^ . / ' • •

AMINO ACID AVAILABILITY IN IN VITRO GRAIN SORGHUM

ENZYMATIC HYDROLYSATES

by

MARGARET ELIZABETH WILLIS BRILEY, B.S. in H.E., M.S. in H.E

A DISSERTATION

IN

AGRICULTURE

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for

the Degree of

DOCTOR OF PHILOSOPHY

Approved

August, 1973

^01

T 3

No. ^8 n ^

ACKNOWLEDGMENTS

I am deeply indebted to Dr. Leland F. Tribble for his direc­

tion of this study and to the other members of my committee. Dr. Willis

L. Stames, Dr. Dale W. Zinn, Dr. Robert C. Albin, Dr. Jerry D. Ramsey,

Dr. S. P. Yang, and Dr. A. Max Lennon, for their helpful criticism.

^

ii

CONTENTS

ACKNOWLEDGMENTS ii

LIST OF TABLES v

LIST OF FIGURES vi

CHAPTER

I. INTRODUCTION 1

II. EXPERIMENTAL METHODS AND PROCEDURES 8

A. Amino Acid Composition of Grain Sorghum 8

Collection and Milling of Grain Sorghum 8

Total Protein Determination 8 Acid Hydrolysis of Grain Sorghum Samples 9 Amino Acid Analyses 10 Method for Correction of Serine and

Threonine Content to Zero Hydrolysis Time .10

Method for Correction of Valine and Isoleucine to Infinite Hydrolysis Time 11

B. Gel Filtration Chromatography for the Separation of Amino Acids from Grain Sorghum Hydrolysates 16

Column Construction 16 Sample Application 17 Ninhydrin Procedure 18 Test of Column for Separation of Amino

Acids from Carbohydrates 18

C. Enzymatic Digestion of Grain Sorghum 18

Preparation of Pig Enzymes 18 Preparation of Semi-Purified Enzymes 20 In Vitro Digestion 20 Gel Filtration Chromatography 22 Amino Acid Analyses 22

iii

III. RESULTS AND DISCUSSION 23

A. Amino Acid Composition of Grain Sorghum 23

B. Gel Filtration Chromatography and the Partial Purification of Amino Acids from Grain Sorghum Hydrolysates 25

C. Enzymatic Digestion of Grain Sorghum 30

IV. SUMMARY 36

REFERENCES 38

APPENDIX ^1

iv

LIST OF TABLES

1. LITERATURE VALUES OF AMINO ACID COMPOSITION OF GRAIN SORGHUM 6

2. AMINO ACID COMPOSITION IN GRAIN SORGHUM AS DETERMINED BY ACID HYDROLYSIS 24

3. AVERAGE NANOMOLES AND PERCENT RECOVERY OF AMINO ACIDS FROM GRAIN SORGHUM SAMPLE I WITH SEPHADEX COLUMN AFTER ACID HYDROLYSIS 28

4. AVERAGE NANOMOLES AND PERCENT RECOVERY OF AMINO ACIDS FROM GRAIN SORGHUM SAMPLE II WITH SEPHADEX COLUMN AFTER ACID HYDROLYSIS 29

5. . AMINO ACID VALUES RECOVERED FROM SEMI-PURIFIED ENZYMATIC IN VITRO DIGESTION OF GRAIN SORGHUM SAMPLE I 31

6. AMINO ACID VALUES RECOVERED FROM PIG ORGAN HOMOGENATES IN VITRO DIGESTION OF GRAIN SORGHUM SAMPLE I 32

7. PERCENT RECOVERY OF AMINO ACIDS FROM GRAIN SORGHUM BY IN VITRO DIGESTION WITH SEMI-PURIFIED AND PIG ORGAN HOMOGENATES 33

LIST OF FIGURES

1. Correction of Serine Content for Grain Sorghum, Sample 1 12

2. Correction of Valine Content for Grain Sorghum, Sample 1 14

VI

CHAPTER I

INTRODUCTION

Grain sorghimi, referred to as Sorghum vulgare, has been grown

in the United States since 1957 (King et_ al., 1961). It is econom­

ically important to central United States, but especially important

in Texas where 319.8 million bushels were grown in 1972 (King, 1973).

Grain sorghum will produce more total energy per acre in marginal soil

and climate conditions than other cereal grains. The abundant supply

of this feedstuff has resulted in the rapid development of the swine

industry and of grain sorghum research in the High Plains area of

Texas.

The availability of amino acids has been important to research­

ers since the discovery of their value in animal nutrition. Berg and

Rose (1929) discovered that the frequency of feeding tryptophan to

rats on a tryptophan-deficient ration influenced their growth rate.

Research using the amino acid analyzer developed by Stein and Moore

(1951) has illustrated the varying amounts of each amino acid present

in feedstuffs. Nevertheless, little information exists which shows

the efficiency of release of amino acids from dietary proteins that

are to be absorbed and utilized by a biological system. The biologi­

cal value of a protein is considered an estimate of the effectiveness

with which the body utilizes the amino acids in the protein.

No allowances are generally made for variation in digestibility or

availability of the amino acids. Therefore, the determination of

availability of amino acids from a protein source remains an

important problem.

In vitro and in vivo studies with ruminant animals have

given some indication of the role enzjnnes play in availability of

amino acids. Low growth rates and smaller amounts of the pancreatic

enzymes, trypsin and chymotrypsin, were associated with soy flour

diets fed to cannulated calves (Gorrill et_ a^., 1966). In a second

study, correlation of efficiency of protein digestion in the small

intestine with trypsin and chymotrypsin activity when calves and

lambs were fed different diets showed as the age of both calves and

lambs increased the trypsin and chymotrypsin activity increased.

Protein digestion using in vitro conditions did not simulate condi­

tions of the ±n vivo study entirely (Gorrill et aJL. , 1968).

When casein and whole egg diets were fed to rats to determine

the response of digestive enzymes to dietary protein, an increase of

rate of trypsin and chymotrypsin activity was found as the dietary

protein increased. Furthermore, ±n vitro hydrolysis of egg protein

by trypsin and chymotrypsin proceeded less rapidly than the hydrolysis

of casein by trypsin and chymotrypsin (Snook £t al.., 1964).

An evaluation by Ingram (1949) of nine soybean diets for

chicken growth showed the ±n vitro enzymatic release of amino acids

was not significantly different from the feeding value of the diets.

When different levels of maize diets were fed to cattle,

results showed methionine, isoleucine, and serine were not completely

liberated from the digesta. An increased intake of maize resulted in

a gain of methionine and histidine in the digesta, but a lower level

of phenylalanine and lysine (Neudoerf fer a]^., 1971).

Investigations on human subjects with an intubation technique

were made to evaluate the rate of release of free amino acids in the

intestines after three different protein test meals. Rapid break­

down of protein occurred in the small intestinal lumen. The lack of

methods to differentiate between dietary and endogenous protein made

it difficult to distinguish between the two. In addition, much of

the test protein was hydrolyzed and the products were absorbed before

they reached the sampling area in the small intestine (Nixon and

Mawer, 1970).

Much work in recent years has been devoted to the baby pig

and its nutrition. Studies began in the late 1950's on digestive

enzyme activity in the young pig (Bailey al.^ 1956; Kitts et al.,

1956). Enzymes of the small intestines of weaned and unweaned pigs

were observed to increase in activity with age. By age seven weeks

the enzymes secreted by the pigs were the same level as in the mature

pig (Hartman £t al., 1961). This work agreed with earlier work done

by Bailey eit al. (1956).

Gastric fistulated young pigs four to ten weeks of age were

used to determine the gastric pH. The acidity of the gastric juice

at age four weeks was insufficient for maximal pepsin activity. The

recovered gastric juice was contaminated by saliva at slaughter which

complicated the use of this material in an in vitro method (Maner et

al., 1949).

Baby pigs from birth to day 23 were sacrificed to study the

development of the pancreas and enzyme activity of the pancreas. As

in previous studies the pancreas weight increased with age and pan­

creatic activity increased three-fold. No significant difference

was found due to different sources of protein in the diet. There

was extreme variability among individual animals in enzyme activity

(Pond et al., 1971).

The preceding information suggests many difficulties are

encountered in studying the proteolytic digestive processes of ani­

mals and the amino acid availability from feedstuffs. Specific

problems in these systems related to amino acid availability included

genetic variability of test animals, variable amino acid content of

the gastric juices which interfere with recovery of products from the

exogenous substrate, variation in temperature and pH of the process

from animal to animal and site of hydrolysis within the animal, and

variability depending on sex and age of the animal. These uncer­

tainties for estimation of amino acid availability from a specific

feed are unsatisfactory in many instances, and even when the models

are suitable, the methods employed in most studies make them inher­

ently too cumbersome and/or expensive for routine study of the amino

acid availability.

An in vitro technique with a partial enzjnne system for estima­

tion of the protein quality of human foods was devised by Akeson and

Stahmann (1964). Small sample sizes were used making this method a

fast and much less expensive procedure compared to a feeding trial.

This model employed only pepsin and pancreatic enzymes and did not

account for other enzymes involved in proteolytic digestion. The cal­

culations required were based on biological values reported in the

literature. This study suggested the possibility of simulating a

digestive system ±n vitro, and of using this model system to estimate

amino acid availability from food proteins.

An example of the amino acid content of some grain sorghums

is shown in Table 1 (Waggle a]^. , 1967; Bressani and Rios, 1962).

The data showing grain sorghum amino acid content varies from analysis

to analysis. Fertilization, maturity, available water, length of time

of storage, processing procedure, method of hydrolysis, and the amino

acid analyzing methods influence the grain sorghum composition data.

The amino acid content of grain sorghum has been estimated with micro­

biological assays requiring several different test organisms and media,

and giving only partial information (Bressani and Rios, 1962). Later

more complete information was obtained with automatic amino acid

analyzers (Waggle and Deyoe, 1966).

Most analyses of amino acid content of feed grains are per­

formed on acid hydrolyzed samples of the grain. Under the normal

conditions for acid hydrolysis, serine and threonine are partially

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destroyed, and valine and isoleucine peptides are resistant to hydrol­

ysis (Blackburn, 1968). Therefore, hydrolytic procedures should include

some method to permit correction of the results obtained for at least

these four amino acid residues. Timed hydrolysis studies are done for

this purpose in protein chemistry (Needleman, 1970; Stames, 1973), and

have been incorporated into this study so that the accuracy of the

determination of these residues may be improved. This is particularly

important since at least three of four residues, namely, theronine,

valine, and isoleucine, are considered to be essential in the nutrition

of mammals.

The objectives of this study were as follows:

A. To study the amino acid composition of two sources of grain

sorghum and to report on the refinement of procedures for acid hydroly­

sis which permits correction of serine and threonine content to zero

hydrolysis time and valine and isoleucine content to infinite hydroly­

sis.

B. To study gel filtration chromatography and the partial

purification of amino acids from grain sorghum hydrolysates.

C. To develop an in vitro digestion simulation to determine

the availability of amino acids from grain sorghum for swine with

fresh pig organs or commercially prepared material as sources of

digestive enzymes.

CHAPTER II

EXPERIMENTAL METHODS AND PROCEDURES

A. Amino Acid Composition of Grain Sorghum

The total protein and amino acid composition of two different

mixtures of grain sorghum were used to standardize and calibrate the

separation methods. Sample I served as the standard substrate for

the in vitro proteolytic digestion study.

Collection and Milling of Grain Sorghum

The grain sorghum (Sample I) used in this study was a mixture

of varieties purchased at a local elevator in Lubbock, Texas. Addi­

tional grain sorghum (Sample II) was obtained from DeKalb Seed Company

and was variety C42-Y, a yellow endosperm type. A sampling probe was

inserted into the bin of grain sorghum in twelve different areas to

collect a random sampling of the grain. The samples were mixed and

passed through a Wiley mill until the consistency of whole wheat flour

was achieved. For laboratory analysis the sample was mixed thoroughly

again prior to each weighing.

Total Protein Determination

Kjeldahl determinations were made according to the procedure

of Official Methods of Analysis of the Association of Official Agri­

cultural Chemists (1970). Triplicates were run on each sample and

on blanks.

8

Acid Hydrolysis of Grain Sorghum Samples

Total nitrogen content determined by Kjeldahl analysis was

used to calculate the weight of the grain sorghum sample which would

provide seven mg of protein for acid hydrolysis (Needleman, 1970).

The sample was transferred to a small test tube; 0.5 ml of a constant

source of 6 N hydrochloric acid was added and the contents frozen in

a dry ice-isopropanol bath. The test tubes were constricted, attached

to a vacuum source, evacuated, and sealed. At the end of each of 20,

26, 44, 56, and 70 hours in a 110°C oven two samples were removed,

frozen, the tubes opened, thawed, and the hydrolysates filtered through

a coarse glass filter to remove particulate matter. The tube and fil­

ters were rinsed twice with 0.1 N hydrochloric acid. The filtrate was

2 concentrated in a rotary evaporator under vacuum with the water bath

temperature no higher than 40**C; the samples were removed from the

evaporator with two washes of 0.1 N hydrochloric acid and dried in

vacuo. The samples were then ready for amino acid analysis.

The procedure for Sample I is described. Sample II was hydro­lyzed with samples in triplicate for time intervals of 20, 26, 36, 48, and 72 hours. Time intervals may be arranged conveniently so that the first and second intervals do not vary beyond a lower and upper limit from 16 to 20 hours, and from 24 to 28 hours, respectively, and so that the total interval is greater than 48 hours. Since the correc­tion factor varies from procedure to procedure, and since the content of the sample alters the experimental result, the timed hydrolyses were performed on each sample.

o

This step was omitted with Sample II. The filtrate was col­lected in a large tube and dried iii vacuo over sodium hydroxide and Drierite.

10

Amino Acid Analyses

The amino acid analyses were done essentially according to a

standard procedure obtained from Beckman Instruments (Stein and Moore,

1951; Starnes, 1973). They were done on a Beckman 121-HP automatic

amino acid analyzer programmed for standard four-hour protein hydrol-

ysate analyses. Samples (0.25 ml) of the enzymatic or acid hydrolyzed

3 protein dissolved in buffer (pH 2.2, 0.2 N citrate) were automatically

injected alternately to a 5.0 cm ("short") ion exchange column packed

with Beckman type Pa-25 resin for analysis of the basic amino acids or

to a 55 cm ("long") column packed with Beckman type AA-15 resin for

analysis of the acidic and neutral amino acids. The short column was

eluted with citrate buffer (pH 5.26, 0.02 N), and the long column was

eluted with citrate buffer (pH 3.25, 0.02N) followed 101 minutes into

the program by another citrate buffer (pH 4.25, 0.02 N). Chromatograms

were calculated using the height times the width at the half-height of

the peak obtained for each amino acid as a suitable approximation of

the integral describing the total area under the peak. Constants for

conversion of the area to units of concentration were obtained from

analyses performed on a Beckman Standard containing a mixture of the

18 common amino acids.

Method for Correction of Serine and Threonine Content _to Zero Hydrolysis Time

Upon calculation of the content of each amino acid, two ratios

were calculated as follows: the content (in nanomoles) of four stable

3 Citrate buffers used are those prepared by Beckman Instruments

11

amino acids were summed and divided by four. The content of glycine,

alanine, phenylalanine, and leucine were used. The serine content and

the threonine content were each divided by the average of four stable

amino acids for each of the various analyses at separate times of

hydrolysis. A plot of the ratio thus obtained versus the time of

hydrolysis usually shows the first order decay curve illustrated for

serine with grain sorghum Sample I in Figure lA. It is impossible to

estimate the value for serine or threonine content at zero time from a

curve of the type in Figure lA. However, if the decay is first order,

a plot of the logarithm of the ratio versus time will be linear.

Figure IB illustrates this transformation of the data in Figure lA.

The method of least squares (unweighted) was used to estimate the

ratio (actually, the logarithm of the ratio) at zero time (Stames,

1973). The appropriate value obtained from similar plots for each of

the residues were used to calculate the corrected serine or threonine

content at zero hydrolysis time. The zero time ratios were multiplied

times one-fourth the sum of the content of glycine, phenylalanine, ala­

nine and leucine for each completed analysis.

Method for Correction of Valine and Isoleucine to Infinite Hydrolysis Time

Ratios were computed for each value of valine and isoleucine

content at each hydrolysis time as they were for serine and threonine

and plotted versus time of hydrolysis. Figure 2A shows the results

obtained from grain sorghum Sample I for valine.

12

V

Figure 1.—Correction of Serine Content for Grain Sorghum, Sample I.

A. A plot of ratio of serine content vs . time of hydrol­ysis. The bars represent range of calculated serine.

B. A plot of the logarithm of the ratio v£. time. The line is unweighted least squares fit. The value of the intercept is -.924 and the calculated (corrected serine content ratio) value is 0.397.

13

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14

Figure 2.—Correction of Valine Content for Grain Sorghum, Sample I.

A. A plot of ratio of valine content vs_. time. The bars represent ranges of the calculated values for ratios.

B. A plot of the reciprocal of ratio vs_. the reciprocal of time. The data are taken from plot A. The line is unweighted least squares fit. The value of the intercept is 2.52 and the calculated (correct valine content ratio) value is therefore 0.397.

15

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16

Theoretically, the curve should be a cubic hyperbola proceed­

ing from the origin and approaching an asymptote (the correct ratio

for valine) at infinite hydrolysis time. Assuming this is true, a

plot of the inverse ratio versus inverse time will permit the estima­

tion of the value of the correct ratio for valine from the intercept

at zero inverse time. Zero inverse time is obtained at infinite time

of hydrolysis, and the value of the inverse ratio will be equal to the

value of the inverse asymptote of the dependence in Figure 2A.

Figure 2B shows this type of plot for the data shown in Figure

2A. The value at the intercept was obtained here by an unweighted least

squares treatment of the data. Once the correct ratio was obtained from

an intercept of this type plot, the correct value for valine or isoleu­

cine content was obtained for each complete analysis as it was for

serine and threonine.

B. Gel Filtration Chromatography for the Separation of Amino Acids from Grain Sorghum Hydrolysates

Column Construction

4 Eighteen grams of Sephadex G25-80 beads were allowed to swell

in boiling deionized water overnight. Equilibration with buffer was

achieved by decanting six times with buffer A (Appendix A). The gel

was suspended in a quantity of buffer A and was poured into a funnel

attached to the upper end of a vertically aligned glass column (40.0 x

2.5 cm) filled with buffer A. The suspension in the funnel was stirred

Sephadex G25-80 was purchased from Sigma Company, St. Louis,

Missouri 63178.

17

to permit an evenly distributed gel. The final height of the gravity

packed column was 22 cm. The column was fitted with a stopcock to

control the flow rate. The invasion of the gel bed by air had to be

prevented. Therefore, the gel was never allowed to become dry.

Buffer A was the eluting buffer and the column was permitted

to flow under a gravity head at approximately 0.66 ml per minute.

Fractions of 10 ml were collected. The fractions were monitored for

protein and amino acids with the ninhydrin procedure described below.

A solution of blue dextran (one percent in buffer A) was used to deter­

mine the void volume.

Sample Application

Before the sample application the remaining buffer above the

gel was removed almost in its entirety. The sample was gradually

applied against the side of the column so as not to disturb the top

layer of the gel. As soon as the sample had completely entered the

column packing, the top of the gel bed was rinsed twice with a volume

of buffer identical to the sample volume, this buffer was permitted

to flow into the gel, and then elution was continued.

Glycine (molecular weight 75) and tryptophan (molecular

weight 204) were used to calibrate the column so that the volume

at which amino acids would be eluted could be predicted.

18

Ninhydrin Procedure

One ml of sample was transferred from each 10 ml aliquot to

a tube; 0.5 ml sodium citrate buffer (Appendix B), and 0.5 ml three

percent ninhydrin solution (Appendix C) was added in that order. The

tubes were mixed, capped, and heated in 100°C boiling water for 15

minutes. All fractions that contained ninhydrin positive material

were pooled and lyophilized. The development of a purple color

(Blackburn, 1968) afforded a qualitative measure of the presence of

amino acids in the aliquots from the Sephadex column. Since the

amino acids were later to be quantitatively analyzed, color intensity

was not considered important here.

Test of Column for Separation of Amino Acids from Carbohydrates

An enzymatically hydrolyzed sample of grain sorghum was chro-

matographed, the ninhydrin positive fractions were collected, evaporated

in vacuo, and the weighed residue suspended in one ml of citrate buffer

(pH 2.2, 0.2 N). An Ames Clinstix specific for glucose was moistened

with the solution, and color on the stick was developed for one minute.

The intensity of the color was determined with an Ames reflectance

meter calibrated to read in mg per 100 ml of solution.

C. Enzymatic Digestion of Grain Sorghum

Preparation of Pig Enzyme

The collection of the enzyme-containing material from the pig

took place in the laboratory on animals that had been left on full

19

feed until kill time. As soon as the pigs were stunned, they were

opened immediately and the stomach, pancreas, and small intestines

were removed. These organs were quickly carried to a cold room (-3°C)

where they were either handled immediately or placed in pans on dry

ice to cool until cleaned. All equipment for grinding was pre-

cooled.

The pigs' stomachs were inverted and washed with cold deionized

water. The fundi were removed, scraped with scalpel to remove mucin,

separated, weighed, and chilled in small pieces prior to grinding in a

Virtis homogenizer (300 mg/ml, 0.1 N hydrochloric acid). After mixing,

the material was placed in small whirl plastic bags, sealed, and frozen

in dry ice-acetone bath prior to being placed in a freezer.

The same treatment was given the pancreata as the stomachs,

although here fatty and connective tissue were removed prior to weigh­

ing. Each were mixed after grinding (0.73 mg/ml, 0.25 M sucrose

Q

solution ), placed in whirl bags, and immediately frozen.

Approximately the first six feet of the small intestine were

removed and handled as the previous organ. After mixing (1.8 mg/ml,

0.25 M sucrose solution) these were immediately frozen.

grams

All animals were crossbreds weighing approximately 100 kilo-

Autolysis is retarded with cooling and large ice crystals are prevented with rapid cooling.

The fundi appeared slightly darker than surrounding tissue due to many blood vessels.

Q

The 0.25 M sucrose solution was used since it is considered isotonic.

20

Separate stock solutions were prepared of pepsin^ (40 mg/ml,

0.1 N hydrochloric acid), pancreatin''- (73 mg/ml, buffer E), and

peptidase (1.8 mg/ml, buffer A).

Preparation of Semi-purified Enzymes

Separate stock solutions of amylase (one ml/25 ml, buffer D),''"^

pepsin (40 mg/ml, 0.1 N hydrochloric acid), pancreatin (54 mg/ml,

buffer E), and peptidase (1.5 mg/ml, buffer A) were prepated."'"

In Vitro Digestion

The digestion procedure was identical for both pig organ

homogenates and semi-purified enzyme preparations, with the exception

of the amount of stock solution added. One ml stock solution was used

9 The source was pig stomach. An additional 9 ml 0.1 N hydro­

chloric acid was added to maintain a slurry mixture.

The source was pxg pancreas.

The source was pig small intestine; an additional 9 ml of buffer A was added to each flask.

12 Alpha Amylase No. A-6255 from Hog Pancreas, Type 1-A, Sigma

Chemical Company, St. Louis, Missouri 63118. Two ml were used for each digestion flask. Hydrolysis with amylase releases some peptides bound to carbohydrates and exposes others for proteolysis (Starnes, 1973).

Since pig amylase cannot be extracted easily, semi-purified amylase was used in both the pig organ homogenates and the semi-purified enzjnne procedures.

13 Pepsin No. P-7000, pancreatin No. P-1750, and peptidases

No. P-7500 were purchased from Sigma Chemical Company.

21

in each instance except for amylase (2 ml) and pancreatin pig organ

homogenate solution (10 ml). Triplicate samples of grain sorghum

weighing 250 mg, 500 mg, and 1000 mg were prepared and placed in

125 ml Erlenmeyer flasks with screw tops. Enzyme blanks containing

no grain sorghum were prepared identically. The enzjnne levels were

kept constant since previous studies in this laboratory had shown

these levels were adequate to produce a measurable rate of hydrolysis.

Four ml of buffer A were placed in each flask with two ml of the amy­

lase mixture. These mixtures were incubated (37°C, 2 hours) in a

shaking water bath. The pH was monitored periodically and maintained

at pH 6.9 (with 0.1 N hydrochloric acid). Flasks were removed and

the pH changed to 1.5 (0.1 N, hydrochloric acid). Pepsin was added

and digestion continued (37**C, 3 hours), with the pH being monitored.

The pH was changed to 7.9 (0.2 N, sodium hydroxide), and the pancrea-

14 tin enzymes, with one drop each of potassium cyanide (one yg/ml)

and capyrlic acid (100 percent) added. Incubation continued for

eight hours with the pH being monitored. The pH was changed to 7.5

(0.1 N, hydrochloric acid), two drops CaCl^ (0.8 M) were added, and

incubation and pH monitoring continued for an additional eight

hours.•'• The pH was changed to 7.2 (0.1 N, hydrochloric acid), the

peptidases were added and incubation continued for five hours with

•'"Potassium cyanide was added to prevent bacterial growth.

Capyrlic acid was added to inhibit mold formation.

CaCl« was source of calcium ions needed by enzymes. Cal­cium precipitates at pH 8.0, thus the need for pH change.

22

the pH being monitored. Enzymatic action was stopped by placing each

flask in boiling water (lOO^C) for ten minutes.

The digestion mixture was centrifuged for 30 minutes to

remove undigested protein and larger peptides, vacuum filtered through

No. 3 Whatman paper in a Buchner funnel, measured, and frozen.

Gel Filtration Chromatography

The chromatography was performed as previously described.

Five ml of the clarified sample were chromatographed, the ninhydrin

positive fractions were collected and lyophilized. The residue was

analyzed for amino acids.

Amino Acid Analyses

The amino acid analyses were performed as they previously

were described.

•"• International Centrifuge, size 2, model K, 251 swinging

bucket head was used at maximum RPM.

CHAPTER III

RESULTS AND DISCUSSION

A. Amino^ Acid Composition of Grain Sorghum

The results of the determinations of amino acid content by

the amino acid analyzer are shown in Table 2. Grain sorghum sample I

was 9.45 percent protein by Kjeldahl and 8.81 percent protein by amino

acid analysis while grain sorghum sample II was 8.17 percent protein

by Kheldahl and 6.05 percent protein by amino acid analysis. Compari­

son of these results with those in Table 1 show that the samples of

grain sorghum used in this study were not unusual in amino acid content.

A total of 20 amino acid analyses were made of the composition of grain

sorghum. The statistical variation (standard deviation) observed in

this study varied from ±15 to 30 percent of the average content. The

amino acid analyzer usually provides results with a ±10 per cent stand­

ard deviation (Blackburn, 1968). The additional variation is perhaps

best explained by variation of the grain sorghum sample and by the

many steps involved in the procedure.

The appropriate extrapolation of serine, threonine, valine

and isoleucine content to correct for time of hydrolysis showed that

as much as 66 percent error could be introduced into the content of

a specific residue (for instance, isoleucine sample I). Figure 2A

(Chapter II) shows that the smallest number obtained for the content

23

TABLE 2

AMINO ACID COMPOSITION IN GRAIN SORGHUM

AS DETERMINED BY ACID HYDROLYSIS

24

Amino Acid

Lysine Histidine Ammonia Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Half Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

Sample I n = 15

%

.15

.19

.28

.27

.72

.27

.28 1.85 .50 .26 .78 .74 .38 .10 .27

1.12 .22 .43

S.D.

.05

.05

.09

.08

.11

.05

.04

.48

.21

.06

.14

.12

.12

.01

.09

.22

.03

.09

Sample II n = 5

%

.14

.15

.19

.19

.48

.21

.23 1.26 .50 .28 .54 .052 .30 .07 .28 .75 .13 .30

S.D.

.02

.02

.02

.02

.07

.03

.03

.06

.04

.03

.04

.03

.01

.03

.08

.01

.04

"74.07 mg grain sorghum.

'Error in analysis.

25

of valine at 20 hours is about 58 percent of the corrected valine

content. The correction for serine and threonine was not so drastic.

The maximum correction was about 30 percent for serine in sample I.

However, errors in serine content in the absence of extrapolation to

correct for destruction are commonly greater than 50 percent (Stames,

1973), and it is likely that earlier sampling time (12 to 16 hours)

would provide a better estimate of the rate of serine destruction.

B. Gel Filtration Chromatography and the Partial Purification of Amino Acids from Grain

Sorghum Hydrolysates

Ion exchange chromatography (AG 2X8, 200-400 mesh) has been

used for some time to separate amino acids from hydrolysates of

proteins from a large number of plant and animal sources, and quan­

titative recovery of amino acids from the hydrolysates has been

reported (Akeson and Stahmann, 1964). In this laboratory, this pro­

cedure proved nonquantitative and cumbersome. Preliminary work

illustrated that the charged amino acids (pH 7.2) were not recovered

quantitatively, and in fact, were recovered in very low yield such

that the precision of measurement of the quantities recovered was

below a reasonable margin. Furthermore, following enzymatic diges­

tion some preliminary separation of dissolved non-hydrolyzed protein

originating in either grain sorghum or the pig organ homogenates

proved necessary. The extra procedures involved in these separations

increased the potential margin for error and reduced the number of

samples that could be handled in a reasonable length of time.

26

Gel filtration chromatography on Sephadex (cross-linked dex­

tran) residues theoretically should permit the separation of the

amino acids from carbohydrates and from non-hydrolyzed proteins and

peptides in one step. Less time is required than the time required

for ion exchange procedures (Determann, 1968). The work done was

preparation, calibration and testing of a Sephadex G-25 column to

see if the theoretical predictions may be observed in practice under

the conditions of acid hydrolysis and enzymatic proteolysis to be

used, and to predict the efficiency of recovery of amino acids from

the column.

The effectiveness of the Sephadex column in the separation

of the amino acids from carbohydrates and non-hydrolyzed protein

and peptides was satisfactory as the glucose analysis showed that

the pooled ninhydrin positive fractions did not contain more than

1,0 percent glucose. The absence of non-hydrolyzed protein and pep­

tides was confirmed by the amino acid analyses of the samples from

the column. Short peptides should appear as spurious peaks and sig­

nificant amounts of non-hydrolyzed proteins would cause mechanism

failures of the analyzer. Spurious peaks were absent and no mechan­

ical failures could be attributed to sample quality. These negative

data are in agreement with the theoretical predictions concerning

the behavior of gel filtration columns.

Aliquots of grain sorghum acid hydrolysates prepared for 20

amino acid analyses were chromatographed, and the pooled ninhydrin

27

positive aliquots were analyzed for amino acids (Table 3 and Table 4).

Table 3 shows the average nanomoles of residue of each amino acid with

standard deviation and percentage recovery of amino acids from 74.07

mg grain sorghum using the Sephadex column after acid hydrolysis for

grain sorghum sample I. The column was calibrated from the results

of the 15 analyses of grain sorghum sample I (Table 3) and the 15

amino acid composition analyses of grain sorghum (Table 2). An addi­

tional five analyses of grain sorghum sample II were passed through

the column, analyzed for amino acids, and percentage recovery calcu­

lated (Table 4). These analyses with another variety of grain sorghum

show comparable recovery from the column and further support the reli­

ability of the column. The statistical deviation is based on a small

number of samples and these values are included to indicate the vari­

ance of the samples. Ideally, an efficient procedure for separation

of the amino acids from the other products of the acid or enzymatic

hydrolyses would permit recovery of 100 percent of each liberated

amino acid residue free of any contaminant.

In most cases the reliability of the mean percent recovery

from sample I and sample II is as good as the reliability of the

analyzer (±10 percent). The uncertainty in the yield of proline is

expected since its analysis is always a great deal more uncertain

than the other residues (Starnes, 1973). The uncertainty in serine,

glycine, methionine, and tyrosine yields is more difficult to explain.

Overall average recovery of amino acids from the column was approx­

imately 74 percent. The percentage recovery of each residue is

28

TABLE 3

AVERAGE NANOMOLES AND PERCENT RECOVERY OF AMINO ACIDS FROM GRAIN SORGHUM^ SAMPLE I WITH SEPHADEX COLUMN AFTER ACID HYDROLYSIS

Amino Acid Nanomoles of Amino Acid

Mean S.D. Percent Recovery

Lysine Histidine Ammonia Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

893 1006 12184 1278 4615 1983 2426 10593 3818 3373 8108 536 2829 640 1762 7354 977 2171

269 240 3970 382 729 360 390 2774 1605 791 1453 84 940 92 644 1467 139 446

74 70 84 76 64 80 100 84 100 72 75 60 71 62 77 72 45 67

74.07 mg grain sorghum,

•n = 1 5 .

29

TABLE 4

AVERAGE NANOMOLES AND PERCENT RECOVERY OF AMINO ACIDS FROM GRAIN SORGHUM SAMPLE II WITH SEPHADEX COLUMN AFTER ACID HYDROLYSIS

Amino Acid Nanomoles of Amino Acid'

Mean S .D. Percent Recovery

Lysine Histidine Ammonia Arginine Aspartic Acid Threonine Serine Glutamic Acid Pr5line Glycine Alanine _ Half-Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

661 704

10245 969 2974 1596 2419 8907 3897 2426 6098

2007 394 1361 5268 441 1449

179 164 1124 228 790 439 407 1624 793 530 1104

607 69 437 1597 87 349

76 79 83 83 75 77 77 81 69 90 72

77 86 77 83 62 73

"74.07 mg grain sorghum.

"n = 5.

Error in analysis.

30

reasonable for a procedure of this type, but further refinement of the

detection procedure for amino acids eluted from the Sephadex column

will probably improve the yields and reduce the statistical variation.

The yields for grain sorghum sample I (Table 3) were used to correct

all amino acid analyses performed on enzymatic hydrolysates passed

through the column.

C. Enzymatic Digestion of Grain Sorghum

Feeding trials with swine provide some answers concerning the

nutritional quality of a protein. However, feeding trials present two

basic problems, namely, cost and time. An in vitro digestion simula­

tion that could substitute for a feeding trial would be valuable to

the swine industry. With pig organs for sources of enzymes as well as

semi-purified enzymes, and grain sorghum as a substrate, work began in

this laboratory to develop such a technique. A total of 35 amino acid

analyses were made in this enzymatic study with an additional 25 analy­

ses being made in preparation for the study.

Table 5 shows the amino acid values with semi-purified enzymes

at each level of grain sorghum sample I. Table 6 shows the corrected

amino acid values with pig organ homogenate enzymes recovered at each

level of grain sorghum sample I. All values in the table are corrected

for yield from the Sephadex column. The number of samples were few and

the standard deviations were given to indicate variation. The 250 mg

level of grain sorghum with semi-purified enzymes gave a better overall

percentage recovery as shown in Table 7. Threonine and serine were not

31

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33

TABLE 7

PERCENT RECOVERY OF AMINO ACIDS FROM GRAIN SORGHUM BY IN VITRO DIGESTION WITH SEMI-PURIFIED ENZYMES

AND PIG ORGAN HOMOGENATES1

Amino Acid Semi-Purified Enzymes

250 mg 500 mg 1000 mg

Lysine Histidine Ammonia Arginine Aspartic Acid Threonine Serine Glutamic Acid Proline Glycine Alanine Half Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine

32 57 ——

—

1 15 —

—

39 35 41 —

91 —

56 35 —

36

33 .71 31 .83 7

6 34 42 51

87 20 49 39 15 46

18 7 9

,42 2

4 19 25 27

39

24 25 2 34

Pig Organ Homogenates Enzymes

189 101 27 78 48 281

54 7

236 121 8 17 12 56 32 476 47

721 123 66 358 34 274

46 26 140 97 7 62 22 96 50 357 88

250 mg 500 mg 1000 mg

38 84 28 214 9

174

22 12 64 58

33 60 55 32 265 68

"Grain sorghum sample I

34

resolved by the amino acid analyzer. Efforts to separate the two peaks

under normal conditions failed. The value reported here as threonine

is the sum of the threonine and serine.

The absence or almost complete absence of glutamic acid has

been noted by other researchers. Hankes £t al. (1948) pointed out

that the extent of liberation of glutamic acid from proteins by

enzymes is not strictly comparable to that obtained on acid hydroly­

sis, since in enzyme hydrolysates any glutamine released is released

as glutamine; whereas, in acid hydrolysis, the glutamic acid released

is the combination of glutamine and glutamic acid. Also, in enzyme

hydrolysis asparagine is not converted to aspartic acid while in acid

hydrolysis the two are both represented in aspartic acid. Furthermore,

glutamyl and aspartyl peptides are resistant to hydrolysis by proteo­

lytic enzymes (Starnes, 1973).

The slow release of methionine and cystine can be partially

accounted for due to oxidation. Denton and Elvehjem (1953) reported

methionine was liberated more slowly from casein and zein than from

ground beef which could indicate the grains may also have a factor

present that slows methionine's release.

Some plants are known to contain structural analogues that

block specific enzyme activities. This has been suggested (Wohl and

Goodhart, 1972) to explain the low yield of tyrosine, and may explain

the low yield of tyrosine from grain sorghum here.

Lysine was found to be absent in the 500 mg and 1000 mg

sample size analysis. Blom et al. (1967) showed that the loss of

35

lysine increased with sample size. Martinez (1970) reported the

unavailability of lysine which is thought to result from the reac­

tion of the free epsilon amino groups of lysine and the aldehyde

groups of certain active meal components such as pentoses, hexoses,

and pigments. Acid hydrolysis was found to release both available

and unavailable lysine and provide a measure only of the total

lysine content.

The rate of release of amino acids during an enzjnnic diges­

tion could account for the difference in the biological value of a

protein. This theory was suggested by the work done by Melnich ex

al. (1946).

The pig organ enzjnne system recoveries exceed theoretical

amounts in some instances as shown in Table 7. The excess must

arise from uncontrolled degradation of peptides and proteins in

the tissue preparation used. Therefore, the pig organ enzjmie system

is not suitable for simulation of the digestive process. To simulate

a pig's digestive tract one would need to use semi-purified enzymes

in proper proportions for maximum hydrolysis rates. Also, in both

systems, it appears that either the rate of mixing or substrate

inhibition controls the rate of release of specific amino acid prod­

ucts from grain sorghum.

The decreased yields of amino acids at high substrate levels

(Table 7) support the concept that a high feeding rate can reduce

the availability of amino acids obtained by proteolytic digestion

in animals.

CHAPTER IV

SUMMARY

The amino acid composition of grain sorghum as determined

with the amino acid analyzer of the 20 analyses used in this study

were comparable to those found in the literature (Waggle £t al.,

1967; Bressani and Rios, 1962). The study showed with an addi­

tional 20 analyses the corrections for serine and threonine to zero

hydrolysis time, and valine and isoleucine to infinite hydrolysis

time as much as 50 percent error can be eliminated in calculated

values of these four amino acids.

The use of the Sephadex column for separation of amino acids

from carbohydrates and non-hydrolyzed protein and peptides was a

success as only 1.0 percent glucose was found in the ninhydrin pos­

itive fractions, and the amino acid analyzer confirmed the absence

of the peptides and non-hydrolyzed protein. Overall average recov­

ery of amino acids from the column was approximately 74 percent.

This is satisfactory for a column of this type. Improvement in

percentage yields may be possible with a more sensitive measure

than the ninhydrin technique.

The enzyme system involved an additional 60 amino acid

analyses, but was unsuccessful with the pig organ homogenates

due to uncontrolled degradation of peptides and protein in the

36

37

tissue preparation used. Data indicated the possible use of the

semi-purified enzymes to simulate the pig*s digestive system and

as a technique to determine the amino acid availability in feed­

stuffs. However, more work is needed to determine the proper

proportion for maximum hydrolysis rates as well as improved

laboratory techniques.

REFERENCES

A.O.A.C. 1970. Official Methods of Analysis (11th ed.). Association of Official Agricultural Chemists. Washington, D. C.

Akeson, Walter R. and Mark A. Stahmann. 1964. A pepsin pancreatin digest index of protein quality evaluation. J. Nutr. 83:257.

Bailey, C. B., W. D. Kitts and A. J. Wood. 1956. The development of the digestive enzyme system of the pig during its pre-weaning phase of growth. Can. J. Agr. Sci. 36:51.

Berg, C. P. and W. C. Rose. 1929. Tryptophan and growth. I. Growth upon a tryptophan-deficient basal diet supplemented at varying intervals by the separate feeding of tryptophan. J. Biol. Chem. 82:479.

Blackburn, S. 1968. Amino Acid Determination Methods and Techniques. Maral Dekker, Inc., New York.

Blom, L., D. Hendricks and J. Caris. 1967. Determination of available lysine in foods. Anal. Biochem. 21:382.

Bressani, Ricardo and Berta J. Rios. 1962. The chemical and essential amino acid composition of twenty-five selections of grain sorghum. Cereal Chem. 39:50.

Denton, A. E. and C. A. Elvehjem. 1953. Enzymatic liberation of amino acids from different proteins. J. Nutr. 49:221.

Determann, Helmut. 1968. Gel Chromatography. Springer-Verlag, New

York.

Gorrill, A. D. L. , J. W. Thomas, W. E. Stewart and J. L. Morrill. 1966. Effect of soybean flour on pancreatic secretion by calves. Fed. Proc. 25:676.

Gorrill, A. D.. L. , D. L. Schingoethe and J. W. Thomas. 1968. Proteo­lytic activity and in vitro enzyme stability in small intestinal contents from ruminants and non-ruminants at different ages. J. Nutr. 96:342.

Hankes, L. V., W. H. Riesen, L. M. Henderson and C. A. Elvehjem. 1948. Liberation of amino acids from raw and heated casein by acid and enzyme hydrolysis. J. Biol. Chem. 176:467.

38

39

Hartman. Paul A., Virgil W. Hays, Roy 0. Baker, Lyle H. Nagle and Damon V. Catron. 1961. Digestive enzymes development in the young pig. J. Animal Sci. 20:114.

Ingram, G. R., W. W. Riesen, W. W. Craven and C. A. Elvehjem. 1949. Evaluating soybean oil meal protein for chick growth by enzymatic release of amino acids. Poul. Sci. 28:898.

King, J. G., J. R. Quinby, J. C. Stephens, N. W. Kramer and K. A. Lahr. 1961. An evaluation of parents of grain sorghum hybrids. The Agricultural and Mechanical College of Texas. Texas Agricultural Experiment Station. College Station, Texas.

King, Jack. 1973. Personal Communication.

Kitts, V. D., C. R. Bailey and A. J. Wood. 1956. The development of the digestive enzjrme system of the pig during its pre-weaning phase of growth. A. Pancreatic amylase and lipase. Can. J. Agr. Sci. 36:45.

Maner, J. H. , W. G. Pond, J. K. Loosli and R. S. Lowrey. 1972. Effect of isolated soybean protein and casein on the gastric pH and rate of passage of food residues in baby pigs. J. Animal Sci. 21:49.

Martinez, Wilda H. 1970. Relationship of chemical analysis to bio­logical activity-proteins and amino acids in oilseed meals. Proc. Nut. Council. Am. Feed. Mfg. Assn. Chicago, 111.

Melnich, D. , B. L. Oser and S. Weiss. 1946. Rate of enzymatic digestion of proteins as a factor in nutrition. Science 103:326.

Needleman, Saul B. 1970. Protein Sequence Determination. Springer-Verlag. New York.

Neudoerffer, T. S., P. A. Tedbeater, F. D. Homey and H. S. Bayler. 1971. The influence of level of grain intake on protein digestion in the intestine of cattle. Brit. J. Nutr. 25:

343.

Nixon, S. Elizabeth and G. E. Mawer. 1970. The digestion and absorption of protein in man. Brit. J. Nutr. 24:241.

Pond, W. G., Jean T. Wombley Snook, Deborah McNeill, W.I. Snyder and B. R. Stillings. 1971. Pancreatic enzyme activities of pigs up to three weeks of age. J. Animal Sci. 33:1270.

40

Snook, Jean T. Wombley and J. H. Meyer. 1964. Response of diges­tive enzymes to dietary protein. J. Nutr. 82:409.

Stames, Willis L. 1973. Personal Communication.

Stein, W, H. and S. Moore. 1948. Chromatography of amino acids on starch columns. Separation of phenylalanine, leucine, isoleucine, methionine, tyrosine, and valine. J. Biol. Chem. 176:337.

Waggle, D. H. and C. W. Deyoe. 1966. Relationship between protein level and amino acid composition of sorghum grain. Feedstuffs Dec. 24:18.

Waggle, D. H. , C. W. Deyoe and P. E. Sanford. 1967. Relationship of protein level of sorghum grain to its nutritive value as measured by chick performance and amino acid composition. Poul. Sci. 46:655.

Wohl, M. G. and Robert S. Goodhart. 1970. Modem Nutrition in Health and Disease. Lea and Febiger. Philadelphia.

APPENDIX

A. Buffer A

B. Buffer B

C. Ninhydrin Solution

D. Buffer D

E. Buffer E

41

SiEv

42

APPENDIX A: BUFFER A

Sodium Phosphate Buffer 0.2 M pH 7.2

A.) Prepare 27.8 g/1000 ml sodium phosphate monobasic to

make a 0.2 M solution.

B.) Prepare 53.65 g/1000 ml sodium phosphate dibasic to

make 0.2 M solution.

C.) Of the above solutions use 28 ml sodium phosphate

monobasic solution, and 72 ml sodium phosphate dibasic

solution and build to 200 ml with deionized water.

APPENDIX B: BUFFER B

Sodium Citrate Buffer

Dissolve 1.96 gm. sodium citrate in 60 ml water.

Bring to pH 3.25 with concentrated hydrochloric add.

Bring to 100 ml with deionized water.

Adjust pH to 3.25.

43

44

APPENDIX C: BUFFER C

Ninhydrin Solution

To 75 ml methyl celloslove add 25 ml sodium acetate buffer.

Stir with magnetic stirring device bubbling nitrogen gas through

the liquid for 15 minutes.

Add two grams ninhydrin, 2 per cent.

Stir for 10 minutes keeping solution under nitrogen.

Add 0.04 grams stannous chloride. Stir three to eight minutes while

solution is under nitrogen.

Place in black bottle and refrigerate.

• Sodium Acetate Buffer:

Dissolve 54 grams sodium acetate in 100 ml water.

Add 10 ml acetic acid. Mix.

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APPENDIX D: BUFFER D

Sodium Phosphate Buffer 0.2 M pH 7.0

A.) Use 39 ml 0.2 M sodium phosphate monobasic solution.

B.) Add 61 ml 0.2 M sodium phosphate dibasic solution.

C.) Mix; check pH.

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APPENDIX E: BUFFER E

Sodium Phosphate Buffer 0.2 M pH 8.0

A.) 5.3 ml 0.2 M sodium phosphate monobasic solution.

B.) 94.7 ml 0.2 M sodium phosphate dibasic solution.

C.) Mix; check pH.