Amino Acid Composition and Nutritional Value Of

Home , Soybean sprout, Sprouting

AMINO ACID COMPOSITION AND NUTRITIONAL VALUE OF

SOYBEAN SPROUTS

—__

—.

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v Thesis for ”19 Degree of M. S. MICHIGAN STATE UNIVERSITY Wanchai Somchit I977

Thai ‘~ 4- 9

610.2034

ABSTRACT

AMINO ACID COMPOSITION AND NUTRITIONAL VALUE OF SOYBEAN SPROUTS

Wanchai Somchit

Soybean sprouts were prepared from mature dry

soybeans. Chemical analysis of the sprout and bean

indicated slight decreases in lipids and carbohydrates

in the sprouts when compared to the bean. Total crude

protein showed insignificant differences. Apparently,

a portion of the amino acids from disintegrated protein

bodies of the beans were liberated and translocated to

the growing tissue which was accompanied by an increase

in the amount of crude fiber.

Amino acid analysis showed that there was no

significant change in the sulfurecontaining amino acids

between beans and four-day germinated sprouts. However,

sprouts contained higher concentration of lysine and

tryptophan and lower concentration of isoleucine, leucine,

phenylalanine, valine and threonine. Sprouts germinated

in the dark showed a slight increase in sulfur-containing Wanchai Somchit amino acids, lysine, histidine, leucine, valine and threonine but lower concentration of isoleucine and phenylalanine than found in the light.

Results of feeding trials conducted with Sprague

Dawley male rats indicated that the growth response from sprouts were slightly greater than that from beans but statistically insignificant. The PER value of sprouts was 1.97 compared to a standard casein, viz., PER 2.5.

The process of germination to sprouts offers a practical method for improving desirable flavor and acceptibility rather than an increase in the nutritional value of soybean proteins. AMINO ACID COMPOSITION AND NUTRITIONAL VALUE

OF SOYBEAN SPROUTS

Wanchai Somchit

A THESIS

Submitted to Michigan State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1977 ACKNOWLEDGMENTS

The author wishes to express his sincerest appreciation to Professor J. R. Brunner of the Food

Science and Human Nutrition Department, Michigan State

University for his kind guidance and encouragement throughout this study.

Thanks are due to Drs. C. M. Stine, M. R. Ben- nink and D. R. Dilley for their advice in preparation of the manuscript.

The author especially thanks Ms. Ursula Koch for amino acid analysis and her aid and counsel on numerous technical matters. .

Appreciation and thanks to the U.S. Agency of

International Development for providing the fellowship which made this study possible.

Last, the author is especially grateful to his wife, Saisuda, for her love, understanding and encouragement throughout the course of this study.

ii TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

REVIEW OF LITERATURE ...... 3

Overall Changes in Soybean During Sprouting . . 3 Protein and Amino Acids Changes ...... 7 Biological Changes ...... 12

EXPERIMENTAL ...... 18

Preparation of Soybean Samples ...... 18

Ungerminated...... 18 Germinating Process ...... 18

Chemical Analysis...... 19

Nitrogen ...... 19 Moisture ...... 20

A811. 0 O O O O O O O O O O O O O 20

crude Fat. 0 O O O O O I O O O O O 21 Acid Detergent Fiber ...... 21 Total Carbohydrates ...... 22 Amino Acid Determination...... 22 Methionine and Cystine Determination. . . . 23 Tryptophan Determination...... 24 Chemical Score ...... 26

Biological Methods ...... 26

In-Vitro Digestibility of Soybean Sprout . . 26 Protein Efficiency Ratio Test (PER) . . . . 26

Test diets ...... 26 Experimental animals ...... 28 Assay period ...... 28 Calculations ...... 29

Apparent In Vivo Digestibility...... 29

iii Page

RESULTS 0 O O O O O O O O O I O O O O 30

Fresh Weight Changes ...... 30 Changes in Chemical Composition ...... 34 Amino Acid Composition ...... 34 Nutritional Quality Evaluation...... 37

DISCUSSION ...... 45

Compositional Significance ...... 45 Biological Significance ...... 49

CONCLUSION O O O O O O I O O O O O O O 5 3

APPENDIX ...... 54

EMORANDUM o o o o o o o o o o o o o o 5 7

BIBLIOGRAPHY ...... 58

iv LIST OF TABLES

Page

1. Vitamin content of soybeans ......

2. Mineral content of soybeans ......

3. Trypsin inhibitory activity (TIA) of mature, soaked and germinating soybeans . . 16

4. Chemical characteristics of soybean oil . . . 16

5. Ingredients of the three test diets . . . . 27

6. Distribution of protein in soybean fractions during early stage of germination , , , , 33

7. Composition of freeze-dried ungerminated and

4-day germinated SOYbeanS o o o o o o o 35

8. Distribution of amino acids in soybean cotyledons and sprouts at various stages during dark germination ...... 36

9. Amino acid content of light and dark germinated soybean sprout, whole soybean and

hulls. O O O O I O O O I 0 O O O 38

10. The PER-value of casein, bean and sprouts . . 41

11. Apparent digestibility, in vitro digestibility, PER and chemical score—3f casein, bean and sprout ...... 41 LIST OF FIGURES

Figure Page

1. Protein, ether extractable components and sugar content of soybean cotyledons from time of planting to senescence . . . . . 4

Change in the components of the cotyledons during germination: A--change in the total carbohydrate and reducing sugar and B-- change in ether extractable components . . 6

Vitamin content of mature and sprouted soybeans at l, 2, 3 and 4 days after soaking in water ...... 9

Soybeans at various stages of germination: (a) dry soybean, (b) after 5 hr soak, (c) first day, (d) second day, (3) third day, (f) fourth day (consumption stage) of germination ...... 31

Fresh weight of soy soybean seedling and its component part at various stages of germination (average value for 10 plants) . . . 32

The essential amino acid pattern of soybean sprouts compared with whole egg protein . . 39

Average weight of rats in each of three groups fed casein, whole soybean and sprouts. Basal diet contained 10% total protein...... 42

Standard curve of tryptophan determined by scanning spectophotometer at 590 nm of known concentration of tryptophan . . . . 55

In vitro digestibility of beans and sprouts at 10 hr digestion ...... 56

vi INTRODUCTION

The explosive growth in world population, the accelerating depletion of natural resources, and a severe food scarcity in a large area of the world are factors influencing the new emphasis on the production, processing, storage, transportation and distribution of foods, as well as on the study of their nutritional qualities.

Plant proteins are considered to be the best remaining source of protein to narrow the gap between insufficient and adequate dietary protein because animal protein is in short supply or forbidden by cultural or religious practices. Legumes, particularly soybeans, are widely utilized in many food forms and are accepted as human food in most parts of the world.

About one-fourth of the world soybean production is produced in Asia but consumption remains at a higher level than production (United Nations, 1974). The forms of soybean products commonly utilized are adamame, tofu, soybean milk, various kinds of fermented products and soybean sprouts. Although most soybean products are accepted among Asian people, protein deficiency still exists. From a nutritional standpoint, the protein quality of soybeans is quite low compared to animal protein. Additionally, soybeans contain antinutritional factors which require appropriate processing to improve nutritional value. In general, the quality of soybean proteins is limited by a low concentration of sulfur- containing amino acids. Supplementation of these par- ticular amino acids, either by direct addition or by combination with other compensatory foods, would improve the quality of foods prepared from soybeans.

The purpose of this study was to compare the amino acid profile of soybeans and soybean sprouts as well as the nutritional quality of their respective protein complements. REVIEW OF LITERATURE

Overall Changes in Soybean During SproutIng

Soybeans contain large amounts of protein and oil. The protein content averages more than 40% and oil more than 20% of the dry weight of mature beans. Lipids consist mostly of neutral triglycerides--non-triglycerides may amount to 5 to 10% of the total lipid materials--of which about 80% of the fatty acid moiety is unsaturated

(B113, 1962). During germination these components contribute the major source of energy and serve as precursors of structural constituents such as carbohydrates.

McAlister and Krober (1951) observed a rapid decrease in protein, oil, sugar and changes in some mineral concentrations during germination of the cotyledons. They reported that oil decreased to one-half of its original concentration at the time of seedling emergence and to

10% 6 days after emergence. Protein decreased to 70% of its initial value at emergence and to 25% 9 days later

(Fig. l).

About 80% of the total phosphorus and potassium was translocated to the seedling within 6 days of germination. Magnesium and calcium contents did not I j T T I T j 30 r \\.\ "—'—' Protein

25 I“ ‘ ‘ a c a\\ Ether o extract

U . I I g 20 - \\ Sugar q >1 ‘3 0 15 "‘~A\\ \\ ‘ mH ‘ \\ 0' 1° " \ . 1 2‘ 4 ‘ \

5 v- \ \. q _.\ ‘\ \. > \.

.\ k._‘__.

0 4 gtﬂrkhWL l 2 3 4 10 15 20 25 30 35 40

Days after planting

Fig. 1. Protein, ether extractable components and sugar content of soybean cotyledons from time of planting to senescence (from McAlister & Krober, 1951). show significant changes at time of emergence. The mineral content of mature soybean and sprouts was given by Smith and Circle (Table 2); Carbohydrates in mature soybeans consisted of 5 to 9% sugar, about 5% pentosans,

5% crude fiber and 15 to 18% holocellulose; starch is absent at maturity (Rubel, 1972; DeMan, 1975). Changes in the characteristics of soluble carbohydrates were studied by Abrahamsen and Sudia (1966) who concluded that reserve carbohydrates present in cotyledons, primarily low molecular weight oligosaccharides, decreased rapidly during germination and showed no difference between light- and dark-germinated sprouts after the

first 7 days (Fig. 2). The reducing sugar content in both cotyledons and embryo increased significantly during the early stage of germination (Van Ohlen, 1931).

Because the oil fraction of soybean cotyledons may contribute to the carbohydrate pool, many investiga- tors have studied the rate of oil depletion, fatty acid characterization and composition. Abrahamsen and Sudia

(1966) observed that by the fourth day after germination a rapid decrease in oils occurred in sprouts grown in the

light, whereas, for sprouts grown in the dark, the rate of depletion was much slower. Eyster (1938) observed no apparent change in the iodine number of soybean oil during seed germination. Brown et a1. (1962) studied 1966). germi- 1 N q H and

a Sudia,

a O during

& H (day)

q sugar / q 00

q time

q \O B

q cotyledons reducing

q V‘ Abrahamsen

V the and I q N

"\ \ (from O. «4 of I Germination ,\ 1

O O KO N Q‘ O N H H °I£qoo go noequa Jeqqa %

I carbohydrate

I components

components 12

sugar (day)

the 10 total

I CHO in

8 time the

I {lb—OM“.

A Reducing

in Total extractable

Change

.._.. °“~' \2‘

I 2. ether ’0 / 2 \

’1. Germination in .1 J A-change Fig.

LL 1 --

‘ +\ ' Alb O

KO NO

14 18

22 (burIpaeS°g°s EmQOI/bm) Jebns nation: B-change changes in the fatty acids during germination of soybean seeds up to a period of 12 days, observing little change in the composition of reserve triglycerides during the stage of their most rapid depletion from the cotyledons.

The chemical characteristics of soybean oil were given by Murata (1965); see Table 4.

Burkholder and McVeigh (1945), Beeskow (1943), Wu

(1953), Smith and Circle (Table 1) reported that an appreciable increase in riboflavin, niacin and reduced and total ascorbic acid accompanied during germination

(Fig. 3). It is believed that ascorbic acid is most abundant in tissue in which there is a high metabolic activity. Increases in the concentration of these vitamins were also noted in sprouted wheat (Lemar & Swanson,

1976).

Protein and Amino Acids Changes

Soybean protein, 80 to 90% of which is soluble, contains some 20 amino acids. Glutamine and asparagine account for about a third of the total protein nitrogen.

Van Ettenet al. (1959) studied the amino acid composition of three protein fractions; soluble protein

(> 95% of total protein in soybean), acid precipitable protein (represents about 60% of soluble protein) and heat coagulable protein (approximately 7-9% of the soluble protein). They reported that the heat coagu-

1ated fraction contained markedly higher concentrations of nutritionally essential amino acids. 12 Cu

0.4

0.2 ------

Acid 32 Mn

Ascorbic

------

- mg/kq--- 100 Fe

3.4 90-150

Choline ----- 37 Zn --

Inositol 109-206

------mg/g

205-309

3.7

2.3 .0008

Folic

Acid .0019

1.1-1.7 0.6 Biotin

0.03 —— Pyri-

6.4 doxine 14.1-17.7

0.24

0.24 Acid

thenic Panto- 12.0 18.8-34.4

1.83 . -25.9 ------‘----__-..------—------20 29.9-48.0 Niac1n

Mg .

.22-.24

1972.

f1av1n Ribo- 4.8-7 2.3

soybeansa

soybeansa ------ug/9----=

.42-.82 -17.5

Circle, Circle, 11 11.9-21.9

and and Thiamin

content

.40

.16-.47 ......

Smith

- tene B-Caro-

Vitamin ------0.2-2.4

Mineral

aFrom aFrom

TABLE Soybean

-m

Soybean Sprout

Mature Sprout TABLE Mature

2.0“. 40 )-

Ascorbic acid Pantothenic acid 1.5 - U1 30 p s 01 \ 2: E 1.0 '- 20 I. o

54/: I I 1 l l A 0 1 2 3 4 0 l 2 3 4

Days Days

80 P 8 :- Niacin Riboflavin 60 — m 6 T U) \ o

E 40 '- /0/° g? 4 " /°/ 1 2 Pro/0 20 I/o/o L F

0 1 l I 1 O I J I l

0 1 2 3 4 0 1 2 3 4 Days Days

Fig. 3. Vitamin content of mature and sprouted soybeans at 1, 2, 3 and 4 days after soaking in water (from Burkholder and McVeigh, 1945). 10

Storage proteins are stored in the protein bodies

(diameter of 2-20 u) and are composed principally of globulins (Tomb, 1967; Sudamadji, 1975). Tomb (1967) reported that the protein bodies (content about 70% of the protein of soybean) can be fractionated by density gradient centrifugation, yielding light and heavy fractions which contain 97.5% and 78.5% protein, respectively.

Lipids, RNA and phytic acid were also present. Quanti- tative and qualitative changes in the cotyledonary proteins occurred during sprouting, presumably due to an increase in proteolytic activity. Soon after seeds are imbibed, their respiratory activity increases and the protein- and carbohydrate-synthesizing systems become active. As concluded from electron microscopy, the protein bodies became more granular in appearance and sometimes appeared to coalesce in the epidermal layer and around the vascular bundles, particularly in the vicinity of the embryo.

Catsimpoolas et a1. (1968) followed the changes in storage proteins by the techniques of disc electrophoresis and immunoelectrophoresis, concluding that the protein bodies consisted of at least 6 immunochemically different components, two of which were the principal

118 and 78 components. The 118 component is the major protein of the protein bodies. During germination, the 11

118 component appeared to be intack even after 16 days of germination, whereas the 7S component disappeared after the ninth day. N-terminal amino acid analyses indicated that the 118 component contained 12 polypeptide chains, 8 of which terminated in glycine, 2 in phenylalanine and 2 in either leucine or isoleucine. The molecule exists in a complex quaternary structure, stabilized through hydrophobic bonds, in which tyrosine and tryptophan residues are buried in hydrophobic regions.

Koshiyama (1970) reported that the N-terminal residues of the 7S component, in moles per 180,000 MW, were one mole each of aspartic acid, alanine, glycine, valine, tyrosine, glutamic acid, leucine (or isoleucine) and 2 moles of serine.

Changes in the amino acid composition during germination of soybeans was studied by Kasai et a1. (1966) who reported that the free amino acid content and composition were different in each tissue fraction. Aspara- gine was found in all fractions in far more abundance than glutamine. More glutamine was found in cotyledons than in other tissue fraction. Their results also revealed that a-glutamyltyrosine and a-glutamylphenyla- lanine were found in high concentration in dry beans and in sprouts in the initial stage of germination but disappeared after 70 hours of germination. 12

Hwang and Bushuk (1973) reported some changes in endosperm proteins during the sprouting of wheat and that the proteolytic activity of flour increased 17-fold after 8 days of sprouting. A marked decrease in the amount of insoluble protein and an increase in the number of amino groups occurred which was taken as evidence of proteolytic cleavage. They also indicated that

SH and S-S groups did not show any significant changes when wheat was sprouted for 8 days from which they concluded that SH and S-S groups do not appear to be involved in the observed degradation of the insoluble endosperm proteins. The increase in the content of lysine and tryptophan during germination of wheat, maize, rice, oat was studied by Margaris and Thanos (1974),

Tsai et a1. (1975), Dalby and Tsai (1976). In maize, prolamine (zein) acts as a precursor of lysine and tryptophan, whereas little or no change in these two amino acids occurred in germinating rice which contains a very low level of prolamine.

Biological Changes

It has been recognized that factors controlling biological value of soybean protein are: the presence of enzyme inhibitors and/or enzyme resistant peptides which affect digestibility and protein utilization, and limiting concentration of essential amino acid for tissue protein synthesis, particularly, the sulfur-containing 13 amino acids. Attempts to improve the biological quality of soybean protein have been studied extensively by many researchers. Blending with other complementary food proteins was found to enhance the protein quality

(Sarwar et a1., 1975).

Trypsin inhibitor, the protein commonly present in a number of plant proteins, inhibits the action of trypsin. The molecule is characterized by a high content of unavailable cystine (Kakade, 1974). The trypsin inhibitor is found in the ZS fraction of the water- extractable protein and is located in the cytoplasm rather than in the protein bodies (Smith & Circle, 1972). The effect of trypsin inhibitor on utilization of protein depends upon the experimental condition and species of animals involved (Kakade, 1974). In chicks, it inhibits the intestinal proteolysis by reducing the effective level of trypsin to form an inactive trypsin-trypsin inhibitor complex. In rats, the inhibitor increases the requirement of sulfur-containing amino acid, thus magnifying the deficiency of these amino acids which already exist in the plant proteins in limiting concentration. Relative to the effect of using heat to inactivate soybean trypsin inhibitory activities (TIA), experiments with chicks were performed by Evans et a1. (1946).

Their data showed that moderate heating of raw soybean meal increased the nutritive value for growing chicks 14 but, when autoclaved at 130°C for 30 or 60 min, the nutritive value was decreased. They also studied the

digestibility of autoclaved meal (100°C for 30 min) by

different enzymes and enzyme combinations, observing

that, when trypsin or trypsin and erepsin were employed,

a marked increase in digestibility resulted. When

treatment with pepsin preceded these digestions, no

enhancement in digestibility was observed. For all

combination of enzymes, the liberation of amino groups was decreased as a result of autoclaving.

Hemagglutinin is another antimetabolite commonly

found in legumes and derives its name from its ability

to agglutinate red blood cells. The role of hemagglutinin was reported by Jaffé (1969) who proposed that hemagglutinin combines with the cell lining of the intestinal wall,

causing interference of intestinal absorbtion of all nutrients.

Sandal (1963) studied the nutritional value of

the protein of oriental soybean foods, reporting that

green soybeans (Adamame) had PER and NPU of 2.27 and 73,

respectively, which are the highest values reported

among the soybean products. Concurrently she showed that

the PER and NPU values of soybean sprouts obtained from

various markets in Hawaii were 1.36 and 56, respectively.

Holm et a1. (1973) found differences in the net protein

utilization, true digestibility and biological value 15 for two soybean flours whose chemical score was similar.

Kakade (1974) suggested that a high level of heat—stable trypsin inhibitor activity could have been a causative factor for the differences.

In human studies, flatus factors or gastrointestinal gas are encountered when soybeans are consumed. Steggerda et a1. (1966) indicated that low molecular weight carbohydrate fraction, primarily sucrose, raffinose and stachyose are gas-producing factors. Soy- bean hulls, fat, water-insoluble polysaccharide (residue product) and proteins are not associated with flatulence to any significant degree. They also mentioned that the main types of flatus are nitrogen, carbon dioxide, methane and hydrogen gases, depending upon the individual diet and microflora spectrum in the intestinal tract. Some of the phenolic acids, i.e., syringic and ferulic acid, are effective gas inhibitors i§_ziE£g and in intestinal segments of dogs (Rackis, 1970).

Collins et a1. (1976) determined the TIA of nature, soaked, germinating soybean (Table 3) and followed the TIA after immersion of beans in boiling water for different times. They found that dehulled beans which were held for 2.5 min in boiling water had 97-98% of the extractable TIA destroyed. Sarwar et a1. (1975) used a boiling water dip for 2 min to destroy anti- nutritive factors. Albrecht et al. (1966) reported 16

TABLE 3. Trypsin inhibitory activity (TIA) of mature, soaked and germinating soybeansa

Time Var1et1es

Treatment Per1od Kanrich Soylima Dare (hr) ------mg/g---—------

Mature 0 22.8 i 0.5 17.2 i 0.5 26.9 i 0.8 Soaked 0-24 22.6 t 0.9 16.5 t 0.8 26.9 i 0.6 Germinating 24-48 21.4 i 0.7 16.8 i 0.6 26.4 i 0.9 48-72 20.5 i 0.6 16.2 i 0.3 25.2 i 0.8 72-96 19.8 i 0.6 16.9 t 0.5 24.7 i 0.4

aFrom Collins and Sanders, 1976.

TABLE 4. Chemical characteristics of soybean oila

Acid value 1.02 Refractive index 1.47 Iodine value 128.50 Saponification value 191.10 Composition of fatty acid C16:0 10.4% C18:0 5.1% C18:1 26.8% C18:2 50.0% C18:3 7.8%

aFrom Murata, 1965. 17 that, when the moisture of beans was raised to 60% or more by soaking, boiling for only 5 min was sufficient to inactivate the inhibitors.

Because soybean products have limiting amounts of sulfur-containing amino acids, it would seem feasible

to supplement these products with the free amino acid, methionine. However, the free amino acid is easily lost during the subsequent pod processing. Moreover, the

free amino acid may degrade or react with food components, viz, Strecker degradation or amino-carbonyl reaction, to affect food qualities such as flavor and color. To remove or lessen the above defects, Yamashita et a1.

(1970 A—C) introduced a method for incorporating free

amino acids into food proteins or peptides by means of

the so-called plastein reaction. The concept was illus-

trated by mixing soy protein hydrolyzate with albumin hydrolyzate or wool keratin hydrolyzate, both of which

are rich in sulfur-containing amino acid residues. The hydrolyzate mixture was incubated with a microbial pro-

tease to obtain soy plastein. Soy protein thus improved by supplemented plastein were higher in nutritional quality as determined through rat-feeding experiments. EXPERIMENTAL

Preparation of Soybean Samples

Dry soybeans [Glycine max (L.) Merrill] obtained from a local elevator were used for this study.

Ungerminated

A quantity of selected, high quality beans was washed once with tap water then soaked in room temperature water for 5 hr. The soaked beans were boiled in water for 5 min to inhibit trypsin inhibitor activity and cooled immediately in cold water. These beans were ground coarsely in a Waring blender and freeze-dried.

The dry bean flour was ground further to a fine powder for chemical determinations and subsequent feeding trials.

Germinating Process

Selected seeds were washed once to remove dirt and soaked in room temperature tap water for 5 hr. The imbibed seeds were placed on a perforated stainless steel tray which was covered with cheese cloth and paper towels. The bean layer was about one inch deep and covered with wet paper to prevent rapid evaporation of water. To simulate conditions employed in Thailand for

18 19 the production of commercial sprouts, germinations were carried out in normal daylight at 24-28 C for a period of 4 days. Germinated seeds were sprayed three times daily with tap water. Upon termination of the germinat— ing period, using the length of roots as a criterion, a representative sample of seedlings was collected for analysis. Seed coats or hulls were removed and the sprouts were boiled in water (steam-jacketed kettle) for

5 min and cooled with cold water. The cooled sprouts were ground and freeze-dried in preparation for analysis.

Chemical Analysis

Tripicate sample of freeze-dried ungerminated soybean flour and first-, second-, third- and fourth-day sprout flour were analyzed.

Nitrogen

A.micro-Kjeldah1 method was used to determine total nitrogen (Mangino, 1973). Samples of about 15-30 mg

(moisture free) were weighed and placed in micro-Kjeldahl digestion flasks. Digestion took place in the presence of 4 ml of a digestion mixture, consisting of 5 g

CuSO4°5H20, 5 g SeO2 in 500 ml conc. H2304, over a gas flame. After one hour of digestion, the sample was cooled to room temperature, one ml of 30% H202 was added and digestion continued for another hour. Digests were allowed to cool for 15 min, neutralized with 25 m1 20 of 40% NaOH and steam distilled into 15 ml of 4% boric acid solution containing a few drops of indicator solution. The indicator solution was made by dissolving

100 mg of methyl red and 30 mg methylene blue in 60 ml of 95% ethanol and made up to 100 ml with water. Dis— tillation was continued until a final volume of 60 ml was reached. The ammonium-borate complex was titrated with 0.020 N HCl. The percentage of nitrogen was calculated as follows:

%N = (ml HC1-ml blank)(N)(l4.007)(100)/mg sample.

Protein content was estimated by multiplying percentage of nitrogen by 6.25.

Moisture

Three grams of ground sample were weighed into aluminum dishes and dried at 90-95 C in a vacuum oven

(30 mm Hg pressure) overnight, cooled in a desiccator and reweighed at room temperature.

Ash

Approximately two grams of sample was weighed into previously ignited porcelain dishes, charred on a clay ring over a Bunsen burner and ignited overnight in the muffle furnace at 550 C. The ignited samples were cooled in a desiccator and weighed after reaching room temperature. 21

Crude Fat

A Soxhlet petroleum ether extraction method was used (AOAC, 1975). Two grams of sample (known moisture content) were weighed into extraction thimbles and topped with cotton balls. Extraction of oil was achieved by percolating petroleum ether over the sample for about

12 hr. Excess ether was evaporated and the residual oil was dried in a 60 C vacuum oven for 3 hr, cooled in a desiccator and reweighed.

Acid Detergent Fiber

Acid detergent fiber was determined by the Acid

Detergent Fiber (ADF) method (AOAC, 1975). One gram samples, ground to pass a 20-30 mesh (1 mm) screen, were weighed into a spoutless 600 ml Berzelius beaker. Two milliliters of decalin (decahydronaphthalene), and 100 ml acid detergent solution were added. The acid detergent solution consists of 20 g hexadecyl trimethyl ammonium bromide, 28 m1 concentrated H2804 and made up to one liter with distilled water. The mixture was refluxed for exactly 60 min at constant boiling. The solution was filtered by suction through a previously tared Gooch crucible. The fiber remaining in the digestion beaker was removed as completely as possible by washing with hot water (90-95 C). The residue was washed twice with hot water and finally with acetone, the contents of the crucible were sucked dry. The crucibles were dried at

105 C overnight and cooled in a desiccator and weighed. 22

Total Carbohydrates

Total carbohydrate was obtained by the difference between the total component composition and 100%.

Amino Acid Determination

Amino acid analysis was performed on 22 hr acid hydrolysates according to the method of Spackman et a1.

(1958) and Moore and Stein (1954). Samples containing

15 mg of protein were placed into 10 ml glass ampules.

Five milliliters of once-distilled 6N HCl was added to the ampule which were frozen in a dry ice-ethanol bath, evacuated with a high vacuum pump and allowed to melt slowly under vacuum to remove dissolved gases. The contents were refrozen and sealed with a propane flame.

Sealed ampules were placed in a silicone oil bath set at 110 i 0.1 C for 22 hr; Following hydrolysis, the ampules were opened and one milliliter of a nor- leucine solution (2.5 uM/ml) was added to each as an internal standard to measure transfer losses. HCl was removed from the hydrolysate in a 25 m1 spear-shaped flask by evaporation on a Rinco rotary evaporator.

The residue was washed with about 10 ml of deionized water and re-evaporated. The washing procedure was repeated three or four times to remove all acid. 23

Acid-free hydrolysate residues were dissolved in 3-4 ml of diluting buffer (0.067 M sodium citrate HCl, pH 2.2) and transferred quantitatively to a 5 m1 volumetric flask. The solution was filtered through a 0.22 Um millipore filter. Two-tenth milliliter portions of filtrate were applied to the ion-exchange columns of a

Beckman 120 C automatic amino acid analyzer. The result- ing chromatograms were compared to those of a standard amino acid calibration mixture. The ratio of area under the curve of each amino acid for the samples and the standard were compared and converted to give the amino acid composition of the sample. Losses of threonine, serine and tyrosine during acid hydrolysis were corrected according to Moore and Stein (1954).

Methionine and Cystine Determination

Methionine and cystine undergo variable destruction during acid hydrolysis. Therefore, samples were oxidized to methionine sulfone and cysteic acid, respectively by the performic acid procedure of Moore

(1963). Approximately 15-30 mg samples were weighed into 25 m1 spear-shaped flask. Ten milliliters of freshly prepared performic acid was added to each sample (performic acid consists of 1 ml of 30% H202 and 9 m1 of 88% formic acid and allowed to stand at room temperature for 1 hr). The sample flasks were 24 held at 4 C for 17 hrs. Performic acid was removed on

a rotary evaporator. Then, 5 ml of over-distilled 6 N

HCl and 1 ml nor leucine standard were added and the contents were transferred to 10 m1 glass ampule in preparation for hydrolysis as previously described.

Standards containing cysteic acid methionine sulfone and methionine sulfoxide were analyzed. Normalized values

for cysteic acid and methionine sulfone, corrected for

sample weight, nor leucine recovery, volume on the

column, and incomplete oxidation, were substituted for

cystine and methionine respectively, in the data obtained for the unoxidized sample.

Tryptophan Determination

Because tryptophan is destroyed by acid hydroly-

sis, the procedure of Spies (1967) was used for its chemical determination. Three to five milligram samples were weighed into 2 m1 glass vials fitted with screw

caps. One-tenth milliliters of freshly prepared pronase

solution was added to each vial. The pronase solution was made by adding 10 ml of 0.1 M sodium phosphate buffer pH 7.5 to 100 mg pronase. The suspension was shaken gently for 5 min and clarified by centrifugation for

10 min in an International clinical centrifuge. The

closed vials were incubated for 24 hr at 40 i 1 C fol-

lowed by cooling to room temperature in a crushed ice bath. To each vial 0.9 ml of 0.1 M sodium phosphate 25 buffer, pH 7.5 was added and the vial (without cap) was inserted into a 25 ml Elenmeyer flask containing 30 mg p-dimethylamino benzaldehyde and 9 ml of 21.2 N H 2 SO 4 . The combined content of the flasks was mixed quickly by gentle swirling, stoppered and placed in the dark at

25 C for 6 hr; then, 0.1 m1 of 0.045% (w/v) sodium nitrite was added to each flask. After 30 min, the transmittance at 590 nm was measured with a Beckman

DK-2A spectrophotometer. Simultaneously, samples of the pronase solution were treated and analyzed as above.

The tryptophan content of the pronase solution was sub- tracted from the total tryptophan value of the protein sample.

A standard curve covering the range of 0-120 ug of tryptophan was prepared. Thirty milligrams of tryptophan was weighed into a flask containing 250 ml 0.1 M, pH 7.5, phosphate buffer (ratio of 16:84 m1 of sodium phosphate monohydrate to dihydrate). From this standard solution, aliquots were measured and added to phosphate buffer as follows: 0.1:0.9, 0.3:0.7, 0.5:0.5, 0.7:0.3,

0.9:0.1, l.0:0.0, 0.0:1.0. The vials were inserted into

25 ml Erlenmeyer flasks contained 9 m1 21.2 N H2804 and

30 mg of freshly prepared p—dimethylamino benzaldehyde.

The stoppered flasks were swirled and kept in the dark at 95 C for 6 hr; then 0.1 m1 0.045% sodium nitrite was added and the transmittance at 590 nm was read following incubation another 30 min (see Appendix). 26

Chemical Score

The chemical score was calculated on the basis of the ratio of essential to total amino acid (E/T ratio).

The amino acid pattern of egg protein (FAO, 1970) was used as the reference protein. According to the method of Mitchell and Block (1946), the chemical score is equal to the greatest percentage deficit in an essential amino acid of the protein being evaluated.

Biological Methods

In-Vitro Digestibility of Soybean Sprout

Casein, whole soybean and soybean sprout samples

(200 mg samples) were suspended in 20 m1 of 0.2 M KCl buffer adjusted to pH 1.8 with HCl (the method described by Choi, 1975, Sanders et al., 1973). A few drops of toluene and 5 mg of pepsin were added. The mixture was incubated at 38 C and periodically (one or two hour intervals) 5 ml of this solution was pipetted into 5 ml of 20% TCA (final conc. of 10%) and centrifuged at

1000 x g for 10 min. The nitrogen in the supernatant was determined and used to determine protein digestibility (%) as follows: (nitrogen in supernatant)/

(nitrogen in protein concentrate) x 100.

Protein Efficiency Ratio Test (PER)

Test diets. The PER was used to evaluate the

protein quality of bean and sprout flour because of its 27 simplicity and current popularity. The method was described by Campbell (1963). Three feed samples were prepared (see Table 5):

(a) High protein casein (ICN-Pharmaceutical Inc.),

(b) Ungerminated whole soybean prepared as described

above, and

TABLE 5. Ingredients of the three test diets.

Casein Ungerminated Soybean Sprouts Ingredients (9) (g) (g)

Casein 1528 Ungerminated soybean 2618 Sprouts 2521 Cottonseed oil 958 205 233 Salt mixture 600 500 500 Vitamin mixture 120 100 100 Cellulose 120 - 2 Corn starch 8672 6575 6642

Total 12,000 10,000 10,000

All rations were standardized to meet the following composition (AOAC, 1975): 10% protein, 8% oil, 5% salt mixture,

1% vitamin mixture, 1% cellulose, corn starch to make

100%.

The components of the diets and their sources were as follows:

(a) Casein--vitamin free casein (ICN-Pharmaceutical

Inc., Cleveland, Ohio, cat #104520), 28

(b) Oil--cottonseed oil (ICN-Pharmaceutical Inc.

cat. #101419),

Inc.),

(d) Vitamin--vitamin diet fortification mixture

(ICN-Pharmaceutical Inc.),

(e) Cellulose--non nutritive fiber (FMC-Corp.,

Newark, Delaware), and

(f) Cornstarch--starch corn (ICN-Pharmaceutical

Inc. cat #102956).

Experimental animals. Sixty Sprague-Dawley male

rats-—age 21 days, 51 9 average weight--were supplied

by the Spartan Research Animal Inc., Haslett, Michigan.

At the end of a 4-day acclimation period on a standard

casein diet, twenty rats were assigned into each of 3

groups. The average weight of the rats was 68.4 g/rat.

Assay period. Rats were kept in individual cages

and both diet and water were provided ad libitum. Body weight and food intake of each rat was recorded at 3-day

intervals. During the fourth week of the feeding trials,

the feces was collected quantitatively on paper towel

from each group of rats and dried in a vacuum oven for

determination of nitrogen and protein digestibility. 29

The experiment was terminated 27 days from the beginning

of the assay period instead of the usual 28 days because

of the depletion of the test diets.

Calculations. Average 27 day weight gain and

protein intake per rat were calculated for each group.

Protein efficiency ratio (weight gain/protein intake)

were calculated for each group.

Apparent In Vivo Digestibility

True digestibility was not measured because a

nitrogen—free diet was not fed to serve as a basis for

Ithe measurement of the metabolic nitrogen excretion.

Instead, apparent digestibility was estimated by col-

lecting feces quantitatively during the fourth week of

the assay period. Four animals from each group were

selected to determine the apparent digestibility. The

collected feces were dried in a vacuum oven overnight

and the nitrogen content was determined. The apparent

protein digestibility was calculated as follows:

[(N infeed)-(N in feces)] x 100 Protein digestibility (%) = (N in feed) RESULTS

Fresh Weight Changes

Stages of soybean sprouting are shown in Figure 4.

The average fresh weights of tissue fractions of soybean seedlings are illustrated in Figure 5. Generally, factors influencing the rate and extent of germination are temperature,oxygen tension and humidity. Relatively high temperatures (approximately 35 C) and humidity favor germination. Yentur (1976) observed that 20% oxygen tension favored germination, but that elongation rates of the roots show no apparent sensitivity to lower oxygen tension.

The increase in total fresh weight was mostly due to growth of root tissue. According to the compositional analysis of the tissue fractions shown in

Table 6, the dry matter in the sprouted seeds decreased slightly (about 7%), apparently due to loss of carbohydrates during respiration. It was also noted that after the fourth day of germination in the light there was some chlorophyll synthesized in the cotyledons.

30 31

Fig. 4. Soybeans at various stages of germination: (a) dry soybean, (b) after 5 hr soak, (c) first day, (d) second day, (e) third day, (f) fourth day (consumption stage) of germination. 32

N Whole beans E] Cotyledons E Roots

(g)

L; Weight 0 5 l 2 3 4 Time (days)

Fig. 5. Fresh weight of soy soybean seedling and its component part at various stages of germination (average value for 10 plants). TABLE 6. Distribution of protein in soybean fractions during early stage of germination a

Cotyledons Roots Seed Coats Germination Day Mb TSC Protein M TS Protein M TS Protein % % % % % % % %

Whole Soybeans

7.09 92.91 35.59

58.30 41.70 16.79 72.57 27.53 11.76 66.80 33.21 33

61.44 38.56 15.55 86.60 13.40 4.57 75.55 24.45

63.80 36.20 16.46 83.40 10.61 3.98 75.25 24.75

67.20 32.80 13.58 93.17 6.83 2.51 67.10 32.21

aEach value represents average of 4 replicates.

b Moisture

cTotal solids 34

Changes in Chemical Composition

Data in Table 7 show a slight increase in crude protein and a decrease of crude fat and carbohydrates in the sprouts when compared to seeds. It should be recognized that during the germination process protein bodies are degraded and translocated to the growing tissue

(Tomb, 1967; Wolf, 1970). Data from the current study

(Table 6) show that the conversion was highest on the first day of germination when the sprouts contained a maximum amount of nitrogen. Nucleic acid synthesis, an essential function of germination, begins somewhat later, subsequent to the start of imbibition. Thus, it is pre— sumed that the total nitrogen increase in the sprouts was due to protein nitrogen.

Amino Acid Composition

Free amino acids in each sample (cotyledon and sprout) are reported in Table 8. The content of amino acids was higher in cotyledons than sprouts. Aspartic acid increased in the cotyledons and sprouts as germination progressed, but glutamic acid decreased rapidly during sprouting. The greatest reduction was found on the fourth day of germination. During germination, transamination takes place and the accumulation of aspartic acid is believed to be a key intermediate in the biosynthetic pathways of plants. TABLE 7. Composition of freeze-dried ungerminated and 4-day germinated soybeansa

ADP-Crude Samples M C TS d Protein Lipid Ash CHO . (Nx6.25) Fiber (by diff.)

Ungerminated 1.2 98.8 38.72 22.71 4.63 12.41 20.33 35 Sproutsb 1.2 98.8 41.09 22.46 4.15 8.03 23.08

aAverage of 4 samples

bSample part of cotyledons and root only (hull is eliminated).

CMoisture

dTotal solids 36

TABLE 8. Distribution of amino acids in soybean cotyledons and sprouts at various stages during dark germinationa

Amino 1st Day Germ. 2nd Day Germ. 4th Day Germ.

ACIdS Coty. Sprout Coty. Sprout Coty. Sprout

Lysine 6.33 7.89 6.40 7.85 6.08 6.39 Histidine 3.04 3.55 2.84 3.78 3.33 4.05 Arginine 6.96 9.32 7.18 7.38 7.49 4.48 Aspartic acid 11.33 10.83 12.84 14.41 16.75 32.53 Threonine 3.75 4.35 3.99 4.27 3.99 3.63 Serine 4.86 5.03 5.20 4.98 4.97 4.12 Glutamic acid 19.28 14.16 19.40 12.77 15.92 7.93 Proline 9.24 4.59 6.18 5.22 4.84 3.91 Glycine 3.42 3.89 3.36 3.82 3.32 2.69 Alanine 3.84 5.38 3.87 5.23 3.95 4.30 Cystine 1.42 2.43 1.71 1.14 1.67 2.40 Valine 4.19 5.12 4.46 5.48 4.81 5.04 Methionine 1.76 2.25 2.09 1.70 1.77 2.53 Isoleucine 4.53 4.50 4.23 4.70 3.89 3.90 Leucine 7.47 7.51 7.98 7.75 8.20 5.94 Tyrosine 3.67 4.06 3.33 4.06 3.48 2.25 Phenylalanine 4.92 5.14 4.94 5.46 5.54 3.91 Tryptophan ------not determined ——————————————

ag/16 g.N. 37

The amino acid content of whole soybeans and soybean sprouts grown in the dark and in normal daylight are shown in Table 9. There were no major changes in the amino acid pattern with the exception of aspartic and glutamic acids. In the dark, accumulation of aspartic acid was higher and correlated with the reduction in glu— tamic acid. Sprouts germinated in the dark were slightly higher in some essential amino acids, i.e., threonine, cystine, methionine and leucine.

The amino acid composition of soybean hulls was taken from Smith and Circle (1972) and presented in

Table 9. The composition of amino acids in the hulls remained fairly constant during germination.

Nutritional Quality Evaluation

The ratio of essential amino acid concentration

(mg) to the gram total essential amino acids in sprouts compared to the whole egg protein is illustrated in

Figure 6. The ratio of leucine, lysine, phenylalanine and tyrosine in sprouts was higher than in egg, but methionine and cystine were much lower. Therefore, it is apparent that sulfur-containing amino acids limit the nutritive value of soybean protein. Based on the amino acid determinations, the chemical score of soybeans and sprouts was found to be 45 and 43, respectively. 38

TABLE 9. Amino acid content of light and dark germinated soybean sprout, whole soybean and hullsa

Amino Whole Sprouts Hullsb Ac1ds Beans Dark Light

Lysine 5.55 6.24 6.56 7.13 Histidine 2.22 3.43 3.43 2.54 Arginine 8.20 7.60 7.69 4.38 Aspartic acid 12.19 17.57 13.02 10.05 Threonine 3.73 4.08 3.12 3.66 Serine 5.24 5.07 5.93 7.02 Glutamic acid 20.90 16.12 19.34 8.66 Proline 5.13 4.94 4.93 5.76 Glycine 3.07 3.39 2.88 11.05 Alanine 3.62 4.06 3.63 3.98 Cystine 1.38 1.73 1.31 1.66 Valine 4.31 4.94 4.29 4.55 Methionine 1.82 1.83 1.76 0.82 Isoleucine 4.40 3.99 4.34 3.80 Leucine 7.80 8.35 7.72 5.93 Tyrosine 4.10 3.54 3.89 4.66 Phenylalanine 5.79 5.64 5.63 3.21 Tryptophan 0.84 - 0.88 -

ag/16 g.N.

bfrom Smith and Circle, 1972. 39

with Val

compared Try

1965'

sprouts

sprout Thr

pattern,

soybean Cys

Soybean FAO Met

E] E2

pattern Tyr

acid Phe

amino

Lys Leu

essential

The Ile

protein.

100 200 150 (proe ourme Ierquesse quoq b/bm) 01191 a/v

Fig.

egg

whole 40

The in_yi£rg and apparent digestibility of soybean and sprouts are reported in Table 11. In_yi££g

pepsin digestion of soybean and sprouts is considerably

lower than the apparent digestibility, possibly reflect-

ing limited hydrolysis of these plant proteins by pepsin. Also it was markedly different from that of

casein which is a fairly good substrate for pepsin.

Values for the protein efficiency ratio (PER) of sprouts, whole soybeans and standard casein at 10%

protein level, fed for 27 days to male Sparque-Dawley

rats, are present in Table 10. The average weights of

rats in each group (20 rats) are summarized in Figure 7.

The variation in PER values was analyzed statistically.

One-way analysis of variance and Tukey's test were employed as indicated below (Steel & Torrie, 1960):

(a) Total sum of squares (ssy, where Y = PER value)

3 20 ssY = z 2 xi. - Y..2/60 i=1 j=1 3 = 607.89 - 188.652/60

= 14.74

(b) Treatment sum of square (SST)

3 2 2 SS = E Yi./20 - Y../60 T . 1=l

= 603.75 - 593.15

= 10.60 41

(0) Error sum of square (SSE)

SSE = SSY - SST

= 4.1 4

(d) Analysis of variance (AOV)

Ho: uca =uo=u4

Ha: uca # no ¢ 04

AOV

Source of df SS MS F ( 1 late) Variation ca cu

Treatment 2 10.60 SST/2 = 5.3 MST/MSE = 75.7**

Error 57 4.14 SSE/57 = 0.1

Total 59 14.74

** highly significant.

F.01 (2, 57):: 4. 98

SOURCE: From statistics table; Steel & Torrie, 1960.

From the analysis of variance table (Steel &

Torrie, 1960), the F-value for this experiment was very high, therefore the null hypothesis (Ho) was rejected.

That is, there are highly significant differences in PER between the means of the three treatments, viz, Casein control (Ca), ungerminated bean (o) and sprout (4). 42

(e) Specific test (Tukey's)

T = (q0.01' 3, 57) (/ MSE/ZO) 2: (4.28) (0.0592)

= 0.2532

The T-value of PER (comparing casein and beans and casein and sprout):

Casein and beans 3.74 .— 2079 0095

Casein and sprouts 3.74 - 2.94 0.80

Because these T-values are greater than 0.2532, there are significant differences between casein and beans and between casein and sprouts.

The T-value of PER (comparing sprouts and beans):

Sprouts and beans = 2.94 — 2.79 = 0.15

The T-value of sprouts and beans is smaller than

0.2532, therefore no significant difference exists between the protein quality of sprouts and beans. 43

TABLE 10. -The PER-value of casein, bean and sprouta

- For Source Protein gain in PER %. Casein Of . intake (9) weight (g) 27 Casein PER = Protein days PER 2 5

Casein 40.11 i 2.9 150.0 i 1.8 3.7 100 2.5

Whole Soybean 36.83 t 5.1 102.8 i 2.3 2.8 74.6 1.9

Soybean sprout 35.27 i 4.3 103.8 i 1.8 2.9 78.7 2.0

aValues are mean of 20 male rats in each group, level of protein was 10%.

TABLE 11. Apparent digestibility, in vitro digestibility, PER and chemical score of—casein, bean and sprout

. . in vitro Chemical Diet App. Digest. _ai§E§Eb' PER. Score

Whole soybean 97.0 50 1.9 44.6

Soybean sprout 97.3 54 2.0 42.8 Casein 98.9 - 2.5 58.0a

aData were taken from FAO, 1970.

bSee Appendix.

130 ”' "" Casein diet 1 (6) 0....0 Whole soybean ’4555CLS ‘ dnoxb 160 )- i—X Soybean SprOUt ‘/ g/ﬁ __, uses

140 ’ at

\ /;// / ' qurem :91

abexenv 100 801/3 j

60£ 1 I 1 I l J l l l 0 3 6 9 12 15 18 21 24 27 Days Fig. 7. Average weight of rats in each of three groups fed casein, whole soybean and sprouts. Basal diet contained 10% total protein. DISCUSSION

Compositional Significance

The respiration process was accelerated soon after the seed imbibed water. Respiration of seeds during the early stage of germination requires a normal oxygen tension (Yentur & Leopold, 1976). Wilson and Bonner (1975) found that the mitochondria of peanut seeds were lacking in cytochrom c until about 16 hr after the start of imbibition. They indicated that respiratory activity of peanut seeds before that time was an alternate respiration. An alternate pathway of respiration in the earliest stage of seed germination was also found in soybean seeds (Yentur & Leopold, 1976). They stated that the dependence of early germination stages on the alternate respiration is reflected in several types of seed functions, including: subsequent root growth rate, chlorophyll synthesis, and germination itself.

The bitter taste of sprouts can be initiated and/or dissipated during sprouting. The bitter taste is attributed to low molecular weight or TCA-soluble peptides; most are glycine-leucine and leucine-phenylalanine (Yamashita et al., 1970). From their experiments

45 46 with the plastein reaction, they reported that these bitter dipeptides were deprived of their bitterness by

condensing with various peptides. They speculated that other bitter peptides may behave more or less in similar manners to glycine-leucine and leucine-phenylalanine.

Therefore, during sprouting of seeds associated with disintegration and resynthesizing of proteins, some

degree of bitter taste might be encountered.

The results from this experiment showed a slight

increase in total crude protein of sprouts after the

fourth day of germination. Since nitrogen fixation was not in progress at this stage of growth, the increase in

total crude protein could arise from one or more mecha- nisms. Amino acid pools arise almost exclusively from

hydrolysis of the endosperm proteins and are transported

to the root tissue. Data obtained in this study showed

high concentrations of amino acids in the sprouted portion.

Dimler (1975) and Shemer (1974) proposed that heating a

mixture of reducing sugar and amino acids cause an

increased resistance of hydrolysis by pronase enzymes.

Due to the increase in reducing sugars found in sprouts

and the fact that sprouts were subjected to heat, the

amino acids liberated by enzymatic hydrolysis might be

lower than observed for unheated specimens.

The amino acid content of sprouts grown under

light and dark conditions exhibited different amino acid 47 patterns. These results agree with the work reported by Kasai et a1. (1966). In the current study, the major difference was evident in a higher accumulation of aspartic acid and a reduction in glutamic acid during germi— nation in the dark. Because aspartic acid is a key intermediate of biosynthesis, it is presumed that a higher rate of enzyme activity accompanied dark germination. Evidence of enhanced enzymatic activity was shown by Abrahamsen and Sudia (1966) who observed that isocitritase and malate synthetase, the enzymes of the glyoxylate cycle, prosessed higher activity during dark germination. In both light and dark germination there were significant increases in lysine and histidine when compared to the composition of ungerminated soybeans.

During light germination, increases of 18% lysine, 55% histidine, 13% serine, 7% aspartic acid were recorded.

Proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine and phenylalanine were essentially unchanged, whereas there were reductions of about 6% arginine, 16% threonine, 8% glutamic acid.

Dark germination produced increases of 12% lysine, 55% histidine, 44% aspartic acid, 9% threonine, 10% glycine,

12% alanine, 25% cystine, 15% valine and 4% leucine; proline, methionine, serine, phenylalanine were unchanged; while decreases of 7% arginine, 9% isoleucine, 14% tyrosine and 23% glutamic acid were recorded. From these data 48 it was concluded that dark germination resulted in a slightly higher net yield of amino acids, particularly the essential amino acids, i.e., histidine (semi- essential), lysine, threonine, valine, leucine and sulfur-containing amino acids, viz, from 3.2 to 3.56

(or 11%).

Kasai et a1. (1966) reported that there was no significant difference in the amino acid pattern during germination of the two varieties of non-nodule-forming and nodule-forming soybeans. Also they observed that dry beans contained large amounts of two peptides, a- glutamyl tyrosine and a-glutamyl phenylalanine, exclusively in the cotyledons. The content of these peptides did not change during the first 20 hr of germination, but decreased rapidly thereafter and disappeared after

70 hr. The result obtained in this study showed no increase in free tyrosine but an increase in the amount of phenylalanine during germination of soybean cotyledons. The fate of these a-glutamylpeptides is unknown at present.

The availability of amino acids of ungerminated soybean proteins, following an appropriate heat treatment, is in the range of 60-100% as determined with rat assays. But in the unheated soybean proteins less than half of the methionine and lysine are available (Smith &

Circle, 1972). 49

Wu (1953) reported studies on the effect of three different methods of cooking--(a) boiling in unsalted water, (b) boiling in salted water, and (c) sautéing in small amount of salted water and lard--on the retention of tryptophan, lysine and vitamins in soybean sprouts. He found no statistical differences among the methods of cooking. The percentage retentions of lysine, tryptophan, thiamine and ascorbic acid were

89-92%, 77-85%, 62-72% and 27-38%, respectively. Also he found that cooking by sautéing in small amounts of salted water and lard give the highest palatability score. In this experiment the palatability test was not performed, but it is believed that cooking by sautéing in salted water and lard would have produced the highest acceptability for the sprouts among the peOple in Asia because of their familiarity with former items prepared.

Biological Significance

Proteinase inhibitors are important components of plants and can be fitted into 3 categories: (a) maintain dormancy by preventing autolysis, (b) regulate protein synthesis and metabolism, and (c) prevent attack by predatory insects. Smith and Circle (1972) reported that young soybean seeds (3 weeks old) contained trypsin inhibiting activity (TIA) amounting to 50% of the mature seeds. There was no loss of TIA in soybeans germinated 50 up to one week. They stated that the soybean trypsin inhibitors do not inhibit germination, but Kunitz inhibitors (found in almost all soybean varieties,

Singh et al., 1969) strongly inhibit root growth.

Szilagyi (1968) postulated that the inhibitors block the incorporation of amino acids into proteins.

The work done by Collins and Sanders (1976) and

Albrecht et al. (1966) indicated that boiling sprouts, containing 60% moisture, for about 5 min was sufficient to inactivate the TIA. This was explained on the basis that the hydrated protein molecule was more sensitive to heat denaturation.

The PER value of germinated soybean determined in this study agreed with value reported by Smith and

Circle (1972) who obtained PER's for raw germinated and autoclaved germinated soybeans of 1.4 and 1.9, respectively. Sandal (1963) reported a PER-value for sprouts of 1.36 which is lower than the value obtained here.

It is suggested that because the sprouts were dried in a hot air oven for 18 hr, the protein may have been degraded or entered into complex interaction with other components.

The chemical score, based upon the quantity of amino acids which can be recovered in the acid hydrolysate of a protein, assumes that the animal can utilize all of the amino acids present. This assumption does 51 not take into account a number of factors which can alter the physiological availability of amino acids, such as: digestibility of protein by the digestive apparatus of the animal; the rate of which amino acids may be absorbed from gastrointestinal tract, and complex interactions with other nutrients which may affect digestibility and absorbtion. In this study the chemical score of ungerminated soybean and sprouts was essentially similar because the limiting sulfur-containing amino acids did not change significantly during germination.

Also, it corresponds to insignificant differences in the PER value.

The apparent digestibility of both the mature soybeans and sprouts were similar. Actually this value is lower than the true digestibility because the latter value includes the metabolic nitrogen in experimental animals. Smith and Circle (1972) reported a value of 91 as the true digestibility for whole soybeans in human subjects. The in 21252 digestibility of soybeans and sprouts after 10 hr exposure to the activity of pepsin revealed that sprouts were slightly more digestible.

The results of in_yitrg_digestibility are considerably lower than the amino acid profile would indicate. A rationale for these low values might include: (a) the pepsin alone is not an efficient proteolytic agent,

(b) substances such as phytic acid, saponins, phenolic 52 compounds, various sugars and metals, may complex with proteins and interfere with pepsin activity, and/or

The results of this study indicate that no significant difference exists between mature soybean and

4—day germinated sprouts relative to value for PER, apparent digestibility or chemical score. The conversion of seeds to sprouts represents a transition to a food form of equally high quality and enhanced acceptability to the Southeastern Asian culture. Thus, soybean sprouts could serve as an alternate source of relatively high quality protein. When added to traditional staples like rice, soy sprouts would enhance the overall nutritional quality of the diet.

53 APPENDIX Gain

Weight

Date

Obser.

Animal

At date......

Init.

Date

Consumed Observation

Feed LOG

Feed FEEDING Log APPENDIX

Wasted ANIMAL Feeding

Feed

Container

Spent Animal

Feed

Container

Initial

N0. date...... Init.

Feed:. Feed:.

Animal

54 55

I 120 scanning by

1 100 tryptophan. of determined

4 80 concentration

tryptophan

60 Tryptophan

of ug known of curve

l 40 nm 590 at Standard

n 20 8. Fig.

ALLA] #1 0

1_._ spectOphotometer % Digestibility and

60 80

sprouts t b Fig. at 9. Digestion 10 In hr vitro digestion. 56 time digestibility

(hr) Beans Sprouts_ 8 I 10 l of beans MEMORANDUM

During the preparation of this manuscript, an article by Bates et a1. appeared in the J. Food Science,

42:271-272 (1977) on the tOpic "Protein quality of green-mature, dry-mature and sprouted soybean." The results reported were similar to those found in this study.

57 BIBLIOGRAPHY BIBLIOGRAPHY

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Albrecht, W. J., G. C. Mustakar, and J. E. McGhee. 1966. Rate studies on atmospheric steaming and immersion cooking of soybean. Cereal Chem. 43: 400—07.

Association of Official Agricultural Chemist (AOAC). 1975. Official and tentative method of analysis. Washington, D.C.

Beeskow, H. C. 1943. Bean sprouts: Their preparation and properties. M.S.U. Tech. Bull. 184.

8115, R. F., and R. W. Howell. 1962. Biochemical and cytological changes in developing soybean cotyledons.

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Burkholder, P. R., and I. McVeigh. 1945. Vitamin content of some mature and germinated legume seeds. Plant physiol. 20: 301-06.

Campbell, J. A. 1963. In Evaluation of protein quality. Publ. no. 110, N. Acad. Sci-Nat. Res. Council.

58 59

Catsimpoolas, N., T. G. Campbell, and E. W. Meyer. 1968. Immunochemical study of changes in reserve proteins of germinating soybean seeds. Plant Physiol. 43: 799-805.

Cerning, J., A. Saposnik, and A. Guilbot. 1975. Carbo- hydrate composition of hourse beans of different origins. Cereal Chem. 52(2): 125-38.

Choi, Y. R. 1976. Blue green algal (anabaena flosaquae) protein as human food. Ph.D. thesis. Michigan State University.

Collins, J. L., and G. G. Sanders. 1976. Changes in trypsin inhibitory activity in source soybean varieties during maturation and germination. J. Food Sci. 41: 168-72.

Dalby, A., and C. Y. Tsai. 1976. Lysine and tryptophan increases during germination of cereal grains. Cereal chem. 53(2): 222-26.

Dimler, S. R. 1975. In Vitro enzymatic digestion of milk proteins. M.S. Thesis. Michigan State University.

Evans, R. J., J. McGinnis, and J. L. St. John. 1947. The influence of autoclaving soybean oil meal on the digestibility of the proteins. J. Nutr. 33: 661—72.

Eyster, H. C. 1938. Fat metabolism in soybean seed. Am. J. Bot. 25: 125.

F.A.O. 1970. Amino acid content of foods and biol. data on proteins. Rome.

Holm, H., K. Fossum, and Wenche B. Eide. 1973. Chemical and biological evaluation of protein quality of locally produced and processed full-fat soybean flour from three Tanzanian villages. J. Sci. Fd. Agric. 24: 333-41.

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