An in vitro digestion of selected starches
by Ronald Lyle Davis
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Animal Science
Montana State University
© Copyright by Ronald Lyle Davis (1966)
Abstract:
Three vitro digestion trials were conducted comparing 5 barley starches, corn starch, milo starch,
amylose and amylopectin. Within each trial, substrates were subjected to digestion on each of 3
different days. The phenol-sulfuric acid procedure used to analyze for residual starch was compared to
the enthrone method of Moore et al. (1962).
In Trial I the starches of corn, milo, wheat, Oderbrucker barley and Compana barley were compared.
Oderbrucker starch was digested significantly faster (P<.01) or equal to corn starch and the other
starches. In most instances wheat and milo starch were digested slower than corn starch.
The starches of corn and the barley varieties, Compana, Newpaha, Betzes and Unitan were compared in
Trial II, Corn starch digested significantly faster (P<.01) or equal to the other starches in all but one
instance. Unitah starch digested significantly slower (P<.01) or equal to the other starches in all but one
instance.
In Trial III corn starch, Compana starch, Newpana starch, amylopectin and amylose were compared.
Amylopectin digested significantly faster (P <.01) or equal to the other substrates. Amylose tended to
digest faster than the 3 starches at the start of the fermentation period; however at the end of the period
amylose digested slower than the other substrates.
In all trials at the end of the fermentation period, digestion was either greater than or approached 90
percent. All interactions excluding hours x variety for Trials I and II were highly significant (P <.01)
Hours x variety was significant (P<.05) for Trial III.
The correlation between the phenol-sulfuric acid method and the enthrone method was highly
significant (r = .99). AN IN VITRO DIGESTION OF SELECTED STARCHES
by
RONALD LYLE DAVIS
A thesis submitted to the Graduate Faculty in partial
fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Animal Science
Approved:
Head, Major Department
Graduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
December, 1966
-iiiACKNOWLEDGMENTS
My gratitude and appreciation are sincerely expressed to Dr, C. W,
Newman for his assistance in conducting the investigation, and for his
invaluable service in preparing this manuscript,
I am also grateful to
him for his assistance and recommendations in acquiring the assistantship.
which made it possible for me to continue my education.
I wish to thank Dr. 0. 0. Thomas and Dr, R„ L. Blackwell for their
cooperation and constructive criticism in completing this manuscript,
I
also want to thank Mr. Dave Jacobsen for his assistance with the statis­
tical analysis.
The help, understanding and encouragement from my wife. Dona, through­
out my graduate program is sincerely appreciated, for without her this
work would have been much more difficult.
An acknowledgment of indebtedness is made to Dr. K. J. Goering for
separating the starch and for his interest in the project, to Dr. G. 0.
Baker for making available bis laboratory equipment, and to Mrs. Mildred
Latta for proof reading and typing this manuscript.
/
TABLE OF CONTENTS
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ACKNOWLEDGMENTS
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INDEX TO TABLES
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INDEX TO APPENDIX TABLES . .
INDEX TO APPENDIX FIGURES
ABSTRACT
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INTRODUCTION . .
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REVIEW OF LITERATURE . . .
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Correlations Between In Vitro and In Vivo Methods
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Factors Affecting Cellulose Digestion
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Inoculum
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Minerals and vitamins
Carbohydrates
Lignin
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In Vitro Starch Digestion
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General Procedures
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Enzymatic Starch Digestion
PROCEDURES
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Measurement of Starch Fermentation
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In Vitro Procedures „ .
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Phenol-sulfuric acid procedure
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Anthrone colorimetric method
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RESULTS AND DISCUSSION . . .
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Trials I, II, III
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Phenol-Sulfuric Acid - Anthrone Comparison
SUMMARY
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APPENDIX . . . .
LITERATURE CITED
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-viINDEX TO TABLES
TABLE
I.
II0
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PERCENT DIGESTION OF STARCHES IN TRIAL I . . . . . . . .
.
.
.
2 0
ANALYSIS OF VARIANCE OF PERCENT DIGESTION FOR STARCHES
IN TRIAL I (CORN, ODERBRUCKER, COMPANA, MILO9 WHEAT) . . . . .
21
. .
22
PERCENT DIGESTION OF STARCHES IN TRIAL II
. . . . . . . .
ANALYSIS OF VARIANCE OF PERCENT DIGESTION FOR STARCHES
IN TRIAL II (CORN, COMPANA9 NEWPANA9 BETZES9 UNITAN) . . . . .
23
PERCENT DIGESTION OF SUBSTRATES IN TRIAL III . . . . . .
. . .
24
ANALYSIS OF VARIANCE OF PERCENT DIGESTION FOR STARCHES
IN TRIAL III (CORN, COMPANA9 NEWPANA9 AMYLOPECTIN9
AMYLOSE) . . . . . . a . . . . . . . a . . . . . . g . . .
. o
25
COMPARISON BETWEEN THE ANTHRONE METHOD AND PHENOLSULFURIC ACID METHOD USING CORN STARCH ON DAY 3
TR IjAL III . . . . . . . . . . . o o . . . . . . .
. .
28
PERCENT RECOVERY OF ADDED STARCH FOR TRIALS I, II9 III
30
viiINDEX TO APPENDIX TABLES
TABLES
I,
II.
Ill,
Page
PERCENT DIGESTION
I, II, III . . . .
THREE-DAY AVERAGE FOR TRIALS
34
TWO-WAY TABLE SHOWING DAYS X VARIETY INTERACTION FOR
TRIALS I, II, III
36
TWO-WAY TABLE SHOWING HOURS X DAY INTERACTION FOR
TRIALS I , II, III . . . . . . . . . . . . . a . .
37
-viiiINDEX TO APPENDIX FIGURES
FIGURE
Page
1.
PERCENT DIGESTION
— THREE-DAY AVERAGE FOR TRIAL I ..........
38
2.
PERCENT DIGESTION
— THREE-DAY AVERAGE FOR TRIAL II
„ . . . .
39
3„
PERCENT DIGESTION
— THREE-DAY AVERAGE FOR TRIAL III
„
40
4,
PERCENT DIGESTION — COMPARISON BETWEEN THE PHENOLSULFURIC ACID METHOD AND THE ANTHRONE M E T H O D ........ .. . . .
41
-ixABSTRACT
Three
vitro digestion trials were conducted comparing 5 barley
starches, corn starch, milo starch, amylose and amylopectin. Within each
trial, substrates were subjected to digestion on each of 3 different days.
The phenol-sulfuric acid procedure used to analyze for residual starch
was compared to the enthrone method of Moore est al. (1962).
In Trial I the starches of corn, milo, wheat, Oderbrucker barley and
Compana barley were compared. Oderbrucker starch was digested significant­
ly faster (P <f.01) or equal to corn starch and the other starches. In
most instances wheat and milo starch were digested slower than corn starch.
The starches of corn and the barley varieties, Compana, Newpaha,
Betzes and Unitan were compared in Trial II, Corn starch digested signifi­
cantly faster (P<%01) or equal to the other starches in all but one in­
stance. Unitah starch digested significantly slower (P<^.01) or equal to
the other starches in all but one instance.
In Trial III corn starch, Compana starch, Newpana starch, amylopectin
and amylose were compared. Amylopectin digested significantly faster
(P <^.01) or equal to the other substrates. Amylose tended to digest fas­
ter than the 3 starches at the start of the fermentation period; however
at the end of the period amylose digested slower than the other substrates.
In all trials at the end of the fermentation period, digestion was
either greater than or approached 90 percent. All interactions excluding
hours x variety for Trials I and II were highly significant (P <^.01)«
Hours x variety was significant (P<^„05) for Trial III.
The correlation between the phenol-sulfuric acid method and the en­
throne method was highly significant (r = .99).
INTRODUCTION
Barley is Montana's most important livestock feed grain.
Many vari­
eties are grown each year and new varieties are continually being devel­
oped,
There is a need to determine if there is a difference in the
microbial digestion of the numerous barley varieties.
Emphasis on high-energy feeding programs for ruminants has resulted
in widespread use of rations in which starch is the primary source of
energy.
Very little digestibility data, either in vitro or iri vivo, is
available on high concentrate rations.
The in vitro procedure has made a major contribution to the under­
standing of cellulose digestion in the ruminant; however relatively little
attention has been given to the study of starch digestion by rumen micro­
organisms.
Because barley is the major source of energy in Montana's livestock
feeding programs, it was deemed necessary to study starch digestion of
several varieties produced by the Montana Agricultural Experiment Station
at Bozeman.
The major objectives of the trials reported in this manu­
script were to determine if differences exist among barley varieties in
regard to starch digestion by rumen microorganisms, to evaluate the in
vitro method as a basis for comparing digestion of starch and to evaluate
the phenol-sulfuric acid method of starch determination as compared to the
more difficult enthrone method used by Moore et al. (1962).
REVIEW OF LITERATURE
The suggestion that microorganisms were responsible for cellulose
disappearance as feed passed through the alimentary tracts of ruminants was
made as early as 1874-1882, Johnson (1963).
It was not until the early
1940"s that the in vitro digestion technique was developed.
Since that
time, jLn vitro digestion has been widely used in cellulose digestibility
studies.
In vitro techniques have also been used to measure the produc­
tion of volatile fatty acids, to study utilization of various nitrogenous
compounds by rumen microflora and to predict forage nutritive values.
.
Warner (1956) established criteria for the validity of in vitro
studies with rumen microorganisms.
Warner stated that motility of the
bacteria appeared to be a sensitive measure of normal rumen function.
For the in vitro microbial population to remain normal in numbers, it was
necessary to use as test substrates only substances similar to the diet
fed the animal from which the innoculum was taken,
Barnes ejt al. (1964) compared several in vitro methods of cellulose
digestion.
As the length of fermentation increased, the standard devia­
tion between methods decreased.
The variability was therefore associated
with the initial lag phase period of 6 and 12 hour fermentations.
Donefer
et al. (1960) stated that the prerequisite of a useful in vitro technique
was its ability to measure the effects on cellulose digestion of the early
lag differences in fermentation.
Correlations Between In Vitro and In Vivo Methods
In order to effectively evaluate the in vitro method of digestibility,
the in vitro and in vivo methods were compared.
-3“
Barnett (1957) reported that results calculated for cellulose digest-?
ibility coefficients were within —5.6 percent of determined in vivo and in
vitro digestion.
It was shown that the variability associated with! cellu­
lose digestibility in vivo was only slightly smaller than the variability
associated with replicate determinations of cellulose digestibility in
vitro.
Quick (1959) compared iji vitro and jm vivo digestibilities of several
grass hays and legumes.
There was no significant difference between the
two methods.
Forty-eight hour fermentations conducted by Lefevre and Kamstra (1960)
yielded.cellulose digestion coefficients similar to values obtained in vivo
(r = 0.84).
Cellulose digestion in twenty-four hour fermentations was
below that obtained in vivo.
Digestion coefficients obtained in vitro with
sheep and cattle rumen fluids as inoculum were similar.
Correlations between JLn vitro and In vivo measures of digestibility
were conducted by Bowden and Church (1962).
follows:
These correlations were as
(I) in vitro dry matter digestibility and in vivo dry matter
digestibility were correlated (r = 0.93), (2) in vitro cellulose digestion
and in vivo dry matter digestibility were correlated (r = 0.87), (3) in
vitro dry matter digestibility with crude protein of the forage was corre­
lated (r = 0.94), (4) in vitro cellulose digestion with crude protein con­
tent of the forage was correlated (r = 0.93).
Baumgardt et al. (1962b) supported the work by Bowden and Church by
finding that the percent forage cellulose digested in vitro was signifi­
cantly correlated with total digestible nutrients, digestible dry matter
-4and digestible energy determined in conventional digestion trials.
The
correlation between in vitro cellulose digestion and iji vivo digestible
energy was highly significant (r = 0.85).
Hershberger et al. (1959) found
a correlation of 0.92 between cellulose digested iji vitro and the digest­
ible energy of the forage.
Digestible protein was estimated in vitro by Baumgardt et al. (1962b).
When the crude protein was known, the correlation between crude and di­
gestible protein was highly significant (r = 0.999).
Pigden and Bell (1955) found that the artificial rumen procedure, when
used with the proper regression equations, gave estimates of total digest­
ible nutrients; and that digestibility of crude protein from eleven forages
was in close agreement with values derived from conventional digestion
trials using sheep,
Baumgardt elt al. (1962a) conducted research in which
the in vitro fermentation of forage carbohydrate was measured.
Estimates
of total digestible nutrients were significantly correlated with conven­
tional digestibility data; however these estimates were consistently under­
estimated.
High correlations between jn vitro and in vivo digestion indicate
that in vitro fermentation has a definite value in estimating the nutritive
value of forages.
I
.
.
■ . .
1
Factors Affecting Cellulose Digestion
Inoculum
A method for the study of cellulose digestion by washed cell suspen­
sions was developed by Cheng et al. (1955).
Washed suspensions of rumen
-5microorganisms were sensitive enough to detect the effect of small amounts
of unidentified factors stimulatory to cellulose degradation.
Washed sus­
pensions were relatively free from any metabolic end products produced by
microorganisms while still in the rumen.
Church and Peterson (1960) noted that the amount of rumen liquor had
appreciable influence on the extent of in vitro digestion of cellulose or
dry matter.
Marquardt and Asplund (1964) found that cellulose digestion
increased as the volume of inoculum increased.
Increased bacterial numbers
■
and additional nutrients possibly increased the rate of fermentation by
decreasing the initial lag phase of growth and/or by increasing the rate
of growth and metabolism during the logarithmic growth phase.
Small
changes in mineral medium concentrations had only negligible effect on in
vitro digestion of dry matter and cellulose.
Based on the composition of ruminant saliva, McDougall (1949) sug­
gested a mineral medium which adequately supported
hi
vitro digestion.
Several variations of this media have been used and all support in vitro
digestion.
Marquardt and Asplund (1964) noted that unless the nqtrient medium
in artificial rumen studies was completely adequate, standardization of
inocula or, statistical dontrol was necessary.
Cheng iet al. (1955) reported the optimum pH for cellulose digestion
to be from 6.8 to 7.6.
Rumen microorganisms exhibited a greater toler­
ance to a high pH than to a low pH.
Church and Peterson (1960) concluded
the optimum pH was 6.0 to 6,7; however the pH tended to vary according to
substrate and/or source of rumen liquor.
-6Burroughs et: a^. (1950a) noted that cattle and sheep fed rations
of a widely varient nature indicated differences in the predominating
types of rumen microorganisms.
The same general type of organisms were
present in each animal even though the predominating type was different.
Minerals and vitamins
Burroughs et_ al. (1950b) reported that rumen microorganisms have
nutritional requirements which must be fulfilled if they are to digest
roughage efficiently.
It was suggested that these nutrients are found in
ample quantities in certain good quality roughages and are inadequate in
certain poor quality roughages.
Cellulose digestion, using the in vitro
technique, has shown needs for varying amounts of the .elements sodium,
potassium, calcium, magnesium, phosphorous, sulfur and chlorine.
Burroughs
et al. (1951) noted that iron and phosphorous found in the ashes of plants
or plant products have a stimulatory effect upon cellulose digestion in
vitro.
Several workers have observed the effects of water extracts and ash
of good and poor quality roughages.
Marquardt (1964) noted that grass
hay extract supported very poor cellulose digestion, even when it contained
high levels of protein, ash, calcium and phosphorous.
Comparable to other
extracts, the high carbohydrate content may have suppressed cellulose
digestion.
Burroughs et Jd. (1950a) reported a beneficial influence of
the ash of alfalfa upon roughage digestion which has been referred to in
in vivo experiments.
Bentley et a]L. (1951) provided evidence which indica­
ted that the low phosphorous content of the poor quality forage was a
=7“
limiting factor in cellulose digestion ^n vitro.
Hunt, ej: al, (1954) found
that more cellulose was digested and more urea utilized when inoculum was
i
obtained from steers fed alfalfa hay than from steers fed poor quality
hay.
Marquardt (1964) showed that many of the nutrients required by rumen
microorganisms for the fermentation of cellulose were present in aqueous
extracts of forages; however phosphorous, sulfur and in some cases nitron
gen were not present in adequate amounts.
Burroughs et_ a2^. (1951) found that urea utilization like cellulose
digestion was dependent to a large extent upon the nutrient requirements
of rumen microorganisms.
They suggested that a roughage was utilized most
efficiently when each of the nutrient requirements was adequately supplied.
Hunt et^ £l. (1954) observed that microorganisms which synthesize'
riboflavin, niacin and vitamin
also digest cellulose and utilize
urea for protein synthesis have a sulfur requirement.
Bentley ej: al,-
.
(1954) found that biotin, vitamin
para~aminobemzoic acid, xanthine,
uracil, guanine and adenine when added to the basal medium improved cellu­
lose digestion.
The amount of cellulose digested was always less than
that digested in flasks which contained rumen juice supernatant, a water
extract of alfalfa, yeast, or molasses.
This implied that there were
factors in natural products which enhanced the microbial activity in in
vitro fermentation.
Hubbert et, al. (1958) found sulfur, magnesium and calcium to be the
inorganic nutrients most likely to be deficient in a prepared fermentation
medium.
Extremely low levels of copper, cobalt, zinc and boron depressed
cellulose digestion.
”8Vitamin Bg showed stimulatory activity in cellulose digestion accord­
ing to Macleod and Murray (1956).
Carbohydrates
A very important factor affecting cellulose digestibility is the
amount and type of concentrate fed with the cellulose containing material.
Head (1961) reported the main effect of concentrates was due to their con­
tent of starch, which lowered the digestibility of roughage.
Head (1961) and el-Shazly (1961) reported that with low nitrogen
containing cellulose substrates, the organisms digesting sugars or starch
used all the available soluble nitrogen sources in the rumen liquor, thus
starving the cellulolytic organisms of this nutrient.
Mills ej: al. (1941) reported that when adequate fermentable carbohy­
drate was included in the ration, urea was utilized at a maximum rate and
a maximum efficiency in the rumen of the cow.
He suggested the carbohy­
drate served as a readily available energy source for the microorganisms
enabling them to build new protoplasm in which the nitrogen from the urea
was incorporated.
Baker (1942) found that the addition of increasing amounts of starch
to rumen contents incubated jri vitro resulted, in a parallel increase in
the rate of multiplication of microorganisms of all types.
Marston (1948)
noted that in diets containing a high proportion of starch, the rate of
proliferation of the microflora in the rumen was limited usually by the
nitrogen supply.
When additional nitrogen was provided, a large proportion
was converted to protein.
-9Arias et al0 (1951) found that increasing the energy content of the
fermentation medium resulted in an increased urea utilization with all
sources of energy tested.
This was true whether the energy source was a
soluable carbohydrate such as dextrose or sucrose, or whether the carbohy­
drate was more complex such as cellulose.
It was also noted that rumen
microorganisms have definite energy requirements and the degree to which
these requirements are fulfilled has considerable influence upon their
utilization of urea or other ammonia supplying compounds.
Small amounts
of readily available carbohydrate aided cellulose digestion which in turn
increased urea utilization.
Large amounts of readily available carbohy­
drate inhibited cellulose digestion.
Hunt et al. (1954) supported Arias”
work in that rumen microorganisms obtain their energy requirements from
the more readily available source, instead of breaking down cellulose.
Macleod and Murray (1956) noted that glucose stimulated cellulose
digestion at a concentration of .05 percent to 2 percent.
Decreased
digestion was observed at higher concentrations of glucose.
Cline et al., (1958) reported a greater synthesis of valeric acid
from the more readily available carbohydrate.
These workers offered this
as an explanation for increased ^n vitro cellulose digestion observed by
Arias et_ al. (1951) and Hunt £t al. (1954) with a 1:2 starch-cellulose
mixture versus cellulose alone.
(
Belascp (1956) found the optimum starch-cellulose ratio to be 1:4.
Here urea utilization, cellulose digestion and volatile fatty acid pro­
duction reached a peak.
The inclusion of large amounts of dextrose in­
hibited cellulose digestion.
These data also supported Arias et. al. (1951)
-10which showed a need for readily available carbohydrate in the proper pro­
portions,
Salsbury (1961) found that if the proportion of readily avail­
able carbohydrate was too high it was doubtful that cellulose digestion
could be maintained at a significant level.
Johnson et aj^. (1960) noted
that when the starch-cellulose ratio reached 1:1 or higher, cellulose
digestion was completely or severely depressed,
At higher ratios of starch
to cellulose, both _in vivo and iri vitro cellulose digestion was completely
inhibited and was not recovered by additional quantities of nitrogen.
The
effect of high levels of glucose was shown by Huhtanen and Elliot (1956).
They postulated that the inhibitory effect of high levels of glucose may
be explained on the basis of availability of energy sources for the rumen
bacteria.
If enough glucose was present, the organisms used it for a
carbon source, rather than the less available cellulose,
,Kane et al. (1959) in a conventional digestion trial showed that high
levels of corn starch had a depressing effect upon the digestion of alfalfa
hay; however when a preliminary period for adjustment to corn starch was
used, no depression in the digestion of alfalfa hay was observed.
el-Shazly e£ al,„ (1961) proposed several theories to explain the de­
pression of cellulose digestion by starch in a ration.
follows:
These are as
(I) production of an inhibitor by starch digesting microorgan­
isms, (2) decrease in rumen pH due to acids produced from starch fermenta­
tion, (3) competition for essential nutrients with the result that starch
digesting microorganisms proliferate preferentially, (4) predominance of
starch digesting microorganisms in the rumen of animals on high starch
diets.
-11Urea appeared to be the most efficient of all nitrogen supplements
tested by el-Shazly in alleviating the depression of cellulose digestion
caused by starch in a ration.
The degree of lignification of cellulose is an important factor in
cellulose digestion because the digestibility of cellulose is lowered by
the presence of lignin. Head (1961)„
Sullivan (1955) reported that the
percent lignin and the digestion coefficients of cellulose are correlated
from -0.92 to -0.94.
Percentage of lignin is significantly correlated
with digestibility of all insoluble carbohydrates.
Tomlin et. £l- (1965)
found that as grasses and legumes matured the digestibility of cellulose
decreased and lignin content increased.
Gaillard (1962) noted a strong correlation between the digestibility
of organic matter and the percentage crude fiber, percentage lignin and
;
i
percentage cellulose plus lignin.’ A decrease in digestibility of the
plant was observed as the plant matured.
Some evidence has been obtained that lignin itself is digestible under
certain conditions.
Kane et aJL (1951) observed the products of metabolism
of the lignin molecule in ruminant urine.
The amount of lignin actually
digested was very small and no microorganisms capable of attacking lignin
have been found.
The grinding of feeds has been tried as a method of increasing digest­
ibility of cellulose.
The expected effect of grinding is to increase total
surface area ,and give bacteria more surface to attack.
This has not proven
-12too useful due to the fact that smaller particles pass through the digest­
ive system faster and lower digestibility rather than increase it,
Dehority and Johnson (1961) found the total amount of cellulose
digested increased with the time of ball-milling.
Amount of forage cellu­
lose digested by rumen bacteria decreased with increasing maturity and
lignification of the plant.
The increase in Jji vitro cellulose digesti­
bility due to ball-milling became larger as the forage matured and lignin
,
deposition was increased. There was a basic difference in grasses and
legumes in regard to amount of cellulose which could be digested per given
amount of lignin in the plant.
This evidence presented by Dehority and
Johnson substantiated the theory that lignin in forages acts as a physical
barrier between*the cellulose and cellulolytic rumen bacterial.
. Enzymatic Starch Digestion
Balls and Schwinmer (1944) found that grains of corn starch were more
rapidly broken down than grains of wheat starch.
more resistant to enzyme activity.
Potato starch was much
It was also noted that the size of the
starch grains did not appear to be a limiting factor.
Leach and Schoch (1961) explained that high linear corn starch is
least affected by enzyme activity.
They also observed that normal pea
■'
:
starch is attacked more rapidly than high linear starch from wrinkled peas;
therefore the presence.of a linear fraction probably retards solubilization
by enzymes.
i
According to Sandstedt (1955) when amylase acts on raw starch, the
i
enzyme enters at the narrow edge of the granule and following the path of
-13least resistance digests its way into the granule.
Sandstedt (1960) also
; V
noted that starch which has been damaged will digest easier than undamaged
starch granules.
Enzymes do not penetrate freely into the molecular
lattice of the granule, but are limited to certain accessible surfaces or
regions, Leach and Schoch (1961).
In Vitro Starch Digestion
Moore et_ al. (1962) studied the use of the in vitro system of starch
fermentation.
A high speed centrifugal procedure was used to obtain the
predominate rumen bacteria.
*
colorimetrically.
Maximal starch fermentation was measured
'
The difference between concentrations of anthrone re­
.
active material in inoculated flasks at the time of inoculation, and after
a period of incubation was taken as an index of fermentation.
Maximal
starch digestion occurred at about 12 hours which amounted to 90 percent
of the added starch.
Starch was added at the rate of 1.5 grams per 100
ml. of liquid.
Loper jet Al. (1966) used a gravimetric technique to study starch
digestion in vitro.
A series of experiments based on washed cell suspen­
sions' of rumen microorganisms was conducted to develop an accurate, simpli­
fied technique.
metrically.
Starch remaining after fermentation was measured gravi-
Results obtained by this method were highly correlated
(r = 0.94) with the results using the anthrone procedure.
was carried out at a pH of 6.8.
Maximal starch digestion occurred at 12
hours, which amounted to 70 percent of the added starch.
at the rate of 0i5 percent.
The fermentation
Starch was added
-14There has been very limited work conducted on Jrt vitro starch diges­
tion.
The foregoing literature on in vitro starch digestion studies is
the only work reported.
PROCEDURES
General Procedures
Barley varieties were selected on the basis of known or expected
structural differences observed in laboratory analysis of their respective
starches, Goering _et al. (1957) and Goering and Imsande (1960),
Varieties
selected were Compana, hulless Compana (Newpana), Betzes, Oderbrucker and
Unitan,
Corn starch was used as a standard for comparison since corn is
a major source of carbohydrate in livestock rations,
Milo and wheat
starches were also included because wheat is being used in Montana live­
stock rations and milo is used extensively as a feed grain in other areas
of the United States.
Barley starch was separated from barley flour by Dr. K. J» Goering
using the method of Dimler et^ al. (1944),
Barley flour used in the above
procedure was obtained from the Montana State University Cereal Quality
Laboratory.
Three trials were conducted to measure differences in the in vitro
digestibility of starches from selected varieties of grain.
amylopectin were included in one comparison.
were employed in each trial.
follows:
Amylose and
Identical in vitro procedures
Starches compared in this study were as
Trial I - corn, Compana, Oderbrucker, milo and wheat; Trial 2 -
corn, Compana, Newpana, Betzes and Unitan and Trial 3 - corn, Compana,
Newpana, amylose, and amylopectin.
Residual starch was measured by the phenol-sulfuric acid, colorimetric
method for hexoses described by Whistler (1962).
The percent starch di­
gested was calculated using as the initial amount of starch the values
-16obtained from control tubes in which bacterial action was not allowed to
take place.
Absorbances were measured on a Bausch and Lomb Spectronic 20.
Fermentation vessels were analyzed for residual starch every 2 hours
for a maximum of 12 hours.
Blanks consisting of rumen liquor and nutrient
media but without added starch were analyzed at zero time to correct for
starch present in the rumen liquor.
The phenol-sulfuric acid method was compared to the enthrone colori­
metric method described by Moore <2t al„ (1962) using corn starch as the
substrate.
Absorbance curves were prepared with a Beckman DK2 spectrophotometer
to insure maximum absorbance for all substrates.
A 3 x 5 x 6 factorial design was used in all trials as described by
Li (1965).
In Vitro Procedure
Each experiment was initiated at 5:00 A.M.
Rumen cdntents were ob­
tained from a three-year-old fistulated Hereford steer maintained on.
ration consisting of 1/3 steam rolled barley, 1/3 dehydrated alfalfa pellets, and 1/3 beet pulp pellets with salt provided free choice.
was fed this ration 10 days prior to the initial collection.
The, steer
Rumen con­
tents were collected and strained through four layers of cheese-cloth
v ;
r• if
mil
into a 39° C. prewarmed thermos.
Rumen liquor was then centrifuged at
2,000 x G for 20 minutes using an international size 2 centrifuge.
The
supernatant was mixed with pre-warmed (39° C.) carbon dioxide saturated
nutrient medium at a rate of 3 parts rumen liquor to 4 parts nutrient
-17medium.
The composition of the nutrient medium used is as follows;
KHgPO^
0.600 gm/liter
NaCl
4.00
NagHPO^ 1.600 gm/liter
FeSO^./HgO
0.075 gm/liter
NaHCOg
3.500 gm/liter
CaCl,
0.550 gm/liter
KCl
4.00
Urea
2.000 gm/liter
gm/liter
gm/liter
Carbon dioxide was bubbled through the mixture for 10 minutes and the
pH adjusted to 6.8»
Fifty ml. of rumen liquor and nutrient medium was
added to 100 ml. fermentation vessels which were fitted with rubber stop­
pers containing inlet and outlet glass tubing for the introduction of
carbon dioxide.
Starch was added at the rate of 0.5 percent or 250 mg.
starch per fermentation vessel.
Blank vessels containing no added starch
were included to correct for starch added via the rumen liquor.
tion vessels were placed in a 39° C. water bath.
Fermenta­
Carbon dioxide was
bubbled through the tubes slow enough to prevent excessive splashing.
Fermentations were carried out for a maximum of 12 hours.
Measurement of Starch Fermentation
Phenol-sulfuric acid procedure
One ml. of unknown containing between 20 and 50 ug. substrate was
pipetted into a colorimetric tube.
One ml. of 5 percent distilled phenol
solution was added and contents mixed.
acid were added to each tube.
Five ml. concentrated sulfuric
Each tube was agitated during acid addition.
After standing 10 minutes, tubes were shaken apd placed in a 25-30° C.
water bath for 20 minutes.
Absorbances were measured at 490 mu.
After
subtracting the absorbances of the blank, the amount of sugar was deter\
-18mined from a standard curve prepared from corn starch.
Anthrone colorimetric method
Ten ml. of enthrone reagent in a series of test tubes were immersed
in a cold water bath (10-15° C.) and carefully overlayed with 5 ml. of
sample containing from 20-40 ug./ml. of starch.
After the addition of
starch was made, the tubes were agitated and replaced in the cold water
bath.
When the series was completed the tubes were heated for 16 minutes
in a boiling water bath and then cooled.
Absorbances were read at 625 mu.
and after subtracting the absorbances of the blank, the amount of sugar
was determined from a standard curve prepared from corn starch.
RESULTS AND DISCUSSION
Trials I, II and III
The pertinent data of Trials I , II and III showing the percent diges­
tion for the 5 substrates every 2 hours on each of 3 days are given in
Tables I , III and V respectively.
Analysis of variance of the data for
the respective trials are given in Tables II, IV and VI.
Analysis of this data revealed considerable variance and several
highly significant interactions in all trials.
All interactions excluding
hours x variety for Trials I and II were highly significant (P<^. 01).
The hours x variety interaction was significant (P<^.05) for trial III.
Since the subsequent digestion rates of the substrates were significantly
different (P<^.05) and the hours x variety interaction was not significant,
some general inferences can be made concerning the differences in
digestibilities of the starches.
In Trial I, Oderbrucker starch was digested significantly faster
(P<^.01) or equal to corn starch and the other starches.
Oderbrucker digested significantly slower.
In no case was
In most instances wheat and
milo starch were digested slower than corn starch; however on day I wheat
tended to digest faster than corn starch.
Digestion of all substrates was
very rapid on day 3 of Trial I; therefore it is difficult to detect any
real differences.
On days I and 3 at the end of 12 hours fermentation,
the percent digestion had exceeded 95 percent.
digestion was approached at 12 hours.
On day 2, 90 percent
Replications were highly significant
(P<%01) for Trial I indicating that the reproducibility of measuring
residual starch in the triplicate fermentation vessels was unsatisfactory.
-20“
Percent Digestion I/
Hours
Day I
Corn
Oderbrucker
Compana
18, Oa
17.7*
17.9*
18,5*
12.6b
4
37„8a
36.1*
36.3*
36.8*
30.7b
6
4 8 .7 *
60.4C
55.5b
50.7*
52.8b
8
72.9d
87,5C
67.2b
a
86.5
80.6*
C
93.4
10
88.6
94.0C
79.6*
be
9 2 .9
12
99.2a
99.3*
99.4*
99.0*
99.1*
19.0C
18.3C
12.6b
8,0*
2
Day 3
Wheat
2
ab
Day 2
Milo
9-6ab
4
40.6C
36.5bc
33.5*b
31.2*
22,5d
6
44.7C
40.7bc
41.5bc
38.2*b
35.9*
8
60.Oab
63.5b
56.4*
56.3*
50. Oc
10
7 8 . Q3b
84.2°
80.0bC
7 5 .3 *
7 8 . 5 ab
12
90.2ab
92. Ib
89,9*b
8 6 .3 *
92.4b
2
3 7 .2 *
43.3b
43. Ib
33.6*
3 7 .7 *
4
77.5b .
75.5*b
77.8b
71.7*
76.6b
6
9 3 .7 *
91,9a
93.7*
93.0*
91.4*
8
95.4*
96.0*
95.9*
95.6*
94.0*
10
9 5 .8 *
9 5 .2 *
9 5 .7 *
95. I*
95.0*
12
97.5*
96.9*
97.0*
97.1*
96.9*
JL/ Values expressed are an average of 3 replications,
a, b, c, d Means on the same line bearing different superscript letters
are significantly different (P<JJ»01) as determined by Duncan’s
multiple range test.
-21=
TABLE.II„
ANALYSIS OF VARIANCE OF PERCENT DIGESTION FOR STARCHES IN
TRIAL I (CORN„ ODERBRUCKER. COMPANA. MILO. WHEAT),
Source
D.F.
S .S .
M.S.
F.
Replication
2
66.05
33.03
8.00**
Days
2
42,696.55
21,348.28
5,172,80**
Hours
5
164,648.65
32,929.73
20.72**
Variety
4
8913.14
223.29
10
15,891.69
1,589.17
385.06**
8
409.53
51.19
12.40**
Hours x Variety
20
620.63
31.03
1.06
Days x Hours x Variety
40
1,168.82
29.22
7.08**
178
734.66
4.13
269
227,129.72
Days x Hours
Days x Variety
Error
TOTAL
*
**
4.36*
P <.05
P <. Ol
It can be seen from the data of Trial II presented in Table III and
from the analysis of variance (Table IV) that there were large variations
and highly significant interactions; however the hours x variety inter­
action was considerably smaller than in Trial I „
In Trial TI corn starch
was digested significantly faster ( P 01) or equal to the other starches
in all but one instance.
On day 2 at 2 hours, Unitan and Newpana were
digested significantly faster (P-<% 01).
Unitan starch was digested signif
icantly slower (P<^.01) or equal to the other starches; however on day 2
at 2 hours Unitan was digested significantly faster than Betzess Compana
or corn starch.
Compana, Newpana and Betzes starch were intermediate
between corn starch and Unitan; however it is difficult to determine how
they did compare because of day to day and hour to hour variation.
At the
-22-
Percent Digestion JL/
Day I
Hours
Corn
Compana
Newpana
2
16,6b
9.4*
4
22.9b
6
Unitan
6.4*
7.2*
9.0*
18.6*b
15.5*
18.7*b
9.5*
48.6b
37.03
33.9*
3 3 .9 *
11. oc
8
65.2b
5 9 .6 *
57.2*
58,6*
34.4*
10
8 3 .6 *
83.8*
81.4*
80.3*
70.7b
12
96. Ob
95.3ab
94.9ab
94.4*b
91.0*
14.8c d >
O
rCM
T—4
Betzes
18.5d
.a
16.4
20.6
41,3b
37.4ab
33.3*
50.6*b
4 8 .3 *
5 3 . 7 bC
4 8 .5 *
80.2b
74.7*
71.4*
73.5*
6 2 .2 *
12
9 2 . 6b
8 9 .5 *
89. Oa
8 9 .8 *
83.8*
2
17.4d
3.1*
9 .4 *
4
22.4b
23. Ob
1 9 .7 * b
21» 2*b
17.4*
6
36,2bc
3 7 .7 *
31.6*b
32.Iab
2 9 .8 *
8
4 9 .4 *
4 6 .8 *
4 6 .9 *
4 7 .7 *
4 7 .7 *
10
66.0*
67.8a
65.7*
67.3*
66.4*
12
86.9bc
90.8*
83.9ab
84.3ab
8 2 .2 *
2
Day 2
Day 3
\J
8 . 7 ab
b
7 .2 *
23.1
b
4
21.8
6
39.7b
33.8*
8
58.1C
10
8,8bc
23.7
b
ab
4 .2 * b
Values expressed are an average of 3 replications,
a, b, c, d Means on the same line bearing different superscript letters
are significantly different (P\,01) as determined by Duncan's
multiple range test.
-23end of 12 hour fermentations in almost all cases the substrates reached
90 percent digestibility.
It should be noted that replications were not
significantly different in Trial II.
TABLE IV,
ANALYSIS OF VARIANCE OF PERCENT DIGESTION FOR STARCHES IN
TRIAL II (CORN. COMPANA . NEWPANA, BETZES„ UNITAN).
Source
D.F.
S.S.
M.S.
F.
Replications
2
3.63
1.81
.4267
Days
2
988.57
494.28
116.25**
Hours
5
213,830.98
42 ,766.20
244,67**
Variety
4
2,631.77
657.94
4.30*
10
1,747.93
174.79
41.11**
8
1,224.45
153.06
36.00**
Hours x Variety
20
894.66
44.73
.8147
Days x Hours x Variety
40
2,196.05
54.90
12.91**
178
756.81
4.25
269
224,274.85
Days x Hours
Days x Variety
Error
TOTAL
'**
po
Qi
The analysis of variance (Table VI) and the data from Table V show
that for Trial III there were again large variations and highly signifi­
cant interactions.
Unlike Trials I and II, the hours x variety inter­
action was significant (P<^.05) indicating that the substrates did not
digest in the same manner at the different hours. Due to the fact that
all interactions were significant, general conclusions cannot be made;
however from an inspection of the data there are obvious differences,
namely amylopectin and amylose digestion.
In all cases amylopectin
-24TABLE V.
PERCENT DIGESTION OF SUBSTRATES IN TRIAL III.
Percent Digestion _1/
Hours
Corn
2
11,4
Compana
a
11.3
4
2 5 .8 *
2 3 .9 *
6
41.8*
8
a
Day I
Day 2
Day 3
Newpana
9 .9
Amylopectin
Amylose
b
a
b
18.9
20.1
2 4 .1 *
5 6 .7 *
42. Ob
39.8*
3 6 .7 *
75.1C
5 7 . 4b
55.9*
53.6*
56.5*
86.1C
74.4b
10
70.3*
68.1*
71.5*
90.7C
78.6
12
89.6b
91.4b
88.2*b
92,5
2
13.4*
16.2*
18.9*
57. Oc
31.6b
4
24.0*
24.3*
2 2 .7 *
90. Ic
49.9b
6
45.6*
51.0®
4 9 .5 *
95.8*
64.4b
8
81.Oab
83.3b
76.3®
96. Ic
66. Od
10
9 3 .9 *
9 5 .9 *
9 4 .1 *
98.1*
7 9 .7 b
12
9 7 .0 *
98.1*
9 7 .1 *
98.7*
84.2b
2
3 9 .3 *
3 8 .7 *
2 9 .3 *
45.7b
4 2 .5 * b
4
7 6 .9 *
7 5 .8 *
7 4 .6 *
91.5*
48.9b
6
9 5 .9 *
94.9*
9 2 ,9 *
^ 9 5 .7 *
7 7 . 3b
8
9 7 .7 *
98.2*
9 7 .9 *
97.4*
82.2b
10
99.0*
99.1*
9 9 .1 *
9 8 .8 *
86.Ob
12
9 9 .0 *
99.1® '
99.2*
98.9*
90.6b
b
b
. 8 3 .4 *
_!/ Values, expressed are an average of 3 replications,
a, b, c, d Means on the same line bearing different superscript letters
are significantly different (P<<,01) as determined by Duncan’s
multiple range test.
-25digested significantly faster (P<^„01) or equal to the other substrates.
On day I amylose digested significantly faster (P<^.01) than corn, Gompana
or Newpana; however at 12 hours amylose was significantly slower (P<^01)
than corn, Gompana and amylopectin.
On day 2 amylose digested significant=
Iy faster (P<^.01) than corn, Compana or Newpana at 2, 4 and 6 hours; how­
ever at 8, 10 and 12 hours amylose was significantly slower (P<^01).
On
day 3 amylose digested faster at 2 hours but beyond 2 hours amylose diges­
ted slower than the other substrates.
As in Trials I and II, digestion
was either greater than or approached 90 percent at the end of 12 hours
fermentation.
TABLE VI.
Replications were again non-significant as in Trial II.
ANALYSIS OF VARIANCE OF PERCENT DIGESTION FOR STARCHES IN
NEWPANA,
Source
D.F.
S.S.
M.S.
F.
I.
Replications
2
18.82
9.41
Days
2
33,775.36
16,887.68
2 ,3 1 7 .2 d * *
Hours
5
145,119.51
29,023.90
3 5 .5 7 * *
Variety
4
14,605.97
3,651.49
10
8,159.56
815.96
1 1 1 .9 6 * *
Days x Variety
8
7,100.47
887.56
1 2 1 .7 9 * *
Hours x Variety
20
7,226.75
361.34
1 .9 4 * '
Days x Hours x Variety
40
7,456.57
186.41
2 5 .5 8 * *
178
1,297.21
7.29
269
224,760.22
Days x Hours
Error
TOTAL
*■ P <.05
** P < Ol
1.29
4 .1 0 *
'
-26The results of these trials indicate that if the digestibility of
-
starches is to be compared by the in vitro method, improved techniques
must be employed.
It became evident as the trials progressed that an improved inoculum
should be tried which would contain a more representative starch digesting
bacterial population.
Strained, centrifuged rumen liquor as used in these
studies probably contained many types and strains of bacteria plus enzymes
which would digest starch.
Moore et al, (1962) used a differential centri­
fugation to prepare a bacterial sediment that was microscopically representative of the mixed culture found in the whole rumen fluid of sheep.
Loper et al. (1966) used a washed cell suspension following the method of
Cheng et'al. (1955) to obtain a predominate bacterial population.
An in­
oculum similar to that used by Moore and Loper could possibly eliminate
some of the variability due to abnormal bacterial, populations.
however many factors which affect the bacterial populations.
There are
Johnson
(1963) reported that numbers' of species and types of microorganisms which
digest starch in -the rumen are considerably greater than for cellulose.
This is complicated further by the fact that inoculum taken from a given
animal on a standard starch ration may differ from day. to day in predomin­
ating species of microorganisms.
in the amylolytic flora of sheep.
Moore et al. (1962) noted the variability
Variability in the microflora within
the animal can be a result of rumen volume, Purser and Moir (1966a) and
■Purser and Moir (1966b), or ration characteristics, Christiansen et' al,.
(1964) .
By providing the donor animal with a constant ration and sampling
at # constant time after feeding variability in microflora can be reduced.
-27Structural differences between starches should be examined when com­
paring differences in starch digestion.
A microscopic examination can be
made to determine differences in granular size and shape.
It has been
reported that structural differences affect starch breakdown.
Baker et al.
(1950) noted a distinct difference between maize starch and potato starch
when they were subjected to jin vivo digestion by oxen.
Goering _et al.
(1957) reported that the amylose content of 44 different barley varieties
varied from 13 to 24 percent.
Leach and Schoch (1951) found that high
linear corn starch (i.e. corn starch with a high percentage of amylose) is
least affected by bacterial enzyme activity.
Harris (1962) and Greenwood
(1964) stated that starch granules in general contain more amylopectin
than amylose.
These authors imply that starches with a high percentage of
amylopectin will digest faster than those starches with a high percentage
of anylose.
In Trial III, amylopectin digested faster than amylose, indi­
cating that a high linear barley starch may digest slower.
If starches
with high percentages of amylopectin repeatedly digest faster than high
linear starches, then it may be possible that starches can be ranked in
regard to digestibility by their percentages of amylopectin and amylose.
Phenol-Sulfuric Acid ~ Anthrone Comparison
The data for the comparison of the phenol-sulfuric acid and enthrone
procedures is shown.in Table VII,
There was a highly significant cor­
relation (r = 0.99) between the two methods.
The percentage of residual
starch measured was comparable except at the 2 and 4 hour determinations.
These results indicate the phenol-sulfuric acid method used in this study
-28was equivalent to the enthrone method employed by Moore ejt aL. (1962).
TABLE VII.
COMPARISON BETWEEN THE ANTHRONE METHOD
AND THE PHENOL-SULFURIC ACID METHOD
USING CORN STARCH ON DAY 3 TRIAL III,
Percent Digestion
Hours
Anthrone
PhenolSulfuric
Acid
2
29.9
27.9
29.2
37.0
38.0
36.5
4
84.1
82.7
82.6
6
92,8
91,8
93,0
93.7
93.2
94.2
8
98,0
97.8
97,6
95.3
95.4
95.4
10
98.9
99.2
99.2
95.8
95.9
95.8
12
98.9
98.6
98.1
97.4
97.6
97.7
u
77.1
78.5
77.0
r = ,99
The phenol-sulfuric acid colorimetric method has worked very well in
the trials reported in this manuscript,
Dubois et al, (1956) observed
that the phenol-sulfuric acid procedure was simple, rapid and sensitive
and gave reproducible results.
The reagent is inexpensive and stable and
a given solution requires only one standard curve for each sugar.
There are several inherent problems in the enthrone procedure used by
-29Moo re £t al^
(1962)„
The concentration of acid in the reagent and the
manner in which the reagent and test solution are brought together deter­
mine the nature and intensity of the color developed, Johanson (1954).
Morris (1948) described some of the problems associated with the enthrone
procedure.
These are as follows:
(I) the reagent is difficult to prepare,
(2) if cooling is produced before the reaction is finished, error can be
introduced, (3) a known sugar standard must be run with each series of
X
unknowns, (4) fresh reagent must be prepared daily and (5) aging of the
reagent causes a color change.
Moore at al. (1962) using the enthrone procedure recovered 130 it
5.7 percent of the added starch.
Using the phenol-sulfuric acid method
in this laboratory the recovery of added starch was 109 i 6„5 percent
(Table VIII).
With the evidence presented the phenol-sulfuric acid procedure
because of simplicity can be used in preference to the anthrone method for
estimating in vitro starch digestion.
TABLE VIII0 PERCENT RECOVERY OF ADDED STARCH FOR TRIALS I. II. III0
Day 2
Day 3
Day I
Day I
Day 2
Day 3
Day I
Trial II Trial II Trial II Trial III
Trial I
Trial I
Trial I
Day 2
Trial III
Day 3
Trial III
108.7
113.3
108.2
112.8
108.6
112.8
110.1
110.2
112.2
113.6
118.0
106.0
112.6
113.9
110.1
112.8
107.7
107.4
113.6
117,8
103.2
112.8
115.9
112.9
115.3
107.6
109.8
113.7
117.7
108.2
112.4
108.4
112.6
107.6
112.5
107.2
113.7
117.9
105.8
102.8
108.4
110.1
110.1
105.1
109.9
118.6
106.8
110.6
105.3
106.1
105.1
112.9
107.6
109.6
108.7
117.7
115.8
102.6
108.6
110.1
115.3
110.0
102.3
118.8
113.3
113.5
107.7
108.4
107.6
112.7
110.0
107.1
113.8 .
113.3
110.6
102.6
108.4
107.9
107.7
105.0
104.7
108.7
113.3
105.7
108.1
113.3
112.7
105.4
105.2
107.2
118.6
113.4
103.2
102.9
108.5
107.6
107.8 .
107.4
107.5
113.9
113.4
105.7
102.6
106.0
110.1
112.9
107.6
112.3
118.8
113.2
103.3
77.9
108.4
102.9
112.7
112.5
112.3
113.8
113.4
105.9
72.7
103.5
102.7
110.1
105.1
105.0
113.8
117.7
108.4
77.7
106.0
105.3
107.6
107.6
107.3
Mean 109.0.
Standard Deviation 6.5.
/
SUMMARY
Three jin vitro digestion trials were conducted comparing 5 barleystarches (Oderbrucker, Compana, Newpana, Betzes and Unitan) corn starch,
milo starch, wheat starch, amylose and amyIopectin0 Within each trial
substrates were subjected to digestion on each of 3 different days.
The
phenol-sulfuric acid method of starch hydrolysis was used to measure
residual starch in the fermentation vessels every 2 hours up to 12 hours.
The phenol-sulfuric acid and enthrone procedures were compared to
determine if the phenol-sulfuric acid method was comparable to the anthrone
method of-Moore _et al, (1962)„
In Trial I the starches of corn, Odepbrucker, Compana, milo and wheat
were compared,
Oderbrucker starch was digested significantly faster
(P<^,01) or equal to corn starch and the other starches.
Oderbrucker digested significantly slower.
In no case was
In most instances wheat and
milo starch were digested slower than corn starch; however on day I wheat
tended to digest faster than corn starch.
In Trial II the starches of corn, Compana, Newpana, Betzes and Unitan
were compared.
Corn starch digested significantly faster (P<^,01) or
/(
equal to the other starches in all but one instance. On day 2 at 2 hours
'
Unitan and Newpana were digested significantly faster (P<\ 01).
Unitan
starch digested significantly slower (P<^.01) or equal to the other
starches; except on day 2 at 2 hours Unitan was digested significantly
faster than Betzes, Compana or corn.
In Trial III corn starch, Compana starch, Newpana starch, amylopectin
.
and amylose were compared,
Amylopeetin digested significantly faster
(P<^.01) or equal to the other substrates.
On day I amylose digested
-32significant Iy faster (P<%01) than corn, Compana or Newpana; however at 12
hours amylose was significantly slower than corn, Compana or amylopectin.
Amylose tended to digest faster than the other substrates at the start of
the fermentation period.
At the end of the period amylose was digested
slower than the other substrates.
In all trials at the end of the fermentation period digestion was
either greater than or approached 90 percent.
All interactions excluding hours x variety for Trials I and II were
highly significant.
Hours x variety was significant (P<'.05) for Trial
III.
There was a highly significant correlation (r = .99) between the
enthrone method and the phenol-sulfuric acid method.
This indicates that
the two methods are comparable.
Some differences in starch digestion have been indicated from these
i
•.
trials.
,
In order to more effectively compare differences between starches,
improved techniques must be employed to eliminate the variability which
was encountered.
v .
'
...
-
'
. ■
. .
,V
APPENDIX
-34APPENDIX TABLE I, PERCENT DIGESTION — THREE DAY AVERAGE FOR TRIALS
___________________ I a II. III.______________________________________
Percent Digestion \J
Trial I
Hours
Corn
Oderbrucker
Compana
2
21.6*
26.7b
4
52.0C
6
Milo
Wheat
26,4b
21.6*
19.4*
49.4bc
49.2bc
46.6*b
43.2*
62.4ab
64.3b
63.6*b
60.6*b
60.0*
8
76.1*
82.3b
77.3*
73.0*
74.9*
10
87.5*b
91.Ib
89.5*b
85.6*
89,Oab
12
95.6*
96.1*
95.4*
94.1*
96.2*
Compana
Newpana
Betzes
Unitan
Hours
Trial II
Corn
2
14.2b
8.5*
8.1*
9. Sab
10,6*b
4
22.4b
21.6b
19.6ab
18.7ab
15.8*
6
41.5b
36.2*
35.6*
34.4*
24.7C
8
57.6b
52.4*
50,8*
53.3*b
43.5C
10
76.6*
75.5*
72.9*
73.7*
66.4b
12
91.8b
91.9b
89.2ab
89.5*b
85.7*
-35APPENDIX TABLE I,
Trial III
Continued.
Hours
Corn
>
Compana
Newpana
Amylopectin
Amylose
2
21.4*
22.1*
19.4*
40.5*
31.4b
4
42.2ab
41.3*b
40.4®
79.4*
46.9b
6
61.Iab
61.9ab
59.7*
88.9C
66,4b
8
78.2®
78.3®
76.9®
93.4b
74.2*
10
87.7*
87.7*
88.2*
95.9C
81.4b
12
95.2*
96.2*
94.8*
96.7*
86, Ib
_!/ Values expressed are an average of 3 replications on each of 3 days.
a, b, c Means on the same line bearing different superscript letters
are significantly different (P<^.01) as determined by Duncan's
multiple range test.
\
-36APPENDIX TABLE II0
__________________
TWO-WAY TABLE SHOWING DAYS X VARIETY INTERACTION FOR
TRIALS I, II0 III.______________________________ '
Percent Digestion _/
Corn
Trial I
Trial II
Trial III
Oderbrucker
Compana
Milo
Wheat
Day I
60.9ab
65.8d
63.6C
59.8*
61.5b
Day 2
53.8*
56. Od
53.3*
50.Oc
47.9b
Day 3
82.8*
_
- _a
81.0
^ab
81.9
ahc
83.1
be
83.9
C
Corn
Compana
Newpana
Day I
55.5d
50.6b
48.2*
48.8*b
37.6C
Day 2
50.2b
46.5*
48.1*
47.2*
44.5C
Day 3
46.A c
45.8=
40.2*
43.7b
41.3*
Corn
Compana
Newpana
Amylopectin
Amvlose
Day I
49.1*
48.0*
47.8*
70. Ob
59.3C
Day 2
59.2*
61.5*b
59.7*
89.3C
62.6b
Day 3
84.6b
84.3*b
82.2*
86.3b
71.2C
Betzes
Unitan
Values expressed are an average of 3 replications on each of the 6
time periods.
.
a, b, c, d Means on the same line bearing different superscript letters
are significantly different (P<%01) as determined by Duncan’s
multiple range test.
I/
-37APPENDIX TABLE III. . TWO-WAY TABLE SHOWING HOURS X DAY INTERACTION FOR
____________
TRIALS I. II. III.__________ £____________________
Percent Digestion I^f
Trial I
2
4
6
8
10
Day I
16.9C
35.5=
53.6=
77.6=
91.1=
99.2=
Day 2
13.5b
32.9b
40.2b
57.2b
79,2b
90.2b
Day 3
39. Oa
75.8a
92.7a
95.4a
95.4a
97.Ia
Day I
9.7^
17. Oa
32.9b
55.0=
80.0=
94.3=
b
„ ,b
72.4
„b
88.9
/
Trial II
Trial III
Day 2
.a
12.4
21.1
a
b
37.1
,
51.8
12
20.7b
33.5b
47.7*
66.6a
85,6*
14.3C
34.5C '
50.2C
65.3°
75.8C
89. Ob
Day 2
27.4b
42.2b
61.3b
8 0 .5 b
92.3b
95. Oa
Day 3
39. Ia
73.5a
91.3a
94.7a
96.4a
97.4a
Day 3
8.6b
Day I
Values expressed are an average of 3 replications for each of the 5
substrates.
a, b, c Means on the same line bearing different superscript letters are
significantly different (P<^01) as determined by Duncan's multiple
rangfe test.
If
-38-
IOO-
90-
Corn Starch
Oderbrucker Starch
Compana Starch
MHo Starch
Wheat Starch
8070TJ
0)
CO
CD
0)
GO50
2
o
(/) 4 0 3020
-
I O-
4
6
8
IO
Hours Fermentation
APPENDIX FIGURE I
12
PERCENT DIGESTION--THREE DAY AVERAGE FOR TRIAL I
-39-
IOOC o r z ? Starch
Compana Starch
Newpana Starch
Unitan Starch
Betzes Starch
90
80
70
*0
0
4(/)
60
Q)
U>
Q 50
x:
o
v
0
40
0)
30
20
I
IO
2
APPENDIX FIGURE 2
4
6
8
IO
Hours F er mentati on
12
PERCENT DIGESTION--THREE DAY AVERAGE FOR TRIAL II.
-40-
Corn Starch
Compana Starch
Newpana Starch
Amytopectin
Amytose
IOO-
9 0 80
—
7 0 "0
0)
</>
0)
gi
O
4-
_c
2
o
S
60 —
5 0 4 0 3 0 20
-
IO-
2
APPENDIX FIGURE 3.
4
6
8
IO
Hours Fermentation
12
PERCENT DIGESTION--THREE DAY AVERAGE FOR TRIAL III.
-41-
ioo90 80
TO­
GO 5 0 -
Phenol-Sulfuric Acid
Anfhrone
4-0 3 0 20
-
IO -
4
6
8
IO
Hours Fermentation
APPENDIX FIGURE 4.
PERCENT DIGESTION--COMPARISON BETWEEN THE PHENOLSULFURIC ACID METHOD AND THE ANTHRONE METHOD.
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I
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An in vitro digestion of
selected starches