Chemistry and Technology of Soybeans

8/12/2019 Chemistry and Technology of Soybeans

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CH PTER

CHEMISTRY N TECHNOLOGYOF SOYBEANS

W. J. WOLFNorthern Regional Research Laboratory Agricultural Research ServiceU.S. Department Agriculture Peoria Illinois

I INTRODUCTION

In the United States soybeans have emerged from relative obscurity as anoilseed to one of our major cash crops in less than 50 years. Official U.S.epartment of Agriculture estimates of soybean production date back only to1924when harvested production was 5 million bushel. Today this quantitywouldbe enough for only a few days operation of the soybean processing industry andis equal to only about 1 of the soybeans that were exported in 1974.Commercial interest in soybeans initially was concerned with processing into oilfor 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 thedefatted meal is a valuable protein source for poultry and swine as well as forcattle. Today, the major portion of the defatted meal is still used for feeds.Food uses for soybean protein in the United States have developed moreslowly than markets for the oil. For example, in 1973 domestic consumption ofedible soybean oil was 6.8 billion Ib which is equivalent to about 635 millionbushels of soybeans or 41 of the crop grown that year. In contrast, less than 2of the crop (primarily from defatted flakes as the starting material) was convertedinto edible protein products for domestic consumption. The bulk of soybeanproteins is converted into animal proteins which are still preferred over plantproteins. However, conversion of soybean proteins into meat, eggs, and milk isinefficient; as a result, animal proteins are more expensive than those ofsoybeans. Worldwide food shortages in recent years have caused sharp price risesin animal proteins used in the food industry, and at times supplies have beenuncertain. This situation and anticipated future trends have prompted a numberof food companies to begin replacing animal proteins in their products and tolMention 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


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6 dvances in ereal Science and Technologydevelop new items based on soy and other plant proteins.Interest in soybeans for food uses has stimulated research and development inmany laboratories and an increase in published information on theircomposition and properties. My review summarizes much of this informationpublished in the last 5 years. In addition, I have included a brief review ofsoybean production and disposition of the crop to acquaint the reader with themagnitude of the soybean supply available for food use and how it is utilizedtoday. For earlier work, particularly on soybean proteins, the comprehensivereview of Smith and Circle 1972 should be consulted. A complementarymonograph covering agronomic aspects of soybeans genetics breeding,varietal development, management practices, pests, and diseases is alsoavailable Caldwell, 1973 . Recent summaries of food uses of soybeans havelikewise appeared American Soybean Association, 1974; Wilding, 1975; Wolfand Cowan, 1975 .

II, SOYBEAN PRODUCTIONA U.S. Production

Successful development of markets for soybean oil for edible purposes and16

14

12= 1c:E 8c=> ;: ; 6>:::Q ,

4

2

0194 195 196 197e r

Figure J Soybean production in the U.S, for 1940-74. From Agricultural Statistics 1972 andAmerican Soybean Association 1975 ,


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hemistry n Technology of oybeans / 327meal for animal feeds over the past half century has led to a phenomenal growthof the soybean crop in the United States. Starting from a crop of about 5 millionbushels in 1925 production increased to 78 million bushels in 1940. Figure 1shows how soybean production has grown since 1940.The largest crop on record, 1,567 million bushels, was harvested in 1973 as aresult of an unprecedented 296 million bushel increase over the previous year.This expansion is outstanding when one considers that it is greater than theaverage for crops harvested in 1950-53. The increase in 1973, however, wasfollowed by an even greater decrease in 1974, but this was largely the result ofadverse weather wet spring, dry summer, and early frost. Harvested acreage in1974 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 theearly 1950 s:

Economic development in many parts of the world with accompanyingaffluence has caused a shift in diets from plant products such as cereals to moreanimal products, particularly poultry meat.2. After World War II, surplus production of wheat, feed grains, and cottonled t o acreage restrictions for these crops. Consequently, much of this landbecame available for growing soybeans.3. Favorable conditions for world trade have resulted in the development of alarge 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 andmeal for feeding livestock and poultry.The majority of U.S. soybeans are grown in the Corn Belt. Illinois, the numberone producer, is followed closely by Iowa Table I). These two states grew onethird of the total crop in 1974. Sizable quantities of soybeans are also produced inthe S outh where cotton acreage has declined since World War Production of soybeans in the U.S. as compared to the major cereal grains isgiven in Table Soybeans are the third largest grain crop; only corn and wheatare grown in larger quantities. Nonetheless, soybeans outproduced the cereals ona protein basis in 1973 Table II), and the resulting protein was of greaternutritional value because of a better amino acid balance, especially with regard tolysine.

World roductionThe U.S. grows about three-fourths of the world soybean crop Table III). The1974 soybean crop in the U.S. was grown on 52.5 million acres but gave asubnormal yield of 23.7 bushels/acre because of unfavorable weather. Brazil hasshown substantial increases in production over the last two years. Argentinalikewise has greatly expanded its crop but is still far behind Brazil because of thesmaller number of acres planted. Yields per acre are highest in the U.S., CanadaBrazil, and Mexico, whereas some of the lowest yields are reported for the Asiancountries where soybeans were first domesticated.

C. uture roduction TrendsSteepness of the curve in Figure 1 indicates that size of the U.S. soybean crop


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328 / Advances Cereal Science and Technologywill continue to e xpa nd in the future, a lth ou gh it is uncertain whether rate ofgrowth can continue as noted over the past decade. Early in 1973 Kromer madeprojections for the U.S. soybean crop for the 1980 s Table IV). Based on nannual increase in production of 65 million bushels or bout 4 p er year, hepredicted respective crops of 1.8 billion bushels and 2.2 billion bushels for 1980and 1985. These projections were made prior to the record soybean pricesreached in une 1973 and before the energy crisis ofl te 1973. The analysis alsoassumed a continued growth in domestic and foreign demand for food fats andhigh-protein meals for animal feeds. High prices for soybeans since mid-1973 andthe slowdown in economic activity, both domestically and abroad, havedecreased the demand for soybeans. Nonetheless, harvested acreage in 1973 56.4million acres) already equaled the acreage projection for 1980. Failure to reachproduction estimates for 1980 and 1985 will likely be caused by yields below theestimated levels Table IV) rather than reductions in harvested acreage.

TABLE IMajor soybean producing slates 1974

StateIllinoisIowaMissouriIndianaMinnesotaArkansasOhioMississippiLouisianaNorth Carolina

Acreage Harvested1,000 acres8.5007,0704,3503.9104,0404.3003.2002,5501.7601,450

Productionmillion bu213198104988986494235

Source: American Soybean Association 1975).

T AB LE IIU.S. produclion soybeans and cereals r 1973Production

Crop Seedmillion bu Proteinmillion tonsSoybeans I,567Corn 5,643Wheat 1 711Sorghum 937Oats 664Rice 206Rye 26

Seed production data from Agricultural Statistics 1974).

18.815.87.33.31.31.30.40.1


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T LE Acreage yield per acre n production o fsoybeansin major producing countries/or 1972-74

Acreage Yield Pe r Acre Production1972 973 1974 1972 1973 1974 972 973 974Country 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 1,243,921Brazil 5,770 7,524 10,425 23.3 24.4 24.7 134,702 183,719 257,206People s Republic of China 20,756 19,800 11.2 12.4 ... 231,485 246,183 248,020Indonesia 1,693 1,726 11.2 11.3 18,923 19,437 20,209Argentina 68 395 838 7 25.3 20.8 2,866 9,994 17,453Mexico 593 756 605 23.2 24.8 23 13,779 18,739 13,963South Korea 702 77 946 11.7 11.7 12.5 8,231 9,039 11,868Canada 405 474 450 34.0 30.8 24.5 13,779 14,587 11,023Estimated world total 1,755,394 2,127,570 1,891,676Source: American Soybean Association (1975).

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33 / Advances in Cereal Science n TechnologyTABLE IV

U.S. acreage yield. and production of soybeans projectedfor 1980 and 1985

Item Unit 1980 1985Acreage harvested Million acres 56 62YieId per acre Bu 32 35Production Million bu 1.800 2,150Source: Kromer 1973 .

TABLE VDisposition of soybeans 1973 and 1974 and projections for 1980 and 19851973 1974 1980 985

Item million buCrushings 82 725 960 .l00Exports 539 465 750 950Seed 58 67 7

79Other 8 23 29

Total disposition 1,436 1.269 1.800 2,150Source: Fats and Oil Situation 1975 and Kromer 1973 .

TABLE VIDomestic use of soybean oil 1973

UseFoodShorteningMargarineCooking and salad oilsOther

TotalNonfoodPaint and varnishResins and plasticsOther drying oil productsOtherFoots and losses

TotalTotal domestic useSource: Fats and Oil Situation 1974 .

Amountmillion Ib

2,185L5I43,070

26.781

977543277

4937,274


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Chemistry n Technology of Soybeans / 331Soybean production in Brazil is also expected to keep growing and willprobably continue to do so at a more rapid rate than anticipated in Kromer sprojections. Brazilian soybeans compete strongly with U.S. soybeans in theexport markets.Other commodities that compete with soybeans to provide oil and protein are:Philippine copra, Malaysian palm oil, African peanuts, Russian sunflowers, andPeruvian anchovies.

III. DISPOSITION OF THE CROPTable V shows how the 1973 and 1974 soybean crops were disposed of, plusprojections for 1980 and 1985 by Kromer (1973). About 57 of the soybeanswere 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 ) ofthe cropwas exported in 1973 and 1974 as beans; oil and meal were also sold abroad. Inthe 1973-74 marketing year 5.533 million tons of meal (equivalent to 233 millionbushels of beans or 15 of the crop) and 1.435 billion Ib of oil (equivalent to 134bushels of beans or 9 of the crop) were exported. Thus, about one-half of thesoybeans were exported either as beans or processed products.Domestic use of soybean oil is primarily as a food. In 1973 (Table VI edibleproducts accounted for disappearance of 6.781 billion Ib of oil or 93 of thetotal. 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 thanconverted into foods. Direct food use ofprotein in the meal is a small but growingsegment of soybean utilization. Estimates for soybean proteins produced for useas food ingredients in 1974 are given in Table VII. Flours and grits are the majorsoybean protein products added to foods. In terms of soybeans, the total

TABLE VIIEstimates of soybean proteins produceds foo ingrediellls n 1974 and projections for 985

Protein Product million Ib 1974 million bub million Ib 1985 million bub

Totals 1130 26.5

Flours and gritsConcentratesIsolatesTextured productsFlours and gritsIsolates

9007060

901

19.22.34.3

0.7

2000 43.9500-600 16.6-19.9400-500 28.6-35.8

400-500

3300-3600 89.1-99.6Source: Johnson (1975).bExpressed as equivalents of bushels of soybeans.Included in figure for flours and grits in first line.


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332 / Advances in Cereal Science n Technologyconsumption was equivalent to 26.5 million bushels or 2.1 of the 1974 cropTable III). Projections for 1985 are also given in Table VII. Increases of aboutthree- to fourfold are expected; but in equivalents of soybeans, usage is projectedto be less than 5 of the crop predicted for 1985 Table IV). Clearly, diversion ofsoybean proteins from animal feeds to human foods is still on a very small scale.Nonetheless, such diversion will gradually increase as animal proteins continueto rise in price and as soy protein products are improved in flavor, functionality,and nutritive properties.

IV. SEED ULTRASTRUCTUREEarly studies of soybean ultrastructure by transmission electron microscopy

Saio and Watanabe, 1968; Tombs, 1967 have been confirmed and extended bvscanning electron microscopy. .A. Protein Bodies and Spherosomes

When a soybean cotyledon is fractured by freezing in liquid nitrogen and thenexamined in a scanning electron microscope, one observes that much of thefracture surface is covered with a spongy layer of spherosomes and cytoplasmicnetwork Figures 2A and B . the fracture surface is first washed with hexane,the oil in the spherosomes dissolves and is removed, thereby leaving only thehoneycomb-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 variousprocessing conditions are still unknown. Techniques such as were used to studyspherosomes 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 proteinbody fraction contained amorphous material plus spherical particles 1 3 }lm indiameter. Although the starting defatted flour contained numerous proteinbodies larger than 1 3}lm in diameter, none of the bigparticles were observed inthe isolated fraction. Presumably the large protein bodies disrupted and formedthe amorphous material found with the small protein bodies.

B. Location of Cellular ConstituentsLittle is known about the cellular location of various enzymes such aslipoxygenase and urease, the oligosaccharides and minor constituents found in

soybeans sterols isoflavones, and saponins. Tombs 1967) found that trypsininhibitor did not sediment with the protein body fraction; hence, it is probablylocated in the cytoplasm. However, the possibility of leaching out of solubleconstituents such as trypsin inhibitor from the protein bodies during densitygradient centrifugation cannot be ruled out in the light of the instability of thelarge protein bodies discussed earlier.Nonaqueous separation methods would be desirable for isolation of cellularcomponents to minimize migration of water-soluble constituents. Recent use offluorocarbon-hexane mixtures to fractionate ball-milled soybeans Finley et al


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hemistry n Technology of oybeans / 3331974) may be pertinent in this regard. Finley and coworkers centrifuged adispersion of full-fat soy flour in a 9: I mixture of fluorotrichloromethane:hexane containing a trace of acetic acid; the solvent had a density of 1.424. Aftercentrifuging, three fractions were obtained: a floating layer, a supernatant, and apellet layer. he floating layer contained 82 protein which equals the proteincontent of crude protein bodies obtained by sucrose density gradient

Figure 2. Scanningelectron micrographs of soybean cotyledon fracture surfaces showing: A) proteinbody 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 OOOXStructures labeled are protein body PB), spherosomes S), and cytoplasmic network CN). romWolf and Baker 1975).


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20 Mean 7.4 4 1 0.2 3 1 Hymowitz et al 1972aRange of mean 5 6 9 9 2 5 6 5 0 1 0 6 1.9-5.1

20 Mean 7.9 4.8 0.5 2.6 Hymowitz et al 1972aRange of mean 5.9-10.8 3 5 7 6 0 1 0 9 1 9 3.5

20 Mean 8.6 5.4 0.5 2.8 Hymowitz et l 1972aRange 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 1972bRange 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 DeMan et al 1975Range of mean 1.6-9.8 0 5 8 0 0 8 0 2 2 5

TABLE VIIIOl e;osaccharide cOlllellt dillerelll soybeall varieties

StrainsMaturity groups00 and 0

Maturity groupsI and II

Maturity groups and IV

Maturity groupsOO IV

Varieties fromsouthern Ontario

No. LinesAnalyzedTotalSugar Sucrose Raffinosc Stachyose

g/IOO g seed Reference

ww,j:>.::t:t:l\ lSQt:l-..i\;\ lt :>t:ll:l

\ loy


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Chemistry n Technology of Soybeans / 335centrifugation Tombs, 1967). The supernatant contained the oil, whereas thepellet layer appeared to be primarily carbohydrate and contained only 1.4protein. By careful adjustment of density, it may be possible to separate theprotein bodies in a relatively pure form by this method.

V SEED CONSTITUENTS AND THEIR PROPERTIESStudies on soybean composition and characterization of constituents havecontinued in the last 5 years and have ranged from relatively simple moleculessuch as the oligosaccharides to the highly complex polysaccharides and proteins.

A CarbohydratesOligosaccharides. Implication of raffinose and stachyose as causes offlatulence when soybeans or soybean flour are eaten Cristofaro et al. 974;Rackis, 1974) has prompted surveys in search of varieties that are low in thesesugars or completely free of them. Hymowitz et al. 972a) analyzed 6 selectedsoybean lines from Maturity Groups through IV able VIII). None of thelines analyzed were free of raffinose or stachyose. n a companion study,Hymowitz and coworkers I 972b) examined three soybean strains from each ofMaturity Groups to IV grown in different geographical areas Table VIII).Again, all samples contained raffinose and stachyose.More recently, eMan et al 1975) analyzed 55 samples representative ofvarieties grown in southern Ontario. They soaked the soybeans in water forseveral hours and then prepared soy milks from them. Analysis ofultrafiltrates ofthe milks for sugars gave results summarized in Table VIII. They observed fivesamples 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 enzymatichydrolysis during the soaking prior to conversion to soy milk, but it seemsunlikely that these sugars would have disappeared completely in a few hours.Oligosaccharides are still detectable in soybeans after 48 hr of germinationAbrahamsen and Sudia, 1966; East et al. 1972). The work of eMan and

coworkers, if confirmed, offers some hope that soybean varieties low inoligosaccharides may be developed.At present, oligosaccharides are removed from commercial soybean proteinproducts by extraction methods as in the preparation of concentrates andisolates. There is industrial interest in the use of a-galactosidase preparations tohydrolyze raffinose and stachyose, and their application to soybean preparationshas been described Sherba, 1972). A thermostable a-galactosidase was isolatedfrom Bacillus stearothermophilus Delente et al. 1974), immobilized on nylonmicrofibrils, and used in a flow-through continuous reactor to hydrolyzeraffinose in beet sugar molasses Reynolds, 1974). Release of reducing sugars,however, may cause browning reactions and problems with palatabilityCristofaro et al. 1974 .PoZvsaccharides. Kikuchi et al. 97 Ia) isolated the cell wall polysaccharidesfrom 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


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336 / Advances in Cereal Science n Technologyapproximately 30 pectic substances, 50 hemicelluloses, and 20 celluloses.Cooking for 30 min at 12 0 C apparently converted the pectic materials, whichcement together the cell walls, from an insoluble to a soluble form therebvcausing separation of cells from each other. -

In a related study, the three polysaccharide fractions were treated with a crudeenzyme preparation obtained from Aspergillus sojae one of the organisms usedin making soy sauce by fermentation (Kikuchi et al 1971b). The hemicellulosefraction was easily hydrolyzed; the pectic fraction was hydrolyzed to a low degreewhereas the cellulose was not attacked. Polysaccharides found in sov sauceprepared by fermentation apparently are derived from the cell wall p ~ t i n sFurther studies of the effect of cooking soybeans on the polysaccharidefraction revealed that a hot water extract of cooked beans contains anarabinogalactan and two acidic polysaccharides (Kikuchi, 1972). One acidicpolysaccharide contained 29 anhydrogalacturonic acid and is thought to be themain component of the cell wall matrix. The other acidic polysaccharide with a

71 anhydrogalacturonic acid content was assumed to be derived from themiddle lamella between cells.Claims of a causal relationship between a lack of fiber in the human diet and anumber of diseases, especially those of the bowel, have focused attention on thefiber fraction in plant foodstuffs (Burkitt et al 1974; Eastwood, 1974). Soybeanpolysaccharides perhaps could serve as a source of dietary fiber in processedfoods. They are already present in flours and grits, as well as in concentrates, andare obtained as a by-product remaining after aqueous extraction of defattedflakes in the preparation of protein isolates. At present this by-product isdisposed of by adding it to animal feeds.

B ipi sChanges during Development of Soybeans Complex changes occur in thecomposition of fatty acids and lipid classes in developing soybeans from 9 daysafter flowering until maturity (Privett et al 1973; Wilson and Rinne, 1974). Theimmature bean is almost free oftriglycerides, and the major lipids are glycolipids

and phospholipids. The lipid extracts from the developing bean also containappreciable amounts of unidentified materials which decrease in amount as thebean matures. N-Acylphosphatidylethanolamine may be one of theseunidentified compounds; it is themajor phospholipid of immature soybeans, butdecreases rapidly to a low level at maturity (Wilson and Rinne, 1974). Duringdevelopment of the bean, there is a rapid synthesis of triglycerides accompaniedby a drop in the percentage of saturated fatty acids and a rise in content of oleicand linoleic acids. The percentage of linolenic acid in the lipids is high initiallyand decreases as the bean matures. nearlier study byRoehm and Privett (1970)showed that the triglycerides from immature beans contained almost 5trilinolenin, but this molecular species disappears completely by 4 days afterflowering.Composition n Fractionation of Commercial Lecithin Two commercialsoybean lecithins were fractionated by thin-layer chromatography and liquidchromatography Erdahl et al 1973). The lecithins contained about 82phospholipids consisting primarily of phosphatidylcholine, phosphatidylethan-


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Chemistry and Technology of Soybeans / 337olamine, and phosphatidylinositol. The remainder comprised virtually all of thelipid classes found in soybean oil. About 2 dozen components were found bythin-layer chromatography of the polar lipids, and unknown compounds madeup about 10 of one of the lecithin samples. This study illustrates the complexityof 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-acylphosphatidylethanolamine followed by extraction into acetone Aneja et al 1971). Theneutral lipids are also soluble in acetone; hence, the residual lecithin fract ionconsists mainly of phosphatidylcholine and phosphatidylinositol which do notdissolve in acetone.

C rot insStudies on Unjractionated Proteins Hill and Breidenbach 1974a)fractionated the buffer pH 7.6, 0.5 ionic strength) soluble proteins of soybeansby sucrose density gradient centrifugation and obtained separations that agreedwell with those reported by earlier workers using the analytical ultracentrifuge.

The density gradient method however, has the advantage that the fractions areseparated from each other and can be recovered for further characterization. Hilland Breidenbach analyzed their protein fractions by polyacrylamide gelelectrophoresis and found one band for the S fraction but three bands for the7S fraction. They made a surprising observation; when the sucrose densitygradient centrifugation was conducted at 1 ionic strength, the 9S fractionbelieved to be dimer of a portion of the 7S species at 0.5 ionic strength) stillconsisted of three gel electrophoretic components. Apparently, there are threeelectrophoretically distinct proteins capable of dimerizing at 1 ionic strength.Hill and Breidenbach 1974b) also followed accumulation of the major proteinsduring seed development and maturation. The 2S fraction predominated early inseed development; but by 23 days after flowering, the density gradient centrifugepattern closely resembled that of the mature seed.

Comparison of soybean globulins, obtained by isoelectric precipitation, withthe proteins found in the protein bodies revealed no significant differences asmeasured by gel filtration, ultracentrifugation, and isoelectric focusingKoshiyama, 972a).Extraction of soybean proteins from defatted meal at pH 4.5 is possible ifsaltsare added to solubilize the globulins Anderson et al 1973). Proteinextractability increases as the salt concentration is raised until a maximum of65 of the meal nitrogen is solubilized. Maximum extraction occurs with 3Ncalcium chloride or 7Nsodium chloride. Proteins in the 2S and 7S fractions

appear to be insolubilized by the pH 4.5 treatment and they are not solubilizedby salts.In a study of solubility of isolated globulins, van Megen 1974) also observedthat at pH 4.5 the proteins dissolved at salt concentrations above 0.7 sodiumchloride. However, below this salt concentration a two-phase system formed,consisting of a protein-poor upper layer and a viscous protein-rich lower layer.

is well-known that moist heat readily insolubilizes soybean meal proteins.


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338 / dvances Cereal Science and TechnologyHowever, Wang 1975 has recently found that if autoclaved flakes areultrasonically treated during extraction, proteins that presumably are denaturedcan be redissolved. Moreover, the redissolved proteins appeared much like thenative proteins in their sedimentation behavior in the ultracentrifuge. is notclear whether denaturation was reversed or if the proteins in the protein bodiesare comparatively stable to autoclaving but do not dissolve because of a barrierof denatured cytoplasmic or membrane proteins on the outside of the proteinbodies. urther work is needed on this problem.Trypsin Inhibitors Availability of soybean trypsin inhibitors in pure form andtheir unique biological activity have resulted in intensive study of these proteinsin several laboratories. One of the most notable achievements has been in thelaboratories at Niigata University in Japan where Odani and Ikenaka 1973have determined the complete amino acid sequence for the Bowman-Birkinhibitor Figure 3 and Koide and kenaka 1973 have elucidated the completesequence for the Kunitz inhibitor Figure 4 .The Bowman-Birk inhibitor consists of 71 amino acid residues with a site forinteraction with trypsin at Lys 16-Ser l and a reactive center for interaction withchymotrypsin at Leu43_Ser44 . This inhibitor is remarkably stable to heat, acid,and proteolytic digestion because of the seven disulfide cross-links that give themolecule a rigid structure. The molecule is unique in its structure around theproteinase inhibitory sites. The two sites are almost identical. Each site is locatedin a nine-membered peptide loop formed by a single disulfide bridge. This loop isfollowed by another nine-membered loop and then by a ten-membered ring siteof trypsin inhibition or eight-membered ring site of chymotrypsin inhibition .The 181 amino acid residues found in the Kunitz trypsin inhibitor give it amore 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 andIkenaka 1973 . Residues are numbered beginning \vith N-terminal aspartic acid. Disulfide crosslinkages are shown in black between half-cystine residues. Residues at the two reactive sites areshown in bold-faced type and have asterisks adjacent to them. Reprinted with permission fromSpringer-Verlag.


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Chemistry n Technology of Soybeans / 339in the Kunitz inhibitor is c omparati ve ly simple, because there are only twodisulfide bridges located at residues 39-86 and 136-145. The reactive center islocated at the Arg63_Ile 64 bond.Another notable development in studies on trypsin inhibitors is thedetermination of the crystal structure of the complex formed by Kunitz trypsininhib itor and porcine trypsin by X- ray crystallography Sweet et al. 1974 .Figure 5 shows a model ofthe complex made from an electron density map at 5 Aresolution. The inhibitor is nearly spherical in shape and has a diameter ofabout35 A A rema rkable feature of the complex is that only about 12 of the 181residues of the inh ibitor make contact with the trypsin molecule to fo rm anextremely stable complex. It is estimated that within this small region there areover 300 interatomic contacts pairs of atoms within 0.5 Aof the theoretical vander Waals contact distance) of which about 18 may be hydrogen bonds. isbelieved that the binding energy derives from the sum of small energy terms frommany interactions rather than any new or unforeseen type of interaction.Agglutinin. Recent reviews of agglutinins, including that of soybeans, areavailable Sharon and Lis, 1972; Lis and Sharon 1973). A survey of over 100soybean varieties and strains revealed about an eightfold variation inagglutinating activity but all samples were active Kakade et al. 1972 .Consequently, elimination of agglutinins by plant breeding does not lookencouraging at this time.

300

Figure 4. Prima ry struc ture of Kunitz soybe an trypsin inhibitor a cc ording to Koide a nd Ike na ka 1973). Residues are numbered beginning with N-termina1 aspartic acid. Disulfide cross-linkages areshown in black between half-cystine residues. Reprinted with permission from Springer-Verlag.


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340 / Advances in Cereal Science n TechnologyBiological activity of soybean agglutinin is of continuing interest in severallaboratories, and recent studies include agglutination of mouse, rat, and human

cell lines after transformation with viral or chemical carcinogens Sela et al1970 ; binding of agglutinin by red blood cells Gordon et al 1972b ; anddetermination of factors that influence agglutination Gordon and Marquardt1974; Pereira et al 1974 . The biological activity of soybean agglutinin has beenused to advantage in developing affinity chromatography techniques for itspurification Allen and Neuberger, 1975; Bessler and Goldstein, 1973; Gordon etal 1972a . Lotan et l 1974 found that the agglutinin contains four identicalsubunits with a molecular weight of30,000. Each subunit has a carbohydrate sidechain of nine mannose and two N-acetyl-D-glucosamine residues. Four of themannose residues can be oxidized with sodium periodate and then reduced withsodium e borohydride to yield the tritium-labeled agglutinin with fullretention of its biological activity Lotan et al 1975 . This radioactive derivativeshould be very useful in studies of surfaces of cells that can interact with theagglutinin.Although the function of agglutinin in soybeans is still unknown, Bohlool andSchmidt 1974 found that the agglutinin combined specifically with 22 out of25strains of the soybean-nodulating bacterium Rhizobium japonicwn No binding

Figure 5 Crystalline complex of porcine trypsin and Kunitz soybean trypsin inhibitor at 5 Aresolution according to Sweet l 1974 . Reprinted with permission from Biochemistry 13: 42124228 1974 . Copyright by the American Chemical Society.


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Chemistry and Technology of Soybeans 4occurred with 23 other strains of rhizobia that do not nodulate soybeans. Theyproposed that the agglutinin may provide a site on the soybean root surface thatinteracts specifically with the polysaccharides on the surface of the appropriateRhizobium cell as the first step in the formation of the nodule. However, theexistence of agglutinins in the roots of the plant does not appear to have beenreported. Globulin. Koshiyama l972b has described a new method for purifying a7S globulin by suspending acid-precipitated pH 4.5 globulins in 0 6M NaClO.OIN H l at pH 2 and then centrifuging. Under these conditions, the lISglobulin is acid-denatured and insoluble, whereas the 7S globulin is stable andremains soluble. The acid-soluble fraction is then passed successively throughSephadex 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 thetotal 7S fraction in the globulin mixture.The presence of subunits in purified 7S globulin has been confirmed bymolecular weight studies in protein dissociating solvents, although there isdisagreement about molecular weights of the subunits. Vaintraub and Shutov1972 obtained molecular weights of 83,800 in 4M urea and 31,200 in 6M ureaand proposed that the parent molecule contained six subunits. On the otherhand, Koshiyama 1970 obtained a molecular weight of 22,500 for the 7Sglobulin in 8M urea which suggests that about nine subunits make up the parentmolecule in agreement with results of N-terminal analysis. The 7S proteinsamples used in the two studies were not prepared by the same method; hence,different 7S globulins may have been examined. This is plausible because resultsof Hill and Breidenbach 1974a suggest that there may be three 7S proteins withdifferent electrophoretic mobilities but having the common ability to undergomonomer-dimer formation with change in ionic strength.Additional studies on subunit structure of a 7S globulin indicated thatdisulfide bonds were not involved in binding between subunits. Urea andguanidine hydrochloride appear to disrupt the internal structure of the subunitswhen the 7S molecule is dissociated with these reagents Koshiyama, 1971 .Conformational studies of 7S and 11S globulins showed both to be very low ina-helix content; ,B-structure and random coil seem to predominate. Althoughsimilar in structure as measured by circular dichroism in the region between 200and 250 nm, there were distinct differences in the 250-320 nm regionKoshiyama and Fukushima, 1973 . Marked dissimilarities between 7S and II Sglobulin conformations were observed by ultraviolet difference spectra,ultracentrifugation, and optical rotatory dispersion in acid solutions at 0.1 ionicstrength Koshiyama, 1972c .

S Globulin Glycinin . Koshiyama 1972d purified II S protein by gelfiltration and redetermined many of its physical properties Table IX . No majorchanges were obtained from most of the values obtained by former workers, butthe new values are likely to be more reliable than older ones because the proteinpreparation was homogeneous by gel filtration, ultracentrifugation, and gelelectrophoresis.Sedimentation equilibrium molecular weights of the subunits of II S protein inacid solution pH2.6 but unspecified ionic strength and in 4M urea pH 7.4,0.1ionic strength were 63,000 and 31,000, respectively Vaintraub and Shutov,


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342 I Advances in Cereal Science nd Technology1971). Basic subunits isolated by DEAE-cellulose chromatography had amolecular weight of 24,400. Sodium dodecyl sulfate electrophoresis yieldedmolecular weights of 22,300 for the basic subunits and 37,200 for the acidicsubunits Catsimpoolas et al. 1971d). Amino acid analyses of the six subunitsisolated by isoelectric focusing by Catsimpoolas and coworkers revealed nosignificant differences in the ratios of acidic to basic residues. Thus, it seems

TABLE IXPhysical properties of S proteinProperty

IsoeIectric point. pH1


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Chemistry and Technology of Soybeans / 343likely that the acidic side chains in the basic subunits are present as amides inorder to shift their isoelectric points to pH 8.0-8.5. The amino acid analyses alsoshowed that the six subunits were all different. Results from both researchgroups are in agreement with the existence of 12 subunits per 350,000 molecularweight entity.Kitamura and Shibasaki 1975) recently described isolation of four acidicsubunits from 11S protein in contrast to the three isolated by Catsimpoolas et l1971d). Two of the subunits separated in Japan contained phenylalanine and theother two had leucine isoleucine) in the N-terminal positions. Geneticpolymorphism was suggested as a possible explanation for these results.Scanning isoelectric focusing of 11S protein has revealed an extremelycomplex system that is attributed to microheterogeneity possibly arising fromvariation of amide groups on the side chain carboxyl groups of aspartic andglutamic acid residues Catsimpoolas and Wang, 1971).Hydrogen ion titration of 11S protein confirms that alkali and acid causeconformational changes in the protein molecule Catsimpoolas et al 197Ia).Approximately 14 of the total ionizable groups appear to be buried and areaccessible only by titration in disaggregating media such as Mureaor guanidinehydrochloride. Ultraviolet difference spectra likewise revealed the presence ofburied ionic groups tyrosine and tryptophan which are exposed by ureaor guanidine hydrochloride Catsimpoolas et al 1971e). Optical rotatorydispersion and infrared studies also confirmed that the lIS molecule primarilyhas a ,B-conformation plus some disordered regions. Jacks and coworkers 1973)measured the optical rotatory dispersion of lIS protein from 200 to 240 nm andconcluded that the molecule contained 9 a-helix, 33 pleated sheet, and 58unordered structure.Destruction of antigenicity of 11S protein by heating in solution wasinvestigated by radial immunodiffusion, complement fixation, and discimmunoelectrophoresis Catsimpoolas et al 1971c). Antigenic properties areretained on heating for 30 min up to 65C, but are lost rapidly between 70 and90 Loss of antigenicity is associated with destruction of the quaternaryprotein structure and possibly alteration of the secondary and tertiary structuresof the individual subunits.

D. EnzymesEnzymes Other than Lipoxygenase Work on a variety of soybean enzymes hasbeen reported in the past 5 years. Studies are often done on germinated beans orseedling parts, but authors frequently fail to point outwhether the ungerminatedseed contains the enzyme under consideration. Such informationwould be usefulto those concerned with the mature seed. Table X lists some of the seed enzymesstudied during 1970-75. Not included are the lipoxygenases whichmerit separate

discussion below.Lipoxygenase After a number of years of near neglect, lipoxygenase is beingactively studied in several laboratories, and a large literature is developing on thisenzyme. Several major properties of lipoxygenase have been clarified in the pastfew years: a) there is good evidence for at least four lipoxygenases in soybeans, bthe various enzymes show differences in substrate specificity, and c) lipoxygenase


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344 Advances in Cereal Science n Technologyis now known to contain iron as a prosthetic group.The presence of four isoenzymes of lipoxygenase was first suggested bypolyacrylamide gel electrophoresis of soybean extracts (Guss et al. 1967Fractionation studies then soon led to the isolation of a second (Christopher etal. 1970) and a third (Christopher et al. 1972) enzyme which differed from theclassical Theorelllipoxygenase preparation. All three enzymes have molecularweights of 100,000, but they differ significantly in several respects (Table XI)

TABLE XIComparison ofproperties of three lipoxygenases

EnzymeProperty L-l L 2 L-3Isoelectric point 5.68 6 5pH optimum 9 5 6 5Effect of Ca None StimulatoryStabil itv to heat Stable UnstableSubstra te specificity Free acid EsterSource: Christopher et al (1970. 1972): Verhue and Francke (1972).bRestrepo et al (1973).Comparison of linoleic acid and methyl linoleate.

GIUCOSY I-0Yf 0W O H enistin

GIUCOSY I-0Yf 0V Y O H

aidzinGIUCOSYI w

I I CH ---lycitein 7pa glucosideFigure 6. Structures of soybean isoflavone glucosides.

6.155.5-8.0InhibitoryEster


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Chemistry n Technology of Soybeans / 345including their chromatographic behavior on DEAE-Sephadex. Verhue andFrancke (1972) obtained good evidence for a fourth lipoxygenase and found thatof the four enzymes, two show a preference for free linoleic acid and two prefermethyllinoleate as a substrate.When lipoxygenase was crystallized nearly 30 years ago, it was found tocontain less than a stoichiometric amount of iron which was considered to be acontaminant. Lipoxygenase was therefore considered an unusual dioxygenasebecause it apparently lacked a transition metal. This anomaly has now beencleared up by careful reexamination of the purified enzymes and finding of oneatom of iron per molecule (Chan, 1973; Roza and Francke, 1973; Pistorius andAxelrod, 1974). The iron is tightly bound and is only slowly removed from theprotein by chelating agents unless the protein is first denatured. The active stateof the iron is believed to be the ferric form (Pistorius and Axelrod, 1974; deGrootet al., 1975b).Although of great theoretical interest as a dioxygenase, lipoxygenase has alsobecome the focus of attention by food scientists because reaction products ofthepolyunsaturated fatty acid hydroperoxides are believed to contribute to theundesirable flavors associated with raw soy flours and derived protein products.This subject is discussed in Section VII-A.

E inor onstituentslsoflavones. 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 wasreported. Naim et l (1973) isolated the new isoflavone and identified it as 7,4 dihydroxy, 6-methoxyisoflavone. It occurs in soybeans largely as the 7-0-{3glucoside Naim et al., 1974) which has the structure shown in Figure 6Isoflavones in soybeans were determined by grinding and extracting with etherfollowed by methanol and finally hydrolyzing the resulting soy flour residue withacid and extracting with ether. The extracts were chromatographed on apolyamide column and the isoflavones were determined by gaschromatography. Results of the analysis are summarized in Table XII. Over 99of the isoflavones in soybeans occur as glucosides, and genistin is found in thehighest concentration. The isoflavone content of soybeans is about 0.25 .

TABLE XIIIsoflavone content of soybeans

FractionGenistein Genistin Daidzein Daidzin Glycitein

rng/ 100 g soybeansGlyciteinGlucoside

Ether extract 0.2 0.3 0 01Methanol extract 2 157.2 0.3 56 1 0 1 32.1Aour residue 7.2 2.0 1 7Total 1 4 164.4 0.6 58 1 0 11 33.8ource: Nairn et al. 1974).


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346 / Advances in Cereal Science and TechnologyPhytic Acid Analyses for phytic acid in 15 commercial soy products werereported by Ranhotra and coworkers 1974). Their results can be summarized as

follows:PreparationsFull-fat floursHigh-fat floursDefatted floursConcentratesIsolateWhey-soy blend

Phytic Acid0.510.44-0.510.46-0.520.35-0.610.330.36

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, thephytic acid was extensively hydrolyzed; presumably phytases of wheat and yeastcaused hydrolysis. The exception noted was a whey-soy blend which, whenadded to bread, resulted in only 22 hydrolysis of the phytic acid. The highcontent of calcium ion in whey may have inhibited phytase. Further studies arenecessary on phytase activity in breads containing whey-soy blends because theblends are now used extensively in baked goods to replace nonfat dry milk otton, 1974).Interaction of phytate with soybean proteins was described by Okubo et al.1975). Dialysis experiments showed that binding ofphytate to soybean proteinswas minimal at pH 5 but strong at pH 3and at pH 8.5. At the low pH, interactionapparently occurs through the cationic groups of the protein-lysyl arginyl, andhistidyl residues plus the amino terminal groups. At pH 8.5, multivalent cationssuch as calcium and magnesium appear to chelate with the phytate, and thechelate complex binds to the protein. Phytate was removed by dialysis underthree conditions: a) pH 8.5, in the presence of sodium ethylenediaminetetraacetate, b) pH 5 in water, and c pH 3 in 5Mcalcium chloride. The lastcondition appeared to be the most effective of the three.Sterols. A survey of sterol distribution by four classes-free esterified,

TABLE XIIIDistribution of sterol classes in selected U.S. soybean varietieS-Form

Variety andYear of HarvestAcylated

Total Sterolsb _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 16Harosoy 1964 0.17 72 17Shelby 1963 0.15 64 15Shelby 1964 0.14 68 17Source: Hirota et l 1974 .bBased on weight of free sterols plus weights of derivatives.

45203927

603332


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hemistryn Technology of Soybeans / 347glucosides, and acylated glucosides in 19 U.S. soybean varieties was reportedby Hirota et al 1974). Selected data fro m th eir results are given in Table XIII.Free sterols are the major form found and the sterol esters are present in thesmallest amounts. A study of developing soybeans revealed that the four sterolclasses are detectable as soon as the beans are large enough to analyze ~ weeksafter flowering). Su rprisin gly, t he percen tage d istrib utio n between the fou rclasses does n ot change appreciably f rom early development until matu rity Katayama and Katoh, 1973).Acylated sterol glucosides were recently identified as an additional group ofsubstances complexed with isolated soybean globulins; they are extracted fromth e pro teins with aq ueou s ethanol Wolf and Thomas, 1973).Saponins The existence of sap on in s in so ybeans was rep orted almo st a h al fcentury ago, b ut these compo un ds received relatively little att en ti on u ntil the1960 s when extensive studies on their chemical, nutritional, and physiologicalproperties were conducted at the Hebrew University of Jerusalem in Rehovoth.Birk 1969) has reviewed these studies in detail. urtherwork in our laboratoryrevealed that the presence of glucuronic acid residues in the saponins makes itpossible to fractionate the crude mixture by anion exchange chromatography onDowex -l co lumn s Wol f and Thomas, 1971). Twelve fractions were separated,but analysis of their sugar contents suggested that they were still veryheterogeneous. One fraction yielded a previously unreported aglycone whoseinfrared spectrum suggested a similarity to so yasap og en ol D. Comp lex ity of

Rhap a 2Galp {31 2GlcUAp{31

Soyasaponm I

Rhapa l A ap a IcUAp {31

SoyasapoOin II

Galp{31 2GlcUAp{3I

Soyasaponm illFigure 7 Structures of soyasaponins I II, a nd III acc ordi ng t o Kitagawa et al 1974b).


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348 / Advances in Cereal Science n TechnoloK}saponins is not peculiar to soybeans; recent work on alfalfa saponins revealedabout 3 different compounds Berrang et al., 1974Kitagawa et al l974a) isolated three soybean saponins, designatedsoyasaponins I, II, and III, having soyasapogenol B as the common aglycone.Separation was achieved by silica gel column chromatography usingchloroform:methanol:water as the developing solvent. Studies of the threesaponins revealed the chemical structures shown in Figure 7 All threecompounds have the common feature of glucuronic acid attached by beta linkageto the 3-hydroxyl group of soyasapogenol B Soyasaponin III is identical tosoyasaponin I except that it lacks rhamnose.Soybean saponins are toxic to rice weevils Sitophilus oryzae) and thereforeshow some promise as naturally occurring insecticides that appear nontoxic to

warm-blooded animals Ishaaya et al., 1969; Su et al., 1972Polyamines. Putrescine, cadaverine, spermidine, and spermine occur insoybeans at a level of about 13 ppm Wang, 1972; Wang and Selke, 1973;Wanget al., 1975). Their function in soybeans is still unknown. Although thesecompounds are malodorous, they may be present in soybeans at levels that arebelow their odor thresholds.VI. T HNOLOGY

A. Processing Soybeans into Oil and MealThere have been no major changes in the past 5 years in the basic processingused to convert soybeans into oil and meal. Virtually all of the beans processed inthe U.S. are extracted with hexane. Reviews ofthe overall process are availableBecker, 1971; Wolf and Cowan, 1975).Annual crushing capacity of the industry is presently estimated to be about 1 1billion bushels, and historically about 80 of capacity has been used. Thenumber of soybean processing mills has declined, but their average size hasincreased as the industry expanded. For example, in 1951 there were 193processing 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 threefoldto 1 billion bushels American Soybean Association, 1975).B Processing l into Edible Products

The various steps in converting crude soybean oil into edible products werereviewed recently Wolf and Cowan, 1975). New developments are thereforedescribed here only briefly.Recycling of Water in Alkali Refining. Plant-scale tests were carried out inwhich the water used to wash alkali-refined oil was passed through a cationexchange column to remove sodium ions, thereby allowing the water to berecycled Beal et al., 1973). Oil washed with recycled water in a continuous 28day run had a satisfactory low content of sodium, iron, and copper. Bleaching,hydrogenation, and deodorization proceeded normally; the recycle processappears to solve the wash water disposal problem.Copper-Chromite Catalyst for Hydrogenation. Hydrogenation of linolenatewith copper-chromite catalyst was tested successfully in plant trials to prepare


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Chemistry and Technology of Soybeans / 349improved edible soybean oils for frying and salad oils List et al. 1974). Coppercontent of the oil increased as a result of hydrogenation, but bleaching andwinterization reduced it to levels below 0.01-0.02 ppm. Residual copper andchromium in processed oil were concentrated in the stearine fraction bywinterization. Results of organoleptic, oxidative, and room odor tests showedthat oils of good stability can be prepared on a commercial scale by copperhydrogenation and winterization. The copper catalyst must be handled carefullybecause if it becomes contaminated with nickel, it loses its selectivity forhydrogenation of linolenate and behaves as a nickel catalyst Moulton et al.1973).Deodorization. The need for increasing production of oil refining plants andthe growing demand for more soybean sterols have led to a new design of thetrays in the conventional Bailey type deodorizer. The new design incorporates animproved jet stream distributor that permits injection of steam at high rateswithout excess oil entrainment and losses. The new tray replaces two or three ofthe existing trays in the Bailey deodorizer and makes possible rapid removal ofsterols with maintenance of oil quality Lineberry and Dudrow, 1972). Severalnew or modified deodorizers with only four trays instead of the normal six orseven are now in use in the industry.

Antioxidants for Soybean ilPolyunsaturated vegetable oils such as soybeanoil are subject to oxidation; hence, antioxidants are often added as stabilizers.Monotertiarybutylhydroquinone TBHQ was approved as a food grade oilsoluble antioxidant in 1972. Toxicological and biochemical studies conducted toestablish safety of this compound were reported recently Astill et al. 1975Rats, dogs, and humans eliminated the compound mainly in the urine as the 4 -sulfate and the 4-0-glucuronide. Extensive feeding and comparative biochemicalstudies indicate that TBHQ is safe for its intended use at the maximum level 200ppm based on weight of fat or oil) permitted by the Food and DrugAdministration.Butylated hydroxyanisole and butylated hydroxytoluene have been usedextensively as antioxidants. Their toxicological and biochemical properties werereviewed recently by Branen 1975).

Field Damaged Soybeans. Adverse weather during harvesting damagessoybeans with the result that poor quality oil is obtained when the beans areprocessed. Evans et al 1974) studied commercial oils from soybeans that werefield-damaged in North Carolina during the 9 growing season. They foundhigh levels of free fatty acids and iron in the oils, and a significant correlationexisted between free fatty acid and iron contents. The iron apparently originatedfrom the damaged beans as well as from steel processing equipment. The highlevel of iron in the oil from damaged soybeans is believed to be responsible for thelow quality of the oil when it is refined.Metabolism of Unsaturated Fatty id Isomers. Use of copper as well asnickel catalysts to hydrogenate linolenate ester in soybean oil causes geometricisomerization and migration of double bonds from their normal positionsCowan et al. 1973a; Vigneron et al. 1972). Table XIV shows the monoenecomposition of soybean oils hydrogenated with copper and nickel catalysts. Thecopper-reduced oil had a total monoene content of 42 ; yet, only 53 of the


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35 / Advances in Cereal Science n Technologydouble bonds were located in their expected 6 9 position. Likewise, in nickelreduced oil, only 61 of the monoene was in the usual 6 9 location. Becausehydrogenation is widely used in the production of soybean oil and relatedproducts, there is concern about the metabolic fate of the various fatty acidisomers generated during hydrogenation. Mounts et al. 971) have examinedthe incorporation of tritium, carbon-14, an d deuterium-labeled oleate an delaidate esters into egg lipids by the laying hen. The cis isomer was preferentiallyincorporated into the neutral lipids, whereas the trans isomer was preferred bythe phospholipids. Both cis and trans isomers were transferred across the chickenmembranes at ab ou t the same rates. Incorporation of label by the neutral lipidindicated that there was no isotopic discrimination or loss, but loss ofcarbon-14from the carboxyl group in the lipids incorporated into the phospholipd fractionindicates that a more complex biosynthetic route is involved. Furtherexperiments by Mounts and Dutton 976) indicate that the hen does notdistinguish between oleic and linoleic acids in synthesis ofthe neutral lipid of theegg. The phospholipid fraction of the egg, however, revealed a selection oflinoleic acid over oleic acid.

C Processing Soybeans into Edible Protein rodu tsAs pointed out earlier, the use of soybean protein in foods is still quite small ascompared to the oil Table VII). Nonetheless, this segment of the soybeanindustry has undergone the greatest change in the past 5 years. Edible soy protein

products for use as food ingredients were first developed by a few of theestablished soybean processors; but as markets developed, many of the otherprocessors, plus several companies that do not process soybeans, entered themarketplace. Table XV lists the major U.S. manufacturers of soybean proteinproducts. Products range from flours and grits to textured isolates. The newestitems introduced are the textured concentrates; three companies announcedavailability of these products in mid-I975.TABLE XIV -fonoene composition of hydrogenated soybean oils

CatalystCopper Nickel

Position of Double Bond of total fatty acids::> 7::> 8::> 9::> 10::> II::> 12::> 13::> 14::> 15::> 16::> 17

Source Cowan et f l973a).

0.32 122 13 14 92 61.60 90 60 20 2

0.32 126 0

3 23.75 30 80.30 40 1


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Chemistry and Technology Soybeans / 351Full 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). t is recommended that the soybeans be blanched by boiling to inactivate

lipoxygenases before disrupting the cellular structure of the seed. Allowinglipoxygenases to remain active while disrupting the seed is believed to result ingeneration of the beany flavors characteristic of raw soybean products. Wholesoybean food prototypes such as canned chicken and soybeans have beenprepared. 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 soakingsoybeans in tap water overnight in 0.5 NaH 3 solution, boiling (blanching) infresh 0.5 NaH 3 for 30 min, draining, grinding, heating to 2000 F, and finallyhomogenizing 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 thebase.Full-fat flours can be made by extrusion cooking as described by Mustakas etal (1970). Soybeans are cracked, dehulled, and then dry-heated to inactivatelipoxygenases. The heated bean particles are tempered to preferred moisturelevels and extrusion cooked. After cooling and grinding, a full-fat flour isobtained. Nutritional studies indicate that the cooking step inactivatesantinutritional factors. Extrusion cooking to prepare full-fat flours does notappear to be practiced commercially in the U.S. at this time although it is beingused abroad.Extrusion-cooked full-fat flour has been converted into experimentalbeverage bases by two procedures. In the first, the flour is suspended in water,mixed with emulsifier and soybean oil, colloid milled, homogenized, and spraydried. The powder is then blended with sugar, salt, flavor, minerals, and vitaminsto yield the beverage base (Mustakas et al. 1971). For the second beverage

TABLE xv}Y ajor U.S. producers soybean protein products

Producer Grits andFloursTextured Products

Concentrates Isolates Flours Concentrates IsolatesArcher-Daniels Midland Co.Cargill, Inc.Central Soya Co.Dawson MillsFar-Mar-Co.General Mills, Inc.Grain Processing Corp.Griffith LaboratoriesLauhoff Grain Co.Miles Laboratories, Inc.NabiscoNational Protein Corp.Ralston Purina Co.A. Staley Mfg. Co.Swift Co.Mainly pepsin-modified isolates.

T

+


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5 Advances in Cereal Science n Technologyprocess, raw full-fat flour is slurried in dilute sulfuric acid at a final pH of3.5 andheated rapidly with steam, thereby inactivating the lipoxygenases. The lipidprotein-polysaccharide curd is then cooled and washed with water to remove thesoluble sugars in the manner used to manufacture protein concentrates fromdefatted flakes and flours. After washing, the slurry is adjusted to p 9 withammonium hydroxide, and another quick cooking is given to inactivate residualtrypsin inhibitor. The alkaline dispersion is then neutralized, colloid milled,homogenized, and centrifuged to obtain the beverage base Mustakas, 1974 .Defatted Flours n Grits To the best of my knowledge, commercial practiceis still to use hexane as in the conventional process and desolventize underconditions that yield flours and grits with the desired protein solubility WolfandCowan, 1975 .

Hexane is a good solvent for extracting oil from soybeans, but it does notremove all of the lipids. In the past few years, several lines of evidence havepointed to the lipids in soybeans as the source of some of the undesirable flavorsthat are characteristic of raw defatted flakes and products made from them.Extraction of raw, hexane-defatted flakes with azeotropic mixtures ofhexane:alcohol yields additional lipids and also removes most of the beany andbitter flavors Eldridge et al 1971 . Hexane mixed with methyl, ethyl, orisopropyl alcohol can be used, but hexane:ethanol 82: 8vv has the advantagesof being nontoxic, causing a minimum of protein denaturation, and removingonly a few percent of solids. The azeotropic mixture apparently is selective for theresidual lipids and the flavor compounds. Protein isolates of improved flavorwere also obtained when the starting flakes were first extracted withhexane:ethanol. Because hexane:ethanol does not inactivate the trypsininhibitors in flakes, heat treatment is necessary to obtain optimum nutritionalvalue. The effects of hexane:ethanol extraction plus toasting have therefore beenstudied Honig et al in press . The combination of hexane:alcohol extractionand toasting yields defatted flakes with flavor scores equivalent to those forwheat flour.Protein Concentrates Several new methods for making concentrates havebeen 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 theconventional acid-leaching process to reduce loss of acid-soluble proteinsDeLapp, 1973 . The gums form insoluble complexes with the acid-solublewhey proteins Smith et al 1962 and thus are retained in the concentrate.Sair 1972 described a modification of his earlier acid-leach process in whichhe prepares a protein isolate by the conventional procedure and then adds theisolated protein back to the thoroughly washed insoluble residue. The mixture isthen neutralized with alkali and dried under vacuum. The resulting concentrate isclaimed to have a light color and less beany flavor then the product made bydirect leaching of defatted flakes.Another variation oerand Calvert, 1972 of the acid-leach process consistsof slurrying defatted flakes in water to dissolve the proteins. The slurry is thenacidified to p 4.5 to precipitate the proteins. The protein curd and waterinsoluble polysaccharides are recovered by centrifuging, washed thoroughly, andadjusted to p 6.8. The slurry is then quickly raised to 310 F in a jet cooker andheld at that temperature for about 5 sec. Next, the slurry is injected into a vacuum


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hemistry and Technology Soybeans / 353chamber to flash off undesirable flavor components and spray dried. The je tcooking step is probably responsible for alteration of functional properties of theresulting protein concentrate as compared to the conventional acid-leachedconcentrate.Miller and Wilding (1973) described a related acid-leach process in whichdefatted Hakes are stirred inwater for to 30 min and then acidified to pH 4 to 5.The isoelectric slurry is then ground MG Model, Urschel Comitrol with aMicrocut-Head attachment , centrifuged, and washed to remove the solublesugars. After washing, the product can be dried in the isoelectric state orneutralized, heated, and spray dried. Rupture of cell walls and protein bodymembranes is claimed, but the importance of this step is not clear. The cell wallpolysaccharides and the globulins remain in the product, in any event, becausewashing is carried out in the isoelectric region of the proteins.A variation of the alcohol-leaching process utilizes a two-phase solvent systemof hexane:methanol:water in the ratio of 10:7:3 that is applied to full-fat soybeanflakes to simultaneously extract the oil and oligosaccharides plus minorconstituents (Schweiger and Muller, 1973). The product obtained has a proteincontent of 6 to 73 . It is likely that this solvent system will remove lipids notnormally extracted by hexane alone.Hayes and Simms (1973) described the use of hexane:alcohol as a solvent forresidual lipids in hexane-defatted flakes. First, full-fat flakes are extracted withhexane. Desolventization is omitted and ethanol is then added to the hexane-wetflakes. 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 thesoluble sugars as in the usual alcohol-leach process. On desolventizing, a proteinconcentrate is obtained.Protein Isolates Isolates are now available from severalmanufacturers TableXV) and have a variety of physical properties to make them suitable for differentfood applications. The methods employed to modify the isolates are proprietaryinformation, but heat and enzyme treatments are included in many of the recentpatents issued on this topic. For example, Hoer et l (1972) quickly heat slurriesof sodium proteinates in a jet cooker to 2850 to 3200 F which also imparts ashearing force to the slurries. After a short holding time, the slurries aredischarged into a zone of lower pressure to flash off water vapor and volatileflavor compounds. The slurries are then cooled and treated with a proteolyticenzyme for 1 to 30 min. Reheating the slurries inactivates the enzyme, and spraydrying completes the process. High wettability and good dispersibility withwaterare claimed for the modified isolate.A second example is described by Puski (1974). An acid-precipitated proteincurd is washed with water, heated to 800 C, and papain plus sodium bisulfite areadded to a 15 protein slurry. After digesting for 2 hr a t 800 C, the slurry is boiledto inactivate the papain and insoluble proteins are removed by centrifuging. Theacid-soluble fraction is then spray dried to yield a derived protein that is solublein acidic pH 2.6) beverages such as soft drinks and fruit juices.Textured Protein Products Successful introduction of textured soybeanprotein products in the late 1960 s stimulated a large amount of developmentalwork on new and better processes for texturizing flours, concentrates, and


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354 / dvances in ereal Science n Technologyisolates. The spun isolate products were developed first, and the textured flourswere introduced later. Recently, textured concentrates have also becomeavailable, but published information on their preparation is not yet available;patents on processes are pending. Most of the published information ontexturization appears in the patent literature, and many of the U.S. patents onthis subject for 1960-72 were summarized by Gutcho 1973). Processes actuallyused by industry are not generally revealed, but examples will be cited to showthe diversity of approaches being developed.

Textured Flours. A departure from the usual extrusion process has beendescribed in patents by Strommer 1973) and Strommer and Beck 1973). Ablend of defatted flour, protein concentrate and isolate introduced into arotating multichambered device and injected with high-pressure steam whichexpels the materials through a nozzle into a region of lower pressure. Theexpulsion causes puffing and texturization plus flashing off of volatile flavorcompounds. Residence time in the apparatus may be less than a second; hence,the product does not turn dark and a bland flavor claimed. A variation of thisprocess has also been developed in which a slurry of the protein material sprayed into a stream of high-pressure steam. The excess moisture flashed offand a textured product obtained Strommer 1975).

Defatted soy flour can be converted into fibers by making a thick aqueousslurry in the isoelectric pH region and pumping it at high pressure 50-5 000 psi)through a heat exchanger. The hot proteinaceous material then pumpedthrough a small orifice to obtain a continuous filament or small discrete texturedparticles, depending on operating conditions Frederiksen and Heusdens, 1972).Soy flour and related materials can also be textured by making a dough-likemass with water, heating to a high temperature and then quickly releasing thepressure. Loepiktie and Flier 1973) carried out the process in Teflon-coated

aluminum tubes heated in a closed chamber whereas Touba 1974) wrapped thedough in aluminum foil and heated it under pressure between the plates of ahydraulic press. The process probably similar to conditions in an extruderwhen the dough passes through the die.

Textured Isolates. A variation of the classical fiber spinning process haseliminated the need for an alkaline treatment step. Soy protein isolate preparedin the customary way dispersed in water by adding solid salt to a final molarityof 0.4 to 0.5 instead of dissolving it with alkali. The curd does not dissolvecompletely, but the soluble portion or mesophase separated by centrifugingand then extruded into a hot water 80 0 C or higher) bath. The high temperaturecoagulates the protein into a fiber and the salt removed by leaching into thewater Tombs 1972). Formation oflysinoalanine deGroot and Slump 1969) eliminated by avoiding the alkaline spinning dope step.A simplified process for convert ing soy protein isolate into edible fibers

consists of pumping a protein slurry up to 35 solids) at high pressure through aheat exchanger and then expelling it through a small nozzle. The protein emergesas fibers 4-6 cm long) that are cooled by dropping 20 ft through ambient air intoa collecting vessel. Excess water removed by centrifuging Hoer 1972). Asomewhat similar process described by Lange 1974) yields a proteinmonofilament. Isolate mixed with water, sodium sulfite, glycerine, plus an acid


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Chemistry n Technology of Soybeans / 355or base. The mixture is heated until it becomes plastic 1900 to 8 0 F) and then isextruded through 2 1 mil orifices into air. The resulting fiber can be combinedwith binders, flavors, and color to form simulated meats. Both dry spinningprocesses eliminate the preliminary spinning dope step and the acid-saltcoagulating bath employed in the usual fiber spinning operation.Soy protein isolate is a major ingredient in a creping process developed tosimulate meats Liepa and Slone, 1974). For example, egg white solids, soyprotein isolate, vital wheat gluten, starch, shortening, and beefflavors are mixedinto a dough with water. The dough is then mixed more thoroughly by passing itthrough a noodle extruder. As they emerge, the extruded dough strands are cutinto pellets which are passed through smooth rolls to convert the mass into asheet. The sheet is removed from the last roll with a doctor blade thereby crepingor crinkling it. The parallel ridges and depressions of the sheet give it a fibroustexture that is stabilized by heating. By coating the creped sheet with an ediblebinder, folding it upon itself, and then heating it, a slab of cooked meat-likeproduct is obtained.A bacon analog containing soy isolate is described by Leidy et l 1974a). Twoemulsions are prepared. One emulsion forms the lean or red portion of the analogwhile the other duplicates the fatty or white part. The red phase is made bymixing soy protein isolate with water, textured vegetable protein, egg albumin,hydrogenated vegetable oil, flavor, spices, and red food color until a stableemulsion results. The white phase is formed by emulsifying water, egg albumin,textured vegetable protein, hydrogenated vegetable oil, flavor, and spices. Thetwo phases are then alternately layered in a pan and heated in an autoclave. Aftercooling, the resulting loaf is sliced to obtain the bacon analogwhich can be fried,broiled, or cooked in a microwave oven.Sausage analogs are also possible bymaking a gel precursor emulsion and thenautoclaving Leidy et al 1974b). For example, soy protein isolate is mixed withalbumin, textured vegetable protein, soybean oil, water, seasoning, flavor, andcolor to form the emulsion. The gel precursor emulsion is then stuffed intobologna or sausage casings and autoclaved to heat-set it. After cooling andremoving the casings, bologna or frankfurter analogs are obtained.

VII. FACTORS AFFECTING FOOD USES OF SOYBEAN PROTEINSIn contrast to the oil which is widely used as an edible fat, soybean proteins stillface some problems before their use becomes more widespread. Flavor,functional, and nutritional properties are three of the major factors that limit orimpede the incorporation of soybean protein products into many foods.

A. l vorAlthough soybean proteins have made inroads as an ingredient in a number ofprocessed foods, the level of protein that can be added is often limited by residualflavors characteristic of raw soybeans. Flavor is less ofa problem in foods such asmeat analogs that contain spices and seasonings, but it becomes a seriouslimitation in bland foods like dairy products. Nonetheless, progress has beenmade. For example, soy protein isolates are used in several liquid coffee


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356 / Advances in Cereal Science n TechnologJwhiteners, and a major company that traditionally used milk proteins is nowadding isolates to some of its products.Several recent reviews have dealt with the flavor problem of soybean proteinsCowan et al., 1973b; Maga, 1973; Wolf, 1975).Flavor of Commercial Proteins. ommercialsoy flours, concentrates, andisolates were surveyed for odor and flavor characteristics by a taste panel in 1970as summarized in Table XVI. Evaluations were made on 2 dispersions inwater,and scoring was based on a I to scale where I is a strong odor or flavor and is bland. A raw, hexane-defatted flour included for comparative purposes wasscored the lowest. For the flours, the highest scores were obtained for those thathad received the greatest amount of moist heat treatment as measured by thenitrogen solubility index NSI). One of the concentrates received the highestflavor score of the various products tested. Beany, bitter, nutty, and toasted weresome of the flavor descriptions given by the panel. For beany and bitter flavors inthe raw flour, respective threshold concentrations levels at which 50 of thepanel detected the flavors) were 0.03 and 0.04 as compared to 1.25 and 3.0 forone of the isolates. Despite the extensive processing given to isolates, the flavorcompounds are not removed completely.Source of Flavor Compounds. Lipids have been implicated by a number ofworkers as the origin of undesirable flavors in soybean products. For example,Nelson et l 1971) have postulated that intact soybeans are free of undesirableflavors and that the flavor compounds are generated by lipoxygenase-catalyzedoxidation of the oil as soon as the cellular structure is broken. Consequently, theyrecommend blanching the beans before crushing them to inactivate thelipoxygenases and thereby prevent the formation of malflavored compounds.Grosch and Schwencke 1969) observed that incubation of linoleic acid withpartially purified lipoxygenase and oxygen resulted in the formation of 3.4monocarbonyl compounds in the following amounts:

Aldeh.vden Pentanaln Hexanaln-Hept-2-enaln-Oct-2-enaln-Nona-2,4-dienaln-Deca-2,4-dienalUnidentified

Mole, 455

26

Hexanal is the major carbonyl compound found. All of these compounds plusabout 5 others were isolated from soy milk made in the traditional Orientalmanner Wilkens and Lin, 1970). Hexanal comprised about 25 of the volatilefraction isolated from soy milk. Many of the volatile compounds isolated havegrassy/ beany odors and have extremely low odor thresholds.Qvist and von Sydow 1974) identified and quantitated the volatilecompounds in the headspace of a solution of a soy protein isolate that was


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Chemistry nd Technology of Soybeans / 357unheated or heated. They found over 5 compounds in the headspace from theunheated isolate. Major compounds found were:

Unheated HeatedCompound pp ppbEthanal 2500 4250I-Pentanal 68 78Hexanal 26 72-MethyIpropanal 62 232-Methylpropenal 68 212-Propanone 360 44002-Ethylfuran 13 40002 Pentylfuran 95 2700

Ethanal was the major compound found in the unheated protein. Heatingusually increased the concentrations of volatile compounds detected.Two hydroperoxides are possible when linoleic acid is oxidized: 9-D-hydroperoxy- I0, I2-octadecadienoic acid and 13 L-hydroperoxy-9, I I-octadecadienoic acid. Leu (l974a, b) found that Iipoxygenase L-I (Classical Theorellpreparation) formed mainly the I3-isomer at high oxygen concentrations, pH 9,and low temperature. However, at low oxygen concentration l O 2 in gasphase), pH 6.8, and high temperature, about equal quantities of the twohydroperoxide isomers were obtained. Analysis of the volatile compoundspresent in oxidized linoleic acid (catalyzed by lipoxygenase L-I) revealed thatwhen the reaction was carried out in I oxygen, a number of aldehydes andhydrocarbons was observed. Prominent compounds were pentanal, hexanal,and 2-n-pentylfuran. In an oxidation carried out with 100 oxygen, hexanalagain was a major component, but pentanal and 2-n-pentylfuran were formed insmaller amounts than at the low oxygen tension.Volatile compounds derived from the hydroperoxides oflinoleic and linolenicacids are usually regarded as the source of malflavors, but a direct test of this

hypothesis was made only recently. Kalbrener et al. (1974) prepared thehydroxyperoxides oflinoleic and linolenic acids by lipoxygenase catalysis at highoxygen tensions. After purifying the hydroperoxides by silicic acid columnchromatography, they made dilute dispersions of the hydroperoxides in water

T BLE XVIOdor and flavor scores for soybean protein productsProductDefatted floursConcentratesIsolates

Odor Scores

5.8-7.56.4-7.46.8-7.7

Flavor Scoresb

4.1-6.75.6-7.05.9-6.4ource: Kalbrencr et l (1971).bBased on a scale of to 1 where is strong and 1 is bland.


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358 / Advances in Cereal Science n Technologyand submitted them to a taste panel. Linolenic acid hydroperoxide at ppm andlinoleic acid hydroperoxide at 50 ppm were scored the same flavor intensity as0.25 raw defatted soy flour dispersed in water. Grassy/ beany was the mainflavor description given. Minor flavor responses \vere bitter, astringent, rawvegetable, and fruity. Because flavors given for raw defatted flour did not includethe last two terms, the hydroperoxide dispersions did not reproduce the typicalsoybean flavors exactly. Nonetheless, the hydroperoxides seem likely sources forthe grassy/ beany flavors. Furthermore the hydroperoxide flavor profile may besensitive to the oxygen tension prevailing during hydroperoxide formation.Kalbrener and coworkers employed a high oxygen concentration, whereasoxidation under ambient conditions when the flavors in soybean products arelikely to be generated) may give a different spectrum ofvolatile compounds Leu,1974b).Recent work by Grosch and Laskawy 1975) showed that lipoxygenases L-lL-2, and L-3 differ significantly in their abilities to form volatile carbonylcompounds. The L-2 and L-3 enzymes formed greater quantities and a largervariety of carbonyl compounds than L-l. They also presented evidence that thevolatile carbonyl compounds do not arise by decomposition of themonohydroperoxides.The hydroperoxides of linoleic and linolenic acids were reported to be bitterKalbrener et al 1974), but there is some doubt whether this is an importantsource of this flavor in defatted soybeans. Sessa et l 1974) have found thatphosphatidylcholine may be another source of bitterness in soybean flakes.When they autoxidized a purified phosphatide preparation for 2 weeks inaqueous dispersion with 1 ppm of Cu it developed a bitter taste with athreshold of 0.006 . In further work Sessa and coworkers 1975) isolated threephosphatidylcholines from residual lipids found in hexane-defatted soybeanflakes. One of the phosphatidylcholines was weakly bitter when isolated fromfresh defatted flakes, but a comparable fraction 0 btained from l-year-old flakeswas much more bitter. The other two phosphatidylcholines were strongly bitterwhen freshly prepared and tasted at a concentration of 0.05 . Fatty acids fromall of the phosphatidylcholines contained hydroxyl and conjugated keto groupspresumably as a result of oxidation of unsaturated fatty acids. appears likelythat these phosphatides contribute bitterness to soybean flakes.The conventional method for inactivating lipoxygenases in soybean productsis heat treatment, but other methods can also be used. Eldridge et l in press)have found that lipoxygenase was inactivated when intact beans were soaked inaqueous alcohol. When the alcohol was removed by vaporization and the beanswere ground, there was a distinct improvement of the flavor as compared tobeans soaked either in water or pure alcohol. Lipoxygenase measured at pH 9.0with linoleate) was inactivated with 2 to 80 ethanol Figure 8), but the bestflavor scores were obtained over the narrower treatment range of 40 to 60ethanol. The possibility that the lipoxygenases that are specific for esterifiedlinoleic acid remain active above and below 40 to 60 alcohol has not been ruledout. Figure 8 also shows that extensive insolubilization ofprotein occurs with 20to 80 alcohol treatment as measured by the NSI test. Urease is extensivelyinactivated in this alcohol concentration range, whereas soybean trypsininhibitor is fairly stable.


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Chemistry and Technology of Soybeans / 359B Functional Properties

Next to flavor, functional properties of soybean proteins have been one oftheimportant factors limiting their use in certain foods. Consequently, many appliedstudies have been carried out, but published information occurs primarily inpatents. For example, almost all of the studies on texturizing soybean proteinshave been concerned with duplicating the chewiness and mouthfeel of variousmeat products starting with a material with little resemblance to meat. Likewise,several industrial laboratories have expended a great deal of effort to makesoybean proteins soluble in acid solutions so as to permit their addition tocarbonated beverages, fruit juices, and the like. Less effort has been expended onbasic studies related to functional properties.Solubility Hermansson (1973) compared solubilities of a commercial soybeanprotein isolate, sodium caseinate, milk whey protein concentrate, and fishprotein concentrate as a function of pH in 2Msodium chloride. As expected,the soy isolate had a minimum solubility in the pH region of 4 to 5, but in the pHrange of 6.5 to 8.0 only 25 to 30% of the protein dissolved. Only by increasing thepH above 11 was high solubility obtained. She also examined the effect of ionicstrength at pH 7 In water the solubility was about 55%; but when salt was addedto 0 2M solubility decreased to 28%. By comparison, sodium caseinate andwhey protein concentrates were not sensitive to changes in ionic strength up to2M salt.A later series of studies Hermansson and Akesson, 1975a, b; Hermansson,1975) described the effects of heat treatment of soy protein isolate, sodiumcaseinate, and milk whey protein concentrate on solubility and other parameters

1

8

->4


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360 / Advances in Cereal Science and Technologyand 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 waterloss. Heated dispersions of isolate gelled, and gelation showed a high negativecorrelation with water loss from the meat systems. Effects of pH and ionicstrength on solubility of soybean globulins were described by van Megen 1974).He observed an unusual liquid-liquid phase separation below critical values ofionic strength. The upper phase supernatant was low in protein content,whereas the lower phase was viscous and high in protein concentration. Ahomogenous solution was obtained at ionic strengths above the critical value.Tombs 1972) called the two layers mesophases and spun them into protein fibersas discussed earlier.Water solubilities for two commercial protein isolates were reported byHagenmaier 1972). At pH 6.0 one had a solubility of 22 and the other was 40soluble.Water Absorption and Swelling Hermansson 1972) has followedspontaneous water uptake of proteins in an apparatus devised by Baumann1966) and uses the results as a measure of swelling properties. Swelling is limitedfor proteins that do not dissolve; but for proteins that dissolve, swelling isunlimited. The Baumann apparatus is a water-filled funnel containing a frittedglass disk that contacts the water. To run the test, dry protein is sprinkled on awetted filter paper placed over the glass disk, and water uptake is followed as afunction of time by reading a calibrated horizontal capillary tube attached to thefunnel. Swelling of soy isolate is greater than for sodium caseinate or milk wheyprotein concentrate. Although the swelling properties of the proteins did notcorrelate with water binding in model meat systems Hermansson and Akesson,1975a), they did correlate with textural properties of meatballs containing theproteins Hermansson, 1975).

Water vapor sorption by isolated soybean protein is reported for three wateractivities by Hermansson 1973). When the protein was heated, water sorptionincreased very little but swelling increased significantly. Water sorption at 84relative humidity was compared for soy protein isolate plus other plant proteinsand several animal proteins Hagenmaier, 1972). Animal proteins bound morewater than plant proteins, and binding appears to be a function of the polar sidechain content-hydroxyl, amino, and carboxyl groups.

Water absorption of soybean and sunflower flours, concentrates, and isolateswas measured by slurrying them in water or sodium chloride and thencentrifuging Fleming et al 1974). Absorbed water was taken as the decrease involume offree liquid. Among the soybean proteins, isolates had the highest waterabsorpt ions, followed by concentrates which were higher than flours. Oneshortcoming of this test is that if a protein is completely soluble, it will show noapparent water absorption; but if it is incorporated in a food system, it may beinsolubilized or gelled by heating and show excellent water absorptioncharacteristics.Various protein additives, including six soybean proteins, were evaluated asemulsion stabilizers in frankfurters Smith al 1973). Water-holding capacitywas determined under conditions of salt concentration, heat treatment, and pHsimulating frankfurter manufacture. Despite attempts to duplicate

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Chemistry and Technology of Soybeans

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