RHEOLOGICAL PROPERTIES OF YEAST CELL WALLS
by
PEI-SYAN LIU
B.S. National Taiwan University
(1978)
SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE
DEGREE OF
MASTER OF SCIENCE
at the
Massachusetts Institute of Technology
January 1981
Massachusetts Institute of Technology
Signature of Author ...........................
Department
Certified by
..,.........
Nutrition and Food Science
January 16, 1981
........ Professor A.J. Sinskey
Thesis Supervisor
Accepted by ........................................
Chairman, Department Committee
ARCHIVE
MASSACHUSETTS INSTiTUTE
OF TECHNOLOGY
API 1 4 11981
LIBRARIES
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1 -
Rheological Properties of Yeast Cell Walls
by
Pei-syan Liu
Submitted to the Department of Nutrition and Food Science
on January 16, 1981 in partial fulfillment of the
requirements
for the Degree of Master of Science
ABSTRACT
Chemical analysis, rheological measurements and hydrocharacterize
employed to
been
studies have
dynamic
structural and morphological properties of yeast cells and
cell wall components.
Saccharomvces cerevisiae A364A and its morphological
The mutant
mutant JD7 were selected for this study.
exhibits dimorphic phenotypes, i.e. mycelium-like and yeastThe
like, depends on the nutritional status of the medium.
cell wall components, which consist primarily of glucan and
alkali insoluble glycogen, were prepared by heating cells in
Microscopic examination showed that the alkali
alkali.
insoluble residues remain in the shape of whole cells.
The carbohydrate compositions of various morphological
form of yeast were also investigated. The glucan content of
mycelium-like cells of JD7 is higher than that of yeast-like
form,
whereas the mannan content is
higher in
strain A364A.
branched and
indicated that
measurements
Rheological
aerevisia.e JD7
exhibit a
elongated cells
of Saccharomyoes
much higher viscosity than spherical cells of A364A and have
a lower critical concentration,
while the yeast-like cells
viscosities
with a
critical
of
JD7 have
moderate
concentration inbetween the other two morphological types.
cell wall
the viscosity profiles of
In comparing
components and those of whole cells, we found that the cell
wall components exhibit a viscosity 3 to 4 times higher than
This suggests that the number of
that of the whole cells.
cell units in the suspension has a dicisive effect on the
viscosity.
Yield stress of certain types of cell wall components was
to other
hydrocolloidal
comparable
and
extremely high
polysaccharides, such as carboxymethyl cellulose.
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2 -
Hydrodynamic volumes of these materials estimated by
sedimentation studies were high and suggesting that a large
amount of water is associated with the cell wall aggregates
during sedimentation.
The water may either be occluded
within the sac-like cell wall components or be entrapped
between the loosely packed
settling aggregates.
The
thickening effect,
the high yield stress as well as the
large hydrodynamic volume suggest a possible use of yeast
cell wall as a food thickening agent.
Thesis Supervisor: Anthony J. Sinskey
Title: Professor of Applied Microbiology
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ACKNOWLEDGMENT
I would
like to express
advisor, Professor
couragement,
years.
In
A.J.
guidance,
addition,
my grateful appreciation
Sinskey,
his
I wish to thank Professor
also grateful
constant
en-
and understanding over the past two
providing valuble advises for
I am
for
to my
to all
C.K.
Rha for
this research.
of
my lab-fellows
for their
friendships and criticisms.
Mr.
Y.C.
Doo
consulting in
Finally,
deserves
special mention for
computer-formatting
I wish to dedicate
his helpful
this thesis.
this thesis to the love of my
parents.
This
Grant No.
work is
supported
ENG 76-02871
by
National
Science
Foundation
and a grant from ITT Corporation.
TABLE OF CONTENTS
page
TITLEPAGE ..................
ABSTRACT
.
.....
. . . .
.....
.
.
ACKNOWLEDGMENT
...............
.
LIST OF TABLES
2
4
5
.
.................
......
.
.
.
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LITERATURE SURVEY .
.
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LIST OF FIGURES . ..........
.
1
...................
TABLES OF CONTENTS
INTRODUCTION
.
...
.
7
.
.
.
8
.
.
10
.
.
.
.
Applications of Yeast and Yeast fractions .
.
.
.
12
.
12
Application of Conditional Mutants in Single Cell
. .
Protein Production
.
.
.
.
.
.
.
.
14
..
.
15
.
16
. . .
18
Rheological Properties of Yeast Cell Suspensions
effects of volume concentration on the
theological behavior of microbial
.
suspensions
.
.
.
.
.
.
.
.
.
.
.
.
.
effects of shear rate on the flow behavior of
.
microbial suspensions
.
.
. .
. .
effects of morphology on the rheological
....
behavior of microbial suspensions
20
effects of cellular interactions on the
rheological behavior of microbial
.
suspensions
. .
.
. .
.
.. .
.
.
.
.
23
Chemicl and Structural Properties of Yeast Cell
Wall
.
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25
Application of Cell Walls as Food Thickening
Agents
. .
MATERIALS AND METHODS
.
.
.
.
.
......
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.
.
. .
.
.
. .
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. .
.
.
Yeast Strains . . . . . . . . . . . . . . . .
Growth Media
. ..................
Yeast Fermentation . ...............
Recovery and Preparation of Cell Fractions
.
Dry weight measurement
. . . . . . .
. . .
Chemical Analysis . . . . . . . . . . . . . .
-5
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.
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27
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30
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30
30
30
. . 32
. . 32
. . 33
.
protein assay
.
.
.
..
..
. .
.
.
.
.
Measurements of Physical Properties .
carbohydrate analysis
apparent viscosity .
yield stress .
.
.
.
..
Sedimentation properties
.
settling rate
.
.
.
.
hydrodynamic volume
.
.
.
.
RESULTS AND DISCUSSIONS
.
.
..
.
Microscopy
.
.
. . . . . . .
Morphology
Chemical Composition .
Rheological Properties of
viscosity profiles .
.....
yield stress
hydrodynamic volume
CONCLUSIONS . .
FIGURES .
...........
REFERENCES
. .
.
.
.
.
.
.
.
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
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·
33
33
.
34
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.
.9
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.
a
.
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34
35
a
.
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.
.
35
35
.
Yeast
. . .
. . .
. . .
.
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.Q
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36
37
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. . . . 38
Suspensions
.
.
.
.
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.
r·····0
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. 47
. . . . 50
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-
. . . 72
.
6.
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LIST OF TABLES
Table
page
1.
Composition of Media
2.
Morphology of Yeast Cells on Different Growth Media . 38
3.
Chemical Composition of Yeast Cells .........
4.
Chemical Composition of Yeast Cell Wall . ...... . 42
5.
Hydr~lnamic Volume and Mean Aggregate Diameter of Yeast
Cell Walls .......
. . . . ..........
. . . . . . . . . . 46
...............
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7
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31
40
LIST OF FIGURES
Figure
page
1.
Chemical Structure of Yeast Mannan . .......
2.
Schematic Structure of Yeast Cell Wall .
...
3.
Flow Sheet for Experimental Procedures .
..
4.
Extraction of Carbohydrate from Yeast Cells
5.
Three Dimensional Structure of Mycelium-like
Saccharomyces cerevisiae JD7 Aggregates .....
.
.
.
. 51
. 52
...
.
50
.
.
.
53
54
6.
Collapsed Cell Wall of Mycelium-like Saccharomyces
. . . . . . 54
. . . . . . . . ...
cerevisiae JD7
7.
Morphology of Saccharomyces cereviisiae A364A
8.
Cell Wall Residues of Saccharomyces cerevisiae A364A
9.
Morphology of Saccharomyces cerevisiae JD7 grown in
SDC .e e. .
. . . . . ..
........................
10.
12.
14.
.
55
55
.
.
.
.
.
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.
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.
.
.
.
.
.
55
Morphology of Saccharomyces cerevisiae JD7 Grown in
YPD . . . .
....
...................
....
. 55
Cell Wall Residues of Saccharomyces cerevisiae JD7
Grown in YPD
13.
.
Cell Wall Residues of Saccharomyces cerevisiae JD7
grown in SDC
11.
.
. .
.
.
. . . . . . . . . . . . ..
Effect of Shear Rate on Apparent Viscosity of
Saccharomyces cerevisiae A364A Cell Suspensions
55
.
56
Effect of Shear Rate on Apparent Viscosity of Yeastlike Saccharomyces cerevisiae JD7 cell
Suspensions . ...
....
...
...........
.
57
15.
Effect of Shear Rate on Apparent Viscosity of Myceliumlike Saccharomyces cerevisiae JD7 Cell
.
58
Suspensions ...................
16.
Viscosity Profiles of Three Different Morphologies . 59
17.
Apparent Viscosity-Shear Rate Plot of Cell Wall
Suspensions of Saccharomyces cerevisiae
.
A364A
60
18.
Apparent Viscostiy-Shear Rate Plot of Cell Wall
Suspensions of Yeast-like Saccharomyces cerevisiae
JD7 . . . . . . . . . . . . . . . . . . . . . . . 61
19.
Apparent Viscosity-Shear Rate Plot of Cell Wall
Suspensions of Mycelium-like Saccharomyces
cerevisiae JD7
. . . . . . . . . . . . . . .
.
. 62
20.
Summary of Viscosity Profiles of Yeast Cells and Cell
Walls Suspensions . . . . . . . . . . . . . .
. 63
21.
Viscosity Profile of Cell Wall Suspensions of
Saccharomyces cerevisaie JD7 Grown in SDC
.
.
.
.
64
22.
Viscosity Profile of Cell Wall Suspensions of Myceliumlike Saccharomyces cerevisiae JD7 . .......
65
23.
Viscosity Profile of Cell Wall Suspensions of
Saccharomyces cerevisiae A364A
. ........
66
24.
Yield Stress-Concentration Relationships of Myceliumlike Cell Wall Suspensions and CMC
. ......
67
25.
Sedimentation Rate of Yeast-like Saccharomyces
cerevisiae JD7 Cell Wall Residues . ...
. .
26.
27.
Sedimentation Rate of Mycelium-like Saccharomyces
cerevisiae JD7 Cell Wall Residues . . . . . . . .
Q4. 6 5
69
Versus Volume Fraction for Cell Wall Residues of
Mycelium-like Saccharomyces cerevisiae
28.
. . 68
JD7
.
.
. 70
Q4. 65 Versus Volume Fraction for Cell Wall Residues of
Yeast-like Saccharomyces cerevisiae JD7 . .....
. 71
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9
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INTRODUCTION
food,
the variable of
applications in
Protein (SCP)
For sucessful Single Cell
many requirements
However,
safety must all be considered.
nutrition and
technology,
economy,
for SCP applications to food are sometimes incompatible with
the technological constraints of
objective of
the research program
this problem is to use
the size,
low cost production.
shape,
at M.I.T.
The
dealing with
powerful genetic techniques to alter
properties of yeast
and other
cells in
order that both the production process and the functionality
of the SCP and
its components could be
using conditional
stage-fermentation processes
being investigated.
In the first
interest are to be propagated
where
expressed.
A
desirable
stage,
Two-
mutants are
the organisms of
in the most economical manner
possible and then transfered to
reactor
improved on.
a second stage fermentation
phenotypic
properties
novel morphological mutant
may
be
of Saccharomvces
cerevisiae isolated by DeAngelo (1978) is one such potential
This dimorphic strain, Lac-
candidate for this application.
charomrces cerevisiae
cycle mutant,
is a conditional
JD7,
its phenotype
is controlled
nutritional factor.
-
10 -
cell division
by an
unknown
The objective of this thesis is to characterize, compare,
and analyze the
chemical and structural properties
morphological types of yeast cells
ponents.
of some
and yeast cell wall com-
Saccharomvces cerevisiae A364A and its morphologi-
cal mutant, JD7, are employed in this study.
applications of
some yeast cell
walls as
for food systems were also investigated.
-
11
-
The potential
thickening agent
LITERATURE SURVEY
This literature
background informations
survey collects
relevent to this thesis which includes the following:
1.
Applications of yeast and yeast fractions.
2.
Applications
of morphological
mutants in
Single
Cell Protein production.
3.
Rheological properties of yeast suspensions.
4.
Chemical and
structural properties of
yeast cell
wall.
5.
Use of cell walls as a food thickening agent.
ADDlications Qof Yeast Aand
Yeast throughout history,
and to brew ale,
eat fracti.ns
have been used to leaven bread
beer and wines.
yeast from brewing operations and
In the 1930's,
residual
yeast produced as baker's
yeast were dried and used in foods, as a source of vitamins,
minerals,
protein,
proteins and unknown nutrients.
dried food yeast contains
true protein
on a weight basis.
been used at
low levels in bread,
-
12
As a source of
approximately 33 to 40%
As such dried
-
cereals,
yeast has
peanut butter
mixtures and other
food proteins mainly as
vitamin supple-
ments.
fractions
Yeast
nutrients.
have also
Autolyzed
been
yeasts or
used
as a
of
source
disrupted by
yeast cells
sources of such fractions.
mechanical or chemical means are
In these cases, the protein may have been recovered from the
disintergrated yeast cells by alkaline extraction or solubilized by
autolysis due
to proteolytic
enzymes present
in
yeast.
In addition, yeast proteins may be used as a flavoring or
functional
ingredient.
Autolyzed
color and
agent.
for this
hydrolyzed
yeast
flavoring agent to give meaty or
extract has been used as a
nutty flavor in food.
or
Often yeast extract has a dark brown
reason it
is also
used as
a coloring
Yeast proteins may also be used as functional ingre-
dient such as surface active agents (Rha, 1975).
Production of Single Cell Protein (SCP)
of this sort is
one of the most promising new sources of protein that can be
produced
cesses
independently
of tranditional
and environmental
constraints.
world food and protein shortage,
agricultural
The
pro-
specter of
a
the urgent need for energy
and environmental conservation and reevaluation of the effeciency associated with food production have led to a continuing interest
in the possibility
Protein as a source of
of utilizing
food protein.
-
13 -
Single Cell
An additional advan-
Protein from microbial sources may
tage is that Single Cell
from processes
a by-product
result as
being developed
to
feedstocks from a variety
produce chemicals and/or chemical
of substrate, e.g. cellulose, n-paraffin ,etc..
Single Cell Protein
ADDlication of Conditional Mutants in
Production
and Bac-
formation (Stanffer
increasing product
useful in
to be
mutants has been found
Selection of morphological
kus,1974; Spizek, 1965; Blumauerova, 1969) in some fermenta-
employing
A
decrease the
and co-workers
by Miyasaka
fermentation
two stage
morphological mutants
temperature sensitive
been suggested
(1978,1980)
has
to
Single-Cell-Protein produc-
recovery costs in
In the first stage of this process, SCP organisms are
tion.
propagated under
where
Signals
desirable
for
and then
optimal conditions of production
transferred to
organisms are
the
1970).
and Herman,
Kurtzman
(Wiekerham,
processes
operation
in optimizing
and
tions
a
phenotypic properties
expression
of phenotypic
second stage
can
be
reactor
expressed.
properties
can
be
extrinsic parameters, such as temperature or level of oxygen
or intrinsic parameters,
or carbon dioxide,
cific
nutrients
(odo)
mutations,
Saccharomyvces
requirement.
strains, 4471, 374,
and 377,
such as a specerevisiae
harboring cell division cycle
were investigated for this purpose,
all
strains exhibited significant morphological changes when the
growth temperature was
C)
to
a non-permissive
shifted from a permissive
temperature (37 oC)
-
14i
-
due
(25 or 30
to their
inability to
complete the budding
process with
the conse-
quence that the cells enlarged and formed aggregates.
.of
lRheological Properties
i.e.
Rheological properites,
shear dependency,
recognized as
transfer,
types of
nomena in
etc.,
Yeast Cell
yield stress,
viscosity,
of microbial suspensions have been
parameters such
factors influencing
transport and heat transfer
momentum
fermentation operations
turn directly
and these
as mass
in various
transport phe-
ability to
affect the
maintain a
the production organ-
controlled homogeneous environment of
isms.
Suspensions
Furthermore, from the technical point of view, appli-
cation of rheology in microbial processes has special significances, because:
1.
Rheological measurements can
tural characterization
provide gross struc-
which is
not possible
in
complex systems using only biochemical analyses or
microscopic examinations.
2.
Some types
of rheological measurements
are rela-
tively simple and rapid.
3.
Once a correct structural model is developed based
on rheological
ments may
properties,
rheological
be adapted directly to
monitoring for control strategies.
-
15 -
measure-
on-line process
the rheological properties
Unfortunately,
of yeast sus-
pension has received very little attention in recent reviews
the
on
rheological
of
properties
(Blanch et al, 1976; Charles, 1978;
cell
concentration,
In other
Metz, 1978).
cell morphology,
broth,
mycelial
e.g.
microbial systems,
suspensions
microbial
intercellular interactions and rate of
shear are claimed to be
four major factors influencing vis-
cosity of microbial suspensions.
effects of volume concentration on the rheological behavior
of microbial susDensions
The Einstein equation:
fs = no (1 + 2.5 4s)
where
s=viscosity of suspension, no =viscosity of suspend-
ing medium,
cs
of spherical
=volume fraction
cells is applicable to Newtonian,
particles or
rigid spheres suspensions
and it becomes less applicable when concentration of suspensions increases
and the particles
deviate more
from ideal
rigid sphere.
Deindoerfer and West (1960)
of fractional volume
stein's
equation
reported
that at low values
viscosity could be correlated
whereas the
(1906),
equation
(1948):
Is
= To*(1
+ 2.5Ps + 7.25
-
16 -
Sq)
by Einof
Vand
applied to
may be
higher volume
suspensions at
fractions
(>.O.o0).
Many theoretical and experimental
studies have been made
to investigate the validity of the extended Einstein's equations, i.e.,
3
ns = ro (1 + a4s + b4s 2 +
The specific
structure of
these equations
...
as well
as the
value of the parameters were variant among published results
(Saito, 1950; Koga,1959; Aiba et al,
1962).
The discrepency
in the published results may in part be due
to the difficul-
ties in
determination of the
volume fraction
of microbial
cells (Reus, 1979) and the different methods for viscometric
measurements employed.
The higher terms
in these types of
equations bring into account
interactions due to collisions
between the particles in
absence of flocculation (Metz,
the
However, these types of relations are not completely
1978).
satisfactory over a wide range of cell concentration (Lee et
al.,1969;
Rao and Hang 1975;
Shimmons et al.,
influence of particle diameter is
neglected,
1970).
this can cause
large errors.
Mooney equation (1950):
In
=
ars
(1 -
where qp
(nlr
Ps/
Pmax)
=volume fraction
a = Einstein-constant
-
17 -
=
The
ls/l O
)
4max
=maximum packing density
for 4s<0.
2
, a =2.5
is one of the frequently used semi-empirical relations, that
particle diame-
are a function of
contain parameters which
ter.
effects of shear rate on the flow behavior of
microbial
suspensions
of fluid the Power
To quantatively express flow behavior
Law equation,
,n
T =
To + KY
the shear stress (T),
(K),
The equation relates
satisfactory.
is in general the most
yield stress (To),
consistency index
rate of shear (j) and flow behavior index (n).
represent
Newtonian,
the values of con-
shear thickening properties depending on
stants (Charm, 1963;
For dilute
Rha, 1975).
suspensions of
the system
single paramenter,
exhibited
characterics.
The
behavior index of
can be
completely represented
by
the 'Newtonian viscosity'.
Concentrated microbial
generally
ideal flow
In such a case, the flow charac-
unity is usually observed.
teristics of
spherical cells,
yield stress and a flow
behavior with no
and
Pseudoplastic
plastic,
Bingham
It can
systems and
pronounced
non-Newtonian
existing models
-
18 -
mycelial suspensions
rheological
for filamentous
mould
suspensions includes
the following
(Blanch and
Bhavaraju,
1976):
(1)
Bingham model
1961;
(Solomons and Weston,
Deindoerfer
and West, 1960)
T = To + q
In this model, the flow behavior of mould suspension is described
as Bingham
includes the
the fluid
plastic.
This
yield stress which
will flow while the
means
that this
has to be
model
exceeded before
flow behavior at
high shear
stresses is Newtonian.
(2) Pseudoplastic model (Deindoerfer, 1960)
T = K
n
+ To
+
where n<1
This model
implies that
the rate
of shear
than in proportion to the shear stress.
this sort
indicates that continueous
rangement of the structure,
flow (Rha,
Shear dependency of
broken down
or rear-
resulting in less resistance to
1978).
(3) Casson model (Bongenaar et al.,
V/
= /TO + Kc/7
suspensions has
1973;
Reol
been
by Casson (1959)
applied
1974)
19 -
for pig-
successfully to
large variety of suspensions (Kooyman, 1971;
-
et al.,
; K c = Casson Viscosity
This model is originally derived
ment-oil
increases more
Charm, 1963).
a
Because all these models do not follow Newton's Law, i.e.
the rate
of shear
shear stress.
ent viscosity
to the
in proportion
does not increase
It is more convienent to deal with an appardefined as the
which is
ratio of
the shear
a given value of shear rate.
stress to the rate of shear at
useful when it is
The apparent viscosity is especially
det-
ermined at the pertinent shear rate for a practiclal purpose
(Rha, 1978).
effects of morDholoRY on
microbial
It is
A& rheological behavior of
suspensions
well known from the
the particles
literature that the
1960).
of a
on the viscosity
has a profound effect
suspension (Sherman,
shape of
Carilli
et al.
have
(1961)
shown quantitatively that cellular morphology influences the
viscosity
three
of mould
were
morphological types
compared:
small pellets and a filamentous suspension.
viscosity
by a
factor of
niger,
For AsDergillus
suspensions.
three was
large
pellets,
A difference
reported between
filamentous suspension and the large pellets.
in
the
The viscosity
of the small pellet in suspensions was found to be inbetween
the filamentous and large pellets.
Reols et al (1974)
developed a mathmatical model,
based
of Casson model, for the rheological behavior of filamentous
mould suspensions.
They treated the
particles like polymer molecules and
filamentous mycelial
assumed that each par-
ticles was coiled to give a spherical shape.
-
20 -
An additional
assumption
was
that
the spherical
linear agglomerates.
model to
Their assumptions
be applied.
volume' concept
morphology,
The
aggregate
into
allowed the Casson
workers employed
to establish a
volume fraction of
coils
the 'excluded
relation between
the mycelial particles and
the total
the particle
i.e.,
l-Cm
=s
where ýs =volume fraction of mycelial particles as spheres.
61
=morphology
factor
dependent on
Cm =biomass
mycelial particles.
ratio of
the
length-diameter
concentration.
Both Kc and T o are then a function of the volume fraction of
the mycelial particle (ýs) and thus of biomass concentration
and a morphology factor.
A final equation including a fac-
tor for the influence of morphology
of mould and the influ-
ence of the concentration of biomass was then deduced:
/
= /
where f(Cm)= no ½
Cm{l
M" + f(Cm)V/}
1
+ C1}
6 =morphology factor =f(6
)
Cm =biomass concentration
fo
=viscosity of suspending medium
Cl =constant
T =shear stress
I =shear rate
-
21
-
objections against this theoretical
The major
model are
(Metz, 1978):
1.
branched hyphae
between the
Network interactions
should not be considered as negligible.
2.
The
are coiled
the hyphae
assumption that
into
spheres is conflict with many microscopic examinations.
3.
The assumption of Casson that coils arrange in the
form of rouleaux with an orientation is questionable.
4.
The randomizing
effect of Brownian
motion essen-
tial for the validity of polymer rheological theories is absent in mould.
Metz (1978)
the rheology of mould sus-
in his review on
pensions concluded that the theoretical model of Reols et al
has too many objections against its assumptions to be a useful basis for further work,
the introduction of a
however,
morphological factor is of great value.
In Reu8 et al.'s studies (1979) on the viscosity of yeast
suspensions,
experimental results
equation of the form:
ls/To
=
1/(1 -(hs..
-
s)a)
22 -
were
correlated by
an
where parameter 'a'
depends on the morphology
,while h s is a function of the osmotic pressure.
tion suggests that
as h s
approaches a value
Thus, I/h
viscosity approaches infinity.
tated
as
maximum
volume
packing
of
s
of the yeast
The equaof one,
the
can be interpre-
suspensions.
The
of the osmotic pressure indi-
increase in h s at high values
cates that the volume fraction for close packing is decreasing.
effects f. cellular interactions on the
of microbial suspensions
At high concentrations,
in
suspensions
causes an
the rate of
viscosity and yield stress.
at which the viscosity of
infinity,
size,
partical interaction takes place
and further increases in
increase in
rheoloiical behavior
cell concentration
change in
the apparent
The concentration of suspension
the suspension begins to approach
namely critical concentration,
shape and surface nature of
is
function of the
the particle in the sus-
pensions.
The interaction between suspending particles in a suspension may be the result of (Rha, 1978):
1.
Physical
entanglement of
caused by large size and
or irregular shape.
- 23 -
molecules or
particles
high degree of branching
2.
co-valent,
Network formation caused by ionic,
or
secondary intermolecular or interparticle interactions.
3.
Crowding as a
of sol-
results of the elimination
vents.
One of the
most distinctive properties of
pensions is the entanglement of hyphae.
perties often play a role in
mycelial sus-
Visco-elastic pro-
these suspensions at every low
shear rates, while at sufficiently high shear rates the system flow like
a Newtonian fluid.
been proposed by Metz (1978)
behavior
of mold
interactions.
theoretical model has
to explained the non-Newtonian
suspensions
In this
A
based
model,
on the
intercellular
the existence of
a network
structure composed of aggregates is assumed.
Upon shearing,
the aggregates break
At
rates,
pletely,
the
aggregates are assumed
i.e.,
Flocculation is
are pulling
into small fragments.
there is
shear rates
to be broken
down com-
no continuous structural network.
still taking place
the cluster apart as
The viscoelastic behavior
high shear
but the
shear stresses
soon as they
are formed.
of mould suspensions at
can then be explained
network structure.
-
24 -
as a consequence
very low
of the
Chemial And Structural Properties _f Yeast Cell Vall
cell walls such as
Properties of yeast
are undoubtly
tion.
and biological activities
morphological features
strength,
based on
their mechanical
their particular
chemical composi-
Bartricki-Garcia (1968) pointed out that a close corthe
exists between
relation
which
represents 80-90%
wall,
are
composed of
acids,
methyl
1970).
Glucose and
dry matter
of the
amino sugars,
and
pentose,
pentoses
of yeast
hexoses,
of skeletal
charideds, such as chitin and glucans.
present mainly in the form
cell
hexuronic
(Bartnicki-Garcia,
N-acetyl-D-glucosamine(GlcNAc)
chemical elements
represent the
Polysaccharide,
among fungi.
fungal cell wall composition
and
taxonomic classification
usually
wall polysac-
The other sugars are
of various homo- and heteropoly-
saccharides, often in chemical complexes with protein.
Three major types of
polysaccharides that constitute the
yeast cell wall are (Farkas, 1979):
1.
Glucan:
The generic name
'glucan' covers a large
group of D-glucose polymers differing both in type
and in
relative proportions of
sidic bonds.
cell
walls
The most
are those
individual glyco-
abundant glucans of yeast
with
the
(Manner and Masson, 1969, 1973).
wall glucans contain mixed
B -1,3
8 -configuration
Most yeast cell
and 8 -1,6
glyco-
sidic bonds, which constitue the skeletal microfibrillar portions of the walls.
- 25 -
2.
Mannan: Yeast mannan is a polymer composed of protein and
two carbohydrate
moieties differing
in
their structure ane mode of attachment to the peptide (figure
1).
represented by
bone
to
linked
The
an a-1,6-linked
which
short chains
of
mannosyl
units
a-1,3-glycosidic
attached predominantly
a-1,2 bond.
is
polymannose back-
a -1,2- and
together by
bonds are
polysaccharide moiety
by means
The polysaccharide moiety
of an
is linked
via a diacetylchitobiose bridge by an N-glycosidic
bond to an asparaginyl residue in the protien part
of the molecule (Nakajima and Ballou, 1974).
second carbohydrate
sists
of short
moiety of
yeast mannan
mannoolig1saccharides
The
con-
containing
both a-1,2- and a-1,3-glycosidic bonds attached at
their reducing ends by
serine or threonine
an 0-glycosidic linkage to
residues or both in
the pro-
tein (Ballou, 1976; Cabib, 1975).
3.
Chitin:
Chitin,
a
exclusively located
of GloNAc
$-1,4 polymer
in yeast bud scar
is
(Cabib and
Bowers, 1971, 1975).
Based on availble chemical, biochemical,
and cytological evidences, Lampen (1968)
tural model
(figure 2).
for the cell
In his
wall of
model,
-
the
26 -
immunochemical,
suggested a struc-
Saccharomyces cerevisiae
innermost microfibrillar
layer, composed of insoluble ý-glucan, is linked via protein
to the outer wall layer, composed of manno-protein molecules
mutually
linked
by phosphodiester
polysaccharide moeties.
supposed to
tase,
of yeasts has
and others.
been also reported
P-glucan are
within the
enzyme inver-
The periplasmic
to contain
space
a large amount
(Gunja-Smith,1977,1974).
physiochemical properties,
walls.
their
cross-linking is
mannoprotein
The different polysaccharides,
cell
between
barrier that holds
the extracellular
acid phosphotase,
of glycogen
The phosphodiester
form a physical
wall structure
bridges
The
due
fulfill
crystalline
to their distinctive
specific function in the
polysaccharides chitin
the components responsible for
strength of the wall,
polysaccharides,
and
the mechanical
while the amorphous homo- and hetero-
often in association
with proteins,
play
the role of cementing susbstances and constitute the carbohydrate moieties of extracellular enzymes and cell wall antigens (Ballou,1974;
Raschke,
1974;
Gander,
1974;
Lam-
pen, 1968).
Application of Cell Walls as Food Thickening Agents
Many
hydrocolloids are
their unique textural,
istics, e.g.
widely used
in
foods based
on
structural and functional character-
they provide stabilization for emulsions, sus-
pensions, and foams and general
of these materials,
thickening properties.
Most
which are sometimes classified as gums,
-
27 -
are derived from natural sources, although some are chemical
Many stabiliz-
modified to achieve desired characteristics.
such as gum arabic,
ers and thickeners are polysaccharides,
guar gum, carboxymethyl cellulose, carrageenan, agar, starch
functions of
Some specific
and pectin.
hydrocolloids in
food include improvement and stabilization of texture, inhi-
emulsions and foams,
Hydrocolloids are generally
concentration of about 2%
food because
or less in
and the desired func-
many exhibit limited dispersability,
tionality is provided
baked goods,
improvement of icing on
and encapsulation of flavors.
used at
stabilization of
ice),
crystallization (sugar,
bition of
The
at these levels.
hydrocolloids in many applications
effeciency of
is directly dependent of
their ability to increase viscosity.
Use of plant cell wall as food thickening agent has first
Rheological proper-
been suggested by Holmes et al.
(1978).
ties of parenchyma cell wall ,
recovered from pressed cran-
berries are studied by Holmes et al.
The workers
have certain
(1978,
concluded that suspensions of
advantages over other
1977)
in
detail.
parenchyma cells
food thickeners
due to
their high water occluding capacity and large yield stresses
at low cell wall material concentrations (1-2%).
Sucher et al (1975)
proposed that insoluble residues from
yeast protein extraction processes, which consists of a mixture of
walls,
whole yeast
and
cell walls,
fragments of
some proteinaceous material,
-
28 -
yeast cell
have interesting
functional characteristics
that include
holding water
and
imparting thickening properties in aqueous food system.
In
addition,
they discovered that the addition of yeast glucan
to liquid food systems,
in
the proper proportions,
food product a fat-like mouthfeel
tains little or no fat.
low calorie products,
puddings,
sour
cream,
gives the
even when these food con-
This is very useful in formulating
such as salad dressing,
based dip,
foods.
-
29 -
and other
ice cream,
low calorie
MATERIALS AND METHODS
Yeast Strains
cerevisiae
Saccharomvces
A364A
its
and
morphological
mutant, strain JD7, were employed in this study.
stains
possess similar genotypes (a, ade 1,
lys 2, tyr 1, and gal 1),
mutant,
whose
These two
ade 2,
his 7,
while JD7 is a cell division cycle
blocked at
cellular division is
some stage
prior to nuclear division in the cell devision cycle.
This
and characterized by DeAngelo
mutant was orginally isolated
(1978).
Growth Media
Growth medium employed were
chemically defined
medium,
a complex medium, YPD, and a
SDC.
Yeast strains
maintained on YPDA agar slants at 40 C.
have been
The compositions of
media are as shown in table 1.
Yeast Fermentation
The fermentations
were carried out
in fermentor
of 7.2
liter total volume (New Brunswick Scientific, New Brunswick,
NJ).
The working volume was 5 liter and air flow rate was 1
vvm (volume of air per liter per minute).
The cultures were
stirred at 500 rpm with double turbine impeller.
-
30 -
TABLE 1
Composition of Media
Amount (%)
Component
. . . . . . . . . . . .
Dextrose (Bacto)
Peptone (Bacto) . . . . . . . . . . . . .
Yeast Extract (Bacto) . . . . . . . . . .
*pH adjusted to 5.5.
.
Dextrose (Bacto)
Yeast Nitrogen Base
Adenine Sulfite . .
Histidine . . . . .
. . . . . .
Uracil
Tyrosine
. . . . .
Lysine
. . . . . .
. .
W/O
. .
. .
. .
. .
. .
. . . . . .
amino acids
..
. ...
. . .. . .
. . . . . .
. . . . . .
. . . . . .
2.0
2.0
1.0
. . .
.
.
. . .
...
.. .
...
.. .
2.0
0.67
0.002
0.002
0.002
0.003
0.003
.
.
.
.
2.0
2.0
1.0
0.003
2.0
YIDA
Dextrose (Bacto)
Peptone (Bacto) . . .
Yeast Extract (Bacto)
Adenine Sulfite . . .
Agar (Bacto)
. . . .
.
.
.
.
.
.
.
.
............
. . .
. . .
. . .
. . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
+------------------------------------------------------------+
Temperature control was set at 300C, pH was maintained automatically at pH
foam formation,
5.5 by additionng of 1N
small quantities
NaOH.
To prevent
of 5 % solution of Antifoam
FG-10 (Dow and Corning, Midland, MI) were added as required.
Portions of a
glucose solution were added
to avoid Crabtree effect
lowed by
(Schaffeld,1980).
Klett-Summerson colorimeter
660nm.
-
31 -
at a
by batch feeding
Growth was folwave length
of
Recovery and Preparation of Cell Fractions
To collect and isolate cell wall fractions, the scheme as
is outlined
in figure 3
treatment',
washed cells were suspended
was followed.
For
'hot alkaline
in 1N NaOH at con-
centrations of 15-20 mg/ml,
heat for 10,20 or 30 minutes at
100 0C in a water bath.
equal volume of cold buffer was
An
then added to stop the reaction.
Supernantant and residues
were separated by centrifugation.
The residues were washed
once with 0.1N NaOH and twice with distilled water.
alkali results in disruption
Heating cell suspensions in
of cell wall and cell membrane structures,
molecules
within the
cells are
able to
such that macrodiffuse into
the
extracting solution, and also the alkali may solubilize protein within the cellular structure.
lx~
Aeiaht
measurement
Millipore filters (Type GS 0.22 um, Millipore Corp.,
Bed-
ford, MA) were employed in this assay, 2 ml or 5 ml portions
of cell
ters.
suspensions were filtered through
preweighted fil-
The yeast cakes were dried at 60 0C and weighed.
weight concentration of
the samples are expressed
dry mass/liter of suspension.
-
32 -
Dry
as gm of
Chemical Analysis
protein
assay
(Herbert et al,
1971):
Lowry method
assayed by the
wall fractions were
and cell
yeast cells
freeze dried samples of
Protein contents of
a 0.5
ml sample containing 0.5 to
The
1.0 mg of dried mass was mixed with 0.5 ml of 1N NaOH.
suspension is heated
for 15 mins.
2.5 ml of reagent A (*)
cool.
room temperature
hold at
at 100 0 C
and allowed to
was added and the sample is
for 20
minutes.
0.5
ml of
Folin-Phenol reagent was added and rapidly mixed.
ture is allowed to stand at
minutes..
1N
The mix-
room temperature for another 30
Absorbance at 660nm was read with a Gilford Spec-
trophotometer.
Bovine serum albumin
was used for prepara-
tion of the standard curve.
(*)
Reagent
immediately before
A was prepared
use by
mixing a,b,c, in the ratio of 100:0.5:0.5.
(a)4% Na2C04 in 0.1N NaOH
(b)2% CuSO4.5H20 in distilled water
(c)4% NaK tartarate in distilled water
carbohydrate
analysis
Fractions of
carbohydrate were
first extracted
step following the procedures shown in fig. 4.
carbohydrates were then assayed
method
Fractionated
by the Phenol-sulfuric acid
(Trevelyan and Harrison, 1956).
- 33 -
step by
Measurements DI Physical Properties
aparent viscosity
Concentrated yeast cell suspensions and cell wall suspensions were prepared in phosphate buffer (pH 7),
dilutions
were made
concentrate with
sample was placed in
Each
buffer.
from this
bath and
Viscosity
was mea-
sured at 25 oC with Brookfield Rotational
LVY,RVT,
MA).
Brookfield
Readings
Viscometer (model
Engineering Laboratories,
every revolution
were taken for
indicator attained a constant value.
rate was starting with lowest
to the highest speed
C
(for
phosphate
water
a 25
reach thermal equilibrium.
allowed to
and a series
Stoughton,
until the
Viscometer rotational
rotational speed and progress
LVT 0.5-100rpm,
RVT 0.3-60rpm).
The shear stress (T) and apparent viscosity (r)
were calcu-
lated from
Engineering
the following
equation (Brookfield
Lab.)
T
1=-r
shear rate
y
=
2wr
2 2r
ri 2 (r2
shear stress
-r 1 )
= A(FST/100)
2 L'ri 2
where ri = radius of spindle=1.2576cm.
r2 = radius of container=1.3811cm.
w = angular velocity of the measuring spindle.
N = revolution per second.
- 34
-
L = length of spindle=9.2398cm.
FST = full-scale torque
LVT:637.7 dyne/cm.
RVT:7187.0 dyne/cm.
A = dial reading.
y.eld stress
Yield stress of each sample was obtained by applying Casson equation(Casson,1959) which is:
VT =
To
v/o
+ KcA7
= yield stress
Kc = constant
By ploting VT versus •
yield
stress is calculated
as the
square of Y intercept.
Sedimentation properties
settlina
rate
10 ml of dilute suspensions (0.2-0.5 weight %) was placed
in a 10
ml graduated cylinder,
covered
with paraffin film
and allowed to set for 1 hour at room temperature (22-25 0 C).
The suspensions were then inverted
the height
of the interface
between the
and supernatant were recorded as
tling rate
versus time
was measured
10 times and mixed well,
a function of time.
by ploting
and calculated
from the
- 35 -
settling material
the height
Set-
percentage
slope (Michaels,1962;
Callaja et al, 1977).
The height percentage is the ratio of
the height of interface to the height of suspension.
hydrodynamic volume
cell wall
The
fractions
assumed to
were
sediment
as
aggregates composed of cell clumps with occluded and associated water.
which is defined as
The hydrodynamic volume,
the volume of solid mater-
the ratio of aggregate volume to
ial, and the mean aggregate diameter are related to sedimenby employing modified Stokes'
tation velocity
of Michaels and Bolger (1962):
Qo
=
g (Pa - P"
a
(1
18
-
a)
4
4 '
z.
where Qo = initial settling rate
g=
gravitational acceleration
Pa =
cellular aggregate density
Pw =
fluid density
da
mean aggregate dismeter
fluid viscosity
4 a
aggregate volume fraction
Rearrange equation to:
Q0o
-6 5
1
1
= Vsa 4 .6 5 (1 _ Ca/cw m.*cw
- 36 -
s
)
law equation
where Vsa
=
Stokes' settling
and Ca/cwm=a/ý
S
velocity for a single aggregate
And then:
g (Pcws - Pw) d2
Vsa
=
18pw Ca/cwm
Pcws
=Density of cell wall material.
I
If
plots Q4.65
one
volume fraction ( cws)
against
a
the corresponding
straight line
the intercept of the abscissa,
values of
should result.
Ca/cwm can be determined and
from the ordinate intercept, da can be calculated.
sity of cell wall suspension
From
The den-
and wall materials were deter-
mined by pyconometer method.
Microscopy
Light
observed
microscopy:
and
Whole
photographed
cells
with a
and
phase
cell
walls
constrast
were
light
microscope at the magnification of 625 or 1000.
Scanning
electron microscopy:
Samples were
dehydrated
through increasing concentration of ethyl alcohol solutions.
The dried samples were mounted
with gold by a spatter
chester,PA).
on polished stubs and coated
coater (model No.12121,
Pictures were taken
SPI,
with a Scanning electron
microscope (Cambridge Strescan, Cambridge, England.).
- 37 -
West-
RESULTS AND DISCUSSIONS
Morphologv
The strain, Saccharomvces cerevisiae JD7,
previously isoculture of S.
lated by DeAngelo
(1978)
cerevisiae A364A,
has been characterized as a cell division
from a mutagenized
cycle mutant whose division is
tion to the bud neck.
cells as
blocked after nuclear migra-
Morphological properties of the yeast
observed with both
light and
scanning microscope
are described in Table 2.
+-------------------------
-------------------------------------
I
TABLE 2
+
Morphology of Yeast Cells on Different Growth Media
Strain
Growth Medium-SDC
Growth Medium-YPD
Saccharomyces
cerevisiae
A364A
spherical and
yeast-like,
diameter= 5p
same as left
Saccharomyces
cerevisiae JD7
yeast-like,
elongated
aggregated.
mycelium-like,3-5
times longer than
the normal cells,
branched,
entangled
and aggregated.
+------------------------------------------------------------+
- 38 -
Figure 5
shows that the
three dimensional
structure of
strain JD7 in the aggregated mycelium-like state consists of
branches
of undivided
yeast
cells.
With a
fluorescent
nuclear staining technique, DeAngelo(1978) characterized the
mycelium like
cells of JD7
as branches of
undivided yeast
cells containing only one nucleus between the first and second cell.
The nutritional
remains unknown.
signal for
dimorphism of
JD7
The morphology of cell wall residues col-
lected after hot alkaline treatment, corresponded exactly to
those of the whole cells from which they are derived, except
that they appeared
darker in the phase
contract microscope
and no cytoplasmic materials could be detected.
and cell walls of different
morphologies are shown for com-
parism in figures 7 through 12.
scanning electron microscopic
like
cell
wall
material.
Shown in figure 6,
picture of the
The
observed to be collapsed and flat,
cell wall
Yeast cells
residues are empty
cell
wall
is the
JD7 myceliummaterial
was
this suggesting that the
sacs composed mainly
of cell
wall polysaccharides.
Chemical Comoosition
Data obtained as the chemical
composition of yeast whole
cell is summarized in table 3.
The protein and
total carbohydrate content of
strains were similar.
these two
Glycogen and trehalose contents var-
ied between the two strains and the carbohydrate contents of
- 39 -
TABLE 3
Chemical Composition of Yeast Cells
(%)
JD7-YPD*
4.5+0.4
Trehalose
Alkali-soluble 5.4+0.7
Glycogen
Total
17.3
Glycogen
Mannan
12.8+0.1
14.6+0.9
Glucan
Total Carbo50
hydrate
54
Protein
A364A-SDC
JD7-SDC
A364A-YPD
3.2+0.8
10.1+0.6
5.7±0 .2
5
9
18.2
13.8
20.3
11.6+0.4
16.3±0.4
12.1±0.5
49
12.1
11.5
41
6.4+1.1
13.8±o.3
43
45
43
52
* Strain-Medium
+----------------------------------------------------------------------------------+
both
strains
conditions.
were
composition and the
glucan contents of
was higher in
be
dependent
upon
growth
the cell walls and
their dependence on
By comparing the glucan and mannan contents in
these yeast samples,
than that of
to
Of perhaps more significance, are the polysac-
charide contents of
morphology.
found
no firm
conclusion on the relation of
morphology can be drawn,
although the
mycelium-like forms seemed to
the yeast-like forms,
and
the parent strain A364A.
may be due to the changes
be higher
the mannan content
These differences
in the structural build-up within
the yeast cell wall through mutation.
- 40 -
Detailed structural analysis of yeast polysaccharides can
be done by various analytical techniques,
alkaline degradation,
such as selective
selective acid hydrolysis,
selective
acetolysis, enzymatic degradation, NMR spectroscopy, immunoHowever, none of these
logical methods, etc.(Ballou, 1976).
chemical methods
is useful
in quantatively
are considered to be
which
the bulk structural properties,
characterizing
more relevant for some applications.
The protein and mannan contents
of the yeast cells grown
in chemically defined medium, SDC,
were lower than those of
This may be a result of an ami-
cells grown in rich medium.
no-acid limitation in the defined medium.
Chemical analysis
alkaline treatment is
The percentage of
data of cell
wall residues
after hot
Table 4.
shown in
i.e.
recovery,
the ratio
of the dry
weight of cell wall residue to the dry weight of whole cells
before treatment ranged from 33 to
residue
samples were
mainly glucan
found
be high
to
and glycogen.
Protein
detected in these residues.
- 41
All the cell wall
39%.
-
in
carbohydrate,
and mannan
were not
I
I
I
I
TABLE 4
I
Chemical Composition of Yeast Cell Wall
I
I
I
I
I
I
I
I
ND - not detected
I
I
I
39.8
31.2
37.9
40.5
ND
39.5
78
ND
35.6
ND
35.9
75
ND
38.3
ND
38.3
80
ND
Glycogen
Total Glycogen
Mannan
Glucan
Total carbohydrate
Protein
I
I
A364A-YPD
1.6
Glycogen
Acid-soluble
I
I
I
JD7-SDC
1.4
JD7-YPDO
0.7
Composition(%)
Trehalose
Alkali-soluble
1
I
i
'strain-medium
I
I
I
I
I
I
+----------------------------------------------------------+
Rheoloaical Properties
fAYeast SusDensions
viscosity orofiles
To compare the gross structural
pensions,
properties of yeast sus-
viscometric measurement were done as described in
Materials and Methods.
ween viscosity and
In figures 13-15, the relations bet-
shear rate at different
dry weights and
morphologies are summarized.
In all the cases,
thinning effect was
That is,
observed.
as
a shear-
the shear rate
increases, the viscosity decreases and approaches a constant
apparent viscosity.
It was also noted that,
centrated yeast suspensions
reach a constant value.
perhaps being
due to
the more con-
require a higher shear
rate to
This phenomenon can be explained as
the existence
structure in the suspensions,
-
which
42 -
of a
cellular network
is in a dynamic state,
continueously being built-up and
brokendown.
As the shear
rate increases the network structure is completely destroyed
and the system then behaves as a Newtonian fluid.
The viscosity-dryweight relationships for spherical cells
of S. cerevisiae strain A364A, yeast-like
cells of S.
16.
All
phase
c.erevisiae strain JD7 are
these cells were
when
expressed.
to be
collected at
distinctive
hand,
stationary growth
morphologies
a function of the
were
dry weight concentration
yeast cells.
cerevisiae
..
The spherical
fully
Suspensions
exhibited
The viscosity
and mor-
cells of
control
A364A had little contribution
viscosity of suspension up to a
gm/l.
presented in Figure
The viscosity of whole cell suspension was found
phology of
strain
the
and mycelium-like
concentration as high as 70
of mycelium-like
much higher
cells,
on
viscosities than
of mycelium-like
to the
the other
the others.
cells suspension
begins to
increase drastically with increasing
dry weight concentrai-
ton at a
Suspensions of floccu-
concentration of 25 g/1.
lated and elongated
viscosities,
cells of JD7 grown in SDC had moderate
with a critical
tion at which the viscosity
between the
size
other two
and the
concentration (the concentrabegins to approach infinty)
extremes.
extensive intercellular
most important features responsible
The relationships
wall materials
The
considerably larger
interaction are
and shear
alkaline treatment
-
43 -
the
for higher viscosities.
of apparent viscosity
after hot
in
rate of
are shown
in
figures 17-19.
For all the morphologies,
the viscosity of
wall material was much higher than that of whole cells.
The
viscosity of cell wall materials was also found to be dependent upon the concentration of cell walls and the morphology
of the intact cells.
Presented in figure 20 is a summary of
the viscosity profiles of whole cells and cell walls.
noteworthy
is the
observation that
the
Also
viscosity of
the
mycelium-like cell wall suspensions was extremely high, with
a critical
concentration as
low as 10
gm/l.
If
the dry
weight of cell wall material is converted into the weight of
the whole cells from which they
were derived,
a profile is
obtained as shown in the dotted line in figures 21-23.
The
viscosity-concentration relationship can thus be compared on
the basis of the
number or volume of cell mass
in the sus-
pension, assuming equal volumes of whole cells and cell wall
material.
It
was interesting to
profiles of cell wall material
ginal dry
find that
after conversion to the ori-
weights are very similar
This suggested that
it is the cell
determines the viscosity of
thickening effect
to that of
of mycelium-like
144
cell wall
The dramatic
material and
composition suggest its pos-
sible use as a food thickening agent.
-
whole cell.
unit concentration that
cell suspension.
the simplicity in its chemical
the viscosity
-
Yield stress
To compare the thickening
cal food thickeners,
results are shown
mass concentration.
exhibit a high
cells and the
cellulose.
stress of the mycelium-like
the yield
walls were estimated by
cells and cell
and the
properties with other commeri-
figure 24
the Casson equation
as a function
of dry
The mycelium-like cell
wall residues
yield stress when compared to
that of whole
value is comparable to
Yield stress is
that of carboxymethyl
generally employed to approxi-
mate the stress required to start
a flow.
A high value of
yield stress suggests the ability of these materials to stabilize fluids.
hydrodynamic
To
volume
study the
thickening mechanism
of
yeast cell
wall
residues, the hydrodynamic volume (Ratio of aggregate volume
to wall material volume)
was estimated by employing a modi-
fied Stokes' law (Michaels and Bolger, 1962).
tation data for yeast-like and
were shown in figures 25
settling rates (Q o )
mycelium-like JD7 cell walls
& 26,
were
The sedimen-
respectively.
The initial
estimated from the
slope of the
linear regions of these curves.
A plot of Qo
4 .6 5 versus
the
corresponding value of volume
fraction resulted in straight
lines
From the slopes
(figures
27 and 28).
and abscissa
intercepts of these lines, hydridynamic volume (Ca/cwm ) and
mean aggregate diameter (da) were derived.
A summary of results are in Table 5.
-
45 -
The derived mean
aggregate diameters were close
actually observed for
scope.
that one
cell wall materials under
The large values
volume of
the micro-
for hydrodynamic volume indicated
the cell wall
holding a large amount of
to that
material was
capable of
fluid during sedimentation.
fluid may either be occluded
The
in the cytoplasmic cavities or
entrapped between the loosely packed settling aggregates.
+-------------------------------------------------------------+
TABLE 5
Hydronamic Volume and Mean Aggregate Diameter of Yeast
Cell Walls
Morphology
da(O)
Ca/cwm
Mycelium-like JD7
167
139
Yeast-like JD7
113
98
= mean aggregate diameter
C
a/cwm
hydrodynamic
volume
+-------------------------------------------------------------+
The thickening effect
can thus
of the resulting cell
be explained in part
wall materials
as due to their
holding capacity.
- 46 -
large water
CONCLUSIONS
1.
Saccharomyces
cerevisiae strain
JD7 has
similar
chemical composition as its parent strain, Sac.haierevisiae A364A,
romoes
although their cellular
morphologies were appearently different.
2.
The
distinct
strain JD7
morphology
of
the
mycelium-like
due to changes
is most likely
in the
chemical structure of the yeast cell wall polysaccharides, especially glucan.
3.
Yeast cell wall fractions isolated by hot alkaline
were high in carboh-
treatment from either strain
ydrate,
gen.
mainly glucan and alkali-insoluble glycoThe cell wall fractions maintained the same
shapes as the orginal cells,
but contain no pro-
tein or cytoplasmic material and mannan.
4.
The viscosity profile of
depends on
yeast suspensions mainly
the morphology of the
cerevisiae JD7
mycelium-like form results
in suspensions
with higher viscosity than
tion is one of
ever,
Larger
S.
and more branched cells, i.e.
in the
cells.
others.
Volume frac-
the controlling parameters.
interactions between cells
-
47 -
How-
are believed to
play an important role
in creating high viscosity
of cell suspensions.
5.
The viscosity of cell
wall fractions is extremely
high when compared to whole
weight concentration.
cells at the same dry
The critical concentration
of the cell wall fraction
of mycelium-like JD7 is
as low as
The
10 gm/1l (1%).
yield stress
high viscosity and
in such yeast cell
suggest the possibility of
wall preparation
employing this
mater-
ial as a food thickening agent.
6.
By series of observations conducted in this investigation,
it was found that the following factors
are responsible for the
rheological properties of
yeast cells and cell-wall preparations:
a) The three
can,
dimensional structure of
which
is responsible
yeast glu-
for retaining
the
shape of the cells.
b) The volume
The larger
occupied by the
hydrated material.
the hydrated volume the
higher the
viscosity.
c) The surface properties of yeast cells.
Surface
properties result in various interrelationships
between single units of
tion and
cells.
floccuation may take
- 48 -
Cell aggregaplace depending
on a
given surface
results in
property and
a distinctive
consequently
rheological behavior
of the yeast preparation.
d) The ability
to immobilized
occluding water
water,
either
in sac-like structures
by
of the
yeast cell wall or entrapping it in between the
loosely packed cellular aggregates.
- 49 -
1
6
1
6
1
M
--
6
*
1
M
-
6
6
1
M
1
6
--
+
1
6
1
6
I
6
---
+
1
I
4
4
--
-
M
]
*
2 M12
M
12
M
1
M -- P
M
NM
M
M
M
M
M
M
--
1
M
+
M
-
1
Mi
M
12
M
M
M
M
M
-
GNAc
--
e
GNAc
--
s
Ask
tT
tr.
M
Outer chain
Inner core
M 1 2
MM -- M
M--. M--- M-
1 •2M
M1 3M
N- 2M-+
Base-labile
oligosaccharidcs
M: Mannose
GNAc : N-Acetyl-Glucosamine
Detailed structure of the mannan from S. cerevisiae
showing the base-labile oliglsaccharides arrached to
hydroxyamino acids, the inner core and the outer chain.
All anomeric linkages have the e-configuration except
for the trisaccharide unit: @Man(l-.4)@GNAc(l-+4) GNAc
linked to asparagine. (From Ballou, 1976)
Figure 1:
Chemical Structure of Yeast Mannan
-
50 -
er (Thr)
P
ENZYME
M
P~HOSPHATE
= MANN AN
G
6LCAN
P
S-S
CYTOPLASM
Figure 2:
Schematic Structure of Yeast Cell Wall
- 51
-
GROWTH
COLLECT
COLLECT
KALINE TREATMENT
EXTRACT
EES
[1] BIOCHEMICAL ANALYSIS
: PROTEIN & CARBOHYDRATE
[2] RHEOLOGICAL PROPERTIES
VISCOSITY & YIELD STRESS
Figure 3:
Flow Sheet for Experimental Procedures
-
52 -
YEAST CELLS
Extract at O0C w/ Trichloric acid
for 60 mins., centrifuge.
Supernatant
Insoluble residue
TREHALOSE
Extract w/ 0.25M
Na 2 CO 3, centrifuge
%41
Supernatant
Mannan + AlkaliSoluble Glycogen
"-
Insoluble
residue
W
Treat with
Fehling' s
solution
Extract with
0.25M HC10
30mins.,
10 °
F
overnight
ACID SOLUBLE
GLYCOGEN
centrifuge
Precipitated
Mannan-c opper
Complex
Dissolve in
I
Insoluble
residue
Suspend in
2M Na0H
GLUC AN
0.5ml of 1M H2 So04
iS4
MANNAN.
Figure 4:
Extraction of Carbohydrate from Yeast Cells
- 53 -
Fig.7. Morphology of
S .cerevisiae A364A(x1000)
Fig.b. Cell wall residues of
S_. cerevisiae A364A(x1000)
Fig.9. Morphology of
S. cerevisiae JD7(x625)
grown in SDC.
Fig.10.Cell wall residues of
S. cerevisiae JD7(x625)
grown in SDC.
S. cerevisiae JD7(x625)
grown in YPD.
S. cerevisiae JD7(x625)
grown in YPD.
rigo
.
Tnree
i
LilmerlSiorfial•
L
bu'utU
LUre
UI
mycelium-like JD7 aggregates(x2000).
l-g.
0. Uollapsecl mycelium-like cell wall.
Picture taken by scanning electron microscope(x4900) .
DRY WEIGHT (
DRY WEIGHT (GI~L)1
196.0
167.8
121.8
74.9
36.1
U)
0O
(mn
O
z*LJ
my
-
v
SHEAR RATE (1/ Sec)
Figure 13:
Effect of Shear Rate on Apparent Viscosity of
Saccharomyces cerevisiae A364A Cell Suspensions
- 56
6
0-I
01
DRY WEIGHT (GIL)
99.9
63.3
49.3
V)
39,1
CO
0O
ICO
i
23.9
I
azLIJ
w
QL
0.
m.
a- V
-- v1 %r
v
v
w
SHEAR RATE (I/Sec)
Figure 14:
100
!1
Effect of Shear Rate on Apparent Viscosity of
Yeast-like Saccharomyces cerevisiae JD7 cell
Suspensions
- 57 -
.2
I,
DRY WEIGHT (G/L)
38.2
EL
0-I
01
I-.Ul)
0
0U)
H
I-.
z
w
0-
a-J
SHEAR RATE
Figure 15:
(I/Sec)
>
Effect of Shear Rate on Apparent Viscosity of
Mycelium-like Saccharomyces cerevisiae JD7 Cell
Suspensions
-
58 -
20
U)
t
6 15
I
0
0
10
ILU
0.
5
0
0
10
20
30
40
DRY WEIGHT
Figure 16:
50
60
(gm/f)
Viscosity Profiles of Three Different
Morphologies
- 59 -
70
IC
DRY WEIGHT (GIL)
0
10
Iz
w
CC-
SHEAR
Figure 17:
RATE (I/Sec)
>
Apparent Viscosity-Shear Rate Plot of Cell Wall
Suspensions of Sacoharomyces cerevisiae A364A
-
60 -
DRY WEIGHT (G/L)
30.0
18.4
14. 9
9.6
6. 8
10
(I)
0o
U'7
0
0
IL)
<-
w
SHEAR
Figure 18:
.v
-
.w
w
.
.o
RATE (I/ Sec)
Apparent Viscostiy-Shear Rate Plot of Cell Wall
Suspensions of Yeast-like Saccharomyces
cerevisiae JD7
- 61
DRY WEIGHT(G IL)
ale I
15 8
12.5
9. 1
l
o
0
00
0I.0
Ui
CL
Q.
I.-
SHEAR RATE (I/Sec)
Figure 19:
Apparent Viscosity-Shear Rate Plot of Cell Wall
Suspensions of Mycelium-like Sacgharomyces
aerevisiae JD7
-
62 -
-& JD 7-YPD -WALL
-A--t
18
7-Va
l..i' I I
50
60
16
14
U
12
-
10
0
O 8
-6
Z
u.
Cr
4
%ýL
0
10
20
30
40
70
CONCENTRATION (G/L)
Figure 20:
Summary of Viscosity Profiles of Yeast Cells and
Cell Walls Suspensions
-
63 -
-0- APPARENT VICOSITY
VS DRY WEIGHT OF
RESIDUE
VI SCOSITY
-.- APPARENT
VS
DRY WT.BEFORE
HOT ALKALINE
TREATMENT
25
-0-
VISCOSITY PROFILE
OFWHOLE CELL
SUSPENSION
O 20
15
0
<10
a -
20
40 60
80 100 120 140 160 180 200
DRY WEIGHT
Figure 21:
(9m/I)
Viscosity Profile of Cell Wall Suspensions of
Saccharomyces cerevisaie JD7 Grown in SDC
- 64 -
SI
I
i
I
16
I
Q.
I
I
14
-0-APPARENT VISCOSITY
VS DRY WEIGHT OF
RESIDUE
-*-APPARENT VISCOSITY
VS DRY WT. BEFORE
HOT ALKALINE
TREATMENT
-6-VICOSITY PROFILE
OF WHOLE CELL
SUSPENSION
12
'I
0
C>
>
I
10
8
z
W
cc
6
a<1:
I
4I , I I
I
10
20
30
40
DRY WEIGHT
Figure 22:
50
1
60
70
70
(gm/I)
Viscosity Profile of Cell Wall Suspensions of
Mycelium-like Saccharomyces cerevisiae JD7
- 65 -
A
APPARENT VISCOSITY
VSDRY WEIGHT OF
RESIDUE
--0--
60
4 50
APPARENT VISCOSITY
VS DRY WT. BEFORE
HOT ALKALINE
TREATMENT
VISCOSITY PROFILE
OF WHOLE CELL
SUSPENSION
U 40
0
/Ol/
0
<
I
-/
20
CLl 20-
Q10
I
n
0
20
'
si
II
40
DRY WEIGHT
Figure 23:
I
60
I
80
I
100
(gm/I)>
Viscosity Profile of Cell Wall Suspensions of
Saccharomyces cerevisiae A364A
- 66 -
o JD7-YPD-ALL
ACMC
<> JD7-YPD-CELL
E
U
>1
m
%am
(n
w
Jr
w
S2
4
5
CONC ENTRATION ( /o )
Figure 24:
6
-
Yield Stress-Concentration Relationships of
Mycelium-like Cell Wall Suspensions and CMC
- 67 -
I
0
0
I.
IL
w3
TIME (m i n.)
Figure 25:
Sedimentation Rate of Yeast-like Saccharomyces
cerevisiae JD7 Cell Wall Residues
- 68 -
I
3=
_
0
I
TI ME (min.)
Figure 26:
-
Sedimentation Rate of Mycelium-like
Saccharomyces cerevisiae JD7 Cell Wall Residues
-
69 -
2.0
d
I.o
0.000
.002
.004
.006
PCWM
Figure 27:
.008
.10
.-
Q4,65 Versus Volume Fraction for Cell Wall
Residues of Mycelium-like Saccharomyces
cerevisiae JD7
- 70 -
_I &f
d
I(
0.000
.002
.004
kCWM
Figure 28:
.008
.006
.010
-"
Qd4.65 Versus Volume Fraction for Cell Wall
Residues of Yeast-like Saccharomyces cerevisiae
JD7
- 71 -
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