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CHAPTER 38 Fortification of Bread with Soy Proteins to Normalize Serum Cholesterol and Triacylglycerol Levels Reiko Urade Department of Bioresource Science, Kyoto University, Gokasho, Uji, Kyoto, Japan CHAPTER OUTLINE List of Abbreviations 417 Introduction 417 Soy Proteins 418 Isolation of Soy Proteins 419 Physiological Function of Soy Proteins 420 Adverse Effects of Soy Protein Fortification on Rheological Properties of Dough and Bread Quality 421 Technological Issues 423 Summary Points 425 Acknowledgments 426 References 426 LIST OF ABBREVIATIONS ER Endoplasmic reticulum GMP Glutenin macropolymer LDL Low-density lipoprotein LPs Lipophilic proteins PC Phosphatidylcholine SDS Sodium dodecyl sulfate SPI Soy protein isolate TG Triacylglycerol VLDL Very low-density lipoprotein INTRODUCTION Soybean (Glycine max L. Merrill) is a species of legume that originated in East Asia and has been cultivated for approximately 5000 years in northeastern China. Soybean is used to make traditional foods, including tofu, soybean paste, miso, and soy sauce. Soybean was first 417 Flour and Breads and their Fortification in Health and Disease Prevention. DOI: 10.1016/B978-0-12-380886-8.10038-8 Copyright Ó 2011 Elsevier Inc. All rights reserved.

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Page 1: Flour and Breads and their Fortification in Health and Disease Prevention || Fortification of Bread with Soy Proteins to Normalize Serum Cholesterol and Triacylglycerol Levels

CHAPTER 38

Fortification of Bread withSoy Proteins to NormalizeSerum Cholesterol andTriacylglycerol Levels

Reiko UradeDepartment of Bioresource Science, Kyoto University, Gokasho, Uji, Kyoto, Japan

Fl

C

CHAPTER OUTLINE

417

List of Abbreviations 417Introduction 417Soy Proteins 418Isolation of Soy Proteins 419Physiological Function of SoyProteins 420Adverse Effects of Soy ProteinFortification on RheologicalProperties of Dough and BreadQuality 421

our and Breads and their Fortification in Health and Disease Prevention. DOI: 1

opyright � 2011 Elsevier Inc. All rights reserved.

Technological Issues 423Summary Points 425Acknowledgments 426References 426

LIST OF ABBREVIATIONSER Endoplasmic reticulumGMP Glutenin macropolymer

LDL Low-density lipoprotein

LPs Lipophilic proteinsPC Phosphatidylcholine

SDS Sodium dodecyl sulfate

SPI Soy protein isolateTG Triacylglycerol

VLDL Very low-density lipoprotein

INTRODUCTIONSoybean (Glycine max L. Merrill) is a species of legume that originated in East Asia and has been

cultivated for approximately 5000 years in northeastern China. Soybean is used to make

traditional foods, including tofu, soybean paste, miso, and soy sauce. Soybean was first

0.1016/B978-0-12-380886-8.10038-8

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418

SECTION 2Fortification of Flour and Breads and their Metabolic Effects

introduced to Europe in the early seventeenth century and to the United States in the eigh-teenth century, and it has since become an important global crop. Global soybean production

was more than 220 million metric tons in 2007.

Soybeans contain extremely high amounts of protein and oil (30e50% and 13e25% of the

total mass, respectively). Soy protein meets the required amino acid composition for humans

and animals, except for slightly low sulfur amino acid content. However, soy foods have notbeen well accepted in areas other than eastern Asia due to their bitter taste, chalky mouthfeel,

and the “beany” and “greeny” flavors that are generated primarily from linoleic acid by soy

lipoxygenases. For that reason, most fat-free soy meal has been used as the primary source ofprotein for animal feeds or rations.

In the 1960s, a new method became available to extract food-grade soy protein isolate (SPI)from defatted soy meal under lower temperatures. SPI is used in a variety of foods for its

functional properties, including solubility, water and fat absorption, and emulsification. In

addition, soy protein has been shown to lower the risk of cardiovascular disease in humans(Anderson et al., 1995). In particular, one of the major storage proteins of soy, b-conglycinin,

reduces high serum triacylglycerol (TG) concentration and visceral fat in humans (Kohno et al.,

2006). Daily intake of SPI and b-conglycinin is required for this effect; thus, SPI orb-conglycinin supplementation in foods eaten daily is desirable. As such, bread is a convenient

vehicle for SPI and b-conglycinin; however, SPI and b-conglycinin produce adverse effects on

bread making and bread quality. This chapter reviews the features and the physiologicalfunctions of soy proteins as well as the characteristics of bread fortified with soy proteins.

SOY PROTEINSIn the typical soybean, proteins comprise approximately 40% of the total mass; however, both

genetic and environmental factors strongly influence seed composition. The nutritional value

of soy proteins is high; the protein digestibility-corrected amino acid score of SPI is approxi-mately that of egg white. Furthermore, the biological values (i.e., the ability of the body to

absorb and utilize the protein) of whole soybean, soy milk, and SPI are 74, 96, and 91,

respectively. These values are the highest among major edible crops. Adding soy protein tofoods made from crops such as wheat, maize, and rice increases their nutritional value because

soy protein contains relatively high amounts of lysine, which is a limiting amino acid for

complete protein in these crops.

Soybean produces exalbuminous seeds. Within the embryo, storage proteins glycinin and

b-conglycinin are synthesized and stored in cotyledons, where they are subsequently used asnitrogen, carbon, and sulfur sources in embryonic development (Nielsen and Nam, 1999).

These globulin proteins comprise approximately 60% of the total soy proteins (Nielsen and

Nam, 1999); the remaining proteins fulfill protective, structural, and metabolic roles.

b-Conglycinin is composed of three main types of subunits designated a, a0, and b, with

molecular weights of 50e70 kDa. Random combinations of these subunits form seven

heterotrimers and three homotrimers. b-Conglycinin subunits are translated in the roughendoplasmic reticulum (ER) and undergo folding and assembly into trimers in the ER lumen.

Subunits are modified by cotranslational N-glycosylation (i.e., one N-glycan on the b subunit

and two N-glycans on the a or a0 subunit). The assembled trimers are transported via the Golgiapparatus and accumulate in protein storage vacuoles.

Glycinin is composed of five types of subunits designated A1aB1b, A1bB2, A2B1a, A3B4, andA5A4B3. They are categorized into two groups according to amino acid sequence similarity:

group I (A1aB1b, A1bB2, and A2B1a) and group II (A3B4 and A5A4B3). Each glycinin subunit

is synthesized in the rough ER as a precursor protein (molecular weights of approximately50 kDa). They undergo folding, formation of intrachain disulfide bonds, and assembly into

trimers in the ER lumen. Several lines of evidence indicate that the folding is performed with

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CHAPTER 38Fortification of Bread with Soy Proteins to Normalize Serum Cholesterol

419

the aid of molecular chaperones and several members of the protein disulfide isomerase family(Kamauchi et al., 2008; Wadahama et al., 2007, 2008). The assembled trimers are transported

via the Golgi or directly from the ER to protein storage vacuoles. Glycinin precursor subunits in

trimers are cleaved into acidic subunits and basic subunits at a well-conserved AsneGlypeptide bond by a vacuolar processing enzyme and then assembled into hexamers in the

protein storage vacuoles (Nielsen and Nam, 1999). The three-dimensional structures ofb-conglycinin and glycinin are very similar, suggesting that these genes evolved from

a common ancestor gene (Adachi et al., 2003; Maruyama et al., 2004).

ISOLATION OF SOY PROTEINSMost edible soy protein products are derived fromwhite flakes made by dehulling, flaking, and

defatting soybeans by hexane extraction. These products consist of defatted flour (approxi-mately 50% protein), soy protein concentrate (65e70% protein), and SPI (>90% protein).

Recently, the purified soy protein component b-conglycinin isolate has become commercially

available. Soy protein products have many important functional properties, including solu-bility, water and fat absorption, emulsification, and imparting of texture (i.e., gelation,

cohesioneadhesion, and elasticity). Soy protein products are used in a variety of foods,

including beverages, meat products, bakery items, pasta products, cheeses, and simulatedmeats.

Commercial SPI is extracted from white soy flakes with water. b-Conglycinin and glycinin are

soluble in salt solution, and salts are present in white flakes; therefore, these proteins are easilyextracted by adding water. Most proteins can then be isoelectrically precipitated after the

extract is acidified (pH 4.3e4.8). Finally, the precipitated protein curd is neutralized, sterilized,

and dried (Figure 38.1). For a long time, SPI was believed to be composed primarily ofb-conglycinin and glycinin. However, SPI has been found to contain lipids associated with

lipophilic proteins (LPs) such as oil body-associated proteins (Iwabuchi and Yamauchi, 1987;

Samoto et al., 1998). Because n-hexane cannot efficiently extract phospholipids or hydro-phobic membrane proteins, acid-precipitated proteins contain LPs associated with membrane

FIGURE 38.1Schematic diagram depicting isolation of soy protein isolate. Water was added to white soy flakes defatted with n-hexaneto solubilize proteins. Isoelectric precipitation of most proteins can be performed after lowering the pH. The precipitated protein

curd is then neutralized, sterilized, and dried. SPI, soy protein isolate.

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FIGURE 38.2Schematic diagram depicting fractionation of glycinin, lipophilic proteins, and b-conglycinin. A three-step acidification ofthe water extract of defatted soy flour separated three proteins with the nitrogen distribution ratio of 23% (b-conglycinin), 46%(glycinin), and 31% (lipophilic proteins). LP, lipophilic proteins.

SECTION 2Fortification of Flour and Breads and their Metabolic Effects

420

phospholipids from oil bodies and protein storage vacuoles. Compared with b-conglycinin

and glycinin, LPs are difficult to detect by sodium dodecyl sulfate (SDS) polyacrylamide gelelectrophoresis due to lower sensitivity to Coomassie brilliant blue staining; thus, the

importance of LPs in SPI has been overlooked. Samoto et al. (2007) developed a methodfor fractionating acid-precipitated proteins (Figure 38.2). In that study, the nitrogen distri-

bution ratios for the three separated proteins were 23% (b-conglycinin), 46% (glycinin),

and 31% (LPs).

PHYSIOLOGICAL FUNCTION OF SOY PROTEINSApproximately 100 years ago, the cholesterol-lowering effects of soy protein compared withanimal protein were reported in rabbits (Ignatowsky, 1908). Since then, many studies have

reported the effects of soy proteins on serum lipids in humans; however, results have been

inconsistent, possibly because of different experimental conditions, such as soy proteincontent in the diet and degree of hypercholesterolemia in the subjects. In a meta-analysis

published in 1995, Anderson et al. concluded that soy protein consumption significantly

decreased serum levels of total cholesterol, low-density lipoprotein (LDL) cholesterol, and TG,corresponding to the degree of hypercholesterolemia. Based on these findings, the U.S. Food

and Drug Administration granted the following health claim for soy protein in 1999:

“25 grams of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reducethe risk of heart disease.”

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CHAPTER 38Fortification of Bread with Soy Proteins to Normalize Serum Cholesterol

421

Most commercially available SPI products contain significant amounts of genistein,daidzein, and glycitein. These isoflavones have been shown to exert strong biological actions

in animals, such as serum cholesterol lowering, arterial vasodilation, and atherosclerosis

inhibition (Sacks et al., 2006). Hence, these isoflavones were assumed to be largelyresponsible for the beneficial effects of SPI on hypercholesterolemia in humans. Human

studies comparing the effects of casein, animal proteins, and ethanol-washed isoflavone-freeSPI on serum cholesterol levels have demonstrated declines in LDL cholesterol with

isoflavone-free soy protein consumption (Jenkins et al., 2002; Lichtenstein et al., 2002).

Furthermore, studies comparing the effects of SPI with or without isoflavones confirmed thatisoflavones are not responsible for the lipid-lowering effects in humans. However, the soy

protein component(s) responsible for this effect is not known. Candidates include a peptide

derived from glycinin that inhibits reabsorption of bile acid from the intestine (Nagaokaet al., 1997) and LPs that have been shown to reduce serum cholesterol (Kanamoto et al.,

2007). However, these studies were performed in rats; the effects of glycinin and soy LPs in

humans are unclear. Thus, identification of components responsible for cholesterol loweringremains unsolved.

The effects of LP-free b-conglycinin were assessed by supplementation of the diets of adults

with high plasma TG. Intake of b-conglycinin (5 g/day) normalized serum TG and reducedvisceral fat in subjects with body mass indices between 25 and 30 (Kohno et al., 2006). Based

on these findings, in 2007, soy b-conglycinin was approved as a food for specified health use in

Japan.

The plasma TG level is controlled by the amount of very low-density lipoprotein (VLDL)

secreted from the liver and the rate of VLDL-TG catabolism in blood. To determine the effectsof soy b-conglycinin on lipid metabolism, small peptides were derived from LP- and isofla-

vone-free b-conglycinin by protease digestion and used to treat the human hepatocellular

carcinoma cell line HepG2 (Mochizuki et al., 2009). The findings showed that theb-conglycinin-derived peptides suppressed TG synthesis, thereby suppressing the secretion

of VLDL from HepG2 cells into the medium.

ADVERSE EFFECTS OF SOY PROTEIN FORTIFICATION ONRHEOLOGICAL PROPERTIES OF DOUGH AND BREAD QUALITYThe addition of SPI and purified b-conglycinin to foods can increase soy protein consumptionand help achieve a physiologically beneficial intake. Fortification of bread with soy protein has

a long history due to the relatively high amounts of lysine and valine in soy, which are the

limiting amino acids of wheat proteins. Although the nutritive value of the bread rises with thepercentage of SPI (Mizrahi et al., 1967), SPI reduces bread quality. SPI-containing bread was

judged to be firmer, drier, grainier, less tender, and gummier compared to the ideal bread

(Elgedaily et al., 1982). In addition, SPI-containing bread exhibited a strong beany flavor,which curtailed its overall acceptability (Ranhotra and Loewe, 1974). Bread loaf volume also

decreased, and bread crumb firmness and firming rate increased proportionally with the level

of SPI fortification (Chen and Rasper, 1982; Mizrahi et al., 1967; Ribotta, Arnulphi, et al.,2005). An inverse relationship was shown between bread loaf volume and firmness; therefore,

the low specific volume of SPI-containing breads was probably the cause of the increased

firmness values rather than the effect of soy protein per se (Ribotta, Arnulphi, et al., 2005).A strong correlation between dough volume after fermentation and finished loaf volume has

been observed in the presence of SPI. SPI had no effect on CO2 production by yeast; thus, SPI

may decrease the gas retention capacity of dough. Scanning electron microscopy revealedthat gluten containing SPI had a much more porous network compared with that of control

dough lacking SPI (Roccia et al., 2009), suggesting that SPI makes the gluten network brittle

against the pressure of gas. Highly purified b-conglycinin decreases loaf volume more than SPI(Ukai and Urade, 2007; Urade et al., 2003).

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TABLE 38.1 Effects of Soy Protein Isolate on Rheological Properties of Wheat Flour Dough

Property Equipment Parameter Effect of SPI References

Farinographicbehavior

Microfarinograph Water absorption Increase Mizrahi et al. (1967),Ranhotra et al. (1974),Ribotta, Arnulphi, et al.(2005), Roccia et al. (2009),Urade et al. (2003)

Arrival time DecreaseDevelopment time DecreaseStability Decrease

Dynamic viscoelasticproperty

Rheolograph gel Storage modulus (E0) Increase Urade et al. (2003)Loss modulus (E00) Increasetan l (E00/E0) Decrease

Large deformationproperty

Rheoner Force at failure point Increase Urade et al. (2003)Failure point No change

Micro-extensograph Rm Increase Ribotta, Arnulphi, et al.(2005), Roccia et al. (2009)E Decrease

Area DecreaseCreep property Texture analyzer Jo Decrease Roccia et al. (2009)

Jl Decreasemo Increase

E, maximum extensibility; Jo, instantaneous compliance; Jl, retarded compliance; mo, Newtonian viscosity; Rm, maximum resistance to extension; SPI, soy protein

isolate.

SECTION 2Fortification of Flour and Breads and their Metabolic Effects

422

Changes in the gluten network structure were accompanied by changes in the rheological

properties of dough. Farinograph analysis revealed that the addition of SPI reduced arrivaltime, development time, and stability (Table 38.1) (Mizrahi et al., 1967; Ranhotra and Loewe,

1974; Urade et al., 2003). SPI also affected the dynamic viscoelastic properties of dough to

increase storage (E’) and loss modulus (E’’) values and decrease tan l (E00/E0) value, whichrepresents the strength of the gluten network as determined by a Rheolograph gel (Urade et al.,

2003). In addition, SPI affected the large deformation properties of dough. The force value at

failure point obtained using a Rheoner increased after adding SPI to the dough (Urade et al.,2003). When tensile strength test was performed with the Intron Universal Testing Machine,

SPI increased the resistance to extension and the relaxation time (Chen and Rasper, 1982).

Increased maximum resistance and decreased extensibility by SPI fortification were revealedusing a micro-extensograph (Roccia et al., 2009). At the same time, area under the extension

curve, which is a measure of the energy required for extension, was reduced by SPI, and the

area under the extension curve was highly correlated with specific loaf volume. By the creeptest, SPI decreased instantaneous compliance, retarded compliance of the dough, and

increased Newtonian viscosity of the dough (Roccia et al., 2009); the decrease in retarded

compliance reflects the loss of elasticity of the dough. Taken together, the results obtained bythe rheological assays indicated that SPI increases dough firmness and weakens the gluten

network.

SPI appears to weaken the gluten network both indirectly and directly. Soy proteins such

as glycinin and b-conglycinin were detected in SDS-insoluble gluten proteins obtained

from dough (Ribotta, Leon, et al., 2005), indicating that soy proteins tightly associatewith gluten proteins in dough. Hydrogen bonds and hydrophobic interactions between

glutenin and gliadin molecules are essential for gluten formation; soy proteins may

interfere with these interactions. Dough’s elastic properties are related to the quantity ofthe SDS-insoluble glutenin macropolymer (GMP), which is comprised of high-molecular-

weight and low-molecular-weight glutenin subunits linked by disulfide bonds. Although

it seems possible that soy protein containing cysteine residues may decrease GMP contentby thiol exchange or reduction of disulfide bonds among glutenin proteins, GMP content

of SPI-containing dough did not show any significant differences compared with dough

lacking SPI (Perez et al., 2008). Even a small amount of soy glycinin (2.5e8.2% of totalwheat protein) decreased developing time and stability time (Lampart-Szczapa and

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CHAPTER 38Fortification of Bread with Soy Proteins to Normalize Serum Cholesterol

Jankiewicz, 1982); thus, direct interactions with soy proteins may considerably alter thegluten network.

The indirect influence of SPI is related to reduced water availability in the dough. Many studieshave reported that SPI increases water absorption in wheat dough as assessed by farinograph

(Roccia et al., 2009; Urade et al., 2003). Thus, water binding in the dough is increased and

syneresis is decreased (Roccia et al., 2009). Free water is thought to act as an inert filler orlubricant in polymers such as gluten (Masi et al., 1998). Therefore, changes in dynamic

viscoelastic behavior and the relaxation phenomena may be due in part to the decrease in free

water by SPI fortification.

423

TECHNOLOGICAL ISSUESThe lower quality of bread due to SPI fortification is a serious problem. The increased firmnessand smaller loaf volume may be primarily due to the reduced free water in dough and

interference of the gluten network formation. Roccia et al. (2009) showed that adding morewater to the dough attenuated the SPI-induced increase in maximum resistance, but the

additional water had no effect on extensibility or area under the extension curve. Therefore, the

problem of increased firmness in SPI-containing bread can be solved by adjusting the amountof water to be added. In addition, adding more water restored normal instantaneous

compliance and partially attenuated the decrease in retarded compliance on the creep test,

which is an index parameter for elasticity (Roccia et al., 2009).

Several materials, including detergents, have been shown to improve the bread making

properties of SPI-containing dough and the quality of protein-fortified bread. Studies have

reported the effects of sodium stearoyl-2-lactylate (SSL), nonionic hydrophilic polysorbate 60(Tween 60), and nonionic lipophilic sodium tristearate (Span 65) on the rheological prop-

erties of the SPI-containing dough (Chen and Rasper, 1982). These detergents did not alter

water adsorption of SPI-containing dough according to the farinograph assay, nor did theyimprove the increased resistance to extension or the relaxation time according to the tensile

strength test. However, Tween 60 improved gas retention in the dough and increased loaf

volume. SSL and Span 65 also improved gas retention in dough, but they did not completelyrestore the normal loaf volume of bread containing SPI.

Lecithin is a mixture of polar lipids that is permitted for use in all types of bread and bakeryproducts. Lecithin also improves gas retention in dough and sufficiently restores the volume of

dough fortified with SPI (Figure 38.3) or b-conglycinin (Mizrahi et al., 1967; Ribotta, Arnulphi,

et al., 2005; Urade et al., 2003). Moreover, the addition of lecithin produces a thicker, densercrust that tends to retain its crispness. Most lecithin used in the baking industry is derived from

soybeans; soy lecithin is a mixture of phospholipids that includes phosphatidylcholine (PC),

phosphatidylethanolamine, and phosphatidylserine. PC is the active constituent in lecithin(Urade et al., 2003), the function of PC cannot be replaced by other major phospholipids,

phosphatidylethanolamines, or compounds derived from PC, phosphatidic acid, lysophos-

phatidylcholine (lysoPC), or choline. Confocal microscopy revealed that soy PC co-localizeswith gluten in dough but not the starch granules (Figure 38.4). Farinograph behaviors or

viscoelastic properties of dough impaired by SPI were not improved by soy PC. Soy PC exerted

little effect on arrival time, development time, and stability. Nor did soy PC change therheolograph value (E0 and E00) or large deformation properties of SPI-containing dough.

Interestingly, the effect of PC on delipidated wheat flour containing b-conglycinin was lowercompared with its effect on native wheat flour containing b-conglycinin (Ukai and Urade,

2007). Thus, soy PC alone has the ability to increase dough volume, and wheat flour lipids

appear to boost this effect. Among wheat lipids, glycolipids such as monogalactosyldiglyceride and digalactosyl diglyceride best enhance the action of soy PC (Ukai and Urade,

2007).

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FIGURE 38.3Effects of soy protein isolate and soy phosphatidylcholine supplementation on bread quality. (A) Unfortified bread (left),bread fortified with soy protein isolate (SPI) (center), and bread fortified with SPI plus soy phosphatidylcholine (PC) (right). (B)

Scanning electron micrograph of unfortified bread (left), bread fortified with SPI (center), and bread fortified with SPI plus soy PC

(right). For the wheat flouresoy protein combinations, 10% of the wheat flour was replaced with SPI (by weight). PC (2 g) was

added to flour (100 g). PC, the active constituent in lecithin, improves gas retention in dough and sufficiently restores the

volume of dough fortified with SPI. Scale bars ¼ 200 mm. Source: Reprinted with permission from Urade, R., Okamoto, S.,

Yagi, T., Moriyama, T., Ogawa, T., and Kito, M. (2003). Functions of soy phosphatidylcholine in dough and bread

supplemented with soy protein. J. Food Sci. 68, 1276e1282.

SECTION 2Fortification of Flour and Breads and their Metabolic Effects

424

PCs are a class of phospholipids composed of two fatty acyl chains. Fatty acyl chains that arecombined in soy PC are linoleic (18:2), palmitic (16:0), stearic (18:0), oleic (18:1), and lino-

lenic (18:3) acids. Many PC molecular species are possible, and the effect of PC on bread loaf

volume depends on the particular molecular species (Figure 38.5). At one extreme, 1-palmitoyl,2-palmitoyl-PC had no effect on dough volume at 36�C during fermentation. This molecular

species exists in the gel crystalline form at 36�C because its gel-to-liquid crystalline phase

transition temperature is 44.3�C. In contrast, PC molecular species with a liquid crystallinephase transition temperature lower than 36�C increase dough volume, possibly because PC in

the liquid crystalline state improves the gas-retaining ability of dough during fermentation. Soy

PC is composed ofmolecular species with liquid crystalline phase transition temperatures lowerthan 36�C, including dilinoleoyl-PC (35%), 1-palmitoyl, 2-linoleoyl-PC (24%), and 1-oleoyl,

2-linoleoyl-PC (15%) (Urade et al., 2003). Differential scanning calorimetry analysis revealed

the phase of soy PC transited at �48.5 to �13.1�C. In its fluid liquid crystalline state, PC self-assembles into a stable bilayer structure and exists as liposomes or multilamellar vesicles

(lamellae) in water. The most surface-active property of polar lipids is thought to be a bilayer

structure. Thus, a possible mechanism for the beneficial effects of PCmay be its direct influenceon gas cells in dough by increasing foam stability (Urade et al., 2003).

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FIGURE 38.4Distribution of phosphatidylcholine (PC) and gliadin in dough. Dough was made with soy PC containing the fluorescentPC, b-BODIPY FL C12-HPC. Gliadin in dough was immunostained with anti-gliadin rabbit serum, biotin anti-rabbit IgG goat

serum, and Cy 5-streptoavidin. The dough was visualized with a laser confocal imaging system showing (A) PC (white) and (B)

gliadin (white). The fluorescent PC, b-BODIPY FL C12-HPC was associated with protein fibers containing gliadin and yeast

(arrows). Scale bar ¼ 10 mm.

FIGURE 38.5Effects of phosphatidylcholine (PC) molecular specieson volume of soy protein isolate (SPI)-containing doughafter fermentation. Dough was made with 100% wheat

flour or wheat flour in which 10% was replaced with SPI. In

some doughs, the flour (100 g) was further supplemented

with 2 g of PC or phosphatidylethanolamine (PE). Dough

(15 g) was then incubated at 36�C for 40 min. 14:0/14:0,

dimyristoyl PC; 16:0/16:0, dipalmitoyl PC; 18:1/18:1,

dioleoyl PC or PE; 18:2/18:2, dilinoleoyl PC. The liquid

crystalline phase transition temperature of each PC is

shown in parentheses. PC molecular species with a liquid

crystalline phase transition temperature lower than 36�Cincreased dough volume.

CHAPTER 38Fortification of Bread with Soy Proteins to Normalize Serum Cholesterol

425

SUMMARY POINTSl Soy protein isolate is composed of three major proteins: b-conglycinin, glycinin, and

lipophilic proteins.

l Physiological functions of soy protein isolate include the ability to reduce the risk of

cardiovascular disease in humans by decreasing serum cholesterol levels.l Highly purified b-conglycinin normalizes serum triacylglycerol levels in subjects with high

serum triacylglycerol and reduces visceral fat in subjects with body mass indices between 25

and 30.l Both soy protein isolate and b-conglycinin adversely affect bread making and bread quality

by increasing water absorption and interfering with the formation of the gluten network.

l Phosphatidylcholine increases loaf volume without affecting rheological properties ofdough.

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SECTION 2Fortification of Flour and Breads and their Metabolic Effects

426

AcknowledgmentsThe author gratefully acknowledges financial support from the Fuji Foundation for Protein Research, the Elizabeth

Arnold Fuji Foundation, and the Iijima Memorial Foundation for the Promotion of Food Science and Technology. The

author thanks Dr. Makoto Kito, Emeritus Professor of Kyoto University, for suggestions on the manuscript andencouragement.

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