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This is to certify that the

thesis entitled

Effects of Processing and Maturity on
Certain Antinutritional Factors in
Soybeans

presented by
Keshun Liu

has been accepted towards fulfillment
of the requirements for

the Master of Sicifle‘gree in Food Science

 

 

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Major professor

 

5/5/1986

Date

 

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EFFECTS OF PROCESSING AND
MATURITY ON CERTAIN ANTINUTRITIONAL

FACTORS IN SOYBEANS

by

Keshun Liu

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

1986

ABSTRACT
EFFECT OF PROCESSING AND

MATURITY ON CERTAIN ANTINUTRITIONAL
FACTORS IN SOYBEANS

BY

Keshun Liu

Seeds of two soybean cultivars [filyging_mgx; (L.)
Merr.], Beeson 80 and Pella, were harvested at four
maturity stages and subjected to soaking (24 hr), cooking
(20 min), steaming (20 min), and soaking-cooking. Samples
were freeze-dried and analyzed for protein, oil, ash,
trypsin inhibitory activity (TIA), oligosaccharides and
phytic acid. As seeds matured, oil increased slightly,
protein was constant, ash remained constant after an initial
high value and phytic acid increased. TIA increased in
Beeson 80 and decreased in Pella. Oligosaccharides were
present in very low amounts early in seed development.

In mature soybeans, soaking slightly reduced oligo-
saccharides, but had no effect on phytic acid and TIA.
Cooking or steaming reduced oligosaccharides and TIA, but
had no significant effect on phytic acid. Soaking-cooking
increased phytic acid, decreased oligosaccharides greatly
and inactivated TI completely. In immature soybeans, changes
in oligosaccharides and phytic acid were similar to those
with mature soybeans except that steaming slightly increased
sucrose content. TIA decreased more readily upon processing

in immature soybeans than in mature ones.

DEDICATION

To my sisters

ii

ACKNOWLEDGMENT

The author wish to express his sincere appreciation to
Dr. Pericles Markakis, the author's academic advisor, for
his able guidance throughout the course of this work and his
critical advice in the preparation of this manuscript.

Special appreciation is extended to Drs. Thomas G.
Isleib, Ian J. Gray, Mark A. Uebersax and Robert J. Brunner
for their serving in my guidance committee and reviewing
this manuscript.

The author feels deeply grateful to the Chinese
Government for a scholarship to study at Michigan State
University.

Special thanks are also expressed to lab fellows in Room
212 of Food Science building (Suparmo, P. Ogun and S. Alani)
for their friendly help; to arthor's friend, Wenhua Lu, and
members of author's family for their moral support and

encouragement.

iii

TABLE OF CONTENTS

LIST OF TABLES O O O O O O O O O O O O O O O O 0

LIST OF FIGURES . . . . . . . . . . . . . . . .

I NTRO DUCTION O O O O O O O O O O O O O O O O 0

LI TERATIIRE REVIEW 0 O O O O O O O O O O O O O O

I.
II.
III.

IV.

VI.
VII.

Soybeans as a Food Source. . . . . . .
Chemical Composition of Soybeans. .
Factors Affecting Nutritional Quality
of Soybean Products. . . . . . . . . .
Antinutritional Factors in Soybeans. .

1. Trypsin inhibitors. . . . . . . . .
2. Flatulence factors. . . . . . . . .
3. Phytic acid . . . . . . . . . . . .

Elimination of Antinutritional Factors
in Soybeans. . . . .
Measurement of Antinutritional Factors
Comparison of the Food Value for
Immature and Mature Soybeans . . . . .

1. Chemical composition. . . . . . . .
2. The essential amino acid pattern

of proteins . . . . . . . . . . . .
3. Antinutritional factors . . .

4. In vivo studies of protein digestibility.

5. Organoleptic properties . . . . . .
6. Difference in variety---Garden Type
soybeans. . . . . . . . . . . . . .

MATERIALS AND METHODS . . . . . . . . . . . . .

I.
II.
III.
IV.
V.
VI.

Soybean Cultivation. . . .
Seed Harvesting. . . . . .
Fresh Seed Processing . .
Sample Preparation . . . .
Analysis of Raw Samples. .
Analysis for Trypsin Inhibi

to cry

iv

0 O O O 0

page
vi

viii

11
14

19
19

21
21
23
24

24
25

25
25
27
27
28

IX.

RESULTS AND DISCUSSION. . . . . . . . . . . . .

I.
II.
1.
2.
3.
III.
1.
2.
3.
4.
5.
IV.
1.
2.
3.
CONCLUSIONS
APPENDIX. .

Activity (TIA) . . . . . . . . .
VII. Analysis for Oligosaccharides. .
VIII.Analysis for Phytic Acid . . . .

Statistical Analysis of Data . .

Compositional Change during Maturation .
Trypsin Inhibitory Activity. . . . . . .

Discussion on the modified procedure
for TIA assay . . . . . . . . . . . .
Changes in TIA during maturation. . .
Effect of processing on TIA in

different maturity soybeans . . . . .

Oligosaccharides . . . . . . . . . . . .

HPLC chromatogrphy. . . . . . . . . .
Changes of oligosaccharides during

maturation. . . . . . . . . . . . .
Effect of processing on sucrose in
different maturity soybeans . . . .

Effect of processing on stachyose in
different maturity soybeans . . . . .
Effect of processing on raffinose in
different maturity soybeans . . . . .

Phytic ACid. O O O O O O O O O O O O O 0

Comparison of two assay methods for
phytic acid . . . . . . . .

Change of phytic acid during maturation

Effect of processing on phytic acid in

different maturity soybeans .'. . . .

BI BLI OGMPHY O O O O O O O O O O O O O O O O O O O

28
30
32
34
35
35
37
37
43
47
48
48
48
51.
51
54
57
57
6O
63
65
67

69

LIST OF TABLES

Table page
1 Classification of oriental soyfoods. . . . . . . . 4
2 Composition of soybeans and their fractions. . . . 6
3 Antinutritional factors in soybeans. . . . . . . . 7
4 Essential amino acid pattern of proteins

from immature and mature soybeans. . . . . . . . . 22
5 In vivo (rats) studies on the nutritive value

of soybean proteins. . . . . . . . . . . . . . . . 23
6 Harvest date, total solids and color

characteristics of maturing Beeson 80 soybeans . . 26
7 Harvest date, total solids and color

characteristics of maturing Pella soybeans . . . . 26
8 Change of chemical composition in developing

Beeson 80 and Pella soybeans . . . .‘. . . . . . . 36
9 Degradation of BAPNA activity with time in

term of trypsin units. . . . . . . . . . . . . . . 38
10 Values of trypsin inhibitory activity (TUI/ml

extract) in relation to levels of inhibitor

SOlution O O O O O O O O O O O O O O O O O O O O O 4 1
11 Effect of maturity and processing on trypsin

Inhibitory Activity in Beeson 80 Soybeans. . . . . 44
12 Effect of maturity and processing on trypsin

inhibitory activity in Pella soybeans. . . . . . . 45
13' Comparison of two assay methods for phytic

acid content in soybeans . . . . . . . . . . . . . 58
14 Effect of maturity and processing on phytic

acid content in Beeson 80 soybeans . . . . . . . . 61
15 Effect of maturity and processing on phytic

A1

acid content in Pella soybeans . . . . .

Colorimetric assay for trypsin inhibitory
actiVity O O O O O O O O O O O O O O O O

LIST OF FIGURES

Figure
1 Structures for oligosaccharides . . . . . . . . .
2 Proposed structure for phytic acid. . . . . . . .
3 Effect of heat treatment on trypsin inhibitory
activity and protein efficiency ratio of
soybean protein . . . . . . . . . . . . . . . . .
4 Scheme for approaches to phytic acid analysis . .
5 Trypsin inhibitory activity (TUI/ml extract)
in Relation to Inhibitor Solution . . . . . . . .
6 Distribution histogram for percentage of trypsin
inhibition at the lowest TUI/ml value of four
portions of the extract . . . . . . . . . . . . .
7 Correlation of trypsin inhibitory activity with
protein content in two cultivars of soybeans
harvested at four different maturity stages . . .
8 HPLC chromatograms for sugar analysis . . . . . .
9 Changes of sucrose, stachyose and raffinose
contents in maturing soybeans . . . . . . . . . .
10 Effect of processing on sucrose in Beeson 80
soybeans of different maturity . . . . . . . . .
11 Effect of processing on sucrose in Pella
soybeans of different maturity. . . . . . . . . .
12 Effect of processing on stachyose in
Beeson 80 soybeans of different maturity. . . . .
13 Effect of processing on stachyose in
Pella soybeans of different maturity. . . . . . .
14 Effect of processing on raffinose in

Beeson 80 soybeans of different maturity. . . . .

\dii

Page
10

10

13

18

4O

42

46

49

50

52

52

53

53

55

15

16

17

A1

Effect of processing on raffinose in
Pella soybeans of different maturity.

Proposed structure for ferric phytate
(4:6 Fe:P). . . . . . . . . . .

Proposed structure for ferric phytate
(6:6 Fe:P). . . . . . . . . . . .

Standard curve for Fe measurement . .

55

59

59

68

INTRODUCTION

Direct consumption of fresh immature soybeans [glycine
max; (L.) Merr.] is very popular in China and in some other
oriental countries. In Chinese, the immature soybean is
called "Mao Dou" or "Qing Dou", meaning green bean. In
Japanese, it is called "edamame". Steamed or boiled in water
before or after shelling, traditionally for not less than 20
min, these fresh green beans can be served either as a
delicious and nutritious green vegetable with the main meal
or as snacks with wine and beer.

In the West, due to increasing production of soybeans
and popularization of vegetarian life-styles, development of
acceptable food products from green immature soybeans has
been suggested to further increase the variety of soybean
products. In fact, frozen and canned immature soybeans have
appeared in Western markets.

In general, immature soybeans have some advantages over
mature ones. These include higher content of ascorbic acid
and B-carotene, lower amounts of antinutritional factors and
higher protein availability. Furthermore, their green color
and soft texture enhance their appeal as a vegetable.

One of the major factors that limit use of soybeans in

human diet is the presence of certain naturally occurring

1

substances, termed antinutritional factors. These
substances, including trypsin inhibitor, oligosaccharides
(mainly, raffinose and stachyose) and phytic acid, can cause
adverse physiological responses or diminish availability of
certain nutrients to animals and humans. For example,
trypsin inhibitors reduce the digestibility of soybean
proteins; oligosaccharides cause flatulence associated with
soybean consumption, while phytic acid decreases the
availability of certain minerals by chelating them. There
are numerous reports in the literature concerning this
subject, including the presence of these antinutrients in
soybeans, the analytical methods to measure them and the
proper processing treatments to reduce them. However, most
of these reports have been limited to mature soybeans.

There are no data available on the oligosaccharides in
developing soybeans, and little information on the effect of
processing treatments (soaking, cooking, steaming and
soaking-cooking) on trypsin inhibitors, oligosaccharides and
phytic acid in immature soybeans. The purpose of this

investigation was to provide information on these subjects.

LITERATURE REVIEW

I. Soybeans as a Food Source

With a steady increase of the world population, demand
for both protein and edible oil is ever increasing. Because
soybeans contain about 40% protein and 20% oil, they have
become one of the most promising crops to fulfil this need.
Indeed, the world production of soybeans has steadily been
increasing. For example, in 1970, the world total soybean
production was 32 million metric tons, while in 1985, it
reached 90 million metric tons (USDA, OCS-8 1985).

Throughout history, the Chinese and other oriental
people have developed various forms of soybean foods.
Traditionally, oriental soybean products can be classified
into two groups, non-fermented and fermented soyfoods. For
both kinds, the name, the brief description and the use for
each variety are presented in Table 1.

For people in western countries, due to the relative
short history of soybean use, the flavor and texture of
soybean products are comparatively strange. Therefore, in

the West, the use of soybeans as a food is not as extensive

3

Table 1. Classification of oriental soyfoods *

 

Oriental Non Fermented Soytoode

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SOWOOOS LOCAL NAMES DESCRIP‘HON USES
Freeh green eovheene MID-I0" ICMneeel Picked trelore meture. Boiled or eteemed In the
Ederneme IJepeneeel line. bright green m end eheled. Served
ee here d’eeuvre I tten
Wm“; ma"...
eerved ee Ireeh green
men-prom Hum-tournament mmmm Served eeveoetebteln
De 3-“!!! eproute In eeled
(Jeeerteeel tperoetledl
Sevnute W mm Mute Served ee enect
trI-meme JeoeneeeI '0' cotyled‘ovm by deep-
rvlng or heet.
Seeeoned “It. north
or eoeted :Iyttt cheeotete
m Tou or tounel m m dam-l. Served hot ee breeltleet
TI . W covfe ml:u drI'r'Ikdodrved cold ee
o-nw etch. muted eo
Mm Ilevoured
Soytleur menu-Immune» Gmma Ueedeet mercoettng
devour tor
Wit—On Ton-Mot w W M Cooked end need ee meet
80: Yuae Ueeerteeel ol over he uleee with chewy te re.
end motet eheete. tlehee
or etidte
mm TMer‘l’eo-NFhe-t WNteeurdcubee Servedeedietteewtttter
Um from m wtthout Mther eoeflno.
Tutu tttoreenl who e eeIcIte-n or
new (SE Aete) eett eott to
tlrm (by proceed and»
mu - - A - , A
SOYFOOOS LOCAL NAMES ORGANISMS USED DESCRIPTION USES
Seuoe I tattneeel or Out: brown I Id.
80v CHI-reg“ . Asp-vellum w m ”a taunt-"v
KetIep lndoneetenl Torutooete end met 0! meet extr e
Ken Moreen; Seccheromvcee flevoun t.
TM. too-bu (S. . Aete)
Mleo Tou-chlon. wet-chow Aeper Illue. eovboen. Hoe ' ht II t dork
(ChtnoeeI tsertevg reddten brownpeete. “9 " ow o
Mbo lJepeneeel Pedioooccue emooth or chunky. Ieltv
Teu (K ) Torulopete end mm eoen' t.
Soybeen Pesto Streptococcus
Tom h Tempeh (IndoneeIenI Rhleopue Whole eovbeene Cooked eon beene bound
9', Tempet tJepeneeeI may by mycetle ee
e dun treen end
WM
end eerved ee metn dleh
Netto Netto IJepeneeeI Bedllue Netto Whole eovbeene Cooked beene bound
eu~nou or Thue-no end covered
ugli'mig’ II betence. produced by
me In
090 the beetede. umnonlurn
odour. muetv flavour.
wIth cooked rice
ee e
Fermentedtotu Twila-ru'orTeo-ru Acttnermoer, Soyeeencwdltlnnw ammu-
Fu-nw 0w Muou Am eervedm
gotta undeneelenl , «WWW
Odrteee dteeee
Numete l eta-duh mm) Nee-m thole eoveeene Nee»; bled to. beat. my
Soy ] eo-eI (W, m Wheet Item tteveur
' eucot I Am
I

 

as in the Orient. However, this situation is now beginning
to change. The move is towards products made from tofu,
tempeh and miso, and the emphasis is on more imaginative
uses of soybeans in modern prosessing systems such as
snackfood products. With advancing technology, it is not
surprising that more and more edible products can be seen in

western markets, which contain soybean derivatives.

II. Chemical Composition of Soybeans

Soybeans are composed of approximately 8% hull, 90%
cotyledon, and 2% germ (hypocotyl and plumule). The chemical
composition for soybeans depends on variety, soil and other
factors. Table 2 gives the proximate composition for whole
beans and their three fractions. Carbohydrates in the table
include polysaccharides, and oligosaccharides such as

sucrose, raffinose and stachyose.

Table 2. Composition of soybeans and their fractions *

 

.Protein Fat

Fractions (Nx6.25) (%) (%)

Carbohydrate Ash

(%)

(%)

 

Whole bean 40 21
Cotyledon 43 23
Hull 8.8 1
Hypocotyl 41 11

34

29

86

43

 

Moisture free basis (from Kawamura, 1967).

The nutritional quality of soybean products is affected

III. Factors Affecting Nutritional Quality of

Soybean Products

by several factors:.

1) Contents of protein, oil and other nutrients.

2) Quantity and availability of the amino acids which

3)

4)

make up the protein of such products.

Contents in certain chemical substances, which cause
adverse physiological responses in man.
they are termed antinutritional factors.

Degree of elimination of these antinutrients by

Commonly,

certain processing treatments such as heating,

germination etc.

IV. Antinutritional Factors in Soybeans

Liener (1981) classified antinutritional factors into
two groups according to their reponses to heat: heat-labile
and heat-stable (see Table 3).

Among these antinutrients, trypsin inhibitors,
flatulence factors and phytates may be considered as key
determinants to the nutritional quality of soybeans for
humans.

An attempt will be made to evaluate their nutritional
significance and biologically detrimental effects,
particularly in human diet. Later on, in the following
sections, discussion will be made on how these effects may
be eliminated to some extent by appropriate processing

methods.

Table 3. Antinutritional factors in soybeans

 

 

Heat-labile Heat-stable
Trypsin inhibitors Saponins
Hemagglutinins Estrogens
Goitrogens Flatulence factors
Antivitamins Lysinoalanine

Phytates Allergens

 

1. I I . 1.1.!

Trypsin inhibitors (TI) are proteinase inhibitors. They
belong to a family of proteins which inhibit a wide
variety of proteinases, mainly trypsin.

Osborne and Mendel (1917) made the first significant
observation that soybeans had to be heated in order to
support the growth of rats. TI proved to be responsible
for reducing digestibility of proteins by inhibiting trypsin
activities. Later, a second observation was made by
Desikachar and De (1947), and Liener (1949) that
preparations of TI were capable of inhibiting growth even
when incorporated into diets containing predigested protein
or free amino acids. This indicated that inhibition of
proteolysis by TI was not the sole factor responsible for
growth depression. Subsequently, another significant
finding was made by Chernick et a1. (1948). They found that
raw soybeans and the TI itself could cause hypertrophy of
the pancreas, which is accompanied by an increase in the
secretory activity of the pancreas. This led to the
suggestion that the growth depression caused by the TI might
be the consequence of an endogenous loss of essential amino
acids being secreted by hyperactive pancreas.

More recent experiments suggested that protein from raw
soybeans is in itself refractory to enzymatic attack unless

denatured by heat (Green et al., 1973). Therefore, it would

appear that the TI and the refractory nature of the soybean
protein act through a common mechanism to inhibit the growth

of rats.

2. Elatulense_fa2ter§

One of the important factors limiting the uses of
soybeans in human diet is the flatulence associated with
their consumption. Steggerda et al. (1966) conducted an
experiment in which four male adults consumed various
toasted soybean products commercially manufactured. They
revealed that flatulence resulted mainly from the low-
molecular-weight carbohydrate fractions of soybeans
containing a—galactosidic and B-fructosidic linkages, namely
raffinose and stachoyse (Figure 1). In other kinds of
legumes, verbascose may be included. Hymowitz et al. (1972)
analyzed soybean seeds of as many as 60 selected lines for
sucrose, raffinose and stachyose contents. They found that
ranges in values (expressed as g/100g seed) were 2.5-8.2 for
sucrose, 0.1-0.9 for raffinose and 1.4-4.1 for stachyose.

Flatulence is generally attributed to the fact that man
is not endowed with the enzyme called a-galactosidase
necessary for hydrolyzing the a-galactosidic linkages of
these oligosaccharides to yield readily absorbable sugars
(Gitzelman and Aurricchio, 1965). Consequently, the intact
oligosaccharides enter the lower intestine where they are
metabolized by microorganisms, resulting in the production

of such gases as carbon dioxide, hydrogen, nitrogen, methane

10

C on CH CH
HO "2 O 0/ a O 0/ a 0 no“; 0
no 0%
H10.
01 0'!
l J

Sucrose
I 4

Rottinoee
l J

Stochyose

 

Figure 1. Structures for oligosaccharides

emu, 090,01,

 

C) G)
opo,ua
C)
tqmao cwogt <3 '
G) ceHNQKHt
crowz ‘IOHh
ANDE N STR T NEUBERG STRUCTURE

 

Figure 2. Proposed structure for phytic acid

11

etc., depending on the individual diet and microflora

spectrum.

3- Ehxtis_asid

Phytic acid, the hexaphosphate of myoinositol (Figure
2), is found mainly in plant seeds, where it functions as a
reserve material for phosphorus. In soybean seeds the amount
of phytic acid present varies with variety, ranging fron
1.01-1.47% (Lolas et al., 1976). To plants themselves, the
presence of phytic acid is desirable, but when these plants
are used as food, it is undesirable, because it readily
chelates with such di- and tri-valent metal ions as Ca, M9,.
Zn, Cu and Fe, to form insoluble salts. Such complexes are
poorly absorbed from the intestine, resulting in reduced
availability of these minerals from soybean products (Jaffe,
1981).

In addition to binding with minerals, phytic acid was
found to bind strongly with proteins at certain pH values.
Therefore, it can affect the properties of proteins (Okubo

et al., 1976).

V. Elimination of Antinutritional Factors in Soybeans

1. Trypgin inhihiggzg
Since TI are proteins, they are readily inactivated by

various heat treatments. These include live steam (a process

called toasting), heating in boiling water, dry roasting,

12

dielectric heating, microwave radiation, micronization and
extrusion cooking (Liener, 1981). Figure 3 shows the effect
of general heat treatment on trypsin inhibitory activity
(TIA) and protein efficiency ratio (PER) of soybean
proteins.

Other methods such as soaking and germination were also
reported to eliminate TIA in soybeans to some extent
although they were not as effective as heating (Ologhobo and
Fetuga, 1983)

Recently, Orf and Hymowitz (1979) discovered a soybean
line from Korea which lacked TI. This might provide an.

approach for eliminating TI through genetic breeding.

Z-W

Flatulence factors are heat-stable, and heat treatments
cannot reduce their contents. There are certain techniques
that have been reported to reduce the content of oligo-
saccharides. These include soaking, enzymatic hydrolysis and
‘germination (Liener, 1981).

The fact that there is a considerable variation in
oligosaccharide content of different soybean varieties may
give the hint that genetic changes by plant breeding is, at

best, only a long-term solution to the problem.

3.2mm
Phytase can hydrolyze phytic acid, but unfortunately,

monogastric animals like man have little or no intestinal

13

 

\.

Protein Efficiency Ratio (PHI)

1
N
Le

A / ' int:

20- \o

l

O

1 L
N
Le

‘
c
.I
0/
g1
‘

 

Trypsin Inhibitor Activity [Till/mg.)

L
0
l
at

 

 

_ I I I
0 2 it 6 to 20
Minute: at 100°C.

Figure 3. Effect of heat treatment on
trypsin inhibitory activity and protein efficiency
ratio of soybean proteins

(From Rackis, 1972).

14

phytase activity. Therefore, it would be advantageous to
develop methods to eliminate it. However, since phytates are
tightly bound to soya proteins, no commercial methods have
been developed to eliminate them completely. At a laboratory
scale, several procedures have been used such as
precipitating phytates as the barium salt, dialyzing them
against a sodium chloride solution, treating them with a
strong anion exchange resin, and controlling the pH during
preparation of soybean isolates (Jaffe, 1981).

Certain microorganisms containing phytase could cleave
phosphates from phytic acid during soybean fermentation

(Erdman, 1979).

VI. Measurements of Antinutritional Factors

l-Wmmm

Although various methods of column chromatography,
affinity chromatography and electrophoresis have proved
valuable for the isolation and characterization of diverse
TI, these methods are not suitable for quantitation. At
present, methods for TIA analysis are mainly colorimetric,
based on interaction among TI, trypsin and a substrate.
There are two kinds of substrates used, natural substrates
like casein and synthetic substrates like benzoyl-DL-
arginine-p-nitroanilide (BAPNA).

The common method employing casein is the one originally

15

described by Kunitz (1947); it involves the spectrophoto-
metric determination of the breakdown products produced by a
given concentration of trypsin in the presence and absence
of the inhibitor. However, the rate of hydrolysis of casein
by trypsin was later reported not to follow zero order
kinetics under the condition defined by Kunitz (Jacobbson,
1955). This poses a question of reliability.

Erlanger et al. (1961) first introduced a synthetic
substance, BAPNA. They found that hydrolysis of BAPNA by
trypsin not only followed a zero order, but also can be
directly traced colorimetrically. Later on, Kakade et al.
(1969) reported that the use of BAPNA is preferable over
casein in tern of simplicity and accurancy, provided that
the competitive nature of the inhibition was taken into
consideration. Subsequently, a collaborative study by a
Committee on Soybean Trypsin Inhibitor Analysis resulted in
several modifications of the original procedure (Rackis et
al., 1974 and Kakade et al., 1974).

However, Hamerstrand et al. (1981) found that the
extrapolation procedure for data interpretation suggested by
Kakade et al. (1969) could lead to an erroneously high value
for TIA in soybean products, since this extrapolation used
data that are not in the region in which zero order kinetics
are followed. In order to obviate this problem, they
developed a further modified procedure in which TIA was
determined from a single dilution of a sample extract that

inhibited trypsin in the range of 40-60%.

16

1W

Reports on the determination of oligosaccharides in
legumes involved a number of techniques. These include
chemical and enzymatic methods, the colorimetric method, the
gravimetric method, thin layer chromatography (TLC), paper
chromatography (PC), gas-liquid chromatography (GLC), and
liquid chromatography (LC) (Cegla and Bell, 1977). However,
most of these methods have their limitations. The enzymatic
procedure is highly specific, but the reagents required for
large numbers of analysis are expensive. The colorimetric
method is applied to pure solutions or mixtures of sugars.
The gravimetric method does not distinguish among different.
carbohydrates. TLC and PC are useful only for qualitative
determination. GLC requires derivatization of the sugars,
although it is one of the most accurate methods. For LC, it
takes a long time to finish a single separation.

Conrad and Palmer (1979) reported a procedure for
analysis of carbohydrates in legumes by high pressure liquid
chromatography (HPLC). This method has then been considered
as an adequate means for rapid analysis of soluble sugars.
However, because soybeans contain many substances other than
sugars, there is a strong interference with separation and
quantitation of sugars. Also there is a high risk of column
contamination.

In order to separate sugars from other components,
several methods have been reported including precipitation

of proteins with 15% trichloroacetic acid (TCA) or with lead

17

acetate solution and purification of sugars on TLC or on
preparative column of hydroxylapatite. Cegla and Bell (1977)
concluded that TLC was the best method to separate unwanted
compounds from sugars although it was very tedious.
Therefore, development of an accurate and simplified
extracting procedure with least risk of column contamination

for HPLC analysis of sugars still remains a challenge.

$me

Figure 4 summarizes the various methods developed for
analysis of phytic acid in soybeans as well as in other
materials.

Most assays for phytic acid employ ferric chloride to
precipitate ferric phytate. The determination may be based
on the analysis of either phosphorous or iron in isolated
ferric phytate (Nahapetian and Bassir, 1975), or indirectly
on the determination of the residual iron (Young, 1936) or
the residual phosphorus (Tangkongchitr et al., 1981) in the
'supernatant after precipitation. Alternatively, sodium
hydroxide may be used to convert the precipitate to sodium
phytate and ferric hydroxide. It is convenient to analyze
for iron after taking up the ferric hydroxide in acid
(Wheeler and Ferrel, 1971).

Most of these methods are time-consuming. Furthermore,
interpretation of the iron content either in the precipitate
or in the supernatant into phytate content is based on

assuming certain molar ratio of iron to phytate phosphorus

18

 

 

 

 

 

Sample
EXTRACTION
(hydrolysis and . (HPLC phytate
it
inositol analysis CENTRIFUGATION analysis)
"/’////’,,-Extract
(P analysis) /////////
FeC13 PRECIPITATION ION EXCHANGE
(residual Fe (thorium Phytate-a(phytate
analysis) titration) analysis)
I I
I
CENTRIFUGATION ACID DIGESTION
(P analysis)
Supernatant Ferric hytate
\
(residual P NaOH ALKALIFICATION DIGESTION

analysis) l ’/////

CENTRIFUGATION (Fe analysis) (P analysis)
Supernatant (Na phytate) Fe(OHk3Precipitate
(hydrolysis and

inositol analysis) (P analysis) (Fe analysis)

Figure 4. Scheme for approaches to phytic acid analysis

l9

(ranged from 3:6 to 4:6 as reported previously), resulting
in poor reliability and repeatability.

An Anion exchange separation of phytate from the extract
was reported accurate (Harland and Oberleas, 1977).

Recently, a HPLC system has been developed to quantitate
phytic acid (Knuckles et al., 1982). It has proved to be a
rapid method with more reproducible results as compared with
traditional methods.

For extracting phytate from soybeans, different acid
solutions were used, including 3% trichloroacetic acid (TCA)
(Wheeler and Ferrel, 1971), 0.5M HCI (Makower, 1970), and

1.2% HCI + 10% Na $0 or 3% TCA + 10% Na SO

2 4 2 4 (Thompson and

Erdman, 1982).

VII. Comparison of the Food Value for Immature and

Mature Soybeans

The high consumption of immature soybeans in China and
other parts of the world can not be explained by tradition
alone. It is likely that nutritional considerations justify

the popularity of this foodstuff.

1- Chemical_semnesitien
1) Ereen_Eeight_and_Ietal_§elidsl According to Rackis
et al. (1972), the maximum fresh weight of soybeans was

reached 44 days after flowering, when these seeds were still

green but the pods began to yellow. The dry matter

20

accumulated with maturation, more slowly at early stages and
faster at later ones. '

2) Protein; On a dry basis, protein content was
constant regardless of maturity, while on fresh weight
basis, it accumulated with maturation (Rubel et al., 1972).

3) Qil_and_gil_§gmpg§itign& On a dry basis, crude oil
increased with maturation, faster at early stages and more
slowly at later ones. Regarding oil composition, it
appeared that with maturation, palmitic, stearic and
linolenic acids decreased while oleic and linoleic acids
increased. However, the ratio of saturated vs. unsaturated
fatty acids remained the same (Rubel et al., 1972).

4) Minerals; Limited data are available concerning the
effect of maturity on ash content in soybeans. Rasmussen
(1978) reported that lower content in boron and phosphorous
and higher content in zinc were noticed in-meal prepared
from immature soybeans compared with that prepared from
mature soybeans.

5) Vitamins; Perhaps, one of the major nutritive
advantages of immature soybeans over mature ones is the
higher contents in ascorbic acid and g-carotene in immature
soybeans (Bates and Matthews, 1975).

6) Qazbghygzatggg It has been found that during
maturation, large amounts of starch accumulated and reached
a maximum at the green mature stages. During subsequent
development to full maturity, starch practically

disappeared and the sugar content increased (8115 and

21

Howell, 1963).

Z-EWIMWM

In general, the essential amino acid pattern of soybean
proteins is considered rich in lysine and poor in
sulfur-containing amino acids, that is, methionine and
cysteine. Table 4 shows the essential amino acid composition

of proteins from both immature and mature soybeans for

comparison.
3. 't'o a ac o s
1) Tryp§1n_Inhibitg;§; Reports on the change of TI in .

developing soybeans are somehow contradictory. Collins and
Sanders (1976) found that as soybeans matured the amount of
TI in the extracts of beans increased, while Yao et al.
(1983) reported that TI remained constant during the
development of soybeans. The divergent results may be due to
the different varieties of soybeans used by the two groups.
2) Qligggaggharigggy No data have been reported on the
changes of flatulence factors in developing soybeans.
However, a study made on peas by Gautier (1979) showed that
total galactoside content was correlated with increase of
total solids during maturation of peas; raffinose appeared
first, followed by stachyose and verbascose. Furthermore, in
fresh peas the amount of these oligosaccharides was too low

to cause digestiVe disorder.

22

Table 4. Essential amino acid pattern of

proteins from immature and mature soybeans

 

Amino Acid (Gram amino acid per 16 grams N)

 

Immature a Mature b
Lysine 8.5 6.5
Methionine 0.8 1.0
Cysteine / 1.2
Tryptophan 1.0 1.8
Threonine 1.9 3.7
Isoleucine 6.6 4.2
Leucine 7.1 8.0
Phenylalanine 5.0 74.9

Valine 5.6 4.9

 

a. From Standal, 1967.

b' From Evans and Banserner, 1963.

23

3) Phytig_AQiQL Phytic acid content was reported
increasing with maturity of soybeans from 0.81 to 1.26%, on

a dry basis (Yao et al., 1983).

4W

Perhaps, the most effective and reliable method to
evaluate the nutritional quality of soybeans is in vivo
studies. Table 5 shows that immature soybeans appeared to
supply protein of higher nutritive value than that of mature
soybeans both before and after autoclaving (Everson et al.,
1944). Standal (1963) found that net protein utilization
(NPU) and protein efficiency ratio (PER) of immature
soybeans, as measured with rats, was comparable to that of

casein and lean beef.

- Table 5. In vivo (rats) studies on a,b

the nutritive value of soybean proteins

 

 

Soybeans Raw Autoclaved
Mature 0.484 1.700
Immature 1.101 2.013

 

a. From Everson et al., (1944).

b' Values represent protein efficiency ratio (PER)

24

5.0—mew

One of the major constraints in the use of soybeans is
their characteristic flavor, described as beany and bitter.
Fresh immature soybeans may have an advantage over mature
ones because of their lower flavor intensity and softer
texture (Rackis et al., 1972). Since the lipoxygenase
activity was found to be related with beany flavor (Mattick
and Hand, 1969), the lower lipoxygenase activity in immature
soybeans might partly explain the lower flavor score of

immature beans as compared with that of mature beans.

6.21fferensLiLzaWbeans

Usually, soybeans for direct consumption are the
varieties called "Garden types". Garden type soybeans are
not basically different from field varieties but generally
larger in size, higher in protein and lower in oil and
yield. They also have a better flavor and texture than

regular field beans (Smith and Circle, 1978).

MATERIALS AND METHODS

I. Soybean Cultivation

Two soybean cultivars, "Beeson 80" and "Pella" were
obtained from the Department of Crop and Soil Sciences,
Michigan State University. Seeds were sown in the open

field on June 6, 1985. No fertilizers were used.
II. Seed Harvesting

The seeds were harvested at four different maturity
stages on the basis of both the solid content and the
organoleptic properties of the beans. The beans were
respectively defined as Immature I, Immature II, Immature
III and mature beans.

Tables 6 and 7 show the dates of harvest, the total
solids contents and the color characteristics of the beans
at each stage of maturity for two cultivars. The total solid
content was determined by drying the seeds in a vacuum oven
at 60C for 24 hr. The mature seeds were harvested when
thoroughly field-dried.

Only the seeds from the middle of the plants were picked

25

26

Table 6. Harvest date, total solids and color

characteristics of maturing Beeson 80 soybeans

 

Stage of Date of Total Solids Color description

 

Maturity Harvest (%) Pods Seeds
Immat. I 9/9 30.0 Green Green
Immat. II 9/17 35.3 Green Green
Immat. II 9/26 38.4 Yellow Light-green
Mature 10/18 92.2 Brown Buff-brown

 

Table 7. Harvest date, total solids and color

characteristics of maturing Pella soybeans

 

Stage of Date of Total Solids Color description

 

Maturity Harvest (%) Pods Seeds
Immat. I 9/9 28.6 Green Green
Immat. II 9/22 35.1 Green Green
Immat. III 9/30 38.6 Yellow Lightgreen,dark spots

Mature 10/23 91.6 Brown buff-brown,dark spots

 

27

up and transported to the laboratory for immediate hand
shelling. Within 6 hours the fresh seeds were subjected to

various processing treatments.
III. Fresh Seed Processing

Immature II, III and Mature seeds were subjected to the
following processing treatments. Immature I seeds were too
small for processing.

1) Soaking for 24 hr;

2) Steaming over boiling water for 20 min:

3) Cooking in boiling water for 20 min;

4) Soaking for 24 hr and then cooking in boiling water

for 20 min.

Each treatment was duplicated. The size for each
independent sample was adjusted to about 35.9 on a dry
basis. Each weighed sample was wrapped in cheese cloth and
marked before processing. The processed samples were

'drained and cooled before freezing.
IV. Sample Preparation

The frozen raw and processed samples were first
freeze-dried in a freeze dryer for about 3 days and then
ground in a blender to pass through No. 20 mesh. Finally,
the ground sample was stored in a plastic bag until

analyzed.

28

V. Analysis of Raw Samples

Nitrogen content was determined by micro-Kjeldahl (AOAC,
1980). The factor 6.25 was used for converting N-content to
protein content. Oil and ash contents were measured

according to AOAC (1980).

VI. Analysis for Trypsin Inhibitory Activity (TIA)

6.059 tris (hydroxymethylamino methane) and 2.949 CaC12‘2H20

were dissolved in 900 ml of water. The pH was adjusted to
8.2, and the volume was brought to 1 liter with water.

2) gubgtrat§_§213tign; 40 mg of benzoyl-DL- arginine-
p-nitroanilide (BAPNA) hydrochloride were dissolved in 1 ml
of dimethyl sulfoxide and diluted to 100 ml with tris-buffer
prewarmed to 37C. The solution was prepared before each run
of measurements and kept at 37C immediately after prepar-
ation.

3) Irxpgin_figlg§igg& In 200 ml 0.001M HCl Solution, 4
mg of accurately weighed trypsin were dissolved. The
solution could be stored in a refrigerator for about two
weeks without considerable loss of activity.

4) WW 500-0 m9
of soybean sample (50 mesh) were extracted with 50 m1 of

0.01N NaOH for 2 hr on a mechanical shaker. The pH of the

29

suspension was in the range of 8.4-10.0. After the extract
was allowed to stand for 10 minutes, a 10 ml aliquot was
pipetted into a 100 m1 volumetric flask and diluted to the
mark. For raw soybean samples, 0.8 ml of the diluted
suspension could inhibit trypsin in the ranges of 40-60%.

5) QQIQI.M§B§QI§E§DE1 Portions (0.0, 0.4, 0.8, 1.2 and
1.4 ml) of each soybean suspension of three independent
samples were pipeted into 17 test tubes and adjusted to 2.0
ml with water. The 16th tube was another trypsin standard
corresponding to 0.0 ml sample suspension. The 17th tube
was a reagent blank. After 2 ml of trypsin solution was
added to each tube, the tubes were placed in a waterbath at
37C. Two min later, 5 ml of BAPNA solution previously warmed
to 37C was added to each tube at exact intervals of 10 sec.
The tubes were then shaken for mixing. Exactly 10 min
later, the reaction was terminated by adding 1 ml of 30%
acetic acid solution to each tube at the same time intervals
(10 sec). After thoroughly mixing, the content of each tube
was filtered (Whatman No.5) and the absorbance of the
filtrate was measured at 410 nm against a reagent blank. The
reagent blank was prepared by adding 1 ml of 30% acetic acid
to test tube containing trypsin and water (2ml each) before
5 ml of BAPNA solution was added.

6) Ezp;gggign_gfi_59tiyity; One trypsin unit (TU) is
arbitrarily defined as an increase of 0.01 absorbance at 410
nm per 10 ml of the reaction mixture under the conditions

used herein. TIA is expressed in terms of trypsin units

30

inhibited (TUI). The TIA in soybeans is expressed as TUI/mg
dry sample by averaging the results of 4 portions of diluted

inhibitor solution taken for assay.
VII. Analysis for Oligosaccharides

As pointed out earlier, HPLC for the measurement of
oligosaccharides is convenient and accurate, but
contamination of the column remains a problem. The HPLC
system originally reported by Conrad and Palmer (1976) was
therefore modified as follows:

1) Extrastins_the_§2xbsan_§sueles1 2-2 g dried soyflour
in a 40-50 ml round bottom polyethylene centrifuge tube were
first defatted by treating it with 40 ml hexane, followed by
centrifuging. This was repeated twice with 20 ml hexane.
Then the defatted flour was dried in a vacuum oven at 35C
overnight before it was refluxed with 25 ml of 75% alcohol
solution in a waterbath at 80C for 4 hr with occasional
shaking. The mixture was allowed to cool before centrifug-
ing. The supernatant was saved and the precipitate was
washed twice with 10 ml alcohol solution. Then, 1 ml 10%
lead acetate was added to the conbination of supernatants
before holding them in the waterbath for 8 min. The tube
was then centrifuged and the precipitate was washed once.
This was repeated once to precipitate the remaining
proteins. After this step, about 0.4 ml 10% oxalic acid was

added to the mixture to precipitate the excess lead (this

31

was optional). The supernatant was now concentrated at
temperature below 50C to a volume of approximate 15 ml,
using a vacuum evaporator. The concentrated solution was
then transfered to a 25 ml flask and diluted to mark with
water.

2) fl£LC_§y§§gm‘ The HPLC system was assembled from a
Waters Model M-45 pump, a Waters RI-401 differential
refractometer detector, a Guard-Pak procolumn module with
silica cartridge in it (Waters) and a Waters u-Bondapak/
carbohydrate column (3.9mm x 30cm). A Kontes model
100 recorder was used for detector response reading.

Samples were loaded onto the column through a Waters
7010 injector with a 20 ul loading loop. The bi-solvent
system, acetonitrile/water of 80/20 resulted in good
resolution. The flow rate was adjusted at 3ml/min.
Detector attenuator was held constant at 4X and chart speed
of recorder was kept at 0.5 cm/min.

3) Ware... ' Immediately before
injection, the sample extract was passed through a 0.22 um
membrane filter (Millipore Corp.)with a prefilter, utilizing
a syringe and then passed through a Waters Sep-Pak C18
Cartridge for final sample clean-up.

4) Stangazg_§ugaz_figlutign§‘ For identifying the
soybean sugars, a standard mixture of sucrose (0.3%),
raffinose (0.05%) and stachyose (0.1%) was used. For
quantifying, the same standard sugar mixtures were prepared

with four concentrations of each standard sugar in the range

of 0.1-

32

0.6% for sucrose, 0.02-0.10% for raffinose and 0.03-

0.21% for stachyose.

VIII. Analysis for Phytic Acid

A modified procedure for analysis of phytic acid was

introduced here according to the method by Wheeler and

Ferrel

method

1.

1)

2)

3)

4)

5)
5)
7)

3)

(1971) for extraction and precipitation and to the

by Makower (1970) for color measurement.

E ! !' 3 i '! !'
Weigh 1.2 g finely ground (20 mesh) soybean sample

into a 125 ml Erlemeyer flask

Extract sample with 40 ml 3% TCA + 10% sodium sulfate
solution on a mechanical shaker.

Pour into a 40-50 ml round bottom polyethylene
centrifuge tube, centrifuge the suspension at
20,000xg for 15 min.

Transfer 10 ml aliquot of suspension into another
tube to which 3 ml ferric chloride solution (made to
contain 6 mg ferric chloride/ml in 3% TCA solution)
has been added (note the order is important).

Heat in boiling water for 50 min.

Cool in cold water for 30 min after heating.
Centrifuge for 15 min at 20,000xg.

Wash the precipitate twice by dispersing it in

20-25ml 3% TCA + 10% sodium sulfate solution and then

9)

10)

11)

12)

13)

14)

1)

2)

1)

2)
3)
4)
5)

6)

33

heating for 7 min, cooling for 10 min.

Repeat washing once with water.

Coagulate ferric hydroxide by adding 4 ml of 1N NaOH
to the precipitate and heating for 40 min.
Centrifuge for 15 min.

Wash precipitate with 15ml deionized water.

Disolve precipitate in 3 ml of 1N HCl with heating
for 15 min.

Transfer to 100ml volumetric flask and make to

volume with 0.1N HCl solution.

Wen
Make iron stock solution by dissolving 0.0484 g

Fe2C13’6H20 (analytical reagent grade) in 100 ml
volumetric flask. Make to volumn with 0.1N HCl

solution. This stock solution contained 100 ug Fe/ml.
Dilute to obtain 0, 5, 10, 15, 20, 25 and 30 ug Fe/ml

with 0.1 N HCl solution.

Ween;

Transfer lml aliquot of standard solution or unknown
sample solution to a 25ml Erlemeyer flask.

Add 9 ml 0.1 N HCl solution.

Add 1 ml 10% hydroxylamine HCl solution.

Add 10 ml 2M sodium acetate solution.

Add 1 ml 0.1% orthophenanthroline solution.

Mix and let stand for 5 min.

34

7) Pour into two 10 ml uniform and transparent glass
test tubes (Pyrex) for duplicate color reading at
510nm in a spectrophotometer (Spectronic 70, Bausch

and Lomb Co.)

IX. Statistical Analysis of Data

Data were analyzed using analysis of variance at 5%
level. A completely randomized design and a factorial design
were used (Bhattacharyya and Johnson, 1977).

If the F-test proved significant, the Least-Significant-
Difference (LSD) procedure was applied to determine whether
a significant difference among means of different levels

existed (Li, 1964).

RESULTS AND DISCUSSION

I. Compositional Change During Maturation

Changes in total solids, protein, oil and ash contents
during seed development of two soybean cultivars are given
in Table 8.

1) Igta1_§glig§& As seeds matured the total solids
increased significantly. This result is expected because
nutrients will accumulate as seeds grow. It was also noticed
that the rate of accumulation varied with stages of
maturity, slower at early stages and faster at later ones.
These results agree with findings of Rackis et al. (1972)
and Urbanski et al. (1980).

2) §I2Q§_2111 On a dry basis, the crude oil content in
two cultivars slightly increased with maturation. This
result agrees with the data of Yao et al. (1983).

3) Ergtginy In Beeson 80, the protein content was
constant, but in Pella, it decreased at the initial stage
and increased at the final stage. It was also found that
Pella showed a slightly lower protein content (mean of four
maturity levels: 37.9%) than that of Beeson 80 (mean of

41.7%).

35

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37

4) Ash; The ash content was approximately 5% at the
final three stages of maturation for both soybean cultivars
but Immature I beans had a higher ash content than in the
following stages. Limited data are available in the

literature concerning this subject.

II. Trypsin Inhibitory Activity

1. Qisgusgion on the modified procedure fig: 11; assay

If the modified procedure for the measure of TIA in
soybeans was compared with the method described by Kakade et
al. (1974) and with the method modified by Hamerstrand et
al. (1981), the following four main points were made.

1) Although previous methods called for preparation of
the substrate BAPNA solution daily, the degradation of the
reaction activities between the substrate and trypsin in the
solution could not be neglected even in a short time. The
'results showed that on the average, the activity of BAPNA
solution decreased at a rate of 5.5% reduction of TU/hr.
(Table 9). Therefore, it is suggested that preparation of
the substrate solution be made for each run of measurements.

2) Since TIA values (expressed as TUI/ml extract) for
all portions of diluted inhibitor solution taken for assay
are interpreted against the trypsin standard, it is
necessary to run multiple trials of the trypsin standard in

order to obtain a reliable result. In the method modified by

38

Table 9. Degradation of BAPNA solution

activity with time in terms of trypsin units

 

Time after TU reduction TU reduction per hr.

 

 

Preparation (%) 1 (%/hr)
1 hr 30 min 9.2 6.1
2 hr 50 min 14.6 5.1
4 hr 21.7 5.4
Average 5.5

Hamerstrand et a1. (1981), this point seemed neglected. In
this modified procedure, quadruplicate trypsin standard
tests were made for each run.

3) For comparison of effects of the particle size of
the soybean sample and extraction time on the final value of
TIA, the samples with mesh size of 20, 50 and 100 were used
and extracted for 1 and 2 hr. Results showed that there was
no significant difference between samples with mesh sizes of
50 and 100 as long as the extraction time was increased from
the original 1 hr to 2 hr. For immature and heat-treated ‘
soya samples, there were even no signficant differences
among samples with mesh sizes of 20, 50 and 100. Thus the

use of the samples with 50 mesh size minimized the labor for

39

preparing the samples with 100 mesh size.

4) As noticed earlier by Rackis et al. (1974), when TI
activity (TUI/ml extract) was plotted as a function of the
volume of inhibitor solution, three different curves
resulted ( Figure 5). Since curve A (negative curve) was
limited only to extracts of raw soybean samples, the
extrapolation procedure for data interpretation of the raw
sample was suggested. However, according to the observations
in this experiment with as many as 23 two-sample replicate
sets of raw or soaked soybean samples, only a few negative
curves were obtained (curve D in Table 10). Most curves
were of A or B types.

Furthermore, if the distribution histogram was plotted
for the percentage of trypsin inhibition at the lowest value
of TUI/ml extract among four portions (0.4, 0.8, 1.2 and 1.4
ml) of diluted inhibitor solution (Figure 6), it was found
that the highest frequency distribution was in the range of
40-60% trypsin inhibition, that is, at this range, the TIA
value tended to be the lowest. Therefore, although the
extrapolation procedure of data interpretation in the
original method tended to give a high value of TIA, the
determination of TIA from a single dilution of a sample
extract that inhibited trypsin in the range of 40-60% as
suggested by Hamerstrand et al. (1981) could go to the other
extreme---to give the lowest value of TIA. As a result, we
suggested that averaging the results from four levels of

inhibitor provide the most reliable estimated value of TIA.

 

7O ‘1' -.
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‘ I ' I ' I ' I ' I
0.2 Oh4 0.6 0.8 llO
Volume of Extract (m1)

Figure 5. Trypsin inhibitory activity
(TUI/ml extract) in relation to inhibitor solution

(From Rackis, 1974.)

41

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[ .
.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0
Percentage of Trypsin Inhibition

Figure 6. Distribution histogram for

1 ‘.
90431001)

percentage of trypsin inhibition at the lowest TUI/ml

value of four portions of the extract

43

(For the details refer to Appendix, Table A1).

AW

Tables 11 and 12 show the TIA in raw and processed
soybean samples of two cultivars harvested at different
maturity stages. Beeson 80 showed an increase in TIA from
59.5 to 64.2 TUI/mg with a significant difference between
Immature II and Immature III, while Pella showed a decrease
from 59.4 to 56.0% TUI/mg with a significant difference
between Immature I and Immature II. If the TIA for four
maturity stages was averaged, the two cultivars appeared to
be slightly different: 61.1 TUI/mg for Beeson 80 and 55.5
TUI/mg for Pella. .

Divergent values and change patterns in TIA during
maturation among soybean varieties were also reported by
other workers (Collins and Sander, 1976; Yao et al. 1983).
All these differences might be attributed to the different
varieties used in different study groups.

It is interesting that the change in TIA is parallel to
the change in protein content during maturation for the two
cultivars studied here. In fact if TIA is plotted against
protein content (Figure 7), a high correlation is obtained,
with r = 0.9069. This relation may be explained by the fact

that trypsin inhibitors are proteins.

Table 11. Effect of maturity and processing on

trypsin inhibitory activity in Beeson 80 soybeans a,b

 

Date of Stage of Trypsin inhibitory activity (TUI/mg)

 

Harvest Maturity Raw Soaked Steamed Cooked S-cooked

 

9/9 Immt. I 59.5 b

 

9/17 Immt. II 58,2 b 57,; b 0.0 0.0 0.0
9/26 Immt. III 62.0 a. 63.3 a 0.0 0.0 0.0
10/18 Mature 6 .3 4.0 a 13.1 9.2 0.0

 

Means in the same vertical column bearing different
subscripts differ significantly (P < 0.05).

Means with a common underline in the same horizontal
row do not differ significantly (P > 0.05).

45

Table 12. Effect of maturity and processing on

trYPSin inhibitory activity in Pella Soybeans a,b

 

Date of Stage of Trypsin inhibitory activity (TUI/mg)

 

 

Harvest Maturity Raw Soaked Steamed Cooked S-cooked
9/9 Imm. I 59.9 b
9/22 Imm. II 52.6 5 55.8 a 0.0 0.0 0.0

9/30 Imm. III 55.1 5 55,7 5 0.0 0.0 0.0
10/23 Mature 55.9 a 55.5 g 6.3 6.6 0.0

 

Means in the same vertical column bearing different
subscripts differ significantly (P < 0.05).

Means with a common underline in the same horizontal
row do not differ significantly (P > 0.05).

46

 

66

t? " '1
> 644 _
.,..|
t), "‘ ..
g A62— _

0
3‘3. .
3 s60~ _
*. a
:9. "’ ‘ '
.2 i€58~ _
H H. ‘1‘" -
.5 £3.56- -
m
Do
6'
B

 

 

 

 

52 r l T I I 1 I I I I I F
36. 37. 38. 39. 40. 41. 42. 43. 44.
Protein Content (%)

I I I

Figure 7. Correlation of trypsin inhibitory
activity with protein content in two cultivars of soybeans
harvested at four different maturity stages

47

3. E e t o c 55' on h
a ' s s

As shown in Tables 11 and 12, in both cultivars,
steaming or cooking for 20 min completely inactivated the TI
in immature soybeans. For mature beans these heat treatments
reduced their TIA to a very low level. Soaking for 24 hr
did not reduce TIA significantly, but it was necessary prior
to cooking in order to bring a complete elimination of TIA
in mature soybean samples.

Heat treatments are commonly used to inactivate the
protease inhibitors of soybeans and other legumes (Liener,
1981). As for the effect of soaking on the reduction of TIA,
striking differences were noticed among various legumes (Al-
Bakir et al., 1982). These differences may be attributed to
differences in chemical composition and seed coat texture.

In order to effect a complete inactivation of TI in
mature soybeans, soaking was necessary prior to cooking,
while for immature soybeans soaking was not necessary. This
indicates that higher moisture content renders the TI more
sensitive to heat. Results here seem to agree with data of
Collins and Beaty (1980), that heating fresh green soybeans

in boiling water destroyed their TIA rapidly.

Si

Pe
ra
im
ma‘

9t

48

III. OLIGOSACCHARIDES

1. oma _

Figure 8(A) shows the HPLC separation of three standard
sugars: sucrose, raffinose and stachyose. Figure 8(B) shows
the HPLC chromatogram of the sugars present in a 75% ethanol
extract of soybeans. Comparison of the two chromatograms
indicates that soybeans contained sucrose, raffinose and
stachyose in large amounts. Cegla and Bell (1977) obtained

the similar results.

$me

Quantitative results showed that during the maturation
of two soybean cultivars, all three sugars increased (Figure
9). Sucrose appeared early in the seed development,
followed by raffinose and stachyose. In young seeds, both
cultivars contained a considerably low amount of oligo-
saccharides, while in full mature seeds, Beeson 80 contained
7.00% sucrose, 3.18% stachyose and 0.52% raffinose and
Pella contained 7.20% sucrose, 2.71% stachyose and 0.64%
raffinose. No data on the oligosaccharide contents in
immature soybeans were found in the literature, but for
mature soybeans our results agree with findings of Hymowitz

et a1. (1972).

 

49

 

 

 

 

 

 

 

 

 

 

1 2
1
2
A. Standard Sugar B. Soybean Extract
Solution
1. solvent front
1. solvent front 2. sucrose
2. sucrose 3. raffinose
3. raffinose 4. stachyose
4. stachyose
4
3 4
3 u
w ' 8 3
['3 'fi
: c
H ~r-1
0 1 2 3 4 0 1 2 3 4
l—L-W-a—l-s-J I_A_L_.I_‘.—L——l—l—-J

 

Figure 8. HPLC chromatograms for sugar analysis:
Waters carbohydrate column; Solvent, acetonitrile/water
(80/20); Flow rate, 3.0/min; Attenuation, 4X; Chart
speed, 0.5/ cm/min.

 

50

 

 
 
 
  
  
    
 
   

 

m

4.)

c 7

3 A:EMaame

g B: Stadhyose

U 6 C: Raffinose

§ "Beeson 80"
.5 ----"Pella"

UI

U

(% dry basis)
5° . f‘ . . .
Il1l?lll?llL?lll?lll?lLL?ljlql

?‘

llllll‘lLl[IllllllllllllALlL

Sucrose, Stachyose and Raff

 

 

 

1 II II II II I! II I
9 17 22 2630 - 18 23

Skpt. Oct.

Inmamnz Manna

Date of Harvest

Figure 9. Changes of sucrose, stachyose
and raffinose contents in maturing soybeans

51

3. o r s' o su s ' ' a u 't
soybeans

Figures 10 and 11 show the sucrose contents in raw and
treated soybeans of two cultivars harvested at different
maturity. Generally, upon all kinds of processing, sucrose
content decreased by 7.9-46.6% in both cultivars except for
steaming, which appeared to increase the sucrose content by
4.5-31.8% in young seeds. The combination of soaking and
cooking caused the highest decrease in sucrose content.

The increase of sucrose content in young seeds as a
result of steaming may be due to enhancement of certain

enzymatic activities or due to some other factors unknown.

£an
W
Among the three sugars, stachyose may play the key role

in causing flatulence because of its relatively high
content. Figures 12 and 13 show the stachyose content in
raw and processed soybeans of two cultivars harvested at the
final two maturity stages: Immature III and Mature. Since
the Immature II seeds contained considerably low amounts of
stachyose no processing effect on stachyose could be
derived.

In general, under various processing treatments,
stachyose content in both cultivars harvested at the final

two maturity stages was reduced by 12.0 - 49.4%. Soaking

Sucrose Content (%dry basis)

Sucrose Content (%dry basis)

52

 

.5?

O O
O" \J
l 1 l

 

 

    
   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Iflflfil hummunalI':

_ m Immature III...

-— ._ :2 Name 1

3 L

3 ./ _, .

3 34 / —

3* ¢ -

3 «7 -:

3 3% v -

3 w 4 ~

Raw Ekmked (knked

Figure 10. Effect of processing on sucrose

in Beeson 80 soybeans of different maturity

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fl

 

 

 

 

 

 

 

 

Figure 11.

fixfied

Effect of processing on sucrose

Sasmed

 

in Pella soybeans of different maturity

 

53

 

 

 

 

 

33‘ 3 61 -
E ' q [ZZZ] Innatm-e III:
3 3'2: - :1 Made 1
be 2.8— —
E 2.4-: F T I— L
g 2.0‘: .:
oILG-d [_- _
8 .. .4
a 1.2: _:
$3; 0.8: » -

0.4- P ' ..

0.01 . - EL a -

 

 

 

 

 

 

 

 

 

Raw Soaked Steamed Cooked Soak-cooked

Figure 12. Effect of processing on stachyose
in Beeson 80 soybeans of different maturity

 

3.6 r '
d , Immture III

E: Mature

5"?!

(”N
lgil
1

1"
.p
L

2i)? 7' -1
1.5-j
1J2:
0.8"

0.4 :_Q _ . a m

0.0 ..
Raw ' fixfied . Suumnd Caflmfll Ekmkmaxked

Staohyose Content (7. dry basis)

 

 

 

llLlLJJ!L|1Lillll.l

 

 

 

 

 

 

 

 

 

 

Figure 13. Effect of processing on stachyose
in Pella soybeans of different maturity

54

soybeans for 24 hr and then cooking for 20 min in boiling
water tended to give the greatest reduction (28.7-49.4%),
while the remaining three treatments seemed to cause
approximate equal reduction in stachyose content (12.9-
34.7%). I

From Figures 12 and 13, it was also found that these
treatments could not completely eliminate the stachyose in
Immature III seeds although at this stage the original

content was very low.

S-WMW
i be s
Raffinose content of raw and processed soybeans of two
cultivars harvested at the final three maturity stages is
given in Figures 14 and 15. Like the other two sugars in
soybeans, raffinose appeared to decrease to some extent
under various treatments. Soaking-cooking caused the largest

reduction in raffinose content.

The effects of various processing treatments on
oligosaccharides in mature soybeans have been studied by
several investigators although so far no data are available
concerning immature soybeans. Bianchi and Silva (1983)
reported that soaking promoted no reduction in oligo-
saccharide contents regardless of the soaking time (3, 6,
12, 18, and 24 hr); cooking in an autoclave at 120C reduced

the oligosaccharides but soaking plus cooking resulted in

55

 

 

Raffinose Content (% dry basis)

 

 

§fl§§|lmmmunaII «

 

 

 

 

 

 

 

 

m Inmature III-
1:: Mature '
11f q
_q, _
4
uni
I"
1
j / _ , /
Raw ikmfied Saumed ‘Oxfled fixkraxked
Figure 14. Effect of processing on raffinose

in Beeson 80 soybeans of different maturity

 

'6 .
~3CL7—
I! ..
‘n()61
5' 4
L“10.54
€0.4—
0

t; 1
80.3:
0
30.24
5 a
230.14
M ..
a:

 

 

 

\\\\\\\V Imam II

 

 

 

 

 

 

 

 

 

 

'—"1 .-

E:2:Z hmmmunaIIId

E: Mam -

' u-i

"—1 __l _1 q

A J ,/ j ‘

new Soaked Steamed Cooked Soak-cooked
Figure 15.3 Effect of processing on raffinose

in Pella soybeans of different maturity

56

the greatest removal of oligosaccharides.

Kim et al. (1973) found that soaking soybeans in water
for 15 hr reduced oligosaccharide content by about 7%; Lo et
al. (1968) also found that as the soaking time for soybeans
increased, large quantities of water-soluble solids
including oligosaccharides leached into the soak water.
Recently, Savitri and Desikachar (1985) reported that there
was an apparent increase in the measurable total
oligosaccaharides in Kalatur soybeans after cooking.

These divergent results might not be suitable for
comparison with each other because of the different
varieties of soybeans and different processing conditions
used in each study.

The fact that similar reduction patterns of oligo-
saccharides upon various processing methods were found
between immature and mature soybeans in this study might
indicate that higher moisture content and softer texture of
young seeds could not provide the more favorable condition
for the reduction and elimination of their oligosaccharide
content. Nevertheless, the amount of these oligosaccharides
in young green soybeans might be too low to cause digestive

disorders in humans.

57

IV. Phytic Acid

1. £9m2aris2n_2f_twe_assax.msthgds_fer_nn2§is_agid

In method I, 3% TCA was used as an extractant and the
4:6 ratio of Fe:P was used for calculation of phytic acid
content. In method II, 3% TCA + 10% Nazso4 was used as an
extractant and the 6:6 ratio of Fe:P was introduced in
calculating the phytic acid content. The results obtained by
the two methods were not statistically different ( Table
13). However, by using the modified procedure (method II),
the sample extracts were clearer and therefore were more
amenable to decantation of the supernatant. Furthermore, the
results by the modified method were more reproducible; the
average relative difference of 9 samples was only 2.3% as
compared with that of 6.9% by method I. (For the standard
iron curve used in this colorimetric measurement refer to
Appendix, Figure A1).

Thompson and Erdman (1982) hypothesized the structure
shown in Figure 16 for the ferric phytate precipitate and
the assumption of 4:6 molar ratio of Fe:P. According to
this model, eight ferric irons interact with one phytate
molecule.

Now for the assumption of 6:6 molar ratio of Fe:P
introduced herein, it is proposed that in the presence of

excess Na+ and SO= the Neuberg-based structure dominate. As

4!
a result, additional four ferric iron could be incorporated

58

Adams

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.033 .839: mumh one no Eaugmmm no.3 ufiuomuuxo mm 8.338 <09. Nm mam: "H oofig

 

 

 

 

 

.333 as .N. ”.838 Boo 393d M
8.0.. m.~ mom; . «mmé 034 do «34 wflmé RNA owwuflé
8.0.. o.o 34 .3A 34 wé SA :4 84 a
36- m6 mmé cm; .82" 5m «NH 34 84 w
mod- m.~ oné wmé 3A wfi , BNA «m4 «Na 5
2.9. Wu $4 $4 , mmA md on; 3.." 3.." o
3.? c3» omé mmé mmA o.m umé $4 8.." m
8.9. n.~ on; mmé 3A od .34 RA 9.." a
86- ad on; mmé 84 «d NNA RNA SA m
No.0- md 84 omA mm; ed and 3..“ wmé N
oo.o H.N .36 cad wad ~.m 3.0 84 mad H
HH can H at ofiwwwwwo mwmugm 5.» menu 5H oma ARV mowwwwwwm owwuopm 5H pom m5.“ um.” moan—Hm
moocuos
c832. HH oozed: H 85oz mo .2
oucououudo

 

msmonhom CH usoucoo pace Owuann you
moonuoa human 03» no souwuomaou .na manna

59

 

 

/’ P °\\~‘ o o-——Fb
o/ \o I "So/é \
F o——ji——O ‘1:::?~0>//
._ -
I .7 Pkg-“\P/o—F'
F0 0 0 o
| \\Fb”

 

 

 

Figure 16. Proposed structure for ferric phytate (4:6 Fe:P)
(From Thompson and Erdman, 1982)

 

l
\ / )e
o/ \ o H
\ f>a\ a \ .'o—-p"
Ire—o o—— \g \o
O—l-‘e
‘\\\ t \\\\ o-li/l
I o I o o.
/0\\/‘°\l|, \Fe/
—F.\o/ \ / ‘0
o O \) \Fe I
L \ /' I \
1" Fe
/ \ |

 

 

 

Pigure:r7. Proposed structure for ferric phytate (6:6 Fe:P)

60

into the precipitate (Figure 17).

2. C e o t'c ' t'

Data about the effect of maturity and processing on
phytate content in both soybean cultivars are given in
Tables 14 and 15. The results indicated that from Immature I
to Mature stage, phytic acid content increased from 0.84-
1.36% for Beeson 80 and from 0.86-1.39% for Pella. These
results agree with findings of Yao et al. (1983); they
reported that the phytate content in Williams soybeans
increased from 0.866 to 1.26% during the final three harvest
stages (Sept. 20, 26 and Oct. 3).

Welch and House (1982) studied the effect of maturity of
soybeans on availability to rats of zinc from the seeds.
They found that phytic acid concentration in seeds of
several species increased with seed maturation and zinc
availability to rats decreased, as determined by the
absorption of radiozinc.

As for the availability of iron, a study by Welch et a1.
(1975) showed that iron from the mature seeds was more
available than that from immature seeds even through the
mature seeds contained approximately three times as much
phytate. So immature soybeans might contain a factor that
depressed iron availability.

Therefore, mineral availability in beans may depend not
only on stage of maturity and their content of phytic acid,

but also on the individual mineral and other factors.

61

Table 14. Effect of maturity and processing

on phytic acid content in Beeson 80 soybeans a,b

 

Date of Stage of Phytic acid content (%)

Harvest maturity Raw Soaked Steamed Cooked S-cooked

 

9/9 Imm. I 0.84 c

 

 

9/17 Imm. II 0.21 6 0,22 c 0.88 c 0.89 c 1.28 c

9/26 Imm. III 1,16 b 1,17 b 1,12 2 1,15 9 1.28 b

10/18 Mature 1,36 6 1,37 6 1,62 6 1.16 a 1.50 a,
a.

Means in the same vertical column bearing different
subscripts differ significantly (P < 0.05).

Means with a common underline in the horizonal row
do not differ significantly (P > 0.05).

62

Table 15. Effect of maturity and processing

 

 

on phytic acid content in Pella soybeans a,b
Date of Stage of Phytic acid content (%)
harvest maturity Raw Soaked Steamed Cooked S—cooked
9/9 Imm. I 0.86 d

9/22 Imm. II 0.97 C 0.98 C 0.98 $110.97 C 1.04 C

 

9/30 Imm. III 1.16 b 1.16 2 1,17 2 1,16 9 1.29 b
10/23 Mature 1,62 6 1,51 6 1,35 6 1,16 6 1.57 a
a.

Means in the same vertical column bearing different
subscripts differ significantly (P < 0.05).

Means with a common underline in the horiZonal row
do not differ significantly (P > 0.05).

63

3. Effect of processing on phytig acid in different
ma u ' so b

As shown in Tables 14 and 15, changes in phytic acid
content upon processing differed with individual treatment.
In general, soaking for 24 hours had little or no effect on
phytic acid content, while cooking or steaming in boiling
water for 20 min had a slight decreasing effect. All these
changes were not significant at 5% level. However, soaking
plus cooking appeared to increase phytic acid content
significantly.

Several reports regarding effects of various processing
treatments on phytate contents in soybeans have appeared in
the literature. Sudarmadji and Markakis (1977) reported that
overnight soaking did not reduce the phytic acid content of
soybeans while boiling for 30 min reduced it by 14%.
Ologhobo and Fetuga (1984) stated that apart from
germination, Soaking soybeans for 3 days, or autoclaving at
105C for 15 min. were not very effective in lowering their
phytate content. It seems that our results agree with these
findings.

The fact that a significant increase in phytic acid
content was observed upon soaking-cooking might indicate
that this treatment resulted in higher extractability of
phytic acid from bound forms of the latter.

The statistical analysis of data showed that no

interactive effect of processing and maturity on changes in

64

phytic acid content existed. This might indicate that the
higher moisture content in immature soybeans could not
possibly facilitate in lowering their phytic acid contents

upon various methods of processing.

CONCLUSIONS

Studies with two soybean cultivars, Beeson 80 and Pella
showed that, on a dry basis, immature soybeans contained
similar amounts of protein, oil and ash, but considerably
lower amounts of oligosaccharides (sucrose, raffinose and
stachyose) and phytic acid than mature soybeans. The
trypsin inhibitory activity (TIA) of immature Beeson 80
soybeans was slightly lower than that of mature ones. The
opposite was true for the Pella cultivar.

The effects of a) soaking (24 hr), b) cooking (20 min),
c) steaming (20 min), and d) soaking (24 hr) plus cooking
(20 min) on the antinutritional factors of soybeans were as
follows: Cooking or steaming completely inactivated the
trypsin inhibitor of immature soybeans, but only partly in
mature soybeans. For the latter, both soaking and cooking
was necessary for complete inhibition. Regarding oligo-
saccharides and phytic acid, the aforementioned treatments
had similar effects on both immature and mature soybeans.
Soaking, cooking, or steaming alone significantly reduced
the oligosaccharide content, but had little or no effect on
phytic acid content. Soaking plus cooking resulted in even

greater reduction in oligosaccharides, but in a slight

65

66

increase in phytic acid content. Nevertheless, because
immature soybeans contain much lower amounts of these
antinutritional factors as compared with mature ones, the
traditional Chinese cooking of immature soybeans for direct
consumption is sufficient to avoid the detrimental effects

of these antinutrients.

APPENDIX

Table A1. Colorimetric assay for trypsin inhibitory activity

APPENDIX

 

 

a

 

. . . f
sample ifizginsigfi A410 Tub TUI/levequUI/lldaverage :2::P1;
0.0 0.824 82.4
0.4 0.602 60.2 22.2 55.5 26.9
A 0.8 0.426 42.6 39.8 49.8 48.3
1.2 0.096 9.6 72.8 60.6 88.4
1.4 0.009 0.9 81.5 58.2 98.9
56.0
0.0 0.824 82.4
0.4 0.593 59.3 23.1 57.8 28.0
B 0.8 0.383 38.3 44.1 55.1 53.5
1.2 0.056 5.6 76.8 64.0 93.2
1.4 0.008 0.8 81.6 58.3 99.0
58.8
a. W 0.8 m1

of this diluted raw soybean suspension inhibited trypsin
in the range of 40-60%.

One trypsin unit (TU) is arbitrarily defined as an
increase of 0.01 absorbance unit at 410 nm per 10 ml of
the reaction mixture (2ml water and/or soy suspension;
2m1 trypsion solution; 5 ml BAPNA solution and 1 ml
acetic acid solution) under condition herein.

Trypsin units inhibited per level of suspension. Example of
0.4 ml of sample A: 82.4-60.2 = 22.2 TUI/0.4nl

Trypsin units inhibited per 1 ml of suspension,.eg.
22.2 TUI/O.4ll = 55.5 TUI/ 1 :1.

Percentage of trypsin inhibited,'eg. for 0.4ml level of

M
sample A: 0 0 82‘ x 100 = 26.9%.

67

APPENDIX

 

. Fecug) = 95.6564 xASlO - 0.005 .
r =- 0.99986 . ..

Fe Content (ug)

 

 

 

 

0, ' r ' r ' I _
0.000 0.100 0.200 0.300 0.400
Absorbance at A510nm

Figure Al- Standard curve for Fe measurement

68

BI BLIOGRAGHY

BIBLIOGRAPHY

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Occurrence and stability of trypsin inhibitors in Iraqi
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70

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