Chemistry and Technology of Soybeans

CHAPTER 6
CHEMISTRY AND TECHNOLOGY

OF SOYBEANS
W. J. WOLF
Northern Regional Research Laboratory, Agricultural Research Service
U.S. Department of Agriculture!, Peoria, Illinois
I. INTRODUCTION
In the United States soybeans have emerged from relative obscurity as an
oilseed to one of our major cash crops in less than 50 years. Official U.S.
Department of Agriculture estimates of soybean production date back only to
1924 when harvested production was 5 million bushel. Today this quantity would
be enough for only a few days' operation of the soybean processing industry and
is equal to only about 1% of the soybeans that were exported in 1974.
Commercial interest in soybeans initially was concerned with processing into oil
for edible and industrial purposes. The resulting meal was considered a by-
product used for cattle feed and as a fertilizer. In time it was learned that the
defatted meal is a valuable protein source for poultry and swine as well as for
cattle. Today, the major portion of the defatted meal is still used for feeds.
Food uses for soybean protein in the United States have developed more
slowly than markets for the oil. For example, in 1973 domestic consumption of
edible soybean oil was 6.8 billion Ib which is equivalent to about 635 million
bushels of soybeans or 41 % of the crop grown that year. In contrast, less than 2%
of the crop (primarily from defatted flakes as the starting material) was converted
into edible protein products for domestic consumption. The bulk of soybean
proteins is converted into animal proteins which are still preferred over plant
proteins. However, conversion of soybean proteins into meat, eggs, and milk is
inefficient; as a result, animal proteins are more expensive than those of
soybeans. Worldwide food shortages in recent years have caused sharp price rises
in animal proteins used in the food industry, and at times supplies have been
uncertain. This situation and anticipated future trends have prompted a number
of food companies to begin replacing animal proteins in their products and to
lMention of firm names or trade products does not imply that they are endorsed or recommended by the U.S.
Department of Agriculture over other firms or similar products not mentioned.
325
326 I Advances in Cereal Science and Technology
develop new items based on soy and other plant proteins.
Interest in soybeans for food uses has stimulated research and development in
many laboratories and an increase in published information on their
composition and properties. My review summarizes much of this information
published in the last 5 years. In addition, I have included a brief review of
soybean production and disposition of the crop to acquaint the reader with the
magnitude of the soybean supply available for food use and how it is utilized
today. For earlier work, particularly on soybean proteins, the comprehensive
review of Smith and Circle (1972) should be consulted. A complementary
monograph covering agronomic aspects of soybeans-genetics, breeding,
varietal development, management practices, pests, and diseases-is also
available (Caldwell, 1973). Recent summaries of food uses of soybeans have
likewise appeared (American Soybean Association, 1974; Wilding, 1975; Wolf
and Cowan, 1975).
II, SOYBEAN PRODUCTION
A. U.S. Production
Successful development of markets for soybean oil for edible purposes and
1600
1400
1200

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400
200
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1940 1950 1960 1970
Year
Figure J. Soybean production in the U.S, for 1940-74. From Agricultural Statistics (1972) and
American Soybean Association (1975),
Chemistry and Technology of Soybeans / 327
meal for animal feeds over the past half century has led to a phenomenal growth
of the soybean crop in the United States. Starting from a crop ofabout 5 million
bushels in 1925, production increased to 78 million bushels in 1940. Figure 1
shows how soybean production has grown since 1940.
The largest crop on record, 1,567 million bushels, was harvested in 1973 as a
result of an unprecedented 296 million bushel increase over the previous year.
This expansion is outstanding when one considers that it is greater than the
average for crops harvested in 1950-53. The increase in 1973, however, was
followed by an even greater decrease in 1974, but this was largely the result of
adverse weather-wet spring, dry summer, and early frost. Harvested acreage in
1974 was only 6% below that of 1973, but yields per acre were below normal.
Several factors are responsible for the dramatic increases in crop size since the
early 1950's:
1. Economic development in many parts of the world with accompanying
affluence has caused a shift in diets from plant products such as cereals to more
animal products, particularly poultry meat.
2. After World War II, surplus production of wheat, feed grains, and cotton
led to acreage restrictions for these crops. Consequently, much of this land
became available for growing soybeans.
3. Favorable conditions for world trade have resulted in the development of a
large export market for U.S. soybeans. Although some of the exported beans are
used directly in foods, as in Japan, most of them are processed into edible oil and
meal for feeding livestock and poultry.
The majority of U.S. soybeans are grown in the Corn Belt. Illinois, the number
one producer, is followed closely by Iowa (Table I). These two states grew one-
third of the total crop in 1974. Sizable quantities of soybeans are also produced in
the South where cotton acreage has declined since World War II.
Production of soybeans in the U.S. as compared to the major cereal grains is
given in Table II. Soybeans are the third largest grain crop; only corn and wheat
are grown in larger quantities. Nonetheless, soybeans outproduced the cereals on
a protein basis in 1973 (Table II), and the resulting protein was of greater
nutritional value because of a better amino acid balance, especially with regard to
lysine.
B. World Production
The U.S. grows about three-fourths of the world soybean crop (Table III). The
1974 soybean crop in the U.S. was grown on 52.5 million acres but gave a
subnormal yield of 23.7 bushels/acre because of unfavorable weather. Brazil has
shown substantial increases in production over the last two years. Argentina
likewise has greatly expanded its crop but is still far behind Brazil because of the
smaller number of acres planted. Yields per acre are highest in the U.S., Canada,
Brazil, and Mexico, whereas some of the lowest yields are reported for the Asian
countries where soybeans were first domesticated.
C. Future Production Trends
Steepness of the curve in Figure 1 indicates that size of the U.S. soybean crop
328 / Advances in Cereal Science and Technology
will continue to expand in the future, although it is uncertain whether rate of
growth can continue as noted over the past decade. Early in 1973 Kromer made
projections for the U.S. soybean crop for the 1980's (Table IV). Based on an
annual increase in production of 65 million bushels or about 4% per year, he
predicted respective crops of 1.8 billion bushels and 2.2 billion bushels for 1980
and 1985. These projections were made prior to the record soybean prices
reached in June 1973 and before the energy crisis oflate 1973. The analysis also
assumed a continued growth in domestic and foreign demand for food fats and
high-protein meals for animal feeds. High prices for soybeans since mid-1973 and
the slowdown in economic activity, both domestically and abroad, have
decreased the demand for soybeans. Nonetheless, harvested acreage in 1973 (56.4
million acres) already equaled the acreage projection for 1980. Failure to reach
production estimates for 1980 and 1985 will likely be caused by yields below the
estimated levels (Table IV) rather than reductions in harvested acreage.
TABLE I
Major soybean-producing slates in 1974'
State
Illinois
Iowa
Missouri
Indiana
Minnesota
Arkansas
Ohio
Mississippi
Louisiana
North Carolina
Acreage Harvested
1,000 acres
8.500
7,070
4,350
3.910
4,040
4.300
3.200
2,550
1.760
1,450
Production
million bu
213
198
104
98
89
86
77
49
42
35
'Source: American Soybean Association (1975).
TABLE II
U.S. produclion of soybeans and cereals for 1973'
Production
Crop
Seed
million bu
Protein
million tons
Soybeans I,567
Corn 5,643
Wheat 1,711
Sorghum 937
Oats 664
ill
Rice 206
Rye 26
'Seed production data from Agricultural Statistics (1974).
18.8
15.8
7.3
3.3
1.3
1.3
0.4
0.1
r: ... "
TABLE III
Acreage, yield per acre, and production o.fsoybeans
in major producing countries/or 1972-74"
Acreage Yield Per Acre
Production
1972 1973 1974 1972 1973 1974 1972 1973 1974
Country
1,000 acres
bu 1,000 bu
United States 45,755 56,416 52,510 28.0 27.8 23.7 1,282,935 1,566,531 1,243,921
Brazil 5,770 7,524 10,425 23.3 24.4 24.7 134,702 183,719 257,206
People's Republic of China 20,756 19,800 ... 11.2 12.4 ... 231,485 246,183 248,020
Indonesia 1,693 1,726 ... 11.2 11.3 ... 18,923 19,437 20,209
Argentina 168 395 838 17.1 25.3 20.8 2,866 9,994 17,453
Mexico
593 756 605 23.2 24.8 23.1 13,779 18,739 13,963
South Korea
702 771 946 11.7 11.7 12.5 8,231 9,039 11,868
Canada 405 474 450 34.0 30.8 24.5 13,779 14,587 11,023
Estimated world total
1,755,394 2,127,570 1,891,676
"Source: American Soybean Association (1975).
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TABLE IV
U.S. acreage, yield. and production of soybeans projectedfor 1980 and 1985'
Item Unit 1980
1985
Acreage harvested Million acres 56
62
YieId per acre Bu 32
35
Production Million bu 1.800
2,150
'Source: Kromer (1973).
TABLE V
Disposition of soybeans in 1973 and 1974 and projections for 1980 and 1985'
1973 1974 1980 1985
Item million bu
Crushings 821 725 960 !.l00
Exports 539 465 750 950
Seed 58

67 71
79
Other 18 23 29
Total disposition 1,436 1.269 1.800 2,150
'Source: Fats and Oil Situation (1975) and Kromer (1973).
TABLE VI
Domestic use of soybean oil in 1973'
Use
Food
Shortening
Margarine
Cooking and salad oils
Other
Total
Nonfood
Paint and varnish
Resins and plastics
Other drying oil products
Other
Foots and losses
Total
Total domestic use
'Source: Fats and Oil Situation (1974).
Amount
million Ib
2,185
L5I4
3,070
12
6.781
91
77
5
43
277
493
7,274
Soybean production in Brazil is also expected to keep growing and will
probably continue to do so at a more rapid rate than anticipated in Kromer's
projections. Brazilian soybeans compete strongly with U.S. soybeans in the
export markets.
Other commodities that compete with soybeans to provide oil and protein are:
Philippine copra, Malaysian palm oil, African peanuts, Russian sunflowers, and
Peruvian anchovies.
III. DISPOSITION OF THE CROP
Table V shows how the 1973 and 1974 soybean crops were disposed of, plus
projections for 1980 and 1985 by Kromer (1973). About 57% of the soybeans
were processed into oil and meal in the U.S., and it is expected that this use ofthe
crop will closely approach 50% by 1985. A high proportion (37-38%) of the crop
was exported in 1973 and 1974 as beans; oil and meal were also sold abroad. In
the 1973-74 marketing year 5.533 million tons of meal (equivalent to 233 million
bushels of beans or 15% of the crop) and 1.435 billion Ib of oil (equivalent to 134
bushels of beans or 9% of the crop) were exported. Thus, about one-half of the
soybeans were exported either as beans or processed products.
Domestic use of soybean oil is primarily as a food. In 1973 (Table VI) edible
products accounted for disappearance of 6.781 billion Ib of oil or 93% of the
total. The remainder went into industrial products (3%) and foots plus losses
(2%).
In contrast to the oil, most of the defatted meal is fed to animals rather than
converted into foods. Direct food use of protein in the meal is a small but growing
segment of soybean utilization. Estimates for soybean proteins produced for use
as food ingredients in 1974 are given in Table VII. Flours and grits are the major
soybean protein products added to foods. In terms of soybeans, the total
TABLE VII
Estimates of soybean proteins produced
as food ingrediellls in 1974 and projections for 1985'
Protein Product million Ib
1974
million bub million Ib
1985
million bub
Totals 1130 26.5
Flours and grits
Concentrates
Isolates
Textured products
Flours and grits
Isolates
900
70
60
90
10
19.2
2.3
4.3
0.7
2000 43.9
500-600 16.6-19.9
400-500 28.6-35.8
400-500
3300-3600 89.1-99.6
'Source: Johnson (1975).
bExpressed as equivalents of bushels of soybeans.
'Included in figure for flours and grits in first line.
consumption was equivalent to 26.5 million bushels or 2.1 % of the 1974 crop
(Table III). Projections for 1985 are also given in Table VII. Increases of about
three- to fourfold are expected; but in equivalents of soybeans, usage is projected
to be less than 5% of the crop predicted for 1985 (Table IV). Clearly, diversion of
soybean proteins from animal feeds to human foods is still on a very small scale.
Nonetheless, such diversion will gradually increase as animal proteins continue
to rise in price and as soy protein products are improved in flavor, functionality,
and nutritive properties.
IV. SEED ULTRASTRUCTURE
Early studies of soybean ultrastructure by transmission electron microscopy
(Saio and Watanabe, 1968; Tombs, 1967) have been confirmed and extended bv
scanning electron microscopy. .
A. Protein Bodies and Spherosomes
When a soybean cotyledon is fractured by freezing in liquid nitrogen and then
examined in a scanning electron microscope, one observes that much of the
fracture surface is covered with a spongy layer of spherosomes and cytoplasmic
network (Figures 2A and B). If the fracture surface is first washed with hexane,
the oil in the spherosomes dissolves and is removed, thereby leaving only the
honeycomb-like cytoplasmic network (Figures 2C and D).
The spherosomes of soybeans have not been isolated and characterized yet.
Consequently, their structure, composition, and stability under various
processing conditions are still unknown. Techniques such as were used to study
spherosomes from peanuts (Jacks et al., 1967) would appear appropriate.
Protein bodies isolated by sucrose density gradient centrifugation (Tombs,
1967) appear to be modified in the aqueous medium used (Wolf and Baker,
1972). When examined in the scanning electron microscope, the isolated protein
body fraction contained amorphous material plus spherical particles 1-3 }lm in
diameter. Although the starting defatted flour contained numerous protein
bodies larger than 1-3}lm in diameter, none of the big particles were observed in
the isolated fraction. Presumably the large protein bodies disrupted and formed
the amorphous material found with the small protein bodies.
B. Location of Cellular Constituents
Little is known about the cellular location of various enzymes such as
lipoxygenase and urease, the oligosaccharides and minor constituents found in
soybeans-sterols, isoflavones, and saponins. Tombs (1967) found that trypsin
inhibitor did not sediment with the protein body fraction; hence, it is probably
located in the cytoplasm. However, the possibility of leaching out of soluble
constituents such as trypsin inhibitor from the protein bodies during density
gradient centrifugation cannot be ruled out in the light of the instability of the
large protein bodies discussed earlier.
Nonaqueous separation methods would be desirable for isolation of cellular
components to minimize migration of water-soluble constituents. Recent use of
fluorocarbon-hexane mixtures to fractionate ball-milled soybeans (Finley et al.,
1974) may be pertinent in this regard. Finley and coworkers centrifuged a
dispersion of full-fat soy flour in a 9: I mixture of fluorotrichloromethane:hex-
ane containing a trace of acetic acid; the solvent had a density of 1.424. After
centrifuging, three fractions were obtained: a floating layer, a supernatant, and a
pellet layer. The floating layer contained 82% protein which equals the protein
content of crude protein bodies obtained by sucrose density gradient
Figure 2. Scanning electron micrographs of soybean cotyledon fracture surfaces showing: (A) protein
body covered with spherosomes and cytoplasmic network, 5000X; (B) same as A, IO,OOOX; (C)
protein body in fracture surface after washing with hexane, 5000X; (D) same as C, IO,OOOX.
Structures labeled are protein body (PB), spherosomes (S), and cytoplasmic network (CN). From
Wolf and Baker (1975).
20 Mean 7.4 4.1 0.2 3.1 Hymowitz et al. (1972a)
Range of mean 5.6-9.9 2.5-6.5 0.1-0.6 1.9-5.1
Range of mean 5.9-10.8 3.5-7.6 0.1-0.9 1.9-3.5
Range of mean 6.2-10.9 3.8-8.2 0.1-0.9 1.4-4.2
18 Mean 9.4 6.0 0.8 2.7 Hymowitz et al. (1972b)
Range of mean 8.3-10.1 5.1-6.8 0.5-1.0 2.2-3.1
55 Mean 6.9 3.5 0.4 1.2 De Man et al. (1975)
Range of mean 1.6-9.8 0-5.8 0-0.8 0.2-2.5
TABLE VIII
Ol(e;osaccharide cOlllellt ~ dillerelll soybeall varieties
Strains
Maturity groups
00 and 0
Maturity groups
I and II
Maturity groups
III and IV
Maturity groups
OO-IV
Varieties from
southern Ontario
No. Lines
Analyzed
Total
Sugar Sucrose Raffinosc Stachyose
g/IOO g seed Reference
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centrifugation (Tombs, 1967). The supernatant contained the oil, whereas the
pellet layer appeared to be primarily carbohydrate and contained only 1.4%
protein. By careful adjustment of density, it may be possible to separate the
protein bodies in a relatively pure form by this method.
V. SEED CONSTITUENTS AND THEIR PROPERTIES
Studies on soybean composition and characterization of constituents have
continued in the last 5 years and have ranged from relatively simple molecules
such as the oligosaccharides to the highly complex polysaccharides and proteins.
A. Carbohydrates
Oligosaccharides. Implication of raffinose and stachyose as causes of
flatulence when soybeans or soybean flour are eaten (Cristofaro et al., 1974;
Rackis, 1974) has prompted surveys in search of varieties that are low in these
sugars or completely free of them. Hymowitz et al. (1972a) analyzed 60 selected
soybean lines from Maturity Groups 00 through IV (Table VIII). None of the
lines analyzed were free of raffinose or stachyose. In a companion study,
Hymowitz and coworkers (I 972b) examined three soybean strains from each of
Maturity Groups 00 to IV grown in different geographical areas (Table VIII).
Again, all samples contained raffinose and stachyose.
More recently, DeMan et al. (1975) analyzed 55 samples representative of
varieties grown in southern Ontario. They soaked the soybeans in water for
several hours and then prepared soy milks from them. Analysis of ultrafiltrates of
the milks for sugars gave results summarized in Table VIII. They observed five
samples that contained no sucrose and one sample that was free of raffinose.
Some loss of sucrose and raffinose may have occurred as a result of enzymatic
hydrolysis during the soaking prior to conversion to soy milk, but it seems
unlikely that these sugars would have disappeared completely in a few hours.
Oligosaccharides are still detectable in soybeans after 48 hr of germination
(Abrahamsen and Sudia, 1966; East et al., 1972). The work of DeMan and
coworkers, if confirmed, offers some hope that soybean varieties low in
oligosaccharides may be developed.
At present, oligosaccharides are removed from commercial soybean protein
products by extraction methods as in the preparation of concentrates and
isolates. There is industrial interest in the use of a-galactosidase preparations to
hydrolyze raffinose and stachyose, and their application to soybean preparations
has been described (Sherba, 1972). A thermostable a-galactosidase was isolated
from Bacillus stearothermophilus (Delente et al., 1974), immobilized on nylon
microfibrils, and used in a flow-through continuous reactor to hydrolyze
raffinose in beet sugar molasses (Reynolds, 1974). Release of reducing sugars,
however, may cause browning reactions and problems with palatability
(Cristofaro et al., 1974).
PoZvsaccharides. Kikuchi et al. (197 Ia) isolated the cell wall polysaccharides
from defatted soybeans and on hydrolysis found galacturonic acid, galactose,
glucose, arabinose, xylose, and rhamnose as the sugar constituents.
Fractionation of the cell wall polysaccharides indicated that they consisted of
approximately 30% pectic substances, 50% hemicelluloses, and 20% celluloses.
Cooking for 30 min at 120
0
C apparently converted the pectic materials, which
cement together the cell walls, from an insoluble to a soluble form therebv
causing separation of cells from each other. ' -
In a related study, the three polysaccharide fractions were treated with a crude
enzyme preparation obtained from Aspergillus sojae, one of the organisms used
in making soy sauce by fermentation (Kikuchi et al., 1971b). The hemicellulose
fraction was easily hydrolyzed; the pectic fraction was hydrolyzed to a low degree
whereas the cellulose was not attacked. Polysaccharides found in sov sauce
prepared by fermentation apparently are derived from the cell wall p ~ t i n s
Further studies of the effect of cooking soybeans on the polysaccharide
fraction revealed that a hot water extract of cooked beans contains an
arabinogalactan and two acidic polysaccharides (Kikuchi, 1972). One acidic
polysaccharide contained 29% anhydrogalacturonic acid and is thought to be the
main component of the cell wall matrix. The other acidic polysaccharide with a
71% anhydrogalacturonic acid content was assumed to be derived from the
middle lamella between cells.
Claims of a causal relationship between a lack of fiber in the human diet and a
number of diseases, especially those of the bowel, have focused attention on the
fiber fraction in plant foodstuffs (Burkitt et al., 1974; Eastwood, 1974). Soybean
polysaccharides perhaps could serve as a source of dietary fiber in processed
foods. They are already present in flours and grits, as well as in concentrates, and
are obtained as a by-product remaining after aqueous extraction of defatted
flakes in the preparation of protein isolates. At present this by-product is
disposed of by adding it to animal feeds.
B. Lipids
Changes during Development of Soybeans. Complex changes occur in the
composition of fatty acids and lipid classes in developing soybeans from 9 days
after flowering until maturity (Privett et al., 1973; Wilson and Rinne, 1974). The
immature bean is almost free oftriglycerides, and the major lipids are glycolipids
and phospholipids. The lipid extracts from the developing bean also contain
appreciable amounts of unidentified materials which decrease in amount as the
bean matures. N-Acylphosphatidylethanolamine may be one of these
unidentified compounds; it is the major phospholipid of immature soybeans, but
decreases rapidly to a low level at maturity (Wilson and Rinne, 1974). During
development of the bean, there is a rapid synthesis of triglycerides accompanied
by a drop in the percentage of saturated fatty acids and a rise in content of oleic
and linoleic acids. The percentage of linolenic acid in the lipids is high initially
and decreases as the bean matures. An earlier study by Roehm and Privett (1970)
showed that the triglycerides from immature beans contained almost 5%
trilinolenin, but this molecular species disappears completely by 40 days after
flowering.
Composition and Fractionation of Commercial Lecithin. Two commercial
soybean lecithins were fractionated by thin-layer chromatography and liquid
chromatography (Erdahl et al., 1973). The lecithins contained about 82%
phospholipids consisting primarily of phosphatidylcholine, phosphatidylethan-
olamine, and phosphatidylinositol. The remainder comprised virtually all of the
lipid classes found in soybean oil. About 2 dozen components were found by
thin-layer chromatography of the polar lipids, and unknown compounds made
up about 10% of one of the lecithin samples. This study illustrates the complexity
of commercial soybean lecithin that is widely used in a variety of foods.
The phosphatide constituents of lecithin have different physical properties;
hence, it is desirable to fractionate for certain applications. Phosphatidyl-
ethanolamine can be separated from lecithin by conversion to N-acylphospha-
tidylethanolamine followed by extraction into acetone (Aneja et al., 1971). The
neutral lipids are also soluble in acetone; hence, the residual lecithin fraction
consists mainly of phosphatidylcholine and phosphatidylinositol which do not
dissolve in acetone.
C. Proteins
Studies on Unjractionated Proteins. Hill and Breidenbach (1974a)
fractionated the buffer (pH 7.6, 0.5 ionic strength) soluble proteins of soybeans
by sucrose density gradient centrifugation and obtained separations that agreed
well with those reported by earlier workers using the analytical ultracentrifuge.
The density gradient method, however, has the advantage that the fractions are
separated from each other and can be recovered for further characterization. Hill
and Breidenbach analyzed their protein fractions by polyacrylamide gel
electrophoresis and found one band for the II S fraction but three bands for the
7S fraction. They made a surprising observation; when the sucrose density
gradient centrifugation was conducted at 0.1 ionic strength, the 9S fraction
(believed to be dimer of a portion of the 7S species at 0.5 ionic strength) still
consisted of three gel electrophoretic components. Apparently, there are three
electrophoretically distinct proteins capable of dimerizing at 0.1 ionic strength.
Hill and Breidenbach (1974b) also followed accumulation of the major proteins
during seed development and maturation. The 2S fraction predominated early in
seed development; but by 23 days after flowering, the density gradient centrifuge
pattern closely resembled that of the mature seed.
Comparison of soybean globulins, obtained by isoelectric precipitation, with
the proteins found in the protein bodies revealed no significant differences as
measured by gel filtration, ultracentrifugation, and isoelectric focusing
(Koshiyama, I 972a).
Extraction of soybean proteins from defatted meal at pH 4.5 is possible if salts
are added to solubilize the globulins (Anderson et al., 1973). Protein
extractability increases as the salt concentration is raised until a maximum of
65% of the meal nitrogen is solubilized. Maximum extraction occurs with 0.3N
calcium chloride or 0.7N sodium chloride. Proteins in the 2S and 7S fractions
appear to be insolubilized by the pH 4.5 treatment, and they are not solubilized
by salts.
In a study of solubility of isolated globulins, van Megen (1974) also observed
that at pH 4.5 the proteins dissolved at salt concentrations above 0.7N sodium
chloride. However, below this salt concentration a two-phase system formed,
consisting of a protein-poor upper layer and a viscous protein-rich lower layer.
It is well-known that moist heat readily insolubilizes soybean meal proteins.
However, Wang (1975) has recently found that if autoclaved flakes are
ultrasonically treated during extraction, proteins that presumably are denatured
can be redissolved. Moreover, the redissolved proteins appeared much like the
native proteins in their sedimentation behavior in the ultracentrifuge. It is not
clear whether denaturation was reversed or if the proteins in the protein bodies
are comparatively stable to autoclaving but do not dissolve because of a barrier
of denatured cytoplasmic or membrane proteins on the outside of the protein
bodies. Further work is needed on this problem.
Trypsin Inhibitors. Availability of soybean trypsin inhibitors in pure form and
their unique biological activity have resulted in intensive study of these proteins
in several laboratories. One of the most notable achievements has been in the
laboratories at Niigata University in Japan where Odani and Ikenaka (1973)
have determined the complete amino acid sequence for the Bowman-Birk
inhibitor (Figure 3), and Koide and Ikenaka (1973) have elucidated the complete
sequence for the Kunitz inhibitor (Figure 4).
The Bowman-Birk inhibitor consists of 71 amino acid residues with a site for
interaction with trypsin at Lys 16-Ser
l
i and a reactive center for interaction with
chymotrypsin at Leu
43
_Ser
44
. This inhibitor is remarkably stable to heat, acid,
and proteolytic digestion because of the seven disulfide cross-links that give the
molecule a rigid structure. The molecule is unique in its structure around the
proteinase inhibitory sites. The two sites are almost identical. Each site is located
in a nine-membered peptide loop formed by a single disulfide bridge. This loop is
followed by another nine-membered loop and then by a ten-membered ring (site
of trypsin inhibition) or eight-membered ring (site of chymotrypsin inhibition).
The 181 amino acid residues found in the Kunitz trypsin inhibitor give it a
more complex structure than that of the Bowman-Birk inhibitor. Cross-linking
30
Figure 3. Primary structure of Bowman-Birk soybean trypsin inhibitor according to Odani and
Ikenaka (1973). Residues are numbered beginning \vith N-terminal aspartic acid. Disulfide cross-
linkages are shown in black between half-cystine residues. Residues at the two reactive sites are
shown in bold-faced type and have asterisks adjacent to them. Reprinted with permission from
Springer-Verlag.
in the Kunitz inhibitor is comparatively simple, because there are only two
disulfide bridges located at residues 39-86 and 136-145. The reactive center is
located at the Arg
63
_Ile
64
bond.
Another notable development in studies on trypsin inhibitors is the
determination of the crystal structure of the complex formed by Kunitz trypsin
inhibitor and porcine trypsin by X-ray crystallography (Sweet et al., 1974).
Figure 5shows a model ofthe complex made from an electron density map at 5-A
resolution. The inhibitor is nearly spherical in shape and has a diameter of about
35 A. A remarkable feature of the complex is that only about 12 of the 181
residues of the inhibitor make contact with the trypsin molecule to form an
extremely stable complex. It is estimated that within this small region there are
over 300 interatomic contacts (pairs of atoms within 0.5 Aof the theoretical van
der Waals' contact distance) of which about 18 may be hydrogen bonds. It is
believed that the binding energy derives from the sum of small energy terms from
many interactions rather than any new or unforeseen type of interaction.
Agglutinin. Recent reviews of agglutinins, including that of soybeans, are
available (Sharon and Lis, 1972; Lis and Sharon, 1973). A survey of over 100
soybean varieties and strains revealed about an eightfold variation in
agglutinating activity but all samples were active (Kakade et al., 1972).
Consequently, elimination of agglutinins by plant breeding does not look
encouraging at this time.
30 20
Figure 4. Primary structure of Kunitz soybean trypsin inhibitor according to Koide and Ikenaka
(1973). Residues are numbered beginning with N-termina1 aspartic acid. Disulfide cross-linkages are
shown in black between half-cystine residues. Reprinted with permission from Springer-Verlag.
Biological activity of soybean agglutinin is of continuing interest in several
laboratories, and recent studies include agglutination of mouse, rat, and human
cell lines after transformation with viral or chemical carcinogens (Sela et al.,
1970); binding of agglutinin by red blood cells (Gordon et al., 1972b); and
determination of factors that influence agglutination (Gordon and Marquardt,
1974; Pereira et al., 1974). The biological activity of soybean agglutinin has been
used to advantage in developing affinity chromatography techniques for its
purification (Allen and Neuberger, 1975; Bessler and Goldstein, 1973; Gordon et
al., 1972a). Lotan et al. (1974) found that the agglutinin contains four identical
subunits with a molecular weight of 30,000. Each subunit has a carbohydrate side
chain of nine mannose and two N-acetyl-D-glucosamine residues. Four of the
mannose residues can be oxidized with sodium periodate and then reduced with
sodium eH] borohydride to yield the tritium-labeled agglutinin with full
retention of its biological activity (Lotan et al., 1975). This radioactive derivative
should be very useful in studies of surfaces of cells that can interact with the
agglutinin.
Although the function of agglutinin in soybeans is still unknown, Bohlool and
Schmidt (1974) found that the agglutinin combined specifically with 22 out of25
strains of the soybean-nodulating bacterium Rhizobiumjaponicwn. No binding
Figure 5. Crystalline complex of porcine trypsin and Kunitz soybean trypsin inhibitor at 5-A
resolution according to Sweet el al. (1974). Reprinted with permission from Biochemistry 13: 4212-
4228 (1974). Copyright by the American Chemical Society.
Chemistry and Technology of Soybeans! 341
occurred with 23 other strains of rhizobia that do not nodulate soybeans. They
proposed that the agglutinin may provide a site on the soybean root surface that
interacts specifically with the polysaccharides on the surface of the appropriate
Rhizobium cell as the first step in the formation of the nodule. However, the
existence of agglutinins in the roots of the plant does not appear to have been
reported.
7S Globulin. Koshiyama (l972b) has described a new method for purifying a
7S globulin by suspending acid-precipitated (pH 4.5) globulins in 0.6M NaCl-
O.OIN HCl at pH 2 and then centrifuging. Under these conditions, the lIS
globulin is acid-denatured and insoluble, whereas the 7S globulin is stable and
remains soluble. The acid-soluble fraction is then passed successively through
Sephadex G-lOO and G-200 columns to yield the 7S globulin. Yield of 7S
globulin from the acid-precipitated globulins was 16% or about one-half of the
total 7S fraction in the globulin mixture.
The presence of subunits in purified 7S globulin has been confirmed by
molecular weight studies in protein dissociating solvents, although there is
disagreement about molecular weights of the subunits. Vaintraub and Shutov
(1972) obtained molecular weights of 83,800 in 4M urea and 31,200 in 6M urea
and proposed that the parent molecule contained six subunits. On the other
hand, Koshiyama (1970) obtained a molecular weight of 22,500 for the 7S
globulin in 8Murea which suggests that about nine subunits make up the parent
molecule in agreement with results of N-terminal analysis. The 7S protein
samples used in the two studies were not prepared by the same method; hence,
different 7S globulins may have been examined. This is plausible because results
of Hill and Breidenbach (1974a) suggest that there may be three 7S proteins with
different electrophoretic mobilities but having the common ability to undergo
monomer-dimer formation with change in ionic strength.
Additional studies on subunit structure of a 7S globulin indicated that
disulfide bonds were not involved in binding between subunits. Urea and
guanidine hydrochloride appear to disrupt the internal structure of the subunits
when the 7S molecule is dissociated with these reagents (Koshiyama, 1971).
Conformational studies of 7S and 11 S globulins showed both to be very low in
a-helix content; ,B-structure and random coil seem to predominate. Although
similar in structure as measured by circular dichroism in the region between 200
and 250 nm, there were distinct differences in the 250-320 nm region
(Koshiyama and Fukushima, 1973). Marked dissimilarities between 7S and II S
globulin conformations were observed by ultraviolet difference spectra,
ultracentrifugation, and optical rotatory dispersion in acid solutions at 0.1 ionic
strength (Koshiyama, 1972c).
11S Globulin (Glycinin). Koshiyama (1972d) purified II S protein by gel
filtration and redetermined many of its physical properties (Table IX). No major
changes were obtained from most of the values obtained by former workers, but
the new values are likely to be more reliable than older ones because the protein
preparation was homogeneous by gel filtration, ultracentrifugation, and gel
electrophoresis.
Sedimentation equilibrium molecular weights of the subunits of II S protein in
acid solution (pH 2.6 but unspecified ionic strength) and in 4M urea (pH 7.4,0.1
ionic strength) were 63,000 and 31,000, respectively (Vaintraub and Shutov,
1971). Basic subunits isolated by DEAE-cellulose chromatography had a
molecular weight of 24,400. Sodium dodecyl sulfate electrophoresis yielded
molecular weights of 22,300 for the basic subunits and 37,200 for the acidic
subunits (Catsimpoolas et al., 1971d). Amino acid analyses of the six subunits
isolated by isoelectric focusing by Catsimpoolas and coworkers revealed no
significant differences in the ratios of acidic to basic residues. Thus, it seems
TABLE IX
Physical properties of 11S protein'
Property
IsoeIectric point. pH
10/<;
E1 em, 280 nIT:
Intrinsic viscosity, dl! g
sYo,w
Partial specific volume, mIl g
Molecular weight
Gel filtration
Sedimentation equilibrium
Sedimentation-viscosity
Diffusion constant, cm'i sec
Frictional ratio
'Source: Koshiyama (1972d).
Value
4.64
8.04
0.0485
12.2S
0.715
312.000
322.000
309.000
3.48 X 10-
1.40
TABLE X
Summary of soybean enzyme studies, 1970-75
Enzyme
Degree of
Purification Reference
L-Alanine:2-oxoglutarate
aminotransferase
Chalcone-flavanone isomerase
Lactic dehydrogenase
Malate dehydrogenase
0:- D- Mannosidase
Protease
Urease
230-fold Dumitru et al. (1970)
8,300-fold Boland and Wong (1975)
110-fold King (1970)
217-fold Barthova et al. (1973)
1,160-fold Barthova et al. (1974)
Bronovitskaya and Kretovich (1970a)
Bronovitskaya and Kretovich (1970b)
5,500-fold Saita et al. (1971)
2I-fold Catsimpoolas et al. (1971b)
Buttery and Buzzell (1971)
likely that the acidic side chains in the basic subunits are present as amides in
order to shift their isoelectric points to pH 8.0-8.5. The amino acid analyses also
showed that the six subunits were all different. Results from both research
groups are in agreement with the existence of 12 subunits per 350,000 molecular
weight entity.
Kitamura and Shibasaki (1975) recently described isolation of four acidic
subunits from 11 S protein in contrast to the three isolated by Catsimpoolas et al.
(1971 d). Two of the subunits separated in Japan contained phenylalanine and the
other two had leucine (isoleucine) in the N-terminal positions. Genetic
polymorphism was suggested as a possible explanation for these results.
Scanning isoelectric focusing of 11 S protein has revealed an extremely
complex system that is attributed to microheterogeneity possibly arising from
variation of amide groups on the side chain carboxyl groups of aspartic and
glutamic acid residues (Catsimpoolas and Wang, 1971).
Hydrogen ion titration of 11 S protein confirms that alkali and acid cause
conformational changes in the protein molecule (Catsimpoolas et al., 197Ia).
Approximately 14% of the total ionizable groups appear to be buried and are
accessible only by titration in disaggregating media such as 6Murea or guanidine
hydrochloride. Ultraviolet difference spectra likewise revealed the presence of
buried ionic groups-tyrosine and tryptophan-which are exposed by 6M urea
or guanidine hydrochloride (Catsimpoolas et al., 1971e). Optical rotatory
dispersion and infrared studies also confirmed that the lIS molecule primarily
has a ,B-conformation plus some disordered regions. Jacks and coworkers (1973)
measured the optical rotatory dispersion of lIS protein from 200 to 240 nm and
concluded that the molecule contained 9% a-helix, 33% pleated sheet, and 58%
unordered structure.
Destruction of antigenicity of 11 S protein by heating in solution was
investigated by radial immunodiffusion, complement fixation, and disc
immunoelectrophoresis (Catsimpoolas et al., 1971c). Antigenic properties are
retained on heating for 30 min up to 65C, but are lost rapidly between 70 and
90 C. Loss of antigenicity is associated with destruction of the quaternary
protein structure and possibly alteration of the secondary and tertiary structures
of the individual subunits.
D. Enzymes
Enzymes Other than Lipoxygenase. Work on a variety of soybean enzymes has
been reported in the past 5 years. Studies are often done on germinated beans or
seedling parts, but authors frequently fail to point out whether the ungerminated
seed contains the enzyme under consideration. Such information would be useful
to those concerned with the mature seed. Table X lists some of the seed enzymes
studied during 1970-75. Not included are the lipoxygenases which merit separate
discussion below.
Lipoxygenase. After a number of years of near neglect, lipoxygenase is being
actively studied in several laboratories, and a large literature is developing on this
enzyme. Several major properties of lipoxygenase have been clarified in the past
few years: a) there is good evidence for at least four lipoxygenases in soybeans, b)
the various enzymes showdifferences in substrate specificity, and c) lipoxygenase
344! Advances in Cereal Science and Technology
is now known to contain iron as a prosthetic group.
The presence of four isoenzymes of lipoxygenase was first suggested by
polyacrylamide gel electrophoresis of soybean extracts (Guss et al., 1967).
Fractionation studies then soon led to the isolation of a second (Christopher et
al., 1970) and a third (Christopher et al., 1972) enzyme which differed from the
classical Theorelllipoxygenase preparation. All three enzymes have molecular
weights of 100,000, but they differ significantly in several respects (Table XI)
TABLE XI
Comparison of properties of three lipoxygenases'
Enzyme
Property L-l L-2
L-3
Isoelectric point 5.68 6.25
pH optimum 9.5 6.5
Effect of Ca"" None" Stimulatory
Stabilitv to heat Stable Unstable
Substra"'te specificity' Free acid Ester
'Source: Christopher et al. (1970. 1972): Verhue and Francke (1972).
bRestrepo et al. (1973).
'Comparison of linoleic acid and methyl linoleate.
GIUCOSYI-0Yf(0)
W-O-
0H
H
Genistin
GIUCOSYI-0Yf(0)
VY-O-0H
Oaidzin
GIUCOSYI-0w-0
0
I I If OH
CH30 ---
Glycitein 7p-a-glucoside
Figure 6. Structures of soybean isoflavone glucosides.
6.15
5.5-8.0
Inhibitory
Ester
including their chromatographic behavior on DEAE-Sephadex. Verhue and
Francke (1972) obtained good evidence for a fourth lipoxygenase and found that
of the four enzymes, two show a preference for free linoleic acid and two prefer
methyllinoleate as a substrate.
When lipoxygenase was crystallized nearly 30 years ago, it was found to
contain less than a stoichiometric amount of iron which was considered to be a
contaminant. Lipoxygenase was therefore considered an unusual dioxygenase
because it apparently lacked a transition metal. This anomaly has now been
cleared up by careful reexamination of the purified enzymes and finding of one
atom of iron per molecule (Chan, 1973; Roza and Francke, 1973; Pistorius and
Axelrod, 1974). The iron is tightly bound and is only slowly removed from the
protein by chelating agents unless the protein is first denatured. The active state
of the iron is believed to be the ferric form (Pistorius and Axelrod, 1974; deGroot
et al., 1975b).
Although of great theoretical interest as a dioxygenase, lipoxygenase has also
become the focus of attention by food scientists because reaction products ofthe
polyunsaturated fatty acid hydroperoxides are believed to contribute to the
undesirable flavors associated with raw soy flours and derived protein products.
This subject is discussed in Section VII-A.
E. Minor Constituents
lsoflavones. The presence of genistin and daidzin (Figure 6) in soybeans was
reported over 40 years ago, but only recently were the isoflavones reinvestigated.
A new isoflavone was discovered, and the content ofisoflavones in soybeans was
reported. Naim et al. (1973) isolated the new isoflavone and identified it as 7,4'-
dihydroxy, 6-methoxyisoflavone. It occurs in soybeans largely as the 7-0-{3-
glucoside (Naim et al., 1974) which has the structure shown in Figure 6.
Isoflavones in soybeans were determined by grinding and extracting with ether
followed by methanol and finally hydrolyzing the resulting soy flour residue with
acid and extracting with ether. The extracts were chromatographed on a
polyamide column, and the isoflavones were determined by gas
chromatography. Results of the analysis are summarized in Table XII. Over 99%
of the isoflavones in soybeans occur as glucosides, and genistin is found in the
highest concentration. The isoflavone content of soybeans is about 0.25%.
TABLE XII
Isoflavone content of soybeans'
Fraction
Genistein Genistin Daidzein Daidzin Glycitein
rng/ 100 g soybeans
Glycitein
Glucoside
Ether extract 0.2 0.3 0.01
Methanol extract 1.2 157.2 0.3 56.1 0.1 32.1
Aour residue 7.2 2.0 1.7
Total 1.4 164.4 0.6 58.1 0.11 33.8
'Source: Nairn et al. (1974).
Phytic Acid. Analyses for phytic acid in 15 commercial soy products were
reported by Ranhotra and coworkers (1974). Their results can be summarized as
follows:
Preparations
Full-fat flours
High-fat flours
Defatted flours
Concentrates
Isolate
Whey-soy blend
% Phytic Acid
0.51
0.44-0.51
0.46-0.52
0.35-0.61
0.33
0.36
Phytase activity of the soy products was low as compared to that of wheat flour.
Nonetheless, with one exception, when the soy products were added to bread, the
phytic acid was extensively hydrolyzed; presumably phytases of wheat and yeast
caused hydrolysis. The exception noted was a whey-soy blend which, when
added to bread, resulted in only 22% hydrolysis of the phytic acid. The high
content of calcium ion in whey may have inhibited phytase. Further studies are
necessary on phytase activity in breads containing whey-soy blends because the
blends are now used extensively in baked goods to replace nonfat dry milk
(Cotton, 1974).
Interaction of phytate with soybean proteins was described by Okubo et al.
(1975). Dialysis experiments showed that binding ofphytate to soybean proteins
was minimal at pH 5 but strong at pH 3 and at pH 8.5. At the low pH, interaction
apparently occurs through the cationic groups of the protein-lysyl, arginyl, and
histidyl residues plus the amino terminal groups. At pH 8.5, multivalent cations
such as calcium and magnesium appear to chelate with the phytate, and the
chelate complex binds to the protein. Phytate was removed by dialysis under
three conditions: a) pH 8.5, in the presence of sodium ethylenediamine
tetraacetate, b) pH 5, in water, and c) pH 3, in 0.5M calcium chloride. The last
condition appeared to be the most effective of the three.
Sterols. A survey of sterol distribution by four classes-free, esterified,
TABLE XIII
Distribution of sterol classes in selected U.S. soybean varietieS-
Form
Variety and
Year of Harvest
Acylated
Total Sterols
b
__F_re_e E_st_e_ri_fie_d__G_l_u_co_s_id_es__G_lu_c_o_sid_e_s_
% mg/ 100 g soybeans
Harosoy 1963 0.18 67 16
Harosoy 1964 0.17 72 17
Shelby 1963 0.15 64 15
Shelby 1964 0.14 68 17
'Source: Hirota et al. (1974).
bBased on weight of free sterols plus weights of derivatives.
45
20
39
27
51
60
33
32
glucosides, and acylated glucosides-in 19 U.S. soybean varieties was reported
by Hirota et al. (1974). Selected data from their results are given in Table XIII.
Free sterols are the major form found and the sterol esters are present in the
smallest amounts. A study of developing soybeans revealed that the four sterol
classes are detectable as soon as the beans are large enough to analyze ~ weeks
after flowering). Surprisingly, the percentage distribution between the four
classes does not change appreciably from early development until maturity
(Katayama and Katoh, 1973).
Acylated sterol glucosides were recently identified as an additional group of
substances complexed with isolated soybean globulins; they are extracted from
the proteins with aqueous ethanol (Wolf and Thomas, 1973).
Saponins. The existence of saponins in soybeans was reported almost a half
century ago, but these compounds received relatively little attention until the
1960's when extensive studies on their chemical, nutritional, and physiological
properties were conducted at the Hebrew University of Jerusalem in Rehovoth.
Birk (1969) has reviewed these studies in detail. Further work in our laboratory
revealed that the presence of glucuronic acid residues in the saponins makes it
possible to fractionate the crude mixture by anion exchange chromatography on
Dowex-l columns (Wolf and Thomas, 1971). Twelve fractions were separated,
but analysis of their sugar contents suggested that they were still very
heterogeneous. One fraction yielded a previously unreported aglycone whose
infrared spectrum suggested a similarity to soyasapogenol D. Complexity of
Rhap a 1-2Galp {31-2GlcUAp{31-0
Soyasaponm I
Rha pal-2A. ap a 1-2G IcUA p{31-0
SoyasapoOin II
Galp{31-2GlcUAp{3I-0
Soyasaponm ill
Figure 7. Structures of soyasaponins I, II, and III according to Kitagawa et al. (1974b).
348 / Advances in Cereal Science and TechnoloK}'
saponins is not peculiar to soybeans; recent work on alfalfa saponins revealed
about 30 different compounds (Berrang et al., 1974).
Kitagawa et al. (l974a) isolated three soybean saponins, designated
soyasaponins I, II, and III, having soyasapogenol B as the common aglycone.
Separation was achieved by silica gel column chromatography using
chloroform:methanol:water as the developing solvent. Studies of the three
saponins revealed the chemical structures shown in Figure 7. All three
compounds have the common feature of glucuronic acid attached by beta linkage
to the 3-hydroxyl group of soyasapogenol B. Soyasaponin III is identical to
soyasaponin I except that it lacks rhamnose.
Soybean saponins are toxic to rice weevils (Sitophilus oryzae) and therefore
show some promise as naturally occurring insecticides that appear nontoxic to
warm-blooded animals (Ishaaya et al., 1969; Su et al., 1972).
Polyamines. Putrescine, cadaverine, spermidine, and spermine occur in
soybeans at a level of about 130 ppm (Wang, 1972; Wang and Selke, 1973; Wang
et al., 1975). Their function in soybeans is still unknown. Although these
compounds are malodorous, they may be present in soybeans at levels that are
below their odor thresholds.
VI. TECHNOLOGY
A. Processing Soybeans into Oil and Meal
There have been no major changes in the past 5 years in the basic processing
used to convert soybeans into oil and meal. Virtually all of the beans processed in
the U.S. are extracted with hexane. Reviews ofthe overall process are available
(Becker, 1971; Wolf and Cowan, 1975).
Annual crushing capacity of the industry is presently estimated to be about 1.1
billion bushels, and historically about 80% of capacity has been used. The
number of soybean processing mills has declined, but their average size has
increased as the industry expanded. For example, in 1951 there were 193
processing plants with a capacity of 310 million bushels, but by 1973 the number
of plants had dropped to 113 whereas capacity had increased more than threefold
to 1.0 billion bushels (American Soybean Association, 1975).
B. Processing Oil into Edible Products
The various steps in converting crude soybean oil into edible products were
reviewed recently (Wolf and Cowan, 1975). New developments are therefore
described here only briefly.
Recycling of Water in Alkali Refining. Plant-scale tests were carried out in
which the water used to wash alkali-refined oil was passed through a cation
exchange column to remove sodium ions, thereby allowing the water to be
recycled (Beal et al., 1973). Oil washed with recycled water in a continuous 28-
day run had a satisfactory low content of sodium, iron, and copper. Bleaching,
hydrogenation, and deodorization proceeded normally; the recycle process
appears to solve the wash water disposal problem.
Copper-Chromite Catalyst for Hydrogenation. Hydrogenation of linolenate
with copper-chromite catalyst was tested successfully in plant trials to prepare
improved edible soybean oils for frying and salad oils (List et al., 1974). Copper
content of the oil increased as a result of hydrogenation, but bleaching and
winterization reduced it to levels below 0.01-0.02 ppm. Residual copper and
chromium in processed oil were concentrated in the stearine fraction by
winterization. Results of organoleptic, oxidative, and room odor tests showed
that oils of good stability can be prepared on a commercial scale by copper
hydrogenation and winterization. The copper catalyst must be handled carefully
because if it becomes contaminated with nickel, it loses its selectivity for
hydrogenation of linolenate and behaves as a nickel catalyst (Moulton et al.,
1973).
Deodorization. The need for increasing production of oil refining plants and
the growing demand for more soybean sterols have led to a new design of the
trays in the conventional Bailey type deodorizer. The new design incorporates an
improved jet stream distributor that permits injection of steam at high rates
without excess oil entrainment and losses. The new tray replaces two or three of
the existing trays in the Bailey deodorizer and makes possible rapid removal of
sterols with maintenance of oil quality (Lineberry and Dudrow, 1972). Several
new or modified deodorizers with only four trays instead of the normal six or
seven are now in use in the industry.
Antioxidantsfor Soybean Oil. Polyunsaturated vegetable oils such as soybean
oil are subject to oxidation; hence, antioxidants are often added as stabilizers.
Monotertiarybutylhydroquinone (TBHQ) was approved as a food grade oil-
soluble antioxidant in 1972. Toxicological and biochemical studies conducted to
establish safety of this compound were reported recently (Astill et al., 1975).
Rats, dogs, and humans eliminated the compound mainly in the urine as the 4-0-
sulfate and the 4-0-glucuronide. Extensive feeding and comparative biochemical
studies indicate that TBHQ is safe for its intended use at the maximum level (200
ppm based on weight of fat or oil) permitted by the Food and Drug
Administration.
Butylated hydroxyanisole and butylated hydroxytoluene have been used
extensively as antioxidants. Their toxicological and biochemical properties were
reviewed recently by Branen (1975).
Field-Damaged Soybeans. Adverse weather during harvesting damages
soybeans with the result that poor quality oil is obtained when the beans are
processed. Evans et al. (1974) studied commercial oils from soybeans that were
field-damaged in North Carolina during the 1971 growing season. They found
high levels of free fatty acids and iron in the oils, and a significant correlation
existed between free fatty acid and iron contents. The iron apparently originated
from the damaged beans as well as from steel processing equipment. The high
level of iron in the oil from damaged soybeans is believed to be responsible for the
low quality of the oil when it is refined.
Metabolism of Unsaturated Fatty Acid Isomers. Use of copper as well as
nickel catalysts to hydrogenate linolenate ester in soybean oil causes geometric
isomerization and migration of double bonds from their normal positions
(Cowan et al., 1973a; Vigneron et al., 1972). Table XIV shows the monoene
composition of soybean oils hydrogenated with copper and nickel catalysts. The
copper-reduced oil had a total monoene content of 42%; yet, only 53% of the
double bonds were located in their expected .6.9 position. Likewise, in nickel-
reduced oil, only 61% of the monoene was in the usual .6.9 location. Because
hydrogenation is widely used in the production of soybean oil and related
products, there is concern about the metabolic fate of the various fatty acid
isomers generated during hydrogenation. Mounts et al. (1971) have examined
the incorporation of tritium, carbon-14, and deuterium-labeled oleate and
elaidate esters into egg lipids by the laying hen. The cis isomer was preferentially
incorporated into the neutral lipids, whereas the trans isomer was preferred by
the phospholipids. Both cis and trans isomers were transferred across the chicken
membranes at about the same rates. Incorporation of label by the neutral lipid
indicated that there was no isotopic discrimination or loss, but loss of carbon-14
from the carboxyl group in the lipids incorporated into the phospholipd fraction
indicates that a more complex biosynthetic route is involved. Further
experiments by Mounts and Dutton (1976) indicate that the hen does not
distinguish between oleic and linoleic acids in synthesis ofthe neutral lipid of the
egg. The phospholipid fraction of the egg, however, revealed a selection of
linoleic acid over oleic acid.
C. Processing Soybeans into Edible Protein Products
As pointed out earlier, the use of soybean protein in foods is still quite small as
compared to the oil (Table VII). Nonetheless, this segment of the soybean
industry has undergone the greatest change in the past 5years. Edible soy protein
products for use as food ingredients were first developed by a few of the
established soybean processors; but as markets developed, many of the other
processors, plus several companies that do not process soybeans, entered the
marketplace. Table XV lists the major U.S. manufacturers of soybean protein
products. Products range from flours and grits to textured isolates. The newest
items introduced are the textured concentrates; three companies announced
availability of these products in mid-I975.
TABLE XIV
,'-fonoene composition of hydrogenated soybean oils'
Catalyst
Copper Nickel
Position of Double Bond %of total fatty acids
!::>.7
!::>.8
!::>.9
!::>. 10
!::>.II
!::>.12
!::>.13
!::>.14
!::>.15
!::>.16
!::>. 17
'Source: Cowan et af. (l973a).
0.3
2.1
22.1
3.1
4.9
2.6
1.6
0.9
0.6
0.2
0.2
0.3
2.1
26.0
3.2
3.7
5.3
0.8
0.3
0.4
0.1
Full-Fat Products. Development offoods in which the whole soybean is used,
including the seed coat, has been described (Nelson et al., 1971; Shemer et al.,
1973). It is recommended that the soybeans be blanched by boiling to inactivate
lipoxygenases before disrupting the cellular structure of the seed. Allowing
lipoxygenases to remain active while disrupting the seed is believed to result in
generation of the beany flavors characteristic of raw soybean products. Whole
soybean food prototypes such as canned chicken and soybeans have been
prepared. If blanched beans are ground and drum-dried, a flake-like item results;
grinding should give a full-fat flour. A beverage base is prepared by soaking
soybeans in tap water overnight in 0.5% NaHC0
3
solution, boiling (blanching) in
fresh 0.5% NaHC0
3
for 30 min, draining, grinding, heating to 200
0
F, and finally
homogenizing to yield a product containing about 4.8% protein and 2.4% oil
(Nelson et al., 1975). A variety of dairy analogs has been formulated from the
base.
Full-fat flours can be made by extrusion cooking as described by Mustakas et
al. (1970). Soybeans are cracked, dehulled, and then dry-heated to inactivate
lipoxygenases. The heated bean particles are tempered to preferred moisture
levels and extrusion cooked. After cooling and grinding, a full-fat flour is
obtained. Nutritional studies indicate that the cooking step inactivates
antinutritional factors. Extrusion cooking to prepare full-fat flours does not
appear to be practiced commercially in the U.S. at this time although it is being
used abroad.
Extrusion-cooked full-fat flour has been converted into experimental
beverage bases by two procedures. In the first, the flour is suspended in water,
mixed with emulsifier and soybean oil, colloid milled, homogenized, and spray
dried. The powder is then blended with sugar, salt, flavor, minerals, and vitamins
to yield the beverage base (Mustakas et al., 1971). For the second beverage
TABLE xv
}Y/ajor U.S. producers of soybean protein products
Producer
Grits and
Flours
Textured Products
Concentrates Isolates Flours Concentrates Isolates
Archer-Daniels Midland Co.
Cargill, Inc.
Central Soya Co.
Dawson Mills
Far-Mar-Co.
General Mills, Inc.
Grain Processing Corp.
Griffith Laboratories
Lauhoff Grain Co.
Miles Laboratories, Inc.
Nabisco
National Protein Corp.
Ralston Purina Co.
A. E. Staley Mfg. Co.
Swift & Co.
'Mainly pepsin-modified isolates.
T
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+'
+
+
+ +
+
+
+ +
+ +
+
+ +
+
+
+ +
+ +
+
process, raw full-fat flour is slurried in dilute sulfuric acid at a final pH of3.5 and
heated rapidly with steam, thereby inactivating the lipoxygenases. The lipid-
protein-polysaccharide curd is then cooled and washed with water to remove the
soluble sugars in the manner used to manufacture protein concentrates from
defatted flakes and flours. After washing, the slurry is adjusted to pH 9 with
ammonium hydroxide, and another quick cooking is given to inactivate residual
trypsin inhibitor. The alkaline dispersion is then neutralized, colloid milled,
homogenized, and centrifuged to obtain the beverage base (Mustakas, 1974).
Defatted Flours and Grits. To the best of my knowledge, commercial practice
is still to use hexane as in the conventional process and desolventize under
conditions that yield flours and grits with the desired protein solubility (Wolf and
Cowan, 1975).
Hexane is a good solvent for extracting oil from soybeans, but it does not
remove all of the lipids. In the past few years, several lines of evidence have
pointed to the lipids in soybeans as the source of some of the undesirable flavors
that are characteristic of raw defatted flakes and products made from them.
Extraction of raw, hexane-defatted flakes with azeotropic mixtures of
hexane:alcohol yields additional lipids and also removes most of the beany and
bitter flavors (Eldridge et al., 1971). Hexane mixed with methyl, ethyl, or
isopropyl alcohol can be used, but hexane:ethanol (82: 18 vI v) has the advantages
of being nontoxic, causing a minimum of protein denaturation, and removing
only a few percent of solids. The azeotropic mixture apparently is selective for the
residual lipids and the flavor compounds. Protein isolates of improved flavor
were also obtained when the starting flakes were first extracted with
hexane:ethanol. Because hexane:ethanol does not inactivate the trypsin
inhibitors in flakes, heat treatment is necessary to obtain optimum nutritional
value. The effects of hexane:ethanol extraction plus toasting have therefore been
studied (Honig et al., in press). The combination of hexane:alcohol extraction
and toasting yields defatted flakes with flavor scores equivalent to those for
wheat flour.
Protein Concentrates. Several new methods for making concentrates have
been described recently, but they often are variations of presently used processes.
A plant gum such as carrageenan can be added during the washing step in the
conventional acid-leaching process to reduce loss of acid-soluble proteins
(DeLapp, 1973). The gums form insoluble complexes with the acid-soluble
(whey) proteins (Smith et al., 1962) and thus are retained in the concentrate.
Sair (1972) described a modification of his earlier acid-leach process in which
he prepares a protein isolate by the conventional procedure and then adds the
isolated protein back to the thoroughly washed insoluble residue. The mixture is
then neutralized with alkali and dried under vacuum. The resulting concentrate is
claimed to have a light color and less beany flavor then the product made by
direct leaching of defatted flakes.
Another variation (Hoer and Calvert, 1972) of the acid-leach process consists
of slurrying defatted flakes in water to dissolve the proteins. The slurry is then
acidified to pH 4.5 to precipitate the proteins. The protein curd and water-
insoluble polysaccharides are recovered by centrifuging, washed thoroughly, and
adjusted to pH 6.8. The slurry is then quickly raised to 310 F in a jet cooker and
held at that temperature for about 5 sec. Next, the slurry is injected into a vacuum
chamber to flash off undesirable flavor components and spray dried. The jet
cooking step is probably responsible for alteration offunctional properties of the
resulting protein concentrate as compared to the conventional acid-leached
concentrate.
Miller and Wilding (1973) described a related acid-leach process in which
defatted Hakes are stirred in water for 15 to 30 min and then acidified to pH 4 to 5.
The isoelectric slurry is then ground (MG Model, Urschel Comitrol with a
Microcut-Head attachment), centrifuged, and washed to remove the soluble
sugars. After washing, the product can be dried in the isoelectric state or
neutralized, heated, and spray dried. Rupture of cell walls and protein body
membranes is claimed, but the importance of this step is not clear. The cell wall
polysaccharides and the globulins remain in the product, in any event, because
washing is carried out in the isoelectric region of the proteins.
A variation of the alcohol-leaching process utilizes a two-phase solvent system
of hexane:methanol:water in the ratio of 10:7:3 that is applied to full-fat soybean
flakes to simultaneously extract the oil and oligosaccharides plus minor
constituents (Schweiger and Muller, 1973). The product obtained has a protein
content of 65 to 73%. It is likely that this solvent system will remove lipids not
normally extracted by hexane alone.
Hayes and Simms (1973) described the use of hexane:alcohol as a solvent for
residual lipids in hexane-defatted flakes. First, full-fat flakes are extracted with
hexane. Desolventization is omitted and ethanol is then added to the hexane-wet
flakes. The resulting hexane:ethanol dissolves the residual lipids and is removed.
Then the drained flakes are desolventized to selectively take off the hexane. Next,
additional alcohol and water are added to the alcohol-wet flakes to extract the
soluble sugars as in the usual alcohol-leach process. On desolventizing, a protein
concentrate is obtained.
Protein Isolates. Isolates are now available from several manufacturers (Table
XV) and have a variety of physical properties to make them suitable for different
food applications. The methods employed to modify the isolates are proprietary
information, but heat and enzyme treatments are included in many of the recent
patents issued on this topic. For example, Hoer et al. (1972) quickly heat slurries
of sodium proteinates in a jet cooker to 285
0
to 320
0
F which also imparts a
shearing force to the slurries. After a short holding time, the slurries are
discharged into a zone of lower pressure to flash off water vapor and volatile
flavor compounds. The slurries are then cooled and treated with a proteolytic
enzyme for 1 to 30 min. Reheating the slurries inactivates the enzyme, and spray
drying completes the process. High wettability and good dispersibility with water
are claimed for the modified isolate.
A second example is described by Puski (1974). An acid-precipitated protein
curd is washed with water, heated to 80
0
C, and papain plus sodium bisulfite are
added to a 15% protein slurry. After digesting for 2 hr at 80
0
C, the slurry is boiled
to inactivate the papain and insoluble proteins are removed by centrifuging. The
acid-soluble fraction is then spray dried to yield a derived protein that is soluble
in acidic (pH 2.6) beverages such as soft drinks and fruit juices.
Textured Protein Products. Successful introduction of textured soybean
protein products in the late 1960's stimulated a large amount of developmental
work on new and better processes for texturizing flours, concentrates, and
isolates. The spun isolate products were developed first, and the textured flours
were introduced later. Recently, textured concentrates have also become
available, but published information on their preparation is not yet available;
patents on processes are pending. Most of the published information on
texturization appears in the patent literature, and many of the U.S. patents on
this subject for 1960-72 were summarized by Gutcho (1973). Processes actually
used by industry are not generally revealed, but examples will be cited to show
the diversity of approaches being developed.
Textured Flours. A departure from the usual extrusion process has been
described in patents by Strommer (1973) and Strommer and Beck (1973). A
blend of defatted flour, protein concentrate, and isolate is introduced into a
rotating multichambered device and injected with high-pressure steam which
expels the materials through a nozzle into a region of lower pressure. The
expulsion causes puffing and texturization plus flashing off of volatile flavor
compounds. Residence time in the apparatus may be less than a second; hence,
the product does not turn dark, and a bland flavor is claimed. A variation of this
process has also been developed in which a slurry of the protein material is
sprayed into a stream of high-pressure steam. The excess moisture is flashed off
and a textured product is obtained (Strommer, 1975).
Defatted soy flour can be converted into fibers by making a thick aqueous
slurry in the isoelectric pH region and pumping it at high pressure (50-5,000 psi)
through a heat exchanger. The hot proteinaceous material is then pumped
through a small orifice to obtain a continuous filament or small discrete textured
particles, depending on operating conditions (Frederiksen and Heusdens, 1972).
Soy flour and related materials can also be textured by making a dough-like
mass with water, heating to a high temperature, and then quickly releasing the
pressure. Loepiktie and Flier (1973) carried out the process in Teflon-coated
aluminum tubes heated in a closed chamber, whereas Touba (1974) wrapped the
dough in aluminum foil and heated it under pressure between the plates of a
hydraulic press. The process is probably similar to conditions in an extruder
when the dough passes through the die.
Textured Isolates. A variation of the classical fiber spinning process has
eliminated the need for an alkaline treatment step. Soy protein isolate prepared
in the customary way is dispersed in water by adding solid salt to a final molarity
of 0.4 to 0.5 instead of dissolving it with alkali. The curd does not dissolve
completely, but the soluble portion or mesophase is separated by centrifuging
and then extruded into a hot water (80
0
C or higher) bath. The high temperature
coagulates the protein into a fiber and the salt is removed by leaching into the
water (Tombs, 1972). Formation oflysinoalanine (deGroot and Slump, 1969) is
eliminated by avoiding the alkaline spinning dope step.
A simplified process for converting soy protein isolate into edible fibers
consists of pumping a protein slurry (up to 35% solids) at high pressure through a
heat exchanger and then expelling it through a small nozzle. The protein emerges
as fibers (4-6 cm long) that are cooled by dropping 20 ft through ambient air into
a collecting vessel. Excess water is removed by centrifuging (Hoer, 1972). A
somewhat similar process described by Lange (1974) yields a protein
monofilament. Isolate is mixed with water, sodium sulfite, glycerine, plus an acid
or base. The mixture is heated until it becomes plastic (190
0
to 285
0
F) and then is
extruded through 2-10 mil orifices into air. The resulting fiber can be combined
with binders, flavors, and color to form simulated meats. Both "dry" spinning
processes eliminate the preliminary spinning dope step and the acid-salt
coagulating bath employed in the usual fiber spinning operation.
Soy protein isolate is a major ingredient in a creping process developed to
simulate meats (Liepa and Slone, 1974). For example, egg white solids, soy
protein isolate, vital wheat gluten, starch, shortening, and beefflavors are mixed
into a dough with water. The dough is then mixed more thoroughly by passing it
through a noodle extruder. As they emerge, the extruded dough strands are cut
into pellets which are passed through smooth rolls to convert the mass into a
sheet. The sheet is removed from the last roll with a doctor blade thereby creping
or crinkling it. The parallel ridges and depressions of the sheet give it a fibrous
texture that is stabilized by heating. By coating the creped sheet with an edible
binder, folding it upon itself, and then heating it, a slab of cooked meat-like
product is obtained.
A bacon analog containing soy isolate is described by Leidy et al. (1974a). Two
emulsions are prepared. One emulsion forms the lean or red portion of the analog
while the other duplicates the fatty or white part. The red phase is made by
mixing soy protein isolate with water, textured vegetable protein, egg albumin,
hydrogenated vegetable oil, flavor, spices, and red food color until a stable
emulsion results. The white phase is formed by emulsifying water, egg albumin,
textured vegetable protein, hydrogenated vegetable oil, flavor, and spices. The
two phases are then alternately layered in a pan and heated in an autoclave. After
cooling, the resulting loaf is sliced to obtain the bacon analog which can be fried,
broiled, or cooked in a microwave oven.
Sausage analogs are also possible by making a gel precursor emulsion and then
autoclaving (Leidy et al., 1974b). For example, soy protein isolate is mixed with
albumin, textured vegetable protein, soybean oil, water, seasoning, flavor, and
color to form the emulsion. The gel precursor emulsion is then stuffed into
bologna or sausage casings and autoclaved to heat-set it. After cooling and
removing the casings, bologna or frankfurter analogs are obtained.
VII. FACTORS AFFECTING FOOD USES OF SOYBEAN PROTEINS
In contrast to the oil which is widely used as an edible fat, soybean proteins still
face some problems before their use becomes more widespread. Flavor,
functional, and nutritional properties are three of the major factors that limit or
impede the incorporation of soybean protein products into many foods.
A. Flavor
Although soybean proteins have made inroads as an ingredient in a number of
processed foods, the level of protein that can be added is often limited by residual
flavors characteristic of raw soybeans. Flavor is less of a problemin foods such as
meat analogs that contain spices and seasonings, but it becomes a serious
limitation in bland foods like dairy products. Nonetheless, progress has been
made. For example, soy protein isolates are used in several liquid coffee
356 / Advances in Cereal Science and TechnologJ'
whiteners, and a major company that traditionally used milk proteins is now
adding isolates to some of its products.
Several recent reviews have dealt with the flavor problem of soybean proteins
(Cowan et al., 1973b; Maga, 1973; Wolf, 1975).
Flavor of Commercial Proteins. Commercial soy flours, concentrates, and
isolates were surveyed for odor and flavor characteristics by a taste panel in 1970
as summarized in Table XVI. Evaluations were made on 2% dispersions in water,
and scoring was based on a I to 10 scale where I is a strong odor or flavor and 10
is bland. A raw, hexane-defatted flour included for comparative purposes was
scored the lowest. For the flours, the highest scores were obtained for those that
had received the greatest amount of moist heat treatment as measured by the
nitrogen solubility index (NSI). One of the concentrates received the highest
flavor score of the various products tested. Beany, bitter, nutty, and toasted were
some of the flavor descriptions given by the panel. For beany and bitter flavors in
the raw flour, respective threshold concentrations (levels at which 50% of the
panel detected the flavors) were 0.03 and 0.04% as compared to 1.25 and 3.0%for
one of the isolates. Despite the extensive processing given to isolates, the flavor
compounds are not removed completely.
Source of Flavor Compounds. Lipids have been implicated by a number of
workers as the origin of undesirable flavors in soybean products. For example,
Nelson et al. (1971) have postulated that intact soybeans are free of undesirable
flavors and that the flavor compounds are generated by lipoxygenase-catalyzed
oxidation of the oil as soon as the cellular structure is broken. Consequently, they
recommend blanching the beans before crushing them to inactivate the
lipoxygenases and thereby prevent the formation of malflavored compounds.
Grosch and Schwencke (1969) observed that incubation of linoleic acid with
partially purified lipoxygenase and oxygen resulted in the formation of 3.4%
monocarbonyl compounds in the following amounts:
Aldeh.vde
n-Pentanal
n-Hexanal
n-Hept-2-enal
n-Oct-2-enal
n-Nona-2,4-dienal
n-Deca-2,4-dienal
Unidentified
Mole, %
5
45
10
5
5
26
4
Hexanal is the major carbonyl compound found. All of these compounds plus
about 50 others were isolated from soy milk made in the traditional Oriental
manner (Wilkens and Lin, 1970). Hexanal comprised about 25% of the volatile
fraction isolated from soy milk. Many of the volatile compounds isolated have
grassy/ beany odors and have extremely low odor thresholds.
Qvist and von Sydow (1974) identified and quantitated the volatile
compounds in the heads pace of a solution of a soy protein isolate that was
unheated or heated. They found over 50 compounds in the headspace from the
unheated isolate. Major compounds found were:
Unheated, Heated,
Compound ppb ppb
Ethanal 2500 4250
I-Pentanal
68 780
Hexanal 260 1710
2-MethyIpropanal 62 230
2-Methylpropenal 68
21
2-Propanone 360
4400
2-Ethylfuran 130 4000
2-Pentylfuran 95 2700
Ethanal was the major compound found in the unheated protein. Heating
usually increased the concentrations of volatile compounds detected.
Two hydroperoxides are possible when linoleic acid is oxidized: 9-D-hydro-
peroxy- I0, I2-octadecadienoic acid and 13-L-hydroperoxy-9, I I-octadecadien-
oic acid. Leu (l974a, b) found that Iipoxygenase L-I (Classical Theorell
preparation) formed mainly the I3-isomer at high oxygen concentrations, pH 9,
and low temperature. However, at low oxygen concentration (l % O
2
in gas
phase), pH 6.8, and high temperature, about equal quantities of the two
hydroperoxide isomers were obtained. Analysis of the volatile compounds
present in oxidized linoleic acid (catalyzed by lipoxygenase L-I) revealed that
when the reaction was carried out in I% oxygen, a number of aldehydes and
hydrocarbons was observed. Prominent compounds were pentanal, hexanal,
and 2-n-pentylfuran. In an oxidation carried out with 100% oxygen, hexanal
again was a major component, but pentanal and 2-n-pentylfuran were formed in
smaller amounts than at the low oxygen tension.
Volatile compounds derived from the hydroperoxides oflinoleic and linolenic
acids are usually regarded as the source of malflavors, but a direct test of this
hypothesis was made only recently. Kalbrener et al. (1974) prepared the
hydroxyperoxides oflinoleic and linolenic acids by lipoxygenase catalysis at high
oxygen tensions. After purifying the hydroperoxides by silicic acid column
chromatography, they made dilute dispersions of the hydroperoxides in water
TABLE XVI
Odor and flavor scores for soybean protein products'
Product
Defatted flours
Concentrates
Isolates
Odor Scores
h
5.8-7.5
6.4-7.4
6.8-7.7
Flavor Scores
b
4.1-6.7
5.6-7.0
5.9-6.4
'Source: Kalbrencr et al. (1971).
bBased on a scale of I to 10 where I is strong and 10 is bland.
and submitted them to a taste panel. Linolenic acid hydroperoxide at 10 ppm and
linoleic acid hydroperoxide at 50 ppm were scored the same flavor intensity as
0.25% raw defatted soy flour dispersed in water. Grassy/ beany was the main
flavor description given. Minor flavor responses \vere bitter, astringent, raw
vegetable, and fruity. Because flavors given for raw defatted flour did not include
the last two terms, the hydroperoxide dispersions did not reproduce the typical
soybean flavors exactly. Nonetheless, the hydroperoxides seem likely sources for
the grassy/ beany flavors. Furthermore, the hydroperoxide flavor profile may be
sensitive to the oxygen tension prevailing during hydroperoxide formation.
Kalbrener and coworkers employed a high oxygen concentration, whereas
oxidation under ambient conditions (when the flavors in soybean products are
likely to be generated) may give a different spectrum of volatile compounds (Leu,
1974b).
Recent work by Grosch and Laskawy (1975) showed that lipoxygenases L-l,
L-2, and L-3 differ significantly in their abilities to form volatile carbonyl
compounds. The L-2 and L-3 enzymes formed greater quantities and a larger
variety of carbonyl compounds than L-l. They also presented evidence that the
volatile carbonyl compounds do not arise by decomposition of the
monohydroperoxides.
The hydroperoxides of linoleic and linolenic acids were reported to be bitter
(Kalbrener et al., 1974), but there is some doubt whether this is an important
source of this flavor in defatted soybeans. Sessa et al. (1974) have found that
phosphatidylcholine may be another source of bitterness in soybean flakes.
When they aut oxidized a purified phosphatide preparation for 2 weeks in
aqueous dispersion with 1 ppm of Cu++, it developed a bitter taste with a
threshold of 0.006%. In further work Sessa and coworkers (1975) isolated three
phosphatidylcholines from residual lipids found in hexane-defatted soybean
flakes. One of the phosphatidylcholines was weakly bitter when isolated from
fresh defatted flakes, but a comparable fraction 0 btained from l-year-old flakes
was much more bitter. The other two phosphatidylcholines were strongly bitter
when freshly prepared and tasted at a concentration of 0.05%. Fatty acids from
all of the phosphatidylcholines contained hydroxyl and conjugated keto groups
presumably as a result of oxidation of unsaturated fatty acids. It appears likely
that these phosphatides contribute bitterness to soybean flakes.
The conventional method for inactivating lipoxygenases in soybean products
is heat treatment, but other methods can also be used. Eldridge et al. (in press)
have found that lipoxygenase was inactivated when intact beans were soaked in
aqueous alcohol. When the alcohol was removed by vaporization and the beans
were ground, there was a distinct improvement of the flavor as compared to
beans soaked either in water or pure alcohol. Lipoxygenase (measured at pH 9.0
with linoleate) was inactivated with 20 to 80% ethanol (Figure 8), but the best
flavor scores were obtained over the narrower treatment range of 40 to 60%
ethanol. The possibility that the lipoxygenases that are specific for esterified
linoleic acid remain active above and below 40 to 60% alcohol has not been ruled
out. Figure 8 also shows that extensive insolubilization of protein occurs with 20
to 80% alcohol treatment as measured by the NSI test. Urease is extensively
inactivated in this alcohol concentration range, whereas soybean trypsin
inhibitor is fairly stable.
B. Functional Properties
Next to flavor, functional properties of soybean proteins have been one ofthe
important factors limiting their use in certain foods. Consequently, many applied
studies have been carried out, but published information occurs primarily in
patents. For example, almost all of the studies on texturizing soybean proteins
have been concerned with duplicating the chewiness and mouthfeel of various
meat products starting with a material with little resemblance to meat. Likewise,
several industrial laboratories have expended a great deal of effort to make
soybean proteins soluble in acid solutions so as to permit their addition to
carbonated beverages, fruit juices, and the like. Less effort has been expended on
basic studies related to functional properties.
Solubility. Hermansson (1973) compared solubilities of a commercial soybean
protein isolate, sodium caseinate, milk whey protein concentrate, and fish
protein concentrate as a function of pH in 0.2M sodium chloride. As expected,
the soy isolate had a minimum solubility in the pH region of 4 to 5, but in the pH
range of 6.5 to 8.0 only 25 to 30% of the protein dissolved. Only by increasing the
pH above 11 was high solubility obtained. She also examined the effect of ionic
strength at pH 7. In water the solubility was about 55%; but when salt was added
to 0.2M, solubility decreased to 28%. By comparison, sodium caseinate and
whey protein concentrates were not sensitive to changes in ionic strength up to
2M salt.
A later series of studies (Hermansson and Akesson, 1975a, b; Hermansson,
1975) described the effects of heat treatment of soy protein isolate, sodium
caseinate, and milk whey protein concentrate on solubility and other parameters
100
80
-
>-
-
:>
40
<C
20
x_
20 40 60
Alcohol, %
80
J" Lipoxyg",,,
x
100
Figure 8. Effect of alcohol concentration used to soak soybeans on lipoxygenase, urease, and trypsin
inhibitor activities and nitrogen solubility index (NS!). From Eldridge et al. (in press).
and their relationships to water binding properties of model meat systems.
Addition of unheated isolate to the meat systems had little effect on water loss,
whereas addition of the more soluble milk proteins resulted in increased water
loss. Heated dispersions of isolate gelled, and gelation showed a high negative
correlation with water loss from the meat systems. Effects of pH and ionic
strength on solubility of soybean globulins were described by van Megen (1974).
He observed an unusual liquid-liquid phase separation below critical values of
ionic strength. The upper phase (supernatant) was low in protein content,
whereas the lower phase was viscous and high in protein concentration. A
homogenous solution was obtained at ionic strengths above the critical value.
Tombs (1972) called the two layers mesophases and spun them into protein fibers
as discussed earlier.
Water solubilities for two commercial protein isolates were reported by
Hagenmaier (1972). At pH 6.0 one had a solubility of 22% and the other was 40%
soluble.
Water Absorption and Swelling. Hermansson (1972) has followed
spontaneous water uptake of proteins in an apparatus devised by Baumann
(1966) and uses the results as a measure of swelling properties. Swelling is limited
for proteins that do not dissolve; but for proteins that dissolve, swelling is
unlimited. The Baumann apparatus is a water-filled funnel containing a fritted
glass disk that contacts the water. To run the test, dry protein is sprinkled on a
wetted filter paper placed over the glass disk, and water uptake is followed as a
function of time by reading a calibrated horizontal capillary tube attached to the
funnel. Swelling of soy isolate is greater than for sodium caseinate or milk whey
protein concentrate. Although the swelling properties of the proteins did not
correlate with water binding in model meat systems (Hermansson and Akesson,
1975a), they did correlate with textural properties of meatballs containing the
proteins (Hermansson, 1975).
Water vapor sorption by isolated soybean protein is reported for three water
activities by Hermansson (1973). When the protein was heated, water sorption
increased very little but swelling increased significantly. Water sorption at 84%
relative humidity was compared for soy protein isolate plus other plant proteins
and several animal proteins (Hagenmaier, 1972). Animal proteins bound more
water than plant proteins, and binding appears to be a function of the polar side
chain content-hydroxyl, amino, and carboxyl groups.
Water absorption of soybean and sunflower flours, concentrates, and isolates
was measured by slurrying them in water or 5% sodium chloride and then
centrifuging (Fleming et al., 1974). Absorbed water was taken as the decrease in
volume offree liquid. Among the soybean proteins, isolates had the highest water
absorptions, followed by concentrates which were higher than flours. One
shortcoming of this test is that if a protein is completely soluble, it will show no
apparent water absorption; but if it is incorporated in a food system, it may be
insolubilized or gelled by heating and show excellent water absorption
characteristics.
Various protein additives, including six soybean proteins, were evaluated as
emulsion stabilizers in frankfurters (Smith et al., 1973). Water-holding capacity
was determined under conditions of salt concentration, heat treatment, and pH
simulating frankfurter manufacture. Despite attempts to duplicate processing
conditions, the water-holding measurements failed to predict performance
characteristics of the protein additives in frankfurters.
Viscosity. Hermansson and Akesson (1975a, b) measured viscosity of a
commercial soy protein isolate in water and in salt solutions. Addition of salt up
to 1M" sodium chloride to unheated and heated protein dispersions caused a
marked drop in viscosity. Water dispersions containing 10 to 12% protein gelled
when they were heated, and gel strengths were estimated with a Brookfield
viscometer. Weak gels formed when the soy isolate was heated in the presence of
salt. Gelation properties showed a high negative correlation with water loss from
model meat systems.
Viscosities of soybean and sunflower flours, concentrates, and isolates slurried
in water and 5% sodium chloride solutions were measured by Fleming et al.
(1974). Protein concentrations were varied from 5 to 20%. A "pH activation"
treatment was given the samples; it consisted of raising the pH to 12.2, holding
for a few minutes, and then reducing the pH to 6.0. An increase in viscosity
usually occurred after "pH activation." The high pH is likely to cause irreversible
unfolding of the protein molecules.
Emulsification. A model system of water, protein, and corn oil was employed
to measure emulsion capacities of soybean and cottonseed protein concentrates
(Crenwelge et al., 1974). Measurements were made in a Waring Blendor by
adding oil until the emulsion inverted as detected by a sharp drop in current
required by the blender motor. Blender speed, rate of oil addition, pH and
protein concentration were optimized, but the model was not compared to an
actual food system to determine its usefulness.
Emulsifying capacity and emulsion stability for a number of proteins,
including six of soybean origin, were compared in model systems and in
frankfurter formulations (Smith et al., 1973). Results of the model tests generally
were of little value in predicting performance of the protein additives in
frankfurters. Solubility of the protein is not a prerequisite for good
emulsification properties. Fish protein concentrate, in which the protein was
completely insoluble, formed stable emulsions. Apparently, the insoluble
particles were sufficiently small to collect at the oil-water interface and thereby
acted as a barrier to coalescence of the oil droplets.
Film Formation. A cream-yellow film forms at the surface of soy milk when it
is heated in flat shallow pans at about 90 C. The films can be lifted off with glass
rods and hung up to dry to yield yuba or soy milk skin which is a traditional food
made in the Orient. An extensive study of this film-forming process is reported by
Wu and Bates (1972a, b, 1973, 1975). Typical films obtained from milk contained
about 55% protein, 26% neutral lipids, 2% phospholipids, 12% carbohydrate,
and 2% ash on a dry basis. Studies with model systems showed that protein in the
heated solution is essential for film formation. Under optimum conditions of
batchwise film formation, about 70% of the total soybean protein can be
recovered, but 80% protein recovery is possible with semicontinuous film
formation. By applying heat and pressure, the folded films will fuse together to
form a firm, continuous, textured mass that can be sliced. Wu and Bates (1975)
have recommended use of the films as meat substitutes. However, formation of
yuba apparently causes destruction of cystine/ methionine. They obtained a
protein efficiency ratio (PER) of only 1.26 (casein = 2.50) for their yuba
preparations; PER increased to 2.45 when the diet was supplemented with 2.2 g
of methionine per 16 g of nitrogen (Bates and Wu, 1975).
Texture. Work on textured products has been largely concerned with
duplicating meat-like textures and is described primarily in patents as reviewed
earlier (Section VI-C). By comparison, very little has been done to gain an
understanding of the physical and chemical changes that occur at the molecular
level when textured materials are prepared.
Cumming and coworkers (1972) studied the effect of temperature over the
range of 110 to 190 C on extrusion of defatted soybean meal. They determined
the effects of processing temperature on product density, degree of rehydration,
shear force, work of shearing, and breaking strength. Extrusion converts the
soybean meal into a spongy, fibrous mass as revealed by scanning electron
microscopy. Cumming et al. (1973) have also determined the changes in
solubility and gel electrophoretic behavior that occur in the soybean meal
proteins when they are extruded.
Textural properties of meat and spun protein isolates were compared by
Stanley et al. (1972). They rehydrated dry fibers in boiling water and measured
load elongation, breaking strength, break elongation, elasticity, and stress
relaxation. Soy fibers had considerably higher breaking strengths and break
elongations than cooked meat.
Storage of spun soybean protein fibers makes them tough, inextensible, and
turns them from cream-white to gray. Chiang and Sternberg (1974) found that
the content of sulfhydryl groups in the fibers decreases on storage, and they
postulate that formation of disulfide cross-links causes the changes in physical
properties that occur during storage.
Gels with different textures are obtained by heating dispersions of partially
purified 7S and II S proteins. Gels made from crude II S protein are firmer and
more cohesive than gels prepared from 7S protein (Saio et al., 1973). Unique
textured products are obtained when solutions of the two protein fractions are
heated at 90 C, cooled to 70 C, coagulated by adding acid or CaCh, molded in a
wooden press, immersed in a buffer, and fmally autoclaved at about 130 C(Saio
et at., 1974). Crude II S protein yields gels that are more porous and much more
expanded than gels from crude 7S fraction.
Saio et al. (1975) have measured solubilities of the heated gels in buffers
containing sodium dodecyl sulfate and mercaptoethanol and examined the
solubilized proteins by gel electrophoresis and ultracentrifugation. The crude
lIS protein was more readily dissolved than the 7S protein, but the gel
electrophoresis and ultracentrifuge patterns were complex and difficult to
interpret.
C. Nutritional Properties
Nutritional properties of soybean proteins continue to be of interest because
there is concern about nutritional equality when soy products are substituted for
animal proteins such as meat, milk, and eggs. Availability of a variety of soybean
proteins made by different methods also raises questions about adequacy of
processing to inactivate antinutritional factors and the possible loss of essential
amino acids by fractionation during processing. Detailed discussion of
nutritional, biological, and physiological properties is found in recent reviews
(Liener, 1972; Rackis, 1972, 1974). Items selected for discussion here include:
biological effects of trypsin inhibitors and agglutinins; nutritional properties of
cereal-soybean blends, textured products, and infant formulas; and effects of
alkali treatment on soybean proteins.
Trypsin Inhibitors. Analysis of 108 soybean strains and varieties revealed an
almost fourfold range in trypsin inhibitor content, but none were free of inhibitor
activity (Kakade et al., 1972). Genetic variants of the Kunitz trypsin inhibitor
have been found by polyacrylamide gel electrophoresis (Hymowitz and Hadley,
1972). Electrophoretic analysis of 1,595 plant introductions in the USDA germ
plasm collection showed protein bands corresponding to trypsin inhibitor in
every sample. One form of the inhibitor predominated. This variant was found in
1,255 samples (Hymowitz, 1973) and occurs in the major commercial varieties
now grown in the United States (Clark et al., 1970). Attempts to breed out the
inhibitor therefore do not look encouraging at this time, unless trypsin inhibitor-
free germ plasm can be found.
Kakade et al. (1973) selectively removed trypsin inhibitors from soybean meal
extracts by affinity chromatography and fed the extracts to rats. Pancreatic
hypertrophy and growth inhibition still occurred, and the results accounted for
about 60% of the effects noted when trypsin inhibitors were present. Resistance
of the native (uncooked) soybean proteins to proteolytic digestion was
postulated as responsible for the residual pancreatic hypertrophy and growth
inhibition after the trypsin inhibitors were removed.
Because the effect of trypsin inhibitors on humans is still unknown, it is
recommended that soybean protein products be thoroughly cooked during some
stage of processing. Moreover, it was only recently learned to what extent the
inhibitors must be inactivated to make them innocuous to rats. An improved
assay was developed to determine residual trypsin inhibitor activity in heated,
defatted soybean flakes (Kakade et al., 1974). The procedure was then used to
assay for residual inhibitor in defatted flakes that had been steamed for varying
times to provide a series of flakes with graded levels of activity (Rackis et al.,
1975). When the various flakes were fed to rats, no pancreatic hypertrophy
occurred with flakes still containing 31 to 45% of the inhibitor activity of raw
flakes. However, maximum body weights, nitrogen digestibilities, and protein
efficiency ratios were obtained only when the flakes received more heat treatment
and had residual inhibitor activities of only 13 to 21% of the original flakes.
Species differences exist with regard to sensitivity to trypsin inhibitors. In
contrast to rats and other species, dogs can ingest 15% raw soybean meal without
harmful effects on body weights, pancreas weights, or pancreatic secretion
(Patten et al., 1971a, b).
Stability of trypsin inhibitors in various food products is still unknown, but
residual inhibitor appears to be no problem in cooked meat products containing
added soybean protein (Nordal and Fossum, 1974). They found that meat or
meat extracts sensitize trypsin inhibitor to heat. No detectable trypsin inhibitor
activity was found in a mixture of meat and protein isolate heated for only 5 min
at 90C.
Agglutinins. Although once considered as possible antinutritional factors in
soybeans, agglutinins are now of questionable significance in this regard. Turner
and Liener (1975) passed a buffer extract of raw, defatted soybean flour through
a column of Sepharose-bound concanavalin A which removed the agglutinin
activity but had no effect on the trypsin inhibitor activity. They fed the
agglutinin-free extract to weanling rats and found that rate of growth and protein
efficiency ratio were not significantly different from those values obtained with
the original extract that still contained the agglutinins. When they heated the
original extract, it gave a marked improvement in growth response similar to that
obtained by heating the raw soy flour.
Cereal-Soybean Blends. Several cereal-soybean blends have been developed
and evaluated in humans. One of the first of these was corn-soy-milk (CSM).
Feeding CSM (68% corn meal, 25% defatted soy flour, and 5% nonfat dry milk)
to infants showed that the protein blend had a biological value that was about 60
to 65% of that for casein (Graham et al., 1971). When they fed "instant
sweetened" CSM, which contains a higher ratio of soy protein to corn protein
(40% corn meal, 38% full-fat soy flour, 5% nonfat dry milk, and 15% sugar),
Graham and coworkers (1973) found that the blend had an excellent digestibility
and a nitrogen retention comparable to that of casein. The improved biological
value of "instant sweetened" CSM over CSM was ascribed to a more
complementary ratio of corn to soy proteins. Graham recommended the
modified CSM as a major source of protein in the diets of normal and
convalescing malnourished infants and children.
Feeding trials with infants and children revealed that wheat-soy blends had
satisfactory biological values but low digestibilities, whereas oat-soy blends had
excellent biological values but digestibilities only equal to the wheat-soy
products (Graham et al., 1972).
A macaroni fortified with soy flour has been tested extensively in Brazil.
Macaroni containing 60% corn meal, 30% defatted soy flour, and 10% wheat
flour was fed to malnourished children for 4 months. A control group was fed a
normal balanced diet containing 25 to 30% milk protein. The corn-soy-wheat
macaroni was well accepted and supplied 69% of the protein ingested by the
experimental group. Both groups of malnourished children recuperated, but the
control group responded better, possibly because it had a higher caloric intake
than the children on the macaroni diet. The macaroni was recommended as a
protein supplement and shows promise because of its good acceptance in the diet
(Beghin et al., 1973).
Protein supplementation of tortillas with soybeans has also been carried out
successfully (Bressani et al., 1974; DelValle and Perez-Villasenor, 1974). Various
ratios of corn and soybeans were cooked in limewater and converted into tortillas
in the traditional manner. Rats fed the soybean-fortified tortillas responded with
a marked improvement of the protein efficiency ratio. A 72% corn:28% soybean
mixture gave the maximum protein efficiency ratio. This application for
soybeans likewise appears promising because organoleptic evaluations indicated
that soybean levels below 20% were not significantly detected in tortillas.
Kies et al. (1975) carried out human nitrogen balance studies on blends of
textured soy flour and millet or triticale flours. Textured soy flour by itself or
blended with millet gave a negative nitrogen balance, but a blend of textured soy
Chemistry and Technology of Soybeans ! 365
flour and triticale gave a positive nitrogen balance.
Nutritional Value of Textured Products. Textured flours and isolates have
been used extensively in the human diet, and recent studies with humans have
revealed no untoward effects.
Nitrogen balance studies with adult males have compared textured soy flour
(with and without 1% DL-methionine) with beef (Kies and Fox, 1971). The
subjects remained in positive balance with all three diets when they received 8.0 g
of nitrogen per day, but went into a negative nitrogen balance when they ingested
only 4.0 g nitrogen per day. At this low nitrogen intake, beef, textured soy flour,
and textured soy flour fortified with DL-methionine had respective nitrogen
balances of -0.30, -0.70, and -0.34 g N per day. In a second study Kies and Fox
(1973) fed mixtures of beef and textured soy flour in varying ratios at an intake
level of 4.0 g N per day. As the level of textured soy flour increased, a
progressively more negative nitrogen balance resulted. These results confirmed
the nutritional superiority of beef protein over soy flour proteins, but no mutual
supplementation occurred. It is, therefore, likely that methionine is also a
limiting essential amino acid in beef. The comparison is biased in favor of beef
proteins because their nitrogen-to-protein conversion factor is about 6.25,
whereas the factor is only 5.7 for soybean proteins. Consequently, equal amounts
of nitrogen are not equal to the same weights of the two proteins.
Poullain and coworkers (1972) fed 20 g of protein in the form of textured soy
flour in place of meat protein to human subjects on a normal diet. No significant
effect on nitrogen balance was observed, although the meat diet gave a higher
positive nitrogen balance.
Morse et al. (1972) compared spun isolate fibers against casein-lactalbumin,
but adjusted both diets to the provisional FAO essential amino acid pattern by
adding crystalline amino acids. The young men in the tests showed no significant
differences in nitrogen balance; apparently both protein sources were equally
well-utilized by the subjects.
Soybean Proteins in l ~ f n t Formulas. Infant formulas containing soy proteins
have been developed primarily for children that are allergic to cow's milk, but
they have also been tested for treatment of infants and children suffering from
malnutrition. Graham et al. (1970) fed a formula containing isolated soy protein
fortified with DL-methionine in initial therapy of malnourished infants and
children and during their convalescence. The soy-based formula performed well;
growth rates plus absorption and retention of nitrogen were equivalent to those
obtained with children receiving modified cow's milk.
Excellent results with soy formulas have also been obtained with normal
infants. Fomon and coworkers(1973) placed 13 normal female infants from8 to
II days of age on a diet in which 90% or more of the amino acids were supplied by
soy protein isolate supplemented with DL-methionine. For the infants on the
soybean protein diet, clinical observations, serum albumin concentrations, and
growth rates were similar to those of infants on milk-based formulas that
supplied larger protein intakes.
Alkali Treatment of Soy Proteins. Recent work suggests that alkaline
treatment of proteins, as described in many patents on preparation of edible
protein products, causes destruction of certain amino acids and leads to
formation of Iysinoalanine, a new, unusual derivative of lysine.
When soybean meal was treated at pH 12.2 for 4 hr at 40C, deGroot and
Slump (1969) found that there was a loss of lysine and formation of
lysinoalanine. Feeding studies with rats showed that net utilization of the alkali-
treated protein was decreased and that about 40% of the ingested lysinoalanine
was excreted mainly in the feces. They also treated isolated soybean protein with
alkali at different pH values, times, and temperatures and determined the
lysinoalanine content of the protein. They found that only 1 hr at 40 C and pH
12.2 was sufficient to form lysinoalanine. However, no lysinoalanine was found
after treatment for 4 hr at pH 8 and 40 C. Detectable quantities oflysinoalanine
were noted after 4 hr at pHI 0 and 40 C; de Groot and Slump also noted that
some of the female rats developed nephrocalcinosis, but this was overcome by
adding more calcium to the diet.
Formation of lysinoalanine in alkali-treated soybean protein was
subsequently confirmed by Woodard and Short (1973). Earlier Woodard and
Alvarez (1967) observed that when an industrial grade of soybean protein
(modified by alkali treatment to change physical properties) was fed to rats, they
developed a nephrotoxic reaction characterized by enlarged nuclei and increased
cytoplasmic portions of the cells in the pars recta of the proximal tubule. This
renal lesion was designated as cytomegalia. Woodard and Short (1973) found
that commercial edible soybean protein isolate contained no detectable
quantities of lysinoalanine and failed to induce renal cytomegalia. However,
treatment of the edible protein isolate with 0.1 N sodium hydroxide at 60 Cfor 8
hr resulted in the formation of 2.6% lysinoalanine in the protein; and when the
treated protein was fed to rats, they developed cytomegalia.
Subsequently van Beek et al. (1974) fed spun fibers made from soy protein
isolate to rats. The spinning process includes an alkali treatment step and the
fibers contained about 0.2% lysinoalanine. At highest protein level, the diets
contained 0.04% lysinoalanine. No adverse effects were noted except for
nephrocalcinosis in females; this effect was eliminated by increasing the calcium
or decreasing the phosphorus content of the diet. Renal cytomegalia was not
observed in any of the rats.
Woodard and Short (1975) fed synthetic lysinoalanine at levels in the diet
ranging from 0 to 0.3% and found that in 4 weeks renal tubular cells of all rats
exhibited a karyomegalic reaction. At the high levels, cellular necrosis and
tubular destruction were also noted. Apparently, the free amino acid is more
toxic than the peptide-bound form found in alkali-treated proteins. Related
studies by deGroot and coworkers (1975a) showed that diets containing 0.14%
lysinoalanine in the form of alkali-treated soybean meal, isolated soybean
protein, or casein caused no pathological changes in rats. However, industrial
soybean protein isolate incorporated into diets to provide comparable levels of
lysinoalanine induced cytomegalia. Rats fed diets containing either synthetic
lysinoalanine or an acid hydrolysate of alkali-treated soybean protein isolate
exhibited a severe tubular nephrosis characterized by necrosis, regeneration, and
cytomegalia of epithelial cells. Free lysinoalanine appeared to be at least 10 times
more active than the peptide-bound form. The free amino acid is excreted mainly
in the urine.
Toxicity of lysinoalanine is thus confirmed in two laboratories, although there
are still discrepancies in the degree of toxicity observed. Clearly, the use of alkali
in treating soybean proteins should be minimized and carefully controlled.
VIII. FUTURE RESEARCH NEEDS
A. Production
Soybean production was discussed only briefly and merely to indicate the
magnitude of the crop presently available and projected needs for the near future.
Yields of soybeans in the U.S. have increased only about 0.3 bushel per acre per
year, and increased demands in the past have been met primarily by expanded
acreage. Because there is a limited amount ofland available, emphasis must be on
increasing yields; this problem is deservedly receiving much more attention than
it has in the past (Caldwell, 1973; Steyn, 1975).
B. Seed Ultrastructure
Progress has been made in elucidating the gross structure of soybeans, but
many of the fine architectural details of the cotyledon cells are still missing. For
example, where are the various enzymes and other minor constituents (saponins,
isoflavones, oligosaccharides, and phytate) located in relation to the protein
bodies and spherosomes? How are these relationships altered during processing?
A better understanding of the original soybean structure at the cellular and
subcellular level and how it is modified by present processing
practices-cracking, flaking, extracting with hexane, desolventizing-may
provide insights for new approaches to processing. The spherosomes of soybeans
have not been characterized yet. Presumably they are similar to those from other
oilseeds which have been studied; their composition, stability of the membrane
surrounding them, and susceptibility to enzyme attack are still unknown.
C. Seed Constituents and Their Properties
A number of minor constituents of low molecular weight-isoflavone
glucosides, acylated sterol glucosides, phytates-have known structures, but
others such as the saponins are still only partially characterized. These detergent-
like molecules are bound tightly to soybean proteins, but we do not know what
effects they have on the properties of the proteins.
Among the polymeric constituents of soybeans, the polysaccharides are least
understood. Further fractionation of these complex molecules is needed.
Properties such as digestibility, water-holding capacity, and ion exchange
capacity (McConnell et al., 1974) should be determined on purified polysac-
charide fractions as well as on the crude mixture. Extensive testing with humans
is necessary to establish whether soybean "fiber" is a desirable dietary
constituent.
The proteins have received more attention than the polysaccharides, but we
still do not know how many different proteins exist in soybeans. Further
separation, characterization, and quantitation of individual proteins is desirable,
particularly those that occur in the 2S, 7S, and ISS uItracentrifugal fractions.
The latter has been suggested to be a polymer of the I IS protein, but this needs to
be confirmed. Quantitation of individual proteins is important because we do not
yet know how much of the total protein is accounted for by the proteins already
isolated and identified. Although progress has been made by using gel
electrophoresis and isoelectric focusing, there is confusion in the literature about
the interpretation of protein bands detected by these techniques, and it is often
impossible to compare results reported by different workers. Standardization of
reliable techniques would be helpful. Origin of microheterogeneity of soybean
globulins (Catsimpoolas and Wang, 1971) needs to be established; is it an artifact
of the isolation and analytical techniques employed or is it an inherent property
of the native proteins?
Soybeans undoubtedly contain a large number of enzymes, yet we know little
about most of them except for a few such as the lipoxygenases. The proteolytic
enzymes are still a mystery and may be causing unsuspected modification of the
major proteins during their isolation. The high content of protease inhibitors in
soybeans suggests that the proteases are present as inactive complexes with their
inhibitors, but there is little substantial evidence for this view at present.
Because of their implied role in the flavor problem of soybean protein
products, lipoxygenases deserve much more attention. For example, what is the
importance of lipoxygenase catalyzed oxidation vs. autoxidation of
polyunsaturated ~ t y acids? Lipoxygenase L- I has a preference for free linoleic
acid as its substrate whereas the L-2 and L-3 enzymes apparently prefer the
esterified form of the substrate (Table XI). To what degree do the two types of
enzymes contribute to formation of flavor compounds? Is lipase or
phospholipase activity involved prior to oxidation of linoleic and linolenic acids
by lipoxygenase L-l? Answers to these questions would provide a better basis for
developing improved protein products for edible use. Likewise, there is a need to
understand the mechanism whereby volatile carbonyl compounds are formed
from linoleic and linolenic acids under the influence of lipoxygenases (Grosch
and Laskawy, 1975).
D. Technology
A number of protein products, especially meat extenders and the like, have
been developed and introduced, but long-term success of these products will
require further improvements in texture and flavor. Methods for measurement
of textural properties are needed, but studies on the physical and chemical basis
fortexture are almost nonexistent. The problems encountered in the treatment of
soybean proteins with alkali have been discussed, but more information
concerning the possible toxicity of such products to humans is a necessity. The
conditions under which lysinoalanine is formed need further investigation,
particularly in relation to processes where alkali is used.
ACKNOWLEDGMENT
Dr. T. Ikenaka supplied copies of Figures 3 and 4 and Dr. D. M. Blow provided a print of Figure 5.
Chemistry and Technology of Soybeans I 369
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Reprinted from Advances in Cereal Science and Technology
1: 325-377. 1976
Pub.by
of Cereal Chern.,
st. Paul} MiIm.

Chemistry and Technology of Soybeans

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Chemistry and Technology of Soybeans

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CHAPTER 6

CHEMISTRY AND TECHNOLOGY

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