Physicochemical Properties and Isoflavone Content Of

Home , Daidzin

PHYSICOCHEMICAL PROPERTIES AND ISOFLAVONE CONTENT OF

BREAD MADE WITH SOY

Dissertation

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the

Graduate School of The Ohio State University

YU CHU ZHANG, M.S.

* * * *

The Ohio State University 2004

Dissertation Committee:

Dr. Yael Vodovotz, Advisor Approved by Dr. Steven J. Schwartz

Dr. Joshua Bomser

Dr. Joseph Sachleben Advisor Dr. Mark L. Failla Department of Food Science and Technology

ABSTRACT

Epidemiological and experimental evidence suggests that consumption of soybean

products may significantly impact upon health. Bread made partially with soy represents

a traditional alternative for increased soy consumption, and, if successfully formulated

and well accepted, may offer an attractive delivery system for isoflavones and soy

proteins. The overall objective of this research was to assess the impact of soy addition

on physico-chemical properties in fresh and stored soy bread.

Addition of large amounts of soy (60%) significantly increases the bread density due to lack of gluten network formation and smaller air cell structure in soy bread. Total moisture content in soy bread (44.7%) was higher than in wheat bread (39.9%) and the distribution of water was found to be affected by soy addition. Upon storage, soy bread firmed at a lower rate (1.6 times) than wheat bread (6.7 times), which may be attributed to various factors: 1). Water distribution in the soy bread that favored easily removed bulk water pool that can act as a plasticizer throughout storage and maintain heterogeneity of the product, 2). A lack of recrystallized amylopectin, and 3). No change in mobility of liquid-like protons and a decrease in mobility of solid-like protons.

Total isoflavones were found to be stable during bread making, although their

profile was largely altered. The proofing stage in bread preparation was key in the

production of isoflavone aglycones in bread dough through β-glucosidase activity with

48 °C for two hours being optimal for aglycone production. Five percent almond

addition was found to be an effective and economic level to enhance isoflavone aglycones in the soy bread formula.

No significant changes were found between the isoflavone content and composition in fresh soy bread and soy bread stored for 14 days, showing good stability of isoflavones in soy bread under room temperature. Therefore changes in the water distribution and starch and protein mobility during storage did not affect the amount or profile of isoflavones indicating that bread eaten even a week or two after storage will deliver the same enhanced nutrition.

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Dedicated to my loving family

ACKNOWLEDGMENTS

I wish to thank my advisor, Dr. Yael Vodovotz, for her support, encouragement,

and patience that made this dissertation possible.

I am grateful to Dr. Steven J. Schwartz, who provided me with intellectual

guidance for my study on isoflavones.

I would like to thank all of my colleagues for their help and understanding. I

would especially like to thank Stefano Tiziani for his help with NMR experiments,

Elizebath A. Clubbs for her help with thermal analysis, Dr. Torsten Bohn for the stimulating discussion on isoflavones and statistical analysis, and Dr. Jae Hwan Lee for his help with isoflavone analysis.

I also wish to thank my dear friends at OSU during my study, especially Dr.

Josephine Kuo, Dr. Nuray Unlu, and Elizebath A. Clubbs, for their loving care and support.

VITA

July 29, 1972 ……….... Born - Shanghai, People’s Republic of China

1990-1996 ………...... Department of Chemistry East China Normal University, People’s Republic of China

July 1996 …………….. B.S. Chemistry

1998-1999..…………….Department of Food Science Nottingham University, United Kingdom

July 1999 ………………M.Sc. Food Production Management

2001-present……………Graduate Research and Teaching Associate, Department of Food Science and Technology, The Ohio State University, United States

PUBLICATIONS

1. Walsh, K.R., Zhang, Y.C., Vodovotz, Y., Schwartz, S.J., and Failla, M. 2003. Stability and bioaccessibility of isoflavones from soy bread during in vitro digestion. Journal of Agricultural and Food Chemistry. 51(16): 4603-4609.

2. Zhang, Y.C., Albrecht, D., Bomser, J., Schwartz, S.J., and Vodovotz, Y. 2003. Isoflavone profile and biological activity of soy bread. Journal of Agricultural and Food Chemistry. 51(26):7611-7616.

3. Zhang, Y.C. and Schwartz, S.J. 2003. Analysis of isoflavones in soy foods. In: Current protocol in food analytical chemistry. John Wiley & Son. New York.

FIELDS AND STUDY

Major Field: Food Science and Nutrition

TABLE OF CONTENTS

Page Abstract…………………………………………………………………………… ii Dedication………………………………………………………………………… iv Acknowledgment…………………………………………………………………. v Vita………………………………………………………………………………... vi List of Tables……………………………………………………………………... vii List of Figures…………………………………………………………………….. xiv List of Equations………………………………………………………………….. xvii

Chapters:

1. Introduction…………………………………………………………………… 1

2. Statement of Problem…………………………………………………………. 7

3. Literature Review …………………………………………………………….. 10

The effect of soy on physico-chemical properties of fresh bread ………………………………………………………...... 10 Mechanism of breadmaking………………………………………. 10 Addition of soy to baked products………………………………... 13

The effect of soy on physico-chemical properties of bread upon 14 storage…………………………………………………………………….. Mechanism of bread staling………………………………………. 14 The impact of added soy on bread staling………………………… 18

Instrumental analysis for characterization of mechanical, physico- chemical, and molecular properties of bread……………………...... 19 Texture……………………………………………………………. 20 Physico-chemical properties……………………………………… 20 Differential scanning calorimetry (DSC)………………… 23

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Dynamical mechanical analysis (DMA)……………….... 25 Thermogravimetric Analyzer (TGA)……………...... 29 Molecular properties…………………………………………….. 32 NMR methodology……………………………………… 33 Proton NMR ……………………………………………. 33 1. Relaxation times………………………...... 33 2. Cross-relaxation NMR………………………... 34

The effect of breadmaking and storage on isoflavones in soy containing bread…………………………………………………...... 38 Isoflavone stability during bread making……………………….. 38 Enhancing bioavailability of isoflavones in soy bread……………………………………………………... 41 Stability of isoflavones during storage…………………... 43 Analysis of isoflavones in soy foods……………………………. 43 Spectra fine structure of soy isoflavones………………… 44 Solvent extraction of isoflavones from soy foods………. 49 Isoflavone anlaysis using High Pressure Liquid Chromatography (HPLC)……………………………….. 52

4. Materials and Methods………………………………………………...... 54

The effects of soy addition on the physicochemical properties of fresh and stored bread on a structural and molecular level……………………. 55 Bread preparation………………………………………………... 55 Bread storage…………………………………………………….. 55 Instrumental analysis……………………………………………. 59 Tests on physical properties of bread……………...... 59 Loaf volume……………………………………... 59 Bread firmness…………………………………… 59 Moisture content………………………………… 60 Thermal analysis………………………………………… 60 Dynamic mechanical analysis (DMA)…………... 60 Differential scanning calorimetry (DSC)……….. 61 Thermogravimetric analyzer…………………….. 65 Molecular properties using NMR……………………….. 66 Sample preparation………………………………. 66

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Liquid state Proton (1H) NMR…………………... 66 Spin-lattice relaxation time (T1 ) measurement…………………………….. 66 Spin-spin relaxation time (T2) measurement…………………………….. 67 Data analysis…………………………….. 67 Solid state 1H Cross-Relaxation NMR………….. 68 Statistical analysis………………………………………. 69

Isoflavone content and composition in the soy bread and their stability through processing and storage………………………………………….. 69 Extraction of isoflavones………………………………………… 69 Sample preparation for extraction………………………. 69 Solvent extraction of isoflavones in their natural forms……………………………………………………... 69 Isoflavone analysis using HPLC-UV spectrophotometry……….. 70 Sample preparation…………………………………….... 70 Isoflavone quantification………………………………… 71 Stock standard solutions…………………...... 71 Working standard solutions……………………… 74 Absorbance measurement using a UV spectrophotometer……………………………….. 74 Calibration curve preparation using HPLC……… 74 Gradient separation and identification of isoflavones in sample……………………………...... 75 Mobile phase preparation………………………... 75 HPLC set-up……………………………………... 75 HPLC analysis…………………………………… 76 Statistical analysis……………………………………….. 76

The effect of the processing and almond addition on the isoflavone content and composition in the soy bread……………………………….. 79 Dough and bread preparation……………………………………. 80 Isoflavone analysis………………………………………...... 80 β-Glucosidase activity assay…………………………………….. 81 Statistical analysis……………………………………………….. 81

5. Results and Discussion………………………………………………………. 83

The effect of soy on physico-chemical properties of fresh bread……….. 83 Bread Macroscopic properties…………………………………… 83 Loaf volume……………………………………………... 83 Bread firmness from Instron…………………………….. 83 Physico-chemical properties by thermal analysis……………….. 84 Moisture content and state of water (DSC and TGA)…… 84 Dynamic mechanical analyzer (DMA) results…………... 89 Molecular properties by NMR…………………………………... 94 Relaxation times of liquid proton……………………….. 94 1H cross relaxation………………………………………. 96

The effect of soy on physico-chemical properties of bread upon storage………………………………………………………...... 100 Bread macroscopic properties……………………………………. 100 Bread firmness-Instron results…………………………… 100 Physico-chemical properties by thermal analysis………… 100 Moisture content and state of water (DSC and TGA)……. 100 Amylopectin recrystallization by DSC…………………… 105 Dynamic mechanical analyzer (DMA) results…………… 109 Molecular properties……………………………………………… 115 Relaxation times………………………………………….. 115 Cross relaxation………………………………………….. 119 Isoflavones in soy bread…………………………………………………. 122 Comparison of isoflavones in soy ingredients and soy bread…… 122 Effect of a soy bread preparation on isoflavone content and distribution……………………………………………………….. 125 Effect of proofing on isoflavones and β-glucosidase activity in 130 soy bread………………………………………...... Effect of almond addition on isoflavones in soy bread………….. 136 Stability of isoflavones during storage…………………………… 143

6. Conclusions…………………………………………………………………… 145

List of References………………………………………….. 149 x

LIST OF TABLES

Table Page

1 Proximate chemical composition of wheat flour and defatted soy flour……………………………………………………………………. 11

2 Structure and scientific names of twelve naturally occurring isoflavones in soy……………………………………………………… 39

3 UV spectra pattern of twleve soy isoflavones in 80% methanol: 20% H2O…………………………………………………………………….. 50

4 Ingredient list of soy and wheat breads used in this research………….. 56

5 The list of ingredient for soy bread and their manufacturer…………… 57

6 The list of equipment and their manufacturer used for soy bread preparation…………………………………………………………….. 58

7 The weight of isoflavone standards and the approximate concentration of stock standard solutions…………………………………………….. 72

8 Concentration range of isoflavone standard working solution for preparing calibration curve…………………………………………….. 73

9 HPLC mobile phase gradients for isoflavone analysis using a reversed- phase C18 column……………………………………………………… 77

10 DSC and TGA results indicating the changes in moisture content, “freezable” water and “unfreezable” water of wheat and soy breads during storage…………………………………………………………... 103

11 Results of fitting of E’(T) of wheat and soy bread with the modified Fermi equation during storage. “Average” represents the average of at least three individual fitting for each sample during storage………………………………………………………………….. 114 xi

12 Relaxation time determination by 1H NMR for wheat and soy breads during storage. T1: proton spin-lattice relaxation time, T2: proton spin- spin relaxation time…………………………………………………….. 116

13 Isoflavone content (nmol/gram, dry basis) of soy ingredients (soy flour and soy milk powder) and soy bread crumb…………………………… 124

14 Isoflavone content and composition (nmol/gram, total weight) in soy bread dough during preparation (proofed at 48 oC for 1 hour and baked at 160 oC for 50 min) and in soy bread crumb and crust………………. 126

15 Isoflavone β-glucoside : aglycone ratio and β-glucosidase activity in soy bread dough after proofing at 22, 32, and 48 oC for 1, 2, 3, and 4 hours……………………………………………………………………. 127

16 Isoflavone content and composition (nmol/gram, total weight) in soy bread dough after proofing containing 0, 2.5, 5.0, 7.5, and 10.0% almond………………………………………………………………….. 139

17 Isoflavone β-glucoside: aglycone ratio and β-glucosidase activity in soy bread dough with 0, 2.5, 5.0, 7.5, and 10% almond (w/w, flour basis) after proofing at 48 oC for 1 hour……………………………………….. 140

18 Isoflavone content and composition (nmol/gram, dry basis) in fresh (day 0) and stored soy bread at room temperature (day 14)…………… 144

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LIST OF FIGURES

Figure Page

1 Factors contributing to crumb firming on staling……………………… 15

2 Schematic of Instron Universal Testing Machine used for bread 21 firmness analysis……………………………………………………….

3 A typical stress-strain curve of a food material from a compression test……………………………………………………………………... 22

4 Schematic of heat flux differential scanning calorimeter (DSC) instrumentation………………………………………………………… 24

5 Schematic of dynamic mechanical analysis (DMA) instrumentation…. 26

6 A typical DMA thermogram of a food material showing a drop in E’ (storage modulus) and E” (loss modulus) and a peak of tan δ (E’/E”) that reflect a transition region………………………………………….. 27

7 Typical instrumentation of thermogravimetric analysis (TGA)……..... 30

8 Typical TGA thermogram of a food polymer showing a weight loss and derivative weight loss curve of a food polymer…………………... 31

9 Typical relaxation curves from 1H NMR relaxation measurement showing a growth curve for spin-lattice relaxation (T1) and a decay curve for spin-spin relaxation (T2)…………………………………….. 35

10 Results of a cross-relaxation 1H NMR experiment for water and bread. (a) Liquid 1H signal (single resonance) observed at various offset frequencies; (b) Z-spectrum obtained from the normalized amplitude of Figure (a) signals……………………………………………………. 37

11 General structure and ring numbering system applied to the known soy isoflavones………………………………………………………… 45 xiii

12 The UV characteristics of daidzein, genistein, and glycitein in HPLC 46 mobile phases ………………………………………………………….

13 UV Spectra of genistein family in HPLC mobile phases……………… 47

14 UV spectra of daidzein family in HPLC mobile phases………………. 48

15 Schematic experimental representing the design of the research on physicochemical properties and isoflavone content of bread containing soy……………………………………………………………………... 54

16 Fitting of storage modulus (E’) from DMA by fitting the curve with a modified Fermi equation…………………………………………...... 63

17 DMA tan δ fitting using gaussian and asymmetric double sigmoidal curves………………………………………………………………….. 64

18 Gradient HPLC separation of isoflavone standards…………………… 78

19 Schematic shows the experimental design of the research on the effect of processing condition and almond addition on isoflavone content of bread containing soy…………………………………………………… 79

20 A typical TGA thermogram for fresh wheat bread crumb (A) and soy bread crumb (B) showing the percent weight loss as well as the derivative weight loss………………………………………………….. 86

21 A typical DSC thermogram for fresh wheat and soy bread crumb showing a major endothermic transition at ~0oC……………………… 87

22 Typical TGA derivative weight loss curves of fresh wheat bread crumb (A) and soy bread crumb (B). These curves were best fitted with two gaussian curves …………………...…………………………. 88

23 TGA derivative weight loss curves for wheat gluten, soy flour, soy protein, wheat starch and wheat flour hydrated to 50% MC………...... 90

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24 Typical DMA thermograms for the crumb of fresh wheat (A) and soy bread (B) showing the storage modulus (E’), loss modulus (E”), and tan δ……………………………………………………………...... 92

25 Deconvolution of tan δ curves of fresh wheat bread (A) and soy bread (B) showing a two-peak fit for wheat bread (a gaussian function around 0 oC, and an asymmetric double sigmoid function around –13 oC) and a three-peak fit for soy bread (a gaussian function around 0 oC, asymmetric double sigmoid functions around –12 oC and -30 oC…. 93

26 A typical result from proton T1 and T2 measurement of fresh wheat and soy bread showing a fit using a single exponential function……… 97

27 Z-spectra obtained from cross–relaxation 1H NMR experiment for fresh wheat and soy bread……………………………………………... 98

28 Instron results for soy and wheat bread firmness. Increased compression load indicates an increase in firmness…………………… 102

29 Deconvolution results showing the peak temperatures of TGA derivative weight loss curves for wheat and soy breads stored at room temperature for up to 14 days after baking……………………………. 106

30 DSC results of wheat bread during storage……………………………. 107

31 Change in the enthalpy (W/g) of recrystallized amylopectin melting in wheat and soy breads during storage………………………………… 108

32 Results for the amplitude and intensity of the asymmetric double sigmoid curve (centered at about -30oC) used to fit the tan δ (T) curve for soy bread during storage…………………………………………… 111

33 Results for the width of the asymmetric double sigmoid curve (centered at about -30oC) used to fit the tan δ (T) curve for soy bread during storage………………………………………………………….. 112

34 DMA E’ value at 25oC for wheat and soy breads stored at room temperature……………………………………………………………... 113

35 Spin-lattice relaxation time (T1) results for wheat and soy breads during storage…………………………………………………………... 117

36 Spin-spin relaxation time (T2) results for wheat and soy breads during storage………………………………………………………………….. 118

37 Z-spectra obtained from cross–relaxation 1H NMR experiment for wheat bread stored at 4 oC for up to 7 days……………………………. 120

38 Z-spectra obtained from cross–relaxation 1H NMR experiment for soy bread stored at 4 oC for up to 7 days…………………………………… 121

39 Gradient separation using reverse phase HPLC (see conditions in text) of twelve soy isoflavones from soy ingredients and bread samples…… 123

40 The change in isoflavone distribution during bread making of soy dough mix, proofed dough and baked dough…………………………... 128

41 Changes in isoflavones and β-glucosidase activity during proofing at 48oC for 1, 2, 3, and 4 hours…………………………………………… 133

42 Changes in isoflavones and β-glucosidase activity during proofing at 22 oC for 1, 2, 3, and 4 hours…………………………………………... 134

43 Changes in isoflavones and β-glucosidase activity during proofing at 32 oC for 1, 2, 3, and 4 hours…………………………………………... 135

44 HPLC chromatogram of isoflavone authentic standards, soy bread with and without almond during bread preparation…………………………. 138

45 Isoflavone aglyone (nmol/gram, total weight) (bar graph) and β- glucosidase activity (dotted line) in soy bread containing almond after proofing as compared to soy bread without almond before and after proofing………………………………………………………………… 141

46 Isoflavone content and composition in soy bread containing 5.0% almond during bread making (before proofing, after proofing and after baking) as compared to soy bread without almond…………………….. 142

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LIST OF EQUATIONS

Equation Page

1 Moisture content calculation……………….……………………… 60

2 Modified Fermi equation…..………………………………………. 61

3 Asymmetric double sigmoidal curve………………………………. 62

4 Gaussian curve…………………………………………………….. 62

5 Percent “freezable” water calculation……………………………... 65

6 Spin-lattice relaxation time (T1) calculation………………………. 67

7 Spin-spin relaxation time (T2) calculation………………………… 68

8 Isoflavone concentration calculation following the Beer Lambert Law………………………………………………………………… 74

xvii

CHAPTER 1

INTRODUCTION

Epidemiological and experimental evidence suggests that consumption of soybean products may significantly impact upon health (Messina et al, 1994; Setchell, 1998; Birt et al, 2001; Brouns, 2002; Hasler, 2002; Messina and Loprinzi, 2001; Scheiber et al,

2001). The biological activity in soy has been associated, in part, with the presence of proteins and isoflavones (Coward et al, 1993; Fournier et al, 1998; Barnes, 1998; Setchell et al, 1999; Henkel, 2000). In 1999, the Food and Drug Administration declared that 6.25 gram of soy protein per serving included in a diet low in saturated fat and cholesterol may reduce the risk of coronary heart disease by lowering blood cholesterol levels (Federal

Increased recognition of the link between soy and health has resulted in the development of a series of soy-containing functional foods (Zhou, et al,2002; Wildman,

2001; Klein et al, 1995). However consumer acceptability of such foods remains low due to off-flavors and textures of many of these products as well as a need to incorporate a non-traditional food (bars, shakes, Asian foods) into the diet. Bread made partially with

1 soy represents a traditional alternative for increased soy consumption, and, if successfully formulated and well accepted, may offer an attractive delivery system for isoflavones and soy proteins.

Previous studies have shown that incorporating soybean flour into bread formulation resulted in altered sensory and physicochemical properties of the final product. Soy proteins are historically used to improve water absorption and dough handling properties in bakery products at a level of 3% (w/w) or lower (Chen and Rasper,

1982; Brewer et al, 1992). At higher supplementation levels, however, negative effects were observed, including lower loaf volume (Erdman et al, 1977; Fleming and Sosulski,

1977; Buck et al, 1987; Brewer et al, 1992) and less desirable texture (Mizrahi et al,

1967). These deleterious sensory qualities in bread were found to be proportional to the amount of soy addition and correlated to a change in the water absorption of bread ingredients (Doxastakis et al, 2002) and/or dilution of the gluten fraction (Knorr and

Betschart, 1978). However, the effects of significant amount of soy addition on bread characteristics during storage are not well understood.

Staling refers to the undesirable changes that occur (except for microbial spoilage) between the time bread is baked and the time it is consumed (Zobel, 1973;

Maga, 1975; D’Applonia and Morad, 1981; Chinachoti and Vodovotz, 2000). The changes (especially firming) occurring in breads during storage have been attributed to several factors including recrystallization of amylopectin (Maga, 1975; Kulp et al, 1981), moisture redistribution (Leung, 1983; Baik and Chinachoti, 2001), changes in gluten functionality (Maga, 1975; Kulp et al, 1981) and the state of the amorphous phase (Laine and Roos, 1994; Hallberg and Chinachoti, 1992). Additional changes to the bread matrix

2 may result upon soy incorporation. Previous research (Vittadini and Vodovotz, 2003)

found soy addition decreased the degree of amylopectin recrystallization in bread during storage, indicating that soy may play a role in modulating the staling process. During storage, water in the bread becomes less mobile as a network of amorphous and crystalline solids is formed (Chinachoti, 2001). This network formation is accompanied by moisture redistribution between gluten and starch. Since soy proteins associate strongly with water (Snyder and Kwon, 1987), soy addition may affect the dynamics (i.e. mobility and redistribution) of water in the bread and hence the mobility of polymer chains during the firming process. Therefore, the state and mobility of water may be better indicators of bread staling rather than just moisture loss.

Soy bread as an isoflavone delivery system requires a thorough understanding of the stability of isoflavones during bread preparation and storage. Previous studies have suggested that the content and composition of isoflavones are subjected to change by various processing conditions and other dietary components in soy foods (Coward et al,

1998). The potential health promoting activity of soy isoflavones is dependent upon their availability for absorption following soy food consumption. Recent studies observed that the chemical forms and abundance of isoflavones have a significant impact on their bioavailability and biological effects (Setchell et al, 2002). Isoflavone glycosides were not directly transported across the apical membrane of absorptive epithelial cells in the gastrointestinal tract but were converted to their aglycones either by β-glucosidase activity in the small intestine or by microflora in the large intestine (Day et al, 1998;

Setchell et al, 2002). However, it is not yet clear about the relative bioavailability between isoflavone glucosides and their corresponding aglycones. Izumi and coworkers

3 found that isoflavone aglycones were absorbed faster and in higher amounts than their glucosides in humans after a single dose of soybean extract (Izumi et al, 2000). Some other studies suggested that the glucosides were not less bioavailable than the free

corresponding forms after a single dose of pure isoflavones (Setchell et al, 2001; Zubik

and Meydani, 2003). The contradictory findings in bioavailability of isoflavones might be due to the sensitivity of detection method and validity of the experimental design.

However, it also indicates the possibility that there may be a function of differences in the

matrix of foods compared with the pure compounds that need to be determined (Setchell

et al, 2001). Nonetheless, it is important to understand the effect of processing on the abundance and profile of the isoflavones when developing novel soy-containing

functional foods.

Isoflavones belong to a group of plant derived phenolic compounds that exhibit

estrogenic activity. Soybean products are the most prominent sources of isoflavones in food (0.1-3.0 mg/g dry weight) (Coward et al, 1993). Isoflavones exist in four possible chemical forms, the aglycone, the β-glucoside, the malonyl-β-glucoside, and the acetyl-

β-glucosides. The predominant (approximately 95%) isoflavone forms in unprocessed soybeans are their malonyl-β-glucosides (Barn et al, 1994; Coward et al, 1998; Murphy et al, 2002). Bread preparation is a complex physico-chemical process involving major

steps such as mixing, yeast fermentation (proofing), and baking. These various steps may

significantly influence the isoflavone profile in a soy containing bread.

The interest in enhancing isoflavone aglycones in soybean product has led to recent studies on β-glucosidase (EC 3.2.1.21), an enzyme found in many animals, plants,

and microorganisms that hydrolyze aryl- and alkyl-β-D glucosides to release glucose and 4 aglycones (Esen, 1993; Reese, 1977; Sue et al, 2000; de Roode, 2003). The specific β-

glucosidase activity was found to correlate well with the isoflavone aglycone to glycoside ratio in various tissues in isoflavone containing plants, i.e. white lupin and soybean

(Wojtaszek and Stobiecki, 1997; Graham et al, 1990). Isoflavone glucosides may serve

as pools for releasing aglycones by enzymatic activity under favorable conditions (Hsieh and Graham, 2001). Several ingredients used in the current bread formula (soybeans, wheat, and yeast) contain β-glucosidase activity (Hsieh and Graham, 2001; Sue et al,

2000; Matsuura and Obata, 1993). Both soybean and wheat β-glucosidases are capable of hydrolyzing isoflavone glycosides to release the aglycones (Hsieh and Graham, 2001;

Sue et al, 2000). Several attempts have been used in previous work to increase the isoflavone aglycone level in soy foods through the action of β-glucosidase. For example, presoaking soybeans in water at 50 oC and pH 6.0 in soymilk production activated the enzymatic process of β-glucosidase (Matsuura et al, 1989), however, a significant portion of the isoflavones was lost in the soaking medium. Microorganisms, i.e. Lactobacillus casei, Saccharopolyspora erythraea, and Ganoderma lucidum, were inoculated into soybean products. However, additional enzymatic biotransformation were found to act on the liberated isoflavone aglycone molecules during fermentation causing marked loss of these isoflavones in the final products (Matsuda et al, 1992; Hessler et al, 1997; Miura et al, 2002). Therefore, processing temperature, pH, components in the formula, i.e. water content, proteins, acids, and other enzymes, may influence the interaction of β- glucosidase with the substrate (Pandjaitan et al, 2000; Zheng and Shetty, 2000; Matsuura

5 et al, 1995). Almond is traditionally known as a rich source of β-glucosidase (de Roode et al, 2003; Li et al, 1997) yet the effect on isoflavone aglycone content upon addition of almond to a soy bread formula has not been explored.

The overall objective of this research was to assess the impact of soy addition on physico-chemical properties in fresh and stored soy bread. The hypothesis therefore is that processing and storage can modulate the physico-chemical properties by delaying staling and altering isoflavone profiles in soy bread.

6 CHAPTER 2

STATEMENT OF PROBLEM

A number of studies have shown that soy can be successfully incorporated in baked goods at a 15% level (on flour basis). Higher quantities of soy in past formulations have resulted in inferior quality of the final product. However, health benefits arise only if sufficient amount of soy is incorporated in the diet to reach physiological relevant concentrations. Therefore, the challenge becomes to produce highly acceptable baked goods with at least 30% soy. Little work has been done on the effects of soy on physicochemical properties of bread from both structural and molecular level when incorporated at a level as high as 30%. Additionally, previous research suggested that soy may play a role in modulating bread staling but how this outcome is achieved is not well understood. Furthermore, the effects of breadmaking and staling on soy phytochemicals, especially isoflavones, have not been studied and are important in evaluating soy bread as a functional food. This study will address these research questions.

Therefore, the overall objective of this research was to assess the impact of

soy addition on physico-chemical properties in fresh and stored soy bread. The

7 hypothesis therefore is that processing and storage can modulate the physico-chemical properties by delaying staling and altering isoflavone profiles in soy bread. The hypothesis was tested as follows.

The first specific aim involved comparison of the physico-chemical properties of

fresh soy bread with fresh wheat bread to determine the effect of soy on physico-

chemical properties of fresh bread. The questions that were addressed include the

following:

1. Does added soy affect the macroscopic properties of fresh bread as represented by

loaf volume and firmness?

2. Does added soy affect the physico-chemical properties of fresh bread (e.g.

moisture content and state of water, the number and homogeneity of transitions)?

3. Does added soy affect the molecular properties of bread system (e.g. proton

mobility)?

The second specific aim for this research was to determine the effect of soy addition on the physico-chemical properties of bread during storage. Bread without soy served as control. The following questions were addressed:

1. Is there a difference in the macroscopic properties between bread with and

without soy during storage?

2. Is there a difference in the physico-chemical properties between breads with and

without soy during storage?

3. Is there a difference in the molecular properties between bread with and without

soy during storage?

8 The third specific aim for this research was to determine the effect of processing and storage on isoflavones in bread with soy. The questions that were addressed include following:

1. Are isoflavones stable during bread preparation?

2. What are the changes in isoflavone profile in soy bread during different stages of

bread preparation?

3. Is there a correlation between β-glucosidase activity and isoflavone β-glucoside to

aglycone ratio?

4. Can the isoflavone aglycone level be enhanced using a β-glucosidase of a natural

source, i.e. almond, for potential application in food processing?

5. Are isoflavones in soy bread stable through storage?

9 CHAPTER 3

LITERATURE REVIEW

The effect of soy on physico-chemical properties of fresh bread

Mechanism of breadmaking The main bread ingredients are wheat flour, water, yeast, and salt. Other ingredients such as fat, emulsifiers, sugars, and dough conditioner may be added to improve the dough and bread quality. Wheat pan bread is prepared by hydrating dry ingredients, producing CO2 through yeast fermentation (proofing), developing gluten in the flour by chemical and mechanical means, and baking (Chinachoti, 1998).

Wheat flour is composed of carbohydrates (67-70%) and proteins (10-14%) on

“as is” basis (Table 1). Starch, the main carbohydrates in wheat flour, is a mixture of amylose (25%), which is a linear polymer and amorphous in nature, and highly branched amylopection (75%), which is semicrystalline in nature (Anglemier and Montgomery,

1976). The major protein in wheat flour is gluten, a mixture of gliadin (40-45%) partly hydrophilic and partly hydrophobic in nature, glutenin (20-25%), which is more hydrophilic than gliadin, and residue protein (35-40%) called insoluble glutenin (Kasarda et al, 1976). Gluten contains high concentrations of glutamic acid, proline and leucine.

When wheat flour is hydrated and subjected to mechanical shield, the gluten proteins

10 Chemical Composition (%) Defatted soy flourb Wheat floura Moisture 14 9 Carbohydrate (including fibers) 76 30 - Fibers - 3 - 22 Protein 13 53 Fat 1 4 a : Wheat flour: Bakers high gluten enriched bromated flour bleached (General Mills. MI) b : Defatted soy flour: Bakers NUTRISOY Flour (Archer Daniels Midland Company, Decatur, IL)

Table 1. Proximate chemical composition of wheat floura and defatted soy flourb (%, “as is”).

11 absorb water and form a continuous three-dimensional network that can trap gas bubbles

(Hoseney, 1994; Pyler, 1988; Ablett et al, 1986). Hydration and mechanical force also cause the swelling and disruption of starch granules and the exudation of amylose. At a molecular level, the hydration of flour causes the partially ordered structure of the starch to be destroyed and converted into a disordered or amorphous state (Manzacco et al,

2002). During proofing (mild heat treatment with high humidity), the enzymes, e.g. α- amylase, in the hydrated yeast and flour are activated producing carbon dioxide and ethanol from sugars (Pyler, 1988). Subsequently, the dough expands and produces the sensory qualities in the final product. The heat during baking partially gelatinizes the hydrated starch and dehydrates the gluten network (Swortifiguer, 1950). Moisture is driven to the surface and evaporates. Starch gelatinization and protein denaturation are the main modification responsible for crumb structure, depending on the severity and time of heat treatment (Eliasson and Hoegg,1980; Schofiedl et al, 1983; Dreese et al,

1988). Gelatinization is higher near the crust than at the core of the loaf (Giovanelli et al,

1997). When the starch granules swell and partially gelatinize, there is less water to diffuse to the surface causing the surface temperature to rise and undergo Maillard browning (Atwell, 2001). Upon cooling, the starch gel sets and produces the cellular crumb structure of the bread (Pyler, 1988). The baked bread consists of the crust, the darker crispy outer shell, and the crumb, the soft spongy inside portion. Typically, bread contains about 35% water.

12 Addition of soy to baked products

The favorable elastic and extensible properties of bread depend on the quality and

quantity of gluten in bread flour. Soybean flour has a chemical composition very

different from wheat flour (Table 1). Defatted soybean flour, for example, contains a

about 50% proteins (principally glycinin) and about 30% carbohydrates on a “as is” mass

basis. The carbohydrate fraction is composed of soluble sugars (sucrose 17%, stachyose

13%, raffinose 3%) and insoluble “fiber” (67%). Starch comprises a very small portion

of the carbohydrates. While wheat flour contains essentially starch and gluten that are

modified during bread making in the presence of moisture, soy flour undergoes mainly

protein denaturation during heating (Riganakos and Kontominas, 1997; Chen and Rasper,

1982). Particularly, soy protein globular in nature does not form a stable network in wheat bread as does gluten through disulfide bonds. Therefore, incorporating soybean flour into bread formulation results in altered sensory and physicochemical properties of

the final product (Brewer et al, 1992; Dhingra and Jood, 2001; Doxastakis et al, 2002).

In the USA, soy flour is used in bakery products not for nutritional reasons but rather for their functional characteristics. One and half to two percent on flour weight basis of soy flour (sometimes soy protein concentrate) is used in bakery products,

particularly in white bread, as a replacement for nonfat milk solids. Enzyme-active defatted soy flour is used as a bleaching and dough-improving agent. Defatted soy flours

with 50-75% protein dispersibility are extensively used in bakery products to increase the

water absorption capacity of flours in bread dough and cake batters. In cakes, they

improve film forming and even distribution of air cells. As a result, even cake texture

and tender crumb structure are achieved. In all these products, soy flour is used at a level 13 of 2-5%. At higher replacement levels, however, negative effects were observed. They

include lower loaf volume (Erdman et al, 1977; Fleming and Sosulski, 1977; Buck et al,

1987; Brewer et al, 1992) and less desirable texture (Mizrahi, 1967). These deleterious sensory qualities were found to be proportional to soy addition to bread and correlated to a change in the water absorption of bread ingredients (Doxastakis et al, 2002) and/or dilution of the gluten fraction (Knorr and Betschart, 1978). Addition of soy to bread requires added water for adequate dough formation and as a result increases the moisture content of the bread (44%) as compared to a typical wheat bread (35%). As a result,

Vittadini and Vodovotz (2003) observed a higher initial stiffness of fresh bread containing soy.

The effect of soy on physico-chemical properties of bread upon storage

Mechanism of bread staling

Bread represents a heat-set firm foam system and is subjected to physicochemical instability during storage (Taub and Singh, 1998). Staling refers to the undesirable changes that occur (except for microbial spoilage) between the time bread is removed from the oven and the time it is consumed (D’Applonia and Morad, 1981). From a sensory standpoint, staling reduces the springback and softness of the crumb and increases the dry mouth-feel. Staling also reduce the crispness of the crust and increases its toughness. These changes occurring in breads during storage have been attributed to several factors (Figure 1) including retrogradation of amylopectin (Maga, 1975; Kulp and Ponte, 1981; Zeleznek and Hoseney, 1987), moisture redistribution (Leung et al,

Overall Firmness Of crumb

s s

e Starch Retrogradation n m r

i F

Protein Transformation

Moisture Absorption By Starch

Time After Baking

Figure 1. Factors contributing to crumb firming on staling. (modified based on Kulp, K. Ponte, J.P., Jr. 1981. CRC Crit. Rev. Food Sci. Nutr. 15. 1-48.)

15 1983; Baik and Chinachoti, 2000 & 2001; Zeleznak and Hoseney, 1986; Yost and

Hoseney, 1986), changes in gluten functionality (Maga, 1975; Kulp and Ponte, 1981) and the state of the amorphous phase (Laine et al, 1994; Hallberg and Chinachoti, 1992).

Retrogradation is an exothermic process in which starch physically changes from a swollen, gel-like state to a more crystalline state (Krog et al, 1989). Contrary to the development in firmness, no significant changes were observed for the staling endotherm of retrograded amylopectin during the first 10 hours after packing of freshly baked bread

(Slade and Levine, 1991). The initial firmness of newly baked bread is dominated by retrograded amylose (Slade and Levine, 1991). However, progressive increase in crystallization of amylopectin was detected upon further storage (Manzacco et al, 2002).

Yet this re-crystallization was not a return to the same degree of order found in the native starch granule (Zobel and Kulp, 1996). Bread firmness is influenced by changes in crystallization as well as other components, in particular, moisture, in the bread and their interactions with starch (Rogers et al, 1988).

Water diffusion and redistribution among the protein-starch and crumb-crust fraction of bread and water loss by evaporation have been related to staling (Willhoft,

1971b; He and Hoseney, 1990; Piazza and Masi, 1995). When the newly baked bread is removed from the oven, the crust is practically moisture free. During the cooling stage, the moisture content of the crust rapidly increases because of moisture migration from the crumb. Zobel and Kulp (1996) found water migration from the crumb to the crust in the course of aging modified staling kinetics. Larsson and Eliasson (1996) separated four aqueous phases from dough samples by ultra-centrifugation and referred to bread dough as a metastable dispersed system with a large surface across which water can move from

16 one phase to another. Gluten, soluble proteins, and pentosans are responsible for

adsorping much water when mixing dough ingredients with water. Mechanical treatment

affects the water-binding capacity of dough and water partition among various dough

ingredients. When dough is heated during baking, some water is involved in starch

gelatinization but a significant fraction remains with the other polymers and solutes,

which compete for the available moisture and results in phase separation. The water in bread has a high thermodynamic activity (above 95%) suggesting that it should be somewhat free to move along any gradient of the relevant chemical potential throughout the crumb structure (Fessas and Schiraldi, 2000). When polymers are thermodynamically incompatible, they can use the water available within their own phase to sustain conformational and phase transitions. Willhoft (1973) suggested that molecular changes in the gluten or starch fraction may lead to the formation of cross-links. The formation of cross-links could be associated with the production of free water, i.e. the release of water originally bound to the polymer chain (Schiraldi et al, 1996). This can facilitate the formation of new cross-links keeping the relevant binding sites closer (Schiraldi et al,

1996; Zobel, 1973). As a result of the transformation of polymer components (such as those occurring during storage), water molecules remain entrapped within the crystal structure, or more easily released toward phases containing other polymers, possibly in the state of viscous starch gel. It has therefore been suggested that free and entrapped water could play different roles in determining the staling rate.

Rasmussen and Hansen (2001) found that the development of bread firmness during storage was correlated to the changes in the “freezable” water fraction of the bread in non-linear manner. “Freezable” water can be determined by Differential Scanning

17 Calorimetry (DSC) as discussed below. On the other hand, various studies on bread

staling found only a small change (0.02) in water activity (aw) of bread crumb during

storage (Rasmussen and Hansen, 2001; Cuchajowska and Pomeranz, 1989) suggesting

that water activity is not an appropriate indicator of bread staling. Water loss causes

crumb firming but bread staling also occurs at constant moisture (He and Hoseney,1990;

Martin et al, 1991) providing evidence that firmness is not directly related to the loss of

water. Chinachoti and coworkers (1998) found that water in the bread becomes less

mobile as a network of amorphous and crystalline solids is formed during storage. A

study using NMR deuteron relaxation method (Leung et al, 1983) suggested that there is

an increase in water binding and decrease in water mobility in staling bread. Therefore,

the state and mobility of water may be better indicators of bread staling than moisture

content and water activity.

There is less evidence regarding the role of proteins in bread staling. A model proposed by Hoseney and coworkers (1994) related bread firming to formation of H-bond

cross-links between the continuous three-dimensional protein structure and the

discontinuous remnants of starch granules (Peppas and Brannon-Peppas, 1994). Studies

by Levine and Slade (1988) suggested that moisture migrates from gluten to starch,

firming the former and softening the latter, during storage. However, Menon and

Mujumdar (1987) found evidence that moisture also migrate from starch to gluten.

The impact of added soy on bread staling

Studies on the effect of soy on bread staling is very limited. Among them, soy addition was found to affect the dynamics (i.e. mobility and redistribution) of water in the

18 bread and hence the mobility of polymer chains during the firming process, since soy proteins increase water absorption capacity in bread (Selvaraj and Shurpalekar, 1982,

Dhingra and Jood, 2004, Vittadini and Vodovotz, 2003). Vittadini and Vodovotz (2003) found soy addition decreased the degree of amylopectin recrystallization in bread during storage, indicating that soy may play a role in modulating the staling process. Gerrard and coworkers (2001) found that breads made with alternative proteins (soy and milk) increased in firmness during storage at the same rate as traditional breads containing gluten proteins suggesting gluten was not essential to the firming process and starch alone caused the bread to firm over time. Since soy flour contains no gluten and a low level of starch but a much higher amount of soy proteins as compared to wheat flour, the impact of storage on the firmness of bread made with soy needs to be further studied.

Instrumental analysis for characterization of mechanical, physico-chemical, and

molecular properties of bread

The effects of soy addition to bread and the changes in bread during storage take place at both structural and molecular levels. Various analytical techniques are available for monitoring changes at macroscopic, microscopic and molecular levels (Biliaderis,

1990; Roulet et al, 1988). It is imperative to consider the various events taking place concurrently within the bread system. Each event occurs in a distinct timeframe and scale (e.g., macroscopic to molecular). Using appropriate instrumental technique(s) will generate useful data that can lead to a better understanding of the physical properties of bread.

19 Texture

Bread is a spongy material, which tends to harden and crumble upon staling.

Mechanical properties, such as firmness and elasticity of bread are highly dependent on storage time, moisture content and deformation applied (Kou and Chinachoti, 1991).

These texture properties can be characterized using various instruments specifically

adopted for food systems, i.e. Texture Analyzer and Instron Universal Testing Machine

(Canton, Mass., USA, Figure 2). Firmness of bread crumb can be determined using a

compression test and described as the force (in units of pounds or in Newtons) required to

compress bread crumb to a given deformation (Figure 3, Baker at al, 1986). The size

and shape of the sample, the environment (i.e. temperature and relative humidity) in

which the test is conducted, the size of the compression plunger, the rate of compression,

and % of the deformation are all determining factors for a test of hardness using Instron

and need to be controlled. The results from texture analysis need to correlate with results

from other analysis, i.e. thermal-mechanical analysis and sensory analysis, for the

meaningful conclusions.

Physico-chemical properties

Thermal analysis techniques (i.e. Differential Scanning Calorimetry, Dynamical

Mechanical Analysis, and Thermogravimetric Analysis) have been used to understand

bread material and its components (i.e. water, protein, carbohydrates) that change

chemically or physically as a function of temperature (Vodovotz et al, 1996; Fessas and

Schiraldi, 2000; Haines, 1995; Hatakeyama and Quinn, 1994; Hallberg, 1996; Hamann et

al, 1990). 20

Aluminum column Crosshead

Control panel

Compression plunger M otor with Sample position control Load cell

Figure 2. Schematic of Instron Universal Testing Machine (Canton, Mass., USA,

Model 5520) for bread firmness analysis in this study.

Figure 3. A typical stress-strain curve of a food material from a compression test (http://www.instron.com/applications/test_types/compression.asp). The result was from Instron Universal Testing Machine (Canton, Mass., USA). The compression stress (N) at a certain compression strain (%) corresponds to the firmness of the material.

22 Differential scanning calorimetry (DSC)

Many of the physical (e.g. evaporation) or chemical (e.g. decomposition) transformations are accompanied by heat absorption (endothermic reactions) or heat evolution (exothermic reactions). These events are easily detected by a difference in

temperature and heat flow between a sample and an inert reference utilizing a differential

scanning calorimetry (DSC) (Liu and Lelievre, 1992; Sahai and Jackson, 1999).

Different types of DSC exist. In a commonly used heat flux DSC (Figure 4), both sample and reference pans are heated in a common block. The energy (heat flow) needed to keep both sample and reference pans at the same temperature while they are both heated or cooled linearly is measured. This energy is proportional to the heat capacity of the sample and is recorded versus time or temperature in a DSC thermogram. Phase transitions in carbohydrate system, such as starch gelatinization, amylopectin recrystallization, amylose-lipid complex formation and “freezable” water melting and crystallization are recorded as endothermic and exothermic peaks (Figure 4). Integration of the peaks permits quantification of these transitions (Russell, 1983; Vodovotz et al,

1996). Such phase transitions proved important in wheat bread storage studies, identifying an increase in crystalline amylopectin and decrease in “freezable water” with increasing storage time (Vodovotz et al, 1996, Vodovotz, 1996) and in soy bread storage

(Vittadini and Vodototz, 2003).

There are various important parameters to consider when operating a DSC.

Sample size, which is specific for the instrument being used, is important to obtain results of proper intensity and resolution (Wunderlich, 1990; Reid et al, 1993). Representative and homogeneous sample ensure an accurate and consistent representation of the system

DSC thermogram

Figure 4. Schematic of heat flux differential scanning calorimeter (DSC) instrumentation.

24 being studied. In addition, high volume pans with an O-ring are used for samples with

high moisture contents such as bread to avoid the loss of moisture from the sample pan during thermo-scanning. An extensive discussion of DSC parameters can be found

elsewhere (Vodovotz, 1996; Smith, 2003).

Dynamical mechanical analysis (DMA)

Dynamic Mechanical Analysis (DMA) is a micro-rheological technique in which

a dynamic stress is applied at a given frequency to a sample of known geometry (Figure

5). The resulting strain is either in phase (elastic, E’) or out of phase (viscoelastic, E”)

(Wendlandt and Gallagher, 1981). Food polymers are mostly viscoelastic with stress and strain being out of phase with respect to each other by a phase angle (δ). The tan (T)

curve is defined as the ratio of the loss modulus (E'') to the storage modulus (E'), and a

peak in tan (T) often corresponds to a phase change (e.g. glass or melting transition) with a particular temperature range. A typical thermo-mechanical transition of a food polymer sample during heating is depicted in DMA thermogram by a drop in E’ and E”, and a peak in tan δ (T) (Figure 6). The drop in E’(T) at around 0 oC reflects a decrease

in the elastic component as the sample loses its ability to restore the mechanical energy

(Murayama, 1978; Roulet et al, 1988) at this particular temperature, which is consistent

with ice melting transition (Baik and Chinachoti, 2000; Vittadini and Vodovotz, 2003).

The tan δ curve spans over a range of temperature around 0 oC, representing the

broadness of a phase transition. Tan δ curve can be further characterized by using the

least number of curves (i.e. asymmetric double sigmoidal curve or gaussian curve) to achieve a good fit as was done previously on corn tortilla (Clubbs et al, 2002), white bread (Vodovotz and Chinachoti, 1996), and wheat starch (Vodovotz and Chinachoti, 25

Stress Deformation Sensor

Furnace E’, E” and tan δ Control Analyzer

Furnace Sample

Deformation Frequency and

M otor Velocity Control

Strain

Figure 5. Schematic of dynamic mechanical analysis (DMA) instrumentation. (Modified from Wendlandt, W. W., Thermoanalytical Techniques; in Handbook of Commercial Scientific Instruments, Vol. 2, Marcel Dekker Inc.: New York, 1974.)

0.8 1e+5 E' (storage modulus) 1e+4 0.6 ) a

P 1e+3 δ δ δ δ M

E" (loss modulus) ( 0.4

n i l 1e+2 a u T d

o tan δ

M 1e+1 0.2

1e+0 0.0 1e-1 -100 -50 0 50 100

Temperature (oC)

Figure 6. A typical DMA thermogram of a food material showing a drop in E’ (storage modulus) and E” (loss modulus) and a peak of tan δ (E’/E”) that reflects a transition region.

27 1996). The deconvoluted curves give values such as amplitude, peak center, and width at

half height of each curve. Changes in the curves characterize changes in the phase

transition within samples of different treatment (Peleg, 1995; Vodovotz, 1996; Clubbs et al, 2002). Such changes may indicate an increase or decrease in heterogeneity of the sample matrix as well as change in the state of components in the system. These can then be related to stability of the products.

The storage modulus (E’) was characterized by fitting with a modified Fermi equation for various bakery products (Peleg, 1993; Vodovotz et al, 2000; Smith, 2003).

The modified Fermi equation (Eq. 2) characterizes the transition of the E’ curves of different treatments yielding a slope, “a”, and a temperature midway through the transition region, “Tc”. The “a” and “Tc” of the E’ curve were found to correlate well

with the physico-chemical properties, i.e. firmness, homogeneity, mobility, of baked products. A lower “a” value corresponds to a narrower temperature range of a transition indicating a more homogenous sample (Clubbs et al, 2002; Smith, 2003). A lower “Tc” value of the drop in the E’ may suggest a transition occurring at a lower temperature.

Various factors influence the operating efficiency of the DMA, including the choices of sample size, clamp, frequency and heating rate. Since sample geometry is important for determining moduli, sample size and shape must keep consistent for accurate results.

The dual cantilever clamp is suited for elastomers (such as bread) as was used by others

(Smith, 2003; Vittadini and Vodovotz, 2003). Details on the different parameters that affect sample measurement in the DMA can be found elsewhere (Smith, 2003).

28 Thermogravimetric analyzer (TGA)

Thermogravimetric Analyzer (TGA) can be used to understand the distribution of water in a system (Haines, 1995). TGA operates on a null-balance principle (Figure 7).

A low thermal resistance furnace performs the heating of the sample. TGA measures the amount and rate of change in the weight of a material as a function of temperature or time

and generates a weight loss curve and a derivative weight loss curve (Figure 8). The important parameters that influence the characteristics of the recorded TGA thermogram

was discussed in previously published work by Smith (2003) and Fessas and Schiraldi

(2000). Assuming the weight loss was due to moisture loss (Fessas and Schiraldi, 2000),

the moisture content is calculated. The derivative weight loss curve can be further

characterized by decomposing the main transition into its component curves using the

least number of curves as done previously for bread and starch (Vodovotz and

Chinachoti, 1998; Baik and Chinachoti, 2001; Fessas and Schiraldi, 2000). Study on

dough (Fessas and Schiraldi, 2000; Smith, 2003) using TGA found that water was not

homogenously partitioned within dough. A major fraction (80%) was easily removed in the range of 25-85 oC due to a simple diffusion of the water from inner part of the dough toward the sample surface. The rest of the water in the dough was more tightly bound to the bread components (gluten) and could only be removed above 100 oC (Fessas and

Schiraldi, 2000). Smith (2003) compared the TGA derivative weight loss curves of wheat gluten,wheat starch, soy flour, and soy protein isolates and found the requirement of a higher temperature (approximately 140 oC) to release the water in wheat gluten than

balance sample holder

oven

Figure 7. Typical instrumentation of Thermogravimetric Analysis (TGA)

110 1.0

100 derivative weight loss 0.8

weight loss h ) g 90 0.6 i C e s o / W s

% o e 80 0.4 (

L v

i s t t s h a o g v i 70 0.2 i L r e

e w

60 D

% 0.0

0 20 40 60 80 100 120 140 160 180

Temperature (oC)

Figure 8. Typical TGA thermogram showing a weight loss curve and a derivative weight loss curve of a food polymer.

31 those in wheat starch, soy flour, and soy protein isolates (approximately 60 oC),

suggesting a stronger interaction of wheat gluten with water as compared to starch and

soy proteins. A similar result was observed in work of Fessas and Schiraldi (2000).

TGA and DSC are complimentary techniques and are commonly used together to

analyze the water distribution within the same sample and changes as well as reactions

(Karmas, 1980) during heating and storage. DSC provides the information about amount

of “freezable” water and starch crystalline in the sample, while TGA detects mass

changes and the rate of mass change as a function of time or temperature. A study on

corn starch (Brouillet-Fourmann et al, 2002) using both TGA and DSC suggested that the existence of interactions between water and corn starch was weakened for samples containing more than 30% water. Riva and coworkers (2000) found that water in pasta might migrate from stronger to weaker binding sites during storage, while overall water content remained unchanged.

Molecular properties

Changes in the polymer back bone and side change mobility and the role of water molecular dynamics in bread during storage have profound impacts on its stability.

Long-range techniques such as DSC and DMA detect structural changes in a macroscopic scale (Chinachoti, 1994; Vodovotz and Chinachoti, 1996, Vodovotz et al, 2000).

However, information from these techniques does not necessarily reveal the changes occurring at the molecular level. Molecular spectroscopic techniques, such as Nuclear magnetic resonance (NMR), have been applied to investigate the molecular mobility of

32 bread components as well as water upon formula modification and during storage (Jaska,

1971; Derome, 1987, Chen et al, 1997; Boltan et al, 1998; Baik et al, 2003; Li et al, 1998;

Vodovotz et al, 2000; Vodovotz et al, 2003, Kou et al, 2000).

NMR methodology

In principle, NMR investigates the molecular changes based on the ability of nuclei with magnetic dipole moments to absorb electromagnetic energy depending on the

strength of the magnetic field and the chemical and magnetic environment. When the nuclei with magnetic moments are placed in a static magnetic field, most nuclei align in

the same direction as the static magnetic field representing a low energy level, while the

rest aligned in the opposite direction representing a high energy level. The net

magnetization at equilibrium (Boltzmann equilibrium) is proportional to the difference in

population between the two energy levels. When a second magnetic field in the form of radio frequency (rf) pulse is applied to this equilibrium system, the nuclei are excited.

The excited nuclei return to their original equilibrium state by losing energy in the form of rf termed relaxation processes. The radio frequency can be received and recorded by the NMR instrument.

Proton NMR

1. Relaxation times

There are various types of relaxation processes with the most common ones being spin-lattice (longitudinal) relaxation, T1, and spin-spin (transverse) relaxation, T2. The

longitudinal relaxation is a result of interactions of the spins with the environment

(“lattice”) and can be measured by inverting the equilibrium by a 180 o pulse and 33 measuring the recreation of the equilibrium magnetization (inversion recovery

o o experiment, 180 -τ-90 FID). T1 follows a recovery or growth curve (Figure 9). The

transverse relaxation is a result of interactions of the spin with each other and can be measured by applying a 90o pulse and then recording the loss of phase coherence of the spin in transverse (x- or y- magnetization). An example of pulse sequence in measuring

o o T2 is a spin-echo sequence (90 -τ-180 -τ FID) with a variable delay τ. The decay follows

1 a decay curve (Figure 9). Leung et al found both T1 and T2 of H in bread decreased with storage time (Leung et al, 1983). They attributed this decrease to an overall

decrease in water mobility and increase in water “binding”.

2. Cross-relaxation NMR

Proton (1H) is the most abundant NMR detectable specie (99.98% natural abundance) allowing for a relatively easy acquisition and strong signal. 1H NMR can be used to ascertain the physical state of the protons in a system, i.e. solid like (wide) and more liquid like (narrow) components (Wu et al, 1992). However, the 1H relaxation process is perturbed by other phenomenon including cross-relaxation and chemical exchange. Nonetheless, protons associated with the solid state can be studied by solid- state proton NMR such as the water sorption in starch and gluten (Li et al, 1996;

Vodovotz et al, 2000; Wu and Eads, 1992, Ruan et al, 1996 ) and cross-relaxation NMR

Spectroscopy, which is a liquid technique used to determine information on the relaxation of the solid component via the observable liquid spin system. In cross-relaxation NMR, a sample is irradiated with a radio-frequency pulse, which is off-resonance from the liquid signals. Due to the dipolar coupling between the liquid and solid, the amplitude of the

34 T2 (ms)

0 10 20 30 40 50 3 5

8 2 T1 8 0 4 0 1 1

x x

) )

1 1 3 2 T T ( (

y y t t i 0 2 i s s n n e e t t n n

I -1 1 I

k k

a T2 a e e

P -2 0 P

-3 0 2 4 6 8 T (s) 1

Figure 9. Typical relaxation curves from 1H NMR relaxation measurement showing a growth curve for spin-lattice relaxation (T1) and a decay curve for spin-spin relaxation (T2).

35 liquid spectrum will change with the offset frequency, and a Z-spectra is obtained

(Figure 10). The shape, area and width of a Z-spectrum are indicative of parameters increased rigidity, and the area increases with an increase of the solid to liquid proton ratio (Wu and Eads, 1993; Vodovotz et al, 2002). For example, the Z-spectrum for a bread sample is significantly wider than the one of water (Figure 10). Among several bread components, i.e. heated gluten, wheat flour and starch, the line-shape of gluten was found to be the widest indicating a greater rigidity. Vodovotz and coworkers (2002) attributed this phenomenon to a greater affinity of gluten for water. Wu and Eads (1993) observed the increase in the broad component width during storage of waxy starch gel and attributed it to restriction of chain conformation or crystallization measured by DSC.

However, Vodovotz and coworkers did not observe such an increase in the line-width of the spectrum in stored wheat bread despite an increase in recrystallized amylopectin over time (Vodovotz et al, 2002). This suggests a different effect of storage between starch gel and bread.

The results from NMR can then be used in conjunction with thermal analysis , i.e.

DSC, TGA, and DMA, and texture analysis, i.e. Instron, to better understand the complex phenomena occurring in soy containing bread during preparation and storage. Each technique has its unique sensitivity to different motions. The combined results would provide comprehensive information of changes in bread.

In addition, soy added to a bread formula not only changes the physico-chemical properties of bread but also adds in a group of bioactive components of recent interest to health, isoflavones. Previous studies have shown that the food matrix was a determining factor for the stability of isoflavones in food besides processing conditions and their

Figure 10. Results of a cross-relaxation 1H NMR experiment for water and bread. (a) Liquid 1H signal (single resonance) observed at various offset frequencies; (b) Z- spectrum obtained from the normalized amplitude of Figure (a) signals. (From Vodovotz, Y.; Vittadini, E.; Sachleben, J. Carbohydrate Research. 2002. 337. 147- 153.)

37 natural chemical forms (Coward et al, 1998). Since isoflavones contain several hydroxyl- groups in their chemical structures, their relatively high affinity for water may contribute to their stability and changes in profiles in bread system during baking and upon storage.

The information obtained about the physico-chemical properties of soy bread, especially the part on water distribution and mobility within bread matrix would facilitate the study on isoflavones in soy bread upon soy addition and storage.

THE EFFECT OF BREADMAKING AND STORAGE ON ISOFLAVONES

IN SOY CONTAINING BREAD

A. Isoflavone stability during bread making

Isoflavones belong to a group of plant derived phenolic compounds that have structures similar to human estrogen. The predominant isoflavones occurring in soy products are the families of daidzein, genistein and glycitein in four possible chemical forms, the aglycone, the β-glucoside, the malonyl-β-glucoside (“malonylglucoside”), and the acetyl-β-glucoside (“acetylglucoside”) (Table 2). The abundance of isoflavones in soy products ranges from 0.1 to 3.0mg/gram on dry weight basis (King and Bignell,

2000; Barnes et al, 1994). The predominant isoflavones in unprocessed soybeans are the malonylglucosides (∼95%).

Conjugation patterns of isoflavones in soy foods are significantly affected by processing, and the presence of other ingredients (Grun et al, 2001; Wang and Murphy,

1996; Wang et al, 1998). For example, previous studies on soybean processing suggested that the malonylglucosides of isoflavones are heat labile, and the addition of sugar and fat

38 Common Name Chemical Name Chemical Structure

Daidzein 7,4’-Dihydroxyisoflavone

Daidzin 7,4’-Dihydroxyisoflavone 7- glucoside or Daidzein 7-O- glucoside or Daidzein-7-O-β-D- glucopyranoside Acetyldaidzin 6”-O-Acetyldaidzin

Malonyldaidzin 6”-O-Malonyldaidzin

Genistein 5,7,4’-Trihydroxyisoflavone or 5,7-Dihydroxy-3-(4- hydroxyphenyl)-4H-1- benzopyran-4-one Genistin 5,7,4’-Trihydroxyisoflavone 7-glucoside Or Genistein 7-O-glucoside Or Genistein-7-O-β-D- glucopyranoside

Continued

Table 2. Structure and scientific names of twelve naturally occurring isoflavones in soy.

39 Table 2 continued

Acetylgenistin 6”-O-Acetylgenistin

Malonylgenistin 6”-O-Malonylgenistein

Glycitein 7,4’-Dihydroxy-6- methoxyisoflavone

Glycitin 7,4’-Dihydroxy-6- methoxyisoflavone-7-D- glucoside

Acetylglycitin 6”-O-Acetylglycitin

Malonylglycitin 6”-O-Malonylglycitin

40 accelerated the degradation of soy isoflavones in batters during thermal treatment

(Coward et al. 1998). As mentioned earlier, bread preparation is a complex physico- chemical process involving major steps such as mixing, yeast fermentation (proofing), and baking. These various steps may significantly influence the isoflavone distribution in a soy containing bread (Walsh et al, 2003; Zhang et al, 2003).

Enhancing bioavailability of isoflavones in soy bread

The potential health promoting activity of soy foods is dependent upon the absorption of its isoflavones and their bioactive metabolites (Németh et al 2003). Dose and chemical forms of isoflavones as well as the physical properties of soy foods candramatically influence the bioavailability and biological activity of those phytoestrogens following consumption (Day et al, 1998; Setchell et al, 2000 & 2002) .

Previous research showed that soy isoflavone aglycones were absorbed faster and in higher amounts than their glucosides in human subjects (Izumi et al, 2000). Isoflavone glycosides were not directly transported across the apical membrance of absorptive epithelial cells in the gastrointestinal tract but were converted to their aglycones either by

β-glucosidase activity in small intestine or by microflora in the large intestine. Thus the physiological effects of isoflavone glycosides were slower but longer than their aglycones.

The interest in enhancing isoflavone aglycones in soybean product has led to recent studies on β-glucosidase, a type of enzyme that is widely distributed in animal, plant, and microorganisms (McCue and Shetty, 2003). The specific β-glucosidase activity was found to correlate well with the isoflavone aglycone/glycoside ratio in

41 various tissues in isoflavone containing plants, i.e. white lupin and soybean (Pilewska et al, 2002; Wojtaszek and Stobiecki, 1997; Graham et al, 1990; Matsuura and Obata,

1993). Isoflavone glucosides may serve as pools for releasing aglycones by enzymatic activity under favorable conditions (Hsieh and Graham, 2001). Several ingredients used in current bread formula (soybeans, wheat, and yeast) contain β-glucosidase activity

(Hsieh and Graham, 2001, Sue et al, 2000, Matsuura et al, 1989).

Both soybeans and wheat β-glucosidases are capable of hydrolyzing isoflavone glycosides to release the aglycones (Hsieh and Graham, 2001; Sue et al, 2000). Several attempts have been used in previous work to increase the isoflavone aglycone level in soy foods by capitalizing on β-glucosidase activity (Tsangalis et al, 2003; Pandjaitan et al,

2000; Matsuda et al 1992). For example, presoaking soybeans in water at 50 oC and pH

6.0 in soymilk production activated the enzymatic process of β-glucosidase (Matsuura et

al, 1989), however, a significant portion of the isoflavones was lost in the soaking

medium. Microorganisms, i.e. Lactobacillus casei, Saccharopolyspora erythraea, and

Ganoderma lucidum, were inoculated into soybean products (Matsuda et al, 1992;

Hessler et al, 1997; Miura et al, 2002). However, additional enzymatic biotransformation was found to act on the liberated isoflavone aglycone molecules during fermentation causing a marked loss of these isoflavones in the final products (Matsuda, 1992; Hessler et al, 1997; Miura et al, 2002). Therefore, processing temperature, pH, components in the formula, i.e. water content, proteins, acids, and other enzymes, may influence the interaction of β-glucosidase with the substrate (Pandjaitan et al, 2000; Zheng and Shetty,

42 2000; Matsuura et al, 1995). Adding a rich source of β-glucosidase such as almond may affect isoflavone aglycone content in the soy bread formula however these changes have not been explored.

Stability of isoflavones during storage

Most of the published studies focus on raw soybeans and not the processed soy foods. Research on the stability of isoflavones during storage is limited (Eisen et al,

2003). A study on the interconversion of isoflavones in soybeans by Hou and Chang

(2002) suggested soybeans favor cold storage (4 oC versus 30 oC) during long-term storage since the interconversion between aglycones and the β-glycosides was significant at 30 oC (p<0.001). Other factors, such as the enzyme activity naturally occurring in soybeans, the impact of food matrix, packaging methods (i.e. the use of controlled atmosphere; temperature, relative humidity) of soy foods on isoflavones during storage have not been reported. Additionally, since isoflavones are highly polar, the amount and state of water in the bread may play an important role in their stability as discussed previously. A study on isoflavones in soy bread upon storage at room temperature may be directive for determining appropriate storage condition for soy bread.

Analysis of isoflavones in soy foods

Analysis of isoflavones in soybean products is an essential part of any research involving these bioactive compounds. Various processing conditions produce soy products with a wide range of isoflavone content and composition. Recent studies observed that the chemical forms and abundance of isoflavones in soy foods have a

43 significant impact on their bioavailability and biological effects (King and Bignell, 2000;

Izumi et al, 2000; Setchell, 1998, Setchell and Cassidy, 1999). It is thus very critical to avoid altering the natural forms and abundance of the twelve soy isoflavones during extraction, identification and quantification. A reliable analytical method is needed for this purpose.

Spectra fine structure of soy isoflavones Isoflavones comprise two benzene rings (A and B) linked through a heterocyclic pyrane C-ring at 3 position (Figure 11), which differ themselves from flavones (Ingham,

1982). The differences in the spectral characteristics of individual isoflavones are small, but are very important in their identification (Harborne, 1967). Isoflavones can be readily distinguished by their UV spectra, which typically exhibit an intense Band II absorption with only a shoulder or low intensity peak representing Band I. Band I (300-

330nm) absorption involves the B-ring (cinnamoyl system) (Figure 11), and Band II

(240-280nm) absorption involves the A-ring (benzoyl system). The Band II absorption of isoflavones is relatively unaffected by increased hydroxylation of B-ring. Band II is, however, shifted bathochromically by increased oxygenation in the A-ring. In addition to the absorption maxima of the isoflavones, the shape of the spectra provides important

information for identification of pure isoflavone standards and isoflavones in soy extract.

UV spectra of three soy isoflavone aglycones and their conjugates are shown in Figure

12, 13, and 14. Details of the molecular characteristics of isoflavones can be found in

The Chemistry of Flavonoid Compounds by Ollis (1962). Isoflavones in solution obey

the Beer-Lambert Law, where absorbance (A) equals concentration multiplied by molar

Figure 11. General structure and ring numbering system applied to the known soy isoflavones.

Figure 12. The UV characteristics of daidzein ( ), genistein ( ), and glycitein ( ) in HPLC mobile phases (A: 1% acetic acid in water, B: 100% acetonitrile, A:B=85:15) at 25oC monitored at 220-400nm. Numbers in figure correspond to wavelength of the absorption peaks.

Figure 13. UV Spectra of genistein family in HPLC mobile phases (A: 1% acetic acid in water, B: 100% acetonitrile; A:B =85:15) at 25oC monitored at 220-400nm. Numbers in figure correspond to wavelength of the absorption peaks.

Figure 14. UV spectra of daidzein family in HPLC mobile phases (A: 1% acetic acid in water, B: 100% acetonitrile, A:B =85:15) at 25oC monitored at 220-400nm. Numbers in figure correspond to wavelength of the absorption peaks.

48 extinction coefficient (εmax). The molar extinction coefficient is defined as the absorbance of 1 molar solution of isoflavones, in a defined solvent, in a 1-cm path-length cuvette, at its maximum wavelength (λmax). This information can be used to quantify the concentration of a certain isoflavone with known absorbance. Table 3 contains some known values of molar extinction coefficients of soy isoflavone. Variations in laboratory conditions (e.g. different solvents, temperature, absorbance wavelength) may beresponsible for difference in reported extinction coefficients. The actual absorption and fine structure will depend on the composition of the mobile phase. A shift of 2-3nm in the maximum absorption wavelength is usual. The spectrum of a specific isoflavone in soy food should be compared with an authentic pure standard. They should be identical for both the λmax and the fine structure.

Solvent extraction of isoflavones from soy foods

Soybeans and soybean products contain high level of protein, carbohydrates and lipids (Table 1). As minor components of complex mixtures, isoflavones must first be separated from the bulk of the matrix constituents prior to analysis. Efficient extraction methods for isoflavones should account for their diverse structures, chemical properties, and the food matrix of which they are constituents.

Before extracting a compound of interest from a complex food matrix, it is necessary to obtain knowledge about the physical and chemical nature of the sample, i.e. moisture content, stability in acid and base, and thermal stability. The hydrophobicity of the isoflavone forms is aglycone > acetyl-glucoside > malonylglucoside > β-glucoside based on their chromatographic behavior on a reversed-phase columns in the presence of

49 MW Absorption peaks λmax ελmax Isoflavone (nm) (l/mol⋅cm)

Daidzein 254 211.4 249.1 303.6 249 31563

Daidzin 416 216.0 250.3 302.5 250 26830

Acetyldaidzin 458 216.1 249.1 301.5 249 29007

Malonyldaidzin 502 211.4 250.3 302.5 250 26830

Genistein 270 211.4 259.8 327.0 260 35323

Genistin 432 212.6 259.8 327.5 260 30895

Acetylgenistin 474 215.0 260.9 329.8 261 38946

Malonylgenistin 518 215.0 259.8 327.5 260 30895

Glycitein 284 215.0 257.4 321.5 257 25388

Glycitin 446 212.6 258.9 321.5 259 26713

Acetylglycitin 488 212.6 258.6 321.5 259 29595

Malonylglycitin 532 212.6 259.0 321.5 259 26313

Note: The ελmax(l/mol⋅cm) values are from Murphy et al., 2002.

Table 3. UV spectra pattern of twelve soy isoflavones in 80% methanol: 20% H2O.

50 an acid in the mobile phase to protonate the glycosidic isoflavones. The ester bonds of acetyl- and malonyl-glucose of isoflavones are labile at elevated temperatures and under acidic or basic conditions. The aqueous solubility of the isoflavone aglycones are low and are pH dependent due to the acidic nature of their phenolic groups. Conjugation to glucose residues increases the solubility, while acetylation or malonylation of the glucoses reduces solubility.

Extraction of isoflavones using acidified organic solvents releases isoflavones in their natural forms from food matrices based on their polarity and solubility in such solvents. Insoluble proteins, carbohydrates and lipids present in the food are removed from the isoflavone extract using centrifugation steps.

The diversity in polarity of soy isoflavones requires the use of a combination of organic solvent and water for extraction. The organic phase to water ratio (10:5) was established based on Murphy (2002)’s study on solvent selection. Water content in the solvent needs to be adjusted according to the moisture content in soy foods. Freeze drying of samples before extraction simplifies the extraction process, however, this is not a prerequisite. Acetonitrile, acetone, ethanol, and methanol have been used to extract isoflavones from soy foods. Among them, acetonitrile proved to be the most efficient

(Griffith and Collison, 2001; Murphy et al, 2002). 0.1mol/L HCl is added to the solvent to completely deionize isoflavones and release them from protein complexes by denaturing and precipitating proteins. Room temperature is recommended for extraction to avoid alteration of their natural forms. Ultrasound can be used to aid the extraction

51 process by degrading and weakening the cellular matrix. The crude extract needs to be further purified for HPLC analysis. Direct injection of the crude extract into the HPLC would clog the frit and analytical column with precipitated impurities, i.e. proteins.

Isoflavone analysis using High Pressure Liquid Chromatography (HPLC)

HPLC is the method of choice for the analysis of isoflavones in soy products.

HPLC is fast, reproducible, requires small sample sizes and can be used for both qualitative and quantitative analysis as well as for separation purposes.

Most isoflavone analyses are performed using a binary gradient, a reversed-phase

C18 column, and a UV detector. Photo diode array detector is capable of producing

UV/Vis absorption spectrum for each peak and has became the norm of isoflavone analysis. Isocratic elution was reported for isoflavone analysis but is not as effective as a gradient to separate the wide variety of isoflavones within an acceptable elution time.

Typical gradient solvent used with reversed-phase C18 columns starts with a high proportion of polar solvents (i.e. water) and gradually increase the proportion of a less polar solvent (i.e. acetonitrile or methanol). The aqueous solvent is usually acidified to prevent ionization of isoflavone glycosides, which can give multiple peaks for some compounds.

For accurate quantification, a standard curve of peak area vs. concentration should be constructed for each standard using the same chromatographic conditions, e.g. wavelength and solvent, as is to be used for the samples under analysis. The concentration range of standard curves should be determined according to both isoflavone level of soy food samples and dilution factors during sample preparation such

52 that the UV absorbance of injected sample is within a range of 0-1. The appropriate standard curve can then be used to calculate the quantity of isoflavones represented by each HPLC peak. An internal standard can be added to the extraction solvent or to the sample prior to HPLC injection to measure the recovery rate of the chromatographic system. Apigenin, 2,4,4’-trihydroydeoxybenzoin (THB), flavone and equilenin have been reportedly used as internal standards.

Hydrolysis of isoflavone glycosides removes the sugar moiety from the aglycones, thereby enabling quantification of total isoflavones in soy foods when the authentic standards of glycosidic isoflavones are not available. Hydrolysis is also a useful tool for structural analysis when specificity is achieved. β-glucosidase (Emulsin) and cellulase (from Aspergillus niger) have been used efficiently to convert isoflavone conjugates to their aglycone forms (Liggins et al, 1998; Franke et al, 1994). These enzymes remove the β-linked glucose from the 7-hydroxyl group on the isoflavone aglycones with adequate purity. Besides enzymatic hydrolysis, acid hydrolysis and alkaline hydrolysis have also been used. Acid hydrolysis is used primarily for cleaving sugars from glycosides, while alkaline hydrolysis finds application in the specific removal of acyl groups from acylated glycosides to produce β-glucosides of isoflavones

(Klump et al, 2001). However, they are less commonly used due to a variety of reasons from incomplete hydrolysis of the glycosides and degradation of the unconjugated isoflavones (Liggins et al, 1998).

53 CHAPTER 4

MATERIALS AND METHODS

The experimental design is as shown in Figure 15.

Soy Ingredients Non-soy bread + Ingredients

Fresh Soy Breads

Texture Bread Modification Analysis for Enhanced Aglycones (Instron) (Processing Condition Storage & Additive) Study Physicochemical Property study (DMA/DSC/TGA)

Isoflavone Molecular Mobility Quantification Study (HPLC) (NMR)

Sto red Soy Breads

Figure 15. Schematic experimental representing the design of the research on physicochemical properties and isoflavone content of bread containing soy.

54 The effects of soy addition on the physicochemical properties of fresh and stored

bread on a structural and molecular level

A. Bread preparation

Table 4 outlines the formulations of wheat (control) and soy bread. Suppliers of bread ingredient and bread-making equipment are listed in Table 5 and Table 6.

The soy bread preparation first involved mixing the wheat flour, gluten dough conditioner, and 253 grams of water in a 5-quart KitchenAid Mixer for approximately 5 minutes at speed 3 (approximately 3,000rpm). Subsequently, the rest of the ingredients were added and mixed for 30 seconds and then allowed to hydrate for 5 minutes. After hydration, mixing continued for 5 minutes at speed 3. Lastly, dough was kneaded by hand for approximately 20 times or until the dough was firm. Dough was shaped into a two pound-loaf size (approximately 1330 g) and placed in nonstick baking pan. The dough was proofed at 48 oC for 1 hour. A container of water was placed in the proofer to keep a high humidity. The proofed dough was then baked at 160 oC in a jet air oven for

50 minutes to produce bread. Bread was cooled on a cooling rack for 40 minutes before collection samples for thermal analysis. This process and formulation are currently patent pending.

B. Bread storage

The cooled loaves of bread were packed in polyethylene bags and stored at room temperature for up to 14-days. Analysis will be performed on days 0, 1, 2, 4, 7 and 14.

Three breads on each day were analyzed.

55 Ingredients Soy Wheat % (w/w) % (w/w)

H2O 45.3 37.7 Soy milk powder 6.6 0 Soy flour 19.9 0 Wheat flour 17.5 54.3 Pure gluten 2.3 0 D.C. 0.2 0 Sugar 4.5 4.0 Yeast 1.0 0.9 Salt 0.9 1.0 Shortening 1.7 2.1 Calcium Propinate 0.015 0.015 (optional) a. Ingredients were added as is (wet basis).

Table 4. Ingredient list of soy and wheat breads used in this research

56 Ingredient Brand Manufacturer Yeast Red Star Instant Universal Foods Corporation, Active Dry Yeast Milwaukee, Wisconsin, 53202 Sugar Kroger The Kroger Co., Cincinnati, Ohio 45202 Wheat Flour Bakers High Gluten General Mills Operations, Inc. Enriched Bromated Minneapolis, Minnesota, 55440 Flour Bleached Gluten Vital Wheat Gluten Hodgson Mill, Inc. VWG P.O. Box with Vitamin C 430. Teutopolis, Illinois 62457 Dough Conditioner Caravan Products Totowa, New Jersey 07512 Company Soy Milk Powder Devansoy Farms Carroll, Iowa 51401 Soy Flour ADM ADM Protein Specialties Division, Decatur, Illinois 62525 Salt Kroger Iodized salt The Kroger Co., Cincinnati, Ohio 45202 Shortening Crisco All- Procter & Gamble, P.O. Box 5558, vegetable Cincinnati, Ohio 45201 Shortening, 50% less Saturated fat than butter

Table 5. The list of ingredient for soy bread and their manufacturer

Equipment Brand/Model Manufacturer 5-quarter Mixer KitchenAid, KitchenAid Portable Appliance, St. with pedal and 350Watt Joseph, MI 49085 dough hooker Proofer CM2000 InterMetro Industries Corp, Wilkes- combination module Barre, PA 18705 Jet Air Oven Model: JA14, Doyon Baking Equipment, Liniere, Quebec G0M 1J0, Canada Balance Max. Cap. 3100g Mettler-Toledo Inc., Columbus, OH 43240 5-digit Balance Max. 220g, Min. Mettler-Toledo Inc., Columbus, 10mg OH 43240

Table 6. The list of equipment and their manufacturer used for soy bread preparation

58 C. Instrumental analysis

C.1. Tests on physical properties of bread

C.1.1 Loaf volume

Loaf volume was determined by rapeseed replacement (AACC 2000, Method 10-

05).

C.1.2 Bread firmness

Firmness is defined as the amount of force required to compress a product by a preset distance. Bread firmness measurement was performed using an Instron Universal

Testing Machine (5542, Instron Corp., Canton, Massachusetts). On the test days, bread samples of 25-mm thickness were obtained from the center of breads using a bread knife.

The crust was removed. Bread samples (approximate dimensions of 16.58mm × 25mm)

were obtained from the center of the slices using a circular bore cutter. Samples were

positioned in the center of a 5-Kg load cell. The compression plunger (35-mm i.d.) was

positioned about 1mm above the surface of the bread samples. Samples were compressed

to approximately 10 mm (40% compression) at a speed of 100 mm per minute. The

compression curves of the bread crumb (distance vs. force) were recorded automatically by the software (MerlinTM, Version 5, Instron Corp., Canton, MA). The force readings

(in Newton) at 40% compression were taken as a measure of bread firmness in accordance with the AACC method 74-06 (AACC, 1986). Eight slices were analyzed from each loaf. The mean coefficient of variation for the determination of bread firmness determined on different days was lower than 5%.

59 C.1.3 Moisture content.

Moisture content was measured by vacuum oven drying (AOAC 2002, Method

925.09). Triplicate samples were placed in a pre-weighed aluminum pan and then vacuum oven dried at 40oC for 24 hours. The final weight of each sample were

determined and moisture content calculated from the weight loss using the following

equation:

g(original sample) − g(sample after vac. oven drying) % mc = ×100 Eq.1 g(original sample)

C.2. Thermal analysis

C.2.1 Dynamic mechanical analysis (DMA)

On test days, bread samples were prepared for DMA analysis by slicing the bread to approximately 1cm thickness from the center of the bread and then compressed with a

Carver laboratory press (Fred S. Carver Inc., Summit, NJ) to approximately 3 mm thickness. Samples were then cut with a die into the appropriate sample shape (3×10×18 mm). The samples were stored in sealed jars at room temperature to prevent moisture loss.

The sample was inserted into a dual cantilever attachement of a DMA instrument

(DMA 2980, TA instruments, New Castle, DE) in the float mode and locked down with

thumbscrews. The sample was cooled to –80 oC and held isothermally for 5 minutes in the oven of the DMA using liquid nitrogen, and then heated at 2 oC/min up to 120 oC in

60 the bending mode. Testing was carried out in triplicates for three separate breads. The

DMA was calibrated monthly to ensure the accurate control of temperature, force, and

frequency.

Storage modulus (E’), Loss modulus (E”), and tan δ (E’/E”) were recorded and

the transitions analyzed. The storage modulus was further characterized by fitting the

curve with a modified Fermi equation (Eq. 2) as follows (Figure 16):

(1− b) E = + b Eq. 2 n » (x − T )ÿ …1+ exp c Ÿ a ⁄

Where En is the normalized E’, a is the slope of the line, b is a constant, T is the temperature and Tc is the temperature at the inflection point of the curve.

The tan δ (T) was characterized by a deconvolution (Figure 17) using an

asymmetric double sigmoidal curve (Eq.3) and a gaussian curve (Eq. 4).

C.2.2. Differential scanning calorimetry (DSC)

Calorimetric measurement was performed on a Differential Scanning Calorimeter

(2920, TA instruments) equipped with a Refrigerated Cooling System (TA Instruments,

New Castle, DE). The instrument was calibrated regularly using Indium to ensure a flat baseline.

On test days, bread samples (approximately10 mg) were obtained from the center of the bread immediately before analysis. The samples were placed in hermetically sealed aluminum plans (PerkinElmer Instruments) with O-rings to avoid moisture loss during

Asymm etric Double Sigmoidal

» ÿ … Ÿ … Ÿ Eq. 3 … Ÿ … Ÿ a 0 1 y = × …1 − Ÿ ≈ a ’ ≈ a ’ ∆ x − a + 2 ÷ … ∆ x − a − 2 ÷ Ÿ 1 … 1 Ÿ 1 + exp ∆ − 2 ÷ 1 + exp ∆ − 2 ÷ ∆ a ÷ … ∆ a ÷ Ÿ ∆ 3 ÷ … ∆ 4 ÷ Ÿ « ◊ « ◊ ⁄ a = maximum amplitude 0 a 1 = center a 2 = width a = shape1 (> 0) 3 a 4 = shape2 (> 0) fit time index = 3.4

Gaussian (amplitude ) Eq. 4 » 2 ÿ ≈ x a ’ … 1 ∆ − 1 ÷ Ÿ y = a 0 exp − ∆ ÷ … 2 « a 2 ◊ ⁄Ÿ a 0 = amplitude a = center 1 a 2 = width (> 0) fit time index = 1.0 (reference for all functions)

Figure 16. Fitting of storage modulus (E’) from DMA by fitting the curve with a

modified Fermi equation (Eq. 2).

o rig in a l D e c o n v o lu te d T a n δ 0 .6 fitte d a s y m d b l s ig m o id g a u s s ia n fitted results 0 .4

d asym dbl sgmoid u t i l

m 0 .2 a gaussian

0 .0

-0 .2 -2 0 -1 0 0 1 0 2 0 3 0 4 0 T e m p e ra tu re (o C )

Figure 17. DMA tan δ fitting using gaussian and asymmetric double sigmoidal curves.

64 thermal scanning. An empty pan was used as a reference. Each sample was cooled inside the chamber from room temperature to –50 oC, held isothermally for 3 minutes, and then heated to 150 oC at a scanning rate of 5 oC/min. The transitions observed in the

DSC thermogram analyzed using Universal AnalysisTM (TA Instruments, New Castle,

DE). The enthalpy of transition was estimated from the integrated heat flow over the temperature range of the transition, and expressed as joule per gram sample (J/g). The endothermic peak at about 0 oC was attributed to ice crystal melting (Vittadini and

Vodovotz, 2003; Smith 2003) and therefore, the percent “freezable” water, was calculated using the equation:

Enthalpy at 0°C(J / g of sample) % " freezable" water = ×100 Eq. 5 Latent heat of fusion of water (335J / g water)

The endothermic peak observed at about 60 oC was attributed to melting of amylopectin crystals (Vittadini and Vodovotz, 2003; Smith, 2003).

C.2.3 Thermogravimetric analyzer (TGA)

Bread samples (about 20 mg) were collected from the center of the bread

immediately before analysis. Bread sample (flattened to cover the bottom of an aluminum pan) was place in an aluminum pan. The pan was then loaded onto a microbalance of the 2960 TGA (TA Instruments, New Castle, DE). Each sample was heated in the TGA from room temperature (approximately 22 oC) to 180 oC at a rate of 20 oC/min. The weight loss and derivative weight loss (the rate of weight loss) were recorded as a function of temperature. The integrated peak area over the temperature range of the scan corresponds to the percentage of total weight loss of the sample during heating and was used to calculate the total moisture content of the sample (Fessas and 65 Schiraldi, 2000). The derivative of weight loss curve was further characterized by

decomposing the main transition into its component curves using PeakFit Software

(Version 4, Systat Software Inc, Point Richmond, CA). The least number of curves were

used to fit the transition by an asymmetric (Eq. 3) double sigmoidal curve and a gaussian curve (Eq. 4) as done previously for bread and starch (Vodovotz and Chinachoti, 1998;

Baik and Chinachoti, 2001; Fessas and Schiraldi, 2000).

C.3 Molecular properties using NMR

C.3.1 Sample preparation

For Proton (1H) NMR, bread samples (approximately 100 mg) were collected from the center of a cooled fresh bread and immediately packed into a 5 mm i.d. NMR tube to fill about 3.5 cm length. The test tube was then sealed with a cap. Between analysis, the sample tube containing bread was stored in a refrigerator (approximately

4oC) until next test. Bread samples were tested on Day 0 (baking day), 1, 2, 3, 5, 7, and

10.

C.3.2 Liquid state Proton (1H) NMR

NMR spectra was acquired on a Bruker DMX 600 spectrometer (Bruker

Instruments, Billerica, MA) with a 5-mm broad-band 1H /X double resonance liquid-state

probe operating at a 1H frequency of 600MHz.

C.3.2.1 Spin-lattice relaxation time (T1 ) measurement

The inversion recover pulse sequence was used to determine T1. The pulse

o sequence began with a recycle time (>5T1). A 180 pulse was then applied which

66 inverted the magnetization. Varying recovery delays, τ, (10ms, 50ms, 100ms, 250ms,

500ms, 800ms, 1.2s, 1.8s, 2.4s, 4.8s, 6.0s, and 9.0s) followed to allow varying degree of

o T1 relaxation depending on the recovery delays. The final 90 pulse the converted the z- magnetization into observable transverse magnetization, which was detected during the acquisition period immediately following the final pulse.

C.3.2.2 Spin-spin relaxation time (T2 ) measurement

The decay of transverse at x- or y- magnetization (spin-spin relaxation, T2) was

o measured using a spin-echo sequence with a variable delay. A 90 pulse put Mo (the

equilibrium z-magnetization) into the x- or y- direction. The spin isochromats fanned out. A 180 o pulse interchanged slow and fast spins at a variable delay time (150, 200,

250, 300, 350, 400, 450, 500 µs, 1, 2, 3, 4, 5, 10, 25, and 50 ms) followed by refocusing and an echo at two times of the delay time.

C.3.2.3 Data analysis

For T1 determination, the relationship between T1 and delay time were analyzed by fitting the peak height as a function of delay time using the following equation:

τ − T1 M z (τ ) = M 0 (1− 2Fe ) (Eq. 6)

Where Mz is peak height; Mo is the equilibrium magnetization constant; F is the

correction factor; τ is the delay time; T1 is the spin-lattice relaxation time.

67 The free induction decay (FID) obtained from T2 experiment was analyzed by fitting the peak height as a function of delay time using the following equation:

t − T2 M xy = M oe (Eq. 7)

Where Mxy is peak height; Mo is the equilibrium magnetization constant; t is recovery delay; T2 is the spin-spin relaxation time.

C.3.3 Solid state 1H Cross-Relaxation NMR

1H Cross-Relaxation NMR experiments followed the method developed by

Vodovotz et al (Vodovotz et al, 2003). NMR spectra were acquired on a Bruker DMX

300 spectrometer (Bruker Instruments, Billerica, MA) using a 5-mm broad-band 1H/X

double resonance liquid-state probe operating at a 1H frequency of 300.13 MHz. Z-

spectra of water component in samples were obtained using a modified method based on

the one described by Wu and co-workers. First, a gaussian saturation pulse was used to narrow the bandwidth of the saturation pulse. Insignificant water saturation occurs 1 kHz off-resonance from the gaussian pulse. The power of the saturating pulse was adjusted between samples to ensure full saturation of the liquid peak while maintaining its length at 600 ms. A 90o pulse (width 5 µs) following a recycle delay of 1 s was used to acquire spectrum. The offset frequencies were calculated from the difference between frequency of off-resonance preparation pulses used to irradiate the solid component and the frequency of on-resonance 90o pulse of the liquid components.

68 D. Statistical analysis

Each sample for physico-chemical analysis was analyzed in triplicate unless otherwise indicated. Statistical analysis was performed using the SAS software (SAS

Inc, Cary, NC). Analyses of variance (ANOVA) using the general linear models (GLM) were conducted. Difference between the sample means were analyzed by Fisher’s least significance (LSD) test at α=0.05.

Isoflavone content and composition in the soy bread and their stability

through processing and storage.

A. Extraction of isoflavones

A.1 Sample preparation for extraction

Whenever possible, soy bread samples were collected and extracted immediately after bread making. If bread samples needed to be stored for later extraction, they were sealed in a plastic bag and stored at –20 oC or lower to prevent isoflavone loss and degradation. The frozen samples were thawed completely at room temperature

(approximately 2 hours) immediately before extraction. Prior to extraction, bread samples

were ground in a coffee grinder or with mortar and pestle until a fine paste was reached

(can pass through a 50-mesh screen).

A.2 Solvent extraction of isoflavones in their natural forms

Bread samples (0.5 g) were homogenized in a mixture of 0.1N HCl (2 mL, Fisher

Scientific, Fair Lawn, NJ), acetonitrile (10 mL, Fisher Scientific, Fair Lawn, NJ) and

69 water (3 mL) at 10,000 rpm for 1 min using a homogenizer (Polytron, Kinemetica AG,

Littau-Switzerland). The mixtures were then placed at room temperature (≤ 25oC) on a wrist shaker (IEC HN-SII, Damon/IEC Division, Needhamhts, MA) for 2 hrs

(approximately 300 strokes/min, Labline Instrument Inc., IL) to extract the isoflavones.

The mixtures were then centrifuged (IEC HN-SII, Damon/IEC Division, Needhamhts,

MA) for 30 min at 2,500 rpm to obtain supernatant (“crude extract”). The crude extract was kept in a 15-mL test tube at 4oC prior to further purification for HPLC analysis. The

analysis of the extracts by HPLC was conducted within 10 hours of extraction to minimize potential conversion of isoflavones.

B. Isoflavone analysis using HPLC-UV spectrophotometry

B.1 Sample preparation

One mL of crude soy extract (see A.1 Sample preparation) was dried under nitrogen gas stream. The residue was re-suspended in 1mL of 100% methanol (Fisher

Scientific, Fair Lawn, NJ). The mixture was ultrosonicated for 10 minutes to completely precipitate proteins and remove methanol insoluble sugars from bread matrix. The mixture was kept at 4 oC if injection was not directly conducted. Prior to HPLC injection, the mixture was vortexed (model no. 231, Fisher Scientific, Fair Lawn, NJ) for

1 min and filtered through a 0.20 µm filter (0.2 µm syringe filter, Alltech Associates Inc,

Deerfield, IL).

70 B.2 Isoflavone quantification

B.2.1 Stock standard solutions

An analytical balance was used to weigh approximately 1-5 mg of each isoflavone standard crystalline (LC labs, MA). The isoflavone standard crystallines (Table 7) were transferred into individual 50-mL volumetric flasks. Methanol in water (80%, v/v) was added to the flasks to near volume. Dissolution of isoflavone standards in 80% methanol was aided by repeatedly inversion and using an ultrasonic bath. Each flask was then filled with 80% methanol in water to the final volume. Flasks were sealed tightly with a cap, and stored at –20 oC for up to 6 months. Frozen stock solutions were completely thawed at room temperature and then filtered through a 0.45 µm filter before use.

B.2.2 Working standard solutions

From each stock solution bottle, 0.1, 0.3, 0.5, 1, and 2 mL of solution was

accurately transferred into 10-mL volumetric flasks using analytical pipettes (Table 8).

Methanol in water (80%, v/v) was used to dilute each flask to volume. Full dissolution

was aided by repeated inversion of the flasks and ultrosonification. Diluted solutions

were then transferred to tightly capped amber vials that were used for absorbance measurement using UV spectrophotometer and for quantification using HPLC. The working solutions were stored at -20oC and used within 2 months of preparation. The

solutions were completely thawed at room temperature (approximately 2 hours) prior to

analysis.

71 Isoflavones Weight in Each 50-ml Approximate Concentration Stock Solution (mg) (mg/mL) Daidzein 2 0.04 Daidzin 5 0.1 Acetyldaidzin 2 0.04

Genistein 2 0.04 Genistin 5 0.1 Acetylgenistin 2 0.04

Glycitein 1 0.02 Glycitin 1 0.02 Acetylglycitin 1 0.02

Table 7. The weight of isoflavone standards and the approximate concentration of stock standard solutions.

72 Analyte Concentration Range (µg/mL) Daidzein 0.4-8 Daidzin 1-20 Acetyldaidzin 0.4-8

Genistein 0.4-8 Genistin 1-20 Acetylgenistin 0.4-8

Glycitein 0.2-4 Glycitin 0.2-4 Acetylglycitin 0.2-4

Table 8. Concentration range of isoflavone standard working solution for preparing calibration curve.

73 B.2.3 Absorbance measurement using a UV spectrophotometer

The spectrophotometer (HP8453 UV-Visible system, Palo Alto, CA) was warmed up at least 30 minutes prior to analysis and then zeroed with an appropriate blank (i.e.

80% methanol). A Quartz glass curvette containing the standard solution of a specific isoflavone was placed in the chamber of the spectrophotometer, and the reading of absorbance at λmax=260 was taken immediately. The concentration of each isoflavone standard solution was calculated based on the Beer Lambert Law and followed Eq. 8.

The UV spectrum pattern was used for identification of isoflavones.

Absorbance = concentration (mol/L) × cell length (cm) × molar extinction coefficient “ε” (L/M-1. cm-1) Eq. 8

Where: cell length = 1cm

Note: The molar extinction coefficient, “ε” (L/M-1. cm-1), is the absorbance of 1 molar

solution in a 1 cm light path at a specific wavelength.

B.2.4 Calibration curve preparation using HPLC

The HPLC instrument (Waters 2695 separation module, Milford, MA) was

prepared as described in B.3.2. Each standard was injected individually into the HPLC

column. The peak area of each working standard solution at λmax = 260nm was measured.

Aside from the retention time, the UV spectra of each peak was used to confirm the identity of isoflavones.

Calibration curves were prepared by plotting peak areas versus known concentrations for each standard solution. The slope of the line was used to calculate the concentration of the respective isoflavone. Peak area was linearly proportional to analyte 74 concentration (Y=aX +b) with a correlation coefficient >0.98 and intersect very near the

origin. Y=peak area, X=concentration of standard solution, a=response factor between

peak area and concentration, b∼0.

B.3 Gradient separation and identification of isoflavones in sample

B.3.1 Mobile phase preparation

The mobile phase, solvent A and B, was freshly prepared for every sample set.

Five hundred mL of solvent A (1% acetic acid in water, v/v) was prepared by accurately measuring 5 mL 100% acetic acid (reagent grade, Fisher) and mixing it well with 495 mL

HPLC grade water. The pH of solvent A was checked for every preparation to make sure it was above 2.0. Solvent B was 100% acetonitrile (HPLC grade, Fisher). Both solvent

A and B were degassed via an inline vacuum degasser.

B.3.2 HPLC set-up

The UV-VIS detector of HPLC system (Waters 2996 photodiode Array Detector,

MA) was turned on at least 30 minute prior to analysis. The pump was primed and lines were flushed with mobile phase following manufacturer’s manual. The gradient of mobile phase was programmed as listed in Table 9. The pump flow rate was set at 0.6

mL/min, the column oven at 25 oC, injection volume at 10µL, and UV monitor at 260nm.

The system (Waters 2695 separation module with heating and cooling system) and column (Waters Nova-Pak C18 reverse-phase column, 150 × 3.9mm; i.d. 4µm, 60Å pore size) were equilibrated with the starting composition of mobile phase for 10 column volumes (10 × 1.8 mL/column volume) and until the baseline was flat and smooth.

75 B.3.3 HPLC analysis

Individual standards and the standard mixtures were injected by an automatic injection system of the HPLC before sample injection to assure the system was working properly. The standard mixture was prepared by taking a known amount of each standard solution into a sample vial and mixing well. The retention time and UV spectrum of each peak were monitored. The recovery rate of each isoflavone in the standard mixture was calculated. An acceptable recovery rate was 90-110 percent.

Samples were injected into HPLC column for isoflavone separation, identification and quantification. Isoflavone peaks in the sample were identified by comparing their retention times with those of isoflavones in the standard mixture (Figure 18).

Identification was confirmed by comparing the spectrum of isoflavones in bread sample

with those of standards. Isoflavones in the samples were quantified by measuring the area of each isoflavone peak. The concentrations of isoflavones in bread samples were calculated using the calibration curves developed in B.2 and considering the dilution factor during sample preparation.

C. Statistical analysis

Each sample for isoflavone analysis was analyzed in triplicate. Statistical analysis was performed using the SAS software (SAS Inc, Cary, NC). Analyses of variance

(ANOVA) using the general linear models (GLM) were conducted. Difference between the sample means were analyzed by Fisher’s least significance (LSD) test at α=0.05.

Time (minute) Solvent A (%) Solvent B (%) 0 85 15 5 85 15 36 71 29 44 65 35 45 85 15 50 85 15

Table 9. HPLC mobile phase gradients for isoflavone analysis using a reversed- phase C18 column

3 8

35%B 4 6 12 9 29%B 1 11 15%B 7 10 2 5

Figure 18. Gradient HPLC separation of isoflavone standards. Condition: Waters Nova-Pak C18 reverse-phase column (150 × 3.9 mm; i.d. 4 µm, 60Å pore size), Mobile phase (Solvent A: 1% acetic Acid in water; Solvent B: acetonitrile), 0.60 ml/min flow rate, UV detector at 260nm, column temperature at 25 oC). Elution: 1. daidzin, 2. glycitin, 3. genistin, 4. malonyldaidzin, 5. malonylglycitin, 6. acetyldaidzin, 7, acetylglycitin, 8. malonylgenistin, 9. daidzein, 10. glycitein, 11. acetylgenistin, 12. genistein. The dotted line represents the gradient of solvent B.

78 The effect of the processing and almond addition on the isoflavone content and

composition in the soy bread

The experiment design for objective 3 is illustrated in Figure 19.

Bread ingredient Raw alm ond w/o alm ond

M ixing and kneading

ß Isoflavone analysis Bread dough by HPLC Proofing at 22, 32, and 48oC ß β-glucosidase for 1,2, 3, and 4 hrs activity by spectrophotom etry Proofed dough

Baking at 160oC for 50m in Baked dough (bread)

Figure 19. Schematic shows the experimental design of the research on the effect of processing condition and almond addition on isoflavone content of bread containing soy.

79 A. Dough and bread preparation

Soy bread doughs were prepared following the same formulation listed in Table

10. The doughs were proofed at room temperature (22 °C), 32 °C, 48 °C for 1, 2, 3, and

4 hours using a proofer (CM2000 combination module, InterMetro Industries Corp,

Wilkes-Barre, PA), and baked in a jet oven (Model: JA14, Doyon, Liniere, Quebec,

Canada) at 160 oC for 50min. Room temperature (22 °C) was selected to explore the

potential for home preparation of the soy bread. 32 °C was chosen since it is a common

incubation temperature for enzymes and yeast in the food industry (Esen, 1993; Hansson

and Adlercreutz, 1992). Furthermore, Hsieh and Graham (2001) found the optimum

temperature of soybean derived β-glucosidase was 30 oC. 48 °C is a common temperature for proofing in bakery industry.

Raw almond was purchased from local grocery store (Wild Oats Natural

Marketplace, Upper Arlington, OH) and ground into fine powder (can pass through a 50- mesh screen) using a coffee grinder immediately before using. Almond powder was

mixed with other bread ingredients at 2.5, 5.0, 7.5, and 10% levels (w/w, flour basis) to produce almond containing soy bread.

B. Isoflavone analysis.

Soy bread doughs before and after proofing, and bread produced from these

doughs were extracted and analyzed for their isoflavone content and composition using

methods described previously.

80 C. β-Glucosidase activity assay

The β-glucosidase activity in soy bread samples was determined using a modified method based on that of McCue and Shetty (2003). The β-glucosidase activity in soy

bread dough was extracted by mixing two grams of dough sample with distilled water (15 mL) and homogenizing for 1 min at 7,000 rpm (Polytron, Kinemetica AG, Littau-

Switzerland), and then centrifuging at 10,000 rpm at 4 oC for 20 min using a Sorvall

RC5Cplus refrigerated centrifuge and a SS-34 rotor (Ivan Sorvall, Inc., Norwalk, CO).

The supernatant was collected and filtered through 0.48 µm filter before analysis.

Sample mixtures contained 0.1mL of 9 mM p-nitrophenol-β-D-glucopyranoside

(Sigma, St. Louis, MO) in sodium acetate buffer (pH 4.6), 0.8 mL of sodium acetate

buffer (pH 4.6), and 0.1 mL of β-glucosidase extract from soy bread dough. Blank mixture contained 0.1 mL of distilled water instead of the enzyme extract. The reaction tubes were incubated at 37 oC for 30 min. The reaction was then stopped by addition of 1 mL of cold (4 oC) 100 mM sodium carbonate (pH=8). The released p-nitrophenol in each sample was determined by measuring the absorbance of each sample at 400 nm versus. blank using a UV spectrometer. One unit of enzyme activity is defined as the amount of enzymes that released 1 µmol of p-nitrophenol from the substrate pNPG per mL per min

under assay condition.

D. Statistical analysis

Each sample for isoflavone analysis was analyzed in triplicate. Each sample for

enzyme activity was analyzed 6 times. All data were expressed as means unless

81 otherwise indicated. Statistical analysis was performed using the SAS software (SAS

Inc, Cary, NC). Analyses of variance (ANOVA) using the general linear models (GLM)

were conducted. Difference between the sample means were analyzed by Fisher’s least

significance (LSD) test at α=0.05.

82 CHAPTER 5

RESULTS AND DISCUSSION

Aim 1. The effect of soy on physico-chemical properties of fresh bread

A. Bread Macroscopic properties

A.1 Loaf volume

Cooked and cooled control wheat bread was found to weigh about 800 ± 25 gram

and to have a loaf volume of 2700 ± 100 cm3. Cooked and cooled soy bread containing

60% soy flour weighted about 1150 ± 50 gram and had a loaf volume of 2250 ± 100 cm3.

This finding is consistent with prior work comparing the loaf volume of soy and wheat

breads (Vittadini and Vodovotz, 2003; Buck et al, 1987). Smaller volume and heavier

weight of soy bread indicated the higher density (0.51 gram/cm3) as compared to wheat bread (0.30 gram/cm3).

A.2 Bread firmness from Instron

Freshly cooked and cooled wheat bread had a maximum compression load (40%

compression) of 0.63 ± 0.04 N (Newton). Freshly cooked and cooled soy bread had a

83 maximum compression load of 3.62 ± 0.96 N. Fresh soy bread was concluded to be firmer (stiffer) than fresh wheat bread. This may be attributed to the lack of gluten network formation and smaller air cell structure of bread after soy addition

(Bucker et al, 1987; Brewer et al, 1992; Vittadini and Vodovotz, 2003) as confirmed by the higher density of the soy bread.

B. Physico-chemical properties by thermal analysis

B.1 Moisture content and state of water (DSC and TGA)

Figure 20 shows a typical TGA thermogram of fresh wheat (control) and soy

bread crumb heated from room temperature to 180 oC. The weight loss was mostly attributed to the water loss (Rova et al, 2000) and used to determine moisture content.

Moisture content of fresh wheat bread was 39.9% ± 0.2%. Moisture content of fresh soy

bread was 44.7% ± 0.5%. The higher moisture content in fresh soy bread may be

attributed to the additional water added in soy bread formulation due to high water

holding capacity of soy ingredients (Brewer et al, 1992; Porter and Skarra, 1999;

Vittadini and Vodovotz, 2003).

DSC analysis indicated the presence of a major endothermic transition ~ 0oC that

was attributed mainly to ice melting (Vodovotz et al., 1996; Baik and Chinachoti, 2001;

Vittadini and Vodovotz, 2003). Typical thermograms for the ice melting transition in the

crumb of fresh wheat bread and soy bread are shown in Figure 21. Fresh wheat bread

crumb was found to have 16.2% ± 0.9% “freezable” water as analyzed by DSC (Eq. 5).

Fresh soy bread crumb was found to have 21.6% ± 0.1% of “freezable” water. The

84 greater amount of “freezable” water in soy bread crumb was likely from the additional

water added in soy bread formulation as compared to wheat bread formulation. The

percent “unfreezable” water content was calculated by subtracting “freezable” water content from total moisture content of bread. The “unfreezable” water content of fresh

wheat bread and fresh soy bread were the same (23.7% and 23.1%, respectively).

The TGA derivative weight loss curve of water released during heating was

further characterized by fitting with the least number of curves. All fittings resulted in an

R2 ≥ 0.98. Two major gaussian (typical of most phase transition; Rotter and Ishida,

1992) curves were found to best fit the derivative weight loss curves of both fresh wheat

and soy bread. Predominant moisture loss occurred at about ~80 oC (32%) and ~100 oC

(68%) for fresh wheat bread and ~65 oC (90%) and ~120 oC (10%) for fresh soy bread

(Figure 22). Two water populations in breads may be attributed to the association of water with different bread components, i.e. starch and proteins. Loss of majority of water at a lower temperature for fresh soy bread during heating indicates a weaker

association of water with bread matrix for fresh soy bread as compared to fresh wheat

bread. Soy bread contains additional proteins from soy ingredients. The greater water

holding capacity of bread components was increased with the addition of soy proteins at

room temperature (Vittadini and Vodovotz 2003). Denaturation of soy proteins at

elevated temperature may, however, cause a decrease in the water holding capacity of

bread components resulting in a major loss of moisture at ~65oC during TGA thermal

scanning. The removal of moisture at a higher temperature (~80 oC and ~100 oC) for fresh wheat bread may be a result of a more stable network structure in wheat bread,

85 110 1.0 A 100 0.8

t h ) g

90 0.6 i C e s o / W s

o % e

80 0.4 ( L

i s t t s h a o g v i 70 0.2 i L r e e w

60 D

% 0.0

50 0 20 40 60 80 100 120 140 160 180 Temperature (oC)

110 1.0 B 100 0.8

90 h ) g i C

0.6 e s o / W s

80 % e ( L

i s t 0.4 t s h a o g 70 v i i L r e e W D 0.2 60 %

50 0.0 0 20 40 60 80 100 120 140 160 180

Temperature (oC)

Figure 20. A typical TGA thermogram for fresh wheat bread crumb (A) and soy bread crumb (B) showing the percent weight loss as well as the derivative weight loss.

Figure 21. A typical DSC thermogram for fresh wheat and soy bread crumb showing a major endothermic transition at ~0oC.

1.0 )

C o / 0.8

% A (

s s

o 0.6 L

t h

g 0.4 i e W

e 0.2 v i t a

i 0.0 r e D -0.2 0 20 40 60 80 100 120 140 160 180 200

Tem perature (oC)

1.0 ) C o

/ B 0.8 % (

s s

o 0.6 L

t h g

i 0.4 e W

e 0.2 v i t

a v i

r 0.0 e

0 20 40 60 80 100 120 140 160 180 200 Tem perature (oC)

Figure 22. Typical TGA derivative weight loss curves (solid line) of fresh wheat bread crumb (A) and soy bread crumb (B). These curves were best fitted with two gaussian curves (dotted lines).

88 and/or a stronger association of water within wheat bread matrix as compared to fresh soy bread (Renkema, 2001; León et al, 2003). Smith (2003) compared the TGA derivative weight loss curves (Figure 23) of several soy bread components, including wheat gluten, wheat starch, soy flour, and soy protein isolates and found the requirement of a higher temperature (approximately 140 oC) to release the water in wheat gluten than those in

wheat starch, soy flour, and soy protein isolates (approximately 60 oC), suggesting a stronger interaction of wheat gluten with water as compared to starch and soy proteins. A similar result was observed in work of Fessas and Schiraldi (2000). The results from deconvolution of derivative weight loss curves are consistent with the findings that fresh soy bread contains more “freezable” water than fresh wheat bread, which is more easily removed.

B.2 Dynamic mechanical analyzer (DMA) results

A typical DMA thermogram for fresh wheat and soy bread is shown in Figure. A major transition was observed at 0 oC as indicated by the drop in E’ and E” values and a peak in the tan δ (T) curve. Since DSC showed a major transition attributed to

“Freezable” water in the same temperature range, it can be concluded that this transition

was attributed mainly to ice melting, but may have contributions from a second order

transition such as glass transition (Hallberg and Chinachoti, 1992; Vodovotz and

Chinachoti, 1996, Vattadini and Vodovotz, 2003). At temperature above 100 oC,

moisture evaporated from the sample resulting cracking and hardening of the sample causing jagged lines and increase in E’ (data not shown).

89 1.8

1.6 soy protein

) 1.4 C o / g (

1.2

s soy flour s o l

1.0 t

h wheat flour g

i 0.8 e w

e 0.6 gluten v i t a

v 0.4 i r e

D 0.2

0.0 wheat starch

0 20 40 60 80 100 120 140 160 180 200 220

Temperature (oC)

Figure 23. TGA derivative weight loss curves for wheat gluten, soy flour, soy protein, wheat starch and wheat flour hydrated to 50% MC (From Smith, 2003 with permission).

90 Tan δ peak of fresh wheat bread was narrower and higher than that of fresh soy bread suggesting a greater homogeneity of fresh wheat bread (Brouillet-Fourmann et al,

2002; Hatakeyama and Quinn, 1994; Smith, 2003; Vittadini and Vodovotz, 2003).

Another transition was evident between –50 oC and –10 oC and therefore the tan δ peaks

of fresh wheat and soy bread were further characterized by deconvolution (Figure 24)

using gaussian and asymmetric double sigmoidal functions (mathmetically most fitted

function). The tan δ peak of fresh wheat bread (Figure 25A) was best fit with a gaussian

function around 0 oC (area% in total tan δ peak: ~92%) and an asymmetric double sigmoidal function around –13 oC (~8%). The tan δ peak of fresh soy bread (Figure

25B) was best fit with a gaussian function around 0 oC (~60%) and two asymmetric double sigmoidal functions around ~-12 oC (18%) and -30 oC (~22%).

The broadness of the tan δ curve as well as the deconvoluted results requiring

more than one curve for best fit indicated that the tan δ (T) peak resulted from a

combined effect of several transitions in bread during heating. The deconvolution results

show a more homogenous transition from –30 oC to 0 oC for fresh wheat bread as compared to fresh soy bread. This fitting is consistent with the results on water loss from

TGA showing a smaller temperature range of water loss for fresh wheat bread (80 oC and

100 oC) when compared to fresh soy bread (65 oC and 110 oC). The additional asymmetric double sigmoid function at around –30 oC for fresh soy bread suggested an additional transition at this temperature, which may be due to the plasticizing soy components or additional water in soy bread formulation (Laine et al, 1994; Cesaro and

Sussich, 2001; Vittadini and Vodovotz, 2003). A similar lowered transition region was

105 0.8 )

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S 10-3 0.0

-50 0 50 100

Temperature (oC)

105 0.8 )

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s 10 0.2 r o o t L -2 S 10 0.1 10-3 0.0

-50 0 50 100

Temperature (oC)

Figure 24. Typical DMA thermograms for the crumb of fresh wheat (A) and soy bread (B) showing the storage modulus (E’), loss modulus (E”), and tan δ.

Figure 25. Deconvolution of tan δ curves of fresh wheat bread (A) and soy bread (B) showing a two-peak fit for wheat bread (a gaussian function around 0 oC, and an asymmetric double sigmoid function around –13 oC) and a three-peak fit for soy bread (a gaussian function around 0 oC, asymmetric double sigmoid functions around –12 oC and -30 oC)

93 observed in Meal Read to Eat (MRE) bread as compared to white bread, and this phenomenon was attributed to the presence of glycerol, a plasticizer, in MRE bread

(Vodovotz et al, 2002). Such changes at low temperatures may impact the stability of bread stored at low temperatures to extend shelf-life. Further work is required to determine the origin of this transition and stability of frozen breads during storage.

Storage modulus (E’) DMA data were also characterized by fitting the E’(T) curves in the transition region with a modified Fermi equation as previously reported

(Peleg 1993; Vodovotz et al. 2000). The fitted parameters are “Tc”, the temperature at

the inflection point of the curves, and “a”, the steepness or the slope of the curve. “Tc” was found to be –28.7 ± 1.0 for fresh wheat bread and –38.8 ± 0.4 for fresh soy bread.

“a” was found to be 10.8 ± 0.7 for fresh wheat bread and 12.3 ± 0.3 for fresh soy bread.

A lower Tc for fresh soy bread indicates its transition region is at a lower temperature than fresh wheat bread. A larger “a” of fresh soy bread corresponded to a more gradual drop in E’(T) reflecting a greater heterogeneity of this fresh soy bread as compared to fresh wheat bread (Vittadini and Vodovotz, 2003). Both the heterogeneity of the soy bread components and the low temperature transition may impact storage stability.

C. Molecular properties by NMR

C.1 Relaxation times of liquid proton

The spin-lattice relaxation time (T1 ) of the proton in the crumb of fresh wheat and soy bread was determined using the inversion recover pulse sequence. The fitting of these relaxation curves resulted in a single exponential equation (Eq. 5) for both fresh

94 wheat and soy bread (Figure 26). T1 of fresh wheat bread was determined to be 559 ± 9

ms, and T1 of fresh soy bread was 441 ± 2 ms.

T1 relaxation times in the order of tens of milliseconds are expected for most food systems (Belton, 1995; Chen et al, 1997). Protons in the range of T1 may correspond to the portion of “more mobile” water in the food system, i.e. less strongly interacting hydration water or bulk water (Chen et al, 1997; Le Botlan et al, 1998; Ruan and Chen,

2003). However, due to the cross-relaxation and proton exchange, these mobile protons can not be assigned directly to the water protons. A shorter T1 of fresh soy bread

suggested a lower mobility of this “more mobile” water in fresh soy bread as compared to fresh wheat bread. This may have a contrbution from water “weakly” bound to soy

protein in soy bread (Le Botlan et al, 1998). T1 has been found to also correlate well with bread firmness (Chen et al. 1997). Fresh soy bread was found firmer than fresh wheat bread using compression tests.

Proton transverse relaxation times (T2 ) was measured using the spin-echo sequence. Results showed the echo decay curves (Figure 26) from both fresh wheat and soy bread fitted a single exponential equation (Eq. 6). T2 of fresh wheat bread was found

to be 1.67 ± 0.02 ms, T2 of fresh soy bread was 1.75 ± 0.05 ms. Previous studies on T2 in

the food matrix have found it to be in the range of 0.2-20 ms (Leung et al, 1983;

D”Avignon et al, 1990; Chen et al, 1997). Generally, pure water has a T2 of about 1-2 s

(Chen et al, 1997)

Protons in the range of T2 may correspond to the portion of “less mobile” proton

that at least partially belong to less mobile liquid water. These protons may strongly

95 interacted with other molecules by hydrogen bonding, particularly macromolecules such as starch and gluten in bread (Chen et al, 1997; Le Botlan et al, 1998). A shorter T2 of fresh wheat bread suggested the interaction of this portion of “less mobile” protons with bread matrix may be stronger in wheat bread than in soy bread. A similar result was

obtained from TGA study showing that the water in fresh wheat bread required to be

removed at a higher temperature.

The relaxation time results suggest the existence of two main liquid proton types

in bread system, the “more mobile” protons and “less mobile” protons. The “more

mobile” portion of protons in fresh soy bread has a lower degree of mobility than that in

fresh wheat bread. However, the “less mobile” portion of protons in fresh soy bread has

a higher degree of mobility than that in fresh wheat bread.

C.2 1H cross relaxation

Figure 27 show the result of a cross-relaxation 1H NMR experiment for fresh

wheat and soy breads. The Z-spectrum was obtained from the amplitude of liquid 1H

signals (signal resonance) observed at various offset frequencies. The Z-spectrum for the

fresh wheat bread shows a broader curve than the one of the fresh soy bread. The Z-

spectrum reflects the effect of the different bread components on the interaction between

liquid and solid protons. Different line shapes of the Z-spectrum indicate different

mobilities of the solid-proton fraction in samples (Vodovotz et al 2002). The current

result suggests the presence of more mobile solid protons in fresh soy bread than in fresh

wheat bread. The greater degree of rigidity of fresh wheat bread as compared to fresh

96 6 fresh soy bread 4 8 0 1

t 2 fresh wheat bread h g i e H 0 k a e P -2 T1

-4 0 2 4 6 8 10 Time (s)

fresh wheat bread 4 fresh soy bread 8 0 1 3 x

t h g i 2 e H

a 1 e P 0 T2 -1 0 10 20 30 40 50 60

Time (ms)

Figure 26. A typical result from proton T1 and T2 measurement of fresh wheat and soy bread showing a fit using a single exponential function (Eq. 5 and Eq. 6). 97 1.2

1.0 y t i s

n 0.8 e t n I

d 0.6 e z i l a 0.4 m r o

N 0.2 soy wheat 0.0 -60 -40 -20 0 20 40 60 Frequency Offset (kHz)

Figure 27. Z-spectra obtained from cross–relaxation 1H NMR experiment for fresh wheat and soy breads.

98 soy bread may be partially attributed to difference in moisture content (44.7% in soy bread vs. 39.9% in wheat bread) and “freezable” water (21.6% in soy bread vs. 16.2% in wheat bread), and a stronger binding of gluten in wheat bread with water.

As a summary, soy addition significantly changed the physico-chemical properties of fresh bread in all aspects. The effects of soy addition to bread formulation at a 60% flour level as compared to a wheat bread include: a decrease in the loaf volume by

16.7%; an increase in the loaf density by approximately 70%; an increase in the bread firmness by 4.7 times (Instron); an increase in total moisture content by 12% (including the water added for acceptable bread quality, TGA); an increase in the amount of

“freezable” water by 33.3% (DSC); an unchanged “unfreezable” water (DSC and TGA); a more easily removed water by heat as shown by TGA derivative weight loss curve; a greater heterogeneity of water as shown a by broader endothermic transition at 0 oC in

DSC and a broader TGA derivative weight loss curve; a greater heterogeneity of bread system as shown by tan δ peak and storage modulus (E’) at 25 oC (DMA) and an

additional transition at –30oC during cooling (this transition also suggests a greater mobility of bread system at lower temperature); a decrease in the mobility of “more

mobile” water and an increase in “less mobile” (more solid like) water as shown in proton relaxation time determination by NMR; and an increased mobility in solid proton as shown in proton cross-relaxation NMR experiment.

Therefore, the greater heterogeneity of soy bread may be due to the additional domains in the bread upon soy addition. The greater availability of water in soy bread may impact bread microbiological stability.

99 Aim 2: The effect of soy on physico-chemical properties of bread

upon storage

A. Bread macroscopic properties

A.1 Bread firmness-Instron results

The firmness of both wheat bread and soy bread increased during 10-day storage

(Figure 28). The firmness of wheat bread increased from 0.63 ± 0.04 N on baking day

(day 0) to 3.59 ± 0.73 N on day 10, and the firmness of soy bread increased from 3.86 ±

0.57 N to 6.25 ± 1.32 N within 10 days.

For soy bread, there was a significant increase (p<0.05) during the first day after

baking and a gradual increase through day 1 to day 10, however the difference in

firmness between days was not found to be significant (p>0.05). After 10 days, the

firmness of soy bread increased by about 62%. For wheat bread, there was a significant difference (p<0.05) in firmness through day 1 to day 10 almost linearly (R2=0.84) except for the difference between day 1 and day 2. Although the fresh soy bread was found firmer than fresh wheat bread, the firmness of soy bread increased at a much lower rate than wheat bread. B. Physicochemical properties by thermal analysis

B. Physico-chemical properties by thermal analysis

B.1 Moisture content and state of water (DSC and TGA)

Moisture content of the wheat (control) and soy bread crumb during storage at

room temperature was measured by TGA and was found to decrease as expected (Table

100 10). Moisture content decreased from 39.9% to 35.9% during 7-day of storage in wheat

bread crumb and from 44.7% to 42.4% in soy bread. The decrease of moisture content of

bread crumb is a consequence of moisture migration from the moist bread crumb to the

drier bread crust (Czuchajowska and Pomeranz, 1989; Baik and Chinachoti, 2001) and loss of moisture to the air from bread surface. A slower decrease in moisture content for soy bread suggests that soy bread crumb is able to retain water better than wheat bread crumb and may, at least partially, result in a slower rate of firmness upon storing at room temperature.

DSC analysis showed an increase in endothermic transition ~0oC for soy bread and a decrease for wheat bread during storage. The change in this endothermic transition

in soy bread during storage corresponds to an increase in “freezable” water from 21.6% to 27.7%. This increase is unusual for bread system (Baik and Chinachoti, 2000;

Vittadini and Vodovotz, 2003) and is likely caused by the high affinity of soy for water

(Brewer et al, 1992; Renkema, 2001). The percent “freezable” water in wheat bread decreased from 16.2% to 14.9% during storage. Its “unfreezable” water decreased from

23.7% to 21.0%. Little change in moisture content (2.3%), an increase in “freezable” water (+6.1%), a decrease in “unfreezable” water (-7.4%) in soy bread with storage suggests the conversion of “unfreezable” water to “freezable” water in soy bread and that the rate of “freezable” water conversion from “unfreezable” water in soy bread crumb was faster than the loss of water by moisture migration from bread crumb to crust.

Decrease in both “freezable” water (-1.3%) and “unfreezable” water (-2.7%) in stored

101

)

N 8 n (

o i d t a

a soy bread o m L

r 6

o n f o e i d s

s 4 e % r 0 p 4

t m a o 2 C wheat bread

0 0 2 4 6 8 10 Days

Figure 28. Instron results for soy and wheat bread firmness. Increased compression load indicates an increase in firmness.

102

Day Soy Bread Crumb Wheat Bread Crumb

Moisture “Freezable” “Unfreezable” Moisture “Freezable” “Unfreezable” water water water water content content 0 44.7a 21.6a 23.1a 39.9a 16.2a 23.7a 1 44.5a 23.7b 20.8b 39.6a 16.2a 23.4a 2 43.6b 25.3c 18.3c 38.3b 16.1a 22.2b 4 42.8c 26.6d 16.2d 36.9c 15.0b 21.9b 7 42.4c 26.7d 15.7d 35.9c 14.9b 21.0c

Note: Different letters in each row suggest statistically significant difference between data. (p<0.05).

Table 10. DSC and TGA results indicating the changes in moisture content, “freezable” water, and “unfreezable” water of wheat and soy breads during storage.

103 wheat bread suggested that the water is more prone to be lost in wheat bread crumb as compared to soy bread. This result can be explained by the fact that wheat bread crust contains much lower moisture content (<5%) than soy bread crust (~23.4%).

Deconvolution of TGA derivative weight loss curves for stored wheat and soy bread found two peak pattern similar to fresh wheat (~80 oC and ~100 oC) and soy breads

(~65 oC and ~110 oC). For wheat bread stored at room temperature up to 14 days, the peak amplitude, width, and intensity of a gaussion function at ~80 oC was found to be unchanged after storing at room temperature for up to 14 days. However, the temperature of peak center (Figure 29) increased from ~80 oC to~90 oC during the first 6 days and then remained stable until the 14th days. A similar trend of increasing in the temperature

of peak center was found in wheat bread in the second gaussian function at approximately

100 oC (Figure 29). The temperature of peak center increased from 103 oC to 107 oC suggesting the moisture in wheat bread was becoming more difficult to be removed with

storage. For soy bread stored at room temperature for up to 14 days, no major change was detected in peak amplitude, width, and intensity for the gaussian functions at about

65 oC and about 110 oC, similar to stored wheat bread. Contrary to the change in the temperature of peak center for wheat bread during storage, the temperature of peak centers for both gaussian functions decreased during the first two days of storage (Figure

29). For the gaussion function at about 65oC, the temperature of peak center decreases

significantly (p<0.05) one day after baking (-8.0 oC) and then remained unchanged during

the rest of the storage time. For the peak ~110oC, the temperature of peak center decreased during the first two days of storage (-8.6 oC), and remained unchanged from day 2 to day 7 followed by a second decrease (-6.0 oC) from day 7 to day 14.

104 The results show that storing soy bread at room temperature led to greater

removal of the moisture by heating. This result correlates well with the results from DSC and TGA showing an increase in “freezable” water and a decrease in “unfreezable” water for soy bread during storage.

B.2 Amylopectin recrystallization by DSC

The endotherm of amylopectin recrystallization (endothermic peak ~ 40 - 70oC in the DSC thermogram, Figure 30 as found to increase in wheat bread during storage.

The enthalpy associated to amylopectin recrystallization in the wheat bread increased from 0.6 to 3.8 W/g during 7 days of storage at room temperature as was found

previously (Vittadini and Vodovotz, 2003). The amylopectin recrystallization in soy

bread increased very slightly from 0.04 to 0.07 W/g during 14 days of storage at room

temperature, significantly lower than the increase in wheat bread (Figure 31). Factors

that may contribute to this difference include the diluting of amylopectin in soy bread by addition of soy ingredients, a low moisture gradient between soy bread crumb and crust

that may affect staling kinetics and the difference in water distribution among bread

components (Vittadini and Vodovotz, 2003).

Amylopectin recrystallization is commonly associated with bread staling,

especially firming (He and Hoseney, 1990; Martin et al, 1991; Ruan et al, 1996). A

slower increase in firmness for soy bread as compared to wheat bread found by Instron

paralleled with the slowed rate of amylopectin recrystallization in soy bread as shown in

105 100 130 ) )

90 120

~80 C

, , C C o o ( (

80 110 e e r r u u t t a

a o

~100 C r r e e p p 70 100 m m e e T T wheat bread 60 90 0 2 4 6 8 10 12 14 Days

100 130

) soy bread )

90 120

, , C C o o ( ( o 80 ~110 C 110 e e r r u u t t a a r r e e

70 100 p p m m e e T T ~65oC 60 90 0 2 4 6 8 10 12 14 Days

Figure 29. Deconvolution results showing the peak temperatures of TGA derivative weight loss curves for wheat and soy breads stored at room temperature for up to 14 days after baking.

106 0.0

) -0.1 Day 7 amylopectin g

/ melting W

C -0.2 Day 0 S D (

p -0.3 l a h t n E -0.4 wheat bread

-0.5 -60 -40 -20 0 20 40 60 80 100 120

Temperature (oC)

Figure 30. DSC results of wheat bread during storage. Melting of recrystallized amylopectin is indicated in box.

107 1.0 4.0 3.5 )

0.8

)

3.0

wheat bread crumb

0.6 , 2.5

g , / g / 2.0 W (

W 0.4 y (

p y 1.5 l a p l h a 0.2 t h soy bread crumb 1.0 n t E n

E 0.5 0.0 0.0 0 2 4 6 8 10 12 14 16 Days

Figure 31. Change in the enthalpy (W/g) of recrystallized amylopectin melting in wheat and soy bread during storage.

108 DSC, suggesting the increase in firmness may be associated with amylopectin and/or other components interacted with amylopectin, i.e. water and proteins. The addition of soy components to bread formulation may therefore alter the staling process.

B.3 Dynamic mechanical analyzer (DMA) results

DMA results show a major transition at 0 oC, which was attributed mainly to ice

melting as confirmed by DSC, in stored wheat and soy breads as was also found in fresh

wheat and soy bread. Deconvolution of DMA tan δ curve showed a pattern of mixed

functions for both stored wheat and soy bread similar to fresh breads. Tan δ curve of

stored wheat bread consisted of a gaussian function around 0oC (approximately 92%) and

an asymmetric double sigmoidal function around -13oC (approximately 8%). All fitting

resulted in an R2 ≥0.98. For wheat bread, the amplitude, width, and peak center of both

gaussian and asymmetric double sigmoid functions remained unchanged through storage.

These results suggest that although the percent of “freezable” water decreases in the

wheat bread, this change does not impact the micro-rheological thermal transitions.

Similar results were found by Vodovotz and coworkers (1996).

Tan δ curve of stored soy bread consisted of a major peak at about -30 to 20oC,

which was fitted with a gaussian function (about 0 oC) and an asymmetric double sigmoid

function (about -12 oC), and a minor peak at about –30 oC, which was fitted with an

asymmetric double sigmoid peak and may be attributed to the sugar present in the soy

flour. The gaussian function and asymmetric double sigmoid function of tan δ curve of

soy bread remained unchanged during storage. Major changes in tan δ curve of soy bread

during storage took place in the asymmetric double peak around –30 oC. The increase in 109 the amplitude and intensity of this function initiated after soy bread has been stored for 7

days and was tripled by the end of the 14-day storage (Figure 32). The width of the

function doubled after just one day of storage and remains unchanged followed by an

additional increase after seven days of storage (Figure 33). Increase in the amplitude

and intensity of the peak at a low temperature (–30oC) may be due to phase transition of

soluble carbohydrates that undergo a glass-rubber transition at low temperatures.

E’(storage modulus) DMA of wheat and soy bread during storage was further characterized by fitting the storage modulus curves in the transition region with a modified Fermi equation. All fittings gave an R2>0.98. Fitting results of wheat and soy

containing bread during storage are shown in Table 11. The fitted parameter, “Tc” and

“a” were -28.7 ± 1.0 oC and 10.8 ± 0.7 for fresh wheat bread, and -38.8 ± 0.4 oC and 12.3

± 0.3. A smaller “Tc ” for fresh soy bread indicated that the transition occurred in fresh

soy bread at lower temperature than in fresh wheat bread. A smaller “a” (slope) for fresh wheat bread suggests that the transition occurring in fresh wheat bread was more

homogenous than in fresh soy bread. During storage, “Tc” and “ a” did not significantly

change (p>0.05) over time for wheat bread or soy bread as was found previously

(Vittadini and Vodovotz, 2003).

Figure 34 shows the change in E’(T) values at 25oC for wheat and soy bread during storage. E’(T) at 25 oC has been used as an indicator for stiffness. E’(T) fresh wheat bread was found to be higher than E’(T) fresh soy bread. During storage, E’(T) increased for both wheat and soy bread.

110

0.008 0.16 0.14 ) 0.007

)

0.12

0.006

( 0.10

(

0.005 e y d t

0.08 i u t s

i 0.004 l n e p 0.06 t m 0.003 n I

A 0.04

k k a

a 0.002 e

e 0.02 P P 0.001 0.00 0.000 -0.02 0 2 4 6 8 10 12 14 Days

Figure 32. Results for the amplitude and intensity of the asymmetric double sigmoid curve (centered at about -30oC) used to fit the tan δ (T) curve for soy bread during storage.

111 16 )

C 14 o (

g 12 n a R

e 10 r u t a r

e 8 p m e

T 6

4 0 2 4 6 8 10 12 14 Days

Figure 33. Results for the width of the asymmetric double sigmoid curve (centered at about -30oC) used to fit the tan δ (T) curve for soy bread during storage.

112

Figure 34. DMA E’ value at 25oC for wheat and soy breads stored at room temperature.

113

Bread Storage Slope Tc time a (days) average std average std

Wheat 0 10.8a 0.7 -28.7a 1.0 1 11.2a 0.5 -28.9a 1.2 2 11.0a 0.3 -27.4a 0.9 4 11.3a 0.2 -26.6a 2.8 7 11.5a 1.1 -24.8a 3.2 Soy 0 12.3a 0.3 -38.8a 0.4 1 12.6a 0.3 -38.9a 0.5 2 12.5a 0.2 -38.4a 0.2 4 12.4a 0.3 -36.1b 0.3 7 12.3a 0.3 -37.1c 0.3 14 12.3a 0.6 -38.2d 0.6

Note: a, b, c, d - different letters in row suggest significant difference between numbers (p<0.05).

Table 11. Results of fitting of E’(T) of wheat and soy bread with the modified Fermi equation (Eq. 2) during storage. “Average” represents the average of at least three individual fitting for each sample during storage.

114 However, E’(T) of wheat bread increased at a faster rate than that of soy bread during storage at room temperature, which is consistent with the bread firmness results by

Instron and previous work (Vittadini and Vodovotz, 2003).

C. Molecular properties

C.1 Relaxation times

Results from spin-lattice relaxation time (T1) measurement (Table 12, Figure 35)

o show that, during the 10-day storage at 4 C, the average T1 for wheat bread decreased significantly (p<0.05) from 559.4 ms to 532.6 ms but remained mainly unchanged for soy bread. A similar decrease in T1 for wheat breads during storage was also reported by

Leung et al (1979), Wynne-Jones and Blanshard (1986), and Slade and Levine (1991).

These authors proposed that this decrease in T1 was due to the incorporation of the water molecules (probably released from gluten upon stabilization) to the starch crystalline

structure that developed during staling. An unchanged T1 in stored soy bread is in

agreement with DSC results showing that there is less starch crystalline development in

soy bread upon storage.

Results from spin-spin relaxation time (T2) measurement (Table 12, Figure 36)

show that the average T2 fluctuated through storage in wheat bread without significant

change. The fluctuation in spin-spin relaxation time in wheat bread may be due to

moisture transfer between macromolecules over time (Willhoft 1971; Chen et al. 1997).

115

Day Wheat T1 Wheat T2 Soy T1 Soy T2 (ms) (ms) (ms) (ms) 0 559.4 ± 8.7 1.67 ± 0.03 441.8 ± 2.0 1.75 ± 0.05 1 553.0 ± 7.8 1.72 ± 0.05 425.2 ± 2.9 1.75 ± 0.02 2 546.7 ± 8.1 1.70 ± 0.05 427.2 ± 3.4 1.77 ± 0.05 3 545.3 ± 12.2 1.66 ± 0.04 431.2 ± 4.8 1.78 ± 0.05 5 537.3 ± 9.9 1.69 ± 0.01 439.8 ± 2.8 1.66 ± 0.03 7 536.8 ± 11.7 1.67 ± 0.01 440.3 ± 5.4 1.68 ± 0.04 10 532.6 ± 10.6 1.64 ± 0.02 441.4 ± 0.6 1.67 ± 0.03

Table 12. Relaxation time determination by 1H NMR for wheat and soy breads during storage. T1: proton spin-lattice relaxation time, T2: proton spin-spin relaxation time.

116

580

560

) s 540 m (

1 520 T wheat bread

H 1

500

e t

t 480

s - d

i 460

u q i 440 L soy bread 420 400

0 2 4 6 8 10 12

Days

Figure 35. Spin-lattice relaxation time (T1) results for wheat and soy breads during storage.

117 2.0 )

s 1.8 m (

2 T 1.6 H 1

e t a t 1.4 s - d i u q i 1.2

L wheat bread soy bread 1.0 0 2 4 6 8 10 12 Days

Figure 36. Spin-spin relaxation time (T2) results for wheat and soy breads during storage.

118 The average T2 decreased significantly (p<0.05) from 1.75 ms to 1.67 ms for soy bread.

This may be due the decrease in the mobility of “less mobile” proton in soy bread during

storage as indicated by Ruan and coworkers (2000).

C.2 Cross relaxation

Results of cross–relaxation 1H NMR experiment for breads stored at 4 oC for up to 7 days show no change in the Z-spectra lineshape of wheat bread with increased storage time (Figure 37). This result suggests that the mobility of solidlike protons in wheat bread did not change during storage. The narrower line shape of the Z-spectrum of soy bread suggests a greater mobility of solid protons with increasing storage time

(Figure 38). The increase in mobility of the solid protons in soy bread over time may be attributed by the increase in “freezable” water and/or the decrease in “unfreezable” water over time (Table 10).

As a summary, soy addition was found to have a significantly impact on the physico-chemical properties of bread during staling. The effects of soy addition to bread staling using wheat bread as a control include: an increase in bread firmness by 1.6 times in 14 days (6.7 times for wheat bread); a decrease (-2.3%) in moisture content (-4.0% for wheat bread), an increase (+6.1%) in “freezable” water (-1.3% for wheat bread), and a decrease (-7.4%) in “unfreezable” water (-2.7% for wheat bread) in 7 days; a decrease in hardness to remove water by heating over time (an opposite effect for wheat bread); a slight increase (+50%) in recrystallized amylopectin in 14 days (a 533% increase in 7 days for wheat bread); an additional transition at –30oC in tan δ curve of soy bread

suggesting an increase in heterogeneity and mobility of soy bread over time (an increase

119

1.0 0.9

y 0.8 t i s

n 0.7 increase e t storage

n 0.6 I

time

d 0.5 e z i l 0.4 a m

r 0.3 o

N 0.2 0.1 0.0 -50 -40 -30 -20 -10 0 10 20 30 40 50 Frequency Offset (KHz)

Figure 37. Z-spectra obtained from cross–relaxation 1H NMR experiment for wheat bread stored at 4 oC for up to 7 days.

120 1.2 soy bread 1.0 y

t increasing i

s 0.8 storage n

e time t n I

0.6 d e z i

l 0.4 a m r 0.2 wheat bread o N 0.0

-60 -40 -20 0 20 40 60 Frequence Offset (kHz)

Figure 38. Z-spectra obtained from cross–relaxation 1H NMR experiment for soy bread stored at 4 oC for up to 7 days. The gray line corresponds to the Z-spectrum of fresh wheat bread added for comparison.

121 in heterogeneity for wheat bread); an unchanged T1 and a decreased (-0.08 ms) T2 as

compared to a decreased T1 (-4.1 ms) and an unchanged T2 for wheat bread; and a narrowing Z-spectrum (slightly broadening for wheat bread) in 7 days. Therefore, the addition of large amounts of soy (60%) significantly reduces the parameters associated with bread staling, i.e. lower amylopectin recrystallization, less moisture loss, lower firming rate, during storage as compared to wheat bread. All these beneficial effects can be reflected by the great heterogeneity of soy bread matrix that may help stabilize the bread components in the domains.

Aim 3: Isoflavones in soy bread

A. Comparison of isoflavones in soy ingredients and soy bread

The isoflavone concentration (in nmol/g soy bread, dry basis) in the starting ingredients (soy flour and soy milk powder), and the final product, soy bread, are shown

in Table 13. No isoflavones were detected in the wheat bread as expected. Twelve isoflavones (3 aglycones and their corresponding derivatives) were clearly separated, identified and quantified (Figure 39). Isoflavones (715 mg) in soy flour and soy milk powder were largely recovered in soy bread (614 mg/loaf), indicating a general stability of isoflavones during bread making. The minor loss of isoflavones may be attributed mainly to thermal decomposition or irreversible binding to other bread components.

However, isoflavone profile between the soy ingredients and final bread product was largely altered.

The primary source of isoflavones in the soy bread is soy flour (Table 1).

Malonylglucosides (47%) and β-glucosides (40%) were the prevalent isoflavones in soy 122

3 8 11 1 4 9 12 6 7 10 5 2 isoflavone standards

soy flour

soy milk powder

soy bread dough

soy bread crumb soy bread crust

Figure 39. Gradient separation using reverse phase HPLC (see conditions in text) of twelve soy isoflavones from soy ingredients and bread samples. The first chromatogram corresponds to elution of isoflavone standard mix. Elution: 1. daidzin, 2. glycitin, 3. genistin, 4. malonyldaidzin, 5. malonylglycitin, 6. acetyldaidzin, 7, acetylglycitin, 8. malonylgenistin, 9. daidzein, 10. glycitein, 11. acetylgenistin, 12. genistein.

123 Isoflavone Soy flour Soy milk Soy bread powder crumb

nmol/gram, dry basis Daidzin 551±33 866±35 432±20 Genistin 1222±37 1926±12 935±8 Glycitin 120±4 87±8 139±5 β-glucosides 1893 2879 1506 Malonyldaidzin 827±8 315±25 404±1 Malonylgenistin 1259±13 593±2 925±8 Malonylglycitin 141±4 37±1 145±0 Malonylglucosides 2227 945 1474 Acetyldaidzin 236±24 85±1 131±1 Acetylgenistin 111±0 73±5 189±3 Acetylglycitin 106±4 70±6 80±4 Acetylglucosides 453 228 400 Daidzein 39±1 551±11 259±3 Genstein 93±1 1148±46 345±9 Glycistein 7±0 5±0 7±1 Aglycones 139 1704 611 Total 4712 5756 3991 a. Values are means ± SD of three independent determinations.

Table 13. Isoflavone content (nmol/gram, dry basis) of soy ingredients (soy flour and soy milk powder) and soy bread crumba

124 flour with the aglycones representing only 3% of the total isoflavone content. The

isoflavone composition of soy milk powder differed in that β-glucosides (52%) and aglycones (31%) were most abundant. The differences in the isoflavone profile of the soy flour and soy milk powder likely resulted from altered processing of the soybeans.

Soy flour is produced by solvent extraction of the lipid portion followed by grinding soybean flakes into powder, while preparation of soy milk powder involves cooking ground soybeans (95oC, 7min) followed by drying the liquid soy milk (Coward et al,

1998; Wang et al, 1996).

B. Effect of a soy bread preparation on isoflavone content and distribution

The isoflavone concentration and profile in soy bread dough before and after proofing (48 oC, 1 hour) and soy bread after baking (160 oC, 50min) are shown in Table

14. Soy bread crust was separated from crumb, and the isoflavones in crumb and crust

were also analyzed to further study the effect of baking (Table 15).

In the first step of bread making, soy ingredients were mixed with other bread ingredients, i.e. wheat flour, water, and yeast, to form a dough. No significant change in the isoflavone content and composition was observed during mixing and kneading (3,000 rpm on average, 15min). No significant degradation of isoflavones was observed during proofing (48 oC, 1 hour). However, proofing caused significant changes (p<0.05) in isoflavone composition (Figure 40). The changes include an increase in aglycones

(+153%) and a decrease in β-glucosides (-23%) as compared to soy dough before

proofing. Both amounts of acetyl-β-glucosides and malonyl-β-glucosides of isoflavones changed slightly during proofing (+9% and -6%, respectively). The isoflavone β- 125 Isoflavones Before After After proofing proofing baking crumb crust

nmol/gram, total weight Aglycones 125±1 316±8 339±5 470±6 β-Glucosides 612±22 469±17 834±22 1451±31 Malonylglucosides 1330±61 1240±18 814±9 276±23 Acetylglucosides 170±7 185±6 220±6 873±13 Total 2240 2210 2210 3070 a. Values are means ± SD of three independent determinations.

Table 14. Isoflavone content and composition (nmol/gram, total weight) in soy bread dough during preparation (proofed at 48 oC for 1 hour and baked at 160 oC for 50 min) and in soy bread crumb and crust.

126 β-glucosidase Isoflavone Proofing Proofing activity temperature time β-glucosides to aglycone (oC) (hour) (µmol/ml‡ min) ratio N/A 0 27.7 ± 0.6 4.9

22.0 1.0 45.8 ± 2.3 4.7 2.0 43.8 ± 0.4 3.5 3.0 44.8 ± 1.3 2.8 4.0 43.1 ± 0.4 2.3

32.0 1.0 26.6 ± 0.7 2.8 2.0 39.7 ± 0.3 1.1 3.0 38.8 ± 0.4 0.7 4.0 15.1 ± 0.5 0.3

48.0 1.0 51.0 ± 1.0 1.5 2.0 34.4 ± 0.5 0.4 3.0 18.9 ± 0.6 0.3 4.0 0.0 ± 0 0.2 a. Values are means ± SD of three independent determinations.

Table 15. Isoflavone β-glucoside : aglycone ratio and β-glucosidase activity in soy bread dough after proofing at 22, 32, and 48 oC for 1, 2, 3, and 4 hours

127

Figure 40. The change in isoflavone distribution during bread making of soy dough mix, proofed dough and baked dough.

128 glycoside : aglycone ratio changed from 4.9 to 1.5 during proofing. After baking (160 oC, 50min), the changes in soy bread crumb include increases in aglycones (+9%), β- glucosides (+7%), and acetylglucosides (+13%), and decreases in malonylglucosides

(-28%) when compared to soy bread dough before baking. The isoflavone β-glycoside :

aglycone ratio changed from 1.5 to 2.5 during baking for soy bread crumb as a result of further hydrolysis of malonylglucosides to produce β-glycosides.

The moisture content in soy bread crumb and crust was 45% and 23%,

respectively. Soy bread crumb represented about 87% of the total weight of soy bread.

After moisture normalization, the levels of total isoflavones and aglycones were the same

(3.9 and 0.6 µmol/g, dry basis) in soy bread crumb and crust. However, significant

difference in their isoflavone composition (p<0.05) was observed. Soy bread crust

contained a higher level of β-glucosides (+10%) and acetylglucosides (+18%) and a

much lower level of malonylglucosides (-28%) of isoflavones when compare to soy bread

crumb. This result was consistent to the findings of Coward and coworkers (1998) about the major conversion of malonylglucosides to acetylglucosides of isoflavones at severe thermal conditions for soy food of low moisture.

In summary, proofing and baking have important but different roles in changing the distribution of isoflavones in soy bread. The proofing stage in bread preparation is key in the production of isoflavone aglycones in bread dough without degrading the isoflavones. In order to determine the bread proofing conditions leading to optimal isoflavone aglycones production in soy bread, variable proofing times (1-4 hrs) and temperatures (22, 32, 48 oC) and the effect of these conditions on β-glucosidase activity in soy bread were studied. 129 C. Effect of proofing on isoflavones and β-glucosidase activity in soy bread

The β-glucosidase activity in bread ingredients (yeast, soy flour, soy milk powder, and wheat flour) and bread samples was determined using a colorimetric method. The β- glucosidase activity in bread yeast was low (0.66 U/g) as compared to soy flour (10.7

U/g) soy milk powder (6.83 U/g) and wheat flour (4.14 U/g). The results are consistent with results from Gunata et al (1986) which show that yeast (Saccharomyces) have a very

low β-glucosidase activity. Sue and coworkers (2000) observed that the β-glucosidase

from wheat was capable of hydrolyzing p-nitrophenol β-glucosides, as well as flavone

and isoflavone glucosides. Therefore, wheat flour, a major bread ingredient in this soy

bread formula, may also contribute to the total β-glucosidase activity along with the soy ingredients.

The changes in isoflavone content and composition (Figure 41, 42 and 43) in dough proofed for 1-4 hours at 22, 32, and 48oC and the changes in β-glucosidase activity

(Table 15) were studied. The level of β-glucosidase activity in soy bread dough before

proofing was normalized to 100%.

Figure 41 shows the changes in isoflavones and β-glucosidase activity in soy

bread doughs after proofing at 48 oC for 1-4 hours. Isoflavone glycosides and aglycones

were most affected by proofing with β-glycoside : aglycone ratio decreasing from 4.9 to

0.2 after proofing for 4 hrs at 48 oC. A good correlation (R2=0.81) between the changes

in β-glucosidase activity and isoflavone aglycones was found. After proofing at 48 oC for one hour, β-glucosidase activity increased about 84%, and no β-glucosidase activity was

detectable in the dough after 4 hrs of proofing. There was little enzymatic hydrolysis 130 during the last two hours of proofing, therefore proofing for 2 hours at 48 was optimal for isoflavone aglycone production (615nmol/g).

Figure 42 shows the changes in isoflavones and β-glucosidase activity in soy

dough at a proofing temperature of 22 °C. β-glucosidase activity increased about 65.3% during the 1st hour of proofing but remained unchanged through the 2nd to the 4th proofing

hours. The isoflavone aglycones increased by 2 fold almost linearly (297nmol/g), while

isoflavone β-glucosides decreased about 5.5% during the 4hrs of proofing. These changes were reflected in the isoflavone β-glucosides : aglycone ratio that decreased

from 4.9 to 2.3.

Figure 43 shows the changes in isoflavones and β-glucosidase activity in soy

dough at a proofing temperature of 32 °C. β-Glucosidase activity increased about 43.3% during the first two hours of proofing and then decreased during the last two hours of proofing. Isoflavone aglycones increased about 3 fold (383nmol/g) in the first two proofing hours and an additional 2 fold (612nmol/g) in the next two proofing hours.

During the four-hour proofing at 32 °C, isoflavone β-glucosides decreased about 66.7%

linearly, and the isoflavone β-glucoside : aglycone ratio decreased from 4.9 to 0.3.

For the three proofing temperatures, the increased β-glucosidase activity in soy

bread dough was paralleled by a decrease in isoflavone β-glucoside : aglycone ratio. The increase in isoflavone aglycones may also be due to enzymatic hydrolysis of isoflavone

malonylglucosides during prolonged proofing. For example, after two hours of proofing at

48°C, isoflavone β-glucosides remained stable while isoflavone aglycones continued to increase during the next two hours of proofing along with a significant decrease in 131 isoflavone malonylglucosides (Figure 41). Interestingly, the isoflavone malonylglucosides showed a steady decrease throughout proofing for four hours at 32°C (Figure 43) but occurred in two stages at 48°C (Figure 41), indicating the stability of isoflavone

maolnylglucosides during proofing is influenced by proofing temperature. This may be

also contributed from a difference in β-glucosidase activity between the enzyme of soybean origin and an exogeneous source (Pandjaitan et al, 2000; Tsangalis et al, 2003; Mastuura and Obata, 1993). The decrease in enzyme activity after prolonged proofing may be due to the accumulation of fermentation products or enzymatic reaction products, i.e. acids and alcohols (Mateo and Stefano 1997), or the exhaustion of nutrients. β-Glucosidase activity became non-detectable after four-hour proofing at 48°C suggesting a complete loss of

enzymatic activity after prolonged proofing at this temperature.

Among all combinations of proofing temperatures and times studied, β-glucosidase

reached its maximal activity (51.0U) in the shortest time (1 hour) during proofing at 48°C.

At 22 and 32°C, the time required to reach the maximal activity of this enzyme were 1 and

2 hours, respectively, but the activities reached was below 51.0 U. The rate of isoflavone

aglycone production in soy bread slowed significantly (p<0.05) after 2 hours of proofing at

48°C. These results in soy bread dough confirm the observation by Mastuur and Obata

(1993) in soy milk that β-glucosidase activity is time and temperature dependent.

Therefore, proofing at 48°C for 2 hour was considered the optimal condition for high isoflavone aglycone production in soy bread preparation.

In conclusion, β-glucosidase activity in soy bread dough was dependent on the

proofing temperature and duration. Both isoflavone β-glucosides and malonylglucosides were used as substrates for β-glucosidase to produce isoflavone aglycones. However, 132

1400 250 h % , g y u t 1200 i

o malonylglucosides

200 v i d t

) c d a a %

1000 ) e 0 e

r 150 0 s m b 1 a

β-glucosidase r

y 800 d s i

g activity o a / s

l s

100 l

o o

aglycones o c n

600 r i m

t u l n s n ( g e o 50 - n c β β 400 β β

( o e v β−glucosides v a i l t

f 200 0 a o l s acetylglucosides e I 0 R 0 1 2 3 4

Proofing time (hour) at 48oC

Figure 41. Changes in isoflavones and β-glucosidase activity during proofing at 48oC for 1, 2, 3, and 4 hours. The β-glucosidase activity in soy bread dough before proofing was used as a control. Solid lines correspond to the isoflavone content in soy bread doughs. Dotted line corresponds to the relative β-glucosidase activity in these doughs. Values are means ± SD of three independent determinations.

133 250 % h

1400 , g y u t i o 1200 200 v i d malonylglycosides t

) c d a % a

) 0

e 1000 150 e r β-glucosidase 0 s m b 1 a

a activity r y d s

800 i g o a / s

s 100 β-glucosides l

o o o n c r i

600 t m

u l n s n ( g e 50 o - n β β β β C

400 aglycones ( o e v v a i l

0 t f 200 a o l s e

I acetylglucosides 0 R 0 1 2 3 4 Proofing time (hour) at 22oC

Figure 42. Changes in isoflavones and β-glucosidase activity during proofing at 22 oC for 1, 2, 3, and 4 hours. The β-glucosidase activity in soy bread dough before proofing was used as a control. Solid lines correspond to the isoflavone content in soy bread doughs. Dotted line corresponds to the relative β-glucosidase activity in these doughs. Values are means ± SD of three independent determinations.

134 1400 250 %

h , g y t u 1200 malonylglucosides i o 200 v i d t

) c d a a

1000 % ) e e 150 0 r s 0 m b a 1

r d y 800 s β-glucosidase i g o a / s

s 100

activity l o

o o c n

600 r i u m

t l s n n g (

e β aglycones 50 β-glucosides o − − − − n c

400 β β β β (

o e v v a i l 0 t f 200 a o l s e

I acetylglucosides

0 R 0 1 2 3 4 o Proofing time (hour) at 32 C

Figure 43. Changes in isoflavones and β-glucosidase activity during proofing at 32 oC for 1, 2, 3, and 4 hours. The β-glucosidase activity in soy bread dough before proofing was used as a control. Solid lines correspond to the isoflavone content in soy bread doughs. Dotted line corresponds to the relative β-glucosidase activity in these doughs. Values are means ± SD of three independent determinations.

135 isoflavone β-glucosides were preferred to isoflavone malonylglucosides in this enzyme-

substrate reaction. Therefore, the production of isoflavone aglycones could be manipulated

by controlling the duration and temperature of proofing. The proofing temperature at 48 °C

for two hours was found to be optimal in soy bread among the proofing condition studied.

Effect of almond addition on isoflavones in soy bread

To further improve the isoflavone profile in the soy bread, a β-glucosidase-rich

natural food, almond, was incorporated at 2.5, 5.0, 7.5 and 10% levels of the dry bread

ingredients. The effects of almond addition on isoflavone content and composition and

the β-glucosidase activity of soy bread were studied.

Almond addition increased β-glucosidase activity in soy bread dough

dramatically (Table 16). After proofing, the increase in the β-glucosidase activity was

found to be in the range of 16 to 30 times upon almond addition of 2.5% to 10.0%

(Figure 44). Isoflavones content was found to be stable through proofing and baking

(Figure 45). After one hour proofing at 48 oC, the aglycone levels of soy bread

containing 2.5, 5.0, 7.5, and 10% almond were 488, 615, 648, and 664.9 nmol/gram (total weight), respectively, while it was 316 nmol/g in soy bread without almond but proofed under the same temperature and time conditions (Table 16). After baking at 160 oC for

50 min, β-glucosidase activity was not detected. Baking had a similar effect on the

changes in isoflavones for both breads with and without almonds (Figure 46). The

increase in isoflavone aglycones was in the range of 6-9% for soy breads containing these

four levels of almond. Therefore, almond addition increased isoflavone aglycones in soy

136 bread significantly, and the largest increase was in the proofing stage. Five percent almond addition was found to be an effective and economic level to enhance isoflavone aglycones in the soy bread formula.

To further study the effect of almond addition on total isoflavones in soy bread during proofing, the isoflavone β-glycoside : aglycone ratio in soy bread containing different level of almond was calculated (Table 16). The increases in isoflavone aglycones during proofing corresponded to a decrease in isoflavone β-glucosides. The

isoflavone β-glycoside : aglycone ratio after one hour proofing at 48 oC was 1.5 for soy bread without almond. Soy bread containing almond lost about 74% of its isoflavone β- glycosides during proofing after addition of 5% almond. After almond addition, the ratio decreased to 0.3 for soy bread containing 5% almond indicating an almost complete liberation of isoflavone aglycones from their β-glucosides. Increasing the almond

addition to 10% only decreased this ratio to 0.2, further confirming that 5 % rather than

10% almond addition was an optimal level to enhance isoflavone aglycones in the soy bread formula.

In conclusion, isoflavones were stable through bread preparation conditions.

Among the stages of bread preparation, the proofing stage was key in the production of isoflavone aglycones in soy bread. Proofing temperature and duration were important controlling factors in regulating the amount of isoflavone aglycones released with proofing at 48 oC for 2 hours being optimal for the maximum isoflavone aglycone production. Almond added to soy bread formula increased β-glucosidase activity

dramatically without degrading isoflavones during bread preparation. An almond level of

5% was found to be optimal for isoflavone aglycone production. 137

1 3 8 4 6 11 12 7 9 5 10 pure isoflavone 2 standard ) m

n 0

6 soy bread dough 2

( before proofing

t i

n soy bread dough w/o almond after U

proofing e

c soy bread dough w. almond n

a after proofing b r o s

b soy bread w/o almond A after baking

soy bread w. almond after baking

Retention time (Minutes)

Figure 44. HPLC chromatogram of isoflavone authentic standards, soy bread with and without almond during bread preparation. 1: daidzin (β-glucoside), 2: glycitin (β-glucoside), 3: genistin (β-glucoside), 4: malonyldaidzin, 5: malonylglyctin, 6: acetyldaidzin, 7: acetylglycitin, 8: malonylgenistin, 9: daidzein (aglycone), 10: glyctein (aglycone), 11: acetylgenistin, 12: genistein (aglycone)

138

Isoflavones % Almond Addition

0 2.5 5.0 7.5 10.0

Aglycones 316±8 488±18 616±27 648±20 665±10

β-Glucosides 469±17 390±10 160±11 130±6 109±6

Malonylglucosides 1240±18 1134±20 1210±27 1152±23 1094±26

Acetylglucosides 185±6 178±4 177±12 170±6 162±3

Total 2210 2190 2160 2100 2029 a. Values are means ± SD of three independent determinations.

Table 16. Isoflavone content and composition (nmol/gram, total weight) in soy bread dough after proofing containing 0, 2.5, 5.0, 7.5, and 10.0% almond.

139

Proofing Almond β-glucosidase Isoflavone Stage activity β-glucosides to aglycone (%, w/w) (µmol/ml‡ ratio min) Before proofing 0 28±0.6 4.9 After 0 51±1.0 1.5 proofing 2.5 800±12 0.8

5.0 1230±33 0.3

7.5 1420±20 0.2

10.0 1540±46 0.2 a. Values are means ± SD of three independent determinations.

Table 17. Isoflavone β-glucoside: aglycone ratio and β-glucosidase activity in soy bread dough with 0, 2.5, 5.0, 7.5, and 10% almond (w/w, flour basis) after proofing at 48 oC for 1 hour.

140

800 10000

) - - - - ) e

(

d n 600 1000 y a ) o t i e c n r i v y i l b t

m g

c y l a a

m e s e 400 100 /

l , n s o a o g

/ v l d m i a o l s m f ( o m o c n

s 200 10 ( I u l

g - β β β β

0 1 before 0% 2.5% 5.0% 7.5% 10.0% proofing after proofing

Fiure 45. Isoflavone aglyone (nmol/gram, total weight) (bar graph) and β- glucosidase activity (dotted line) in soy bread containing almond after proofing at 48oC for 1 hour as compared to soy bread without almond before and after proofing.

141

Figure 46. Isoflavone content and composition in soy bread containing 5.0% almond during bread making (before proofing, after proofing, and after baking) as compared to soy bread without almond (------).

142 Stability of isoflavones during storage

Table 18 shows the isoflavone content and profile in fresh soy bread and soy bread stored for 14 days at room tmperature. No significant changes were found between the isoflavone content or profile in fresh soy bread and soy bread stored for 14 days,

showing good stability of isoflavones in soy bread under room temperature. This result may also indicate a complete deactivation of hydrolyzing enzymes in soy bread during

baking. Additionally, the changes in the water fraction found in the soy breads during

storage (increase in “freezable”water, decrease in “unfreezable” water) did not impact the

stability of these isoflavones indicating that soy bread consumed up to two weeks after

baking would deliver the same isoflavones.

143 Isoflavone Soy bread Soy bread (day 0) (day 14) Nmol/gram, day basis Daidzin 432±20 443±15 Genistin 935±8 930±7 Glycitin 139±5 141±6 β-glucosides 1506 1514 Malonyldaidzin 404±1 400±5 Malonylgenistin 925±8 922±10 Malonylglycitin 145±0 141±3 Malonylglucosides 1474 1463 Acetyldaidzin 131±1 130±10 Acetylgenistin 189±3 187±6 Acetylglycitin 80±4 78±8 Acetylglucosides 400 395 Daidzein 259±3 254±10 Genstein 345±9 340±13 Glycistein 7±1 10±3 Aglycones 611 604 Total 3991 3976 a. Values are means ± SD of three independent determinations.

Table 18. Isoflavone content and composition (nmol/gram, dry basis) in fresh (day 0) and stored soy bread (day 14) at room temperature.

144 CHAPTER 6

CONCLUSIONS

Aim 1: The effect of soy on the physico-chemical properties of fresh bread

Soy was added to a wheat bread (control) formulation at 60% flour basis to produce soy bread. Soy addition significantly changed the physico-chemical properties of fresh bread.

Upon soy addition, a decrease in bread loaf volume (-16.7%) was observed along with an increase in weight (+44%). Smaller volume and heavier weight of soy bread indicated a higher density (+70%) as compared to wheat bread. Fresh soy bread was found to be more than 5 times firmer than fresh wheat bread by compression test. This increase may be attributed to the lack of gluten network formation and smaller air cell structure of bread after soy addition as confirmed by the higher density of the soy bread.

Total moisture content in soy bread (44.7%) was higher than in wheat bread (39.9%) due to the additional water in soy bread formulation required to adequately hydrate the soy protein. Soy bread also had a greater (+33.3%) of “freezable” water content that was more easily removed but no difference in “unfreezable” water as compared to wheat bread indicating a more mobile water pool in soy bread. This difference in water

145 association may be due to the proteins since gluten in wheat bread was found to have a stronger interaction with water as compared to soy protein in soy bread. On a molecular level, the more mobile liquid protons decreased while the less mobile liquid protons increased upon addition of soy to bread which may correspond to the impact of soy protein protons on the total proton pool. An additional thermal transition at a lower temperature was visible for soy bread which may impact low temperature storage.

Aim 2: The effect of soy addition on the physico-chemical properties of bread

during storage

Soy bread was stored at room temperature for up to 14 days. Wheat bread without soy served as control.

Soy added to bread was found to have a significant impact on the physico-

chemical properties of bread during storage. Both wheat and soy bread firmed over a 14-

day period of storage. However, soy bread firmed at a lower rate (1.6 times) than wheat

bread, whose firmness increased by 6.7 times. The lower rate of firming in soy bread

may be attributed to various factors: 1). Water distribution in the soy bread that favored easily removed bulk water pool that can act as a plasticizer throughout storage and

maintain heterogeneity of the product, 2). A lack of recrystallized amylopectin, and 3).

No change in mobility of liquid-like protons and an increase in mobility of solid-like

146 protons. Therefore, the addition of large amounts of soy (60%) significantly reduces the

parameters associated with bread staling, i.e. lower degree of amylopectin recrystallization, less moisture loss, and lower firming rate, during storage as compared to wheat bread.

Aim 3: The effect of processing and storage on isoflavone profile

of soy bread

Total isoflavones were found to be stable during bread making, although their profile was largely altered.

Both proofing and baking affected isoflavone profile in soy bread. The proofing stage in bread preparation was key in the production of isoflavone aglycones in bread dough through β-glucosidase activity. The β-glucosidase activity in soy bread dough was dependent on the proofing temperature and duration, with 48 °C for two hours being optimal for aglycone production. Both isoflavone β-glucosides and malonylglucosides were used as substrates for β-glucosidase to produce isoflavone aglycones. However, isoflavone β-glucosides were found preferred to isoflavone malonylglucosides in this enzyme-substrate reaction. Low moisture and high temperature also affected the isoflavone increasing acetylglucosides at the expense of malonylglucosides with no change in aglycone fraction.

Almond addition increased β-glucosidase activity and isoflavone aglycones in soy bread dough significantly, and the largest increase was in the proofing stage. Five percent almond addition was found to be an effective and economic level to enhance

147 isoflavone aglycones in the soy bread formula. These findings provide a food process to potentially alter the level of bioactive components and bioavailability of isoflavones in soy bread and other soy foods.

Future studies need to focus on microbial stability of the soy bread as well as the exact mode of action of soy in a bread system.

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