
in
Figure
shown
as
2.
of
gave a similar
unresolved
suitable
for
further-investigations.
PAGE was for
of purified
location
and then
definite
bands.
invertase.
location
with
oxidase
by using
andinvertase
picric
of an orange
on gels
acid
This
technique
the only
use
in unstained
gels.
napthalene
black
stain
was also
grade
oxidase
glucose
for
observed
the
sharp
and
a mixture
The position
enzymes.
from
in
resulted
top of
containing
and distance
the gels
9.
of enzyme activity
in honey applied
as an indiactor
standard
protein
enzymes on polyacrylamide
laboratory
due to diastase,
to polyacrylamide
as described.
red band due to the formation
to which
high
grade
bands were measured
the presence
the
in cases
Also,
relatively
samples
8.
as most of
of enzyme activity
moved by each of
Moreover,
in Table
given
Therefore,
laboratory
in Table
in the honey
due to overload.
not
honey and the standard
and have been given
serum albumin
laboratory
band pattern
these
the calibration
4 cm long.
These enzymes were diastase,
Similar
was
bovine
present
band about
became stained
straight
from
was disappointing
gel
The application
gels
gels
concentration
The concentration
protein,
and are
the whole
was not
of
reference
honey Code No. 209 and other
China buckwheat
honeys
(1951).
were determined
were evaluated
The use of polyacrylamide
after
of ultrafiltration
The amount of protein
to concentration
honeys
technique
in the honey residues
obtained
prior
the honey residues
by the
in Honey
of Proteins
by the method of Lowry et aZ.,
proteins
curve
in
of proteins
a sample of honey (25g)
determined
of
and Estimation
grade
of picramic
gels
However,
acid
enzymes diastase
glucose
was examined
the presence
was detected
and invertase
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103.
TABLE 8
Honey concentration
by ultrafiltration
Honey source
Weight
(Code No. )
(g)
Australia
Banksi. i
from
(194)
Canada (199)
(214)
Chile
China
Buckwheat
(209)
(229)
New Zealand
Hungary
Polyflora
Yellow
Mexico
Yucatan
(222)
Box (246)
(227)
English
Staffordshire
Determined
milligram
protein
(249)
concentration*
(mg) per 25 g of honey
(and per 100 g of honey)
25 g of honey
1.322
58 (240)
1.660
106 (424)
0.462
13 (52)
0.238
7 (28)
0.253
2 (8)
0.245
3 (12)
0.294
58 (240)
0.244
4 (16)
1.705
by the Lowry*et
equivalent
Protein
of concentrate
al.,
of bovine
132 (528)
method
values
serum albumin
represent
used as reference
104.
TABLE 9
Detection
of enzymes in honey by polyacrylamide
of presence
gel
electrophoresis
Orange Red Band**
from
Distance
Presence
top of gel (cm)
Enzymes*
Substrate,
(1%)(w/v)
Diastase
Starch
Yes
Glucose
No
Sucrose
Y910
es
Glucose
oxidase
2
Invertase
laboratory
Standard
grade
4.4
to 4.9
0.5
to 0.8
from
enzymes obtained
microorganisms.
The reaction
of pic. ric
conditions
Average
black
resulted
in
length
for
gel
stained
position
of
stained
with
the
with
glucose
formation
both
cm.
acid
Also,
bands were similar
black
naphthalene
alkaline
under
of picramic
picrIc
was 10.1
gels,
these
acid
acid.
and napthalene
that
the
on gels
which
were
stain.
The presence of enzymes diastase
and invertase
were not
in honey but only in honey to which laboratory
detected
enzymes had been added.
This
mixture
then
analysed
I
by electrophoresis.
2
No orange
red bands were detected
enzyme glucose
activity.
oxidase
oxidase
with
of presence
(3.6-4.5,5.0-5.5
napthalene
laboratory
the absence
of glucose
Whereas preliminary
detection
bands
suggesting
standard
for
black
of
tests
the enzyme resulted
and 6.8-8.0
stain.
cm) after
for
the
in
three
staining
105.
had been applied
but not however
to which
on gels
glucose
the orange
red band was not
honey was applied,
the orange
red band was however
to which
containing
both
Although
applied.
a mixture
enzymes were applied.
The position
red bands were similar
to those
black
napthalene
technique
dodecyl
of polyacrylamide
treated
the
electrophoresiS
being
this
bands were labelled
The protein
bands corresponded
between
standard
60,000
the cathode
to proteins
daltons
pattern
3 was observed
on the
of
protein).
The unusual
The polyacrylamide
gels
scanner
onto
at wavelength
a chart
paper
for
been shown in Figures
the protein
those
already
after
pattern
staining
bands have also
shown in Figure
been labelled
3.
the
the
top
These
top.
reference
bands from
gels
that
of national
Code No. 209 and soyolk
has been shown in Figure
4.
were also
scanned on a gel
A typical
scan recorded
the 5% and 10% polyacrylamide
5 and 6 respectively.
from
after
were largely
SDS-polyacrylamide
560 nm as described.
both
from
the protein
show sample Code No. 252, China buckwheat
(soyabean
10 and 11.
compared against
honey
bands
end as shown in Figure
weights
given
sodium
heat
appeared
were measured
in Figure
of
were either
alphabetically
daltons,
by the
the protein
3 and Tables
or the negative
An unusual
proteins.
was detected
that
whose molecular
and 20,000
with
9.
bands which
gels
orange
in the presence
samples
moved by protein
laboratory
stained
and staining
honey
to which
and distance
gel,
(2g)
on gels
moved by these
in Table
electrophoresis
on 5% and 10% polyacrylamide
staining
3.
gel
detected
as gels
have been shown in Figure
or otherwise
The position
of
After
on gels
appeared
which
bands obtained
in raw honey
of proteins
sulphate.
and distance
to which
on gels
the honey and standard
These have been given
stain.
The presence
evident
was
oxidase
The relative
alphabetically
gels
have
position
of
according
to
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00
A
10
10
C
Co
CVI
.M
00
0
W
P.o
tri
0C0
107.
TABLE 10
Protein
bands
a honey
sample
which
appear
on a 5% polyacrylamide
with
bands
Protein
(Code No. )
Honey source
gel
PQ
R
S
T
+-
-
+
+
-+
+
+
-+
-
+
--
+
-+
-
+
-+
+
+
-+
-
+
-+
+
+
Cbina light
--
+
+
English
amber (211)
(249)
Staffordshire
--
+
+
English
Chorley
++
+
+
Protein
bands P to T, refer
Australia
(194)
Banksia
Canada (199)
(214)
Chile
(227)
Mexico Yucatan
(222)
Hungary Polyflora
(229)
New Zealand
Box (246)
Yellow
(208)
China acacia
+-
to Figure
3.0
bands which were present on a ge l
bands which were not present on a gel
Protein
Protein
*English
(261)*
+
to heat
honey was subjected
Chorley
treatment
described.
as
,
TABLE 11
Protein
bands
a honey
sample
which
appear
on a 10% polyncrylamide
(Code No. )
Honey source
Canada (199)
(208)
China acacia
English
(249)
Staffordshire
amber (211)
Pure honey (PHFJH)(260)
China light
(259)
Ireland
Northern
Lincolnshire
(258)
Lancashire
(Tarieton)
Australia
Chile
Banksia
(257)
(194)
(214)
Protein
(227)
Hungary Polyflora
(222)
(229)
New Zealand
Yellow
Box (246)
English
Chorley
Protein
bands A to K, refer
(261)*
F
G H
I
J
K
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
..
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
++
+
-
+
-
+
+
-
+
+
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
.
.
.
.
.
.
.
.
.
..
to Figure
3.0
Protein
bands which were present
--
Protein
bands which were not present
Chorley
as described.
bands
C D E
+-
*English
with
AB
.
Mexico Yucatan
Rel
honey was subjected
on a gel
on a gel
to heat treatment
108.
FIGURE 4
An unusual
protein
band pattern
ABC
honey show honey
A-
National
B-
Soyolk
C-
China buckwheat
(Soya bean protein)
honey
on 10% polyacrylamide
gel.
109.
FIGURE 5
Gel scan of Canada sample No. 199 on 5% polyacrylamide
P, Q, R, S, T-
refer
to Figure
3.
gel.
110.
FIGURE 6
Gel scan of Canada
A to K-
refer
sample No. 199 on 10% polyacrylamide
to Figure
3.
gel.
ill.
Analysis
of Ninhydrin
and fifty
technique
of
isolation
the
were:
analyses
the ninhydrin
the
of
the appropriate
thirteen
determination
The abbreviations
utilised
positive
ninhydrin
for
used
were
codes
as and when they
of paper
showing
identified
were allocated
from
which
the d'oncentration
as the area under
analyser;
individual
the
of
content
of amino acid
by a computer
programme.
the amino or imino
in
detected
all
of
in Table
12.
these
ninhydrin
These have
the
spot.
were unidentifiable
a symbol and were labelled
complexes,
by the
technique
chromatogram
complexes
have
have been labelled
complexes
and by conventional
samples
These numerical
the ninhydrin
A typical
the
or
honey
these
separation
The ninhydrin
outside
acids
and coded numerically.
subsequent
of
7.
spot
concentrates
the amino acid
analysis
the position
position
on a line
complexes
determined
and chromatography.
the
and identification
in the amino acid
have been given
after
in Figure
in
separation
the pollen
are
order
labelling
occurred,
the relative
by numbers
ninhydrin
that
standards
electrophoresis
been illustrated
those
for
in an alphabetical
been listed
the
by the
amino acids
Substances
substances
or used as reference
free
and chromatography;
statistical
evaluated
Positive
These analyses
the
recordings
of
the
of Ninhydrin
Analysis
both
the chart
two hundred
the
on each of
present
amino acids
measurements
concentration
of
electrophoresis
samples and finally
honey
and Pollens
were collected.
substances
peak of
the microscopical
out
chromatography;
positive
by the method of paper
measurements
that
and concentration
ion-exchange
of
were carried
of honey
samples
six
Substances
in Honey of_Known Origin
Present
The following
Positive
abbreviated
name of
The low concentration
and which
inside
the
may be pepýides
spot.
112.
TABLE 12
List
of ninhydrin
their
positive
substances
detected
and
abbreviations
Code No.
1
Ninhydrin positive
substances
Abbreviations
2
a-alanine
0-alanine
3
a-aminobutyric
acid
a-Abu
4
y-aminobutyric
acid
y-Abu
5
Arginine
Arg
6
Aspargine
Asn
7
Aspartic
8
Cysteine
Cys
9
Clucosamine
Cluc
10
Clutamic
Glu
11
Glutamine
Gln
12
Glycine
Cly
13
Histidine
His
14
Isoleucine
Ile
15
Leucine
Leu
16
Lysine
Lys
17
Methionine
Met
18
Methyl-Histidine
Me-His
19
Phenylalanine
Phe
20
Proline
Pro
21
Serine
Ser
22
Threonine
Thr
23
Tryptophan
Trp
24
Tyrosine
Tyr
25
Valine
Val
26
Hydroxypipecolic
27
Hydroxyproline
Hyp
28
Pipecolic
Pip
29
P
P
30
x
x
34
C
C
35
ff
7r
36
w
w
39
a
a
40
11
ot-Ala
$-Ala
Asp
acid
acid
H-Pip
acid
acid
31
32
33
37
38
*1-28
**29-40
identity
identity
1
0
confirmed
not proven - could
include
peptides
113.
FIGURE 7
Amino acids
map of
standard
reference
ninhydrin
Ile
14
15
0
Leu
Qý)
0(
substances.
positive
19
0
Phe
x
Met
17
5
Val
e
23
p ip24
Tyr4
a-Abu
I
-Trp
4
(A)
c0
F_ý
%I
4
. _'.
y-Abu(
-Al
1
2
20
Pro
2-6
H-Pip
11
$-Ala,
7r
22
Thr
10
Gln
-
Glu
Gly
SezeN7
t,
Me-His
5
\
0
Asp
ñPro
ý)
Gluc
Asn
sn
Arg
:x
04
pq
ý-01
9
(D(.
u
3Lys-ý16
(25-His
pH 1.9 Electrophoresis
Cys
buffer
Origin
114.
A series
of experiments
the amino acid
of
from
solutions
5, and from
were determined
measurements
on chart
obtained
for
and for
as peak height
of
example
chart
was correlated
usual
to carry
its
All
these
use
possible
For
reasons
of
per
g of
nanomoles
internal
alternative
discussion.
the
of
of
honey
are
of
merits
only
are
final
the
presented
this
for
It
and then
the
a further
standard
procedure
in
amino
and
in
with
results
all
was
internal
dealt
Each
III.
solutions.
samples
standards
brevity
at half-height
to a constant
were correlated
such a triangle
see Appendix
f ive
area
was estimated
The area of
two or more calibrated
then
curve
The initial
amino acid
calculations,
run
concentration
the response
by the width
multiplied
Discussion
of
a given
to Table
refer
analyser.
a triangle.
use and for
with
(norleucine).
for
of
out a calibrating
calibration.
the area under
curve
of amino acids
The amino acid
the amino acid
to that
an equivalent
was calculated
level
from
paper
of amino acids,
samples.
from
peak response
a specific
as being
known amounts
chosen honey
randomly
the precision
method used and the recovery
extraction
containing
for
out tn check
were carried
the
terms
acid
analyser
determinations.
The amino
have been given
of clarity
less
given
than
values
samples
the honey
were rounded
500 nmoles
and honey
13 to 22.
of presentation
of all
analysis
in Tables
honey
three
and percentage
solutions
recovery
and percentage
values
concentration
the amino acid
each of
to the
acid
are
it
for
samples
values
the amino acid
in Tables
was considered
examined,
to the nearest
the average
whole
added
For reasons
that
in the replicate
concentration
for
number
per g of honey and to the nearest
6
concentrations
23 to 29.
necessary
for
in Table
listed
known amounts of amino acids
given
samples
off
Also
recovery
ten unit
values
for
115.
TABLE 13
Quintiplate
of the thirteen
analysis
Amino Acids
amino acids
Concentration
to experimentation
prior
of amino acids
(nMoles per ml)
W*
Al**
I A2**
A3**
A4**
AS**
Average***
394
415
402
415
414
416
412
418
430
423
427
410
430
424
Threonine
502
506
496
500
503
495
500
Serine
380
385
382
387
387
397
388
596
607
604
600
594
592
600
Lysine
Aspartic
acid
Clutamic
acid
Proline
4,960
5,022
Glycine
618
633
645
623
620
645
633
Alanine
474
477
473
480
466
464
472
Valine
324
320
317
316
327
316
320
Isoleucine
398
408
417
420
413
410
414
Leucine
388
403
407
400
403
400
403
Tyrosine
650
647
660
638
640
640
645
Phenylalanine
814
816
817
820
800
796
810
1
W-
1
amount of standard
1
analyses
technician
1
for
for
thirteen
determined
1
4,990
1
5,030
A determined
by
A using
solution
and for
Average concentration
reference
and
standards
These conditions
of '15
are used
to Table 6.
details
experimental
of the five
from five
amino acids
by the autoanalyser
nanomoles of each amino acid.
throughout
5,047
in solution
amino acids
Al to A5 - are the values
replicate
5,088
to Table 5.
refer
weight,
5,010
refer
A2, A3, A and A5.
Al,
TABLE 14
Recoveries
of
amino
in
acids
Percentage
Amino acids
Al
A from
solution
recovery
automatic
of amino acids
A4
A5
amino
acid
(%)
A2
A3
105.3
102.0
105.3
105.0
105.6
104.6
102.8
101.2
102.2
98.0
102.8
101.4
Threonine
100.8
98.8
99.6
100.2
98.6
99.6
Serine
101.3
100.5
101.8
101.8
104.5
102.0
101.8
101.3
100.7
99.7
Proline
101.3
101.0
102.6
101.8
100.6
101.5
Glycine
102.4
104.4
100.8
100.3
104.4
102.4
Alanine
100.6
99.8
101.3
98.3
98.0
99.6
Valine
98.8
97.8
97.5
101.0
97.5
98.5
Isoleucine
102.5
104.8
105.5
103.8
103.0
104.0
Leucine
104.0
105.0
103.0
104.0
103.0
103.8
Tyrosine
94.5
101.5
98.2
98.5
98.5
99.2
100.2
1
100.4
1
100.7
1
98.3
1
97.8
99.5
Lysine
Aspartic
acid
Glutamic
acid
Phenylalanine
Average percentage
also
values
refer
of the five
to Table
determined
13 for
Al,
99., 5
Average
100.6
A2, A3, A4 and A5,
corresponding
from the autoanalyser.
concentration
analyser
on solution
A
116.
TABLE 15
quintiplate
analysis
of the thirteen
acids
recovered
Concentration
Amino Acids
after
solution
evaporating
Al
of amino acids
(nMoles per ml)
W*
El**
E2**
E3**
E4**
I
Average***
E5**
394
385
386
383
388
387
386
418
408
412
408
410
414
410
Threonine
502
493
496
492
498
494
495
Serine
380
372
375
376
374
377
375
596
590
592
588
593
590
590
Lyiine
Aspartic
acid
Glutamic
acid
4,860
Proline
4.960
Glycine
618
606
605
608
612
610
608
Alanine
474
464
465
467
470
464
467
Valine
324
320
318
317
320
322
320
Isoleucine
398
394
395
396
392
394
394
Leucine
388
388
385
385
386
387
386
Tyrosine
650
635
640
643
645
642
640
Phenylalanine
814
808
806
803
810
805
806
W-
1
amount of standard
refer to Table 5.
The series
4,870
1
amino acids
El to E5 are the values
4,830
1
4,850
1
in solution
Al determined
in replicate
for
4,860
4,880
by weight,
the thirteen
amino
Al after evaporation
acids of solution
and subsequent determination
(see Table 13).
by the autoanalyser
under standard conditions
Average concentration
of the five El, E2, E3, E4 and E5.
TABLE 16
Recoveries
of amino acid
in solution
Pe centage
Amino Acids
Al from automatic
recovery
amino acid
of amino acids
analyser
(Z)
El
E2
E3
E4
E5
Average*
97.7
98.0
97.2
98.5
98.2
98. o
97.6
98.6
97.6
98.0
99.0
98.2
Threonine
98.2
98.8
98.0
99.2
98.5
98.5
Serine
98.0
98.6
99.0
98.4
99.2
98.6
99.0
99.3
98.7
99.5
99.0
99.0
Proline
98.0
98.2
97.4
97.8
98.4
98.0
Glycine
98.0
98.0
98.4
99.0
98.7
98.4
Alanine
98.0
98.0
98.5
99.2
98.7
98.5
Valine
98.8
98.0
97.8
98.8
99.4
98.6
Isoleucine
99.0
99.2
99.5
98.5
99.0
99. Q
100.0
99.2
99.0
99.5
99.7
99.5
97.7
98.5
99.0
99.2
98.8
98.6
99.3
99.0
98.6
99.5
99.0
99.0
Lysine
Aspartic
acid
Glutamic
acid
leucine
Tyrosine
Phenylalanine
L
1
1
1
1
Average percentage of the five El, E2, E3, E4 and E5, also refer
to Table 15 for corresponding
concentration
values determined
from the autoanalyser.
117.
TABLE 17
quintiplate
of the thirteen
analysis
after
amino acids
ion-exchange
chromatography
A2
solution
Concentration
Amino acids
W*
Il**
of amino acids
(nMoles per ml)
12**
13**
1
14**
Average***
15**
394
343
340
346
345
340
343
418
425
417
423
426
420
422
Threonine
502
492
488
485
498
487
490
Serine
380
378
372
380
374
370
375
596
590
588
596
590
588
590
4,830
4,830
Lysine
Aspartic
acid
Glutamic
acid
4,960
Proline
4,830
4,820
4,840
4,820
Glycine
618
600
605
602
613
608
606
Alanine
474
467
468
475
480
473
473
Valine
324
316
315
318
320
322
318
Isoleucine
398
415
412
414
411
410
412
i, eucine
388
394
395
395
405
392
396
Tyrosine
650
633
637
640
647
636
638
Phenylananine
814
810
815
805
807
804
808
W-
amount of standard
refer
in solution
amino acids
A2 determined
by weight,
to Table 5.
for the thirteen
to 15 are the values in replicate
amino
A2 after ion-exchange chromatography and subsequent
acids of solution
by the autoanalyser
(see Table 13).
determination
under standard conditions
The series
Il
Average concentration
of the five
11,12,13,14
and 15.
TABLE 18
Recoveries
of
in
amiTTo acid
solution
Percentage
Amino acids
A2 from
recoveries
automatic
amino
acid
of amino acids
analyser
(2)
11
12
13
14
15
1 Average*
87.0
86.3
87.8
87.6
86.3
87.0
101.7
99.8
101.2
102.0
100.5
101.0
Threonine
98.0
97.2
96.6
99.2
97.0
97.6
Serine
99.5
98.0
100.0
98.4
97.4
98.7
99.0
98.7
100.0
99.0
98.7
99.0
Proline
97.4
97.2
97.6
97.2
97.4
97.4
Glycine
97.0
98.0
97.4
99.2
98.4
98.0
Alanine
98.5
98.7
100.2
101.3
99.6
99.6
Valine
97.5
97.2
98.0
98.8
99.4
98.2
Isoleucine
104.3
103.5
104.0
103.3
103.0
103.5
Leucine
101.5
101.8
101.8
104.4
101.0
102.1
Tyrosine
97.4
98.0
98.5
99.5
97.8
98.2
Phenylananine
99.5
100.0
1
99.0
99
98.3
99.3
Lysine
Aspartic
acid
Clutamic
acid
Average percentage
to Table
17 for
the autoanalyser.
of the five
corresponding
11,12,13,14
concentration
10
and 15, also
values
refer
determined
from
of
118.
TABLE 19
quintiplate
analysis
of carbohydrate
of the thirteen
amino acids
ion-exchange
after
chromatography
B
soluti2n
Amino acids
Concentration
W*
of amino acids
(nMoles per ml)
B3**
B4**
B2**
Bl**
Average***
BS**
394
350
360
344
362
353
354
418
433
433
430
426
430
430
Threonine
502
490
500
493
497
490
494
Serine
380
380
383
387
376
390
383
596
582
583
580
592
580
583
4,900
4,870
Lysine
Aspartic
acid
Glutamic
acid
4,960
Proline
4,910
4.930
4,900
4,880
Glycine
618
620
620
615
607
612
615
Alanine
474
472
466
464
460
468
466
Valine
324
323
320
320
318
315
320
Isoleucine
398
402
395
405
412
400
403
Leucine
388
394
390
390
397
392
393
Tyrosine
650
640
630
640
650
660
644
Phenylananine
814
805
812
814
810
803
810
W-
1
amount of standard
refer to Table S.
1
amino acids
1
1
in solution
1
B determined
by weight.
for the thirteen
Bl to B5 are the values in replicate
amino
B after ion-exchange chromatography and subsequent
acids of solution
(see Table 13).
by the autoanalyser
determination
under standard conditions
The series
Average concentration
of the five
values
Bl,
B2, B3, B4 and B5.
TABLE 20
Recoveries
Amino
of
amino
in carbohydrate
acids
Percentage
B from
solution
of
B2
B3
B4
B5
88.8
91.4
87.3
92.0
89.6
89.8
103.6
103.6
103.0
102.0
103.0
103.0
97.6
99.6
98.2
99.0
97.6
98.4
100.0
100.8
101.8
99.0
102.6
100.8
97.7
97.8
97.3
99.3
97.3
97.8
Proline
98.8
98.2
99.0
99.4
98.4
98.8
Glycine
100.3
100.3
99.5
98.2
99.0
99.5
Alanine
99.6
98.3
98.0
97.0
98.7
98.3
Valine
99.7
98.8
98.8
98.0
97.2
98.5
Isoleucine
101.0
99.2
101.8
103.5
100.5
101.2
Leucine
101.5
100.5
100.5
102.3
101.0
101.2
Tyrosine
98.5
97.0
98.5
100.0
101.5
99.0
Phenylalanine
99.0
99.8
100.0
99.5
98.6
99.4
Bl
Lysine
Aspartic
acid
Threonine
Serine
Glutamic
acid
Average percentage
refer
to Table
determined
1
1
of the five
19 for
Bl.
corresponding
from the autoanalyser.
1
acids
Average*
B2, B3, B4 and B5, also
concentration
amino
(Z)
recoveries
acids
amino
automatic
values
acid
analyser
119.
TABLE 21
Quintiplate
analysis
carbohydrate
solution
of the ihirteen
amino acids
ion-exchange
after
chromatography
BI
Concentration
Amino acids
of
(Moles
W*
amino
per
ac6,
ml)
BS1**
BS2**
BS3**
BS4**
BSS**
483
450
430
428
440
433
436
513
527
532
530
528
535
530
Threonine
616
608
600
597
605
604
603
Serine
466
463
460
458
468
460
462
730
722
724
720
716
720
720
Lysine
Aspartic
acid
Glutamic
acid
6,080
Proline
of
5,920
5,970
6,020
6,040
Average***
5,990
5,990
Glycine
757
740
744
742
750
748
745
Alanine
580
567
576
580
570
573
573
Valine
397
388
395
392
390
387
390
Isoleucine
488
493
Soo
490
496
495
495
Leucine
476
482
486
478
480
484
482
Tyrosine
797
790
787
786
780
790
787
Phenylalanine
998
993
990
996
983
986
990
amount of standard amino acids in the mixture
(0.6 ml) determined by weight, refer to Table S.
The series BSI to BS5 are the values in replicate
W-
Bl and Sl
of solution
for
the thirteen
amino
BI plus solution
Sl (0.6 ml) after ionacids of the mixture of solution
by the autoanalyser
exchange chromatography and subsequent determination
(see Table 13).
conditions
under stindard
Average concentration
of the five
BS1, LS2, BS3, LS4 and BS5.
TABLE'22
Recoveries
of amino acids
in carbohydiate
Percentage
Amino acids
solution
Bl from automatic
(%)
BSI
BS2
recoveries
I
BS3
93.2
89.0
88.6
91.0
89.6
90.3
102.7
103.7
103.3
103.0
104.3
103.4
Threonine
98.7
97.4
97.0
98.2
98.0
97.9
Serine
99.4
98.7
98.3
100.4
98.7
99.0
99.0
99.2
98.6
98.0
98.6
98.7
Proline
97.4
98.2
99.0
99.3
98.5
98.5
Glycine
97.7
98.3
98.0
99.0
98.8
98.4
Alanine
97.7
99.3
100.0
98.3
98.8
98.8
Valine
97.7
99.5
98.7
98.2
97.5
98.3
Isoleucine
101.0
102.5
100.4
101.6
101.4
101.4
Leucine
101.3
102.0
100.4
100.8
101.7
101.2
Tyrosine
99.0
98.7
98.6
98.0
99.0
98.7
Phenylalanine
99.5
99.2
99.8
98.5
1
98.8
99.2
Lysine
Aspartic
acid
Glutamic
acid
of amino acids
BS4
BS4
amino acid
Average*
Average percentage of the five BS1, BS2, BS3, BS4 and BS5, also
refer to Table 21 for corresponding
concentration
values
determined from the autoanalyser.
analyser
120.
TABLE 23
Quintiplate
amino acid
of U. K. survey
analysis
honey sample code No. 174 after
ion-exchange
chromatography
Amino acids
Concentration
(Moles
.
C
B
A
of amino acids
per g of honey)
D
E
Average*
37
33
40
36
36
36
197
203
200
198
200
200
Threonine
150
154
146
152
146
150
Serine
355
348
350
358
348
352
412
410
416
414
41o
412
6,600
6,610
Lysine
Aspartic
acid
Glutamic
acid
6,610
Proline
6,586
6,620
6.640
Glycine
48
45
44
45
42
45
Alanine
165
167
180
175
173
172
Valine
125
120
114
123
120
120
Isoleucine
82
85
83
90
92
86
Leucine
56
60
58
60
65
60
Tyrosine
563
560
556
566
558
561
Phenylalanine
334
330
322
320
328
327
'Weight of honey
*.
1
1
1
10.0 g
of the five
Average concentration
A, B, C, D and E.
TABLE 24
Quintiplate amino acid analysis of U. K. survey honey sample code No. 174 plus 0.63 g of
solution Sl after ion-exchange chromatography
Concentration
Amino acids
of amino acids
1
(nMoles per g of honey)
2
3
4
5
142
130
136
134
145
137
317
320
313
310
308
314
Threonine
288
292
284
288
286
288
Serine
448
440
443
437
432
440
565
560
558
550
550
557
Lysine
Aspartic
acid
Glutamic
acid
Proline
7,637
7,653
Clycine
221
223
218
226
218
211
Alanine
300
306
294
298
304
300
Valine
214
212
204
206
208
209
Isoleucine
195
200
195
200
193
197
Leucine
173
170
170
167
170
170
Tyrosine
718
718
716
712
710
715
Phenylalanine
548
546
544
544
Total
*.
weight
of honey plus
Average concentration
7,600
1
542
0.63 g of solution
of the five
7,620
Average*
7,686
540
1
1
S1 - 10.7 g.
1,2,3,4
and 5.
7.640
121.
TABLE 25
Quintiplate
amino acid
of China light
analysis
amber honey'sample
code No.
_211
after
exchange chromatography
Amino acids
Concentration
A
of amino acids
(nMoles per g of honey)
C
B
D
E
170
176
174
170
175
173
Threonine
19
21
18
21
19
20
Serine
92
94
90
90
94
92
298
302
296
300
294
298
Proline
1,777
1,830
1,800
1,837
1,850
Glycine
60
62
62
60
58
60
Alanine
86
88
90
90
87
88
Valine
35
34
38
33
35
35
Isoleucine
63
60
62
.
60
64
62
Leucine
82
78
80
83
80
80
Tyrosine
105
108
106
107
110
107
Phenylalanine
535
537
530
524
525
530
Lysine**
Aspartic
acid
Glutamic, acid
1
1
1-
1
1
Average*
1,820
1
Weight of honey - 10.0 g.
Average concentration
of the five
A, E, C, D and E.
This could be because lysine,
vo value for lysine was recorded.
always
low, was in fact below the limit
of detection
or that the very large
ammonia peak had overshadowed lysine and other basic amino acids.
These amino acids occurred infrequently
to resolve
and were difficult
for
on the autoanalyser
14owever, the paper
did so resolve
combination
reason
stated.
and chromatography
amino acids but only trace amounts (1 nanomole)
electrophoresis
basic
these
were present.
TABLE 26
Quintiplate
analysis
of China
Sl after ion-exchange
amino acid
0.65 g of solution
light
amber honey sample code No. 211 plus
chromatography
Concentration
Amino acids
1
of amino acids
(riMoles per g of honey)
2
3
4
5
108
107
118
113
107
110
286
290
295
283
290
290
Threonine
172
175
166
172
170
171
Serine
198
200
203
204
195
2oo
450
468
462
456
454
458
Lysine
Aspartic
acid
Clutamic
acid
3,256
Proline
3,197
3,183
3,142
3,250
Average*
3,206
Clycine
248
250
242
245
240
245
Alanine
230
228
222
228
223
226
Valine
132
128
130
134
130
130
Isoleucine
180
175
184
178
178
180
Leucine
195
197
194
194
190
194
Tyrosine
303
296
297
307
294
300
Phenylalanine
750- 1
744
743
737
740
743
Total
*.
weight
of honey plus
Average concentration
1
0.65 g of solution
of the five
1
Sl - 10.7 g.
1.2,3,4
and 5.
ion-
122.
TABLE 27
Quintiplate
amino acid
analysis
of Mexico Yuctan honey sample code No. 227 after
exchange chromatography
Amino acids
Concentration
of amino acids
A
(nMoles per g of honey)
B
C
D
E
414
422
420
425
420
420
Threonine
230
220
225
228
222
225
Serine
204
200
207
198
200
202
1,256
1,264
1,183
1.173
1.190
1.213
4,512
4,518
4,445
4.504
4,478
4,490
Lysine**
Aspartic
acid
Glutamic
acid
Proline
Average*
Glycine
68
66
65
66
65
66
Alanine
230
236
232
227
224
230
Valine
115
112
116
118
110
114
Isoleucine
80
78
80
75
72
77
Leucine
64
60
62
62
58
61
Tyrosine
180
173
177
177
176
177
Phenylalanine
1
1.684
1
1,643
1
1,652
1
1,668
1
1,636
1
1,657
71
of honey - 10.0 9
Ueight
Average concentration
of the five A, B9 C, D and E.
Analysis of lysine difficulties,
refer to Table 25.
TABLE 28
Quintiplate
amino acid
0.63 g of solution
analysis
of Mexico Yuctan honey samples code No. 227plus
ion-exchange
S2 after
chromatography
Concentration
Amino acids
1
of amino acids
(nMoles per & of honey)
2
4
3
5
100
106
96
94
108
101
516
518
522
514
528
520
Threonine
360
354
358
364
362
360
Serine
307
300
295
306
302
302
1,287
1,317
1,323
1,315
1,332
1,315
5,720
5,665
5,672
5,716
5,647
5,685
Lysine
Aspartic
Glutamic
acid
acid
Proline
Average*
Glycine
24o
238
242
244
247
242
Alanine
364
358
352
356
354
357
Valine
204
202
208
200
206
204
Isoleucine
186
196
190
192
186
190
Leucine
166
177
175
173
168
172
Tyrosine
363
360
353
350
357
357
1,788
1,810
1
1,762
1
1,800
1,840
1,800
Phenylalanine
Total
*-
weight
of honey plus
Average concentration
1
0.63 g of solution
of the five
1
Sl - 10.63 g
1,2,3,4
and 5.
ion-
123.
TABLE 29
Recoveries
in solution
of amino acids
Sl after
honey samples:
with
mixing
U. K. survey
amber code No. 211 and Mexico Yuctan code No. 227
code No. 174, China light
Amino acid
Percentage recoveries of aminý acid*
(Z)
UK
CLA
MY
89.2
92.0
96.6
101.6
101.1
100.4
100.1
99.4
99.8
99.2
98.8
99.4
98.0
99.1
98.5
Proline
100.2
99.9
99.4
Glycine
97.8
loo. 6
98.2
Alanine
99.8
99.9
97.7
Valine
101.6
98.9
100.9
99.8
101.3
99.7
100.1
101.3
98.9
99.5,
100.2
100.0
Lysine**
Aspartic
acid
Threonine
Serine
Glutamic
acid
Isoleucine
Tyrasine
Phenylalanine
*ý
The percentage
values
given
1
values
obtained
in Tables
23,25
28, respectively.
where HS - the total
refer
and 27 with
The following
to footnotes
and
100
the amount of solution
24,26
to Tables
24,26
was utilized
equation
of honey plus
weight
the concentration
in Tables
those given
HS x IA -HxIx
.
sl
rercentage
recovery
of amino acid
by using
were calculated
and 28 for
weight
Sl added,
values
each honey sample.
used for
IA - individual
average amino acid
concentration
value
S1 was added, refer
sample to which solution
in each honey
to Tables
24,26
and 28.
H-
the weight
weight
I-
values
individual
refer
of honey used,
used for
23,25
SI - the concentration
Analysis
of lysine
to Tables
23,25
and 27 for
each honey sample.
average amino acid
to Tables
solution
refer
concentration
value
in each honey,
and 27.
of each amino acid
calculated
in the amount of
Sl added to each honey sample from those given
difficulties,
refer
to Table 25.
in Table 5.
124.
than
greater
values
Replicate
average
500 nmoles per g of honey.
recovery
±1% to ±5% of
to within
agreed
analysis
the
values.
Mellissopalynology
The pollen
as described.
The pollen
for
for
Sciences
Social
statistical
analysis
The numerical
for
in Table
grains
pollen
grains
grains
were photographed
in the United
Amino Acids
in Order
Arranged
After
four
evaluation
frequency
two honey
ninty
pollen
multiple
coded as
Package for
grains.
names, English
names
and secondary
the complete
Some of
at X400 magnification
grains
list
of
identified
the
and these
pollen
identified
pollen
have been shown
8.
in Figure
into
VIII.
frequency
different
between
and for
in Appendix
given
of
of pollen
Statistical
-
of predominant
30,
VIII.
and characteristics
classes
the botanical
list
a partial
have been given
are
package
with
in terms
and were numerically
ease of differentiation
codes together
family
and plant
software
at both
usually
The data
frequency
microscopically
7 and Appendix
and presented
and secondary
by the computer
required
counted
sample as described.
the predominant
was utilised
in Table
as detailed
were then
each honey
were identified
which
grains
and genus level
grains
for
class
from
The pollen
family
plant
sample was determined
of a honey
content
for
samples
groups.
of Pollen
and subsequent
classes
of
Types
the United
type.
of
classification
each sample of honey,
These were:
and the unidentified
Kingdom Samples
the one hundred
Kingdom survey
the predominant,
the pollen
were grouped
secondary,
grains
and
into
secondary
125.
TABLý 30
List
pollen
Code
No.
of pollen
grains
identified
in honey and the numerical
codes allocated
to the
rains
Botanical
name' of Genus*
English
I
name of species
Plant
family
301
Brassica
Oilseed
Rape
Cruciferae
302
Hypericum
Rose of
Sharon
Guttiferae
303
TiZia
Silver
304
Inpatiens
Policeman's
305
Aeaculus
Horse Chestnut
Hippocastanaceae
306
IZex
Holly
Aquifoliaceae
307
TrifoZiwn
praten8e
Red Clover
Leguminosae
308
TrifoZiwn
repens
White Clover
309
Vicia
Broad or Field
310
Rubus
Blackberry
Rosaceae
311
Prunus/pyrus
Plum/pear
to
313
Epilobium
Rosebay Willowherb
Onagraceae
314
Anthz-iscue
Cow Parsley
Umbelliferae
315
Heracleum
Hogweed
316
Urtica
CommonNettle
Urticaceae
317
CornjZus
Hazel
Corylaceae
318
Castanea
Sweet Chestnut
Fagaceae
319
CaZluna
Ling
Ericaceae
320
Ligustrwn
Privet
Oleaceae
321
Teucrium
Wood Sage
Labiatae
322
AchiZlea
Millfoil
Compositae
323
Carduu8
Welted Thistle
is
324
Taraxacum
Dandelion
to
325
Lotus
Birdsfood
326
Mematis
Traveller's
327
Cotoneaster
Cotoneaster
Rosaceae
328
114yosotis
Forget-me-not
Boraginaceae
329
Linaria
CommonToadfax
Scrophulariacea
330
AZnus
Alder
Betulaceae
331
Banksia
Honeysuckle**
Proteaceae
332
Eucalyptus
Aromatic
Myrtaceae
333
Fagopyr-um
Buckwheat
Polygonaceae
334
Echium
Viper's
Boraginaceae
335
Eucalyptus
Eucalyptus
Myrtaceae
336
Relianthus
Sunflower
Compositae
337
Mezilotus
Yellow
338
Onobrychis
Sainfoin
It
339
Robinia
False Acacia
to
340
SaUx
Willow
341
Viguiera
'Tah'
342
Ahjrtaceae
Eucalyptus
343
ReZZeborus
Christmas
344
Fragaria
Carden Strawberry
1
Pendent Lime
Helmet
Balsaminaceae
if
to
Bean
to
Trefoil
Joy
Gum
Bugloss
(Sallow)
Railway
Leguminosae
Ranunculaceae
Leguminosae
Melilot
Salicaceae
Daisy
Compositae
Myrtaceae
Rose
and Crane, Walker and Day (1984).
Due to difficulty
of
of pollen identification
of some plants especially
in Table 7 and also Appendix VIII,
for example, which is detailed
only
name is given under Botanical
species to be found
column and the likely
under English column.
in
identified
all the
Australian
For complete list
the
of all
pollens
fifty
VIII_.
six samples examined, refer_to
__
_Appendix
These are according
Tiliaceae
Ranunculaceae
Rosaceae
to Sawyer (1981)
the genus Brassfca
the genus latin
in honey is given
two hundred and
126.
FIGURE 8
identification
Microscopical
and foreign
and commercial
of pollens
honey
in
present
U. K.
the
survey
Olt
lb
00e
Ilb
dio
d,
400 410 olb V
117
40
J6
Brassica
U. K. Code No. 27
AP'losotis
U. K.
Code No.
106
. 0& %ý
Ti Zia
U. K. Code No.
-
Ligustrum
23
English
Code
No.
p
Castanea
U. K. Code No. 13
Rubus
U. K. Code No. 105
25 ijm
250
4k
127.
8 cont'd
Figure
cri
V* 4-
0
;
6.1w
40
MeliZotus
Canada
Code No.
7-
EucaZyptus
Australia
203
--
Code No.
197
r
dO
$
.
10
0
0
Taraxacum
China Code No.
HeZianthus
France Code No.
208
218
I
'0
Echium
Yellow
Box
Code
No.
246
Loranthus
Australia
Code No. 197
128.
8 cont'd
Figure
Ißin
, a
I
it
if
qll
I
"A
A
TrifoZium
English
repens
Code No.
248
Fagopyrum
China
Code
Purus
U. K. Code No.
120
Viguiera
No.
209
Cayman
Island
Code
No.
207
129.
Figure
8 cont'd....
I-
di
A
CaZLuna
English Code No. 251
Acacia
Australia
Code No. 195
fA. 41
31
do
w
*4
Banksia
Australia
Vicia
U. K. Code No. 12
Code No. 194
25 im
130.
Predominant
type
pollen
Honey samples containing
is,
that
those
There were one hundred
nine
Brassica,
TrifoZium
one hundred
for
responsible
The honey
in a decreasing
of pollen
order
to questionnaire
the response
to these
five
Itosotis
pollen
The amino acid
From the amino acid
following
ninhydrin
standards
in each honey
acid,
glucosamine,
glycine,
and the
acid,
amino acid
five
and
31,34,
and the responses
measurements
in the order
arginine,
lysine,
proline,
of ninhydrin
groups.
were identified
each of
of
aspartic
acid,
glutamic
threonine,
spots
positive
substances
were evaluated
For reasons
within
of clarity
and 'p'.
groups.
a- and y-
cysteine,
hydroxyproline,
histidine,
tryptophan,
as 'x'
labelled
acid,
reference
pollen
phenylalanine,
methyl-histidine,
methionine,
serine,
the five
7 the
using
a- and $-alanine,
were:
asparagine,
glutamine,
shown in Figure
to that
substances
substances
maps and these
pollen
and
CaUuna
of Tables
and 6 have been given
sample within
two unidentified
in occurrence
the
positive
leucine,
pipecolic
content
Castanea,
in the order
maps similar
positive
aminobutyric
isoleucine,
were arranged
and 44.
32,35,38,41
These ninhydrin
sources
each sample belonging
for
concentration
3,5
questions
for
were
The pollen
repens,
have been lised
groups
to questionnaire
Tables
TrifoZium
floral
major
groups
pollen
percentages.
4,
accounted
category.
samples.
the five
question
named Brassica,
and 43.
37,40
in each of
samples
honey
and six
that
five
pollen,
to this
and ltosotis
CaUuna
'Castanea,
repens,
from
Pollens
pollens.
class
were allocated
honey samples
and thirteen
floral
predominant
45% pollen
over
containing
frequency
the predominant
tyrosine,
valine
Differences
were observed
on the
and between
each of
of presentation
it
was
(next on page 162)
131.
VVV
Irr%
MAVTV
')I
In the key listed
honeys,
1.
2.
that
is,
and 4 are common to all
below,
notes 1,2,3
31,34,37,40,46,49
Tables
the pollen
of the U. K. survey
tables
and 52.
location
in the United Kingdom, refer to
number and approximate geographical
location
of apairy not given to maintain confidentiality).
The percent pollen quoted is the numerical value of the number of pollen grains of an identified
floral
by the number one hundred and then divided by the total number of polyfloral
source multiplied
For questionnaire
appendix I (exact
pollen
grains
Thus, the pollen
counted.
sample was grouped into
Predominant
frequency
following
the appropriate
(>45%)
p9llen
values
percentage
for
each floral
source
in each honey
classes:
Secondary pollen (16% - 45%)
Important Minor pollen (3% - 15%)
Minor pollen (>13%)
3.
Genus quoted only.
also
4.
the plant
In Appendix
family
RQQNo4- response
VIII
are given
and genus are quoted
to questionnaire
question
the common English
No. 4-
G-
C
Clover
Pr
-
Pears
NS -
Not specified
PM
-
Plums
S
-
Sycamore
D-
L
-
Lime
CN
B
-
Blackberry
W-
F
-
Fruit
A
-
Apple
rape
pears,
plums,
etc)
Gooseberry
Dandelion
-
(horse or sweet chestnut
specified)
Chestnut
Willows
Py
-
Pyrus
SP
-
Soft
LG
-
Local
(peach,
fruit
CP -
Carden flowers
Sn
-
Sainf. oin
Pv
-
Privet
Bn
-
Beans
GR
-
Golden rod
Honeydew
Ba
-
Blossom
White Clover
Ch
-
Cherry
Mixed (whether flowers,
shrubs or trees
not specified)
Rw
-
Ragwort
LBs
-
Late beanstick
HD WC Hx
-
(Roses,
Dandelions,
etc)
blossom
Spring
H-
Hawthorn
Rbw -
Rose Bay Willow
Bulbs
Hz
-
Hazel
DN -
Dead Nettle
Ce
-
Catoneaster
FB
-
Field
G-
Hr
-
Heather
-
Bean
(ling,
gorse, broom,
billberry)
clover
(Rosebay)
FW
-
Firwood
FT
-
Field
Thistle
Heather
Willowherb
Br
Bracken
BH
-
Bell
WB -
Water balsam
Sh
-
Sea Lavender
Bm -
Bramble
WS
-
Wood sage
Hw -
Hedgerow (type
Bu
-
Bugoss
P-
Pasture
Tr
-
Tree (Elm,
Balsam
CV
-
Calluna
Eucalyptus
SPF -
Spring
Rb
-
Rowanberry
Is
-
Industrial
UF
-
B1
-
ELg
-
Ling
Fb
-
Fruit
blossom
HC -
Horse Chestnut
Sh
Shrubs (not
-
and species
specified)
not
specified)
etc)
Gardens
Wh -
pear,
gardens
SB Bb
and
these were:
oilseed
(apple,
species
therein.
OR -
the likely
names for
Birch,
Marple,
vulgaris
flowers
area
(not
Urban flora
specified)
etc)
not
132.
Key toTable 31 Contd
The responses
by
the
and
of
plants,
it
was
shrubs
generally
A full
the samples
(some
list
with
the
all
examined
column entries
species
concerned.
were
quoted
in
are given
the
those
known
given
genera
that
plant,
in
and
species
these
cases
which
one
would
and
trees
observed
in
shrubs
been
VIII.
in Tables
no pollen
vague,
parenthases).
have
in Appendix
quoted
were
type
In
were
been
is
responses
that,
was necessary
where there
the
specified.
not
common
floral
it
of
those
were
page
responses
have
examples
of
Some
the
in presentation
clarity
omit
that
previous
definition,
trees
and
associate
U. K.
the
in
unclear
assumed
the
on
beekeeper.
participating
ambigucus
of
listed
For reasons
of
31 - 60 to
representation
of
the
133.
TABLE 31
Frequency of distribution
of pollen
grains
honeys (ex.
in samples of Bras8ica
Pollen
Floral
genus
M
U. K. survey)
2.3
Honey sample code No
27
108
58
56
61
26
64
180
66
8
67
153
155
150
151
82.6 82.2
M
Acer
89.0
92.5
B=88ica
89.7
88.0
88.0
87.0
86.4
86.3
85.8
84.7
84.7
84.1
83.4
3.8
4.7
2.4
0.2
5.8
6.2
4.9
0.3
1.2
Castanea
o. 3
Her-acleum
0.9
Hyperiom
0.1
Liguatrwn
onobrychi8
Pinus
1.7
Prunus/Pyru8
o. 7
Rubus
4.1
2. o
1.7
2.2
3.0
1.0
1.1
4.4
Taraxacum
TrifoZiwn
pratense
Trifolim
repene
1.2
o. 6
o. 8
1.1
Urtica
6.7
Vicia
Shrivelled
pollen
unidentified
Total
pollen
5.2
5.7
6.5
4.6
7.0
11.5
7.1
7.5
3.0
5.7
6.1
6.5
8.0
11.8
0.8
1.7
1.2
1.7
3.0
1.7
0.9
2.7
1.7
8.0
2.6
4.1
3.8
4.5
1.5
548
345
577
460
408
756
339
337
359
419
387
295
337
288
338
OR
OR
OR
OR
OR
OR
OR
OR
OR, S
OR
NS
NS
C
WS
S, OR
C, L,
B
counted
grains
RQQNo4
pollen
6.0
4
The notes
to key 1,2.3
and 4 are given
on the key sheet.
134.
Table 31 cont'd
Pollen
Floral
genus
M
2,3
Honey sample code No
182
140
135
54
68
143
118
138
137
132
145
124
114
115
80.0
79.3
78.7
78.1
77.6
77.3
75.4
74.4
73.9
73.0
72.8 67.5
2.9
0.9
175
Acer
81.6
Bras8ica
81.3
80.3
2.2
Ca8tanea
HeracZewn
Hypericum
Ligu8trwn
onobrychi8
Pinu8
5.1
Prunuslpyru8
2.7
3.8
10.5
6.9
Rubus
o. 4
Taraxacum
o. 4
Trifolium
pratense
TrifoZium
repen8
4.0
6.4
16.7
11.3
7.6
6.9
4.7
3.5
0.9
4.4
2.8
o. 4
2.2
2.9
Urtica
0.9
Vicia
Shrivelled
Unidentified
pollen
pollen
Total pollen
grains counted
4
RQQNo4
1.2
6.8
11.3
11.0
7.7
10.0
9.9
9.7
14.8
16.4
10.4
4.9
8.7
10.3
20.3 14.2
4.2
4.7
4.8
1.8
2.3
4.4
3.5
3.8
5.3
1.9
3.9
6.9
5.3
4.7 11.0
354
257
290
506
261
272
310
344
225
317
305
230
300
320
317
OR
F'S,
OR
A, G,
OR
Pr, Pm
D
OR
OR
OR
OR
OR
OR
OR
OR
OR
OR,CN, S, py
S, W,A OR
135.
Table 31 cont'd
Pollen
Floral
(Z)
2,3
1
Honey sample code No
genus
92
88
51
188
Acer
162
74
11
42
131
144
110
185
179
87
56.1
56.0
55.2
54.1
53.0
51.2
47.9
45.2
45.1
5.3
66.9
Bras8ica
66.0
64.5
63.5
60.3
4.6
1.8
Ca8tanea
2.4
Heracleum
2.1
Hypericwn
Ligustrwn
5.8
onobrychis
11.3
0.4
Pinus
10.0
Prunuslpyrus
3.8
Rubus
2.4
3.2
11.4
26.9
28.9
4.0
0.6
20.1
6.8
17.8
3.2
2.5
TrifoZium
praten8e
TrifoZium
repene
5.3
1.3
3.4
4.4
Urtica
Vicia
Unidentified
1.8
5.9
Taraxacm
Shrivelled
3. o
pollen
pollen
Total pollen
grains counted
4
RQQNo4
2.7
17.5
11.4
11.3
6.4
23.2
22.5
15.6
7.1
23.0
14.6
5.2
2.0
1.8
7.2
6.5
2.5
5.7
5.2
290
406
482
293
262
237
421
221
OR,S
OR
3.7
OR
B
o. 8
S
26.0
18.4
C'L.
OR
SF, GF
Sn
11.7
13.4
3.0
5.4
20.4
13.7
12.1
23.8
17.3
5.1
16.0
5.5
6.4
18.2
3.0
294
328
324
313
369
466
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139.
TABLE 33
Occurrence
of ninhydrin
positive
Ninhydrin
positive
substance
substances
in Brassica
honeys
Sample numbers in which given compound
was found not* to occur
O-Ala
8,58,61,64,
a-Abu )
y-Abu.
67,118,153
67, 74,87,124,143
Arg
54,56,58,66,
124
Asn
8,11,27,42,
108,110,115,
56, 58,61,64,66,92,
180
Cys
8,11,58,67,
74, 118,124,132
Glu
8,11,26,27,
175,179,180
67, 88,92,108,124,135,
His
88
Ile
Leu
132
Me-His
26,58,66,67,
Trp
8.11,67,88,
179,182,188
92, 114.138,140,151,153,
1XV
8,27,51,54,
92,110,114,1
56, 58,64,67,68,74,87,
40, 162
In addition
to the other
88, 110,114,115,118,
74, 92,124,140
amino acids
found in all
samples,
the following
These were:
hydroxyproline
positive
substances were also present.
ninhydrin
in sample numbers 74,1099
114 and 124; pipecolic
acid in sample numbers
42,61 and 64 and the unidentified
spot 'p' in sample numbers 27,66,68,
114 and 162.
The presentationof
give
every amino acid
an excessively
long list
However, unless stated
found in all samples:
acid,
proline,
glycine,
and phenylalanine.
by the sensitive
present
in all
which was considered
the samples would
not justifiable.
the following
common amino acids were
otherwise
lysine,
glutamic
acid, threonine,
serine,
aspartic
isoleucine,
leucine,
tyrosine
valine,
alanine,
Small amounts of amino acids
paper electrophoresis
used and not by the amino acid
Tables 36,39,42,45,48,51,54,56,59
analyser.
were often
and chromatography
These conditions
and 62.
detected
technique
apply
to
140.
FIGURE 9
Amino acid
from honey U. K. sample No. 91 (Brassica).
map obtained
(14
Ile
15
Leu
(
19
-Phe
@1:!
et
Val
25
(3
)
23
Trp
24
Tyr -(
a-Abu4
a-Ala
0(
20
)
Pro
y-Abuý
)_
Thr-ý
Gly
22
1
12
Gln
Glu
O-Asp
0
21
rn
Ser
.e
- N%"
Me-His4ý1'83
16
Gluc
'*,.
---ýýAsn
Arg
Lysý
&
OHis
pH 1.9 Electrophoresis
&
buffer
Cys
0
Origin
141.
FIGURE 10
Amino acid
from honey
map obtained
U. K. sample No. 131 - Brassica
Q
14
Ile
F-Leu
15
19
Phe
x&
@
17
-Met
-Val
5
(
G24
23
Trp
Tyr
a-Abu-ý
20
a-Ala
Pro
1
y-Abu
2
(9-Gln
$-Ala
22
Thr-
1
)
Gly4
(D
12
Glu
a
21
r-4
0
cn
Asp
Ser
GlGluc
Me-His _6
8
Sý
Asn
T Arg
Lys
16
S
(2
r His
pH 1.9 Electrophoresis
Cys
bu
Origin
142.
TABLE 34
Frequency of distribution
of pollen
Rrains
in samples of Trifolium
Pollen
M
repens
honeys (ex.
U. K. survey)
2.3
1
Honey sample code No
Floral
genus
142
47
102
141
158
186
157
82
164
81
-
5.2
0.6
184
174
45
44
160
Acer
Aesculus
3.3
Brassica
1.1
Calluna
9.2
15.7
14.7
9.4
1.5
0.2
Corduus
3.8
Castanea
3.1
1.4
2.5
1.5
10.0
Cotoneaeter
0.3
gpilobiwn
1.6
1.4
HeracZeum
2.3
o. 2
0.3
o. 6
2.1
6.1
3.1
1.3
Impatiens
Ligustrum
o. 2
Phlox
o. 8
Pmnus/pyrus
9.8
1.7
Rubus
1.5
7. o
2.6
10.8
2.3
1.9
Sazix
1.3
Taraxacwn
M
TiZia
TrifoZivm
repens
78.1
73.6
70.7
70.7
68.8
67.6
Vicia
Unidentified
62.7
62.5
61.8
60.4
60.2
59.3 59.3
14.0 29.4
1.2
o. 4
pollen
pollen
Total pollen
grains counted
4
RQQNo4
For notes
63.4
-
Viola
Shrivelled
67.1
11.4
11.5
8.4
2.5
297
488
NS
to key 1,2,3
Hr
18.8
16.6
19.2
12.5
22.8
25.1
22.7
18.6
18.3
22.7
2.3
7.1
5.1
6.6
10.9
2.7
5.8
3.5
16.1
7.4
1.1
1.9
5.1
263
293
343
395
432
254
343
344
280
230
457
464
194
Wh
C, L,
B
H
S, C Wh, B
2o. 1
C, B
and 4 refer
A
B, Wh,
C
to Table 31.
NS
Hr, Br
Hr, C B, Wh S, C
143.
Table 34 cont'd
Pollen
Floral
genus
(Z)
2,3
Honey sample code No'
117
103
101
35
34
20
183
98
154
Acer
163
116
134
192
100
1.8
Aeeculua
9.7
Brassica
5.8
CaUuna
Carduua
0.7
Castanea
2.0
8.9
1.8
5.0
3.2
8.3
Cotoneaster
5.8
8.8
9.5
o. 3
EpiZobium
1.3
Heractewn
0.9
0.7*
4.3
1.4
0.9
1.7
1.4
0.3
0.6
2.5
2.3
Impatiens
Ligustrum
1.3
PhzOx
Prunus/pyru3
3.4
Rubus
2. o
7.6
16.4
10.8
6.8
2.0
3.6
5.0
Salix
6.8
1.8
4.7
47.1
47.0
2'. 2
9.2
10.7
Taraxacum
TiZia
Trifolium
0.7
repens
58.6
55.9
Vicia
52. o
52.9
51.4
51.3
48.6
0.5
46.4
46.3
45.9
1.3
45.9
45.7
9.5
0.9
VioZa
Shrivelled
Unidentified
pollen
pollen
Total pollen
grains counted
4
RQQNo4
30.1
24.6
31.6
32.9
45.0
18.7
20.1
33.2
28.7
30.9
45.4
31.9
24.6
39.7
3.4
1.7
2.5
1.0
1.0
6.3
7.3
6.6
4.0
11.3
3.3
4.4
11.1
4.5
292
236
434
395
321
450
383
274
300
278
361
342
305
339
W,D,
C
11W.
P
NS
S, DR
L. C
NS
B
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H: L
B, WB,
Wh
B, C Bm,C Mx
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c--4
r.
0.
4)
41
ae
HM
41
Co
e0v
cn p=
r
ýi
0 lu
bo 4)
:10
U) w
146.
TABLE 36
Occurrence
of ninhydrin
positive
substances
Sample numbers in which given
was found not* to occur
Ninhydrin
positive
substance
$-Ala
82,100',
Y-Abu
158
100,141,157,158,160,163,184
Asu
45,81,82
Cys
158
Gluc
34,35,100,117,158,160,117
Glu
100,158,160
Gln
154
His
134
Phe
158
Trp
158,160
following
to the other
ninhydrin
pipecolic
were:
amino acids
positive
acid
in
spot
unidentified
Details
in sample numbers
spot
'E'
was present-in
as per Table
in all
were also
the unidentified
100,101,163
33.
honeys
compound
samples,
present.
the
These
sample numbers 34,44,81,98,100,102,
47,134,141,160,174,175,184
numbers
'p'
found
substances
and 164;
116,121,142,163
repens
101,102'g'134
Arg
In addition
*-
in TrifoZium
spot
IxI
in sample
and 186 and unidentified
and 164.
One other
sample numbers 44 and 164.
147.
PTr. TTRV. 11
Amino acid
map of U. K+' survey
No. 44 - Trifotium
14
repens.
Ile
15
Leu
19
Phe
-riet
-Val
JLi
&23Trp
)
P:Lp--e
Tyr-
24
a-Abu-0
20
e
Pro
&Gln
y-Abu
U
U
12
Gly-
Ser
-21
i
27
G-Asp
-4
0
ý1:
&
H-Pro
Me-His -G) 18
w
Gluc
(SLAsn
5
Lys -6
Arg
Gý--Cys
16
(EýýHis
origin
pH 1.9 Electrophoresis
btiffer
-
148.
FIGURE 12
Amin acid
No. 164 - TrifoZium
map of U. K. survey
repens.
Ile
14ý5
-ýl
Leu
4
19
Phe
Met
-Val
923-Trp
(
pip
f)
e
24
Tyr
a-Abu4
1.e
F-
t
2o-
a-Ala
Pro
G*
y-Abu
ß-Alal
2
Glu
Thr_(ýýGlu
Gly
4
aAsp
7
C21
12
0)
Ser.
cn
&Gluc
Me-His
m
18
Asn
5
Lys
Arg
T
His
pH 1.9 Electrophoresis
Gý-Cys
buffer
origin
149.
TABLE 37,
Frequency of distribution
of pollen
grains
in samples of Castanea honey (ex.
%213
Pollen
Floral
genus
U. K. survey)
Honey sample code No
91
39
89
178
36
97
29
84
43
AchiZZea
AlZiwn
o. 2
Brasaica
2.2
0.8
0.2
CaUuna
o. 2
Carduue
94.6
Castanea
86.7
85.3
0.1
81.9
81.4
79.4
75.4
74.3
72.3
CoryZue
0.5
Epilobiwn
o. 7
HeracZeum
o. 3
0.5
o. 4
0.9
3.3
2.5
6. o
7. o
15.1
16.1
Ligustrwn
7.9
Lotus
Malva
Prunualpyrus
o. 4
Rubus
0.5
Sanbacus
o. 2
Tilia
Trifolium
1.5
1.8
6.7
repens
0.5
0.5
Vicia
3.4
9.6
10.8
8.9
15.8
12.2
18.2
0.4
2. o
0.5
2.5
2.0
0.0
2.4
o. 7
1.5
Total pollen
grains counted
445
871
389
282
183
189
578
397
838
4
RQQNo4
SC
B
NS
Bm,C,
Shrivelled
Unidentified
pollen
pollen
B, SCO L, B
S, L, C,
Wh,
CF,
Hr
B
BA
Hr
E
Cont'd....
For notes
to key 1,20
3 and 4 refer
to Table 31,
150.
Table
37 cont'd
Pollen
Floral
genus
(Z)
2,3
Honey sample code No
83
28
90
2
AchilZea
25
107
59
23
1.2
AZZiwn
5.8
Braseica
13.0
3.2
o. 6
CaZIuna
Carduue
0.6
68.2
Ca8tanea
63.7
61.5
60.4
59.4
57.9
Corylus
57.6
53.2
o. 4
Epilobium
o. 2
Heraclewn
M
1.3
Liguatrum
2.0
Lotus
6. o
MZva
o. 3
1.7
Prunue/pyrus
2.5
Rubua
1.2
14.6
S=bacue
M
4. o
TrifoZiwn repena
1.3
3. o
12.4
8. o
1.5
Vicia
Unidentified
1.4
4.7
3.0
TiZia
Shrivelled
0.9
5.4
pollen
pollen
Total pollen
grains counted
4
RQQNo4
2.0
20.2
9.9
1.7
1.3
5. o
16.2
19.2
11.5
8.5
14. o
31.0
14.6
23.5
o. 4
1.6
5.2
8.5
1.2
9.6
2.5
6.7
444
511
348
270
762
145
465
718
L
Sc, L,
B
'Wh
B,
H
L
B, L
B
Bm,L
Vl%
o,
ei
r4
"
00
r,0
10
-4
rm
CYN
10
In
0
't 1
10
CM
0
r,
't
l
fCh
t
U»,
Co
C)
irl
00
ýT
-4
rý
le
m
10
t"
e4
ýd
rý
CO 10
4n
10
ý4
0
0)
tr,
(:
ý4
Co
ý
0
Z
'tKn
Co
0
Z
0)
>4
0
Z
9)
0
>4
Z
>4
Z
Z
151.
0
Co
0%
in
%D
4
14
Co
11
r4
ý4
-4
0
pl
"
ein
w%
t4
ý4
ýd
LM
cl%
r,
LM
ý4
0
t4
-4
ý4
14
11
4
ý4
r-
-7
r4
Co
r4
0%
t>
wl
"4
r4
A
.t
f4
,4
4
ý
ý4
cla
f4
CYN
cm
0
Co
Co
10
(21
ý7
-4
0
-4
Co
ri
r4
Co
0
%0
17
0
10
10
10
CA
10
r,
c4
cýJ
10
t4
-4
e4
cn
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Co
ým
ý4
Co
qi
Co
C,4
00
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r%
e4
c4
Ch
0
-
cli
t"
en
c4
rq
0
0
0
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0
0
t
ý4
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0
M
r-
ID
Co
IT
ý4
m
-4
V)
0
U'%
10
00
-4
0)
01
co
r-
Ch
el
't
Co
cu
qi
in
0
-f
ý-4
00
cq
r,
a
0
Co
w
M
02
E-4
cm
e4
e% 0
le
ýD
'tr-
Co
tn
0
". 4
r4
(2% Ch
11
4)
P.
rq
Co
11
00
"4
rý
cý
--4
4n
en
(3%
0
ri
0
-4
CIN c4
f4
't
ý4
i
cm cm
tm
m
cm
9%
r4
C-4
14
ý4
(71
ON
4
.4
CY, f
LM 't
,
.4
c4
93
'. , , , -M0
(Y,
ei
C,4
Co
4m
Co
M
gd
841
0
rý
Co
m
"
.4
ý-4
"
.4
cm
't
17
e4
IT
ulb
rý
ý
0
ýn
0
ýn
cm
ri
"
0
re4
ým
r-
0
-4
Co
-4
f4
ý4
ým
oý
t
cm
0
0)
>-
Z
c4
Co
c4
CO
"
00
0
0)
Co
cn
e
-4
0
10
u, %
LM
t
11
tn
%D
c%
"
0
w
-4
00
0
ll
Z
:
Co
0
Z
0
Z
0)
Z
0
0
0
C"
,4
(>
10
CI)
,
r4
tn
0 ri
CY% 0
4
10
10
00
ID
0
-4
Co
g"
Co
ý4
0
""
-,
10
*t
00
0
cm
0
0%
0
0
ul%
r,
10
en
r,
cm
ri
ý4
c)
.n
(1)
e4
ý4
-q
0
Co
C,4
U')
10
0
r4
10
Co
ý
IT
W
0
90
Co
-1
0
Z
90
0
0
44
0
17
Co
r-
lý
Co
0
N
CY% r,
0
(71
M
"4
cm
m
M
'N
cy% rý
(4
Co
'm
ýn
(4
Q)
0
41
U)
cu
44
>,
gi
CY,
-
ti
43
c2
cm
ri
cm
't
0
C,4
C.-4
en
r,
rý
0%
fl
0
cm
0%
ri
0
10
Vi
r.
KM
-4
ult
r-
-4
rý
ým
c9
U'%
%0
ON
rý
c4
CY% t
kn
ýI
CY%
,4
u
Co
Co
cm
,
0
Z
c4
"
1.4
cq
-4
Ul
r,
0
cy,
en
ým
10
0
r4
rn
C%
t
10
en
0%
0
r-
f4
ý4
e4
fn
't
10
00
cq
.4
- 4
CYN
ý4
r4
tr)
rpl
0
17
-to
0
10
0
w
trN
0%
ý4
r- 4
c4
Co
1-1
0
Z
0
Z
0
M ÖD
ý4
00
w
Z
0
0
p4
fý 0)
0
Ad
(A
't
0
E-r
CYN
rz
., l
Ei
4)
0
en
0
Co
c4
th
-ci
ý4
00
4
60
IttM -ý
-0
m
.2
te
bo 0
0
I
13
152.
TABLE 39
Occurrence
of ninhydrin
positive
in Castanea
substances
Sample numbers in which given
was found not* to occur
Ninhydrin
positive
substance
ß-Ala
28, 59,83,84
y-Abu
28
Arg
2, 36,39,83,84,90,178
Asu
36,
Gluc
36, 43,178
Gln
23,
91
Me-His
59,
84
Trp
28
to the other
In addition
following
ninhydrin
hydroxyproline
were:
59,83,84,89,91
amino acids
positive
in
found
substances
sample numbers
and 97;
*=
in
the unidentified
Details
sample number 107.
as per Table
33.
compound
89,90,91,96
in all
were also
samples,
present.
the
These
25,28,29,36,39,43,
spot
2,23,25,29
178
and the unidentified
and
numbers
42.
39
One other unidentified
and
numbers
sample
present
Honeys
'x'
in sample
'p'
in
spot
spot
'e'
was
153.
FIGURE 13
Amino acid
map of U. K. survey
No. 25 - Castanea.
Ile
Leu
?he
17
Met
25
Val
pip---o
t
'.
24
Tyr4
23
Trp
ot-Abu -(
a-Ala
20
Pro
y-Abu11
O-Ala-oý
1
Gln
Glu
22
Trhr
Gly4
12D
21
7
Asp
Ser
0
09
Me-His-(
18D
Gluc
rn
m
pq
Asn
5
OHis
4
Lys
Arg
acys
16
pH 1.9 Electrophoresis
buffer
origin
154.
TABLE 14
Amino acid
No. 107 - Castanea.
map of U. K. survey
14
Ile
15-
Leu
k
19
Phe
(
17
Met
Val
25
-Trp
24
Tyr -(E)
ý3)
a-Abu
a-Ala
F-
-Pro
&
y-Abu--
Gln
Glu
Thr
&7Asp
GlyýCU
>
0
W
27
Se)O(
e
Me-His.
b
5
a
H-Pro
CD
pq
Gluc
-Asn
I
Arg
&-i
Lys -0 16
(5-
His
pH 1.9 Electrophoresis
Cys
buffer
'
a-
Origin
155.
TABLE 40
Frequency
honey
of distribution
(ex.
Pollen
in samples of CaZluna
grains
of pollen
U. K. survey)
Honey sample Code. NJ
M23
152
41
31
o. 4
Brassica
91.0
CaZZuna
86.1
147
57
187
80.6
3
23.9
3.0
84.3
32
74.7
58.7
57.0
52.5
1.4
carduus
M
Castanea
13.0
1.3
0.9
Conpostae
Hypericum
M
Ligustrwn
0.7
23.2
Lotus
M
Phlox
2.2
Taraxacum
Trifozium
repens
o. 4
4.7
11.2
21.7
3. o
Urtica
Vicia
o. 4
Shrivelled
pollen
6.6
5.1
Unidentified
pollen
1.6
0.5
225
NS
Total, pollen
grains
counted
4
RQQNo4
For notes
to key 1,2,3
13.0
12.2
9.4
16.5
4.5
o. 4
2.0
0.2
8.7
2.5
2.0
800
22j-
268
376
286
242
284
Lg, Hr
NS
Hr
Hr
NS
CV
Lg
15.0
4,
refer
and
to Table
31.
156.
TABLE 41
Free amino acids
in CaZZuna honey
Concentration
Amino
Acids
1
(nMoles
per
g of honey)
152
41
31
147
57
187
32
3
Lys
13
143
95
17
102
T
120
133
Asp
77
125
63
71
380
59
112
101
Thr
25
51
32
30
50
19
39
93
Ser
49
65
69
57
60
45
51
98
Glu
99
121
110
124
88
120
176
170
Pro
1350
1470
1310
1540
3240
2350
1470
2220
Gly
26
58
71
32
45
51
32
49
Ala
28
81
64
53
85
82
65
100
Val
T
43
35
T
50
32
43
61
Ile
T
50
26
T
31
48
31
47
Leu
T
41
89
T
35
33
24
44
Tyr
77
156
47
104
T
307
146
Phe
T
117
191
115
142
180
124
Mo
.
Sample
Date
Heating
ill
T
2
3
Un
9/81
Un
Un
9/81
Un
12/81
9/81
Un
No
Un
Un
No
Un
No
No
Un
Yes
Un
Un
Yes
Un
No
No
4
Sugar
Feeding
Notes
to key 1,2,3,4,
T and Un - refer
to footnote
to Table
32.
157.
TABLE 42
Occurrence
I
of ninhydrin
positive
substances
in CaZZuna honeys
Sample numbers in which given
was found not* to occur
Ninhydrin
positive
substance
$-Ala
3,32,57
a-Abu
y-Abu
152
Arg
57
Asn
31,32,57
Gluc
147,152
Gln
152
I.X 1
3,32,57
In-addition
following
were:
spot
to the other
amino acids
ninhydrin-positive
pipecolic
'PI
Details
in the
acid
in
found
substances
were also
samples,
present.
sample number 32 and the unidentified
sample numbers 3 and 31.
as per Table
in all
compound
33.
the
These
158.
FIGURE 15
Amino acid
map of U. K. survey
No. 41 - CLMuna.
14
Ile
15
Leu
19-
::
17
Phe
Met
25-
Val
&
25
Trp
Tyr-624
a-Abu
CD
a-Ala
20
1
y-Abu-(ýý
Pro
qGln
2
ß- la
.
Thrý<22
10
Glu
U
: )S
(3-Asp
Gly -t
w
0
99Gluc
Me-His _S 18
Lys
ýD
ýAsn
)-Cys
allis
pH 1.9 Electrophoresis
buffer
origin
159.
FIGURE 16
Amino acid
No. 187 - CaUuna.
map of U. K. survey
n19
14
--3
15
4
Leu
Ile
Phe
17
25
x
Met
Val
&Trp
23
Tyr-6
3
a-Abuý
20
a-Ala
Pro
12
y-Abu-1ý4
&
Gln
O-Ala
10
'22
Th
Glu
12
Gly
17
-4
0
Asp
Ser
18
Me-His
Lys
16
)
(
6A
Gluc
nc-
(ýý
)His
pH 1.9 Electrophoresis
buffer
Cys
'Origin
160.
TABLE 43
Frequency
of distribution
(ex.
honey
of pollen
grains
in
samples
of Myosotis
U. K. survey)
Pollen
Floral
genus
(%)
2,3
Honey sample code No
106
120
121
125
1.0
Brassica
15.0
o. 4
Castanea
1
170
171
28.0
5.7
5.7
3.1
17.4
p
129
8.9
Cotoneaster
1.8
HeZleborus
0.8
IZex
95.0"
AV080tis
95.0
1.0
Prunuslpyrus
73.0
8. o
63.7
95.0
95.0
95.0
95.0
3.2
37.3
30.4
5.7
p
11.4
49.6
p
6.8
Rubus
3.5
Salix
TrifoZium
repens
1.8
11.1
Shrivelled
pollen
1.0
21.6
13.5
8.2
17.8
25.4
19.1
Unidentified
pollen
1.0
6.2
1.6
7.7
10.2
9.7
11.0
Total pollen
grains counted
VE
72
299
282
4
RQQNo4
For notes
microscope
VE - visually
pollen
and 4-
to key 1,2,3
220
126
ORpC,
B, Wh
SqFB
The amount present
p-
177
FB, C
HC, S
refer
were visually
LGF
SF
to Table
estimated
eyepiece.
estimated.
grain
present
but
not
counted.
225
GF,
L
B, Pv
F, L,
Bs, S,,
Wh, GR H, A,
PrCh
31.
as seen through
VE
the
161.
LM
r,
1-
0ý0
CD
c4
rýA
ýt
'm
-
(A
CY%
t
r,
rq
r,
-4t
vi
%0
rq
CY%
CD
r-A
(1q
00
Tý-
cm
0
Co
-0
r-.
ýt
Ln %o co %o t
01
rl%
%In
-t
c114 r-
%0
ZT
LM
Co
00
CN
0
J%
vi
ZD
m
Lr%
co %o -t
0
CN
Lei
r,
Co
Ln
C%
M
c*II
t
r,
-
Co Z0
-t
LM
OD
Lei
-t
CY%
c4
00
Co
00
Z
0)
00
r14
0
W4
0
ei
0
44
0
'I'll
C14
%0
Co
r*ý
(D
Co
%0
CA
r-
%0
tn
0
CO
Co
CD
r%
r-4
%0
r-
c4
r, -
r-4
rý.
ci
-
r-t
Ln
CN
Cq
Cm
0Z00
C%
"t
ý
cq
00
Z0
c*II
Co
r*%
ri
LM
(14
en
C'4
,Ný 00
LM cn
rl%
0
4,J
e4
00
c% 00
- LM
ý
ýt
N
ZZ
Q)
41
0
0
41
0
0
4-4
%D
-r4
--00
ýt
-t
$4
ýt
%D
ýt
c>
ýt
%o cq
Ze
CO
c114
co
00 >40w
00
0
Aj
Co
1.14
0
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LM
0
in
't
A
%0
r,
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CD
0
fi
%D
Ln
rm
CO
rrý.
("
M
M
t
ci
Cý4
0
C%
C,4
00
cm (D
00
t
0
,
r%
r,
r.
CO
(D
rh
0)
00
CN
%0
0
Z
(D
r,
0
Co
t
LM
LM
LM
c4
00
00
10
r.
C13
0
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cn
r4
0
r.
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cn
C'4
0)
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04
co
-
r. 0
. r4 . "4
ýu
E-4
C)4
ta
ý
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04
tn
>%
$4
4W
4
14
w0
;j0
P-4
>ý
14
134
ý4
04>
Cd
r-4
9-4
ca
4)
-4
1-4
00
0)
ý4
>.
*
E-4
.0
N
cd
Ul
60
0
4)
41
co
%I
00
r.
%'T
-, 4
-A
"
Cd
(U
=
CIO
0 4.4
C/3
0
41
W
0
z
162.
to list
considered
necessary
particular
amino acid
in excessively
positive
long
and 45.
Also
at
Typical
analyser.
of
the pollen
that
groups
four
is,
Inpatiens,
AescuZus,
predominant
The pollen
in Table
and responses
in Table
47.
acid,
tryptophan,
in occurrence
tyrosine,
questions
ninhydrin
acid,
isoleucine,
pipecolic
valine
of ninhydrin
major
for
each
for
the
The format
previously.
a- and y-
acid,
leucine,
lysine,
acid,
proline,
spot
been given
were detected
substances
histidine,
4 have
measurements
and 6 have also
aspartic
of
question
concentration
susbtances
even
sources,
same as described
3,5
histidine
amino acid
floral
accounted
samples.
and unidentified
positive
electrophoresis
9 to 18.
questionnaire
glycine,
32,36,
chromatograms
a- and 0-alanine,
asparagine,
of ninhydrin
by the automatic
positive
These were:
glutamic
phenylalanine,
to
The amino acid
arginine,
hydroxyproline,
histidine,
48.
maps.
was the
result
would
on chromatograms
thenine
seven
responses
The following
glutamine,
amine,
46.
only
a
such as arginine,
Mewtis
and
samples
to questionnaire
on the amino acid
aminobutyric
of
the
of
groups
Vicia
honey
and
content
been listed
pollen
pollen
these
of
arrangement
detected
two amino acid
of
present
of Tables
amino acids
not
contain
the paper
of
have been shown in Figures
The remaining
remaining
acid,
examples
sensitivity
were more evident
of concentration
not
in occurrence
in the order
basic
combination
such as methionine
low levels
as those
These differences
due to the
did
which
group
have been listed
and chromatography
and others
samples
a pollen
within
lists.
substances
39,42
those
cysteine,
glucos-
hydroxypipecolic
methionine,
serine,
Ix'.
methyl-
threonine,
The differences
have been given
in Table
163..
TABLE 45
Occurrence of ninhydrin
Ninhydrin
positive
substances
positive
substances
120,
Cys
120
Gluc
170,
171
Gln
120,
170
txt
106, 120,121
In addition
to the other
the ninhydrin
* Details
honeys
Sample numbers in which given compound
was found not* to occur
Arg
unidentified
in Myosotis
positive
spot
'e'
as per Table
171
found
amino acids
substances
were present
33.
in all
hydroxyproline
in
samples,
and
sample number 121.
164.
FIGURE 17
Amino acid
map of U. K. survey
No. 106 - AVosotia.
Phe
P Met
Val
Trp
Tyr
Ql
a-Abu-,
-(D
ß-Ala
-e
(D
9ý,
a-Ala
y-Abu
24
20
Pro
: E) 2
(3-
,
Gln
Glu
Thr
4J
Gly -ý
P
Ser
aAsp
0
21
Me-His
Gluc
"*ýýAsn
5
Lys
16 1,
-Arg
G-cys
(Dý--His
1.9
Electrophoresis
pH
buffer
Origin
165.
FIGURE 18
Amino acid
No. 121 - AVosotie-
map of U. K. survey
ý
14:::
'15
Ile
Leu 19 -4-
Phe
Cl
7
-Met
- Val
___25
3ý-Trp
(
Pip
24
3).
a-Abu4
Tyr
.0
C20)
a-Ala
1
y-A
-
Pro
2
O-Alý
Gln
10
22
Thr
Glu
)Ser
&
Gly
#0
1%
21
27
-C.
".
Me-His Is 18
-5
16
-I
0
Asp
H-Proc
GJ;.
Ly s-(
cl
9
Gluc
:3:
ýcc
go
AsnO
Arg
Cys
(:
lD3ý-His
pH 1.9 Electrophoresis
buffer
Origin
166.
TABLE 46
of distribution
Frequency
pollen
honey
(ex.
of. pollen
grains
in
samples
of predominant-
U. K.. survey)
Pollen
Floral
genus
(7 )2,3
Honey sample code No
13
156
1
159
105
12
1.0
AchiUea
87.0
AescuZus
55.3
32.0
2.2
Brassica
3. o
58.4
Mematis
13.8
Cotoneaster
o. 2
HeracZeum
12.0
2. o
1.0
Impatiens
54.9
Ligustrum
4.3
8. o
Lotus
Prunus/pyrus
o. 6
1.5
1.3
o. 3
Quercus
68.8
Rubus
60.7
19.8
o. 8
M
TiZia
Trifolium
70
3.8
o. 7
repens
1.4
4.3
23.2
19.0
Urtica
56.9
Vicia
1.0
Shrivelled
pollen
9.0
6.5
9.0
18.9
13.7
5.6
9.7
Unidentified
pollen
2.2
6.5
3.2
4.7
3.9
3. o
3.7
Total pollen
grains counted
809
273
311
275
233
418
349
LBS9.
SF
GB,
Rbw
GF
4
RQQNo4
For notes
RwjWh
to key 1,2,3
4and
refer
to Table
Rbw,
WB
31.
Bn
L, Bm,,C
Ijz, Ch, W
167.
CD
Co
%D
c>
%0
cl
00
cm
ýo
LM
-
Co
c4
CO
In
CD
00
rý
C,4
C)
LM ý0
CY% -t
--
cyý
ON
%0
D
(:
Lri
en
't
LM
a
z
r4
00
_T
0
c>
ý
't
cn
c>
Cl
%0
r-
V)
-
Q)
;r
C%4
%D
cz
M
44
a
r
00 0
-ýr r,
%D -a-,
a% Ln r,
Ln
t Ln 0o ,Zt
%.
ON cn rý
Ln cn rý
Co
rq
%D
't
cm
(D
00
LM
00
0
CA
4)
>4
co
T
r-.
Co
C14 0
00 z
't
%0
r'D
%ID
00
cm
t
00
cn
00
-t
_T
c4
4)
CN
00
0
(0
(L)
0
V-1
0
0
4-4
r.
0
D
N
00
c4
--N0
%0
cm
-
-CN
CM
Co
C%
%0
C%
r-
tt0
Co
0
00
0-
%D
-t
0
0
rlý
cm
0
C,4
Co
44
%0
CYI
00
rl
r- 00 r,
cm
C:
-r4
Co
CD
A
r,
%t
CD
M
(*,tj
CN
tn
en
rý-
0
cl% r,
0
ON
LM
t
-,
LM
t
%0
r,
C%
Lti
-
CM
rý
ý
(IN
CN
%0 %0 LM Ln cl% 00
Lri
Cl
-
cm
CD
"D
00
vi-0
ZO c% r,
cm
Co
1. ý
Co
ri
cn
10
-, 4
Co
0
u
co
0
P:
cm
0
94
cn
rA
E-4
rz4
04
Co
ww
il.
:i
0)
$4
E-4
cn
P-4
V-4
C<
to
p-,
--4
tu
(L)
:ip
cu
4
12,4
P-4 0
0,41
9
(13
p4
41
ce
ÖD
W Iti
cu 0)
bo gi
ý
0)
4.
0
Z
168.
TABLE 48
occurrence
of ninyydrin
type
pollen
positive
substances
honeys
Ninhydrin
positive
substances
Sample numbers in which given compounds
were found not* to occur
O-Ala
13,156
Cys
105
Gluc
159
Gln
1,159
Me-His
1,12,13,70
Trp
70,159
Ixt
1,70,105
In addition
following
were:
acid
to the other
ninhydrin
amino acids
positive
hydroxyproline
in
70 and the unidentified
as per Table
found
substances
sample numbers
in sample number 70; pipecolic
* Details
in the predominant
spot
33.
'pl
acid
in all
were also
samples,
present.
the
These
70 and 156; hydroxypipecolic
in
sample numbers 1 and
in sample number 105.
169.
Secondary
type
pollen
Honey samples
is,
that
those
in this
floral
Trifolium
The format
before.
The pollen
in
honey
and the responses
and 6 have been listed
in the order
The ninhydrin
substances
positive
a- and 0-alanine,
were:
phenylalanine,
methyl-histidine,
tyrosine,
tryptophan
in occurrence
The differences
in Table
listed
51.
positive
substances
labelled
'0'
as
spots
other
Secondary
and between
within
were evaluated
Further,
samples
which
accounted
lysine,
acid,
glycine,
methionine,
threonine,
serine,
'x'
spots
positive
group
asparagine,
acid,
proline,
two unidentified
maps
and
substances
and these
have been
unidentified
ninhydrin
of occurrence
position
relative
were
to the
19.
type
pollen
were allocated
arginine,
glutamic
were two other
have been shown in Figure
pollens
50.
on the amino acid
acid,
each pollen
questions
in sample code number 169 and these
Their
Honey samples which
question
The amino acid
in Table
of ninhydrin
there
detected
'a'.
and
multiple
detected
leucine,
and the
same as described
to questionnaire
as given
pipecolic
valine
49.
glutamine,
isoleucine,
histidine,
hydroxyproline,
I pl,
glucosamine,
and AZnue.
to questionnaire
a- and y-aminobutyric
cysteine,
acid,
aspartic
Brassica,
were:
samples was the
in Table
as given
for
accounted
Teucrium
and the response
the order
measurements
concentration
Castanea.,
these
contents
4 have been listed
3,5
Rubus,
of
of arrangement
that
one samples
pollen,
were allocated
These seven pollens
pollens.
repens,
class
16% and 45% pollen
There were twenty
seven secondary
frequency
secondary
between
containing
category.
Aesculus,
the
containing
contained
to this
for
category.
twelve
frequency
two or more secondary
pollen
There
groups
were forty
listed
nine
class
honey
in the order
170.
TABLE 49
Frequency
U. K.
of
distribution
of
grains
pollen
in
samples
of
secondary
type
pollen
honeys
(ex.
survey
Pollen
Floral
Genus
(Z)
2.3
Honey sample code no
76
69
AchiZZea
176
24
9.9
1.9
30
62
1
79
133
127
99
80
10.1
Aewulu8
44.8
Brassica
36.5
4.4
1.9
7.3
4.4
2.3
1.3
12.6
6.2
CaUuna
Caotanea
40.5
38.0
3.6
4.0
9.0
28.0
6.6
8.6
Cotonea8ter
3. o
YeracZewn
1.4
Ligu8tz, um
Prunuelpyrua
Rubus
12.7
11.6
6.6
3.2
5.0
5.2
1.4
M
TiZia
Trifolium
pratense
2'rifoliwn
repens
11.5
4.5
9.5
5.3
4.1
2.8
1.5
10.7
2.8
15.4
44.3
44.1
42.0
38.5
37.9
34.4
12.0
pollen
pollen
Total pollen
grains counted
4
RQQNo4
For notes
8.6
0.8
8.2
Vicia
Unidentified
13.7
13.4
7.6
Tar=acum
0.3
3.8
15.0
MeliZOtU8
Shrivelled
1.8
to key 1,2.3
18.5
14.0
27.9
37.0
23.2
18.5
33.8
35.7
23.9
35.5
30.5
5.8
4. o
8.5
6.6
3.8
5.1
6.6
9.4
9.3
5.1
8.6
243
370
365
213
289
136
255
226
335
151
NS
NS
L
NS
Wh,B
OR
NS
P, GF
B, Ce
C, F,
Ce
and 4-
refer
to Table 31.
97
NS
171.
TABLE 49 Cont'd.
Pollen
Floral
Genus
149
14
19
Honey
sample
17
148
(2)
2.3
code
is
no
1
104
AchiZZea
5
55
2.0
Aesculus
13.9
33.2
Alnus
38.0
Brassica
9.5.
6.0
CaZZuna
14.0
0.9
10.9
0.7
Castanea
5.6
13.0
Carduus
0.8
9.1
0.7
Chenopodiwn
3.7
Endynion
6.2
Epizobium
3.0
HeracZeum
0.8
Hyperioum
3.0
0.5
2.0
3.0
5.5
11.0
Ilex
6. o
Impatieng
1.5
Ligustrum
11.0
Lotus
0.8
5.0
8.0
Iva Iva
0.5
Myrtaceae**
12.6
Prunus/pyrus
11.8
4.7
Rubus
11.0
6.5
6.5
12.0
1.5
1.5
Teucrium
33.6
TiZia
5.0
Trifolium
repene
Urtica
30.2
26.8
4.7
5.0
23.9
22.1
21.6
20.9
10.1
3.6
Verbascum
4.0
Vicia
14.0
Shrivelled
pollen
Unidentified
grains
3.4
27.7'
Taraxacum
Total
169
pollen
pollen
counted
4
RQQNo4
** - Identified
38.2
46.4
32.2
7.1
2.0
4.5
338
610
C
at plant
family
B, Fw
level
36.2
45.0
31.3
23.7
34.4
15.1
4. o
5.0
3.0
16.6
16.0
6.7
15.1
472
290
342
522
L
FT, B
C, BII
B
only.
5.62
5.5
B Fw
,
211
202
Bs
B, C,
SL
119
324
Ws. Bm
Bu
Sn
172.
0
00
ei
Kri
V-4
.t
P-4
-i
c4
-,t
A
9-4
"-4
aý
A-4
0
rq
IT
ei
F-4
t
0%
-te
-t
P-4
V-1
P-4
00
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P-4
CY% %0
c%
ZT
r,
a%
ZO
im
LM
0
-t
rý
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00
00
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tri
00
r,
P-4
t
C'4
-t
cylt
0
9-4
P-4
CIM r.t
r0
c*II
0
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rý
r, ý
0%
CY%
Ul
Ul
V-1
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C-4
00
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00
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00
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00
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r-
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cm
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0%
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00
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0
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4)
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0
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m
Co
00
C%
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r, %
Co
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r.
elli
P-4
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CD
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rý
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r-4
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c%
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00
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>d
00
P-4
CN
CD
C%
CY)
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61
Lt)
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(VI
CO
cl%
rý
00
rý
cri
00
N'm
F-4
ci
rA
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Lri
t
00
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c4
%0
0
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(V
:
C14
Ln
w
1-41
Co
Co
CD
P-4 c4
>. 4
c4
c"i
CM
>4
--.
cq
P-4
rq
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ýt
r.
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0)
"p4 .4
Eci
rz4
14
Q)
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0
w
P4
>%
r-4
0
Cd
ý4
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P-4
4)
f. -4
HP
14
>,
4)
4
P4 In
4)
Aj
Cd
"o
.
41
cl
Q)
=
-P4
10
Cd Q)
00 a)
:1 44
En
U
0
0
173.
th
vi
1Lri
CY% E-4 H
Ln
cq
0%
Lr)
cn
r,
,
00
c,4
C% (D %0
%0 Ln
ý
CD
cyl
00
CD
CN
t
t
1
C,
r-ý
0-% ci
tr%
rq
LM
Cl
(D
r4
LM
cq
0
E-4 'i0
C-4
%0
rm
ZD
r.
Z:)
E-4 0%
Co
elli
Co
1-.
0
Z
C,4
Ln
e4
,t
cq
rý
CD
cm
Co
-
te)
0
: 131
tn
t
t
Co
t
CD
-
(D
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00
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r.
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t, 1
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rt
c%
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%D rý
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00
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C%
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ri
V) CN
rq
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J'.
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c4
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00
0
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r.
:Z)
rý
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r:
0
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0
Co
. r4 ., 4
ý
E-4
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cu
rA
>b
4
g2.
in
9
s4
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si
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rn
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P. 4
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P-4
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v--1
<
CO
>
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cn
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a)
=
:3 44
cn
174.
TABLE 51
Occurrence
of ninhydrin
positive
substances
in
secondary
tyPe honeys
pollen
Ninhydrin
positive
substances
Sample numbers in which given
was found not* to occur
ß-Ala
24, 79, 80
Arg
17, 55,
69,76,99,104,127,169
Asn
55, 69,
76,104
Cys
55
Gluc
14,15,104
Glu
5,14,15,19,176
Me-His
799 80
Trp
112b
Ixt
30,55,169
1p1
76,176
In addition
following
to the other
ninhydrin
hydroxyproline
acid
in
amino acids
substances
were also
in sample numbers
sample numbers 62,80
found
present.
17,79,99
unidentified
number 169.
* Details
as per Table
33.
spot
spots
samples,
the
These were:
and 148; pipecolic
and 99; unidentified
sample numbers 149 and 176; unidentified
number 56 and two other
in all
compound
lp'
spot
'x'
in
in sample
16' and 'a'
in sample
175.
FIGURE 19
Amino acid
map of U. K. sample No. 169
14
Ile
15
Leu
a
19
Met
/25
17
Phe
Val
23
Trp
Try
C20
Pro
a-Ala_ý
&
11
$-Ala _(D
'
Thr _ý2.
Gln
Glu
U
aJ
Asp
D
(
Me-H
NIJ
Gluc
'Asn
His
pH 1.9 Electrophoresis
ne
-4
0
/-\
u
ý-71)
Cys
buffer
origin
176.
in Table
as given
the pollen
the
in the format
The amino acid
questionnaire
shown in Table
The ninhydrin
acid,
aspartic
lysine,
belong
those
appropriate
of
the
secondary
insert
secondary
on two samples which
Unidentified
pollen
allocated
extraction
to
between
this
frequency
minor
pollen
group
were inadequate
group
rather
two
of ninhydrin
and these
samples which
class
Code Nos.
list
acid,
and the
because
category,
3% and 15% pollen.
two samples
leucine,
in occurrence
type
glycine,
pollen,
It
that
did
these
is,
was considered
16 and 33 at
the end
than base a section
in number for
floral
categorisation.
did
contain
enough honey
type
pollen
There were eight
for
important
multiple
valine
There were two honey
pollen
acid,
pipecolic
each pollen
within
multiple
these
tyrosine,
maps
asparagine,
isoleucine,
The differences
were evaluated
containing
to
53 in the
arginine,
glutamic
histidine,
tryptophan,
54.
acid,
phenylalanine,
and 'p'.
contained
samples
in Table
to
on the amino acid
glutamine,
hydroxyproline,
'xl
to the
two samples
detected
glucosamine,
in Table
have been listed
in Table
and the responses
and 6 have been given
substances
threonine,
substances
positive
contents
4 have been listed
measurements
methyl-histidine,
spots
The pollen
previously.
a- and y-aminobutyric
acid,
serine,
unidentified
not
positive
methionine,
proline,
arranging
52.
cysteine,
hydroxypipecolic
in each sample,
was used for
value
question
3,5
a- and 0-alanine,
were:
described
concentration
questions
identified
two pollens
percentage
to questionnaire
and responses
order
From the
the highest
with
samples
52.
52.
honey
samples
and subsequent
category.
which
not
identification
The amino acid
and these
concentration
were
measurements
177.
TABLE 52
Frequency of_distribution
honeys (ex. U. K. survey)
of pollen
grains
in samples of secondary
Pollen
(A
pollen
type
52 . 531 168
94
multiple
2,3
lioney sample code no
Floral
Genus
711
9
146
173
139
221
1.5
AchilZea
Aesculus
31.5
Bras8ica
16.6 43.2 39.4 34.4 28.9 16.8
1.6
CalZu
46
75
10
9.4
21
0.5
0.6
5.2
8.0
1.0
19.4
3.2
9.0 36.1 35.3 32.7 32.5 26.5 19.5
15.3
0.8
0.5
Epilobium
18.4
Fagopyr,um
18.2
Fragaria
2.0
Heraclewn
0.5
0.7
6. o
o. 8
Hypericum
0.3
IZex
1.0
1.2
Ligu8trwn
Myrtaceae**
3.3
Fý,unuslpyrus
8.7 10.0
Rubus
2.0
2.0
2.9
1.3
17.8 26.4 32.3 30.3
1.5
15.0
5.0 35.1 25.5 31.8 18.8
17.3
pollen
Unidentified
pollen
Total pollen
counted
4
RQQNo4
grains
18.6
8.0
31.9
13.7 12.0 14.8 16.2 18.4 47.5 18.0 17.6 18.0 43.0 16.6 29.5 35.7 26.3 26.9 35.0 26.0
4.6
5.0
9.4
6.8
3.9
6.0
1.2
5.4
6.0
0.6
2.7
2.0
1.8
2.9 10.4
2.2 13.0
241
643
297
340
76
463
496
239
361
479
373
346
283
406
489
200
BM
C, B
Wh
L, He
at plant
4.6
3.9
1.3
Vicia
2.0
0.2
25.0
Urtica
18.5
2.9 11.2 18.6 11.0
1.5
TiZia
TrifoZium repens
3.2 16.7 19.0
2.6
1.0
0.3
Taraxacwn
Identified
112
43.9 42.7 35.7 32.1 35.9
0.1
Ca8tanea
**
122
0.1
Cardicue
Shrivelled
96
V, L Tr, L
family
level
C, B FB
only
lis
C, L
'
G
Hr
CV
CV
Hr
W ttx'
L, I%L NS
G: Ce
268
NS
178.
TABLE 52 Contd.
M
Pollen
Honey
Floral
sample
2,3
2
code
no
WOULMU
72
85
Ill
190
50
60
113
161
63
4
123
18
119
191
Achi I lea
2.4
1.5
8.9
Brassica
23.3
21.3
3.0 23.0 20.8 14.4 17.7
2.6
22.2
Calluna
20.8 21.4
1.5
Carduue
1.7 24.7
Castanea
15.9
7.9
16.0
Chrysanthenwn
24.7
Cotoneaster
16.8
5.3
5. o
Heraclew? i
2.1
12.0
0.5
17.9
Helloborus
24.3
Hypericum
8.3
Vex
.Impatiens
31.9
Liguatzrum
25.2
4.5
LiZium
25.5
Linaria
7.2
Melilotus
22.7
Prunus/pyrus
41.5 38.8 34.2 33.8 25.2 24.8
15.6
13.2
5.5
Quer=8
Rubus
3.2
52lix
3.6
10.1
7.0
7.7 35.6 18.2
2.5
5.6
0.8
Tar=acum
6.0
11.5
0.7
Tilia
Trifolium
inc*
TrifoZiwn
repene
pollen
pollen
Total pollen
grains counted
4
RQQNo4
* inc - incarnatwn
0.3
19.6
12.7
14.3
5.6
Vicia
Unidentified
37
9.0
Acer
Shrivelled
65
23.2 24.6
6.0 22.8
13.9
0.5
21.0
15.7 40.8 33.3 40.1 35.1
2. o
21.0 15.8 17.8 18.1 14.2 18.3
6.4 21.8 35.9
27.7 28.9 31.0 19.9 18.7 21.3 13.7
2.4
4.1
5.8
8.8
4.6
2.8
6.6
9.4
7.7
0.1
4.3
11.0
251
266
259
342
394
465
361
266
117
205
346
127
NS
L, Pr
CF
B, H
A, H C, Hr V, Mx
NS
FB
S
SPF
,
F
D
3.1
7.2
1.8
0.6
287
390
389
713
Hr
Lg
L, B Bn, DR
NS
Rb
WB
179.
TABLE 52 Cont'd.
Pollen
2.3
(%)
Honey sample code no'
Floral
Genus
38
167
172
128
48
93
86
AchiZZea
49
78
771 181
189
136
1091
1.5
16.7
CaUuna
6.9
3.7
3.5 10.8 40.6 24.7 26.3 18.4
0.7
7.6
0.9
Carduus
2.1
19.6 31.3 31.5
Castanea
3.2
4.8
Endynion
1.0
2.2
o. 3
EpiZobium
2.6
HeracZezan
2.1
2.8
Ilex
4.6
rmpateins
1
5.4
Ligustrwn
0.7
2.2
4.3
MeZitotus
26.7 35.7 17.7 17.1 24.1
0.8
Prunualpyrus
1.6
Rubus
4.4
7.6
0.5
Tar=acum
2ýrifoliwn repens
5.1
3.9
4.2 25.0 13.6
18.6 17.2
6. o
5.0
9.5
14.0
6.0
1.6
42.1 37.7 36.0 37.9 37.5 34.4 34.2 29.9 34.9 34.4
12.6
5.2
Vrtica
13.2
2. o
Vicia
Unid6ntified
33
3.7
Brassica
Shrivelled
16
pollen
pollen
42.0 35.7 26.9 21.2
26.5 24.9 27.5 18.9 16.9 1348 31.5 31.5 35.2 25.8 18.6 1249 10.7 26.4 66.0 65.0
2.3
5.3
4.5
9.5
3.0
8.5
6.3
0.5
3.5
3.4
5.0
9.8
5.8
4.4
8.0
5.9
Total pollen
grains counted
667 358 2oo 116 361 305 222 187 338 314 362 356 308 250 278 158
4
RQQNo4
D,
NSF,
Ch
C, B NS
NS
Hr
UF, BMMxCF
C, WL C, Ia
FB
not C Sh
C
FB
B, L, MxL, B
C, Bm
Wh WB
180.
f-
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1.4
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0%
0
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c4
11
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0
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CK)
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(21
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19)
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rý
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cm
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w
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to
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(L)
x
bo 0
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ch w
182.
fn
m
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r,
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in
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m
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0
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Ln
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m
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rl
C14
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co
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eq
ell
rý
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CN
10
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r,
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CF%
'T
Ln
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eq
ý
rl
10
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11
c7%
in
r,
rl
4
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Ln
4m
co
C"
rl
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Ch
m
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0
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to
4)
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N
co
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ta
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ýo N
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in
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f%
00
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co .4
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cn
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a%
r-4
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f-,
C%
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cn
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cl
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co
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cn
co
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WN
cn
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co
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0
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co
Zo
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rr4
ý-4
in
Go
rý
co
CYN 0
41% 10
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co
C4
co
00
4
m
a
cn
- C14
co
z
co
co
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cli
N
'o
r4
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C4
0
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co
0
z
4)
co
tvi
t. 0
t. 0
4
4)
A
z
0 0)
93 -0
.4
0
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S.
4
-4
w
Q)
En
i
k
4)
g
4
4
Id
.
ý
0
0
0
w
o
P,
w
:1
P
u
N
0
<
ý-
H
w
P.
ý4
R '"
.W
'd %I
U3
p
4:
'1
4
"
00
DOT
'Co
,
a,
M:
m
'
4)
94
183.
TABLE 54
Occurrence
pollen
of ninhydrin
positive
in
substances
secondary
multiple
honeys
Ninhydrin
positive
substances
Sample numbers in which given compound
was found not* to occur
B-Ala
37,38,53,65,72,75,128,190
Arg
16,37,48,49,52,53,63,65,93,94,
109,111,119,122,123,139,161,167,
168,181,189,190
10, 21,37,
Asn
49, 52,65,72,85,86,93,
112 , 161
Cys
9, 53,60,
85, 93,119,139,161
Gln
4, 72,78,
94, 139,167,168,173,189
Gluc
48, 499 72, 94, 122,161,167,168,172
Hyp
50, 113,16 1,1 18
Me-His
38,50,78
Pip
4,16,37,50,63,72,85,93,94,128,
146,161,167,168,172,190,191
53,72,77,167,168,173,189
Trp
1xv
37,38,65,71,77,78,85,123
Ipt
119,191
In addition
foll
to the other
owing ninhydrin
were:
amino acids-found
hydroxypipecolic
in sample numbers
substances
positive
acid
71,138
in
in other
were also
the
samples,
These
present.
sample number 71; hydroxyproline
and 172;
the unidentified
spot
'xl
in
and 190 and the unidentified
sample numbers 33,46,128,161,168
'p'
in sample numbers 9,10,16,93,113,122
spot
and 123.
* Details
as per Table
33.
184.
and responses
to questionnaire
in Table
The ninhydrin
55.
and 0-alanine,
isoleucine,
glucosamine,
leucine,
'x'
spots
unidentified
from
the combs for
in
Amino Acids
The foreign
sugar
products
sixty
four
rather
into
for
Samples
were categorised
processing
three
suspect
origin
according
and commercial
pollen
into
in this
category
one samples
country
groups
I.
The pollen
these
The amino acid
were grouped
There were sixteen
forty
IB in Appendix
to each of
analysis.
The
content.
and these
categories
and English
commercial
Type
of origin.
sixteen
enough honey
Sources
their
of
because
types.
Honey samples
accounted
on the basis
of ninhydrin
as described
or pollen
samples
commercial
unknown country;
products
Known Country
country
than
and the two
There was not
Country
honey samples were allocated
were known country;
and sugar
of origin,
phenylalanine,
56.
and Commercial
honey
histidine,
in occurrence
amino acid
Stated
of
aspartic
glycine,
valine
analysed
comb.
the Foreign
and commercial
known country
their
of
either
in Order
Arranged
to
in pieces
honey was received
extracted
in Table
sample Code No. 126 was not
Moreover,
this
tyrosine,
a-
asparagine,
acid,
The differences
have been given
substances
positive
were:
methyl-histidine,
tryptophan,
and 'p'.
arginine,
glutamic
methionine,
threonine,
serine,
proline,
acid,
glutamine,
lysine,
identified
substances
a- and y-aminobutyric
cysteine,
acid,
positive
and 6 have been listed
3,5
questions
sixteen
country
concentration
groups
of
order
them they
each honey
and the
in Table
as listed
sample belonging
have been given
measurements
stated
These samples were arranged
in an alphabetical
contents
and between
countries
of honey.
to their
according
in Table
information
57.
regarding
185.
'10
0
%.
1 CD
M
-t
Cy% CD
Ln
-
c4
%D
0
(D
0
C,,
(N
C,4
rý
%0
ýt
ýt
rq
(A
rý
(Ylt
00
M
Ln
as
%D
-e
CD
rý
OD
C%
ýt
r_
0
-t
CD
cm
(A
E-4
cl
t
CYN
cq
E-4
C4
r4
00
Z1
%D
0
00
CY%
N0
00
Z
m
r-t
0
CD
c4
OD
0
Z
LM
1--
ll-ý
>1
0
%ID
rlý
t
-00M
r-.
%0
r*.
cm
cyý
Co
LM
OD
44
0
to
w
0)
914
00
ýT
00
N
W
0i
0
("
r,-
ril
0)
1 c> c% %m cn
cm r,
-o
r.
tvi cm
Le)
Ln
CN
00
-
co cn c%
rý r,
Ln o
00 ZZ
00
U)
00
%0
0
0
rlý
LM
%0
C,4
ý
CM
0
-, 4
41
co
p
1-4
0
ý
rq
ý
'm
00t0
rý
ýt
rl
c41
rn
10
ci
-, 4
114
-, 4
ot
%Im t*%
C-4
0
rA
(A
-t
0
C-)
rý
Ul
:t
0
Cl
-
0
00
%D -t
%t
0
-
CM r%
ýt
CO
C,4
CD
ýt
km
e
"
ý
t
(:
0
%,
%D
Cl
N
cl
CD 00
CN
cm
t4
-
00
It
%0
LM
-
e
c4
0
Ln
0
ri)
CL
E-4
>%
A<H
to
40
ýd
cn
u
ýA
ý4
g24 u9>
v--4
Ce
P-4
0
N-4 4
>%
c
E-4
P-4
P-4 0
41
cu
cu -0
cn
r4
41
cu
W "0
cu 0)
ÖD 0
0)
:)
=
U)
4-4
186.
TABLE 56
Occurrence
positive
of ninhydrin
substances
in unidentified
honey samples
Ninhydrin
positive
substance
Sample numbers in which given
was found not* to occur
a-Ala
7,40,166
A-r0,
6,40,95,165,166
Asn
40,73
Cys
7,40
Gluc
95,130
Trp
165
-o
165,166
IXI
to other
In addition
the ninhydrin
in
other
positive
as per Table
amino acids
substance
sample number 73.
* Details
compounds
33.
found
hydroxyproline
in all
samples,
was present
187.
TABLE 57
Frequency
distribution
of
of
pollen
grains
in
samples
of
known
type
country
honeys
(ex.
foreign
and commercial).
Pollen
Floral
Genus
(Z)
2,3
Honey sample code No
193
194
195
196
197 i 198
199
200
201
202
203.
204
Acacia
206
0.3
Bank8ia
21.9
0.3
Brassica
3.9
5.2
0.6
2.0
46.8
27.7
61.5
70.7
25.3
4.1
1.7
55.3
63.9
Castanea
2.9
Cireiwn
Echiwn
1.1
6.0
78.2
73.8
8.0
53.3
2.7
4.9
40.5
21.6
5.6
14.1
3.2
13.9
1.9
Erica
L'ucaZyptus
12.1
10.0
5.2
0.3
Liliwn
Loz-anthua
8.7
Myrtaceae**
TrifoZium
pratense
Trifolim
mpene
Shrivelled
18.3
1.6
3.2
11.8
44.7
13.0
9.1
19.5
13.2
14.7
8.7
11.5
9.8
13.2
10.0
8.6
20.6
7.6
3.3
6.7
8.3
14.7
2.6
5.4
3.8
1.9
1.0
7.4
6.0
374
383
298
329
323
347
312
404
314
317
296
351
347
and 3-
refer
to Table 31.
pollen
but not present
to plant
family
level
grain
85.8
7.0
Sample examined for
Pollen
13.6
7.3
Total pollen
grains counted
Identified
21.6
10.4
pollen
to key 1.2
25.7
33.1
1.6
pollen
Unidentified
For notes
P
34.3
Melizotue
P
205
present
only.
but not counted.
0*
188.
TABLE 57 Cont'd
Pollen
Floral
Genus
(Z)
2.3
honey sample code Noý
207
208
209
210
211
212
Acacia
213
214
215
216
217
218
0.3
Aesculue
79.8
Brassica
2.3
13.9
23.3
77.6
Castanea
Cir8iuM
11.8
15.3
15.8
8.8
18.2
6.5
21.4
1.2
Erica
26.3
Fagopyrium
0.3
ReZianthus
Lilium
64.9
5.4
18.1
3.5
0.7
53.0
Lotus
45.0
35.1
Myrtaceae**
9.0
Prunuelpyrua
8.1
Robinia
3.8
Taraxacum
4.3
Trifozium
pratense
Trifolium
repens
6.8
4.3
9.1
5. o
1.2
Tilia
1.5
10.9
1.3
43.0
75.2
30.0
28.4
68.5
Urtica
Vicia
1.3
0.4
3.9
17.4
56.2
viguirea
Unidentified
5.6
7.2
Echium
Shrivelled
219
pollen
pollen
Total pollen
grains counted
3.0
11.8
18.8
15.1
23.0
5.2
9.8
8.5
17.4
8.1
20.7
26.7
8.5
9.5
29.5
5.6
13.6
20.4
4.9
4.6
4.9
43.5
6.1
9.3
8.4
2.9
1.9
329
394
338
270
346
306
305
344
353
367
376
317
299
189.
TABLE 57 Cont'd.
(2)
Pollen
Floral
Loney
220
221
222
223
224
225
sample
226
2.3
code
227
No
228
Acacia
1.7
Banksia
0.8
Brassica
229
15.1
2.0
Bruseraceae**
5.1
7.3
Centaurea
230 1 231
8.8
0.3
19.4
13.1
6.2
2.9
Echim
3.3
10.9
Erica
11.0
Eucalyptus
42.4
Fagopyrw? z
3.7
22.8
6.4
24.9
Helianthus
30.8
Lotus
0.3
41.9
17.9
17.4
Melilotus'
65.0
22.6
Onobrychis
7.7
Robinia
3.8
Roamarinue
2.9
2.1
SaZix
47.5
2.4
TaraxaZum
.
TiZia
1.7
Trifolium
pratensa
Trifozium
repens
4.8
14.2
79.2
Viguirea
pollen
pollen
Total pollen
gra i ns coun t ed
Pollens
28.0
8.8
Ci8tus
Unidentified
233
14.0
Cir6iwn
Shrivelled
232
present
the literature
6.9
8.4
6.7
3.2
16.4
9.5
19.8
12.5
14.9
20.6
9.5
16.3
14.9
15.8
23.4
15.1
6.0
37.4
17.5
3.0
11.3
28.9
8.7
20.5
380
393
349
361
407
329
379
273
family
level
367
in
31.0
59.0
308
these three
resources
337
000
0
samples but were not identifiable
available.
17.4 '24.1
even at plant
with
190.
the samples were commercially
whether
in Table
have been given
otherwise
From the amino acid
following
ninhydrin
standards
in each honey sample within
These ninhydrin
butyric
positive
acid,
glutamic
glutamine,
pipecolic
phenylalanine,
of ninhydrin
maps and these
histidine,
positive
other
compounds which
could
not be established
using
reference
amounts which
could
of
the paper
chromatograms
for
However,
mass spectroscopy.
eq w, w, X, e, a
complexes
Country
and SI.
Origin
Unknown,
Honey samples
and these
bottle
were:
label
in this
known. by their
was suspected*to
position
category
floral
of origin
donors
compounds
from the
off
by
as C, 6,
of
these
20 to 25.
Honey_Types
into
was not
from comb of unspecified
by their
group.
were
identification
and English
names; samples whose identity
be difference
these
of occurrence
were allocated
samples whose country
or were obtained
there
Also
not be eluted
subsequent
Origin
in the amino
whose identity
complexes
have been shown in Figures
Suspect
Differences
each country
compounds were labelled
The'relative
identified
to those
of
these
tryptophan,
Moreover,
standards.
were present
methyl-histidine,
were observed
59.
positive
acid,
and Ip'.
and between
in Table
in trace
cut-outs
IxI
spots
within
gave ninhydrin
glucosamine,
threonine,
serine,
substances
were evaluated
cysteine,
methionine,
groups.
a- and y-amino-
hydroxypipecolic
lysine,
proline,
have been listed
These differences
acid,
reference
country
a- and $-alanine,
aspartic
leucine,
acid,
or
7, the
using
the sixteen
each of
and the two unidentified
valine
in occurrence
acids
glycine,
isoleucine,
hydroxyproline,
tyrosine,
acid,
were identified
were:
asparagine,
shown in Figure
to that
substances
substances
arginine,
bottled
58.
maps similar
positive
is,
that
processed,
three
stated
origin
of country
groups
on the
or were
source
and samples of English
191.
58
KEY - To Table
below
The key given
is
1.
CNo - Code No.,
2.
CP - Commercially
T-
Trace
Up -
Raw hive
also
refer
61.
common to Table
to Appendix
produced
I for
country
source.
or otherwise.
amounts.
honey or comb honey,
or processed
by beekeeper
or honey strained
or bulk
honey
by cloth
from shipment
container.
Pr -
Commercially
+-
Information
otherwise
processed
regarding
was not
into
the
available.
bottles,
commercially
Jars,
etc.
processing
or
192.
0
C,4 '
--t
M
LM
CN
0
P-ý
4
C,
0
cq
%D
cq
r-
It
LM
%0
H
r-
1--f
r1.
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r-
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V)
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0
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0
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rý
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Cy%
Co
cq
LM
't
0
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LM
00
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A
0
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r-.
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T-4
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-t
LA
-t
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r_
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Co
r-
cm
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(D
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LA
rq
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cm
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r%
CYN
V-4
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Co
M
C%
r-
r%
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00
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V-4
00
Cl%
0
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H
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41
rCY%
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414
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ci
194.
cm
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H
cm
0
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0
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0
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00
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r.
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c4
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00
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00
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r-
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cm
cm
%D
V-4
r-
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c4
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CD
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00
%0
r1.
1.-4
C,4
-t
vi
LM
00
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cyl
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r 4 Ili
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ai
-2
Co
196.
TABLE 59
Occurrence
of ninhydrin
positive
substances
in known country
honeys
Sample numbers in which given compound was found
not* to occur
Ninhydrin
positive
substances
cc-Abu
217, 218
y-Abu
215, 217, 218, 225
O-Ala
196, 201, 202, 204, 206, 209, 212, 215, 217. 220,
221, 224, 225, 227, 228, 229
193, 196, 205, 206, 207, 210, 212, 213, 214, 215,
Arg
216, 217, 218, 220, 224, 225, 228, 229, 231, 232,
233
Asn
201, 202, 204v 209, 212, 215, 221
Cys
196, 197, 201, 202, 204, 208, 220, 221, 226, 227,
231,
Gluc
216, 232
Gln
199, 200, 201, 202, 204, 208, 224. 227, 232
His
218, 224, 225
H-pip
193, 199, 201, 202, 203, 204, 205, 207, 208, 209,
211, 212, 213, 214, 215, 216, 217, 218, 219, 222,
229, 232, 233
Me-His
204, 208, 219, 222, 226
Pip
201, 204, 210, 212, 214, 215, 216, 217, 220, 224,
225, 232
201, 204, 208, 215, 216, 217
Trp
ext
217, 118
193, 195, 197, 198
p
I
In addition
substances
to the other
were also
amino acids
present.
found
These were:
in all
samples,
hydroxyproline
the following
positive
ninnydrin
in sample numbers 197,222
213,214,223;
spot 'x' in sample numbers 193,206ý
and 223; the unidentified
225; the unidentified
spot
spot 'p' in sample numbers 199,201,208,224'and
the unidentified
10
in
spot 'A' in sample numbers 223,225
and 223; the unidentified
spot 'C' in sample numbers 209,210
and 212; the unidentified
and 231; the unidentified
in sample
spot 'a' and V
spot '61 in sample numbers 209 and 211; the unidentified
spot
spot 'A' in sample number 211 and the unidentified
number 209; the unidentified
sample numbers 211,222
'e'
in sample number 214.
* Details
as per Table 33.
197.
FIGURE 20
No. 194 - Banksia.
from
Australia
hone'y
map of
Amino acid
Leu
Ile
(
19
Phe
125
17
Met -
Val
Pip -e 28
a-Abu-(
Tyr
3
cL-Ala
y-Abu --0
20
(9
51
1
Gln
26
Pip
O-Ala-0,
-u
Ser
12
Gly
(D
76
21
e
18
Me-His
5Lys
Asp
I-I
0
G
9
(DAsn
Gluc
Arg
(Sý
16
pq
Cys
C
His
1.9
Electrophoresis
pH
buffer
Origin
198.
FIGURE 21.
Amino acid
map of honey from
Canada No. 199
14
Ile
15
Leu
139 -Phe
17
Met
25
Val
Pip -G
Trp
Tyr-(5 24
3
a-AJ
a-Ala
20
D
Y-Abuja
Pro
$-Ala -0 2
10
22
Thr
Gly
97
12
21
(
sier
(:
16
0
rn
0
D:
94
pq
Gluc
(D
Arg
(:
Asp
SlAsn
\ý
6
Lys
7
18
Me-His
5.
5
Glu
31-
pH 1.9 Electrophoresis
His
Cys
buffer
Origin
199.
FIGURE 22
Amino acid
map of honey from
China No. 209 - Buckwheat.
Ile
Leu
e)
The
0
Met-7(225
Val
Pip -e 28
Try
24
23
)9
-Trp
a-Abu -3)
a-Al
20
Pro
1
y-Abu-e4
(aGIn
10
Thr-1(22
Glu
1.1
Gly
12
w
J
-4
0
Asp
Se
21
: 3:
44
PO
18
9
Me-His
Gluc
Arg
a
ýu
Lys
13
Electrophoresis
.9
8
His
buffer
Cys
origin
200.
FIGURE 23
map of honey from
Amino acid
14
No. 225 - Gujarat
India
2.
Ile
Leu
&19
Phe
Met
Val
&23Trp
Tyr
a-Abu-
3
Q
Pro
a-Ala
O-Gln
26-2ývH-Pin
Thr-_,
Gly
Glu
O-Asp
12
w
21
0
Ser
Me-His
CD
18
Ast
16
Lys
pH 1.9, Electrophoresis
Cys
buffer
9
8
(D
Gluc
0
origin
201.
FIGURE 24
map of honey from Mexico
Amino acid
Ile
No. 227 - Yucatan.
1
15
Leu
Q
19
Met--
iv
Pip
Phe
25
Val
4)
Trp
Thr
30
a-Abu-
11
i
20
a-Ala
Pro
4
y-Abu-ý
26=;
7r %
%
%.
_ýI
Thr
)
H-Pip
10 -f-
Glu
t<22
U
Gly
7
0
Asp
)-Gluc
Me-His
18D
m
;C4
pq
6D
Jý,
Asn
5
Lys
Arg
16
Cys
His -(ý13:
pH 1.9 Electrophoresis
buffer
Origin
202.
TIGURE 25.
knino
,
from
New
Zealand
honey
of
map
acid
No. 230 - Clover.
@
14
Ile
15
Leu
Phe
(
17
Fet
25
Val
G2)3-Trp
Pip -
28)
C)
24
Tyr
3
a-Abu-6
a-Ala
G
20
Pro
1
-y-Abu -0
H-P, P&Gln
__26
ß-AlalDThr
10
22
Glu
.1-I
G)-Asp
12
Gly-o
Ser
Me-His -6 18
5
Lys-Q
16
-4
0
21
J-Asn
Gluc
'Arg
GHis
pH 1.9 Electrophoresis
Cys
buffer
Origin
203.
honey that
were processed
There were twenty
one honey
The pollencontent
of each sample of honey
in Table
given
information
bottled
60.
the
The following
the
each of
within
were:
three
phenylalanine,
methyl-histidine,
tyrosine
tryptophan,
and between
in Table
each of
three
refined
for
and accounted
positive
samples
substances
arginine,
leucine,
The differences
sugar
asparagine,
acid,
lysine,
proline,
glycine,
methionine,
threonine,
serine,
in occurrence
of
maps were evaluated
These differences
the
wihin
have been given
Code No. 256 was a standard
These sugar
retailers.
concentration
There were no ninhydrin
amino acid
maps of
substanceswere
sugar
syrup
as man made honey.
sugar
syrup
did not
sold
this
positive
identified
and
The other
at various
contain
have been given
category
The amino acid
detected
on the replicate
reference
sample
61.
The following
256
No.
Code
using
sample
from
commercial
pollen.
in Table
substances
255.
Code
No.
sample
detected
into
were allocated
sold
samples
measurements
products
one sample Code No. 255 was derived
two samples,
and was being
starch
the
the honey
of
glutamic
acid,
on the
Type
Comm rcially
of
glutamine,
groups.
is,
that
62.
Sugar Product
potato
the
and the
were identified
acid,
on the amino acid
substances
have been
groups
processed,
in-each
pipecolic
and valine.
positive
ninhydrin
substances
isoleucine,
acid,
groups.
61.
a- and y-aminobutyric
hydroxypipecolic
histidine,
three
measurements
These ninhydrin
glucosamine,
producers.
these
the three
in Table
standards
groups.
cysteine,
acid,
aspartic
positive
reference
a- and 0-alanine,
in
samples were commercially
ninhydrin
maps using
for
accounted
concentration
have been given
or otherwise
amino acid
that
samples
The amino acid
whether
or by commercial
by the beekeeper
on the replicate
ninhydrin
positive
amino acid
standards.
maps
These
2o4.
TABLE 60
Fr"uency
of
distribution
of
pollen
in
grains
country
of
now known,
origin
suspect
origin,
and
honeys and sugar products.
English
Z23
Pollen
Floral
Cenus
honey sample code No
234
.
235
236
237
238
239
241
240
1.0
Acacia
242
246
1.5
Banksia
M
4.5
1.9
Braasica
20.5
19.4
16.8
1.2
17.2
10.2
21.5
61.2
Castanea
5.9
10.0
14.9
13.4
35.4
40.5
Echium
BucaZyptua
4.5
57.2
37.1
65.0
4.5
12.4
5.3
1.1
Helianthue
4.6
0.5
Loranthus
14.6
Lotus
6.3
14.6
5.8
5.5
Myrtaccae**
14.0
Robinia
4.1
8.0
19.9
12.5
Rosmarinus
2.4
repens
Urtica
Shrivelled
pollen
Unidentified
to key 1,2
0*
0*
and 3
refer
Sample examined for
subjected
Identified
pollen
to high pressure
at plant
family
1.3
14.4
12.7
16.0
12.9
11.9
20.2
10.1
14.3
8.9
7.7
11.9
8.8
6.2
M
0.6
381
363
377
373
410
392
327
336
0*
to Table 31.
but none present,
filtration
level
8.2
15.6
16.7
pollen
Total pollen
grains counted
For notes
1.9
7.2
Taraxacum
Trifolium
247
only.
for
it
is suspected
the purpose
that
these
of clarification.
three
samples were
205.
TABLE 60 Cont'd.
Pollen
Floral
Genus
M
2,3
Honey sample code No.
Brassica
243
244
245
32.4
17.9
10.8
253
39.7
254
124.6
248
'
249
250
74.0
3.1
Echium
11.8
21.5
6.0
16.2
11.8
3.6
8.8
Beracleum
4.7
1.3
5.1
6.9
6. o
3.0
Impatiens
2.2
Ligustrum
56.7
Loranthu8
2.9
Lotus
5.8
3. o
onobrychis
0.7
1.9
8.6
PhZox
0.7
6.2
Prunualpyru8
1.5
3.7
Robinia
0.8
Rowkwinua
0.5
2.5
TiZia
2.5
TrifoZium pratense
15.4
TrifoZium repen8
14.6
35.8
17.7
pollen
pollen
Total pollen
grains counted
Sample
0.5
62.9
12.7
14.5
4.2
Vioia
+-
7.9
1.2
Eucalyptus
Unidentified
255
68.7
Epilobium
Shrivelled
9.0
12.1
0.6
252
74.8
CaZluna
Ca8tanea
251
1
examined
for
13.5
12.9
21.1
21.1
10.1
23.7
28.5
18.7
10.2
7.5
8.8
6.1
8.8
5.8
6.0
19.9
6.1
4.6
9.5
0.7
364
396
397
345
385
430
256
236
322
428
pollen,
but
none
present,
0+0+
256
206.
03 1
r-
rq
00
en
Co
%D
-t
-t
M
r-
00
in
P-4
0
7-4
%0
CD
r-i
r_T
rý
0
P-4
000
%D
00
r_
_T
C%
r4
C*4
-t
rr-
0
LM
LM
p4
in
in
%D
cq
w
P.4
0
Cj%
CD
rH
0
cq
%0
%0
r-i
N0
w
Ici
0
N4
0N
C'4
C%i
Co
eq
-: r
04
v-4
_d*
(D
1-1
CIM
Ln
Co
cm
-t
rq
E-4
(DN
rHM
r-
CM
cm
LM
0
9-4
M
Co
M
ýT
00
c4
in
%D
H
LM
C%
Ln
CD
CY%
%0
CY%
%0
%0
in
00
-e
M
00
ri
%D
NH
rl%
%0
%T
cq
-t
CVI
0
H
%D
im
c4
c4
V-4
00
V-4
0
00
en
c>
0
%D
_T
km
P-4
Ow
ZD
0
A
10
r.
co
a
044
0
C14
0
P.
Ln
od
0
14
C7%
m
C*4
ý4
0
0)
a)
Ln
00
cq
%M
CD
r-
CD
C%
C%
0
00
M
LM
it
C%
C%
%0
-7
10
P-4
P-4
0
-H
41
co
w
w
0
44
0
%0
cyý
r-
C, 4
ci
C%
cq
0
04
CVI
-t
cli
ZD
e
0
4-4
0
Co
r4
%0
r-
00
CD
Ln
%D
_T
clq
V-1
H
P-1
%0
Co
ul
CVI
CIN
cm
vi
0
rF-4
cn
r_
Ln
V-4
CD
H
r-t
%D
-t
vi
%0
0%
ri
CD
't
Ln
92.4-1
ci
r.
0
ci
%D
cm
ed
tn
C,4
0
Lr%
M
0
Co
Iti
H
u
0
Co
C%
r-i
V-1
tn
%0
r-
0
C,4
00
%0
00
Co
CD
00
Co
P-f
P-1
c"i
vi
0
ci.
Ici
ci
Co
1.4
P.,
CD
LM
00
Ln
MH
rA
V-1
00
NID
%D
7-4
ýo
tn
cm
r-
Ln
1-1
r*ý
(D
cm
CY%
r-i
ci
00
%D
r-4
V-4
CN
04
Co
r-
V-4
1-4
01
t2.
P.
v-;
0
P-4
%0
0
41
9
rA
0
ß4
e Co
00
4
el
cn
:100
W-4
0
wH
914
9
u1>
ce
1-4
1-4
AH
>b
4
p4
207.
%0
tn
c4
Lt)
LM
r-A
eli
LM
1
r-i
cm
Co
r-
0
A
P-1
gn
C,4
r00
0
00
M
LM
4
C,
%D
C%
V-1
00
E-4
0
r_
en
Co
C,4
ýt
00
-t
00
V-1
Le)
C%
clq
M
C%
P-1
(n
C,4
0
00
rlý
clq
0
r-
00
r-i
P-1
00
0
M
c14
00
V-4
P-A
M
clq
Ln
Co
H
rq
_T
ýT
%D
P-4
%D
r4
CO
e
CN
0
-rq
Ai
r-
cq
M
Co
r-
P-4
%D
0
e4
C%i
ci
r_
%D
cm
0
0
ýT
P-4
r00
c4
%D
%D
vi
1
P-1
E-4
Kri
N
V-1
0
-t
--t
-t
C,4
_T
0
Lri
C,4
0
r_
0%
H
H
0
%0
%0
ri
+
C7N
Ln
Ln
Co
V-4
%D
r-
M
M
cli
%D
-zt
Co
km
%D
P-4
C%
0
%0
r-
P-l
Lri
ei
cm
r-
cm
tri
CD
CD,
%
P-4
-t
CY%
wi
Cj%
00
0
%0
t
(3%
M
Lri
0
f-4
IT
r14
. %0
rCN
%D
C,4
1-4
00
0
P-4
C,4
C,4
CN
Co
C%
0
zr
CN
P-4
Cl%
rM
r4
H
00
r-
0
rý
r-
cq
cm
-4
-e
N
00
0
cm
P-4
0%
C*4
N4
1
M
V)
rý
M
r-
1
-t
(2%
C*4
Q.
w
4)
1-4
0
1
_T
44
0
r-
0
1
1
%D
rlý
14
P.4
E-4
tn
1
LM
00
Co
E-4
cq
c4
0
r-4
(D
00
CD
cm
E-4
rLn
cm
cm
00
Lm
-t
fl-
C%
0
cyl
H
c%&
r-
10
0
0
0
C%
93.
ZD
w
w
P. 4
93-
4)
0
u
M
't
C,4
cq
(:
cm
c4
tn
r-
LM
rý
0
-t
--1
0%
LM
C,4
V-4
r, Co
0.
:D
92.
:D
t2.
Im
IM
ai
r4 lii
>%
A
ril
<
19
0
rn
:1
0
P-1
0
ý4
p4
Co
P-4
u
H
9
r-4
Co
0
V-4
H
:1
0
e2
:,
E-4
4
p4
p4
ci
208.
TABLE 62
Occurrence
suspect
of ninhydrin
positive
Ninhydrin
positive
substances
in unknown country,
honeys
and English
origin
substances
Sample numbers in which given
was found not* to occur
a-Abu
241,248
y-Abu
248
a-Ala
237,240,241,243,244,247,248,249,
compound
251
235,236,237,238,239,240,241,242,
Arg
248,249,251,252,253,254
Asn
245,249,252
Cys
237,245,248
Gluc
250
Gln
245
H-pip
234,237,240,241,244,245,246,247,
.
248,249,250,251,252,253
Me-His
234,246,248
Pip
237,239,240,241,242,244,246,247,
248,249,250,251,252
1XI
234,235,237,238,240,241,246,247
In addition to the other amino acids found in all samples, the
following
These
ninhydrin positive
substances were also present.
'p'
in
the
spot
sample number 234; the unidentified
unidentified
were:
'c'
1XI
in
246
in
the
and
unidentified
sample number
spot
spot
sample numbers 243 and 245.
* Details
as per Table
.
33.
209.
positive
ninhydrin
acid,
substances
asparagine,
The relative
in Figure
concentration
were much lower
than
those
into
groups
pre-selected
to utilize
preferable
The statistical
by the
of officially
recognised
standardised
the
data
It
ANALYSIS! using
the
SPSS from
standards.
responses
using
observed
there
Moreover,
status.
and described
data
the experimental
pollen
analyses
of
statistical
the amino
information.
in default
standards';
As a consequence
However,
when any such results
the
the available
the given
were some agreement
'MAHAL'
SPSS.
'reference
reference
package.
statistical
and figures
tables
the analytical
SPSS were
was considered
software
data
as to
of computer
were obtained.
SPSS programme gave a misclassification
to the experimentally
available
analytical
(SPSS) on the PRIME computer
sciences
by the
in
given
samples
procedure.
text,
products
honey
six
the appropriate
evaluating
selected
selected
In some cases
of
to by the abbreviation
The standards
misclassification
social
used for
were randomly
programming
with
the data
the amino acid
of
sugar
Data
'MAHAL' method has been defined
be referred
will
methionine,
sample.
and fifty
statistical
In the following
The standards
criteria.
for
statistical
VI(a).
in Appendix
acids
on the basis
performing'DISCRIMINANT
This
package
two hundred
the
aspartic
sample Code No.
in these
and classification
a computer
package
for
used
was
method.
of Analytical
the use of a complex
necessitated
map for
to the honey
compared
each of
methyl-histidine,
the amino acids
of
allocating
31 to 62 for
Tables
leucine,
acid,
26.
Computer Processing
In evaluating,
a-aminobutyric
The amino acid
and valine.
256 has been illustrated
a-alanine,
isoleucine,
glycine,
threonine
serine,
were:
in respect
in most cases
selected
of misclassification
were re-examined
of
group
or the
became
in conjur)ction
210.
FIGURE 26
Amino acid
map of Sainsbury's
golden
syrup
No. 256.
0
pH 1.9 Electrophoresis
buffer
Origin
211.
the paper
with
which
electrophoresis
The experimental
becuase
to the computer
given
were not
and chromatography
take
origins
account
amino acid
their
of
patterns,
qualitative
nature.
of any such reassessment
and are
as stated.
The merits
dangers
SPSS predictive
this
of
by the
of misclassification
and the present
classification
SPSS have been dealt
in
with
the
discussion.
Analyses
Computer
Samples from
of
of honey
placements
Honey samples of*ýthe
analysis
using
statements
55 to 63 of
the
A selected
range
SPSS as detailed
the predominant
of
for
the amino acids
were calculated
and then
These amino acids
analysis.
alanine,
glycine,
proline,
and a constant
derived
four
honey
samples
into
their
respective
-The
amino acid
groups
containing
Hence,
five
pollen
and of
the Fisher's
which
amino acids
nine
classification
groups
by the
isoleucine,
only
discriminant
linear
acid,
on the
into
serine,
leucine,
functions
the discriminant
acid,
glutamic
phenylalanine
The SPSS selected
one hundred
45.2% were correctly
and
classified
groups.
measurements
inadequate
processed
were entered
aspartic
SPSS.
these
were allocated
The predicted
into
IV(b).
These samples were classified
valine,
pollen
groups
VI(b).
were:
by the
in Appendix
the number of variables
63.
these
in control
measurements
honey samples
type
in Table
SPSS have been given
basis
concentration
in Appendix
pollen
given
for
were selected
listed
file
Honeys
groups
type
pollen
SPSS control
of amino acid
pollen
specifications
number so as to reduce
an arbitary
of
the
Kingdom Survey
into
samples
predominant
discriminant
by the
the United
were modified
as described
sample numbers were ommited
thought
to contain
adequate
and the pollen
from analysis.
sample numbers
212.
%0
CY)
P-4
r4
000000
0
Ln
V-4
9-4
9-4
040000
-x
0
v-4
V-4
-: T
r-4
Nt
C14
C14
-T
P-4
00
9-4
cm
V-4
92.
Co
r4
cm
Ixi
gi
00
V--f
-t
0
-ýT
-T
0
P-4
V-4
000
9--4
000
z
19
%M
P-4
P-4
000000
-t
"4
ri
Ln
r-4
1-4 r-q
14
0
44
T-4
a
44
0
0
zr
rP-4
00
c4
N
00
%0
P-4
1-4
- V-4
Z
r-4
.0
Cd
E-4
0
Aj
44
0)
tn
v-4
r-4 0
0w
P., to
W4
ic
"4
0
cli
«
«
00
0
cyl
«
Co
P-4
cyl
«
(n
P-4
M
00
CN
cn
-x
0
en
0
cli
c4
C"
4c
213.
The unifloral
were examined.
for
the four
each of
pollen
Castanea
and CaUuna,
Myosotis
did
predominantly
The results
for
subclass
the point
at
classification
the above pollen
groups
for
80% and over,
over,
the pollen
each of
been shown in Figure
honey
samples by the
for
subclasses
of
27.
the Brassica
as those
The classification
The honey
VI(c).
into
two subsets
discriminant
linear
acids
which
valine,
twenty
correctly
the honey
the modified
samples
functions
into
defined
samples
the five
In one subset
were calculated
derived
in this
subset
samples
classified
into
have been shown in Table
their
64.
respective
for
file
pollen
five
for
and of
pollen
groups.
groups
were divided
samples
the Fisher's
the following
proline,
amino
glycine,
The SPSS selected
SPSS.
these
two
in Appendix
groups
These were:
by the
pollen
pollen
given
of honey
the
subclass
the four
into-the
the ungrouped
to that
pattern
the unifloral
and over
and a constant
for
The peaks of
a similar
the
group has
the classification
follow
the analysis.
example
the Brassica
SPSS control
in each of
by the SPSS.
honey
nine
70% pollen
of
were entered
isoleucine
having
Thus,
fýr
group
75% and
at
As a typical
been shown.
samples
containing
out using
of
SPSS have also
samples.
samples
was carried
groups
a histogram
However,
Brassica
and Castanea
70% and over.
each
was eliminated,
repens
The results
the ungrouped
two subclasses
were:
at
into
samples
These points
in the form of
obtained
results
group
samples were
to be similar.
were found
the
group,
of one species.
groups.
TrifoZium
and for'CaUuna
of honey
between
the overlap
at which
for
varied
the computer
of
95% pollen
repens,
pollen
the honey
since
than
greater
TrifoZium
The fifth
previously.
sub-classification
being
unifloral
were determined
subclAsses
namely Brassica
groups
as detailed
require
not
and multifloral
only
groups.
89.7% were
These results
214.
ý.o
r-4
04
9-4
Qo
%T
0
0
P.
V-1
W
w
0
r-i
4-4
u
-H
44
. r4
ji
cli
Ici
0
r-i
4-4
0
H
r-q
r.
0
. r4
z
4.4
0
Ici
4)
0
PA
0
t) -
0
Co
0
0
Co
u
PCZ
0
0
44
0
,0
0
ce
12
ei
Co
Co
P-ý
Co
N4
0
P-4
44
cn
cn
00
H
44
0
41
r-I
C'4
9
>-.
'l
bo 0
0 od
C%
In
00
V-4
C,4
00
H
A:)uanbaza
-t
a-
215.
TABLE 64
Classification
by the
of
into
the U. K. honey samples
five
pollen
groups
SPSS
SPSS predicted
Pollen
groups
No. of
samples
pollen
group membership
301
308
318
319
328
A
301
17
17
0
0
0
0
308
1
0
1
0
0
0
318*
6
1
0
3
0
2
*
319
2
0
0
0
2
0
328*
3
0
0
0
0
3
301
12
11
0
0
0
308
2
0
1
1
0
0
318
3
0
0
0
1
2
319
3
1
0
0
1
1
328
4
1
1
2
0
0
B
Refer
to Table
30.
A=
Samples used for
discriminant
B=
Samples used for
estimating
function
calculation.
misclassification
rate.
216.
honey samples
There were six
the SPSS misclassified
of Brassica
presence
the
samples
SPSS for
54.2% of
in
pollen
sample Code No. 89 but
the
of honey samples,
subset
predicted
the
was incorrect
for
four
twenty
the misclassification
estimating
samples were correctly
There were eleven
samples were used
rate
into
classified
these
and of
their
only
respective
and 177,
84 and 91 did not
Graphical
agree
1 against
The samples
SpSS.
The computer
in Table
given
discriminant
identified
above as
in
65.
the present
The SPSS selected
calculating
amino acids
were entered'into
threonine,
acid,
glutamic
leucine,
tyrosine
were all
100.0% correctly
and a constant
The SPSS then
misclassifiation
proline,
classified
selected
rate
derived
and of
ninteen
these
into
their
samples
only
into
the
for
by the
of honey
for
lysine,
aspartic
isoleucine,
The honey
samples
pollen
groups.
respective
which
major
and the following
alanine,
SPSS.
three
Kingdom have been
These were:
by the
discriminant
have been outlined.
samples
glycine,
57,
samples
2 was derived
functions
the analysis.
acid,
the honey
the canonical
the United
eighteen
discriminant
linear
the Fisher's
of
samples
of
samples
Code Nos.
of
_misclassified
survey
Code
to be present.
function
of honey
these
samples
predicted
scatterplot
canonical
classification
identified
pollens
This
and the SPSS
samples
which
the classification
of
28.
for
three
as to the pollen
representation
has been shown in Figure
function
The remaining
to contain.
subset
in eight
the pollen
of
39,41,102,106,118,121,171
were predicted
in this
samples misclassified
the presence
predicted
correctly
"II
and
groups.
pollen
Nos.
The SPSS correctly
samples.
group
Code No. 28 and 36.
In the other
by the
three
in the Castanea
selected
it
estimated
68.4% were correctly
the
classified
217.
FIGURE 28
The five
pollen
group
scatterplot,
by the
SPSS.
8
6
4
rd
0
.41r4
u
.Ni
Z
0
0
r.
-4
-6
-8
Canonical
Discriminant
Function
1
218.
TABLE. 65
of. the U. K. honey samples
Classification
by the
into.
three
pollen
groups
SPSS
SPSS predicted
No. of
samples
Pollen
groups
301
pollen
*
308
group membership
*
319
*
A
301
15
15
0
0
308
1
0
1
0
319
2
0
0
2
301
14
10
1
3
308
2
0
2
0
319
3
2
0
1
B
TABLE 66
of the U. K. honev samvles
Classification
calendar
year
Year
groups
into
the
1981 and 1982
group s, by the. SPSS
No. of
samples
SPSS predicted
year
membership
-group
1981
1981
Percent of group
correctly
classified
by SPSS
A
1981
33
30
3
39
9
30
1981
33
24
9
1982
63
24
39
1982
83.3%
B
These notes
are comon, to both
Refer
to Table
30.
A-
Refer
to Table
64.
B-
Refer
to Table
64.
Tables
65.6%
65 and 66:
219.
into
their
respective
pollen
There were six
the presence
and 152 for
118,147
Nos.
The remaining
three
to the pollen
predicted
number 3 the
into
the
following
aspartic
acid,
derived
by the
SPSS.
groups
pollen
only
three
of
samples
statements
given
were calculated
were:
tyrosine,
by the
for
differed
phenylalanine
groups
which
of
pollen
the amino acids
and a constant
derived
each of
of
the
Trifozium
67.
discriminant
functions
These differences
used were valine,
by the
five
seventy
in Table
groups.
the
were obtained
were Brassica,
linear
pollen
(dii)
The results
classification
the
the
for
criteria
and these
three
for
and a constant
VI part
groups,
groups.
the Fisher's
in Table
These were:
valine
in Appendix
SPSS have been given
in these
group,
alanine,
year
samples
classification
the analysis.
these
satisfied
The predicted
selected
in the Brassica
the five
groups
were calculated
into
proline,
two calendar
honey
to allocate
functions
threonine,
the pollen
The amino acids
as
question
samples have been given
were entered
which
the
and Castanea.
repens
honey
which
samples
into
agree
to questionnaire
The predicted
discriminant
the control
SPSS allocated
not
1981 and 1982 calender
recieved
selected
and 5 representing
1,2,3,4
and 145 did
as described
groups.
eight
linear
lysine,
for
year
amino acids
Using
the responses
with
and sixty
into
samples
SPSS was specified
The Fisher's
66.
to contain.
to be present.
two calender
one hundred
66,115
Code
samples
samples were predicted
Code Nos.
of honey
Computer placements
these
SPSS
and the
subset
in three
the pollen
of
which
samples
In accordance
in this
samples misclassified
predicted
correctly
groups.
isoleucine,
SPSS; in the
220.
TABLE 67
Classification
pollens
into
of the U. K.. honey samples
1981 and 1982 calendar
Year
groups
No. of
samples
year
representing
groups
by the
SPSS predicted
year
group membership
1981
1982
the three
SPSS
Percent of group
correctly
classified
by SPSS
Brassica
A.
1981
6
5
1
1982
16
4
12
1981
2
0
2
1982
15
7
8
1981
8
8
0
1982
5
0
5
1981
2
1
1
1982
6
2
4
1981
7
7
0
1982
1
0
1
1981
4
2
2
1982
3ý
3
0
77.3%
B
TrifoZium
47.1%
repens
A
100.0%
B
62.5%
Castanea
A
100.0%
B
Refer
to Table
64.
Refer
to Table
64.
28.6%
- 221.
TrifoZium
glycine,
the amino acids used were lysine,
repens group,
leucine,
alanine,
tyrosine
and in the Castanea group,
acid,
threonine,
proline,
serine,
derived
and a constant
by the SPSS
the amino acids used were lysine,
tyrosine
valine,
proline,
aspartic
derived
and a constant
by
the SPSS.
No distinct
was noted in the samples that
pattern
were misclassified
in Tables 66 and 67.
Computer
of honey
placements
In accordance
samples which
the responses
with
5
SPSS
the
was specified
number
and 'not
heated'
one hundred
68.
calculated
for
the
analysis.
These were:
a constant
derived
given
one honey
and seventy
in Table
The Fisher's
following
substituted
part
(dii)
pollen
were obtained
for
into
selected
which
by the
were
into
were entered
phenylalanine
and 'not
'heated'
year
groups
only
three
and 'not
pollen
classification
SPSS have been given
the three
linear
pollen
discriminant
groups.
five
the
the
and
for
groups.
and these
of
in Table
functions
the
69.
groups
in Appendix
pollen
criteria
heated'
groups
heated'
as given
these
satisfied
'heated'
the
the Fisher's
in each of
the
which
The predicted
and Myosotis.
of
SPSS have been
functions
glycine,
serine,
5
and
representing
samples
for
by the
selected
which
'heated'
classification
discriminant
linear
the
question
by the SPSS.
1,2,3,4,
groups
The predicted
amino acids
the two calendar
SPSS allocated
into
samples
samples
threonine,
The specifications
for
to allocate
or otherwise
to questionnaire
received
ais described.
groups
were heated
VI
The
groups.
each of
the
The results
were Brassica,
Castanea
four
samples
sixty
honey
The amino acids
were:
for
differed
were calculated
These differences
were
in
the
222.
TABLE 68
Classification
the
of
U. K. honey
into
samples
'heated'
and
'not
heated'
groups by the SPSS
Beat
groups
No. of
samples
SPSS predicted
heat
group membership
heated
Percent of group
correctly
classified
by SPSS
not heated
Aheated
17
10
7
not heated
77
19
58
heated
is
4
11
not heated
62
15
47
72.3%
B
66.2%
TABLE 69
Classification
groups into
Heat
groups
of the U. K. honey samples representine
three
'heated'
and 'not heated' groups by the SPSS
No. of
samples
SPSS predicted
heat
group membership
heated
Dollen
Percent of group
correctly
classified
by SPSS
not heated
Drassica
A
heated
not heated
770
12
94.7%
1
11
B
heated
not heated
312
17
55. OZ
7
10
Castanea
A
heated
110
not heated
808
100.0%
B
heated
211
not heated
615
75.0%
Myosotis
A
heated
110
not heated
505
100.0%
B
heated
0-00
not heated
202
These notes are common to both Tables
ARefer to Table 64.
B-
Refer
to Table 64.
100.0%
68 and 69.
223.
Brassica
the amino acids
isoleucine,
used were aspartic
tyrosine
the amino acids
by the
SPSS.
feeding
used was glutamic
the responses
with
SPSS was specified
fed'
'not
and the
sugar-fed'
of
classification
into
the
by the
for
of
sugar
(dii)
groups
for
samples
which
the
SPSS have been given
differed
in the Brassica
samples
linear
amino acids
selected
discriminant
which
isoleucine
threonine,
'sugar-fed'
the
satisfied
'sugar-fed'
of
for
The predicted
The Fisher's
year
and 'not
groups
and 5 representing
classification
were calculated
lysine,
'sugar-
the
were entered
and a
SPSS.
1,2,3,4
into
70.
question
into
samples
one honey
and seventy
the two calendar
The amino acids
were:
valine,
derived
to questionnaire
honey
the following
These were:
derived
SPSS allocated
predicted
were a result
received
in Table
for
were calcualted
were substituted
pollen
acid,
and a constant
as described.
groups
SPSS have been given
The specifications
VI part
acid
to allocated
the one hundred
the analysis.
constant
glutamic
group,
or otherwise
6
the
number
functions
the Castanea
by the SPSS and in the A&osotis
derived
of honey samples which
placements
In accordance
by the
threonine,
acid,
leucine,
proline,
serine,
SPSS; in
by the
and a constant
group,
Computer
derived
and a constant
phenylalanine
used were lysine,
the amino acids
group,
'not
and
the ninety
in Table
which
five
as given
the five
pollen
criteria
for
sugar-fed'
honey
in Appendix
The
groups.
each of
groups.
samples
groups
the
The
selected
by
71.
the Fisher's
in the five
group,
these
sugar-fed'
pollen
the amino acids
linear
groups.
discriminant
functions
These differences
used were lysine,
serine,
224.
:6
cu
10
gi
X
cu
ci
uw
k4
ei
94
%Z
Ln
cn
Q) CL4
cn
w
0
u
0)
44
1
w
ce
0
c4
C,4
P%
en
0%
1
P.4
%0
c4
M
ý
0
C,4
cm
"d
""
(IM
ýt
ýt
to
41
. ýi ,,
0
ý;
,Ni w
C',
Qi Q)
54 0
gl.
En
wý
co
a)
0
m
tio
.0
V-4
LM
ýc
%0
12
c13
1-4
iz
m
&4
ce
0
.
p4
10
Q)
cli
rA
w 12.
cu =
ýi
E--4
u=
0
cJ
c
jj
0
225.
TABLE 71
Classification
fed'
of
and 'not
U. K. honey
the
sug ar-fed'
No. of
samples
Sugar
groups
samples
representing
five
pollens
into
groups by the SPSS
SPSS predicted
sugar
group membership
sugar-fed
Percent of group
correctly
classified
by SPSS
not sugar-fed
Brassica
A
12
sugar-fed
not sugar-fed
5
11
1
94AZ
05
B
13
sugar-fed
not sugar-fed
Trifolium
9
11
2
68.2%
54
repens
A
sugar-fed
770
not sugar-fed
514
91.7%
B
sugar-fed
880
not sugar-fed
752
66.72
Castanea
A
sugar-fed
3
3
0
not sugar-fed
2
0
2
10
8
2
2
1
1
I
1
0
2
0
2
sugar-fed
I
1
0
not sugar-fed
0
0
0
Sugar-fed
3
3
0
not sugar-fed
1
0
1
sugar-fed
3
2
1
not sugar-fed
I
1
0
100. U%
D
sugar-fed
not sugar-fed
75.0%
Calluna
A
sugar-fed
not iugar-fed
IOU. 0%
B
100.0%
Myosotis
A
100.0%
B
A-
Refer
to Table 64.
B-
Refer
to Table 64.
'sugar-
50.0%
226.
glutamic
alanine,
acid,
the TrifoZiwn
repens
SPSS; in the CaUuna
by the
used were serine,
acids
in the
of
were calculated
serine,
by the
glycine,
for
tyrosine,
'not
and
the
one honey
samples
linear
selected
discriminant
These were:
amino acids.
derived
and a constant
phenylalanine,
SPSS was specified
sugar-fed'
SPSS have been given
The amino acids
for
were calculated
differed
in the Brassica
group,
threonine,
glycine,
which
select
honey
samples
the
'sugar-
the five
pollen.
The
samples
selected
by the
linear
the Fisher's
groups.
discriminant
isoleucine,
leucine
repens group,
and a constant
derived
the amino acids used were proline,
functions
These differences
the amino acids used were lysine,
alanine,
into
73.
in four pollen
by the SPSS; in the TrifoZium
isoleucine
the ninety
in Table
to
representing
groups
of
classification
predicted
group,
The autumn
and the
group
The Fisher's
following
and
SPSS-
As before
lysine,
the
72.
SPSS.
The predicted
way.
and seventy
in Table
by the
manner.
sugar-fed'
in the usual
the one hundred
SPSS have been given
functions
'not
the amino
group,
in the winter
in the following
the
performed
analysis
classification
lysine,
into
were allocated
statistical
by the
to the honeybees
months were assessed
spring
samples
sugar
acid
used was glutamic
derived
and a constant
valine
of feeding
The results
the amino acid
group,
derived
and a constant
alanine
serine,
tyrosine
the amino
group,
by the SPSS and in the Myosotis
derived
and a constant
red'
acid,
used were aspartic
acids
used were leucine,
SPSS; in the Castanea
by the
by the SPSS; in
derived
and a constant
the amino acids
group,
derived
and a constant
fed
leucine
aspartic
and a constant
were:
acid,
derived
the amino acids used were
by the SPSS; in the Castanea
glycine,
alanine,
leucine,
227.
0
w
cu
".
44
0
C
V
N
CY)
9
Ln
41 tn
Ir
Ln
M
cn
"l. -
:3
00
.
0)
4
Ici
a)
0
%0
:i
. f4
li J.,
a) rA
41
0
r.
Ei
10
lii
41
cn 0
P.4 $W
cn to
10
r.
44
0
rn
00
-t
z
ON cn
c'c'
ull
7-4
(7%
C14
LM rý
ýT
c4
tn
10
bo
:i
rA
W
9-4
lu
U-
0)
10
gi
Ici
w
,
10
0)
4.4
tv
C14
U)
rZ
rx
ct
E-4
Ici
9)
4.4
to
:3
W
E-4
00
ý-4
ce
rA
14
E-4
44
a)
pel
4-4
CL)
Pd
41
0
$4
bo
PQ
rn
r,
.94 pq
228.
TABLE 73
Classification
'sugar-fed'
of the U. K. honey samples representing
and 'not sugar-fed'
groups by the SPSS
Sugar
groups
No. of
samples
four
SPSS predicted
sugar
group membership
sugar-fed
not
sugar-fed
--
pollens
Percent of group
correctly
classified
by SPSS
Bras8ica
A
6
6
0
17
2
15
3
1
2
13
9
4
sugar-fed
2
1
1
not sugar-fed
8
1
7
3
1
2
sugar-fed
not sugar-fed
91.3%
B
sugar-fed
not sugar-fed
Trifolium
31.3%
repens
A
80.0%
B
sugar-fed
64.7%
14
4
10
sugar-fed
4
4
0
not sugar-fed
3
0
3
sugar-fed
2
1
1
not sugar-fed
7
3
4
sugar-fed
1
1
0
not sugar-fed
2
0
2
sugar-fed
1
1
0
not sugar-fed
4
2
2
not sugar-fed
Castanea
A
100.0%
B
55.6%
AV030tis
A
100.0%
B
A-
Refer to Table 64
B-
Refer
to Table 64.
into
60.0%
229.
SPSS and in the Myosotis
by the
used was tyrosine
the amino acid
group,
derived
and a constant
phenylalanine,
derived
and a constant
by the
SPSS.
The effects
imino
of
to those
proline
acid
The autumn,
was evaluated.
file
The SPSS control
the control
of
These were:
were calculated.
derived
of
fed'
fed'
visa
and
sugar-f
In the
'heated
and
the
combinations
linear
which
in Tables
sugar-fed'
SPSS selected
alanine,
derived
by the SPSS.
The SPSS selected
sugar-fed'
edl
samples
The following
discriminant
ratios
functions
isoleucine/proline
were both
heated
into
and the
valine,
ninety
'not
heated
75,77,79
and the
seventy
and
and
combinations
-and
'not
nine
heated
sugar-
and
not
sugar-
for
analysis.
honey samples
were calculated
isoleucine,
not
and 81.
leucine
samples
and sugar-fed'
for
These were:
the analysis.
two honey
heated
the four
all
'not
functions
were entered
serine,
for
and the
discriminant
glycine,
not
74.
linear
which
classification
groups
amino acids
honey
versa
have been given
The Fisher's
The predicted
glycine/proline,
of honey samples
The SPSS predicted
the
by using
by the SPSS.
Computer placements
'heated
in Table
amino acids
was modified
three
and seventy
the
samples were used.
VI(eii).
the Fisher's
which
twelve
fed
(c)
VI
in Appendix
listed
for
the amino acids
or
in Appendix
SPSS have been given
by the
sugar-fed
winter
of
concentration
individual
'spring
and
the one hundred
of
a constant
on the relative
the other
of
given
statements
classification
selected
feeding
sugar
for
the following
lysine,
and a constant
the
'heated
combination.
and
The
230.
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00
W CO
60 r-4
44
0
Aj
r4
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ce
Co
to
0
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P-4
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ri
cq
rl%
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P-4
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r-4
rý
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cn
tn
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41
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r3
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lu
Q) Q)
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Ci.
cn :i
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9)
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C14
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10
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P-4
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E-4
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Cd
E-4
00
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41
ti, 4uI
(1)
Pd
0)
P4
-4
pq
Ai
231.
TABLE 75
Classification
and not
of the U. K. honey samples into
sugar-fed'
Heat and sugar
groups
'heated
and sugar-fed'
and 'not-heated
groups by the SPSS
SPSS predicted
heat and
sugar group membership
No. of
samples
HT+SF
NHT+NSF
11
3
Percent of group
correctly
classified
by SPSS
A
HT+SF
14
NHT+NSF
24
5
HT+SF
12
57
NHT+NSF
29
9
79.0%
19
B
61. OZ
20
TABLE 76
Classification
and not
of
sugar-fed'
Heat and sugar
groups
the Bra8sica
honey samples into
'heated
and suRar-fed'
and 'not-heated
groups by the SPSS
SPSS predicted
heat and
sugar group membership
No. of
samples
HT+ST
NHT+NSF
Percent of group
correctly
classified
by SPSS
Bra88ica
A
HT+SF
8
71
NHT+NSF
7
16
HT+SF
0
00
NHT+NSF
5
14
86.7%
B
These notes are common to Tables
A-
Refer
to Table 64.
B-
Refer
to Table 64.
HT
-
NHT SF NSF +-
to Table
Refer
Refer
Refer
to Table
Refer
Refer
to Table
to Table
75.
75.
75.
75.
to Table 75.
75 and 76.
80.0%
232.
TABLE 77
Classification
of the U. K. honey samples into
and sugar-fed
groups by the SPSS
Heat and sugar
group
No. of
samples
'heated
and not
SPSS predicted
heat and
sugar group membership
HT+SF
sugar-fed,
and not heated
Percent of group
correctly
classified
by SPSS
NHT+NSF
A
HT+NSF
3
2
1
NHT+SF
43
9
34
HT+NSF
3
2
1
NHT+SF
43
7
36
78.3%
B
82.6%
TABLE 78
Classification
of the Bras8ica
not
and 'not
sugar-fed'
heated and sugar-fed'
No. of
samples
Heat and sugar
group
and Trifolium
repens honey samples into
'heated
groups by the SPSS
SPSS predicted
heat and
sugar group membership
HT+SF
Percent of group
correctly
classified
by SPSS
NHT+NSF
Bras8ica
A
HT+NSF
1
1
0
NHT+SF
7
0
7
HT+NSF
1
0
1
NHT+SF
10
1
9
HT+NSF
1
1
0
NHT+SF
7
0
7
HT+NSF
0
0
0
NHT+SF
7
0
7
100.0%
B
2'rifoZium
81.8%
repene
A
100.0%
B
These notes
are common to both Tables
A-
Refer
to Table 64.
B-
Refer
to Table 64.
HT NHT SF NSF +-
to Table 75.
Refer
Refer
Refer
to Table
Refer
Refer
to Table
to Table
75.
75.
75.
to Table 75.
77 and 78.
100.0%
and
233.
TABLE 79
of the U. K. honey samples into
Classification
'not
heated and not sugar-fed'
Heat and sugar
group
'heated
and not sugar-fed'
and
groups by the SPSS
heat and
SPSS predicted
sugar group membership
No. of
samples'
HT+NHF
NHT+NSF
Percent of group
correctly
classified
by SPSS
A
32
HT+NSF
97.0%
30
NHT+NSF
30
0
B
312
HT+NSF
76.9%
4
23
NHT+NSF
19
TABLE 80
of the Brasaica
Classification
'not
heated and not sugar-fed'
Heat and sugar
group
No. of
samples
honey samples into 'heated
groups by the SPSS
SPSS predicted
heat and
sugar group membership
-
NHT+NSF
HT+NSF
and not sugar-fed'
Percent of group
correctly
classified
by SPSS
Brassica
A
HT+NSF
220
NHT+NSF,
404
100.0%
B
HT+NSF
000
NHT+NSF
835
A-
are common to both Tables
Refer to Table 64.
B-
Refer to Table
These notes
HT
64.
Refer to Table
75.
NHT
Refer
to Table 75.
WSF
Refer
to Table
+
Refer
75.
to Table 75.
62.5%
79 and 80.
and
234.
discriminant
linear
Fisher's
glutamic
acid
acid
functions.
discriminant
'not
and
heated
samples.
The
honey
nine
linear
the Fisher's
calculating
aspartic
phenylalanine
the amino
SPSS.
sugar-fed'
fifty
These were:
tyrosine,
proline,
acid,
were used for
amino acids
by the
and not
the SPSS selected
sugar-red',
following
'heated
of
for
were calculated
derived
and a constant
For the combination
and not
functions
acid,
serine,
glutamic
derived
and a constant
by the
SPSS.
In the final
'not
and
fed'
isoleucine,
honey
the four
samples
'heated
the
and sugar-
one hundred
S'.PSS selected
discriminant
functions
amino acids.
These were:
lysine,
phenylalanine
and a constant
linear
and
were
serine,
derived
the four
of
have been given
in Table
by
76.
In this
linear
calculated
the following
phenylalanine
In the
and these
SPSS selected
nineteen
were calculated
were:
honey samples
for
the
group
the
functions
twenty
were
lysine,
These were:
aspartic
by the SPSS.
were obtained
classification
and TrifoZium
In the Brassica
and the Fisher's
following
The results
only.
SPSS selected
the Brassica
78.
select
above the predicted
discriminant
derived
in Table
have been given
The results
functions
groups
group
the predicted
second combination,
two pollen
listed
amino acids.
and a constant
to
groups.
the Brassicq
samples and the Fisher's
for
pollen
combinations
for
was obtained
classification
honey
the five
SPSS was specified
the
combinations
representing
In the first
for
is,
that
SPSS.
For each of
acid,
the
The Fisher's
the following
valine,
glycine,
the four,,
of
and sugar-red',
samples.
for
calculated
the
heated
honey
twelve
combination
group,
linear
amino acids.
repens.
the
discriminant
These were:
235.
threonine,
acid,
aspartic
derived
by the SPSS.
fifteen
honey
for
Brassica
fourteen
SPSS selected
functions
aspartic
acid,
by the
SPSS-
In the fourth
groups
for
which
been given
honey
calculated
of
the four
the predicted
in Table
samples
for
and a constant
82.
and the Fisher's
derived
were calculated
aspartic
acid,
by the
SPSS.
serine,
linear
amino acids.
by the
SPSS.
in Table
linear
these
group,
the
proline,
leucine,
The
discriminant
derived
two pollen
These were:
two pollen
groups
SPSS selected
have
twenty
discriminant
functions
were
These were:
alanine,
tyrosine
In the Castanea
the following
80.
These were:
were only
group,
by the SPSS and the Fisher's
for
was obtained
were obtained.
for
SPSS.
and a constant
there
combinations
threonine,
by the
amino acids.
phenylalanine
In the Brassica
honey samples were selected
functions
the following
functions
classification
and, the Fisher's
The results
the following
derived
and a constant
SPSS selected
These were:
classification
and Castanea.
the Brassica
five
for
isoleucine,
glycine,
discriminant
have been given
honey samples
were calculated
linear
and a constant
The results
group only.
the
group,
the predicted
combination,
tyrosine
repens
amino acids.
phenylalanine
In the third
for
In the TrifoZium
the following
tyrosine,
leucine,
valine,
samples and the Fisher's
were calculated
alanine,
proline,
amino acids.
tyrosine
thirteen
linear
discriminant
These were:
and a constant
derived
236.
TABLE 81
of the U. K. honey samples into
Classification
'not
heated and suear-fed'
Heat and sugar
groups
No. of
samples
'heated
and sugar-fed'
and
erouvs bv the SPSS
beat and
SPSS predicted
sugar group membership
HT+SF
NHT+SF
Percent of group
classified
correctly
by SPSS
A
HT+SF
13
10
3
NHT+SF
29
5
24
HT+SF
13
58
NHT+SF
57
81.0%
B
58.6%
21
36
TABLE 82
Classification
sugar-fed'
of the Brassica and Castanea honey samples into
and 'not heated and sugar-fed'
g roups by the SPSS
Heat and sugar
groups
No. of
samples
heat and
SPSS predicted
sugar group membership
HT+SF
NHT+SF
'heated
Percent of groups
correctly
classified
by SPSS
Bras8ica
A
HT+SF
3
2
1
NHT+SF
9
0
9
HT+SF
5
1
4
NHT+SF
8
4
4
HT+SF
3
3
0
NHT+SF
4
0
4
HT+SF
0
0
0
NHT+SF
6
4
2
91.7%
B
38.5%
Caetanea
A
100.0%
B
These notes
are common to both Tables
A-
Refer
to Table 64.
B-
Refer
to Table 64.
HT NHT SF +-
Refer to Table
Refer
Refer
Refer
75.
to Table 75.
to Table
to Table
75.
75.
81 and 82.
and
33.3%
237.
Analyses
Samples from
of
Discriminant
analysis
foreign
allocated
the criteria
specified
The predicted
selected
by the
and English
each of
the
statements
classification
of
five
the Australia,
country
groups
group
83.
to
in Appendix
honey
three
samples
New Zealand
The honey
into
were divided
The SPSS
54 to 60 listed
twenty
four
sixty
according
Canada, Mexico,
in Table
have been given
groups
country
the
the
of
was performed
'MAHAL' method.
the
in the control
SPSS into
groups
measurements
to the appropriate
samples
VII(a).
concentration
Honeys
the U. K. survey
of
honey samples using
and commercial
honey
country
to that
similar
amino acid
and Cammercial
into
of honey samples
Computer placement
on the modified
the Foreign
samples
two subsets
in
by the
SPSS.
the Fisher's
In one subset,
for
the
following
amino acids
which
lysine,
serine,
glutamic
and a constant
derived
by the
were:
by the
linear
SPSS were 100.0% correctly
glycine,
valine,
honey
The twelve
into
classified
functions
into
were entered
acid,
SPSS.
discriminant
were calculated
the analysis.
leucine,
samples
their
These
tyrosine
selected
appropriate
country
groups.
In the other
subset,
rate
the misclassification
by the
SPSS into
their
There were six
misclassified
from
respective
samples
Australia,
predictions
from Australia,
mistake
and of
these
(Code Nos.
because
Canada,
suggested
Mexico
that
groups
from
estimating
45.5% were correctly
classified
groups.
194,195,203,204,229
England,
five
for
were selected
were experimentally
and New Zealand.
the pollen
only
country
by the SPSS and these
countries
computer
honey samples
eleven
Mexico
samples
This
families
and 249)
shown to come
and New Zealand;
excluding
204 originated
is a reasonable
Cruciferae
the
predictive
and Leguminosae
238.
are
in fact
common to all
The following
predicted
when the English
commercial
linear
Fisher's
classified.
The following
These were:
lysine,
selected
by the SPSS for
42.9% of
the
It
SPSS.
samples
should
by the
SPSS.
not
to those
out
to that
expected
When the China honey
SPSS.
The nine
from
functions
discriminant
amino acids
acid,
by the
into
valine,
tyrosine,
alanine,
SPSS.
the misclassification
classified.
if
rate
their
the predicted
selected
for
the analysis
There were thirteen
and of
and-only
and 228) misclassified
composition
predicted
and pollen
origins
predicted
in the above analysis
and these
these
pnly
linear
The following
were:
glutamic
and a constant
samples
by the
the Fisher's
classified.
honey
were
were correct.
85 were obtained
phenylalanine
of
countries
patterns
calculating
were 100.0% correctly
were entered
rate
The computer
respectively.
shown in Table
honey samples
alanine,
There. were seven samples
the pollen
that
samples were included
classifications
the predicted
the analysis.
proline,
195,202,227
be pointed
expected
selected
classified.
227 was from Canada and 228 from New Zealand
dissimilar
into
the misclassiciation
(C6de Nos.
Canada and New Zealand,
samples
analysis.
were 100.0% correctly
were entered
acid,
SPSS
the previous
honey
calculation
glutamic
derived
estimating
195 and 202 were similar
namely
acid,
samples were correctly
There were four
from
The nine
function
aspartic
by the
was obtained
84.
amino acids
and a constant
phenylalanine
present.
samples were omitted
discriminant
sample 204 was
omitted
pollen
classification
have been shown in Table
The results
by the
The
countries.
shown to have no detectable
experimentally
for
these
selected
for
derived
estimating
15.4% were correctly
239.
TABLE 83
Classification
of the foreign
and commercial
honey samples into
five
country
groups by the SPSS
Country
groups
SPSS predicted
No. of
samples
Australia
country
Canada
group membership
Mexico
New Zealand
English
A
Australia
2
2
0
0
0
0
Canada
5
0
5
0
0
0
Mexico
2
0
0
2
0
0
New Zealand
1
0
0
0
1
0
English
2
0
0
0
0
2
Australia
2
0
0
1
0
Canada
3
1
1
0
0
Mexico
0
0
0
0
0
0
New Zealand
1
1
0
0
0
0
English
5
1
0
0
0
4
B
TABLE 84
Classification
of the foreign
and commercial
honey samples into
four
country
groups by the SPSS
Country
groups
SPSS predicted
No. of
samples
Australia
country
group membership
Canada
Mexico
New Zealand
A
Australia
3
3
0
0
0
Canada
4
0
4
0
0
Mexico
0
0
0
0
0
New Zealand
2
0
0
0
2
Australia
1
0
1
0
0
Canada
4
0
3
0
Mexico
2
0
New Zealand
0
0
B
These notes
are common to both Tables
A-
Refer to Table 64.1
B-
Refer
to Table 64.
83 and 84.
0
0
0
0
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241.
honey samples misclassified
There were eleven
samples had pollen
four
these
seven honey samples had pollen
The remaining
by the
predicted
No. 197,
204,
and
Code No. 228.
dissimilar
composition
China
Australia
were:
These seven samples were:
200,203
they
to
Australia
Code Nos.
Code
208 and 209
Code No. 227.
honey
The five
discriminant
by the
linear
The following
classified.
SPSS-
rate
the misclassification
honey
and of
for
were 100.0% correctly
into
the analysis
derived
and a constant
samples
these
the above
selected
functions
isoleucine
alanine,
There were twenty
samples
were entered
amino acids
threonine,
were:
discriminant
in
shown in Table
classifications
The seven honey
SPSS.
included
were also
and the predicted
the Fisher's
calculating
from France
samples
analysis
86 were obtained
by the
SPSS.
Canada Code Nos.
and Mexico
and these
samples
195 and 196, Canada Code No. 201 and Mexico
Code Nos.
SPSS and of
to the country
similar
These four
to have originated..
were predicted
that
composition
by the
for
selected
estimating
35.0% were correctly
only
classified.
There were twelve
these
six
samples had pollen
were predicted
Code No. 196,
218.
honey
that
predicted
Nos.
194,195
Canada Code No. 202, France
six
by the
samples had pollen
SPSS.
These six
and 197 and China
In the above analysis,
were replaced
These six
by those
from
the honey
India
samples were:
dissimilar
composition
208,211
samples
Australia
and
to
Code
and 213.
of Mexico
and the predicted
they
Australia
215,216,217
Code Nos.
samples were:
Code Nos.
SPSS and of
to the country
similar
composition
to have originated.
The remaining
by the
samples misclassified
and New Zealand
classifications
242.
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obtained
by the
samples
selected
for
calculation
into
were entered
isoleucine,
glycine,
SPSS.
leucine,
There were fifteen
rate
misclassification
had pollen
four
had
pollen
samples
Nos.
glutamic
acid,
honey
samples
selected
these
40.0% were correctly
dissimilar
Australia
samples were:
derived
the
estimating
SPSS and of
they
to that
by
classified.
these
were predicted
215 and 216.
Code Nos.
proline,
Canada Code Nos.
samples were:
composition
for
by the
to the country
similar
Code No. 208, France
China
These four
were:
amino acids
and a constant
These five
206,
discriminant
phenylalanine
and of
composition
to have originated.
SPSS.
linear
honey samples misclassified
There were nine
five
and these
honey
The eleven
The following
classified.
the analysis
87.
the Fisher's
of
were 100.0% correctly
functions
the
SPSS have been shown in Table
200 and
The remaining
by the
predicted
Code No. 197,
Canada Code
202 and 203 and China Code No. 209.
Discriminant
European
The predicted
88.
in
Table
shown
The following
by the
The seven samples
amino acids
acid,
glutamic
threonine,
SPSS.
functions
valine,
rate
and of
into
by the
expected
from
only
(Code Nos.
SPSS, however
their
selected
predicted
calculation
of
classified.
These were:
derived
for
the
estimating
71.4% were correctly
pollen
origins.
the
and a constant
216 and 232) which
their
France
SPSS have been
the analysis.
phenylalanine
these
for
by the
and the
from
were 100.0% correctly
were entered
There were two samples
to that
selected
the English
the honeys
obtained
There were seven samples
misclassification
misclassified
included
samples
classifications
discriminant
linear
out between
was carried
The European
sample.
and Spain.
Fisher's
analysis
composition
classified.
were
was similar
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to
$4
Itl
4-J
CA
(Is
0
0,
Cd
co
ci
Cd
0
0
P4
-P4
4
Cd
ý4
10
r.
CO
W
41
0
ce
lu
cu
cu
rý
.
p4
u
9
ce
., 4
10
-4
pq
245.
The SPSS was specified
to their
given
The predicted
functions
into
100.0%
were
of honey
Computer placements
The control
the
The three
serine
amino acid
The misclassified
the pollen
predicted
Including
analysis,
from
that
the Leguminosae
by the
rate
acid,
two
discriminant
which
were
entered
phenylalanine
groups
in Appendix
the honey
for
selected
derived
as
into
samples
calculation
these
of
the
the Fisher's
The
classified.
by the SPSS were entered
honey samples
New Zealand
VII(a)
by the SPSS have been shown in
were 100.0% correctly
and of
were used in
VII(bvii)
given
of
groups
honey samples
only
selected
for
estimating
66.7% were correctly
sample Code No. 230 did
into
not
classified.
contain
by the SPSS.
the honey
is,
those
classification
and a constant
the misclassification
pollen
in Appendix
given
There were three
the analysis.
acids
glutamic
into
samples
functions
discriminant
linear
were:
the
linear
The Fisher's
amino
into
divided
SPSS.
and MeZiZotus
repens
as described.
VII(bvi)
and then
following
by replacing
The predicted
90.
Table
statements
analysis
described.
TrifoZium
by the
statements
by the SPSS have been shown in
selected
the
according
samples
the control
in Appendix
given
The amino acids
derived
and a constant
by replacing
classified.
for
calculated
the analysis.
following
correctly
source. groups
honey
and allocate
obtained
classification
were
subsets
by those
VII(a)
the honey
groups
The ten honey samples
89.
Table
to recode
honey source
appropriate
in Appendix
into
of honey samples
Computer placements
samples
discriminating
plant
containing
the Lotus
between pollen
family.
SPSS have been shown in Table
in the above
groups originating
The predicted
91.
pollen
The three
classification
samples
obtained
selected
246.
TABLE 88
Classification
Country
groups
of the English
and European honev samples bv the SPSS
SPSS predicted
No. of
samples
country
France
group membership
Spain
English
A
France
3
3
0
0
Spain
1
0
1
0
English
3
0
0
3
B
France
2
Spain
1
0
0
1
English
4
9
0
4
0
TABLE 89
Classification
of the floral
SPSS predicted
No. of
samples
Floral
groups
A
Honeydew
honey samples by the SPSS
floral
group membership
Honeydew
Nectar
220
Nectar
202
Honeydew
1-10
Nectar
505
TABLE 90
Classification
of
the
Trifoliwn
and Melilotus
SPSS predicted
No. of
samples
Pollen
groups
repen8
pollen
honey
samples
by
the
SPSS
group membership
308*
337*
A
308*
2
20
337*
1
01
B
308*
2
337*
1
01
TABLE 91
Classification
of
the
honey
belonging
SPSS predicted
No. of
samples
Pollen
groups
samples
325*
to
pollen
the
group membership
337*
A
325*
1
100
337*
0
000
308*
2
002
325*
1
001
337*
2
101
308*
2
002
B
These notes
are common to Tables
A-
Refer
to Table 64.
B-
Refer
to Table
*-
Refer
to Table 30.
64.
88,89,90
Papilionaceae
and 91.
308*
plant
famlly
by-the
SPSS_
247.
for
linear
Fisher's
discriminant
The amino acid
classified.
correctly
into
SPSS were entered
by the
samples
for
selected
There
honey
There were five
rate
did
203,209
Code Nos.
these
and of
and 226 which
the predicted
contain
not
of honey samples
Computerplacements
derived
and a constant
the misclassiciation
samples
and these
were misclassified
glycine
-
classified.
honey
were three
were 100.0%
calculations
the an4lysis.
estimating
40.0% were correctly
only
function
pollen.
into
of unknown origin
country
groups
The appropriate
country
and the predicted
classifications
The eighteen
honey
discriminant
functions
glutamic
into
acid,
the misclassiciation
correctly
five
These five
for
Fisher's
calculating
VII(a)
and these
and of
aspartic
leucine,
There were thirteen
rate
these
92.
linear
were:
alanine,
was used
The following
classified.
glycine,
by the SPSS.
acid,
phenylalanine
samples
selected
46.2% were
only
classified.
There were seven honey
these
and amino
have been shown in Table
proline,
for
content
obtained
the anlysis
and a constant
pollen
in Appendix
were 100.0% correctly
derived
estimating
their
of
samples
detailed
selected
samples
were entered
amino acids
threonine,
file
The SPSS control
maps.
acid
by evaluation
source
of unknown country
to honey
code number was allocated
had pollen
composition
samples were:
and 205,
New Zealand
remaining
two honey
samples misclassified
Australia
to that
similar
Code No. 196,
Code No. 230 and Russia
samples,
the pollen
204 was not known and the pollen
SPSS and of
predicted
by the
Canada Code Nos.
Code No. 231.
composition
composition
by the
SPSS.
200
In the
of Canada Code No.
of Yellow
Box sample Code
248.
No. 246 was not similar
Computer
to that
of the Canada honeys.
of honey samples ,that
placements
were commercially
processed
or otherwise
The control
54 to, 58 of
statements
the
as des,aribed.
Appendix
VII(a)
were modified
obtained
by the
SPSS have been shown in Table
selected
for
80.0% correctly
the
rate
the misclassification
honey
four
and of
glycine
these
classifications
honey
functions
samples
were
were entered
for
selected
into
derived
and a constant
samples
in
given
The twenty
amino acids
threonine,
There were thirty
SPSS.
by the
were:
93.
discriminant
ý The, following
classified.
and these
anlaysis
The predicted
linear
of Fisher's
calculation
file
SPSS control
estimating
64.7% were correctly
only
classified.
No distinct
by the
misclassified
Evaluation
Computer
of
of
and commercial
combined
as described
by the
of
the
Data
honey
and
the
SPSS control
previously.
of
Honey
subset,
calculation
and twenty
of Fisher's
79.2% correctly
that
From All
into
foreign
and commercial
file
the
detailed
The predicted
were divided
one hundred
Samples
samples
SPSS have been shown in Table
two groups
the samples
were
United
Sources
Kingdom
and
groups
The U. K., survey
and the
in
SPSS.
Analytical
placements
Foreign
became evident
pattern
classified.
linear
data
in Appendix
The combined
into
two subsets
five
honey
discriminant
The following
VI(c)
was modified
of honey
classification
94.
files1were
honey
samples
by the SPSS.
samples
In one
were selected
functions
amino acids
samples
and these
for
were
were entered
into
249.
TABLE 92
Classification
country
of the unknown country
groupsby
Country
groups
of origin
honey samples into
the_SPSS
No. of
samples
'
SPSS predicted
Australia
country
Canada
group membership
Mexico
New Zealand
A
Australia
7
7
0
0
0
Canada
4
0
4
0
0
Mexico
5
0
0
5
0
00
New Zealand
B
Australia
6
4
2
0
0
Canada
3
2
1,
0
0
Mexico
3,
1
1
0
New Zealand
1
0
0
0
TABLE 93
Classification
'processed'
Process
groups
of the foreign
and 'unprocessed'
No. of
samples
and commercial
groups
by the
SPSS predicted
honey samples
SPSS
process
Processed
group membersh.ip
Unprocessed
A
Processed
'Unprocessed
11
10
9-
1
36
B
Processed
19
15
4
Unprocessed
15
8
7
These notes
are common to Tables
A=
Refer
to Table
64.
B=
Refer
to Table
64.
92 and 93.
into
250.
and these
the analysis
alanine,
tyrosine,
valine,
lysine,
were:
estimating
rate
No distinct
samples
explain
the results.
and foreign
these
derived
by the
for
selected
72.5% were
only
The SPSS was specified
Canada,
Australia,
China,
to
France,
classifications
obtained
by the
one hundred
and fourteen
function
discriminant
classified
into
their
acids
were entered
acid,
threonine,
isoleucine,
spSS.
estimating
serine,
leucine,
74.6% of
country
glutamic
acid,
in Table
There were one hundred
rate
of Fisher's
The following
alanine,
valine,
derived
selected
by
for
57.3% were correctly
and of
these
of all
the misclassified
only
amino
aspartic
and a constant
samples
The
were correctly
glycine,
and ten honey
95.
calculation
were lysine,
proline,
phenylalanine
as detailed
The predicted
the samples
groups.
Kingdom,
the United
groups
for
and these
could
groups
VII(cii).
selected
samples
the analysis
the misclassification
samples
honey
which
into
and English
India
only
tyrosine,
honey
SPSS have been given
respective
into
groups
the country
in Appendix
statements
honey
into
select
listed
the control
in the misclassified
and commercial
of honey samples
Comput r placements
the
and of
became evident
pattern
of U. K. survey
linear
glycine,
classified.
correctly
in
adid,
one honey samples
and thirty
the misclassification
glutamic
and a constant
phenylalanine
There were one hundred
SPSS.
threonine,
classified.
.
The predicted
were similar
to
pollen
composition
the allocated
country
groups,
by the
SPSS.
samples
251.
The SPSS was specified
TrifoZium
forty
selected
seven honey samples
discriminant
to perform
samples
obtained
by the
selected
for
and these
isoleucine,
by the
derived
estimating
correctly
the misclassification
rate
were
were entered
glutamic
acid,
and a constant
honey
samples
these
only
selected
for
68.0% were
classified.
of TrifoZium
repens
samples misclassified
pollen
in the U. K. sample Code No. 178.
survey
Code Nos.
pollen
composition
samples.
and of
two samples
functions
threonine,
six
classifications
amino acids
five
and fifty
The twenty
phenylalanine
There were twenty
There were seven honey
presence
tyrosine,
96.
discriminant
lysine,
were:
leucine,
SPSS.
linear
The following
classified.
the analysis
alanine,
Fisher's
calculating
The SPSS
The predicted
analysis.
the Brassica,
two hundred
the
of
into
as described.
groups
out
groups
samples
SPSS have been shown in Table
100.0% correctly
into
pollen
pollen
honey
to allocate
and Castanea
repens
into
of honey samples
Computer placements
was correctly
The remaining
26,39,64,68,102
predicted
did
by the
SPSS and the
predicted
six
samples,
to be present
U. K.
and the Canada Code No. 202 the
not
agree
with
that
present
in
these
252.
TABLE 94
Classification
of
and commercial
groups by the SPSS
Country
groups
the
honev
combined
samples
into
U. K.
the
and
foreiRn
SPSS predicted
country
group membership
No. of
samples
U. K.
F. C.
A
U. K.
97
83
14
F. C.
28
12
16
U. K.
96
74
22
F. C.
35
14
21
B
TABLE 95
Classification
Country
groups
of
the
honey
combined
samples
into
seven
SPSS predicted
No. of
samples
Australia
U. K.
country
country
Canada
groups
by
the
SPSS
group membership
China
France
India
English
A
99
71
1
14
0
60
7
Australia
3
0
3
0
0
00
0
Canada
1
0
0
1
0
00
0
China
3
0
0
0
3
00
0
France
3
0
0
0
0
30
0
India
2
0
0
0
0
02
0
English
3
1
0
0
0
00
2
92
59
3
12
1
82
7
Australia
1
0
1
0
0
00
0
Canada
7
6
0
0
0
10
0
China
3
1
0
0
1
10
0
France
2
1
0
0
0
10
0
India
1
0
0
0
0
01
0
Enlish
4
3
0
0
0
10
0
U. K.
B
U. K.
TABLE 96
of the combined honey samples into
Classification
SPSS predicted
Pollen
groups
No. of
samples
301*
15
15
three
Rroups by the SPSS
pollen
pollen
group membership
308*
318*
A
301*
308*
20z0
318*
5005
00
B
404
18
301*
308*
3210
318*
4112
These notes
are common to Tables
A-
Refer to Table
B-
Refer
94,95
and 96.
64.
U. K.
-
to Table 64.
F. C.
-
United Kingdom
Foreign and commercial
Refer
to Table 30.
ISCUSS10N
CHAPTER 4
253.
ISC
U'S
S10N
CHAPTER 4
PRELIMINARYSUBJECTIVE EXAMINATION OF HONEY SAMPLES
of honey is
The quality
The light
and flavour.
colour
flavours.
strong
However,
these
factors.
storage.
It
heating
of honey under
the darkening,
hydroxymethy1furfural
of honeys
Blending
(1939)
Echigo,
that
also
combination
storage.
The formation
heating
and caramelisation
as the non-enzymic
which
reactions
such as Schiff's
considered.
derived
in unheated
are
its
derivatives
the production
The enzyme browning
nectar
reaction
would
The absence
also
of
by Milium
iron
from
and changes
due to
Also,
factors
the Maillard
the
such
and other
intermediates
should
be
also
enzymes and phenols
be expected
significant
(1978).
during
reactions
using
Kushnir
and White
of reactive
further
its
formation
factors.
involving
in
in
White,
with
in colour
contributing
that
results
was reported
and polyphenols
bases and subsequent
honey samples.
It
results
and
and an increase
colour.
reactions
usually'lead-to
from the original
flavour
formaldehyde
browning
conditions
(1974)
equipment
of
researchers
Takenaka and Ichimura
tannates
of
by various
were:
effects
and processing
containers
of
by a
processing
These researchers
content.
(1964);
and Subers
loss
influenced
'handling,
unfavourable
to
by the honeybee.
gathered
of honey are greatly
reported
honeys with
class
colour
was originally
has been generally
in
are usually
a honey are different
These are beekeeping,
and storage
an increase
the nectar
two properties
number of
the dark
while
and flavour-of
The colour
from which
the plant
honeys
class
of its
on the'basis
assessed
colour
flavour
a mild
with
associated
generally
to contribute
amounts of
the
254.
aromatic
honeys
class
such as tryptophan
amino acids
and their
by Phadke (1962).
honeys
It
gave intense
some of the
such as alkaloids,
components
alcohols,
in honey by Ten-Hoopen
of amino acids
reactions
such as methyl
honey which
gives
influence
could
could
be introduced
honeybee
the
dioxide;
or sulphur
an increase
from addition
during
bottled
burnt
(1965).
Most of
or
from orange
and lavender
aroma.
Other
factors
These factors
of nectar
by the
of honey and the manipulations
extraction
These factors
could
be compounds
such as smoke, benzylaldehyde
repellants
wax resultant
of
with
heating
excessive
in hydroxymethy1furfural
honeys
present
Compounds
of honey.
and colour
or blending
esters,
of honey.
characteristic
product.
honeybee
of water
honeydew or foreign
flavgur
the ripening,
final
such as pesticides;
their
Other
degradation
the honey from the gathering
into
and during
to achieve
causes
the
aldehydes,
of
constituents
have been isolated
non-
of honey.
have been reported
the process
of
other
honeys
these
and minor
and Cremer and Riedmann
with
anthranilate
also
fermentation
content;
high
and even lubricating
yeast
which
content
oil
also
occuring
honeys,
from machinery
processing.
In the present
Mexican,
china,
(1963)
compounds are the resultant
these
polyphenolic
tanins
compounds,
alcohols
coloured
named above such as
acids,
aromatic
and benzyl
phenylethyl
for
colour
dark
that
the aroma and flavours
towards
contributes
volatiles
honeys were noted
class
constituents
amino and other
carbohydrates,
various
colour
when tested
reactions
colour
in
by Browne (1908)
was reported
Further,
compounds.
dark
in
presence
light
and tyrosine
had very
Yellow
study
high
most of
phenylalanine
Box and samples
the
foreign
content
of Gale honey.
honeys,
followed
Also,
especially
by Australia,
some of
the
the
255.
U. K. survey honey had high phenylalanine
1729 1739 1749 183,184
sample nos. 53,1580
could be a result
high content
heating
honeys.
The depression
reaction
condensation
sugars.
It
conditions,
of the other
the foreign
amino acids could be the result
from the genetic
the phenylalanine
of
the
especially
these high phenylalanine
not be consumed by persons who suffer
which affects
This phenylalanine
especially
other honey constituents
with
is suggested that
phenylketonuria
and 191.
breakdown due to excessive
of protein
in unfavourable
and storage
These were:
content.
honeys should
disorder
of
metabolism.
ASSESSMENT OF PRESENT WORK
-Proteins
Isolation
Due to the very viscous
(1: 2) with
had to be diluted
standard
honey and these
distilled
water before
and then isolate
be made to concentrate
for
techniques
of honey a given sample of honey
nature
separating
dialysis,
were:
chromatography
and ultrafiltration.
of application
of each of
the proteins
proteins
gel
for use on
(sephadex),
filtration
could
Several
present.
were considered
ion-exchange
and disadvantages
The advantages
these
any attempt
methods were evaluated
for
honey.
Dialysis
The advantages
process
which
usually
results
However,
with
can,
in
this
the
of
if
dialysis
desired,
a2-3
method
fold
the
are
procedure
be conducted
increase
disadvantages
at
of
the
that
a low
it
a gentle
temperature
solution.
protein
outweigh
is
the
advantages
and
256.
in
that
the procedure
and thus,
another
The complete
four
to separate
due to microbial
risks
length
a long
solutions
final
the
volume.
of days,
smaller
molecular
has to be concentrated
solution
time
of experimental
of the dialysis
contamination
of
in order
time
from dialysable
the protein
This
step.
as a separate
volumes
needed to reduce
takes
proteins
and then
material
weight
of dialysis
procedure
five,
to
method is
separate
large
the use of
requires
also
presents
tubing
and the
honey solutions.
diluted
Gel filtration
The technique
considered
to'be
separation
of proteins
method
of
amounts
pure
of
quantities
but would
solutions
in
result
polysaccharides
Furthermore,
amounts
Small
errors
in
the
final
this
as protein
sephadex
gel
requires
small
different
a number of
of
of honey was a must and therefore,
are needed for
according
create
the
sephadex
calculations
This
under
bed.
Further,
the gel
size
liable
increased
quantities
would
recoveries
this
of
of protein
to irreversible
and losses
of
pressure
and presence
such as distortion
is
large
can be
filtration
to molecular
problems
milligram
filtration.
of gel
of
sized
Due to the presence
positions.
of proteins
approached
the
of
as well
ideally
containing
the compression
would
in
result
of using
technique
the technique
separates
method only
elution.
mixtures
as medium is
The advantage
desalting
enables
this
at different
by performing
overcome
and would
the disadvantages
the dilution
carbohydrates
protein
it
that
protein
eluting
molecules
of
is
on honey are that
filtration
sephadex
from carbohydrates.
However,
concentration.
using
technique
an efficient
dialysis
over
filtration
of gel
absorption
as the amount of
lead
to significant
(Washington,
1966).
257.
Ion-exchange
chromatography
of honey a large. mesh size
Due to the viscosity
to be advantageous
Thorough
washing
carbohydrates
acids
the
in the separation
binding
after
to be eluted
final
Therefore,
extract.
ion-exchange
would
be the poor absorption
sizes
of
charged
the protein
the
Moreover,
in
the elution
separating
This
carbohydrates.
Furthermore,
if
cross-linked
resin
the more rigid
Also
they
separation
would
was reported,
after
the polysaccharides
lead
to trailing
preclude
of resin
only
available
grgde
use for
by Schepartz
oxidase
ion-exchange
media
at all
with
respectively.
Therefore,
of
for
resin
these
high
losses
the
were binding
a loosely
loosely
than
amino acids.
cross-linked
techniques
resolution
that
column.
needed rather
of approximately
(1964)
result
and
used for
honey as done in
and Subers
enzymes such as glucose
is
molecules
would
to be efficient
routinely
applications
are weakly
or alkaline
throughout
due to
partly
the protein
just
x8% cross-link
are often
resin
acidic
is
by
difficulty
this
be it
of proteins
be present
proteins
of
stability
of x2 or x4% divinylbenzene
chromatographic
utilizing
factor
would
due to the restricted
resins
the
media whether
of proteins
because
and also
were
of proteins
for
reason
the ion-exchange
onto
molecules
molecules.
in
Another
only
would
isolation
total
the amino
of
proteins
amino acids
the
be difficult.
would
If
the
of-all
the elution
solution.
inevitably.
than
and peptides.
in the loss
enabling
7M ammonium hydroxide
found
chromatography-was
of amino acids
results
thus,.
and proteins
using
required
in
of ion-exchange
and the technique
required
would be
resin
400 mesh.
This
It
the present
study.
some of
the honey
irreversibly
or not
binding
such as DEAE- and CM-cellulose,
similar
to that
encountered
with
basic
258.
here.
amino acids would also result
Ultrafiltration
For the
for
selected
advantages
reason
normal
for
of a filter
membrane with
molecular
a specific
initial
used for
but
is
there
in
during
to four
2.
stirring
the fact
that
possessed
It
was also
calibration
it
enabled
the
size
so that
molecules
that
selection
above
the proteins
were similar
to those
of honey,
was observed
that
through
could
the pores
the
be due to:
the UM10 filter
to minimise
dilution
viscosity
resulting
filterable
soluble
in honey, even after
of
the
was much slower,
of all
inclusions
considerable
Assuming
the amicon cell
this
even after
The main
point.
the removal
for
solutions
it
can be retained.
was available
clarification,
membrane even
this
effect;
the honey solution
when compared
still
to water
or
solutions.
found
that
due to microbial
even though
procedure
the ultrafiltration
and the outlet
was that
of large
be blocking
dilute
technique
in
The reasons
though
at room temperature.
pore
The
of protein
solutions
a specific
was
seems to be reasonably
and the process
ultrafiltration
days for
the presence
could
the volumes
on this
no evidence
components.
1.
size
the honey solution
three
are that
industrial
Howevert
put of
this
choosing
in honey.
present
unchanged
protein
of ultrafiltration
the proteins
technique
this
of
for
quick
concentrating
approximately
remain
above. the technique
given
reasons
contamination
tubing
usually
were taken
precautions
at a temperature
of
the
occurred
inner
after
of
to perform
40C discolouration
walls
of
the amicon
the
third
day of
cells
the
259.
An odour similar
experiment.
buffer
to that of alcohol
were added to the concentrated
solutions
was evident
when
in the
solution
amicon cell.
The residue
whose average
limit
molecular
filter
the
of
if
especially
hygroscopic
concentrates
which
polysaccharides
was determined
concentration
protein
a week. after
within
Concentration
protein
(1951)
response
these
of
reasons
these
and this
errors
proteins
did
this
of
that
(1951)
the
method
(1976)
was taken
in a sample relative
labile
reagents,
at equal
searched
and was successful
up commercially
was extensively
many years
unstable
give
concentration
for
a different
in developing
as kits.
to a standard,
disadvantages
of different-composition
the standard
Bradford
for
method has several
this
of complicated
more importantly
than
due to traces
perhaps
by the Lowry et aZ.,
concentration
However,
albumin.
that
the protein
Measurements
used to measure
including
the
freeze-drying.
The method of Lowry et aZ.,
usually
some of
was becau. se of
It
important
to this
of moisture
precautions
may be present.
membrane
in a dessicator
to store
access
gummy and sticky
were usually
off
freeze-drying
After
was necessary
Even with
material.
cut
and shape are equally
size
to prevent
pentoxide
phosphorous
over
it
dalton
may have a molecular
are Involved.
polysaccharides
of those molecules
on how the filter
retained
and molecular
above the membrane,
residue
Depending
the material
all
above 10,000,
weight
was above the 10,000
size
membrane.
not
was calibrated
by the UM10 disc consists
retained
colour
and
a very
different
technique
a dye-protein
Typical
For
of-proteins.
kits
free
method
such as that
260.
by 'Pierce'
are one-step
for
responses
similar
method relies
free
as being
extremely
by interferring
was
and is
erratic
showed that
inconsistent
absorbance
with
On the basis
measurements.
unsuitable
be that
could
explanation
reacting
fushin.
The differences
for
responsible
the inconsistent
3) however,
(Figure
bands precluded
using
that
absorbance
or spores.
blue
for
being
of
one
could
be
of basic
manner to that
absorbance
sporopollinin
species
could
be
measurements.
very
it
by PAGE or SDS-PAGE gave protein
in
the variation
this
sample source
Furthermorev
(1986)
on replicates.
reagent
brilliant
different
of honey concentrates
Examination
detecting
method
Gel Electrophoresis
Polyacrylamide
bands
pollen
coomassie
between
exine
valves
as
be due to honey components
in amount and distribution
of pollen
surface
could
in a similar
exine
latter
the
the Pierce
wax,
residual
the acidic
the pollen
with
on the
for
The reason
such as gum, polysaccharides,
methods
the Lowry method was adopted
use on honey concentrates
for
unaffected
and reproducible
gave reasonable
of this
honey
concentrates.
on
use
regarded
months.
concentrates
the Lowry reagent
In contrast
is
reagent
concentrations,
for
stable
The
Coomassie Brilliant
of the Lowry and dye-binding
comparisons
atypically
This
to low protein
sensitive
and giving
colour
of the acidic
bound state.
to protein
to the honey protein
applied
a stable
even at low concentrations.
in absorbance
substances
Preliminary
giving
most proteins,
on a shift
G250 from
Blue
procedures,,
technique
chemically
recently
was possible
it
for
rather
the position
honey proteins
than
by pollen
was shown by Reeder,
to distinguish
Richie
of
the protein
as a means of
estimation.
and Guenther
honey based on geographical
261.
location
of
advanced
technology
collection
laser
focusing,
incorporated
scanner
computer.
Moreover,
nationally
or more likely
of
to form a data
data
accumulated
commonly used laboratory
aims of
the present
study
a crystal
of presenting
as pollens
clear
In the absence
appearance.
is
the precision
considered
of
present
filtration
in
This
the
of
existing
such as PAGE or investigate
techniques
removed by pressure
completely
it
Therefore,
is not
because
such equipment
improve
centres
a few honeys
of honey such as amino acids.
constituents
minor
at
based research
to only
bank.
to either
necessary
economically
may be available
at internationally
not justify
isoelectric
programme on a mainframe
technology
such technology
This
technology.
layer
ultra-thin
form-of
but, would
uneconomical
the art
of
and a chemometric
this
and the application
only
state
using
other
the
was one of
can be partially
or
the commercial
interest
product
of extended
of pollen
conventional
shelf-life
and good
identification
is
impossible.
of honey for
Heating
usually
present.
results
This
in
the
liquification
coagulation
the 5% and 10% SDS-PAGE gels
samples
fact
that
and, the
to Tables
extraction
commercial
band pattern
samples
10 and 11,
of the not
(fourteen)
suggests
was effecting
of
that
the nature
denaturation
heat
of
the heat
the normal
the proteins
observed
3.
treated
this
treatment
and heat
treated
see Figure
used in
of
commercial
band patterns
honey No. 261,
Chorley
the protein
and/or
in the protein
was evident
of English
or other
on
treated
Further,
the
sample No. 261
analysis,
processing
the honey proteins.
refer
method after
Furthermore,
262.
of heating
the effects
honey were observed when the China Buckwheat
honey sample Code No. 209 of necessity
in order
receiving
The prepared sample was applied
and wax.
The polyacrylamide
gels loaded with
showed a very high background of protein
of
honey applied.
the original
were infact
proteins
This
followed
precipitating
precipitates
concentrates
were examined by applying
suggests
reproducibility
were either
examined
the
5% and 10% SDS-PAGE gels
suggesting
heating
the honeys
that
The scanning
Tears
simple.
recognised.
obtained
in gels
the
stained
Gels scanned against
to Figures
to being
prior
5 and 6.
honey
of
the honey
at
some
bands obtained
in
on
the patterns
to some degree
rece ived
in
of
the laboratory
honey.
polyacrylamide
were usually
due to the protein
in
lack
had occurred
the protein
showed consistency
China buckwheat
of
This
examined were subjected
heating
and excessive
as was the. case with
refer
However,
the
PAGE, the protein
present
or proteolysis
of
the raw honey-and
accurately.
the proteins
coagulated
sample preparation.
of
stage
that
the coagulated
by dissolution
to straight
not be reproduced
could
sufficient-dilution
that
When both
electrophoresis.
to the
China Buckwheat
even after
suggests
during
bands obtained
upon
and debri
to remove glucose monohydrate crystals
such as bee fragments
SDS-PAGEgels.
to heating
was subjected
recorded
a clear
gels
as peaks and were easily
background,
bands were usually
was relatively
sharp
the peaks
and well
defined,
263.
Enzymes
For convenience
dealt
the
proteins
below.
with
is retained
was used to particularly
the
sort
in honey,
is,
that
diastase,
successful.
for
gels
1.
The laboratory
standard
have different
characteristic
in honey,
2.
although
by these
Even if
their
during
explain
and invertase
enzymes
was not
reasons:
from micro-organisms
found
to those
same substratp.
at
although
limit
the volume
handled
enzyme or enzyme aqitvity
by the technique
in
have leached
out
employed.
the amount of honey
from
their
position
incubation.
why the presence
its
and
enzyme
oxidase
the
enzymes were present
the enzymes could
on the gels
could
presence
sufficient
applied,
This
(100 pl)
with
laboratory
compared
properties
catalyse
gels
the following
the following
may not have sufficient
gels
indicate
to
for
Their
of honey enzymes on PAGE
enzymes obtained
they
The raw honey applied
oxidase
technique
compounds.
with
of
existence
detection
have been successful
may not
latter
the completed
studies
pilot
glucose
looked
The sort
This
alone.
by incubating
the definite
enzymes were successful,
in SDS-PAGE b; It
are denatured
enzymes and other
Although
substrate.
enzymes are
powers namely
on PAGE gels
separate
was subsequently
appropriate
catalytic
Enzymes and proteins
enzyme activity
existence
with
activity
of
the
were not
laboratory
standard
detected
glucose
on the appropriate
gels.
Furthermore,
after
the U. K. survey
the completion
samples were not
of amino acid
and pollen
was scheduled
from
to PAGE
subjected
for
analyses
the
following
reasons:
1.
The U. K. survey
1982 but
very
even with
erratically
pre-paid
mailing
and a substantial
October
service
1981 through
the
samples
number were still
to October
in
came
coming
in
264.
even as late
2.
It
as 1984.
to attempt
was not possible
between variables
correlation
the samples had been analysed and the final
all
involved
of variables
major variables
for
considered
measurements for
thirteen
the qualitative
or confirmed
of under-
presence
addition
of sugar
sources as suggested by the beekeepers,
floral
type including
pollen
analysis,
the dangers
as given by Sawyer (1975),
or over-representation
information.
and other
or otherwise
commercial processing
concentration
of honey or otherwise,
heating
of sugar to honeybees or otherwise,
by pollen
The
individual
were the quantitative
amino acids,
compounds such as syrups,
number
had been determined.
correlation
or absence of seventeen amino acids,
feeding
finite
until
This
could not be handled by a simple micro-computer
range of factors
a Prime mainframe computer
such as an Apple or BBC1, therefore,
had to be used.
3.
in each sub group
numbers
small
package
had to be made initially
further
modifications
of
weighting
4.
due to the large number of factors
Furthermore,
to the
that
The modifications
treatment
dealt
be
not
could
that
of
was being
the
used.
statistician
orientated
with
statistics.
to
statistician,
and subsequently
within
Therefore,
of
data
the
to prevent
under-
involved.
became necessary
the programme had been running
SPSS
modifications
by the
sub groups
small
special
and consequent
both
for
some time
the
simple
initially
and after
were-such
that
operating
I had to await
who was experienced
in
they
programme
the availability
the aspects
of computer
265.
N*i'nhydrin
Covered
was because
It
in the
of
The sequence and order
out.
protonation
to the resin,
evaporation
and then
removal
under
ion-exchange
the amino acids
the amino acids,
of
vacuum,
under
by paper
amino acid
analyser,
of honey necissitated
The feasibility
the isolation
for
resin
re-constitution
electrophoresis
pollen
analysis
the use of a large
of ninhydrin
of column chromatography
of
the large
mesh resin
and fifty
six
in the order
excessive
dilution
necessary
to be able
large
samples.
by evaporating
under
chromatography
technique
the
tubing,
to be too
nylon
slow
in view
ten times
was found
batchwise
of
flow
to be prone
two
was used
This
rate.
to be
also
to be concentrated
would be required
Furthermore,
of
the
or more was found
a reasonable
mesh filter
substances.
The procedure
of
pressure.
mesh cation
stirred
column chromatography
of eluents
reduced
or continually
If
to establish
volumes
Positive
was considered.
was assessed
chromatography
of
elution
were analysed
automatic
The viscosity
clogging
were:
Chromatography
Ion-exchange
meant that
procedure
analysis.
and computational
hundred
be carried
of
desiccation
pressure,
Extracts
chromatography,
column
binding
resin,
of carbohydrates,
reduced
storage.
operation
the ion-exchange
of
analysis
could
and quantitation
ion-exchange
the
of
so that
amino
given
procedures
were necessary
stages
explained
the free
with
the following
that
identification
such as detection,
plus
this
of
and order
sequence
work was concerned
including
amines
For reasons
polypeptides.
the present
most of
previously
Substances
are the -amino acids,
section
amides and small
amino sugars,
acids.
this
under
Positive
the column
to leakages
and the resin
and especially
column
itself
266.
by large
particulates
inclusions
time
normally
hand the
dilution
sufficient
positive
ninhydrin
The ion-exchange
determination.
The final
reproducibility
using
known amounts
of
fructose
lysine,
to ensure
amino acids
and phenylalanine;
and these
so as to represent
the concentration
The loss
honey.
the
isolation
binding
the resin,
carbohydrates
resin,
re-constitution,
paper
and other
storage
electrophoresis
automatic
components,
and evaporation
collection
amino acid
may occur
sugars
at
- glucose
and chromatography
glutamic
leucine,
in a typical
found
samples,
stages
operational
and operational
of
transfer
washing
pressure,
tyrosine
standardised
of amino acids
extract,
and
The thirteen
the following
reduced
Further,
media.
threonine,
to the resin,
and
contained
honey samples.
elution
of prepared
analyser.
These solutions.
of honey
under
and
the precision
of amino acids
the amino acids
of
by Siddiqi
were deliberately
preparation
procedure:
devised
isoleucine,
solutions
of amino acids
accurate
serine,
valine,
obtained
enabled
for
plus
acid,
required
those
of
of
optimum quantitative
for
standard
aspartic
and
binding
in the appropriate
amino acids
aianine,
glycine,
technique
A2 and Sl.
a suitable
were:
proline,
acid,
thirteen
to provide
amino acids
out.
method was assessed
B and BI contained
solutions
to be carried
solutions
On the other
the volume of eluents
also
chromatography
to be a
controlled
effective
turn,
and improved
was modified
enabled
than one-fifth
is
This
determinations
quantitative
(1981)
or less
were about
chromatography.
column
Thus,
and other
was considered
to enable
stirring
dust
soot,
of components.
extraction
substances.
evaporation
for
with
This
of loss
risk
with
of batchwise
technique
spores,
in honey.
present
process
consuming
for
such as pollen,
to
to remove
from
the
desiccation,
loss
loss
for
for
the
267.
Due to the multiple
of
carbohydrates,
it
covered
each stage
which
and total
recovery
These aspects
13 to 29 show, with
were satisfactory
mentioned
Once these
recovery
experiments
These recovery
a few exceptions,
recoveries
be reviewed
that
separately.
were prepared
it
by weighing,
had occurred
was essential
and concentrations
For this
acid analysis
which had been made ready and pre-calibrated
reason these were submitted
as described.
and Stein
due to dampness and incorrect
(1951).
Furthermore,
in the various
The standards
examined and were controls
Due to the varying
of
samples
operated.
of
step
within
the guidelines
the work load
periodic
of Moore
for
was satisfactory
in honeys.
individually
checks
recovery
related
schedule
were carried
out
to the numbers
could
on all
not
be
aspects
procedure.
was found
extract
were not
extracts
only
of amino acids,
present
of
nature
However,
was not
commercial
each stage.
per week a rigid
The final
these
with
media and stages were likewise
for
amino
weighing were not presentl
the autoanalyser
received
the final
to automatic
of the amino acids expected
the range and concentration
were
A was shown to be satisfactory
This solution
of 101.3% were obtained
and recoveries
98.8%.
of amino acids
standards
and gross errors
the
than
greater
as expected.
standards
effects
adverse
as possible.
with
above will
that no gross errors
to verify
as far
separately
determination
Quantitative
and possible
to formulate
was essential
Tables
experiments,
involved
stages
furthered
unnecessary
other
ninhydrin
in such extracts,
to be free
with
of protein
to a deproteinisation
but
had the danger
positive
step.
of carrying
substances
the precipitate.
and therefore,
and other
This
down traces
components
268.
Preparation,
application
from 'the resin
and'elution
and concentration
stages
The individual
the net
from
recoveries
the acceptable
for
recoveries
The problem
range.
amino acids
especially
Stein (1951)
was also
As a formal
in the present
found
statement
the variable
of
(±11%)
was
also
lysine
the
of
for
are given
figure
the
with
of
histidine
Choice
(Stage
of Desiccants
little
Normally
adverse
experiences
context
the choice
is
thought
Siddiqi
Although
sulphuric
several
concentrate,
sodium hydroxide
in
conditions
foreign
the presence
origin
precluded
predominantly
arginine,
cysteine,
study.
traces
of moisture.
dehydrating
of
However,
have indicated
study
that
importance.
the use of concentrated
for
hydroxide
because
honey amino acid
were examined
preliminary
concentrate
of concentrated
accidental
Mexican
pentoxide,
for
suitability.
experiments
became discoloured
sulphuric
spillage.
it
certain
in this
that
compounds such as phosphorous
was undertaken
which
to the choice
was suitable
the honey amino acid
a blackmass
contrasted
in the present
given
reported
and potassium
evaluation
shown that
(1981)
other
97, and these
solutions
data
relevant
is of more than normal
desiccant
as a desiccant
acid
for
(1981).
included
in the present
of
found
5)
used to remove the final
agents
This
were not
and methionine
by Moore and
respectively,
the amino acids
elsewhere
of basic
recoveries
recoveries
in Table
94.6% found by Siddiqi
discussed
For reasons
were within
as noted
individual
98.9% and 98.7%,
analysis
and
study.
A2, B and Bl were 98.4%,
quintiplate
four
to stage
one through
stage
were negligible
each stages
acid
had
into
and under
In a few samples
was noticed
that
these
of
were
269.
TABLE 97
Average
recovery
of
analysis
of
thirteen
amino acid
synthetic
after
quintiplate
solution
Amino acid
(10 mls) or
solutions
honey sample (10g)
No.
amino acids
Addition
of Sl*
(MIS)
2
AAAA
IxC
Percentage
recovery#
M
1
Solution
A
+
101.3
2
Solution
Al
+
98.8
3
Solution
A2
+
+
98.4
4
Solution
B
+
+
98.9
5
Solution
Bl
o. 6
+
+
98.7
6
U. K. survey sample
Code No. 174
o. 6
+
+
99.0
China Light Amber
Code No. 211
o. 6
+
+
99.5
Mexico Yucatan
Code No. 227
0.6
+
+
98.4
7
8
For notes
to key *,
The average
recovery
of
the
**,
1 and 2 refer
percentage
percentage
values
values
quoted
obtained
13 amino acids, of amino acid
The concentration
amino acid
solutions
measurements
are given
6.
to Table
are
after
those
the average
quintiplate
solutions
and percentage
in Tables
of
given
analysis
in Table
recovery
13 to 29.
for
6.
these
270.
in
stored
in the presence
vacuo
may be due to volatile
concentrated
sulphuric
as desiccant
under
This
could
acids
produced
the
dehydration
and other
(Partington,
on the
1961).
Due to the risk
factors
environmental
adverse
were taken
many precautions
for
unstoppered
when not
pressure
and the use of a thoroughly
prevent
sulphuric
to
the closed
dehydrating
This
days.
However,
it
vessels
became discoloured
could
be one aspect
was found
At
residue
all
the desiccation
concentrated
available
surfaces.
stage
in one area of
and other
atmosphere
from
after
precautions
access
that
was essential
the vessel
when
this
seven
above.
were encountered
was used in the present
to
of ammonia
of about
used in the pellet
reduced
trap
with
the phenomenon mentioned
it
were:
under
chromatograms,
a period
taking
and no vessels
was observed
preventing
paper
hydroxide
sodium hydroxide
pentoxide
by dust
desiccator
It
no such problems
or potassium
of choice
the end of
that
of
of polyphosphoric
evaporation
cleaned
storing
agent
hydroxide
sodium
a matter
used for
action
uncovered
parafilm,
was used for
acid
of reactions
phosphorous
Additional
when re-pressuring.
entering
concentrated
with
vessels
series
such contamination
in use.
the reaction
concentrates.
in the laboratory
to prevent
covering
dust
be used
no honey sample was left
example,
left
were
should
of pollution
present
and
pentoxide
complex
the excess
of
with
neither
of honey or the catalytic
surface
colour.
reasons
honey amino acid
for
sugars
original
For these
nor phosphorous
vacuum condition
unexpected
involve
place;
acid
their
interacting
present
pentoxide.
the whole
pentoxide
removed from
being
matter
by phosphorous
dehydrated
being
far
became purplish-red
concentrate
This
of phosphorous
with
form.
either
As
study.
to have the dry
and not
spread
over
271.
(Stage
Re-constitution
A bacterial
6)
resistant
media - propan-2-ol/HC1
and steps were taken to ensure that
Also,
and transferred.
the dry residue
was uniformly
wetted
there was no loss of small volumes used
that
It
elsewhere on the glass surface.
used in the extraction
was used
mixture
and transfer
to ensure all
was essential
glassware
grease free.
were completely
Storage (Stage 7)
The use of bacterio-stat
bacterial
to prevent
sufficient
technique
ninhydrin
concentration
the major
amongst
levels
discolouration
some of
found
220C and
0
28 C.
of ninhydrin
the usual
the residual
in the
Usually
spots
of
the amino acid
The faint
spots
which
were sometimes
background.
'The
paper
effects
maps becoming
temperature
conditions
resulted
difficult
to appear.
by presence
Even with
great
and the relevant
affected
the mild
slight
maps and the need
laboratories.
maps were adversely
complexes
pink
the amino acid
low
selectivity
of
the low concentration
when the laboratory
especially
of
the effects
in analytical
were re-examined.
in the backgrounds
and greater
were evident
extracts
of
of ammonia were compounded further
the effects
some amino acid
extracts
the detection
However,
the background
8)
and chromatography
substances
amino acids.
ammonia generally
care
sensitivity
positive
for
overnight
Sometimes
of
of
(Stage
in
checks
was noted.
electrophoresis
in
the final
ammonia
of
to wait
of paper
greater
enabled
of the extract
and Chromatography
The combination
Frequent visual
or mould growth.
deterioration
no
and
made
were
Paper Electrophoresis
of 00 to 40C was
media at temperature
often
pink
resulted
after
staining,
high,
were
between
due to low concentrations
to distinguish
electrophoresis
from
and chromatography
272.
0
150C and 20 C, for
between
or overlapping
occurring
but
first
acid
aspartic
this
so after
Therefore,
fifteen
to the
the paper
in
resulted
improved
'PI
the
0-alanine,
'xI
and
concentrations
such as pipecolic
concentrations
whilst
It
solvent
under
two thirds
staining
complexes
took
usually
acid
was present
resolutions,
and it
in large
staining
with
complex
the ninhydrin
the
in high
very
were
spots
laboratory
to
near
the
conditions
solvent
These conditions
Also
took up to two hours
for
Some such as glucosamine,
acid
visible
banksia
approximately
spots
respectively.
one to three
a day before
Whereas in the case of Australia
of
distance.
this
and hydroxypipecolic
took
intervals
temperature
hour
go undetected.
had migrated
front
to appear.
to become sufficiently
in the first
that
was observed
usually
characteristic
in the movements of
a- and y-aminobutyric
usually
acid
after
serine,
was present,
by its
the ninhydrin
cold
of
and impaired
impaired
the coloured
glutamine,
spots
only
temperatures
some of
front.
solvent
travelled
for
was variation
conditions,
top of
hours
three
but
would usually
at
and
complexes
of proline
maps were checked
the RF values
as there
calculated
front
presence
the first
Further,
temperature
cold
for
minutes
relative
the
its
the amino acid
reagent.
not
to that
at
acid
separated
recognised
similar
then
staining,
problems
hydroxyproline
If
was not
complex
complex
coloured
yellow
or
ninhydrin
always
of closely
was the case for
of ninhydrin
were not
be outlined.
could
presented
threonine-glutamic
The cluster
and asparagine
conditions
the recognition
This
methionine-valine,
boundaries
if
complexes
experience.
arginine-lysine.
sometimes
then
ninhydrin
temperature
Also,
comparison.
were overcome with
isoleucine-leucine,
their
at low laboratory
were repeated
experiments
and the
days depending
on their
to be outlined.
acid
the
two unidentified
if
spots
present
Others
in low
became visible.
sample No. 194, hydroxypipecolic
amounts and became visible
only
two hours
273.
This
staining.
after
threonine
pioline,
technique
of paper
identification
the
of
o-esters
amines,
CO
C",
in
the ninhydrin
this
the
study
identity
the
the
procured
were
compounds
kynurine,
these
for
reference
the unknown and their
peptide
by the fact
present
nature.
that
with
other
studies
Moreover,
precluded
estimated
this
low concentration
the trace
kynurine
standard
spots
unidentified
other
reference
compounds included
acid.
The relative
to those
of
from the paper
to confirm
visually
compared to the other
level
labelled
extraction
Hence, the spot was observed to develop slowly
faint
very
spots were
maps obtained
correlate
positive
positions
substance
the identification
these unknown ninhydrin
detected
that
and these
hydrolysis
Ip',
was thought
amounts of
did
not
compounds
low levels
in low concentrations
it
and 0-aminobutyric
total
and
subsequent
chromatograms
their
for
small
similar
ornithine
citrulline,
of
position
Similarly,.
and In'
positive
ninhydrin
IwI and also
spot
be confirmed.
not
could
of
(1981)9
were made
spots
the relative
When co-developed
be kynurine.
complex
on the amino acid
Siddiqi
the unidentified
for
identity
those
and with
of
IwI
could
as
substances
positive
Attempts
'a'
'G'j,
IV,
likely
compounds such as
and other
By comparing
maps.
the amino acid
on some of
of
I .ff 19, IwI,
ICI,
sample,
glutamine
is
It
extract.
the unknown ninhydrin
of
one week per
asparagine,
may be present.
and peptides
the identity
to determine
IxIq
and threonine,
serine
amino sugars
maps that
in the final
present
were also
and tryptophan
that
from amino acid
became evident
It
and
be examined.
more than one sample per week could
although
The entire
staining
about
required
spots
of ninhydrin
20.
and chromatography,
electrophoresis
of
spots
surrounding
as shown in Figure
acid
and glutamic
the
with
overlapped
spot
or otherwise
was further
compounded
substances were always
to be about 0.5 nMoles.
after
identified
staining
and the
compounds.
At
curve peaks of those unidentified
274.
ninhydrin
chart
autoanalyser
amino acid
was because
and diffusion.
also
Hence,
to judge.
difficult
of
were cut-out
The invisible
to the
subjected
(1981).
Siddiqi
these
technique
The most likely
electrophoresis
paper
Those compounds that
when examined
spots
under
not
could
be that
they
ultraviolet
be reproduced
Moreover,
in
the
the presence
on the paper
in
colour
technique
of
of pyridine.
after
and fluorescent
of these
manner to that
of
the*presence
chromatograms
The pattern
in a like
by
identity
as to the
by performing
light.
acetone.
no compounds were present
were yellow
reagent
with
as described
are peptides.
were separated
spots
the acetone, was
off
explanation
and chromatography
ninhydrin
with
complexing
that
compounds were detected
like
of peptide
indicated
closely
was often
complex
ninhydrin
evaporating
after
of
question
maps and eluted
amino acid
This
due to overlapping
in
of the spot
honey
the normal
successful.
spots
of mass spectroscopy
The result
residues.
the
of all
complexes
in overlapping
some of these
obtained
times
were not
resulted
the outlining
ninhydrin
unidentified
to twenty
only
streaking
the overloaded
compounds could
these
not
However,
residue
fifteen
on the
obtained
none were detected.
had been applied,
the overloading
spots
occurring
in
maps to which
but
these
some of
concentrates
amino acid
the baseline
and therefore
recording
to isolate
Attempts
from
would mearge into
substances
of
separated
the amino
acids.
Automatic
Amino Acid
From the present
Carter,
in honey,
positive
1983)
it
Analyser
9)
work and recent
on the world
past
literature
has been reported
substances
(Stage
that
reviews
on the
upto
have been identified
thirty
free
(Siddiqi,
amino acids
two different
to be present
1981 and
present
ninhydrin
in honey.
275.
However,
in
some of these
occurrence
could
system
auto-analyser
honey
the
technicon
became obvious
were not
methionine
peaks Just
tiny
about
half
2 nanomoles
lysine
was always
small
Owing to the nature
are
two columns
basic,
very
amino acids
large
on which
the order
acid,
into
The other
threonine,
proline,
for
is
are
are:
amino
that
sample.
final
there
dispensing
is
the
for
and also
the longer
for
separated
aspartic
alanine,
and
measurements.
and arginine
cartridge
glycine,
level,
in every
One cartridge
histidine
These amino acids
At this
the auto-analyser
cartridges
amino acids
gave
although
concentration
of
and
present
the amount of
of
it
manner,
some thirteen
present
account
for
amino acid.
the peak height
peak size;
the two columns.
and neutral
of elution.
serine,
filled
such as lysine,
ammonia peak.
the acid
into
the construction
and two resin
if
of
left
corresponding
histidine
cysteine,
This
always
the auto-analyser
of
solutions
amino acid
and not
on the
on the chart.
and chart
in
to
level,
a preliminary
the measurements
error.
was taken
Due consideration
concentrate
in
and
by the
as standards
sample and often
cm above the baseline
concentration
used in
in
arginine,
in every
present
of amino acid,
of reasonable
basis
25 nanomole
at the
amino acids
was grossly
peak width
acids
for
0.5
electrophoresis
from 7 cm to 15 cm depending
four
that
the remainder
number of amino-acids
used routinely
auto-analyser
2
CuJarat
Some of
on a quantitative
the honeys had been examined
When some of
in Indian
by paper
The remaining
employed.
peak height
to a chart
all
to the seventeen
approximated
calibrate
and not
the unidentified
example,
as'Ofound
semi-quantitatively
technique
chromatography
for
infrequent
are very
in low concentration.
and always
be detected
only
substances
one sample,
labelled
substance
225,
No.
sample.
positive
in only
perhaps
positive
ninhydrin
ninhydrin
and eluted
acid,
cysteine,
the
a
column
in
glutamic
methionines
276.
leucine,
isoleucine,
liquid
tyrosine
added to each cartridge
a factor
is,
that
loaded.
of three
distortion
gross
Hence,
scale.
for
amino acids
it
Also,
was normal
internal
standard
was found
internal
of
in
terms
pattern
does appear
is
the
short
in
use of other
pairs
and cysteic
appear
the
at
acid
the extremes
sets.
elution
it
that
This
is
of pump
minor
or
used and the
and is
for
the
to the
therefore
common amino
the aminoacid
the use of known
is
composition
standards
of
has the advantage
hydrolysate
the
are
calculated
This
and tryptophan
of
mixture
predicted
is
peak is
of correction
internal
this
and other
norleucine
protein
25 ill
example,
be either
not
it
study,
recordings.
the mid position
amino acid
of
for
case of honey where
of a typical
of defined
honeys
These would
type
an average
that
column
the chart
all
at approximately
However,
not
artificial
possible
for
equivalent
pattern.
could
for
the analysis.
of ninhydrin
the eluted
of area under
assumed to provide
acid
which
of
an
with
standard
variations;
of reaction
with
the present
chart
in
present
off
per cartridge.
solution
20 ul
applying
In the above study
controlled.
norleucine
it
the extent
irregularities
instrumental
that
and not normally
being
consideration
concentrate
of an internal
can be used to correct
it
diameter,
response
value
in
in acceptable
the requirements
general
has a constant
closely
25 V1 of the final
resulted
and
amount to compensate
Therefore,
and then
sample
resolution
to be up to 30 ul
of constant
norleucine
every
with
to mix the amino acid
procedure
60. pl,
of
amino acids
limit
upper
was considered
the cartridges
to each of
because
ammonia and major
norleucine
standard
gave decreased
the particular
by mixing
that
was used for
which
of 40.1il
amount of
limit
exceed an upper
that
due to instrumentation.
variations
In
times
due to high
the chart
other
cannot
sample loading
Since
The total
and phenylanine.
The
essential.
for
such as arginine
for
the other
pattern
which
column.
is
277.
desirable
but
internal
not
standards
was not
Considerations
the height
resulted
in
the amplification
significantly
The average
percentage
the moisture
that
used in
in
sealed
would
bvman factor
in
use,
to ensure
for
the normal
in
the
residual
achieve
and
extract.
of 10 pmoles
and therefore
are
in
the
which
the
final
residual
already
eluted'a
taken
from
extract
solids
in
supplied
explained,
amino acids
of
similar
be due to
by the
in
was not
continual
samples
was considerable.
the basic
also
One
be ensured.
departmental
and the down time
The conversion
ion-
not
practised
sufficient
the precautions
of
could
reason, could
procedure
separated
thirteen
honey solutions
The other
were never
the
before
some seventeen
artificial
column on which
and arginine
spite
of
operational
maximum efficiency
final
be
not
laboratory
dose samples
For reasons
the auto-analyser
as there
ammonia in
could
the amino acid
of
purified
firm.
sample.
The auto-analyser
histidine
but
as due to two factors.
A, B and_Sl
25 nanomoles
the use of
especially
however,
question
for
obtained
to the auto-analyser
specially
to prepare
to the real
in
This,
in a general
value
of solutions
containing
composition
technician
amplification
or noise
and the homogeneity
to the
be suitable
not
recovery
by the commercial
standards
baseline
can be explained
content
contrast
vials
study.
the peaks in
only
operation
A applied
the preparations
is
This
of not
paired
age.
chromatography
exchange
in the present
generalised
of solution
amino acids
with
to the use of electrical
due to its
reduced
to instrument
also
the
This
control
peaks on the baseline.
of small
the baseline.
additional
possible
were. given
to boost
also
This
possible.
to
lysine,
amino acids
very
eliminate
large
its
peak
presence
a 7M.ammonia solution
proved
ammonia always
to a
to be impossible
present
adversely
to
278.
not
affected
of histidine
detection
and not
concentrations
at about
present
For this
of
the
was far
in
also
was found
be measured
Another
reason
these
basic
their
R-groups.
Table
98 the degree
amino acid
Hence,
were
in perhaps
Histidine
to be present
with
9.31
of
for
situation
lysine
the histidine
the ammonia
the honey samples
and
for
of estimation
low concentration
the relatively
in
final
the
and extent
with
to effects
its
a pI R value
and finally
tend
extracts
this
the ammonia solution
(1981).
binding
of
the
levels
of each basic
from
be followed
pI R value
ion-exchange
concentration
to
in
given
and elution
will
of
be attributed
the R-group
of 10.77
to
could
the pI R values
which
histidine
to adhere
would
used by Siddiqi
The error
quantitatively.
amino acids
reason
side
in many of
can be seen from
As it
likely
arginine
of PI R value
for
amino acids
is
on either
was
rarely
the peak for
Furthermore
closely
level
one quarter
and the
charts
technique
to be ±11.0%.
was found
lysine
been mentioned.
seen on the chart
histidine
was comparable.
was
or less.
work by the
This
technique
of paper
arginine
methyl-histidine
the auto-analyser
peaks occurring
Lysine
could
that
ever
the present
by this
technique
removed from the ammonia peak as compared against
and arginine
peak.
and in
the amino
2 nanomoles
was hardly
the
also
in low
only
was estimated
of approximately
and methyl-histidine
has already
arginine
It
and chromatography.
electrophoresis
be identified
could
honey sample by the
arginine
(1981)
by Siddiqi
samples
to be present
and to some extent
low and was detected
always
of
the peak for
Histidine
recordings.
paper
in every
a concentration
reason
identified
were found
but
however,
Fortunately,
and chromatography..
electrophoresis
of lysine
of estimation
and arginine.
and histidine
arginine
acids
the accuracy
only
7.59.
resin
was raised
the resin.
by lysine
These basic
and it
over
was for
that
279.
TABLE 98
The pI
and
pI
values
I
R
of
Amino acids
a-Alanine
O-Alanine
the amino acids
pKa 1
(aCOOH)
pKa2
(aNH+
3
2.34
9.69
6.02
3.60
10.19
6.90
6.08
a-Aminobutyric
acid
2.55
9.60
y-Aminobutyric
acid
4.23
10.43
2.17
Arginine
pKa 3
(Rgroup)
PI*
PI**
7.33
9.06
12.48
5.62
10.77
Aspartic
acid
2.09
9.82
3.86
5.96
6.84
Glutamic
acid
2.19
9.67
4.25
5.93
6.96
Glutamine
2.17
9.13
-
5.65
-
Glycine
2.34
9.60
5.97
Histidine
1.82
9.17
6.00
5.50
Hydroxyproline
1.92
9.73
-
5.83
-
Isoleucine
2.36
9.68
6.02
-
Leucine
2.36
9.60
5.98
Lysine
2.18
8.95
9.67
9.31
Methionine
2.38
9.21
-
5.80
-
Phenylalanine
1.83
9.13
-
5.48
-
Proline
1.99
10.60
-
6.30
-
Serine
2.21
9.15
-
5.68
-
Threonine
2.63
10.43
6.53
Tyrosine
2.20
9.11
10.07
Valine
2.32
9.62
-
5.97
The pKa values
from Dawes (1972)
PI
were obtained
pKa 1+
2
pKa 2+
PI R2
pKa 2
pKa 3
5.67
5.66
and Dawson et aZ.,
7.59
9.59
-
(1969).
280.
On the
broad
curve
high
in
low concentration-amino
that
observed
in
observed
and leucine.
and isoleucine
were found
pairs
present
high
in very
The eluted
209
but
No.
sample
from
since
technique
of amino acid
peak height
the measured
was
examined.
in China buckwheat
only
in
was evident
electrophoresis
of paper
amino
proline
the samples
in low concentrations
presence
these
of
acid
all
was observed
cysteine
determination
The quantitative
effected
its
by the
in-nearly
peaks was
and alanine,
recovery
The imino
present
the chart
glycine
total
to be consistent.
peak for
chart
many samples
The average
concentrations
amino acids
amino acids'on
and threonine,
serine
of small
of elution
overlapping
eluting
closely
These were:
recordings.
acid
for
samples
for
was true
Some slight
concentrations.
was generally
were indicative
acids
the reverse
whereas
peaks,
it
of the auto-analyser
recordings
chart
and chromatography.
methionine
could
to be below
was observed
be
not
0.5
cm
in most of the samples.
the baseline
Pollens
The microscopical
by Louveaux,
Imellissopalynology'
evaluated
Maurizo
majority
using
the technique
and Vorwohl
of
(1970).
the isolated
and/or
osmosis.
classical
pollen
analysis
the more difficult
methods
expertise.
acetolysis
required
prior
Further,
for
pollen
Maurizo
pollen
were distorted
not
analyses
knowledge
the application
in peat
be applied.
of botany
of
this
the
authors
reason
Also,
carried
which
specialised
are designed
was
due to the process
due to
were not
termed
and Louveaux,
by these
quoted
was partly
could
(1970)
and Vorwohl
For reasons
It
in honey
present
by Sawyer (1981)
described
pollens
diffusion
of
of pollens
examination
at
methods
my
such as
type
some
these
since
was not within
to remove that
the
that
attempts
out
of
of
281.
from pollen
contamination
honeydew honeys,
and therefore
respect
(Moar,
1985).
If
fragile
pollen
grains
then
thus
of
large
the
the casual
essential
to use a simple
Louveaux,
Maurizo
but
similar
contents
that
isolated
of
The degree
pollen.
with
became apparent
stained
Loranthus,
EucaZyptus,
to
difficult
castanea.,
concerned
stained
were unavailable.
Castanea
and Lotus
features.
This
of TrifoZium,
with
The
species
the
genera
were
as those
pollen
family
Vicia
It
evident
perfect
was noticed
were
and MeZiZotus
CaUuna,
pollen
pollens
in a particular
pollen
that
troublesome
for
of
of
ease.
of pollen
since
to
no pollen
to contain
EpiZobiwn,
relative
were particularly
was also
fushin
and some of the unidentified
be distinguished
always
basic
of Myrtaceae
of Brassica,
those
at a
identification.
of almost
For example,
was
A
identification.
slides
and generally
grains
was done.
from different
pollens
of different
The identity
not
Pinus
Prunus/pyrus
honeys
microscope
early.
very
whereas
Tilia,
foreign
some
which
Pollen
stain.
lightly
stained
could
two or three
required
which
It
recommended by
of pollen
eventual
that
or
as to source.
work utilised
from
ranged
pollen
in view
to verify
and advocated
the purpose
and then
damage
Furthermore,
and hence this
in the present
this
further
method such as that
1981 for
characterise
to the already
sustain
by beekeepers
(1970)
acetolysis
in
had to be examined
that
reliable
to enhance recognition
stain
would
method was advertised
The method outlined
limited
of identification.
and Vorwohl
at Cardiff
sumner school
is
technique
these. grains
observations
simplified
which
cells
these. methods were applied
number of samples
otherwise
of algae
this
the possibility
reducing
Furthermore,
so obtained.
in the destruction
results
procedure
samples
from
flowers
of Aescuzus,
pollens
as these
of Echiwn
genus
have similar
genera
in the
282.
foreign
samples.
pollens
were quoted
in order
Therefore,
by their
genus,
to prevent
7 and Appendix
in Table
as given
the identified
confusion
VIII.
The classification
to that
sample was according
(1970).
Vorwohl
identified
of
by U. K. and EEC regulations
not
there
it
Such doubts
slide
content
pollen
limited
counting
for
obtained
the
the decimal
after
is
percent
likely
Maurizo
and
doubt
frequency
for
computer
were not
The accuracy
to be in error
for
samples
sent,
of the
content
factor.
time
pollen
grains
was experienced.
visually
estimated
high
report.
However,
since
percentage
adjusted
of the
for
values
any digit
frequency
expressed
class
2% to
of not more than
a range
with
was
one amino acid
because
out the average
classes
point.
amount of
pollen
count
was carried
required
to re-evaluate
regarding
or an adverse
is
counting
practice
normal
each
per sample as recommended
grains
and also
low pollen
were either
by Louveaux,
material
estimation
if
slide
in
of the small
sufficient
was considered
on a microscope
counted
where triplicate
and one pollen
Further,
only
only
was sometimes
extraction
of 1,200
was because
This
out.
carried
suggested
The counting
grains
pollen
4%. This
honeys
as they were sent
or port
from wild
true
only
was the
situation
of departure.
by their
Most of the commercial
colonies
of the major
floral
retrospective
somewhat complicated
and known only
semi-wild
and/or
knowledge
same but
guideline
species
was that
were indexed
The major
species
practical
work and most of the
but
this
thesis
could
given
work
had been collected
labourers
so that
the
not be obtained.
by Crane,
did
foreign
description
commercial
samples
by unskilled
for
not
Walker
appear
had been completed.
The
and Day (1984).
till
all
the
283.
This comment could be remedied if
and documentation
bibliography
Bee Research
International
is
this
a long
bank of floral
in
future.
the
desirable
of
listed
hive
have enabled
is
Hive
below
that
there
the study
available
at
having
sometime
Campus
period
it
should
be an acqiiisition
on the University
sited
for
reason
on University
investiation
the
such an ideal
towards
another
composition
of'Experimental
reasons
an experimental
would
1.
for
and full
of the present
onset
is moving
and it
had an efficient
Although
souces.
Association
sources
Procurement
At the
of floral
way from realisation
a data
every country
was thought
Such a hive
campus.
of:
The effects
of environmental
composition
and production
and seasonal
of honey over
on the
changes
a period
starting
from 1981 - 1984.
2.
Whether
the
secretions
on the overall
of honeybee have a significant
ninhydrin
substance
positive
effect
composition
of
honey.
3.
Whether
the ninhydrin
contribute
to the
substances
positive
final
ninhydrin
positive
of pollen
injested
substance
composition
of pollen
supplement
substance
and also
of. honey extracted.
4.
To what degree
contribute
pollen
5.
Feeding
and extent
to the
composition
a sugar
and determining
of honey thus
final
of
product
does the
ninhydrin
produced.
positive
honey.
extracted
containing
the ninhydrin
feeding
known amounts
positive
substance
of amino acids
composition
284.
To establish
by honeybee
v1sited
combs during
from nectar
Whether
the pollen
amino acid
composition
latter
work
of Carter
sources
in cells
and of
similar
to that
this
over
was not
contribute
carbohydrate
solution
a honey of known
and/or
of one to two years.
a period
because
liability
if
passersby
done because
over
as the urbanised
the previous
of
proved
The very
few possible
or
and the University
e in any legal
against
prudent
on
very
of bee access
The Head of Department,
liabl
a hive
questions-about
by lack
either
and collectively
were naturally
but
were stung.
impractical
for
sites
raised
neighbourhood
or open to vandalism.
be separately
suitable
of years,
a period
or students
were also rendered
authority
traps
(1983).
difficult
the
free
can be devisable
item
investigated
were
campus
could
season,
from pollen
33% fructose)
plus
This
However,
obtained
to an amino acid
(33% glucose
forage
deposited
and at the end of the
amino acids
sites
floral
of the
any,
by Adams, Smith and Townsend (1979).
reported
7.
if
the variation,
any future
action
and
adverse
circumstances.
Computation
Compilation
orderly
fashion
of
in
into
an SPSS data
the results
obtained
statistical
methods
overcome with
University
the data
of all
Execution
attention.
and Statistical
the help
Mathematics
of
the
Analysis
gathered
file
statistical
required
maticulous
extensive
to computer
of a statistician,
Department.
each honey sample
It
Dr.
care
in
an
and
and interpretation
analysis
demanded prior
in relation
for
specialised
programming.
Pemberton
was upon his
knowledge
This
of the
recommendations
was
285.
following
the
that
1.
the
removal
2.
the
transformation
of groups
the
less
containing
their
random selection
square
roots
of samples
for
were carried
out
and
These were:
analyses.
of the individual
into
measurements
3.
the present
to throughout
adhered
and improvements
modifications
than
ten
sample numbers;
amino acid
concentration
and natural
logrithms,
and
of misclassiciation
estimation
rate.
The accuracy
of the predicted
the
SPSS increased
is,
from
square
of
Modifcation
logrithm
transformation
enabled
and to stabilise
sample variation
data,
of the amino acid
terms
in
that
is,
that
than
rather
the minimisation
variation
from
obtained
increased,
per group
of the concentration
transformation
root
natural
as the number of samples
ten onwards.
results
classification
the effects
of
the amino acid
data,
itself.
From a biological
amino acid
event
due to the
data
of a continuous
These factors
all
view
complicated
due to the free
instinctive
visitations
of
several
use of
during
honey
of
up to the
acid
time
composition.
calculations
performed.
ranging
floral
One should
also
source
consider
and processing
of analysis
and the effects
was because
such as "student-t"
the
situation
of the
of
this
cells
a comb,
is more
bee and
The pattern
the effects
followed
over
of
a period
of manipulation
by storage
the analysis
reason
of
and the
foraging
is maintained
of
the unlikely
was available.
at her whim.
extraction
It
life
nature
source
to a particular
hours.
The real
floral
in
the combs from a hive
all
hives.
combination
the
several
source
of a comb, all
cell
the combs from a super,
of
even if
of one floral
supply
in -the
be variation
should
factors
following
a single
are:
there
point
that
or the "Hotterlings-T"
leading
on the amino
statistical
were not
286.
Floral
Source
Pollen
The initial
SPSS indicated
the
using
as that
63,
of Dr.
Pemberton
groups,
refer
five
the
success
of
refer
agreed
to contain
the re-definition
pollen
contain
inclusion
the
refer
and upon the
distance
between
the honeys.
influenced
Most of
indicated
in
with
for
that
these
which
the
they
which
SPSS
the
floral
the
evaluating
in
samples
to some extent
This
predictions
by two. factors:
the unifloral
90% and over,
given
and improvements
of
content
The accuracy
fifty
thenumber
as to the pollen
determined
of
of
of
by the SPSS.
a honey sample.
upon is
such an analysis,
28.
64,
to some extent
are accurate
such
and improvements
in
to Table
groups
data modification
such modifications
the classiciation
samples
be
relied
can
2.
groups,
rate
were predicted
1.
of
pollen
expertise
the modifictions
due to the non-uniformity
misclassified
of
statistical
64 and 65 and Figure
to Tables
pollen
predictions
amino acid
nine
of the minimum Mahalanobis
the maximisation
However,
the
between
The outcome of
The effects
above were utilized.
in
IV(b).
the need for
suggested
suggestions
resulted
the need for
in Appendix
given
to Table
source
at discriminating
attempt
pollen
subclass
group
to
and
or more unifloral
samples
per pollen
group.
The reason
pollen
they
for
were predicted
in
the similarity
product
in
of:
misclassified
to comprise
the amino acid
samples with
misclassified
similarity
the remaining
the amino acid
those
samples
of by the
concentration
of
the predicted
concentration
did
not
SPSS could
measurements
pollen
measurements
the
contain
be due to
of
group.
could
the
This
be the
287.
1.
honey gathered
and/or
from another
and nectar
It
was noted
from
analysis.
of
the beekeeper
question
so as to retain
should
complications
that
of merging
in Tables
2.
that
of
of
spring
early
fascination
is
frames
a good crop
or hives
representing
to questionnaire
and 52
have not
beekeepers
been
two added
could
analysis
to some extent
is Brassica,
and autumn harvests
as was the case with
such as Brassica
result
only
one
some of
near
available
on the
repens
SPSS package
the
three
and Castanea
which
These were:
Canada,
following
countries.
France
and the United
Kingdom.
The significance
because
observations
between
to be common to
to question
or
the hive.
may occur
in differentiating
Trifotium
or TiZia
in misleading
found
96 can be given
if
and
of TrifoZium
The use of discriminant
in Table
flowers
specific
In addition,
some crops
the honeybees
even if
pollens,
to
observations
31,34,37,40,43,46,49
practised
of
for
that
of pollen
be considered:.
CaUuna
was successful
technique
The replies
of participant
beekeepers,
the participant
when whole
confidentiality.
per year
collection
flights
the
all
the honest
many of
are examined.
the name and addresses
disclosed
that
honeybee
as to source
4 have been given
by the
to
received
in nearly
observations
suggests
of flights
many thousands
the replies
significantly
of an individual
be misleading
could
but
difference
one plant
*
that
study
from
pollen
species.
4 differed
the microscopical
This
foraging
plant
the present
question
questionnaire
samples
in
unifloral
and
hive
from a single
each of different
hives
sources,
multifloral
honeybees
2.
from several
of the results
there
were only
were
China*
obtained
two samples
288.
for
available
basis
this
of
Kanematsu
between
(1982)
et aZ.,
pollen
species.
(1981)
with
Further,
some success
combination
ratios
during
study
this
the interim
ratios
in view
However,
period.
of
by Siddiqi
utilized
1981-82
the same
contained
were also
report
of
to distinguish
but
ratios
in her analysis
instability
and the
acid
amino
in
the amino acid
those
ratios
countries
On the
countries.
agree with
who used amino acid
from different
samples
three
do not
the findings
result,
honey
samples
in each of the first
analysis
with
applied
sent
to the beekeepers
the large
number of honey
of the variance-covariance
matrix
their
use for
during
computation
precluded
survey
showed that
the amino acid
other
the
of
SPSS
predictions.
or honeydew
Nectar
A literature
honeydew
in
both
as compared to that
terms
obvious
honeydew or nectar
and furthermore,
ideal
from
state
the
refer
groups
of
these
other
seasonal
is not
yet
two limited
to Table
groups
89.
However,
numbered ten
the usefulness
honeydew and nectar
a wide
resulted
of
However,
in
chemical
honeys.
the nectar
statistical
due to the available
technique
of
its
possible
samples,..
consistently
honeys.
Such an
analysis
of honey
discrimination,
samples
can be utilized
in
it
of honey provenance
the successful
the SPSS classification
of the
range
in honeydew honeys
variations
possible.
indicate
could
of many specimens
the increase
that
that
provided
variations
and yearly
seasonal
exceed
origins
Therefore,
of amino acids.
the honey amino acids
that
this
of
to be higher
reported
was generally
of nectar
of number and concentration
from
composition
distinguishing
in both
these
indicative
as
between
is
289.
Source
Geographical
When discriminating
been ideal
if
analysed.
This
the
of which
for
the authenticity
verified
Nonetheless,
originated.
results
classification
and 96 indicate
88,92,95
basis
floral
the
of
of
be
to have
the predictive
samples
than
rather
in default
not
they were alleged
the samples
only
available.
could
the honeys were classified
that
origin
were not
by the SPSS as shown in Tables
obtained
for
available
samples
samples
the available
with
due to
However,
reference
reference
of
were available
above which
as to the country
certainty
with
these
of
honeys.
as to source
samples mentioned
statistically
verification
honey samples
These were used for
have
would
were-first
by statistical
information
reliable
official
Moreover,
samples
number of foreign
it
countries
the reference
samples with
one or two samples.
of verified
reference
then be followed
would
limited
relatively
analysis
verified
authentic
unknown or suspect
from different
honeys
83 to
on the
geographical
source.
The statistical
that
suggests
present
work
factors
mentioned
Moreover,
as suggested
for
genuine
predictions
the
useful
with
investigation
before,
a data
and indexing
searching
its
between
in this
undoubted
aspects
In general
system
the
assessment,
section.
the
of all
the
be effectively
many unifloral
so as to provide
giving
highly
a
accurate
potential.
of assessment
self
the
of a honey sample.
bank containing
modified
in
Thereaftert
thesis.
this
the origins
SPSS programme howsoever
The boundary
are dealt
in
to determine
and fulfilling
somewhat arbitary.
a need for
is
there
SPSS progranne
the
from such an SPSS programme could
Analyst
Public
by
a
used
from
obtained
above and elsewhere
classification
predicted
honeys
results
criticism
and evaluation
and likely
Whereas the
were
errors
evaluation
of
290.
the
of
results
present
Present
for
are reserved
Work.
Where necessary
following
the
'Evaluation
section,
literature
the relevant
was quoted.
EVALUATION OFTRESENT WORK
Proteins
At the
time
showed that
However,
is
which
of
the
of
layer
this
favoured
was not
survey
prepared
possible
by ultrafiltration.
(1951)
Lowry et aL.,
per
This
This
average
honey reported
being
of proteins
and then
the
concentration
of nitrogen
method.
of honey was obtained
value
out
honeys.
geographical
by using
highly
technology
such as ultra-thin
programme
to a
was applied
However,
region.
and a less
form
this
of
but more
expensive
on raw honey and protein
was followed
and Guenther
sophisticated
and chemometrics
scanner
source
because
possible
Reederg Richie
authors
techniques
Salford
survey
concentrates
by examination
using
gels.
ultrafiltration
loog
at
was carried
The concentration
after
this
with
aims but was not
only
and contained
SDS-PAGE on cylindrical
types
included
laser
Also,
computer.
restrictive
which
focusing,
isoelectric
technology
objective
technology
geographically
characterising
The American
conditions.
accomplished
on mainframe
been used for
one of the present
course
and expensive
literature
of honey protein
correlation
present
(1986)
had not
the proteins
the
investigation,
of honey protein
compares
by White
in
freeze-drying
was determined
by the
value
of 172 mg of protein
from nine
samples
of honey examined.
suitably
and Rudyj
that
with
of
(1978).
Dialysis
employed
to date
were compared by White
during
obtained
The average
two most common methods
components
the honey concentrate
(1978),
the period
169 mg per 190g of
and ultrafiltration
for
honey protein
to determine
of operation
of
the loss
these
two
291.
He reported
procedures.
by Bergner
10,000
of
between
daltons
(1967b)
loss
the
study
of
and Diemar
(1975a).
calibrated
pore
of
was not
Bagdanov
that
been of value
methods
of
time
during
the
(1981)
protein
of
per
and it
was found
Therefore,
study.
reported
by White
that
samples
was not
This
(1978)
have
available
later
chemical
on
analysis
that
months work
has been discussed
the
at
in
the
changed to an examination
the average
by Bagdanov
binding
would
evaluated
three
after
the emphasis
and Rudyi
It
the aim to utilise
of
defined.
However,
amino acids.
100g of honey reported
that
comparison
analysis
of two such diverse
the nine
using
because
the
free
size
protein-dye
in honey.
suitability
period
were not uniquely
the
the molecular
honey protein
of the present
the
This
source
previous
by Bergner
membrane with
to determine
concentration
determination
investigation.
section.
ultrafiltration
encountered
had used the Bradford/Bio-rad
to determine
proteins
40% - 60%
In the present
during
the use of a filter
time
to have assessed
the research
used by
between
components.
components
procedure
in honey.
known at the
of protein
the soluble
technique
to have losses
those
with
can be employed
size
to determine
technique
nitrogen
molecules
components
55% of
that
the dialysis
nitrogen
Therefore,
present
proteins
It
filterable
employed
by the ultrafiltration
was estimated
soluble
of nitrogen
calculated
However,
to be comparable
indicated
were
total
of
as a percentage
(1978)
(1934).
et aL.,
and Kushnir
loss
encountered
White
technique
membranes to retain
components were lost
nitrogen
used by Paine
filter
using
and above,
33% - 45%.
filterable
White
(1975a)
and Diemar
of
the ultrafiltration
that
(1981)
value
is
of 129 mg of proteins
rather
and to that
low. compared to
found
in
the present
292.
Band Patterns
Protein
band pattern
The protein
raw honey
had been applied
No. 261 (English
sample
fourteen
coagulation
on the prior
providence
them indicated
treatment
heating
that
presents
a barrier
but
to, the determination
the
the
also
in
that
Figure
3 could
of
for
such honey,
flavour,
can be presented
if
This
for
more likely
that
for
financial
This
of honey proteins
poster
session
Society,
I in
at
London
the'Fifth
(1986).
level
identification
Moreover,
4, as compared
Figure
of
to those
to uncharacteristic
with
then
this
and reputable
prcbable
events
be verified
would
implicate
substitute
in this
such
case
to soya. protein.
on PAGE gels
Meeting
the owner
or
advantages,
was sought,
of
such as National
not
were fed on a pollen
possibility
addition
and delicate
quality
at
could/could
the unusual bands weremot-due
These results
receiving
on the 10% SDS-PAGE gels
competition
possibility
honeybees
as soya bean protein.
before
samples
in honey.
of transleucent
the outcome was true
in malpractice
of
at an industrial
of clarification
so that
because
fourteen
and/or
be due to two reasons:
the purpose
shows.
treated
The lack
geographical
observed
samples
the remaining
samples.
of enzymes present
flavours
honey
for
sample No. 252 had been subjected
this
heating
2.
to source
honey show sample No. 252,
national
1.
these
or pasteurisation
only
commercial
of denaturation
these
of
band pattern
protein
unusual
observed
not
in
present
to which
on the heat
and also
the likelihood
the proteins
of
honey)
Chorley
fifteen
the
observed
consistency
indicated
samples
in
was consistent
The band pattern
examined.
on the SDS-PAGE gels
obtained
of
the
were presented
International
as a
Electrophoretic
293.
Other
unusual
associated
with
evaluated
briefly
Current
peak
with
resolved
by gradient
proteins
of
shows that
weight
a molecular
Also
weight.
have been
and more recently
which
of about
50,000
into
other
as one
migrate
was infact
two closely
associated
proteins
unusual
Salford'.
at
proteins
pore electrophoresis
similar
dependent
1987)
are the proteins
honey -and these
(1944)
by Pryce-Jones
on sephadex
being
in heather
thixotropy
(Hamilton,
work
have been noticed
that
proteins
can occur
of the bee or honey to unusual
on the exposure
conditions.
Enzymes
Generally,
of
total
the
honeys
the proteins
1944)
characterised
as mentioned
and these
enzymes
and lengthy
quantities
active
unique
similar
enzymes from other
honey
and unusual
enzymes under
were:
diastase,
and Maherv
Rinaudo
Furtherv
1953;
et aZ.,
connection
of
and invertase.
these
been
low level
proteins
elaborate,
of
namely
maticulous
to ensure
adequate
active
the
in
of hydroxymethy1furfural
The
same substrate.
general
(White,
in Table
1966;
White,
and Echigo,
of honey.
would
4
1952; White
1975).
enzymes in honey would
processing
to
compared
1964 and 1966;
and Takenaka
due to the commercial
the presence
active
properties
described
and Suber,
and (b)
1973(a)
this
are necessary
catalysing
sources
oxidase
Schepartz
the presence
to be minimal
characteristic
investigation,
glucose
of
of
(1.5%)
protein
honey enzymes have been shown to
enzymes since
display
because
in honeydemandan
These procedures
procedures.
of
is
levels
even lower
detection
their
that
It
1%
than
Thixotropic
have infact
proteins
specific
above.
and consequently
et aZ. . 1962).
and manuka have additional
heather
(Pryce-iones,
less
honey constitute
(White
of honey
composition
predominantly
proteins
of normal
be expected
Hence,
be a crude
in
that
294.
of heating.
indicator
had not
could
were not
to, which
have been discussed
this
would
which
For this
source.
present
in
amounts or trace
small
the
results
heating
indicated
and other
Therefore,
that
geographical
centred
The range
of
a chemical
selected
proteins
to be of value
on amino acids
for
method
honey as to
of honey such as
of honey
constituents
for
this
were too
in
both
worthy
of
purpose
and
susceptible
the present
floral
to
context,
and
determinations.
Ninhydrin
amino acids
for
The reasons
amounts were considered
the native
pre-treatments
interest
However,
were initially
Proteins
investigation.
was
section.
components
investigated.
were not
carbohydrates
Such activity
even a pollen-free
the major
reason,
on PAGE gels
oxidase,
work was to develop
characterise
adequately
standard
As expected
properties.
glucose
in the previous
Suitable
had to be made using
honey was applied.
only
The main aim of the present
honey Code No. 261
honey enzymes because
characteristic
for
by the
enzyme activity.
enzymes were applied.
to which
on gels
of
comparison
except
laboratory
Chorley.
commercial
Hence,
was observed,
standard
observed
not
not be made with
available.
enzyme activity
loss
so as to minimise
enzymes of different
laboratory
honey was obtained
a special
whose sample - English
been heated
comparisons
these
Croft
of Dr.
courtsey
Therefore,
of
Positive
compounds within
could
Substances
this
not be examined
in very low and erratic
amounts.
overall
quantitatively
title
with
the exception
and were present
295.
Ion-exchange
size
Chromatography
The viscous
nature
resin
after
which
from
regeneration
for
but
respectable
(1981)
for
the thirteen
could
study
Her not
particles
mesh
followed
by
to be suitable
The technique
by Bosi
in
when regulating
to be slow compared
employed
in
loss
for
in
of
to the Dowex
and in
excess
ammonia.
the
air
to facilitate
was found
and was also
this
to sample
prone
was found
procedure
low pressure
and which
to be liable
evaporation
was not
method
to
subject
or. contaimination.
(1981)
did
the effects
on the amino acid
effects
not
of honey namely
she assessed
the
of
Moreover,
analysis
the
assessing
elution
(1978)
procedure
to the enclosed
of honey constituents
adverse
this
flow.
air
predominant
effects
the removal
of precautions
sugars
However,
binding
after
and pressurized
spite
the present
Siddiqi
Further,
effects
for
solution
and Battaglini
However,
evaporation.
sample
hydroxide
substances
employed
contamination
loss
(98.6%) obtained
amino acid mixture
used a steam bath
Siddiqi
to
as
mixture
investigation.
present
rapid
amino acid
by
reported
be due to:
positive
as described
(.94.6%)
values
synthetic
7M a=onium
using
ninhydrin
resin
recovery
the same thirteen
present
the
2.
resin
+
Na form to the H+ form was found
The lower
for
compared
1.
of fine
removal
a large
use on honey.
Siddiqi
the
its
of honey demanded the need for
of
assess
on addition
of glucose
the predominant
of
and fructose,
glucose
analyser
the effects
the
of amino acids
(20% w/v)
results'.
sugars
of
and also
to honey.
no
ashaving
Moreover,
in
of honey and honey
296.
on amino acids it
constituents
components of honey slightly
from
Electrophoresis
The method
staining
of paper
electrophoresis
and chromatography
was used for
the qualitative
determination
However,
instrument.
was not
technique
detected
substances
the
of paper
method
of both
origins
the
(1981)
paper
was able
acids
honeys
examined.
ninhydrin
positive
procured
previous
variation
section.
and that
previous
sect. ion.
identified
and/or
and pollen
could
standards
not
for
unidentified
positive
to assess
analysis
honey
three
samples.
-Siddiqi
ninhydrin
separated
from
the
the presence
of
0-aminobutyric
The presence
of
these
this
given
in
the
was due to biological
identical
positive
four
by co-developing
already
can be no guarantee
ninhydrin
Therefore,
was used as
be confirmed
the reasons for
there
(1981).
was considered
technique
forty
reasons
samples were not
Hence,
pollen
the
and ornithine.
substances
the
it
thirty
of
the
of
by Siddiqi
containing
she identified
However,
estimation
and chromatography
as compared against
reference
and chromatography
under-estimation.
with
the presence
kynurine
citrulline,
acid,
pollen-free
by the automatic
and chromatography
in conjunction
Moreover,
detected
quantitatively
the ninhydrin
and/or
over-
electrophoresis
to detect
not
section
of the amount of
to both
those
as evaluated
the previous
followed
of a wide
only
semi-quantitative.
electrophoresis
substances
positive
in
to and/or
an alternative
others
substances
determination
lead
not
electrophoresis
for
given
could
but
the paper
positive
already
visual
Using
substances
utilised
ninhydrin
For reasons
that
positive
by the auto-analyser
determined
with
of the-amino
and Chromatography
of ninhydrin
range
the
enhanced the recovery
these
the Dowex resin.
Paper
by
Table 97, that
was observed,
in
as discussed
that
the
these
substances
would
be
297.
in every
present
samplq and was in fact
in
particularly
individual
Acid
AutomAtic'Amino
presence
of
co-elute
with
amides,
particularly
threonine
these
out
the
of
it
columns
histidine,
proline,
leucine,
and those
glycine,
tyrosine
quoted
determined
were
and phenylanine,
in the previous
for
only
thirteen
(1975)
to the columns
out because
carried
system
dust*and
pollen,
could
block
the following
similar
the
analyser
threonine,
valine,
the order
used
seventeen
serine,
methionine,
given.
quantitative
amino acids
to
was able
These amino acids
acid,
section
carried
and seven unknowns.
to detect
in
observed
but
The amino acid
cysteine,
glutamine,
to some extent
Davies
yeast,
programmed cycle.
alanine,
was
the
clarification
aspartic
could
the auto-analyser
operating
the
although
honey was not
was calibrated
arginine,
was not
Davies
two amino acids
the analyser.
on a two hour
amino acids
acid,
of
This
work,
calibration,
even after
that
Peptides,
honey solutions
such as spores,
study
the present
lysine,
twenty
present
and tubings
in this
overlapped
After
by the technician
inclusions
large
often
of diluted
honey constituents
that
in
of
This
one peak.
types
the asparagine,
distinguished.
analyser.
application
was feared
in that,
the diluted
of
the presence
direct
This
(1975),
elutions
application
amino acid
detect
amino acids.
be clearly
peaks could
direct
thirteen
system employed
and serine
is possible
and small
into
eluted
it
substances
different
amino sugars
by Davies
and serine
threonine
positive
the other
any of
the auto-analyser
with
such as honey containing
amines,
noted
rarely,
samples.
fluids
of ninhydrin
and amounts
very
Ahalyser
biological
With
occurring
with
were:
glutamic
isoleucine,
For this
reason
measurements
the exclusion
of
298.
histidine,
arginine,
cysteine
and methionine.
P6110-ns
The diversity
in
British
the
of
variety
of the floral
for
Isle
different
The major
and rare
agreement
with
floral
and those
examined.
The presence
from
the honeys were stated
which
three
commercial
(248)
and English
as of
English
(254).
clover
of
in honeys
not
carried
out
of
stabilisation
of
this
study
dividing
content
and then multiplying
square
root
the
transformation
the variance-covariance
in
was evident
agree
English
pure
those
with
had been imported
from
it
matrix
measurements
concentration
and Davies
because
individual
was the
the
(1981)
did
the variance-covariance
of
samples
Analysis
the amino acid
et al.,
in
the
the world.
The transformation
by Gilbert
TrifoZiwn,
samples were described
content
that
to
common to the country
to have originated
pollen
Statistical
utilised
are not
These three
and their
origin
countries
which
by Crane,
refer
in most of
was evident
samples.
in good
are
such as Brassica,
(Code
MR68
No. 243),
samples namely
by Sawyer (1975)
identified
other
of pollen
study
by Sawyer (1981),
of pollen
and MeZiZotus
this
of honey as listed
given
the
in the U. K. survey
in
sources
The occurrence
Cirsiwn
EucaZyptus,
identified
identified
types
pollen
the major
VIII.
Appendix
genera
by the honeybees
utilized
of honey can be seen from
the production
pollen
(1984),
Day
and
Walker
sources
not
matrix.
amino acid
in the
result
This
in
measurement
and coupled
the present
with
pollen
was
successful
transformation
by the number one thousand.
adopted
(1982)
and Harris
by the
total
Howevero
analyses
analysis
stabalised
enabled
299.
the
correct
of honey samples without
classification
by Gilbert
as reported
outliners
1.
the
redution
in the number of
2.
the
defining
of unifloral
3-
the
random selection
These modifications
Kanematsu
over
advantage
boundaries
round
out
that
few
experimental
and
all
since
small
could
the
not
SPSS packageg
used large
variables
sub-groups
analysis.
with
study.
It
should
of unconfirmed
floral
lead
be
sources
In the present
study
independently
was
to a multiplicity
of
data,
To overcome
consequential
on
defined
so as to produce
randomness
work.
the amino
such a way that
two dimensions.
These factors
Beekeepers
in this
and each sub-group
and a consequent
modified
in
were considered.
be drawn in the present
SPSS was heavily
in
groupings
were considered
by pollen
verified
of
factors
used data
The use
to have any statistical
analysis
statistical
to
group
used by the Japanese
Therefore,
transformation.
for
sample groups.
the SPSS.
those
found
were not
the minimum
in the British
as described
sample groupings
they
of
capability
and co-workers
standardised
a substantially
pointed
adopted
Gilbert
Furtýhermore,
(1982)
the square root
were not
ratios
acid
et aL.,
classification
samples per
1981 - 1982 and also
report
of
between
number of
was attempted
interim
Association
predictive
rate.
discrimination
the predictive
ratios
acid
is,
for
group
the maximisation
and inconsistent
limited
some extent
authors
overall
and
subclass,
samples per
improved
that
limited
the
pollen
of
vastly
distance,
Mahalanobis
of
Other
sample groups,
of misclassification
and estimation
amino
(1981).
et aZ.,
of
such as:
modifications
However,
the ommission
so that
the lack
improvements
boundaries
of boundaries
in
300.
discrimination
but still
to draw the 95% confidence
not significantly
boundary.
Nonetheless,
the predictive
and misclassification
classification
the honeys in this
computed by the SPSS programme suggests that
investigation
than geographical
rather
those
by pollen
donors.
their
but relied
analysis,
is a well
It
deliberately
can often
the containers;
explain
upon the description
between
distinguish
and also
findings
were in conformity
(1982).
Further,
the
English
English
the
of honey
amino acids
source.
However,
foreign
to obtain
honeys
lack
verified
honey,
samples
knowledge
these
findings.
research
These
between
especially
the composition
that
extent
88.
94,
and Harris
by Davies
composition
to a large
from
to Table
refer
reported
of background
samples
for
to Table
refer
seems to indicate
seem to disprove
that
matrix
SPSS programme.
and foreign,
of-pollen
determined
as outliners.
SPSS programme was also able to
those
with
similarity
is
the
honey would
official
by their
and European
and European
could
by these authors
classified
the U. K. survey
between
the labels
This in turn
of variance-covariance
the modified
study,
of
source of the honey in
pollen.
and Canadian origins,
In the present
samples
given to them by
et aZ. , (1981) were omitted
the U. K. samples were incorrectly
of Argentinian
of their
Penine Heather honey sample No.
75% Brassica
the instability
Also coupled with
could be
this
sales appeal and therefore,
why some-data of Gilbert
with
commercial labelling
that
example, English
251 was found to contain
of sample
do not confirm
origins
mislead as to the true
for
origin
The reason for
the floral
known fact
has an eye for
honey containers
(1981).
et aZ.,
did not verify
et aZ.,
of floral
These findings
sources.
by Gilbert
reported
Gilbert
that
on the basis
were classified
by its
related
It
stations
floral
to each
was not
possible
of each
301.
country
to be attempted
programne
Nevertheless,
the
that
of
time
nor was sufficient
for
of Brassica
success
in
Further,
of the lack
SPSS classifications
this
application
indicates
by the number
were governed
for
example,
the
sample
size.
the
sample numbers per
group
the potential
use and
indication
of
to various
technique
chemical
such a
thesis
was due to the
of adequate
gave every
this
analysis,
a particular
honey classification
spite
of
in
work embodied
of the SPSS predictions
available
for
available
how desirable.
no matter
the present
credibility
samples
or resources
of honey
aspects
production.
is
It
method.
official
with
those
collaborating
with
such a chemical
with
research
Analyst's
technique
however,
is
as an
A great
and this
body of
in other
workers
then
would
to
necessary
two or more independent
investigation.
by various
Public
chemical
is
there
use of computers
can be done it
this
in this
utilised
this
of
relationship
be compiled
can then
of
Before
a reciprical
establish
the increased
with
the application
to validate
a need
that
suggested
methods
evidence
laboratories
justify
the use
technique.
ENVIRONMENTAL AND PROCESSING FACTORS AFFECTING HONEY
following
The
are:
Major
variations,
Isles,
British
will
now be considered
Sugar-feeding
Adulteration,
Seasonal
in
topics
honey
honeys
Toxic
of
honeybees.
sources
and
and Honeybee
individually
- Heating
agricultural
of
and
these
honey,
practices
species.
Adulteration
It
is known that
it
because
market
temptation
English
is considered
of adulteration
honeys commanda high price
as being unique.
via adulterants
in the honey
This has lead. to the
such as commercial invert
302.
and
glucose
honeys.
-to
used
.
1.
Various
detect
the presence
are:
Monitoring
increased
known that
The detection
of blending
Ratio
Doner
1978).
plants
which
the
product
from
in
However,
of pollen
In
from C3 plant
(Edwards
the British
removal
analysis
suspected
qualitative
the absence
of pollen
to have been similarly
amino acid
analysis
in
and
C3 and C4
Honey is
obtained
1983).
of pollen
not
invalidating
producers
the use
1969).
204
honey
No.
of
sample
no pollen
234,235
Both
This
was present.
to high
sample Nos.
processed.
White
by commercial
(James,
to have been subjected
Stable
by Sawyer (1975).
Thus,
analysis
that
1977;
HFCS is
the presence
filtration
of pollen.
revealed
rise
syrup
namely
pathways.
has been reported
pressure
corn
1983 and Prince,
can reveal
pollen
gives
between
whereas
and Walker,
on such honeys
study,
(Canada Samantha Clover)
Further,
can differentiate
secretions,
Isles
the use of high
sample was suspected
fructose
high
photosynthetic
analysis
is
It
also
and White
employ different
the present
conditions
and
1980a).
honeys with
technique
syrup
and Gomez, 1986).
(Doner
the total
enables
invert
technique
This
analysts
(HMF) and
commercial
and storage
foreign
The most commonly
the use of an expensive
The use of pollen
found
of
Mass Spectrometry
C4 plants
in honey.
(Serra
(White,
in honey
by interested
of hydroxymethylfurfural
heating
to HMF levels
(HFCS) requires
3.
levels
respectively
adverse
and cheap low grade
syrup
adulterants
due to the addition
syrups,
Isotope
corn
have been developed
of these
techniques
glucose
fructose
techniques
maltodextrin
2.
high
syrups,
pressure
filtration.
and 236 were
the quantitative
of sample No. 204 revealed
and
low levels
303.
amino
of
acids
by Drs.
Inquiries
honey
as compared against
Croft
by another.
and was purchased
blended
then
to
compared
do not
syrups
of
the available
foreign
other
honey.
adulterated
known to be reduced
are
Furthermore,
of
being
analysis,
spSS.
It
English
amino acid
was found
but
honey
All
these
action
court
the
termed
were later
by Public
commercial
Analyst
producers
foreign
The result
machinery
pollens
been processed.
resulted
involved.
in English
of unfortunate
after
confirmed
by Sawyer
as misrepresentation
these
English
samples
a batch
in
mixing
of honey
the
by the
estimated
Whereas,
found
not
sample
in Australian
be established.
and were sold
sample donors
the successful
as
and
prosecution
(1975).
The possible
could
has been
presence
be due to:
leftover
from a particular
of
to be one-half
normally
English
aid
by the
classification
form of fraudulence
with
added.
suspected
This
honeys
sugar
levels
with
Mexican.
pollen
as being
of
concentrations
honey added could
honeys were quoted
These findings
such.
of
four
four
investiga-
composition
and sample No. 244 was also
the amount of Australian
be
cheaply
constituents
honey was established
to contain
not
amount of adulterant
and three-quarters
253 and 254 were found
Nos.
could
amino acid
maps and predictive
Mexican,
English
to be one-quarter
sample 204 was
sample No. 243 was estimated
that
and one-half
of
foreign
with
amino acid
upon the
origins
quality
known from the present
the amino acids
depending
the true
adulterated
pollen
of
Howevert
particular
relatively
and sold
the overall
affect
significantly
origin.
low grade
its
whose identity
It. is
that
this
that
was suspected
resouces
(1975)
that
for
retailer
honeys.
of Davies
and that
tions
It
of Canadian
revealed
a sugar based syrup
with
with
established
and Washington
by a supermarket
was rejected
honeys
other
honey in
the
country
has
304.
The fact
2.
that
not
The delibrate
In
from
of honey containing
by pollen
analysis
comb was thought
from
Australia.
from
the
suspected
samples.
technique
could
and for
is
Also,
feeding
for
these
this
in
the jar
be confirmed
not
(1981)
authenticity
that
this
that
of
chemical
of blending.
this
in nature
'thin
honeys
could
not
runny'
Further,
could
(Croft,
syrup
this
is
normally
nectar
1986).
however,
source
Although
can lead
to other
question
honeys.
6
it
number
honey had been the product
it
was considered
of
as
were observed
received
compared
this
to
honey can be classed
be the result
be verified.
sugar
in available
honeys
to questionnaire
to honeybees.
runny'
with
weather
beekeepers
and runny'
most of
sugar
honey but
the degree
and the resultant
received
replies
'thin
The honey in
and that
could
the
deficiencies
of bad
periods
'thin
be unusually
feeding
92.
by Siddiqi
were noted
Some of the U. K. survey
adulterated.
noted
determining
of honeybees
for
to compensate
of overfeeding
that
sources
by the
also
to Table
suggested
indications
a recommended practice
From the
that
be used to determine
prolonged
temptations
of these
agree with
The artificial
carried
maps but
was confirmed
of Honeybees
Feeding
out
This
from Mexico
to have originated
can be used for
amino acids
Sugar
were different.
honey
that
was noted
producer.
These findings
honey
comb it
and amino acid
The identities
commercial
cut
advantage.
by the SPSS, refer
classification
predictive
the
in the jar
comb and that
only
not
for. financial
mislabelling
a sample
the
out can be successfully
from the filters.
removed
3.
filtered
the pollens
all
collecting
that
was
of
some of
unripened
to
305.
The reason
for
the limited
of feeding
were products
which
products
sugar
the
and 73.
feeding
of
effects
of honey-obtained
composition
3:t was known from
commercial
Davies
(1975)
effect
on the overall
amino
the sugar
fed samples
This
This
expected.
Bergner
and Rahn (1972)
far
largestýcompared
the
honeybee
and later
The general
is
to honeybees
not
guidelines
both
to which
practice
for
honey
practically
probatily
This
sugar with
closely
akin
is
proline
suggested
by Croft
would
but
lead
vastly
to sugar
(1986)
fed,
obtained
although
this
'honey
yield'
one to believe
syrup
but was
sugar
fed.
74, which
was not
that
or no intrinsic
in
composition.
of
an amount by
meZZifera
on feeding
However,
and recommended.
to quote
reduced
of
was
study
by the Apia
amino acids
(1981).
is
this
added to honey in
by Siddiqi
per hive
autumn feeding,
per hive.
in Table
to recommend a product
10 kg of sugar
in
was
was based on the findings
evaluation
to be encouraged
This
and type
extent
of
to the other
relative
examined
obtained
to the other
reported
worthy
considered
that
it
of concentration.
upon the degree,
on the results
was reflected
as entirely
terms
of
that
Further,
proline
in
to be dependent
noted
acid
74.
to Table
have a significant
not
of amino acids.
imino
of
to
of amino acids
should
be higher
in most of
evident
of sugar
of
of establishing
61, and from
Table
study,
proportion
the content
should
acids
this
types
refer
refer
determination
the feeding
that
that
expected
in
syrups
were
on the amino acid
from such colonies,
the quantitative
sugar
the purpose
to honeybees
syrup
sugar
that
and those
beekeepers,
hindered
This
honey samples
in amount and different
by the participating
utilized
70,71,72
Tables
discriminating
to honeybees
sugar
due to the variation
was mainly
not,
in
success
from
is
sugar
it
is
a colony
a reconmended
of 20 kg of
such honeys
value
In
this
at all
are
and
connection
306.
the
ready
blending
illegal
or addition
acid
amino
Heating
rise
indicated
as
from
was noted
U. K.
by
evident
that
the
processing
techniques
industrial
level.
that
of
heating
temperature
in
result
reputation
honey.
due to
of
be detected
honeys
of
measurements.
to
to
honeys
which
the
to
beekeepers
However,
it
a flavourless
of beekeepers
analysis
of sugar
by predictive
the inconsistency
sample numbers per group,
the
hours
but
that
was
to
as compared
to
the
general
nature
to
of
at
number
5
recommendations
treatment
X2 would
also
in
at
not
a
only
on the
reflect
country.
could
classification
of heating
refer
honey,
This
question
heat
that
some fifty
of samples
feeding
to
the
Further,
subjected
questionnaire
adhere
poor quality
in this
usually
5,
was apparent
was due
This
are
heated.
aniline
acid
be evaluated
not
heating.
with
amino
question
it
samples
minimal
was noted
(120 0 F) for
could
were
the
discriminant
questionnaire
formation
colour
effect
random
following
on its
some effect
examined
subject
by
However,
this
to
samples.
of
has
of
commercial
of 48.9C
were a product
the
The responses
most
Statistical
certainty
of
Analyst.
mass spectral
determined
usually
received
were
amount
with
obtained
suggest
expensive
honey
screening
honeys
survey
is
and extent
replies
.
qua litative
selective
the Public
the U. K. could
(HMF) content,
which
with
non-uniformity
the
honey
heating
that
The degree
composition.
it
heating
of
5-hydroxymethylfurfural
analysis
in
temptations
can offer
to confuse
of pollens
or by very
composition
extent
in
syrup
Honey
of
The
fructose
they become prevelant
if
Such practices
by
of high
availability
subjected
to both
not be evaluated
aspects
of
75
to
with
the SPSS.
and sugar-feeding
to Tables
heating
82.
and
any
This
and also
of
was
307.
Seasonal
Variations
Changes in seasonal climatic
to
can influence
year,
and their
pollen
be a function
by
honeybee.
the
from pollen
acids
amino
in
a hive.
It
from
honey
different
floral
flowering
extended
period
can be acheived
honey
is
(1958),
by Mittler
of honey is
during
required
but
negligible
may be present
that
unifloral
the deposition
seasonal
of honeydew and its
an
of unifloral
can be
changes
constituents
(1973)
that
changes may be
seasonal
before
Noda, Sogawa and, Saito
the
of
and Townsend (1979)
Similarly,
on a comb.
of honey
ingested
the contribution
the comb to become nearly
for
on the production
prevalent
in a jar
sources
on a comb and that
present
that
by Adams, Smith
was observed
has
It
of pollen
the amount of pollen
when considering
significant
out
pointed
present
1980).
the amino acid composition
of the amino acid composition
He also
year
on the production
reflects
1980c and Maurizo,
(1983) that
that matter
and to some extent
of nectar
This in turn
(White,
quality
by Carter
been reported
could
the availability
constituents.
of honey and its
and for
conditions
as reported
and Hertel
and Kunkel
(1977).
Discriminant
did
and 1982,
it
the
could
total
not
content
It
was also
which some of
of
composition,
whether
noted
the
statistical
from newly
established
analysis.
control
the calender
from
refer
seasonal
of
played
This
hives
of
seasonal
changes
the different
that
years
the
1981
changes
66 and 67.
to Tables
the available
the U. K. survey
the honeys were subjected
in
the possibility
or proportion
each honey sample of
regarding
outcome
confirm
be established
amino acid
in honey.
present
honey
to some extent
of honey harvested
the honey amino acid
influencing
Also,
analysis
influenced
amino acids
information
conditions
an important
part
to
in the
can be overcome by obtaining
that
have had no
honey or
308.
pollen
and has a fresh brood chamber.
stores
be placed
alongside
monitored
with
existing
to the solar
over a period
activity
for
constituents
of
This would
cycle.
of seasonal changes influencing
of honey but also. its
production
hives
annual changes of the control
the pattern
establish
should
and both these should be carefully
colonies
eleven years attributed
perhaps
hives
Such control
not only the
the major honey sources
worldwide.
Honey Sources
Major
Referring
out
and Agricultural
to the major
in
identified
the U. K. survey
honeys
changes
in
changes
have been shown in Table
both
identified
in
pollens
types
honeys
good agreement
catalogued
These
of pollens
as given
in
between
the
by Crane,
Honeys
The most likely
the
plant
and storage
In
and also
the United
petioZasis,
source
- nectar
AescuZus especially
toxic
list
the
foreign
honey sources
and the major
pollen
(1984).
Day
and
Walker
from
is
the U. K. carried
Isles.
the British
and the
Isles
have been a significant
Comparing
there
in
predominant
there
in
99.
the U. K. survey
identified
that
practices
can be seen that
it
VIII
Appendix
Toxic
the agricultural
the British
of honey sources
survey
from
be
the
seen
can
it
by Deans(1957)
in
Practice
TiZia
of
compounds in honey could
toxic
and honeydew or during
from
processing
the environment.
Kingdom honey from nectars
AescuZus hippocastanum
OrbicuZar
extraction,
originate
and TiZia
Nectar
honey from these species are reported
and TiZia
plant
or
such as Tilia
tomentosa are known to contain
have
been
reported
which
compounds
honeybees.
humans
and
consumers -
of Rhododendron
to be injurious
from TiZia
to be harmful
to both
and also the
to bees.
Palmer-Jones
309.
TABLE 99
Mal or honey
sources
in the United
Kingdom
Deans
Plant
Species
England
1
1952
Scotland
1
1952
Howells
Wales
2
1969
452
Brassica
1
4
CaUuna
4
7
Castanea
3
5
Ivosotis
prunualpyrus
2
2
3
Rubus
3
3
4
1
1
12
8
10
Trifolium
.
Mistry
U. K.
2
1987
repens
Vicia
Aescu4us
7**
6
-
-
importance
in survey
1-
Ranked as plant
2=
Ranked as percentage
of honey
samples
Ranked in position
with
AescuZus
Ranked in position
with
Impatiens
6*
recieved
plant
and Mematis
plants.
310.
(1947)
limes
from
to
the
strong
for
used
smoke
can also
of honeys.
high
Honey with
The use of chemicals
have
also
This
is
has
evident
especially
residues
to be bitter
1974).
tasting
1983).
to control
during
and insects
pests
in
in
the mortality
spraying
and after
contrary
to the advice
of Ministry
the present
investigation,
there
compounds
and
1976 and Morse,
been reported
to the increase
contributed
or
benzaldehyde
be
(Hooper,
extraction,
taste
from comb or metallic
containers,
content
Madau and Solinas,
(Campus,
to the bitter
These compounds could
in metal
phenol
honey during
into
contribute
honeybees
repelling
of storage
resultant
are known
aroma and flavoured
and strong
of compounds introduced
and storage
flavour
1980).
(ragwort)
and Senecio
due
is mainly
This
(Crane,
sugars
tasting
Honeydew
1979).
The presence
processing
bitter
to distinctly
(Howes,
honeys
(privit)
honey from Ligustrum
rise
give
to bees.
harmful
of physiologically
(1982).
and Atkins
to be harmful
has been reported
presence
Also
to
(1981)
Howes (1979) , Kerkvleit
,
the
rate
crops
of Agriculture
environment
of honeybees.
with
such
and Food
directives.
In
sample No. 38 was described
survey
flavour
and was suspected
U. K. survey
sample Nos. 89,90
bitter
have
a
to
f our
taste.
EeracZewn
the presence
(Hogweed)
such plants
of other
and TiZia
donor
honeys
of nausea.
The other
could
three
and 91 and were described
of pollen
of Castanea
pollen
(lime)
have been reported
in
to give
rise
honeys were
donor
each of these
pollen.
(privit),
such as Ligustrvm
noted.
cause
by their
(Sweetchestnut)
were also
U.
K.
one
-
to have an obnoxious
compounds which
The distribution
samples showed the presence
Further,
from
to contain
or that
symptoms of poisoning
by its
were four
Honeys obtained
to bitter
flavoured
311.
(Crane, Walker and Day, 1984).
honeys
The amino acid maps, pollen
by the SPSS indicated
to that
composition
content
However, the bitter
are forbidden.
with
experience,
occur
evaluation
Furthermore,
and identification
in bitter
tasting
is only reliable
pallate
and many similar
such observations
panel are not based on one individuals
by a tasting
detection
where such sensory examinations
a non-fatigued
at one time.
samples collected
flavour
days of occurrence
of these compounds with
Honeybee
honey was not carried
that
to
compared
received
gas-liquid
out.
in very
and examined for
to
fresh
the presence
chromatography
technique.
investigation.
Species
The low proline
indication
the sensitive
in the present
This was not possible
The
of the presence of compounds reported
or strong
within
conducted
assessment.
This was because such compounds would only be detected
honeys obtained
in
similarity
study was conducted in a
research
such subjective
Also,
of these four honeys
nature
because the present
Further,
considerable
and 91 displayed
tasting
designed for radioactive
building
in
of U. K. survey sample No. 36 (same donor).
not be confirmed
could
classification
that U. K. survey sample No. 38 was similar
the U. K. survey samples No. 89,90
composition.
and predictive
content
of honey produced
from the Indian
cerena
Amino acid analysis
sub-continent
revealed
of four honeys
that-two
samples Nos.
224 and 225 were of very low proline
content
and the other
Nos. 169 and 233 had proline
similar
to that
by Apia meUifera.
were the product
It
content
is known that
of nectar
indica
was used by Davies (1975) as an
by Apia melZifera
of honeybee species.
by Apia
two samples
of honey produced
both samples, Nos. 224 and 225,
gathered by either
Apia cerena or Apia fZorea.
312.
hand sample No. 233 was known to be an admixture
On the other
other
were not known.
whose identities
products
sample No. 169 was either
by Apia meUifera
or similar
SIGNIFICANCE
In view
honey
to comply with
there
that
aims
floral
the
study
is
their
changing
pattern
discussed
tion
aspect
than
are a possibility
with
present
The present
purpose.
use of amino acids
survey
evaluation
of unifloral
which
- pollen
This
pattern.
and
as this
to
attempted
analysis
correlation
source
analysis
using
It
with
was not
aspect
as close
is
using
the SPSS proved
analysis
boundaries.
pollen
analysis
and
amino acid measurements by the application
was established
the discriminant
on geo-political
honeys
of catogarising
the quantitative
as to source.
of
become of
elsewhere.
then correlating
statistical
this
national
microscopical
due to the lack
for
predominant
the first
is
this
The feasibility
of
of pollen
source.
with
amino acid
corresponding
as desired
with
concerned
conventional
compare
here would
the resolution
source,
the use of proteins
therefore
Even though
to identify
the absence
of proteins
and patterns
and floral
precluded
equipment
described
the
importance.
the separation
geographical
the
such as that
In
in
as stated
technique
a chemical
of honey.
source
can be removed from
pollen
Therefore,
was a need to establish
technique
Although
which
interests.
commercial
analytical
valuable
OF PRESENT WORK
ease with
and geographical
a chemical
for
the relative
of
gathered
of nectar
sample No. 233.
to that
product
was not known whether
It
or the result
an admixture
with
that
the predictive
was based on floral
From the
in the
successful
evaluation
identifica-
classification
source
of
rather
the present
313.
it
work
that
was concluded
is
still
to
differentiate
in
the
the various
processing,
commercial
floral
honeys,
of
it
technique
such a chemical
has the
also
feeding
adulteration
pollen
foreign
with
belonging
species
These aspects
to honeybees,
sugar
especially
to be able
capacity
of honey production.
aspects
of different
sources
but
stage
embryonic
honeydew and nectar
are:
the potential
heating,
honeys
to the
and
same plant
family*
Further,
in association
technique
of
for
the application
honeys.
pollen-free
honeybee Apia
cerena
adulteration
honeys
the
result
A brief
but
foreign
concise
of pollen
of a sample proved
the application
Beekeepers
and Western
honeybee Apia
to excessive
sugar
especially
technique
proved
meZZifera;
products,
feeding
of
- the
species
and
of honeybees.
this
chemical
the 11st Bee Research
at
on this
it
useful,
sugar
of the application
and also
the purpose
honeybee
between
A report
Association
Bee Research
International
this
of
following:
account
1986.
London,
Colloquia'in
for
analysis
honey and to some extent
of moderate
and chromatography
electrophoresis
f or use on honey was presented
technique
British
Also,
the
with
that
source
distinguishing
in
Eastern
with
the geographical
verifying
useful
of paper
Workers
to the
work was presented
has been acknowledged
by the
1986.
Association,
FUTURE WORK
The following
needs to be assessed
On the predictive
suitably
advantage
if
and data
classification
modified
the
SPSS programme,
following
and evaluated:
(a)
Obtaining
honey samples
(b)
Obtaining
large
aspect
of
the
can be used to full
this
conditions
handling
can be achieved
such as:
of known provenance.
sample numbers per
group,
at
least
fifty.
314.
(c)
(d)
Using
honeys
unifloral
one floral
source.
Perfo=ing
quantitative
and identifiable
The advantages
present
containing
dete=ination
ninhydrin
from
the detectable
of all
positive
these
ofensuring
90% pollen
over
substances.
as deduced
conditions,
from
the
would be to:
work,
Improve
the potential
by identification
of sub-group
and correct
followed
allocation
classification
of unifloral.
sources.
(ii)
Ease the recognition
(iii)
Enable
the characterisation
substances
(iv)
Produce
(v)
of causes
with
floral
ninhydrin
positive
and geographical.
significant-information.
statistically
to base predication
a framework
Establish
of unusual
both
source
of misclassification.
of uncatogarised
samples.
However,
the disadvantages
these
of certifying
conditions
would
be:
(i)
The difficulty
of co-ordinating
a uniform
based programme of pre-treatment
colonies
late
(ii)
samples
or seasonal
commercial
practices
The siting
of hives
is
species
a radius
other
species
heather
established
miles
within
to
of one floral
over
an area
from a hive.
honey contains
to perhaps
and
honey.
where availability
five
of early
of
or honey subjected
or cheap foreign
of at least
a good natural
mixing
changes
artificially
feeding
of hives,
to avoid
and monitoring
nationally
10% of
wild
the
covering
Since even
clover
total.
and
315.
The monitoring
(iv)
other
this
of
sort
at a suitably
information
equipped
from a significant
National
and quantitative
number of colonies
from up to one hundred
and concurrently
The data
the qualitative
to monitor
of honeys
elsewhere.
and
centres.
be necessary
same site
of peptides
and analyse
be conducted
and could
research
composition
identification
to handle
The capacity
would
of trace
to enhance the measurement
hydrolysiso
to
compounds.
would
It
to be able
of the amino acid'analyser
overloading
components,
2.
analysis.
The modification
allow
(v)
and recording
for
receiving
before
of sample treatment
similar
sites
can be used to determine
so obtained
on the
the
f ollowing:
(a)
The composition
honeydew and pollens
of nectar,
visited
by
the honeybee.
(b)
The composition
(c)
The changes
through
(d)
of secretions
in composition
to full
ripened
if
The variation
the
lag
time
as discussed
(e)
A quantitative
visited
reported
between
of a given
appearance
honey and subsequent
by Carter
at a given
flowering
nectar
in
capping.
to cell,
Also
important
and first
and pollen
honeybee.
the hive
from cell
site.
time
forager
deposited
of nectar
any in composition
to comb and of a hive
by the
provided
comb
is
and last
source
in the hive
(1983).
assessment
and the comPosition
of
the location
of
the
final
and floral
honey,
by Adams, Smith and Townsend (1979).
species
such as that
316.
(f)
of human intervention,,
The effect
blending
at both
to establish
honeys
(g)
amateur
of variation
due to an apairy,
the
(h)
(i)
average
seasonal
levels
of restricting
especially
sugar
changes,
soil
and/or
example,
composition
to just
source
protein
for
data
of relevant
for
and
fed as supplement.
protein
bee food
products
are mixed.
a common sourceý
of special
The effect
Compilation
with
Furthermore,
of two unifloral
composition
honeys
when these
are additive
and
scales.
and professional
the separate
whether
The effects
honey extraction
major
one,
supplement.
honey
and minor
sources
worldwide.
to the above listed
In addition
reduction
by obtaining
(a)
(b)
in the
of variable
the ninhydrin
positive
harvested
following
the
To what degree
class
pollen'
and influence
affect
of honey
composition
does the contribution
'Important
Minor
the overall
of ninhydrin
and Minor
composition
frequency
of unifloral
honeys?
(c)
Does the feeding
effect
significant
cifsuch
(d)
honeys
pollens
light
substitutes
on the ninhydrin
produced?
The identification
conventional
of pollen
-If
of pollen
microscopy
as appropriate.
so,
positive
if
using
to species
fresh
any,
composition
to what degree
grains
Further,
have,
and extent?
level
by
and reference
examination
the
be accomplished
could
year?
of
affect
be of use if
questions:
pollen
substance
and extent
substances
positive
stored
years
would
data
statistical
to the following
the answers
Does the previous
it
research
of suspect
317.
pollen
fine
the much higher
using
details
surface
the Brassica
of
by using
Thus,
can be done for
for
necessary
enabling
This
of
source
In
floral
present
source,
serve
country
of
data
floral
of this
and/or
chemical
of chemical
regard
to amino acids
with
aims of detection
of adulteration,
and the application
bank containing
samples
per
but
system,
also
geographical
technique.
verification
of
has been accomplished.
misrepresentation
and future
be
will
the world.
reference
as an indexing
the
ascertaining
in honey.
analysis
work the feasibility
especially
be
to
possible
shown
found
on
similar
verified
'only
not
a sample by the application.
the
The other
for
plus
can only
be successful
of an international
a thousand
then
would
then
automated
procedure
pollen
honey producing
the -compilation
system
as a reference
and may not
one country
each individual
on at least
information
country.
pollen
The labour
some extend
This
damaged and uncleaned
osmotically
this
be accomplished.
image retrival.
scanning
so that
identification
species
can be eased and to
be done on perfect
If
for
essential
genus can easily
of such searches
(20K)
magnification
potential
have been
of computers
has been investigated.
The way forward
but
1987,
future
over
achievements
none of
these
perhaps
must await
ideas
have yet
the next
their
ten years
turn.
appeared
has been indicated
Upto the present,
in print.
June
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U. S. Dept.
Bull.
Tech.
Willson,
J.
1-3.
66(l),
White,
F. A.
and Robinson,
(1986)
W. F.
and Jiang,
97,52500k.
H.
(1982)
Linye
Kexue,
18(l),
80-84;
Chem. Abst.,
APPENDICES
330.
APPENDIX I
LIST
OF HONEY SAMPLES
Honey samples
the United
12 months
several
listed
in Table
Kingdom survey
received
over
of
1982.
However,
1983
September
upto
for
available
within
a period
Association.
Beekeepers
eventually
obtained
1981 to October
being
from members of the British
192 honey samples
those
and were received
commencing from October
samples were still
IA are
There were
and these
analysis
were
catalogued.
IA:
In Table
Column one
=
the questionnaire
beekeeper.
individual
Column two
=
the approximate
-
Column four
location
the hive
of
as
by the beekeeper.
supplied
Column three
to each
number allocated
the map grid
reference
of
to each sample
the code number allocated
to identify
and serves
the hive.
the
sample
throughout
the analysis.
Honey samples
or as specified
commercially
packers.
listed
in Table
of
samples were
Nearly
all
these
either
at port
of
shipment
There were 64 samples
available
foreign
those
on the label.
processed
were catalogued.
IB are
for
or at
analysis
origin
the U. K.
and these
331.
In Table
IB:
Column one
=
the name and country
of origin
of a honey
sample.
Column two
=
the name of
specified
the packer
on the
label
or the
supplier
or the name of
as
the
donator.
Column three
-
the code number allocated
serves
analysis.
to identify
the
to each sample
sample
throughout
and
the
332.
TABLE IA
LIST
OF HONEY SAMPLES FOR THE UNITED KINGDOM SURVEY
Questionnaire
Major Lacality
Number
Map Grid*
Code
Reference
Number
3b
Crawtborne
SU 8464
4a
Ruddington
SK 5733
4c
Beeley Moor
SK 2667
7a
Southend-on-Sea
TQ 8885
7b
Great Wakering
TQ 9487
9a
Rothbury
NU 0602
9b
Lesbury
NU 2412
9c
Longhirst
NZ 2389
10a
Newburn
NZ 1865
10b
Font
NZ 1785
loc
West Rainton
NZ 3247
lla
Galleywood
llb
WoodhamWalter
TL 8006
12a
Bideford
SS 4526
12b
Bideford
Ss 4526
13a
Hoggrills
13b
Llanfachreth
SH 7522
13c
Birmingham
SP 0786
14a
Barlaston
SJ 8938
14b
Barlaston
SJ 8938
l6b
Merstham
TQ 2953
18a
Chalfout
19a
Northwood
TQ 1090
19b
Northwood
TQ 1090
20a
Woodbridge
TM 2749
21a
Writtle
TL 6706
21b
Writtle
TL 6706
23a
Dulwich
TQ 3373
23b
Dulwich
TQ 3373
25a
Thulston
SK 4031
25b
Thulston
SK 4031
26a
Exmoor
SS 7642
26b
West Pennard
ST 5438
27a
St.
Day
SW 7242
27b
Truro
28a
Oulton
28b
Otley/Ilkely
281
Oulton
The grid reference
(1) Mason (1981).
(2)
Automobile
(Chelmsford)
TL 7002
end (Coleshill)
St.
SP 2291
Giles
SU 9893
SW 8244
(South Leeds)
SE 3628
SE 2045
SE 3628
(South Leeds)
given
Association
here were according
Great Britain
to:
Road Atlas.
Cont'd
333.
Table 1A cont'd
Questionnaire
Major Lacality
Number
Map Grid*
Code
Reference
Number
30a
Camberley
SU 8660
39
30b
Bagshotheath
SU 9163
0
31b
Leck Fell
SD 6779
41
32a
Wind Rush
SP 1913
42
32b
Thornbury
ST 6390
43
33a
Penrith
NY 5130
44
33b
Lazonby
NY 5439
45
34a
Brampton
NY 5361
46
34b
Brampton
NY 5361
47
35a
Pont-y-Goytre
So 3509
48
35b
Newport
ST 3189
49
36a
Reigate
TQ 2550
50
36b
Gatwick
TQ 2841
51
37a
Egton
NZ 8106
52
38a
Helmsley
SE 6186
53
38b
Fakenham
TF 9229
54
40a
Lydd
TR 0421
55
40b
Ashford
TR 0142
56
41a
Church Stretton
so 4593
57
41b
Market Drayton
S3 6734
58
42a
Rookley
SZ 5084
59
42b
Chillerton
Sz 4883
60
43a
Newbridge
Sz 4187
61
44a
Bonning Gate
SD 4895
62
44b
Stavely
SD 4798
63
45a
Hadden
NT 7836
64
45b
Morpeth
NZ 2086
65
46a
Hexham
NY 9464
66
46b
Hexham
NY 9464
67
47a
Moulton
SP 7866
68
47b
Kingsthorpe
SP 7563
69
48a
Staines
TQ 0471
70
48b
Staines
TQ 0471
71
49a
Yatton
ST 4365
72
49b
Yatton
ST 4365
73
50a
Aycliffe
NZ 2822
74
50b
Pikeston
NZ 0432
75
51a
London
TQ 3079
76
52a
Bristol
ST 5872
77
52b
Weston-Super-Mare
ST 3261
78
54a
Piddington
SP 6317
79
54b
Long Creadon
SP 6908
80
56a
Jarrow
NZ 3265
81
Airport
(Runway)
Moor (Carlton)
Fell
Cont'd
334.
Table TA cont'd
Questionnaire
Major Lacality
Number
Map Grid*
Code
Reference
Number
56b
Woolsingham
NZ 1969
82
57a
Starcross
SK 9781
83
57b
Starcross
SX 9781
84
58a
Bolton
SD 7109
85
58b
Bolton
SD 7109
86
60a
West Hanney
SU 4092
87
60b
Wantage
SU 4087
88
61a
Cobham
TQ 1060
89
61b
Cobham
TQ 1060
90
61c
Cobham
TQ 1060
91
62a
Sawdon
SE 9485
92
62b
Bickley
SE 9192
93
63a
Goring
SU 6080
94
63b
Whitchurch
SU 6479
95
64a
Crowthorn
SU 8464
96
64b
Crowthorn
SU 8464
97
65a
Newton Tracey
SS 5226
98
65b
East Combs
SS 8904
99
68a
Huntshaw
SS 5023
100
68b
Torrington
SS 4919
101
69a
Ottery
St. Mary
SY 0995
102
69b
Ottery
St.
SY 0995
103
70a
Hoddesdon
TL 3709
104
70b
Hoddesdon
TL 3709
105
71a
Cheshunt
TL 3502
106
71b
Waitham Cross
TL 3600
107
72a
Barham
TL 1375
108
72b
Barham
TL 1375
109
73a
Houghton Hill
TL 2972
110
73b
Houghton Hill
TL 2972
ill
75a
Plaistow
TQ 0030
112
75b
Billinghurst
TQ 0825
113
76a
Stevenage
TL 2325
114
76b
Stevenage
TL 2325
115
77a
Craven Arms
SO 4382
116
77b
Craven Arms
so 4382
117
78a
South Cerney
SU 0497
118
78b
South Cerney
SU 0497
119
79a
Findern
SK 3030
120
79b
Littleover
SK 3234
121
80a
Pinner
TQ 1289
122
80b
Pinner
TQ 1289
123
Hill
Mary
Cont'd
335.
Table
IA cont'd
Questionnaire
Major Locality
Number
Map Grid*
Code
Reference
Number
81a
Gt. Dunmow
TL 6221
124
85a
Broadstairs
125
85b
86a
Broadstairs
Lewes
TK 3967
TK 3967
TQ 411o
86b
Lewes
87a
126
127
Birchington
TQ 4110
TR 3069
129
87b
Birchington
TR 3069
130
88b
Broxted
TL 5727
131
89a
Broxted
91a
Rochford
91b
Rochford
TL 5727
So 6268
so 6268
132
133
134
93a
Stainton-Le-Vale
TF 1794
135
93b
Moortown
TF 0699
136
94a
Appleby
137
98a
Bury St. Edmonds
SE 9ý14
TL 8564
98b
Bradfield
99a
Hale
St. George
128
138
TL 9059
ST 4682
139
141
14o
100a
Lowington
100b
Barston
SP 2069
SP 2078
101a
Toton
SK 5034
143
101b
Beeston
144
102a
Bradmore
SK 5336
SK 5831
102b
Nottingham
103R1
103R2
Ollerton
Ollerton
103R3
Ollerton
103R4
Ollerton
103R5
Ollerton
103R6
Ollerton
103R7
Ollerton
103R8
142
145
SK 5640
Si 7776
146
147
SJ 7776
SJ 7776
148
149
150
SJ 7776
SJ 7776
Si 7776
151
152
153
Ollerton
SJ 7776
SJ 7776
103R9
Ollerton
SJ 7776
155
105a
Leyland
SD 5421
156
105b
Waddington
SD 7243
157
105d
Preesall
SD 3646
158
105e
Bolton
SD 7109
159
105f
Mellor Brook
160
105g
Cottam.
SD 6331
SD 4932
105h
Southport
105J
154
161
162
Dalton
SD 3316
SD 4907
105K
Bretherton
SD 4720
164
107a
Hoar Cross
SK 1223
165
107b
Hoar Cross
SK 1223
166
163
Cont'd
336.
Table
IA cont'd
Questionnaire
Major Locality
Number
Map Grid*
Code
Reference
Number
108a
Hewelsfield
SO 5602
167
108b
Hewelsfield
SO 5602
168
108c
Srinagar
Kashmir
(India)
169
109a
Denham
TQ 0386
170
109b
Stamore
TQ 1692
171
110a
Halse
ST 1327
172
110b
Charlton
ST 5228
173
111a.
Wiggaton
SY 1093
174
112a
Feering
TL 8720
175
112b
Feering
TL 8720
176
114a
Tunbridge
TQ 5839
177
114b
Bodiam
TQ 7826
178
114c
Comberton
TL 3856
179
114d
Comberton
TL 3856
180
114f
Trowbridge
ST 8557
181
114g
Trowbridge
ST 8557
182
114k
West Calder
NT 0613
183
1141
West Calder
NT 0613
184
115a
Shrewbury
SJ 4912
185
115b
Shrewbury
SJ 4912
186
115C
Unknown
187
117a
New Mill
SP 9212
117b
Hemel Hempstead
TL 0506
189
118
Clay Cross
SK 3963
190
119
Mylor
SW 8036
191
122
Unknown
-
192
Mackrell
Wells
Bridge
188
337.
TABLE IB
LIST
OF THE FOREIGN AND COMMERCIAL HONEY SAMPLES
Name and Country
Name of packer
Code
or supplier
or donator
of origin
Number
Austria
Marchtrenk,
Australia*
Banksia
donated by Manley Ratcliffe
Oxford (1)
Australia
Bluebell
Crabtree
Australia
Bluebell
donated by Mr. Rex Sawyer (2)
196
Rowse Honey Ltd.,
197
193
and Evelyn,
Ltd.,
Berinsfield,
194
London
195
(H470)
Australia
Argentina
Austria
to
Clover
It
Canada*
donated by (1)
Canada Clover
Rowse Honey Ltd.,
Canada Clover
(H666)
Canada Sunflower
Ewelme, Oxford
198
199
Ewelme, Oxford
200
donated by (2)
(H669)
Canada British
Colombia (H707)
201
of
of
202
it
of
203
Canada Clover
Samantha
D. J. Sales Ltd,,
Canada Clover
Pure Sainsbury's
J.
Canada Clover
Pure Tiptree's
Wilkin
Cayman Island
Coconut
donated by Mr. Harrison
China Acacia*
donated by (1)
Sainsbury
Salford
Ltd.,
2o4
London
and Sons Ltd.,
205
Tiptree
206
of Ashford
207
2o8
China Buckwheat*
It
of
209
China Eucalyptus*
Light Amber
of
to
21o
China Lightolo
Amber
91
to
211
China Whiteot
212
China White
donated by a Beekeeper of Middlesex
donated by (1)
Chileok
France Fl
purchased on site,
Mr. J. M. Carter
La Belliole,
213
214
France,
by
215
France F2
of
It
216
France F3
to
to
217
France A
purchased on site.
Mr. J. M. Carter
to
France F5
These are bulk honeys in steel
as stated on consignment.
Manley Ratcliffe
Ltd.,
Charente,
tt
drums as shipped
Berinsfield,
France,
to
by importers,
by
218
It
219
origin
and information
Oxford.
Mr. Rex Sawyer.
Cont'd
338.
Table IB cont'd
Name and packer
Name and Country
Code
or supplier
or donator
of origin
Thasos,
Halkidiki,
Greece Nectar
Number
220
Greece
North
donated by (2)
221
Hungary* Polyflora
donated by (1)
222
India
Charak
Charak Pharmaceuticals
India
Gujarat
(1)
India
Gujarat
(2)
(H689)
Greece Nektar
PVT Ltd.,
is
by Mr. C. Airey
Mexico Pure
Sainsbury's
J.
London
New Zealand*
donated by (1)
New Zealand Clover
Manley Ratcliffe
Russia White
Parish
Spain Orange-Blossom
The Honeycomb Co.,
Spain Sunflower
Cotswolds
Windrush
Gales Honey
Colman's
of Norwich,
of
purchased
Yucatan
Mexico*
Sainsbury
Gales Honey (A)
to
Gales Honey (B)
to
225
226
227
Ltd.,
228
229
Ltd.,
230
Oxford
and Fenn Ltd.,
231
Surrey
ELLEL. R. P. Lancaster
232
Honey Farms Ltd.,
233
Carrow,
Is
J. Sainsbury
223
224
to
on site,
donated by (1)
Malta
India
by Mr. P. M. Mistry
on site,
of
purchased
of
(India)
Witney
234
Norwich
to
to
235
of
is
236
London
Ltd.,
237
Sainsbury's
Acacia
Pure
Sainsbury's
Clear
Pure
238
Sainsbury's
Set
Pure
239
Rowse's Cut Comb (1)
Rowse Honey Ltd.,
Pure
Ratcliffe's
Clover
of
to
Rowse's Cut Comb (2)
Ewelme, Oxford
go
Ltd.,
Manley Ratcliffe
240
241
242
Oxford
mR68
donated by (1)
243
mR67
it
244
mR66
of
245
to
246
Yellow
Box*
Comb Honey
English
Peirson
Ewelme, Oxford
248
Cheddleton
249
Shaffalong,
Apairy,
Dr. Croft's
Pure
247
Croft
Rowse Honey Ltd.,
Pure
English Pure
Staffordshire
English
by Dr.
supplied
Nr.
English Pennine
Heather
Pennine Bee Farms, Galgate,
English
Honey Show, Caxton Hall,
National
London 1979
English
Pure M8418
English
Clover
Golden
Tarleton
Lancashire
supplied
J.
Northern
Ireland
Pure Honey
English
Chorley
Dr.
Aalyst's
Lancaster
Sainsbury
252
Laboratory
253
to
254
01
255
honey,
256
London
Ltd.,
251
Westminister,
by Dr. Croft
Reference
Linconshire
Public
11
of
M8522
Man Made Honey
Sainsbury's
Syrup
donated by Preston
250
Lancashire
Chorley,
Siddiqui
(1981)
257
11
of
11
258
it
of
of
259
99
it
of
260
Croft's
Apairy,
Chorley,
Lancashire
261
339.
APPENDIX II
QUESTIONNAIRE
Given below
were sent
which
to the beekeepers
who took
Kingdom
0000
Approximate
0.00..
*0000.0.00..
000..
location
of
faili'llwals:
Map Grid
Estimated
main floral
please
yes,
Has this
If
give
please
yes,
previous
state
unable
Any other
Beans. (stick)
softfruit
.. .....
.................
XKX/No
conditions:
0-0
sugar?
M
INo
and form,
the quantity,
0000000
given
during
12 months.
from colony
Yield
000
comb
source:
been fed
colony
0.0900000
..........
of honey from
Has the honey been heated?
If
of a farm
..............................
Reference
Date of removal
00.00.0060a000
the colony:
'outskirts'
Situated
on
oo. 0 ... 00 ...
.....
(7)
in the United
part
...........................................
.0..
(6)
on the questionnaires
the questions
of
Name ..................................................
Address
(2)
the list
For example:
survey.
(1.)
is
to judge
during
but
previous
averaged
5 months:
30lbs
from
For this
.....
analysis
10 colonies.
comments:
000000000000a00000000.0000000a000000000
Note:
beekeeper and the
The name and address of the participant
number have not been quoted so as to retain
questionnaire
The responses to the question
confidentiality.
number are
to those quoted by the beekeeper.
according
340.
III
APPENDIX
IN A HONEYSAMPLE
DETERMINATION OF THE AMINO ACID CONCENTRATION
as described,
was mixed with an equal volume (25 pl) of buffer
concentration,
(20
duplicate
were applied;
one to each of the Technicon resin columns.
norleucine
and
pl) portions
containing
The absorbance of the reaction
mixture and eluate was determined at 570 nm for amino acid and 440 nm for
A honey sample after
The data obtained
proline.
paper as a trace
that is,
of a triangle,
was observed
response
curve whose peak shape approximated
base multiplied
by the perpendicular
on a chart
half
The normal definition
a triangle.
Hence, an equivalent
of the curve limits.
height
could not be applied because of the varying position
by the width at half curve
definition
as curve peak height multiplied
which has been used universally,
to
that
of
peak
height
acid
concentration,
was applied.
-
area
that
k[amino
acid
reaction
amount
of
at a possible
internal
of the standard
The order
arginine,
serine,
acid, thrionine,
aspartic
leucine,
isoleucine,
norleucine,
methionine,
However,
histidine.
arginine,
The concentration
area
the trace
under
of
Derivation
A
be used for
lysine,
special
purposes.
histidine,
ala; ine,
eluted
ammonia,
cysteine,
valine,
and phenylalanine.
that
there
concentrates
very
were naturally
as described
low levels
of
in the methods
the amino
peaks due to these amino acids
and significant
were
of an absorbing
and the measured value of the
the same proportion
of the incident
substance
a given sample always absorbs
is defined by the Beer-Lambert's
Law.
This Law can be expressed
as:
Ccl
A
could
glycine,
proline,
chosen norleucine
amino acids
between the concentration
Equation
.............................................................
absorption
C
molar
C
concentration
1
light
path
and the concentrated
weight
the molecular
2
of radiation
absorbance
When comparing
Thus:
fourteen
and the fact that
of its intensity,
regardless
mathematically
where
only
radiation
radiation
containing
Equation
The relationship
absorbed
standards
the honey amino acid
and also
solutions
were commonly recorded in the honey samples.
honey amino acid sample was evaluated from the
of an amino acid in a concentrated
responce curve using the equation derived below.
Therefore,
observed.
rarely
tyrosine
standard
usually
and ammonia were:
acid,
glutamic
and methionine
cysteine
other
amino acids
of preparing
the ammonia peak was very high
sections
acids
due to the technique
The standard
these variations.
eighteen
of elution
authentic
with
pump tubings and measurements of the
*
to include in each sample a constant
was essential
it
range although
acid
I
heating,
position,
for
to correct
of the eluted-amino
the middle
in local
to the amino
was proportional
Equation
the instrument
by pre-calibrating
non-equilibrium
standard
thus obtained,
....................................
Due to variations
of each amino acid.
colour
near
concentration]
determined
k is a constant
where
25 nMoles
The area under each curve peak,
is
of a substance
-1
1
mol
coefficient
of a substance
(cm).
the absorbance
value
honey amino acid
can be regarded
for
(I mol
amino acid
a particular
sample the molar absorption
as constant
As -ESca16
for
the
standard
AH'EHcH1H
for
the
concentrated
amino
acid
honey
for
present
coefficient,
in both
the standard
constant
measurements at the same wavelength.
solution.
amino
acid
solution.
path length
solution
and
341.
Hence:
ECIc
sa9
EHcHIHcH
By using
Equation
an internal
of constant concentration
standard norleucine
the ratio ASN/A HN derived similarly
as in equation
acid solutions
Thus:
to compensate for changes in instrumentation
parameters.
amino
factor
ACxA
-A
AHcHxA
in both
the standard and honey
3, can be used as a multiplication
SN
s
4 the concentration
of an amino acid in the concentrated
mixture (20 jil) applied to the analyser column can be obtained.
equation
norleucine
4
Equation
HN
Rearranging
standard
3
honey amino acid
- internal
Thus:
csxA
SN xAH......
A
HN xAs
CH
/A is the norleucine
of
equivalent
SN S
of the standard amino acid to that of the
the analysis
by the term NESP, and CS has the value 25 nMoles, that
5
be
Hence.
written
can
equation
solution.
amino acid
A
The ratio
csx
the corresponding
amino acid response curve from
honey amino acid sample and was denoted
concentrated
is the concentration
of each amino acid in the standard
as:
NESP x 25 (nMoles)
AHN
honey amino acid
concentrated
of CH in the 25 pl of the original
(25 pl) can be obtained
internal
standard norleucine
The value
with
the
5
Equation
by multiplying
Equation
5a
solution
prior
to mixing
5a by the factor
equation
Thus:
50/20.
A,, x NESP x 25 (nMoles)
HA
50 (ol)
20-Tpl)
HN
6
Equation
in 10. Og of honey can be obtained
by multiplying
6 by the
equation
The amount of amino acid present
1-H,
(1.5 mls) plus
SM HCJ solution
1OZ (v/v)
PH is the total
the amount
propan-2-ol/O.
weight
of
where
factor
.25
(10.0g).
honey
from
It was found at least
that
disssolved
approximately
concentrated
acid
amino
the
of
lml
of
volume
the
weight
of
(Iji).
Thus:
c
this
concentrated
honey
concentrated
honey
amino
amino
acid
A. x NESP x 25 (nMoles)
ARN
H'
Moreover,
the concentration
by the factor
7 was divided
x
10.0.
50 (PI)
20 7-;jlT
x
was expressed
in
PH (PI)
25 (jil)
1 gram
volumetric
......
in
weight
units,
Equation
and
the
total
microlitres
7
as nMoles per gram of honey.
Therefore,
equation
Thus:
x
AHN
-
(PH)
solution
to
was equivalent
solution
measurements were expressed
AH x NESP x 25 (nMoles)
c
acid
50 jil
10-ju 1
x
PH (lil)
-25 (-ju-IT
f
10.0
Equation
nMoles per gram of honey
8
was noted from the trace response curve chart paper that when two or more trace curves occurred
limits
for
did
trace
by
to
the
each
curve
curve
not always occur on
some
extent
overlapped
or
close
devised
in
Therefore,
these
to determine the height
baseline.
approximations
were
rare
cases
the
N. B. It
and width
at half
occurring
trace
height
response
of curve peak.
curves
throughout
The approximations
the calculations
procedure
was than utilized
of amino acid
concentrations.
for
similar
342.
A Typical
Example of Calculation
The concentration
of aspartic
in the U. K. survey
acid
sample Code No. 27 was calculated
using
equation
8 as follows:
AH x NESP x 25 (nMoles)
CH
where
A
20 (Ml)
x
HN
CH
,
Concentration
AH
-
Area
-
2.0
SN
NESP -A
AS
under
x 25 (MI)
of
the
cm (height)
(jil)
x 50
x 10.0(g)
aspartic
trace
x 0.4
-
AHN -
acid
response
(nMoles
curve
cm (width
for
I height)
per
g of
aspartic
- 0.8
honey).
acid
2
cm
for norleucine
Area under the curve
Area under the curve for aspartic
acid
14.6 cm (h) x 0.4 cm (w1h)
12.5 cm (h) x 0.4 cm (w1h)
PH
x PH (pl)
.
1.17
Area under the trace response
13.7 cm (h) x 0.4 cm (w1h)
2.
5.48 cm
curve
1530 yl
for
in
the
U. K.
standard
2)x1.17
0.8 (cm
2)x
(cm
5.48
-
27.
amino
acid
solution
(1.53g)
for
internal
standard
norleucine
Therefore:
C
H
Sample
x 25 (nMoles) x 50 (pl) x 1530 (ml)
20 (ml) x 25 (pl) x 10.0(g)
65.33 nMoles of aspartic
acid
in lg of U. K. sample Code No. 27.
applied
to U. K. sample 27.
343.
APPENDIX
IV
COMPUTATION
data
files.
in the U. K. survey
of the data arranged
below is the format
Described
and the foreign
and commercial
is as follows:
This format
of the free
from the area under the
used were those calculated
There were
trace response curves on the chart paper output as detected by the amino acid analyser.
whose curve peaks could be adequately measured
seventeen amino acids detected and of these only thirteen
The concentration
These thirteen
were considered.
amino acids
amino acids
were:
lysine,
serine,
glutamic
aspartic
acid, threonine,
isoleucine,
leucine,
tyrosine
and phenylalanine
in that
acid,
proline,
glycine,
alanine,
valine,
order.
as sent to the beekeepers contained eight questions of which the stated responses
'Date of removal of
The relevant
toquestion
were:
questions
numbers, 3,5 and 6 were used here.
'Has the honey been heated'? Yes or No, question 5; and 'Has this
honey from comb'?, question 3;
colony been fed sugar'? Yes or No, question 6.
The questionnaire
of the distribution
The classification
Out of the four
of honey was used.
of
the pollen
classes
pollen
into
grains
frequency
for
classes
each sample
the predominant
and the secondary pollen classes
which occurred over 45% of the total
pollen
only
Honey samples containing
a given pollen
into the predominant pollen class.
Those honey samples whose pollen content
counted were allocated
into the secondary pollen class.
in this case
Furthermore,
ranged between 16% and 45% were allocated
in decending
for honey samples which occurred in the secondary pollen class;
two pollens
only the first
were considered.
All
were considered.
of percentage
order
the two hundred and fifty
or the secondary pollens.
that is, the amount of pollen
content,
samples of honey contained
six
the predominant
either
The pollen
by visual
was determined q'uantitatively
of one to five were selected,
slides
scale
the highest
represented
of low,
contents
pollen
pollen
grains
microscopical
where one represented
content.
medium and high,
The intermediate
on the prepared
present
or zero pollen
and five
the intermediate
content
and 4 represented
numbers 2,3
pollen
Values on an arbitrary
examination.
the lowest
microscope
respectively.
other amino acids besides the thirteen
amino acids which were
presence of fifteen
estimated by the amino acid analyser and common to all the samples were also detected
quantitatively
Besides these twenty eight amino acids
by the technique of paper electrophoresis
and chromatography.
The occasional
complex colour spots whose identity
could not be ascertained
were also occasionally
other ninhydrin
These fifteen
found to be present.
amino acids and the eleven unidentified
ninhydrin
complex colour
The identity
on the computer.
analysis
of the thirteen
spots thus detected were used for qualitative
The other fifteen
to be:
amino acids were identified
o- and y- amino
amino acids has*been given above.
eleven
butyric
histidine,
arginine,
asparagine,
cysteine,
glucosamine,
glutamine,
hydroxypipecolic
0-alanine
acid, methyl-histidine,
and pipecolic
acid,
hydroxyproline,
tryptophan.
methionine,
The unidentified
acid.
as ps Xo Ct as to es now, X9 0. a and 0.
complex spots vere labelled
ninhydrin
for each individual
All this data were compiled in the order listed
above into a data file
twelve
honey
sample.
There were two data files
the foreign
compiled,
Kingdom survey
while
the other
contained
honeys.
and commercial
for each sample of honey the data mentioned above were arranged
In each of these two data files,
The
Each line composed of eighty characters,
four lines.
one character
a column.
representing
into
two lines
first
of the four
lines
the concentration
contained
These two lines
quoted
to two decimal
line.
Each column composed of ten characters.
of
the United
one contained
the first
threonine,
eight
serine,
the concentration
phenylalanine,
places.
amino acids
glutamic
values
in that
proline,
of the remaining
order.
Line
three
glycine
five
line
of
sixteen
contained
the thirteen
columns,
amino acids were:
in that order.
and alanine,
amino acids
of the four
lines
valine,
isoleucine,
was divided
free
into
lysine,
amino acids
columns per
eight
the concentration
These eight
of the thirteen.
acid,
into
were divided
The first
values
values
aspartic
The second line
leucine,
thirteen
tyrosine
acid,
contained
and
columns of five
344.
for
This third line contained
the
column twelve which was of one character.
by the predominant pollen and its
questions 3,5 and 6. This was followed
answers to questionnaire
This was then
secondary pollen and its percentage value.
percentage value and then by the first
followed
by the second secondary pollen and its percentage value.
This was in turn followed by the
characters
each except
and shrivelled
unidentified
pollen
percentage
values,
quoted
to one decimal
place
and then finally
the information
on the presence of the fifteen
amino
into seventeen
line was divided
This fourth
complex spots.
acids and the two unidentified
ninhydrin
in the following
These fifteen
amino acids were listed
order:
columns of three characters
each.
by the pollen
a-.
The last
content.
and y-aminobutyric
acid,
contained
arginine,
cysteine,
asparagine,
hydroxypipecolic
hydroxyproline,
tryptophan,
methionine,
line
acid,
glucosamine,
glutamine,
methylThistidine,
histidine,
0-alanine,
pipecolLe
and the unidentified
spots 'p' and 'x'.
Moreover, in the second data file
which contained
all the data for the sixty four foreign and
manner to that of the United Kingdom
commercial honey samples the data were arranged in a similar
in this second data file
The only difference
was that there were no questionnaire
survey data file.
acid
from these questionnaire
the
questions
all the other data were exactly
These questionnaire
questions
were replaced by
same as those in the United Kingdom survey file.
Hence,
information
the country of origin
and the commercial processing of the honey samples.
regarding
quetions
question
3t 5 and 6 apart
3 was replaced
by the information
the country
regarding
of origin
for
each sample.
The
Question 6 was replaced by the following
the question 5 was left blank.
question:
column representing
Yes or No.
'Has this sample of honey been commercially
processed'?
The code can take the form of words,
It is generally
required by the SPSS to code the data.
letters
or numbers.
in the SPSS require
The words or letters
numerical
codes used in this
The numerical
concentration
complicate
an SPSS command and also
some procedures
Hencet a numerical
coding scheme was adopted.
in the control
statement 'VALUE LABEL' shown in Appendix
data values.
case are given
codes used for identifying
as the amino acid
can often
values
pollen
grains
and the pollen
are given
percentage
in Table
values
30.
Actual
did not require
The numerical
V (b).
numbers such
coding.
345.
APPENDIX V
A DISCRIMINANT
ANALYSIS
below is the order in which the DISCRIMINANT analysis
on the one hundred and
two samples of the U. K. survey honeys, the sixty four samples of the foreign
ninety
and commercial
honeys and also on both the combined honeys was performed using the SPSS.
Described
(a)
United
Kingdom Survey Honey Samples
The results
of the microscopic
and evaluation
examination
each of the samples of the U. K. survey showed that
in the predominant frequency class.
When specified
the distribution
of
there
were nine
major
in
of pollens
pollens
which occurred
one hundred and four
the SPSS allocated
into one of the pollen groups specified.
The remaining
samples of honey which were classified
samples were not dealt with because these had either
missing or out of range amino acid concentration
or they were composed of secondary
I
values,
to improve the percentage
In order
the pollen
of the original
amino acid
the effects
minimise
concentration
of the original
logarithms
were the natural
better
of
the pollen
Therefore,
groups.
the number of pollen groups and to
besides the predominant pollen which may
concentration
values
The square root
logarithms.
the amino acid
the honey samples in each of
These factors
were:
a transformation
of
reducing
pollens
amino acid
than the natural
matrix
The four
valuesq
and the square root.
variance-covariance
the following
were considered.
of
Two transformations
pollen.
classification
the presence of other
between pollen groups.
cause overlapping
discrimination
of correct
a number of factors
groups,
or multiple
This
transformation
data vector
transformation
These transformations
improved
transformation
square root
concentration
the square root
were chosen.
the
makes the
more nearly
equal
was chosen and adhered
across
to throughout
analyses.
pollen
Aesculus,
groups,
Vicia
Inpatiens,
and Clemantes,
contained
only
five
samples
of honey between them. This number of honey samples for the four pollen groups were considered
Therefore,
these four pollen groups
to be inadequate to produce a valid discriminant
analysis.
analysis.
were not included in any of the following
in the predominant
Further,
there
Discriminant
analysis
or the presence
of
into
the other
Preliminary
subclasses.
pollen
groups to discriminate
groups were:
Thus,
groups.
tests
using
between the unifloral
2YifoZiumrepens,
rrassica,
group was defined
subclass
and 852.
pollen
tests
The multifloral
present
of these
minor pollen
the predominant
secondary
frequency
pollen
class
class
were labelled
as the unifloral
the discriminant
analysis
was carried
pollen subclass
group was considered as the unifloral
in each of these honey samples was over 95%.
six preliminary
the presence
These two subclasses
two subclasses.
pollen
pollen
to check whether
in the important
pollens
was performed
between the pollen
the discrimination
In all
were a number of honey samples which
These samples of honey
the predominant pollen as well as the secondary pollen.
to occur in the 45% to the 60% region of the predominant poilen frequency class.
were found
four
class
both
contained
divided
frequency
pollen
were carried
subclass
since
the amount of this
out and in each of these
group was defined
pollen
tests
was further
and the multifloral
out on four
pollen
the unifloral
over 60%, 702,75%,
present
pollen
801
as those honey samples containing
between 45% and 60%, 45% and 652,45Z
The results
showed that
and 85%, respectively.
and multifloral
six tests between the unifloral
were affecting
The
and the multifloral
pollen subclasses.
Castanea and Calluna.
The Ahjosotis pollen
as those honey samples containing
pollen
pollen
and 70%,45% and 75%, 45% and 80% and 45%
there was considerable
overlap in each of the
subclass
pollen
groups.
346.
In order
to minimise
this
group was defined
pollen
between the unifloral
and multifloral
pollen groups, the multifloral
pollen present between 45% and 60Z
as those honey samples containing
overlap
pollen present
group was defined as those honey samples containing
over 702. Intrýduction
of this 10% gap between the two pollen subclasses reduced to a certain
pollen present between 6OZ and 65%,
extent the overiap effect
of the honey samples containing
65% and 70%.
The unifloral
only.
Further
tests
the unifloral
pollen
were carried
out using
and multifloral
pollen
the discriminant
in each of
subclasses
to eliminate
analysis
the four
pollen
betweeý
the overlap
In these
groups.
The unifloral
subclass pollen group was defined as mentioned before.
pollen present over 75%.
subclass pollen group were defined as those honey samples containing
80% and 85%. The results
and the unifloral
showed that as the gap between the multilloral
subclass
Typically,
the overlap between these two pollen groups was eliminated.
pollen groups was increased,
tests
the multifloral
(results)
27
Figure
the histogram
between the two pollen
of overlap
for
obtained
the Brassica
pollen
group show the elimination
groups.
subclass
also showed that as the percentage of the pollen present in the unifloral
the number of samples in each of the four
overlapping
group was increased to eliminate
Therefore,
groups decreased from forty six at 702 pollen to twenty seven at 8OZ pollen.
These results
pollen
pollen
it was considered
between the five pollen groups honey samples which
when discriminating
honeys.
This 70% limit
was
contain pollen present over 70% would be considered as unifloral
honey
five
because
the
of
contained
adequate
number
samples which
groups
pollen
each
of
chosen
described and detailed
in Appendix
file
The SPSS control
analysis.
would give valid discriminant
that
VI (c) was used to initiate
In this analysis.
the honey samples
the SPSS as mentioned previously.
into two subsets.
One subset. was used for calculating
in each of the pollen groups were divided
functions
linear discriminant
the misclassification
and the other for estimating
the Fisher's
however,
rate,
subsets was also used in all
the following
sources of honey.
major
the beekeepers
The honey from these
of the United
Kingdom.
dividing
of
the honey samples into
two
analyses.
Kingdom, honey from the Brassica,
In the United
This
the samples were classified.
all
2!rifoZium
three
Discriminant
repen8 and Catluna
plants
analysis
plants
are the
importance
are of considerable
to
on the honey samples
detailed
in Appendix
was performed
file
The SPSS control
each of these three pollen groups.
representing
the Castanea and Myosotis pollen groups
the SPSS. In this analysis,
VI (c) was used to initiate
58
60
deleted
in
from the SPSS
from
that,
and
control
statements
were
analysis,
ommitted
were
control
file.
question 3 which requested the 'Date of removal
received to questionnaire
of honey from the comb' it was observed that nearly all the honey samples were either harvested
in the crop year 1981 or 1982. Discriminant
was performed to check whether there were
analysis
in the amino acid composition
differences
of the honeys produced during these
any significant
From the response
two crop years.
The SPSS control
file
In this
SPSS as mentioned previously.
by those given in Appendix VI (d)(i).
detailed
in Appendix
analysis,
the control
VI (c) was used to initiate
statements
the
56 to 60 were replaced
of above was also performed on honey samples representatives
in
harvested
individual
the crop years 1981 and 1982. The five
were
the
groups
which
pollen
of
56
60
in
The
to
of
the
control
statements
were
analysis
used.
previous
chosen
groups
pollen
Statistical
results
were obtained
Appendix VI(c) were replaced by those given in part (d)(HA-5.
Similar
for
only
discriminant
three
and Castanea.
of
the years
be computed.
analysis
of these pollen
For the other
specified
to that
groups.
two pollen
and hence,
These three
groups
statistical
pollen
there
analysis
groups were Brasaica,
2ý-ifolium
repene
were not enough samples of honey in either
for
these
two pollen
groups could
not
347.
From the response received
to questionnaire
5 which requested
question
been heated or not.
Discriminant
analysis
its amino acid compositions.
honey effects
to test
was performed
The SPSS control
file
whether the honey had
whether heating a sample of
in Appendix VI (c)
detailed
the SPSS. In this analysis,
the control
was used to initiate
statements 56 to 60 were replaced
discriminant
by those given in Appendix VI (e)(i).
Similar
to that of above were performed
analysis
of the individual
on honey samples representative
pollen groups which were either heated or
not heated.
results
The five
groups chosen in the previous
pollen
for
were obtained
Castanea and 10josotia.
of honey in either of the 'heat'
From the response
In this
6 which
requested
was performed
to test
whether
and spring
groups were:
were not enough samples
for those two pollen
file
whether
had
the colony
feeding
sugar to a colony
composition
of honey
the overall
amino acid
in Appendix VI (c) was used to initiate
detailed
effect
the control
again
analysis,
VI (e) (ii).
in Appendix
analysis
The SPSS control
colony.
question
to questionnaire
received
Discriminant
been fed sugar.
of bees during autumn, winter
SPSS.
pollen
not be computed.
could
from that
These three
groups.
Statistical
were used.
For the other two pollen groups there
groups and hence, statistical
analysis
Braasica,
groups
of these pollen
three
only
analysis
56 to 60 were replaced
statements
the
by those given
of above was performed on honey samples representatives
from colonies
that had been fed sugar or otherwise.
pollen groups which were collected
of individual
Statistical
The five pollen groups chosen in the previous analysis
were used.
results
were
Similar
discriminant
analysis
five
obtained
for
In order
to assess whether
all
to that
groups.
pollen
the honey stores
from colonies
that
in the New Year's
harvested
had been fed sugar
in the autumn
Those samples which were
crop.
months are stored and subsequently
Discriminant
analysis
as
autumn fed were regarded as samples which had not been fed sugar.
mentioned previously
spring fed samples.
control
statements
Similar
discriminant
of
the individual
fed or otherwise.
Statistical
of above was also performed on honey samples representatives
from colonies
that had been either
pollen groups which were collected
sugar
in the previous analysis
The five pollen groups considered
were chosen:
analysis
for
results
to that
the Calluna
pollen
group could
not be computed due to the lack
to colonies
of bees was determined
of honey
of the 'SUGAR' groups.
samples in either
Furthermore,
the autumn, winter and spring fed was carried
out on the winter and
in Appendix VI (C) was used with the modified
detailed
file
The SPSS control
56 to 60.
for
verifýcation
of sugar feeding
by testing
the
The honey
to that of the other amino acids.
of the amino acid proline
concentration
The SPSS control
file
detailed
samples used in this case were the autumn, winter and spring fed.
in Appendix VI (c) was used to initiate
the SPSS. In this case, the control
statements 56 to
Since the results
did not show any
60 were replaced by those given in Appendix VI (e)(ii).
relative
improvements
in this
classification
analysis.
of questionnaire
questions
The analysis
of the five
pollen
groups was not
performed.
From the responses
5 and 6. it
was observed
that
some of
the honey
There are
that had been fed sugar and visa versa.
samples were both heated and from colonies
into two groups.
differentiated
four possible combinations
questions
of these two questionnaire
Discriminant
These four combinations
was performed on
analysis
are given in Appendix VI M.
(c)
detailed
in
file
VI
SPSS
Appendix
The
four
was used.
control
these
combinations.
each of
the control
In these analysis,
statements 56 to 60 were replaced by one of the four combination
statements
Similar
given
in Appendix
discriminant
of the individual
analysis
pollen
VI M.
was performed
groups
for
each of
to that
the four
of above on honey samples representatives
combinations.
348.
(b)
Foreign
and Commercial
Discriminant
in
analysis
Appendix
English
and the
(a)
VII
on the
was performed
New Zealand
Mexico,
Samples
Honey
honey
of
from
originating
Australia.
file
The SPSS control
samples.
commercial
initiate
to
was used
samples
Canada,
and described
detailed
SPSS.
the
Discriminant
In the next analysis.
the English commercial samples were omitted.
analysis
was performed
Canada, Mexico and New Zealand.
The SPSS control
on the four foreign country group samples, Australia,
file
detailed
The control
in Appendix VII (a) was used.
statements 54 to 58 were replaced by those given
in Appendix
Discriminant
VII
VII
(b)(i).
analysis
(a) was used.
From the foreign
included
The control
honey samples that
and commercial
Discriminant
were available.
analysis
(b)(ii).
VII
was performed
These countries
were the same as those mentioned in the
countries.
The control
the France honey samples were also included.
statements 54
in Appendix VII (a) were replaced by those given in Appendix
detailed
selected
that
except
previous -analysis
to 58 of the SPSS control
in the above analysis.
detailed
in Appendix
file
The SPSS control
as mentioned previously.
statements 54 to 58 were replaced by those given in Appendix
was performed
on honey samples from six
VII
the samples of honey from China were also
Further,
file
(b)(iii).
the Mexico and New Zealand honey samples were replaced by those samples originating
file
in Appendix VII (a) were replaced
The control
from India.
statements 54 to 58 of the SPSS control
by those given in Appendix VII (iv).
In the next analysis
From the microscopical
it
and evaluation
examination
th4t
was noticed
the pollen
contents
of the English,
Hence, discriminant
France and Spain honey samples showed considerable
similarities.
analysis
was
in the amino acid composition
between the Enqlish
out to test whether there are any differences
carried
'
file
in Appendix VII (a) were
The control
statements 54 to 58 of the SPSS control
and European honeys.
Also it
in Appendix
by those given
replaced
during
was noticed
cells
algael
contained
VII
(b)(v).
the microscopical
and other
the Austria
that
examination
Thus, discriminant
It
elements.
microscopical
unidentifiable
and Greece honey samples
that
was considered
these
was performed on honey samples of European
Further the honey samples of Austria
Greece and Spain.
and Greece
from honeydew and hence the code for Greece was changed to that of Austria.
were considered to originate
from nectar and hence, the code for
The honey samples of France and Spain was considered to originate
The control
file
detailed
Spain was changed to that of France.
statements 54 to 58 in the SPSS control
samples may be of honeydew origin.
France,
that is, Austria,
origin,
in Appendix
VII
(a) were replaced
by those
given
was also noticed during the microscopical
had very similar
features.
It
and Melilotu8
It
analysis
in Appendix
that
examination
was also
(b)(vi).
VII
the pollen
that
noted
of TrifoZium
grains
these two pollen
grains
repens
belonged
to the
Leguminosae..
Discriminant
analysis was performed to test whether there are any
sample plant family.
in the amino acid composition
The control
differences
of these honeys.
staiemente 54 to 58 of the SPSS
detailed
file
in Appendix VII (a) were replaced by those given in Appendix VII (b)(vii).
control
it
Further,
was also
noticed
that
the pollen
grain
of Lotus
also
included
The honey samples containing
this pollen were also
for the control
statements used.
to Appendix VII (b)(viii)
knowng It
the country
were fourteen
was thought
which
that
samples in the foreign
on the basis
they were most likely
and 242 were allocated
the code for
allocated
the code for
analysis.
Refer
and commercial
honeys whose country
of origin
was not
pollen
these
to that
the appropriate
was suspect
The sample number 234,235
Australia
with
to have originated
and were allocated
the replaced
from.
by the SPSS in a previous
code.
control
Similarly,
allocated
and from its
and 236 from their
the appropriate
statements
samples be allocated
Thus,
the code for
sample numbers 237,238,239
the sample numbers 240,241,243,244
Australia;
of the Mexico samples was also
whose authenticity
content
Mexico and the sample number 247 was allocated
sample number 205 was predicted
and was allocated
of their
to the Leguminosaa plant
in the previous
family.
Since there
belonged
pollen
analysis
the code for
to have originated
sample number 231 whose pollen
the appropriate
content
it
code.
Also,
and 246 were
New Zealand.
from Australia
content
was similar
the sample number 253
the code for
was attributed
Further.
Australia.
from
amino acid maps were thought to have originated
file
The SPSS control
code.
given in Appendix VII (a)
54 to 58 by those
listed
in Appendix
VII
(b)(i)
was used.
349.
Some of the samples received
had been commercially
Discriminant
control
(c)
analysis was performed using
statements 54 to 58 were replaced
Combined Honey Samples of the United
Discriminant
analysis
was performed
processed
the SPSS control
by those listed
and bottled,
file
detailed
in Appedix
while
others
in Appendix
VII
were not.
VII (&)The
(b)(ix).
Kingdom Survey and the Foreign
on the two hundred and fifty
and Commercial
six honey samples to test
whether
in the amino acid composition
of the United Kingdom and foreign
were any differences
and commercial
Hence, in this analysis,
the five 'IF' control
statements 56 to 60 of Appendix VII (a) were
samples.
The SPSS was initiated
replaced by those given in Appendix VII (c)(i).
as mentioned before.
there
Discriminant
France.
analysis
India
and England samples.
From the evaluation
Kingdom survey
was performed
of the distribution
and the foreign
to discriminate
The control
statements
of the pollen
and commercial
between the U. K. survey,
grains
given
it
in Appendix
was observed
honey samples contained
VII
(c) (ii)
that
both
the Braeaica,
Discriminant
subclass group.
and Castanea pollen in the unifloral
analysis
file
detailed
in Appendix VII (a).
In this case, the control
SPSS control
omitted.
Australia,
Canada, China,
were used.
the United
TrifoZium
repene
was performed using the
statements 59 to 60 were
350.
APPENDIX VI
DISCRIMINANT ANALYSIS OF THE U. K. SURVEYHONEYS
(a)
Definition
of Discrimination
in this
Discrimination,
Distance
and Mahalanobis
for
case,
is between the pollen
example,
to one of a number of groups or population
free
honey sample the concentration
of the thirteen
according
allocated
it
to the pollen
For each
is on the basis
contains.
It
were measured.
is allocated
a honey sample of unknown floral
origin
measurements that
of these thirteen
A sample of honey can be
groups.
amino acids
to a pollen
group.
free
For each honey sample the thirteen
dimensional
in a thirteen
amino acid measurements can be thought of as co-ordinates
in one pollen group the honey samples from that particular
Thus,
space.
The problem is to define a boundary in this space to
of points.
Once the boundaries have been chosen a honey sample of unknown
(due
be no true boundaries
Since there will
floral
to a pollen group.
can then be allocated
origin
'over ýap' in this space. errors will
to randomness in the measurements), that is, the populations
The staýistical
methods of 'Discriminant
a honey sample to a group.
sometimes be made when allocating
form a cluster
group will
groups.
separate the different
pollen
have been developed
analysis'
by choosing
a linear
minimise the probability
of making these
function.
This function
is obtained
concentratiors
which defines the direction
of maximal
which will
discriminant
is to use the Fisher's
One such procedure
errors.
boundaries
to select
of the amino acid
combination
in the sense that the means of the projections
of the honey samples from the pollen
(relative
to the variance of the projections
around their
respective
apart
means).
are
maximally
groups
These
A number of linear
co-ordinates
or canonical
combinations
are known as discriminant
variates.
for separating
Usually the first
two or three are sufficient
the
give varying degrees of separation.
group separation
a simple
groups,
which give
variate
these being plotted
picked
observations
stray
The (sample)
on co-ordinate
Not all
out.
distance
Mahalanobis
each sample having
representation,
graphical
;
and
measurements may be used in the analysis.
of the thirteen
between two groups
on each canonical
can be seen immediately
The group separations
axes.
a value
is the square root
of-
Oll
sample means of the two groups and is a matrix of elements describing
where ý19 and x are the (vector)
.2
The SPSS 'MAHAL' option
the variability
of all the measurements (sample variance-covariance
matrix).
the Mahalanobis distance between each pair of groups
selects the amino acids one at a time, calculates
This
the minimum value.
the new variable
and if it has not
Stuart and Ord, 1976).
Kendall,
(b)
SPSS Control
A Typical
This
is
which
starting
field.
of
SPSS control
increased,
the
U. K.
a typical
two
divided
into
composed
of
eighty
characters
one and
ends
from
column
information
This
definition
needed
specification
statements
for
the
field
at
or
columns.
command to
and read data statements.
four
of
operate
the
file
column
control
The control
fifteen
from
for
A field
fields.
column
of an SPSS control
Package
seventy
distinct
starts
new variable
of 1.0 to enter
(Gnandesikan,
is discarded
value
without
1977 and
Honeys
Statistical
contained
is
this
Survey
file
card
of
The control
task
an example
particular
statement
extra
is
below
Given
for
File
the default
is then compared with
and finds
a line.
are divided
statements
being
(SPSS)
Sciences
a part
field
cards.
or
of
contains
and
into
or
functions
ends
three
at
column
types.
control
file.
Each
control
a statement
the
defined
eighty
or
a line
command names
field
The specification
routine
sixteen
Social
contains
by the
of
the
control
a line.
These are data definition,
351.
The data definition
statements
name denoted as Run Name, the list
of the data for
structure
honey samples,
the labels
of the variables
the data
to the SPSS.
In this
case,
the procedure
the file
data, the
which contains the raw-input
of each variable
on them, the number of
each honey sample and the location
the numerical codes used for identifying
each
used to define each variable,
and the missing
The task definition
and describes
inform
statements
of variables,
for
values
define
activate,
data.
incomplete
to be performed
the calculations
and control
on the
to be
were utilized
required
by these concentration
This was due to the fact that the amount of work space specified
values
coded.
for each of the amino acids could not be handled by the SPSS. Hence, the concentration
values were
For example, the control
to the
statement 43 specifies
values 1,2,3
and 4.
coded into four arbitary
data.
In this
the original
case,
amino acid
that
values
concentration
be allocated
the concentration
values between 50 and 99 nMoles per g of honey, inclusive,
for the other concentration
The samples containing
the
range values.
a value of 1. Similarly,
following
pollens which were classed as predominant were selected into nine groups by the nine 'IF'
Brasaica,
Trifolium
These nine pollen groups were:
repens, Chatanea, Calluna,
statements.
control
SPSS that
4oeotie,
AeocuZus, Impatiens,
allocated
into
given
above.
their
appropriate
Canonical
groups using
nine pollen
and Memantia.
Vicia
variates
groups
pollen
These selected
labelled
numerically
was performed using the subprogram 'DISCRIMINANTO on the
The 'MAHALI
codes for the amino acid concentration
values.
analysis
the arbitary
method was used to maximise Mahalanobis distance between the pollen
and the Mahalanobis distance have been defined above, in part (a).
was used and was defined
analysis.
This stepwise
each separate
samples of honey were then
from one to nine in the order
as a default
selection
that
value,
is,
twice
A stepwise
are either
The number of discriminant
step of the analysis.
analysis
selection
procedure
listed
in the
the number of variables
is where variables
procedure
The discrimination
groups.
functions
entered
derived
or removed from
were three
and the
minimum cumalative percentage was defined as 100%. A maximum value of 1.0 was defined for the
functions.
levels of additional
The prior
that is, assigning
the honey
probability,
significance
for which it has the greatest
probability
of membership was defined as the value,
sample to that'group
equal
for
detailed
all
The details
the groups.
extensively
by Nie et al..
of the processing
(1975).
The 'READ INPUT DATA' statement instructs
in this case named as 'DAT1-UK1'.
file
the SPSS to begin
file
has been detailed
The structure
of this SPSS control
was typed in at a visual display unit terminal.
1)
RUN NAME
A TEST OF DISCRIMINANTý
2)
VARIABLE LIST
AA1 TO AA8
3)
AA9 TO AA13
4)
Ql TO Q3,
5)
Al
TO P9,
and optional
reading
statistics
INPUT MEDIUM
[DAT1-UK11
7)
INPUT FORMAT
FIXED(8FlO.
F3.0,2X,
2/5FIO.
below in the order
required
BFC
2/
2(F2.0,3X).
3(F3.0,2X,
F5. l),
2FS. 1. Fl. 0,2X,
17F3.0)
10)
NO OF CASES
192
11)
VAR LABELS
AA1, LYS/AA2, ASP/AA3, THR/AA4, SER/AAS, CLU/
12)
AA6, PRO/AA7. GLY/AA8, ALA/AA9, VAL/AA10,
13)
AA11, LEU/AA12,
14)
Ql, DATE OF REMOVAL OF HONEY FROM COMB/
15)
Q29RAS THE HONEY BEEN HEATED? YES OR NOI
16)
Q3, HAS THIS
17)
Pl, PREDOMINANT POLLEN>45%
18)
P2, PERCENTACE OF Pl/
19)
P3, SECONDARY POLLEN 16% - 45%/
used are
the data from the raw-input
TO A17
6)
_8)
9)
Pl
options
ILE/
TYR/AA13, PHE/
COLONY BEEN FED SUGAR? YES OR NOI
F3.0/
data
by the SPSS which
352.
20)
P4, PERCENTACE OF P3/
21)
P5, SECONDARY POLLEN 16% - 45%/
22)
P6, PERCENTACE OF P5/
23)
P7, PERCENTACE OF UNIDENTIFIED
24)
'P8, PERCENTACE OF SHRIVELLED
25)
P9, POLLEN CONTENT/
26)
BFC, ENCLISH HONEY OR FOREIGN AND COMMERCIAL HONEY/
27)
Al, a-ABU/A2,
28)
A5, CYS/A6, CLUC/A7, GLN/A8, HIS/A9,
29)
A10, TRP/All,
HPRO/Al2,
30)
A15, PIP/Al6,
p/Al7,
31)
VALUE LABEL
Ql
POLLEN/
ARC/A4, ASN/
y-ABU/A3,
(101)JANUARY
POLLEN/
MET/
$-ALA/
MEHIS/Al4,
HPIP/Al3,
X/
(103)MARCH
(102)FEBRUARY
(105)JUNE
(108)AUCUST
(109)SEPTEMBER
32)
(105)MAY
33)
(110)OCTOBER
(111)NOVEMBER
(112)DECEMBER
34)
(201)JANUARY
(202)FEBRUARY
(203)MARCH
35)
(205)MAY
36)
(210)OCTOBER
37)
Q2 (88)
YES (99)
NO (0)
UNKNOWN
38)
Q3 (88)
YES (99)
NO (0)
UNKNOWN
39)
Pl (999)
PRESENT
NOT PRESENT P3 (999)
NOT PRESENT PS (999)
NOT
40)
P2 (100)
PRESENT
NOT PRESENT P4 (100)
NOT PRESENT P6 (100)
NOT
41)
P9 (1) VERY LOW OR ZERO (2)
VERY HIGH (0) UNKNOWN
42)
Al
43)
(207)JULY
(206)JUNE
TO A18
(211)NOVEMBER
PRESENT (77)
(66)
(212)DECEMBER
(99
45)
(249
THRU 499-3)
46)
(499
THRU HICHEST-4)
(99
49)
(249
THRU 499-3)
50)
(499
THRU HICHEST-4)
HIGH (5)
UNKNOWN
THRU 249-2)
AA6(1OOO THRU 1999-1)
RECODE
52)
(1999
THRU 3999-2)
53)
(3999
THRU 5499-3)
54)
(5499
THRU HICHEST-4)
55)
MEDIUM (4)
THRU 99-1)
48)
51)
1982
THRU 249-2)
AA7 TO AA3(50
RECODE
(209)SEPTEMBER
NOT PRESENT (00)
44)
47)
(204)APRIL
(208)AUGUST
LOW (3)
1981
THRU 99-1)
AA1 TO AA5(5O
RECODE
(107)JULY
(104)APRIL
IF
(Pl
EQ 301)
POLLEN-1
56)
IF
(PI
EQ 308)
POLLEN-2
57)
IF
(Pl
EQ 318)
POLLENm3
58)
IF
(Pl
EQ 319)
POLLEN-4
59)
IF
(Pl
EQ 328)
POLLEN-5
60)
IF
(PI
EQ 305)
POLLEN-6
61)
IF
(Pl
EQ 304)
POLLEN-7
62)
IF
(Pl
EQ 309)
POLLEN-8
63)
IF
(Pl
EQ 326)
POLLEN-9
64)
MISSING
AAl
TO AA13
(0)/Ql
65)
DISCRIMINANT
VALUE
CROUPS-POLLEN(l.,
TO Q3(O)/Pl
9. )/VARIABLES-AAl
TO AA131
66)
ANALYSIS-AAl
67)
METHOD-MAHAL/
68)
MAXSTEPS-26/
69)
FUNCTIONS-3,100.,
70)
PRIORS-EQUAL/
TO P9(0)/Al
ld
71)
OPTIONS
5,6,7,8,9,10,11,12,15,18,19
72)
STATISTICS
1,2,3,4,5,6,7,8.9
73)
READ INPUT DATA
74)
FINISH
TO AA13/
TO A17(0)/
353.
In the above list
the 'VARIABLE LIST' statements 2 to 5, have been defined
of control :statements,
in the 'VAR LABELS' statements 11 to 30. The numerical
codes used for Identifying
some of the
have been defined in the 'VALUE LABEL' statements 31 to 42.
Furthers the numerical codes
variables
The structure
the pollen grains have been given in Table
of the data in the
used for identifying
.30.
'DAT1-UK1' have been defined
data file
'INPUT FORMAT' statements
in the
7 to 9, which can be read by
the SPSS.
(C)
Modification
of the SPSS Control
Listed
File
In Part
(b)
file
This modified
Given below is the modified structure
of the SPSS control
given above in part (b).
file
between the
contained modifications
which improved the discrimination
part of the SPSS control
file
listed
in part (b) was modified
from control
The SPSS control
statement 43
pollen groups.
onwards as listed
values into their
below.
These modifications
square roots
were to transform
the original
amino acid
statements 43 to 55.
by control
and are specified
concentration
honey samples which contained pollens whose percentage presence in each of the honey samples
These
statements 56 to 60 were used for the following
pollens.
was greater than 70%, the 'IF' control
TrifoZiwn
By using the control
the Brassica,
repens, Castanea, CaUuna, and Afyosotie.
pollens were:
distributed
random
statement 61, 'SET - TRUNC(UNIFORM(2.0))', the uniform (2.0) generates uniformly
To select
50% can be expected to be between 0 and 1.0.
Of these numbers so generated,
numbers between 0 and 2.0.
SET
The use of the trunc function
Therefore,
give O's for these numbers and I's for the others.
will
in which the expected probability
of the value 0 is 0.5 or some number close
will be a random variable
The SELECT - SET(O) specifies
that only those honey samples for which a set have a value of 0
to it.
function
be used to compute discriminant
will
The other
honey samples for
rate
cation
(Hadlaihull
Some of the processing
were included.
others
options
The structure
of 1, these will
in the SPSS control
specified
Details
of the modified
the samples will
be used for
be classified.
estimating
the misclassift-
1981).
This removed unnecessary
which were required.
other
which SET has a value
and Nie,
but all
coefficients,
analysis
of these options
section
file
not required
are given
of the SPSS control
listed
file
in part
in this
by Nie et al.,
given
below:
43) COMPUTE
LYS-SQRT(AAl)
44) COMPUTE
ASP-SQRT(AA2)
45) COMPUTE
THR-SQRT(AA3)
46) COMPUTE
SER-SQRT(AA4)
47) COMPUTE
GLU-SQRT(AA5)
48) COMPUTE
PRo-sQRT(AA6)
49) COMPUTE
GLY-SQRT(AA7)
50) COMPUTE
ALA-SQRT(AA8)
51) COMPUTE
VAL-SQRT(AA9)
52) COMPUTE
ILE-SQRT(AA10)
53) COMPUTE
LEU-SQRT(AA11)
54) COMPUTE
TYR-SQRT(AA12)
55) COMPUTE
PHE-SQRT(AA13)
56) IF
(Pl EQ 301 AND P2 GT 70.0)
POLLEN-1
57) IF
(Pl EQ 308 AND P2 GT 70.0)
POLLEN-2
58) IF
(Pl EQ 318 AND P2 GT 70.0)
POLLEN-3
59) IF
(Pl EQ 319 AND P2 GT 70.0)
POLLEN-4
60) IF
(Pl EQ 328 AND P2 GT 70.0)
POLLEN-5
61) COMPUTE
SET-TRUNC(UNIFORM(2.0))
62) DISCRIMINANT
GROUPS-POLLEN(1..5. )/VARIABLES-LYS TO PHE/
63)
SELECT-SET(O)/
64)
ANALYSIS-LYS TO PHE/
65)
METHOD-MAHAL/
in part
(b) were omitted
and
case and introduced
(1975).
(b) above has been listed
354.
66)
MAKSTEPS-26/
67)
FUNCTIONS-3,100., l. /
68)
PRIORS-EQUAL/
69) OPTIONS
2,3,5,6,7,8,10,11,12
70) STATISTICS
1.2,3.4,5,6,7,8,9
71) READ INPUT DATA
72) FINISH
(d)
Control
Statements
repens,
Castanea,
Year 1981 and 1982, and the Five Main Pollens
of Calendar
Calluna
of control
were used for distinguishing
year 1981 and 1982 were recoded
that
(ii)
the control
statements
2.
3.
4.
between the twelve
calendar
months.
to represent
These control
each year.
56 to 60 of the SPSS control
file
eiven
recoding
the numerical
the months of the calendar
below
statements listed
in part
(c)
Ql(202-212)
57) RECODE
Ql(102-112)
70) RECODE
Ql(203-212)
58) RECODE
Ql(103-112)
71) RECODE
Ql(204-212)
59) RECODE
Ql(104-112)
72) RECODE
Q1(205-212)
60) RECODE
Ql(105-112)
73) RECODE
Ql(206-212)
61) RECODE
Ql(106-112)
74) RECODE
Ql(207-212)
62) RECODE
Ql(107-112)
75) RECODE
Ql(208-212)
63) RECODE
QI(108-112)
76) RECODE
Ql(209-212)
64) RECODE
Ql(109-112)
77) RECODE
Ql(210-212)
65) RECODE
Ql(110-112)
78) RECODE
Ql(211-212)
66) RECODE
Ql(111-112)
79) RECODE
Ql(212-212)
67) RECODE
Ql(112-112)
80) IF
(Ql EQ 112) YEAR-1
68) RECODE
Ql(201-212)
81) IF
(Ql EQ 212) YEAR-2
by those given
Brassica
of the control
statements
below to specify:
pollen
79)
IF
(Ql
EQ 112 AND Pl
EQ 301)
YEAR-1
80)
IF
(Ql
EQ 212 AND PI
EQ 301)
YEAR-2
Trifolium
repens
pollen
79)
IF
(Ql
EQ 112 AND Pl
EQ 308)
YUR-1
80)
IF
(Ql
EQ 212 AND Pl
EQ 308)
YEAR-2
Castanea pollen
79) IF
(Ql EQ 112 AND Pl EQ 318) YEAR-1
80) IF
(Ql EQ 212 AND Pl EQ 318) YEAR-2
CaZZunapollen
79) IF
ld.ýosoti8
(Ql EQ 112 ANDPl EQ 319) YEAR-1
(Ql EQ 212 ANDPI EQ 319) YEAR-2
pollen
79) IF
(Ql EQ 112 AND Pl EQ 328) YEAR-1
80) IF
(Ql EQ 212 AND Pl EQ 328) YEAR-2
codes
All
69) RECODE
80) IF
5.
which were used for
Ql(101-112)
Given below are the modifications
1.
statements
56) RECODE
replaced
Tri-foZium
and MyosotiB
Given below is the structure
replaced
- Brassica.
abovel
79 and 80 in the above list
and were
355.
(a)
Control
Statements
below
Given
are
of
Heating
modifications
and Sugar
of
the
Feeding
control
79 to
statements
80 from
(d)
to
specify:
Heating
(ii)
(f)
79) IF
(Q2 EQ 88) HEAT-1
80) IF
(Q2 EQ 99) HEAT-2
Sugar
feeding
79)
IF
(Q3 EQ 88) SUCAR-1
80)
IF
(Q3 EQ 99) SUCAR-2
Control
Statements
of the Four Combination
Given below is the list
of Bested and Sugar-fed
Groups
the four various combinations
of the
statements which specified
These control
two questionnaire
questions number 5 and 6 into two groups, that is, 'HEATSF1 and 2'.
control
statements 56 to 60 given in part (c) above.
statements were used to replace the 'IF'
(i)
(ii)
(iii)
(iv)
the 'heated
of control
and sugar-fed'
group and the
tnot
heated
56) IF
(Q2 EQ 88 AND Q3 EQ 88) HEATSF-1
57) IF
(Q2 EQ 99 AND Q3 EQ 99) HEATSF-2
the
'heated
and not sugar-fed'
group and the
'not
heated
56) IF
(Q2 EQ 88 AND Q3 EQ 99) HEATSF-1
57) IF
(Q2 EQ 99 AND Q3 EQ 88) HEATSF-2
the 'heated
and not sugar-fed'
group and the
'not
heated
56) IF
(Q2 EQ 88 AND Q3 EQ 99) HEATSF-1
57) IF
(Q2 EQ 99 AND Q3 EQ 99) HEATSF-2
the 'heated
and sugar-fed'
group and the
'not
and not sugar-fed'
heated
56) IF
(Q2 EQ 88 AND Q3 EQ 88) HEATSF-1
57) IF
(Q2 EQ 99 AND Q3 EQ 88) HEATSF-2
and sugar-fed'
groups:
groups:
and not sugar-fed'
and sugar-fed'
groups:
groups:
356.
APPENDIX VII
DISCRIMINANT
(a)
A Typical
ANALYSIS OF THE FOREICN AND COMMERCIAL HONEYS
SPSS Control
for
File
the Foreign
and Commercial
Honeys
file
Given below is an SPSS control
between country groups from the
that was used to discriminante
foreign and commercial honey samples.
file
is similar
This SPSS control
in structure
to that listed
in Appendix VI (b).
The only differences
are in the 'NUMBEROF CASES's 'VAR LABELS', 'VALUE LABELS'
and 'IF'
to select
control
In this
statements.
the following
case the
'IF'
These countries
countries.
control
were:
the SPSS
statements 54 to 58 specified
Australia,
Canada, Mexico, New Zealand
and England commercial.
The structure
file
of the SPSS control
typed in at a visual
display
is listed
below in the order
required
by the SPSS which was
terminal.
unit
1) RUN NAME
A TEST OF DISCRIMINANT ON FOREIGN AND COMMERCIALHONEYS
2) VARIABLE LIST
W
3)
AA9 TO AA13
4)
Ql TO Q3, Pl TO P9. BFC
5)
Al TO A17
6) INPUT MEDIUM
IFC11
TO AA8
7) INPUT FORMAT FIXED(SF10.2/5FlO.
8)
F3.0,2X, 2(F2.0,3X),
9)
17F3.0)
2/
3(F3.0,2X,
F5.1), 2F5.1, Fl. 0,2X, F3,0/
10) NO OF CASES
64
11) VAR LABELS
AA1, LYS/AA2, ASP/AA3, THR/AA4, SER/AA5. GLU/
12)
AA6, PRO/AA7,GLY/AA8, ALA/AA9, VAL/AA10, ILE/
13)
AA11, LEU/AA12, TYR/AA13, PHE/
14)
Ql, COUNTRYOF ORIGIN/
15)
Q2, /
16)
Q3, HAS THIS SAMPLEOF HONEYBEEN COMMERCIALLYPROCESSED?
YES OR NOI
17)
PI. PREDOMINANT POLLEN>45%/
18)
P2, PERCENTAGE OF Pl/
19)
P3, SECONDARY POLLEN 16%
20)
P4, PERCENTAGE OF P3/
21)
P5, SECONDARY POLLEN 16%
22)
P6, PERCENTAGE OF P5/
23)
P7, PERCENTAGE OF UNIDENTIFIED
24)
P8, PERCENTAGE OF SHRIVELLED POLLEN/
25)
P9, POLLEN CONTENT/
26)
BFC, ENGLISH HONEY OR FOREIGN AND COMMERCIAL HONEY-
27)
Al, a-ABU/A2,
28)
A5, CYS/A6, CLUC/A7, GLN/A8, HIS/A9,
29)
A109TRP/All,
HPRO/Al2,
30)
AlS, PIP/Al6,
P/Al7,
31)
VALUE LABEL
45%/
POLLEN/
ARG/A4, ASN/
y-ABU/A3,
Ql(OO1)AUSTRIA
45%/
MET/
HPIP/Al3,
MEHIS/Al5,0-ALA/
(002)AUSTRALIA
(003)ARGENTINA
X/
32)
(005)CAYMAN
ISLAND
33)
(009)GREECE
(01O)HUNGARY
34)
(013)MEXICO
(014)NEW
35)
(017)UNKNOWN COUNTRY ORIGIN
STARCH (020)GOLDEN SYRUP
36)
Q3,
37)
Pl (999) NOT PRESENT P3 (999)
NOT PRESENT
NOT PRESENT P5 (999)
38)
P2 (100) NOT PRESENT P4 (100)
NOT PRESENT
NOT PRESENT P6 (100)
(44)
(006)CHINA
(007)CHILE
(004)CANADA
(011)INDIA
(012)MALTA
ZEALAND (015)RUSSIA
PROCESSED (55)
(008)FRANCE
(018)ENGLISH
NOT PROCESSED (00)
(016)SPAIN
(019)POTATO
UNKNOWN
357...
39)
P9 (1) VERY LOW OR ZERO (2) LOW (3) MEDIUM (4) HIGH
(5) VERY HIGH (0) NOT POLLEN
40)
Al TO A17 (66) PRESENT(77) NOT PRESENT(00) UNKNOWN
41) COMPUTE
LYS-SQRT(AAl)
42) COMPUTE
ASP-SQRT(AA2)
43) COMPUTE
THR-SQRT(AA3)
44) COMPUTE
SER-SQRT(AA4)
45) COMPUTE
GLU-SQRT(AA5)
46) COMPUTE
PRO-SQRT(AA6)
47) COMPUTE
GLY-SQRT(AA7)
48) COMPUTE
AIA-SQRT(AA8)
49) COMPUTE
VAL-SQRT(AA9)
50) COMPUTE
ILE-SQRT(AA10)
51) COMPUTE
LEU-SQRT(AA11)
52) COMPUTE
TYR-SQRT(AA12)
53) COMPUTE
PHE-SQRT(AA13)
54) IF
(Ql EQ 002) COUNTRY-1
55) IF
(Ql EQ 004) COUNTRY-2
56) IF
(Ql EQ 013) COUNTRY-3
57) IF
(Ql EQ 014) COUNTRY-4
58) IF
(Ql EQ 018) COUNTRY-5
59) COMPUTE
SET-TRUNC(UNIFORM(2.0))
60) DISCRIMINANT
S. )/VARIABLES-LYS TO PHE/
GROUPS-COUNTRY(1.,
61)
SELECT-SET(O)/
62)
ANALYSIS-LYS TO PHE/
63)
METHOD/MAHAL/
64)
MAXSTEPS-26/
65)
FUNCTIONS-3,100., l. /
66)
PRIORS-EQUAL/
67) OPTIONS
2,3,5,6,7,8,10,11,12
68) STATISTICS
1,2,3,4,5,6,7,8,9
69) READ INPUT DATA
70) FINISH
(b)
Control
Listed
Statements
of the Country,
below are the control
of the SPSS control
file
Floral
statements
given
in Part
Source,
(Ql EQ 002) COUNTRY-1
55) IF
(v)
Types and Commercial Processing
which were used for
(a) above.
(1) 54) IF
(111)
Pollen
(11)
replacing
the control
statements
54 to 58
54) IF
(Ql EQ 002) COUNTRY-1
(Ql EQ co4) COUNTRY-2
55) IF
(Ql EQ 004) COUNTRY-2
56) IF
(Ql EQ 013) COUNTRY-3
56) IF
(Ql EQ 006) COUNTRY-3
57) IF
(Ql EQ 014) COUNTRY-4
57) IF
(Ql EQ 013) COUNTRY-4
58) IF
(Ql EQ 014) COUNTRY-5
54) IF
(Ql EQ oo2) COUNTRY-1
(iv)
54) IF
(Ql EQ 002) COUNTRY-1
55) IF
(Ql EQ 004) COUNTRY-2
55) IF
(Ql EQ 004) COUNTRY-2
56) IF
(Ql EQ 006) COUNTRY-3
56) IF
(Ql EQ 006) COUNTRY-3
57) IF
(Ql EQ 008) COUNTRY-4
57) IF
(Ql EQ 008) COUNTRY-4
58) IF
(Ql EQ 013) COUNTRY-5
58) IF
(Ql EQ 011) COUNTRY-5
59) IF
(Ql EQ 014) COUNTRY-6
54) IF
(Ql EQ 008) COUNTRY-1
54) RECODE
Ql(009-001)
55) IF
(Ql EQ 016) COUNTRY-2
55) RECODE
Ql(016-008)
56) IF
(Ql EQ 018) COUNTRY-3
56) IF
(Ql EQ 001) HONEY,l
57) IF
(Ql EQ 008) HONEY-2
(vi)
358.
(vii)
(ix)
(c)
Control
54) IF
(Pl EQ 308) POLLEN-1
55) IF
(Pl EQ 337) POLLEN-2
54) IF
(Q3 EQ 44)
PROCESS-1
55) IF
(Q3 EQ 55)
PROCESS-2
Statements
used for
Distinguishing
Between United
(1) 54) IF
(BFC EQ 000) COUNTRY-1
55) IF
(BFC EQ 001) COUNTRY-2
54) IF
(BFC EQ 000) COUNTRY-1
55) IF
(Ql EQ 002)
COUNTRY-2
56) IF
(Ql EQ 004)
COUNTRY-3
57) IF
(Ql EQ 006)
COUNTRY-4
58) IF
(Ql EQ 009)
COUNTRY-5
59) IF
(Ql EQ 011)
COUNTRY-6
60) IF
(Qj EQ 018)
COUNTRY-7
0
(ii)
(viii)
54) IF
(Pl EQ 308) POLLEN-1
55) IF
(PI EQ 337) POLLEN-2
56) IF
(Pl EQ 325) POLLEN-3
Kingdom and Foreign
Honeys
359.
APPENDIX VIII
IDENTIFIED POLLEN CRAINS
Pollen
grains
have been listed
were identified,
that
The identity
below.
in the two hundred and fifty
as described,
was determined
of each grain
levels
at three
samples of honey
six
namely
of classification,
in that order.
The species of pollen for each genus are given as those
genus and species,
to be present according to those quoted by Sawyer (1975 and 1981) and Crane. Walker and Day
most likely
(1984).
CommonEnglish names equivalent
of the Latin botanical
names have also been included for reference.
family,
plant
TABLE VIII
of Pollen
List
compared with
grains
those reported
Family
Plant
in the U. K. survey and the foreign and commercial honey samples,
by Sawyer (1975 and 1981) and Crane, Walker and Day (1984)
identified
1
2
Genus
Species
(Botanical
Survev
U. K.
honev
3a
and
Comment
(English
name)
name)
samples
Aceraceae
Acer
paeudopZatanue
Sycamore
MT
Aquifoliaceae
IZex
aquifoZiwn
Holly
Mr
Balsaminaceae
Impatiene
glanduZifera
Policeman's
Betulaceae
AZnus
Alder
Boraginaceae
,
14yosoti8
gZutinosa
Arvensis
Forget-me-not
MT
Caprifoliaceae
sambucus
nigra
Elder
MT
Compositae
Carduu8
acanthoidea
Welted Thistle
MT
Helmet
Ox-eye daisy
to
Chrysanthemum
if
Taraxacwn
officinale
Dandelion
Cruciferae
Bras8ica*
napus
Rape
Ericaceae
CaZZuna
vuZgari8
Heather
(ling)
nr
Fagaceae
Ca8tanea
eativa
Sweet chestnut
VMT
to
Quercus
robur
English
Guttiferae
Bypericum
Rose of Sharon
Hippocastanaceae
AeocuZu.9
caZiciuum
hippoca8tanum
Horse chestnut
Labiate
Teucrium
scorodonia
Wood sage
Leguminosae
Lotus
cornicuZatu8
Birdsfoot
MT
oak
Trefoil
I/
Cont'd
KEY
1,2,3
-
title
is
Crane,
according
to
plant
species
Walker
as honey
and Day,
1984 and
the
to other
and Day,
possible
1984.
-
For
to
speices
-
Data
been
-
Data
-
Main
types
of
pollen
found
in
honeys
according
to
Sawyer
1975
and
1981.
Rare
types
of
pollen
found
in
honeys
according
to
Sawyer
1975
and
1981.
RHD
each
For reference
Crane,
Walker
a-
MT
The names below
-
reference
other
on chemical
and physical
Walker
given by Crane,
of
Brassica
of
composition
and Day, 1984.
the physical
on only
composition
by Crane,
Walker and Day, 1984.
given
Honeydew
honey
source.
of
to
refer
Unifloral
sources
Table
same genus
1981.
refer
to
7.0.
honeys
honeys
Unifloral
of
Sawyer,
of
of
this
this
pollen
pollen
source
source
have
have
been
360.
Table
VIII
cont'd
Plant
Family'
Genus
2
Species
(Botanical
Leguminosae
MeZitotua
name)
3a
Comment
(English
name)
aZba
White Melilot
V
VR
to
Onobrychia
viciifoZia
Sainfoin
11
2!r-ifOziW? I
incarnatum
Crimson Clover
it
N
praten8e
Red Clover
MT
to
ff
repene
White
Clover
MT
faba
Field
Bean
to
Vicia
AZliwn
Liliaceae
+ MT
Lily
if
Endymion
non-scriptue
Bluebell
to
Lilium
regale
Royal
Malvaceae
Malva
eyZve8tri8
Mallow
Oleaceae
Ligu8trum
vuZgare
Privet
Onagraceae
Epilobium
augu8tifolium
Rosebay
Pinaceae
Pinus
eylve8tri8
Scots
PolemDniaceae
Phlox
drummondii
Phlox
Polygonaceae
Fagopyrwn
e8cuZentwn
Buckwheat
Ranunculaceae
Mematis
vitalba
Traveller's
it
HelZeborus
niger
Christmas
Rosaceae
Cotoneaster
Lily
MT
Pine
HD
R
Joy
Rose
Cotoneaster
of
Fragaria
x ananassa
Strawberry
it
MaZue
domestica
Apple
of
Frunu, s
domestica
Plum
+
MT
to
Pyrue
commnis
Pear
if
Rubus
fruticosus
Blackberry
Salicaceae
SaZix
caprea
Sallow
Scrophulariacea
Linaria
vulgaris
Common Toadflax
Tiliaceae
Tilia
cordata
Lime
Umbelliferae
Heracteum
ephondyliwn
Hogweed
Urticaceae
Vrtica
dioica
Nettle
Violiaceae
Viola
tricolor
Pansy
English
Commercial
+ MT
(Willow)
+
MT
Samples
VR
Boraginaceae
Schiwn
vulgare
Viper's
Compositae
Helianthus
Qnnuus
Sunflower
R
Cruciferae
Bra8sica
vapus
Rape
MT
Ericaceae
CaUuna
vulgaris
Heather
Fagaceae
Castanea
sativa
Sweet Chestnut
Labialae
Roamrinus
officinaZie
Rosemary
Leguminosae
Lotus
corniculatus
Birdsfoot
of
Robinia
pseudoacacia
False Acacia
to
2rifoZiwn
pratanse
Red Clover
MT
repens
faba
White Clover
MT
to
of
ff
Vicia
Myrtaceae
EucaZyptua
Tiliaceae
TiZia
cordata
Field
Bugloss
(ling)
MT
MT
Trefoil
Bean
MT
Eucalyptus
R
Lime
MT
Cont'd
361.
Table
VIII
cont'd
Plant
Family
1
Genus
2
Species
(Botanical
Foreign
name)
3a
Comment
(English
name)
Samples
America's
Argentina
Compositae
Cirsium
Cruciferae
Braesica
Myrtaceae
Sucalyptus
to
Thistle
napue
Rape
Eucalyptus
-
-
-
Boraginaceae
Echium
vulgare
Viper's
Compositae
Cirsium
Cruciferee
Braseica
Leguminosae
Acacia
Acacia
it
MeZilotus
Melilot
to
Zrifozium
it MT
MT
Canada
to
Myrtaceae
-
napu8
Bugloss
of
Thistle
MT
Rape
MT
pratanse
Red Clover
MT
repen8
White
MT
Eucalyptus
Clover
Eucalyptus
Cayman Island
Bruseraceae
MT
Ericaceae
Tiliaceae
filia
Lime
Chile
Boraginaceae
Echium
pZantagineum
Purple
Bugloss
Bruseraceae
Compositae
Taraxacum
Dandelion
Labiatae
Myrtaceae
Mexico
Bruseraceae
MT
Compositae
Viguiera
Leguminosae
Acacia
It
Robinia
heZianthoidee
pseudoacacia
"Tah" Railway
Daisy
+ MT
Wattle
It MT
False
Acacia
Myrtaceae
Eýucalyptus
Polygonaceae
Fagopyrum
esculentum
Buckwheat
Proteaceae
Banksia
serrata
Red Honeysuckle
Tiliaceae
TiZia
Eucalyptus
Lime
Cont'd
.....
362.
Table
VIII
Plant
cont'd
Family
1
2
Genus
Species
(Botanical
name)
3a
Comment
(English
name)
Asia
China
Compositae
it
Cirsium
Thistle
Yaraxacum
Dandelion
Rape
Cruciferae
Bras8ica
Leguminosae
Acacia
Wattle
go
Lotus
Birdsfoot
It
Robinia
p8eudbacacia
False Acacia
of
2rifoZium
pratanse
Red Clover
repens
White Clover
faba
Field
It
of
of
napus
Vicia
Trefoil
Bean
+
Eucalyptus
Myrtaceae
Eucalyptus
Polygonaceae
Fagopyrum
Rutaceae
Citrus
Citrus
Tiliaceae
TiZia
Lime
Urticaceae
Urtica
Nettle
eacuZentwn
Buckwheat
Australia
Australia
Boraginaceae
Echium
pZantagenium
Purple
Cruciferae
Brasaica
napus
Rape
MT
Leguminosae
Acacia
Wattle
MT
Myrtaceae
EucaZyptus
Eucalyptus
MT
to
Protenceae
Banksia
eerrata
Bugloss
MT
MT
Red Honeysuckle
MT
Thistle
MI
MT
New Zealand
Compositae
Cirsium
Cruciferae
Brassica
napua
Rape
Leguminosae
2yifozium
repens
White
Myrtaceae
Eucalyptu8
MT
Clover
MT
Eucalyptus
Russia
Boraginaceae
Echium
Compositae
Cir8iwn
pZantageniwn
Purple
Bugloss
Thistle
Viguiera
heZianthoides
"Tah" Railway
Daisy
Cruciferae
Bra8sica
napue
Rape
Myrtaceae
EucaZyptus
of
+
Eucalyptus
Cont'd
.....
363.
Table VIII
cont'd .....
Plant
Family'
Genus
2
Species
(Botanical
name)
3a
Comment
(English
name)
Europe
Austria
Compositae
Cirsium
Thistle
Ericaceae
Erica
Heather/Heath
Fagaceae
Ca8tanea
Chestnut
France
Compositae
Helianthus
annuus
Sunflower
Cruciferae
Bra8sica
napus
Rape
Fagaceae
Castanea
Sweet Chestnut
11
Hippocastanaceae
AescuZus
Horse Chestnut
VI
Leguminosae
Acacia
Wattle
of
Rosaceae
IF
2rifoZium
repene
White Clover
Prunus
Fruit
Pyrus
Blossom
Greece
Compositae
Centaurea
Ericaceae
Erica
Leguminosae
Lotus
of
cyanus
Cornflower
Heather/Heath
cornicuZatus
Onobrychis
Birdsfood
Trefoil
Sainfoin
V,
Hungary
Compositae
Relianthua
annuus
Sunflower
of
Taraxacum
officinale
Dandelion
Cruciferae
Brassica
napus
Rape
Leguminosae
2-rifoZium
repens
White Clover
Robinia
paeudoacacia
False Acacia
to
Malta
Labiatae
Ro8marinua
Leguminosae
Lotus
it
Rosemary
corniculatus
Birdsfoot
MeZilotus
Mililot
Cirsium
Thistle
Trefoil
V,
IEILin
Compositae
MT
to
HeZianthu8
annuus
Sunflower
of
Viguiera
heZianthoides
"Tah" Railway
Daisy
Cruciferae
Bras8ica
napus
Rape
Leguminosae
Robinia
pseudoacacia
False
2rifolium
pratense
Red Clover
repene
White Clover
$I
It
IF
Rutaceae
Citrus
Salicaceae
SaZix
Citrus
caprea
willow
+
MT
acacia
ot
MT
rr
364.
APPENDIX IX
LIST
OF PUBLICATIONS
1.
Croft,
L. R.,
'Analysis
2.
session)
International
Electrophoretic
Mistry,
Washington,
R. P.,
'Correlation
at
3.
as poster
the
'lst
Washington,
'Analytic
Association,
at
the
'Fifth
Society'
with
Pollen
Bee Research
Workers
R. J.,
L. R. and Mistry,
Studies
on Honey'
Cardiff.
(1986)
-
of
the
L. R. (1986)
Analysis'
Colloquia'
International
Meeting
in London.
R. J. and Croft,
of Amino Acids
Croft,
R. J.
in Honey by Gel Electrophoresis'
the Proteins
of
(presented
R. P. and Washington,
Mistry,
(lecture)
in London.
R. P.
(1986)
Bee Research
POSSIBLE CONMBUTION TO BEE WORTD
the first
At
Bee Research
19th
London
on
at
held
Colloquium
February 1986j, papers were read by R. J. Washington, R. P. Mistry
S.
on behalf
Slight
of
work
Since 1975,, scine 27 graduate
our
the
with
supervision,
have been involved,
students
analysis
of
hive
the
(Miss M. A. Siddiqui)
levels
acid
(Mr.
R.
ýYs
fOr
with
rxallen-freel
floral
He also
carried
'7bis
technique
BBKA. '
the
amino acid
to
sources and ccrrputer predictions
out
a honey
was -shown
sanple
to
be feasible
the upper limit
is work progressing
even
and mis-
on the toxicity
which has been neglected.
of 9 mg HMF-per ml of fluid
of
He showed that,
prevented all
synthesis.
The mechanism of action
protein
synthesis
Showed, in vitro,
arrested
UK
of
of the protein
in the detection of adulteration
of honey, there
RW (Mr. S. Lee), a field
lens
survey
in heather honey.
as an interest
representation
protein
-source
saffriples.
responsible for thixotrophy
in vitro,
floral
of free amino
quantitation
Progress was also made on the characterisation
As well
under
and of honey (Mr. J. M.
patterns
and,, most recently,,
determined
P. Mistry).
for
of
relationship
of
7he major
products.
eaphasis has been on the amino acids of pollen
Carter),
Departnent
of Salford.
Sciences, University
Biological
by the
out
carried
and
protein
that
is
of bee venom, principally
being investigated
changes to lens structure,,
by Mr. S. Slight.
on
He
injected,
bee
10
per
ml
of
vencm
ug
of
levels
synthesis
melittin,
in
the eye lens
resulting
and caused irreversible
in opacity.
R. J. Washington
L. R. Croft
Washington, R. J., Croft, L. R. and Piistry,, R. P. (1986) Analysis
honey. Paper presented, Iv-don, 19th February
of
Croft# L. R.'j %shingtont
R. J. and Kistryl R. P. J1986) Final report.:
Amlysis
The
Reekeepers'-A"oci&Uon
Yiar
Book,
of honey.
-British
June, p2O
Croft,
L. It-# Kistryt
R. F. and Wshingtont
R. J. (1986) Arýalysis
_' SO Int.
the Proteins
Conf.
of honey by gel electr6phorasis.
Electrcphoresiss
Icndbnl
September (to be- pub: 'Springer-Werlag)
of
ýbri.
the 4,6a-e
Mi
R.
lvl,
P.
M;
s
'07( -re
pill h c4lf-Plo"
te yp. tv, 4.4 4
7W7/f'7
ANALYSIS OF THE PROTEINSIN MONEYBY GEL ELECTROPHDRESIS-.
,
L. R. Croft,
R. P. Pistry
Department
of Biological
Vniversity
of Salford.
and R. J. Washington
77
Sciences,
Salford
N5 4WT. U. K.
I INTRODUCTION
The adulteration and misrepresentation of honey
ari major threats to the viability
of the apicultural industry (1).
At this present time honey may be subject to
adulteration with high fructose syrups.
Although-these may be-detected in honey by
stable isotope analysis. the Instrumentation necessary Is-extremely tostly bindnot*
ily available to manypublic analysts (2).
In addition the technique is not
'read- to detect
able
the presence of high fructose syrups prepared from C3 Plants (3)'.
On the
other
hand.
(4).
but
palynology
sieved
so
that
most
misrepresentation
this
of
the
is
not
pollen
honey
may -be V*odflydetected--by-fwlfsslý-ýI
the honey ýduring
prockssine-hat'b"n"hiolyso if
'has been nmwve;.
of
apýr;
'xiýýiely
0.5% protein. : These.include specific.
Moneyis knownto contain
ihthese
od
hence
i
go
bee,
are
from
of
the
directly
derived
reducedlevels
enzymes
,
also
Other
'These,
present,
has
ate
ptDteins
dication that adulteration
OccurredIn the main, are deri ved f rom the pol I en, or pi ant-i-Octar%,and SWstrve iks 'the- basi s-,
for the determination Of the floral, and so, geogriphic4l to-igin of'the h6nVy.
Our proc*4riA-4j-JwmIYes.
jmAIysis by PAGE
-el ectrophoresi &:of j protei rv concentrates
bdultersti=-`and-`ý
derived from honey and is a simple and effective
means-forldetetting
misrepiesenution.
2 METHODS
The proteins pýesent-16 i-sample of honey are concentrated by ultrafiltration
(Amicon Standard cell fitted
with a UKID membrane).- This process. which is carried
out at 40C, is continued. after repeated dilution -of the swiple, until the
"'*-'
The resulting,
is negative for sugar.
mainly protein toncentiate! iS-freeii-dried.
polyscrylamid,.
sodium
after
dodecyl
destaining
gel
sulphate
electrophoresis
(6).
are scanned
Gels
of the
samples
are stained
is
Cor&cted
with-Commassit.
on on ISCO model 659 scanner.
'-
in, the
presence
brilliant-Blue
'.. , --I
ýt---ý
and
of
-
3 RESULTS
Because of the low levels of proteins in honey it was found to be necessary to
ýý
AX
submit the sWles to a preliminary concentration
.in,
separation of the proteins in the concentrates was ochievedIX-eloctrophoresis.
the presence of SDS, on 10%polyacryl&mide gels.
In this way it was found that as many as eleven protein bonds could be rtsolved
in the honey samples. The presence or absence of any of these bands was used to
j::
*IIir-honey simple with regard to its likely geographical
characterize
origin.
The technique is also &ble. to detect adulteration and in fact is so sensitive
been
had
that
honeys
obtained from bees fed on
detect
to
substitutes.
-pollen
as
4 ACKNDWLEDGEW-WT-ý-ý-u-ýý='ý-ý'-ý
WewDuldlike to thank the British Beekeepers'Asoclation. for fl. n#ncipl support.
5 REFERENCES
(1)
L. R. Craft,
tritish
see a. JOB (19BO) 231-234-
-_ ::
J. W. White, L. W. Boner, J. Assoc. Off. lknal. Chm... fil
J. W. Trumpeterjjwr,
-kt-4.,,
--.
1ý
-. I--
) 7ýk-750.,.
_11978.
a2j (1981) 857-W,,
(4)
R.W. Sawyer, Pollen Identification
1981.
(5)
R-P- MistrY,
(6)
K. Weber,-_,
#L.
for Beekeepers, Ujversity-
Ph.D. Thesis. University
of Salford,
1986..
Chem. 244 (1969). 4406-4412,e.
-ý%-
of-, Cardiff
,_-,
-
I
_ L- .