Physicochemical Characterization and Isoflavone

Home , Glycitin, Saccharomyces cerevisiae

PHYSICOCHEMICAL CHARACTERIZATION AND ISOFLAVONE

PROFILING OF SOURDOUGH SOY BREAD

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of

Science in the Graduate School of The Ohio State University

By GABRIELLE YEZBICK, B.S. Graduate Program in Food Science and Technology

The Ohio State University 2012

Master’s Examination Committee: Dr. Yael Vodovotz, Advisor Dr. Steven Schwartz Dr. Zhongtang Yu

ABSTRACT

Soy isoflavones have been correlated with risk reduction for various diseases, such as hormone-related cancers and cardiovascular disease.

Consumption of soy by the Western population, however, is substantially less than that observed in Asian countries. Given this, a soy-supplemented wheat bread was developed as a familiar delivery vehicle of these health-promoting compounds to the Western population. Only the deglucosylated form of isoflavones is believed to be readily absorbed by the body and, therefore, almond powder, containing the enzyme responsible for this deglucosylation (β- glucosidase), was added to the soy bread to increase the proportion of isoflavone aglycones in the product. Almond, however, is considered a major allergen and requires declaration as such. Therefore, the primary objective of this study was to utilize sourdough fermentation to match or enhance the conversion of isoflavone β-glucosides to aglycones observed upon the addition of almond powder and, thereby, eliminate the need for inclusion of allergenic substances. It was hypothesized that sourdough fermentation would result in an increase in the proportion of aglycones in the final product without negatively impacting the physical and thermal properties, as well as overall acceptability, of the regular

(yeast-leavened) soy bread product.

The physical and thermal properties of sourdough and yeast-leavened soy dough, and fresh and stored sourdough and yeast-leavened (regular) soy bread, were evaluated to determine the effect of sourdough fermentation on the overall loaf quality of soy bread. Sourdough fermentation of soy dough produced a less extensible dough, smaller specific loaf volume, harder bread crumb, and lighter crust color compared to the regular soy bread. When analyzed after 10 days of room temperature storage, the sourdough soy bread was more shelf-stable than the regular soy bread, which displayed signs of mold and bacterial growth.

Sensory analysis revealed participants’ greater overall liking for the regular soy bread compared to the sourdough soy bread and, consequently, indicated preference for this soy bread variety. Participants described the sourdough soy bread as being “very sour”, which may have been a reflection of the overall participant preference for whole wheat and white bread varieties, as indicated in a demographic questionnaire.

The isoflavone profiles of various dough formulations were evaluated during proofing by RP-HPLC. The greatest proportional increase of isoflavone aglycones was observed in soy dough containing Lactobacillus brevis and

Lactobacillus plantarum, suggesting β-glucosidase activity of 1 or both of the bacterial strains. Furthermore, the addition of Saccharomyces cerevisiae to the lactic acid bacteria-containing preferment appeared to hinder the deglucosylation of isoflavone simple β-glucosides.

Regardless, sourdough fermentation by lactobacilli and Saccharomyces cerevisiae successfully resulted in approximately double the proportion of

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isoflavone aglycones observed in the regular soy bread product, with regular soy bread and sourdough soy bread containing 17% and 32% of isoflavones in the aglycone form, respectively. Additionally, the isoflavone profile observed in fresh sourdough soy bread remained stable over 10 days of room temperature storage. Given this, sourdough soy bread may provide a shelf-stable and sustainable delivery of bioavailable isoflavone aglycones for use in human clinical trials.

Dedicated to my continuously supportive and loving parents, Gary and Denise.

ACKNOWLEDGEMENTS

Thank you to Dr. Yael Vodovotz and all individuals in the Vodovotz lab, who provided the support and guidance that enabled me to complete this work. I would like to extend a special thank you to Jennifer Ahn-Jarvis, who shared her expansive knowledge on isoflavone chemistry and gave up her own valuable time to enhance my education here at The Ohio State University. I would, lastly, like to thank all the faculty and staff, especially Paul Courtright and Melody

Leidheiser, whose daily efforts help shape the experiences of all the graduate students within the department.

VITA

May 8, 1987……………………………… Born – Ypsilanti, MI August 2005 – May 2009………………. B.S., Nutritional Sciences, Michigan State University June 2010 – present……………………. Graduate Research Associate, The Ohio State University

FIELDS OF STUDY

Major Field: Food Science and Technology

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TABLE OF CONTENTS

Page

Abstract...... ii

Dedication...... v

Acknowledgments...... vi

Vita...... vii

List of Figures ...... xii

List of Tables ...... xv

List of Abbreviations ...... xvii

Chapters:

1. Introduction ...... 1

2. Statement of the Problem ...... 6

3. Literature Review ...... 9

3.1 Soy Isoflavones ...... 9

3.1.1 Health Benefits ...... 9

3.1.2 Bioavailability ...... 13

3.1.3 Effects of processing on the soy isoflavone profile...... 18

3.1.4 Soy isoflavone HPLC analysis...... 21

3.2 Sourdough Fermentation ...... 22

3.2.1 Microbiotic associations...... 22

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3.2.2 Flavor and aroma development ...... 25

3.2.3 Nutritional benefits ...... 27

3.2.4 Shelf-life extension ...... 28

3.2.5 Beta-glucosidase activity ...... 29

3.2.6 Structural and textural changes ...... 31

3.3 Thermal analysis of bread...... 34

4. Materials and Methods...... 37

4.1 Dough formulation and bread production...... 37

4.1.1 Regular soy bread ...... 38

4.1.2 Sourdough soy bread ...... 39

4.1.3 Dough formulations for HPLC analysis...... 41

4.2 Isoflavone extraction and analysis ...... 43

4.2.1 Isoflavone extraction...... 43

4.2.2 RP-HPLC isoflavone analysis...... 44

4.2.3 Isoflavone quantification ...... 45

4.3 Physical properties...... 45

4.3.1 Dough extension...... 45

4.3.2 Bread crumb hardness ...... 46

4.3.3 Specific loaf volume...... 47

4.3.4 Crust color ...... 47

4.4 Thermal analysis ...... 48

4.4.1 Thermogravimetric analysis (TGA) ...... 48

4.4.2 Differential scanning calorimetry (DSC)...... 49

4.5 Sensory analysis ...... 50

5. Results and Discussion...... 52

5.1 Aim 1: Quantitative and qualitative assessment of the isoflavone profile of

fermenting sourdough, and fresh and stored sourdough soy bread and

regular soy bread by RP-HPLC ...... 52

5.1.1 Isoflavone profile of fermenting soy dough ...... 52

5.1.2 Isoflavone analysis of fresh and stored R-SB and SD-SB...... 60

5.2 Aim 2: Physical and thermal properties of sourdough and yeast-leavened

dough, and fresh and stored sourdough soy bread and regular soy

bread ...... 64

5.2.1 Dough extensibility ...... 64

5.2.2 Specific loaf volume...... 67

5.2.3 Crust color ...... 68

5.2.4 Bread crumb hardness ...... 70

5.2.5 Water properties of dough ...... 74

5.2.6 Thermal analysis of fresh and stored bread ...... 77

5.3 Aim 3: Overall acceptability of sourdough soy bread and regular soy

bread, and preference between the two, as determined by sensory

analysis...... 84

6. Conclusions...... 86

6.1 Aim 1: Quantitative and qualitative assessment of the isoflavone profile of

fermenting sourdough, and fresh and stored sourdough soy bread and

regular soy bread by RP-HPLC ...... 86

6.2 Aim 2: Physical and thermal properties of sourdough and yeast-leavened

dough, and fresh and stored sourdough soy bread and regular soy

bread ...... 87

6.3 Aim 3: Overall acceptability of sourdough soy bread and regular soy

bread, and preference between the two, as determined by sensory

analysis...... 88

References ...... 90

Appendix A: Isoflavone aglycone and simple β-glucoside standard curves acquired with Agilent HPLC system...... 97

Appendix B: Approval letter for exemption from IRB review...... 101

Appendix C: Sensory analysis recruitment letter...... 103

Appendix D: Sensory analysis informed consent form ...... 105

Appendix E: Sensory analysis electronic questionnaire ...... 109

Appendix F: Sensory analysis preference test ballot...... 113

LIST OF FIGURES

Figure Page

1.1 Chemical structures for (a) daidzein (aglycone) (b) daidzin (simple β-

glucoside) (c) 6”-O-acetyldaidzin (d) 6”-O-malonyldaidzin...... 2

3.1 Chemical structures for 17-β-estradiol and the isoflavone aglycones,

daidzein, genistein, and glycitein ...... 10

3.2 Estrogen receptor (ER) subtype (α and/or β) distribution in males and

females ...... 11

3.3 Chemical structure of the isoflavone glucosides...... 14

3.4 Diagram depicting isoflavone metabolism and enteric recycling ...... 17

3.5 Interconversion of the isoflavone forms, using daidzin as an example...... 19

3.6 Carbohydrate metabolism of the obligately heterofermentative Lactobacillus

sanfranciscensis ...... 24

3.7 Extensogram displaying the 5 viscoelastic stages of uniaxial dough

extension...... 34

4.1 Image of optimally active, liquid sourdough preferment...... 40

4.2 Commission Internationale d’Eclairage color model depicting values of

lightness (L), and chromatic components, a* and b* ...... 48

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5.1 Overlay of chromatograms displaying the isoflavone profile of SD-SD at 0,

9, and 24 h of fermentation at 30 °C. Dashed lines highlight the conversion

of the isoflavone simple β-glucosides, daidzin and genistin, to their

corresponding aglycone forms (courtesy of Jennifer Ahn-Jarvis)...... 53

5.2 Isoflavone profiles (aglycones and various glucosides given as % of total

isoflavone concentration) for C-SD, Y-SD, LAB-SD, and SD-SD at the

beginning (T0) and end (T9) of fermentation at 30 ºC...... 57

5.3 Total isoflavone concentration (nmol/g of dough) for SD-SD and Y-SD over

24 h of fermentation at 30 °C...... 59

5.4 Isoflavone concentrations (nmol/g) of aglycones and various conjugates of

SD-SD over 24 h of fermentation at 30 °C ...... 60

5.5 Isoflavone profiles (aglycones and various glucosides given as % of total

isoflavone concentration) for R-SB and SD-SB...... 62

5.6 Overlay of chromatograms (obtained from Agilent HPLC) displaying the

isoflavone profile of fresh and stored SD-SB...... 63

5.7 Extensograms displaying the load (N) versus hook extension (mm) for soy-

supplemented wheat dough obtained by (A) sourdough fermentation and (B)

fermentation with traditional baker’s yeast ...... 65

5.8 Images displaying the crust color of SD-SB and R-SB...... 70

5.9 Image displaying mold growth on RSB after 10 days of storage...... 72

5.10 Images of slices of (A) fresh R-SB, (B) stored R-SB, (C) fresh SD-SB, and

(D) stored SD-SB...... 73

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5.11 Compression curves displaying the compressive load (N) versus time (s) for

(A) fresh SD-SB and (B) stored SD-SB ...... 74

5.12 Typical TGA thermogram obtained for SD-SD, displaying sample weight

loss (%) and the derivative of weight loss (%/ºC) with increasing

temperature (ºC)...... 76

5.13 Thermogram displaying the endothermic peak associated with ice melting

for R-SD and SD-SD ...... 77

5.14 Thermogram displaying the endothermic peak associated with melting of

amylose-lipid complexes in fresh SD-SB and R-SB ...... 81

5.15 DSC thermogram displaying the transition associated with the melting of

amylopectin crystals in fresh and stored SD-SB ...... 82

5.16 Moisture contents and FW and UFW populations (% of total sample weight)

for SD-SD and fresh and stored SD-SB ...... 83

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LIST OF TABLES

Table Page

4.1 Ingredients and formulations (%) for regular soy bread (R-SB) and

sourdough soy bread (SD-SB) ...... 37

4.2 Soy bread ingredients and their corresponding manufacturers...... 38

4.3 Formulation (%) of ingredients for control soy dough (C-SD), yeast soy

dough (Y-SD), lactic acid bacteria soy dough (LAB-SD), and sourdough soy

dough (SD-SD) ...... 41

4.4 Mobile phase elution gradient for isoflavone analysis by RP-HPLC...... 44

5.1 Isoflavone profile (aglycones and various conjugates given as % of total

isoflavone concentration) for SM ...... 54

5.2 Isoflavone profiles (aglycones and various conjugates given as % of total

isoflavone concentration) and dough pH for C-SD, Y-SD, LAB-SD, and SD-

SD at the beginning (T0) and end (T9) of fermentation at 30 ºC...... 56

5.3 Isoflavone composition (aglycones and various glucosides given in nmol/g)

of R-SB and SD-SB, with significance established at p < 0.05...... 61

5.4 Maximum load (N) and extension at maximum load (mm) for R-SD and SD-

SD...... 66

5.5 Specific loaf volumes (cm3/g) for R-SB and SD-SB, with significance

established at p < 0.05 ...... 67

5.6 Values of lightness (L) and the chromatic components a* and b* for the

crust of SD-SB and R-SB, as well as the corresponding dough pH prior to

baking ...... 69

5.7 Bread crumb hardness (N) for fresh (day 1) and stored (day 10) SD-SB and

R-SB, with significance established at p < 0.05...... 71

5.8 Moisture content (%) and water distribution properties for R-SD and SD-SD,

as determined by TGA and DSC ...... 75

5.9 Moisture and thermal properties for fresh and stored R-SB and SD-SB, with

significance established at p < 0.05...... 79

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LIST OF ABBREVIATIONS

Abbreviation Meaning

C-SD Control soy dough (no fermenting

microorganisms)

DAD Diode array detection

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

EPS Exopolysaccharide

ER Estrogen receptor

FQ Fermentation quotient

FW “Freezable” water

HPLC High performance liquid chromatography

IRB Institutional review board

Km Michaelis constant

LAB Lactic acid bacteria

LAB-SD Lactic acid bacteria soy dough

LPH Lactase phlorizin hydrolase

NF-κB Nuclear factor kappa-light-chain-enhancer of

activated B cells

PDA Photodiode array

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pNPG 4-nitrophenyl-β-D-glucopyranoside

PSA Prostate-specific antigen

Rmax Maximum resistance

RP Reverse-phase

R-SB Regular soy bread (yeast-fermented)

R-SD Regular soy dough (yeast-fermented)

SD-SB Sourdough soy bread

SD-SD Soy sourdough

T0 Proofing time-point 0 (h)

T9 Proofing time-point 9 (h)

TGA Thermogravimetric analysis

THB Trihydroxydeoxybenzoin

UFW “Unfreezable” water

UV Ultraviolet light

Vmax Maximum velocity

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CHAPTER 1: INTRODUCTION

Soybeans were first established as a commercial crop in the United States in the 1920’s for the production of soybean oil, with the by-product of the manufacturing process, defatted soybean meal, being utilized as feed for livestock and poultry (Erdman and Fordyce 1989). Since the introduction of soy to the Western diet, epidemiological studies have shown significant differences in the incidence of various diseases amongst different ethnic groups, which are believed to be in part due to varying dietary habits between these groups. These epidemiological observations have lead to research suggesting correlations between soy consumption and risk reduction for various cancers, chronic inflammatory diseases, coronary heart disease, osteoporosis, and menopausal symptoms (Lee and others 2004; Pyo and others 2005). In 1999, the Food and

Drug Administration approved a health claim correlating diets low in saturated fat and cholesterol that include soy protein with reduced risk for coronary heart disease. That same year, soybeans were planted on 72 million acres, accounting for 27% of the United States total crop area, and resulted in approximately 2.74 billion bushels of soybeans valued at 15 billion dollars

(Friedman and Brandon 2001).

While soybeans contain a number of bioactive constituents that may contribute to the overall health benefits of soyfoods, the primary isoflavones,

genistein and daidzein, have been the most extensively studied due to their estrogenic and non-hormonal functionalities that make them likely modulators of disease states. Glycitein, a soy isoflavone of weaker estrogenic activity, is also present, accounting for approximately 5-10% of the total isoflavone content of soybeans (Song and others 1999). Isoflavones may occur in nature as aglycones (daidzein, genistein, and glycitein), however, they are predominately found as higher molecular weight conjugates, including the simple β-glucosides

(daidzin, genistin, and glycitin), 6”-O-acetylglucosides, and 6”-O- malonylglucosides (Figure 1.1) (Mathias and others 2006). Interconversion between the isoflavone forms may occur from chemical and/or heat induced decarboxylation, or enzymatic hydrolysis of the glucose moiety via β-glucosidase.

Figure 1.1. Chemical structures for (a) daidzein (aglycone) (b) daidzin (simple β- glucoside) (c) 6”-O-acetyldaidzin (d) 6”-O-malonyldaidzin (Lodi 2006).

Research suggests that isoflavone aglycones are absorbed by the intestinal epithelium via passive diffusion, with the more hydrophilic conjugates requiring hydrolysis of the glucose moiety for absorption (Xu and others 1995;

Scalbert and Williamson 2000; Setchell and others 2002). Given this, enzymatic hydrolysis of conjugated isoflavones is required for their absorption, exertion of their physiological effects, and derivation of their purported health benefits. One means of achieving this chemical conformation is by applying food-processing techniques that chemically and/or enzymatically hydrolyze isoflavones to their more bioavailable, aglycone form, prior to their consumption. The effects of various processing techniques on the isoflavone profile of soyfoods has been extensively studied, with fermented soyfood products typically exhibiting a significantly larger aglycone pool compared to non-fermented soyfood products

(Coward and others 1993; Pyo and others 2005; Chien and others 2006). Such observations are generally attributed to enzymatic hydrolysis of isoflavone glucosides by bacterial β-glucosidases. Fermented soyfood products such as miso, soy sauce, and tempeh are frequently consumed in Asian countries, however, these high aglycone foods are, to date, not widely accepted amongst the Western population.

Prior to the introduction of baker’s yeast at the turn of the 20th century, bread was leavened by extended fermentation with autochthonous bacteria and yeast resulting in, what is known today as, sourdough bread (Carnevali and others 2006). Sourdough starter cultures are propagated by back-slopping to form a complex ecosystem of lactic acid bacteria (typically Lactobacillus spp.)

and yeasts, which ferment the available flour carbohydrates to acidified end products. Several strains of lactic acid bacteria isolated from sourdough cultures and other fermented food products have been shown to exhibit β-glucosidase activity and, if applied to soy ingredients, could result in the hydrolysis of isoflavone glucosides to aglycones (Sestelo and others 2004; De Angelis, Gallo, and others 2005; Pyo and others 2005; Di Cagno and others 2010; Michlmayr and others 2010). Additionally, sourdough fermentation has been shown to have several beneficial effects on bread quality, including the generation of favorable flavor and aroma compounds (Gobbetti and others 2005; Carnevali and others

2007; Moroni and others 2009), improvement of gas retention and loaf volume

(Gänzle and others 2007; Lacaze and others 2007), reduction of phytic acid levels resulting in increased mineral bioavailability (Gobbetti and others 2005;

Moroni and others 2009), and shelf-life extension by the generation of antimicrobial compounds, retarding of starch retrogradation, and significantly lower pH due to the formation of acids during the fermentation process (Gobbetti and others 2005; Moroni and others 2009).

Sourdough bread is typically made with either wheat or rye flour, however, the application of sourdough fermentation to a soy-supplemented wheat dough may increase the proportion of bioavailable isoflavones present via bacterial β- glucosidases and, thereby, act as an appropriate delivery vehicle for these health-promoting compounds to the Western population. Given this, the general objective of this study was to increase the proportion of isoflavone aglycones in a

soy-supplemented wheat bread by sourdough fermentation, while maintaining the physical properties and overall acceptability of the product.

CHAPTER 2: STATEMENT OF THE PROBLEM

A wheat bread supplemented with soy flour was developed at The Ohio

State University in 2001 as an appropriate delivery vehicle of soy and its bioactive isoflavones to the Western population. This product is now intended for human clinical trials, however, only a small portion (~15%) of the isoflavones in the soy bread are present as the bioavailable aglycones, daidzein, genistein, and glycitein. In order to increase the proportion of bioavailable isoflavones, a soy bread variant supplemented with almond powder (5% w/w) was developed.

Almonds are naturally rich in β-glucosidase activity, and the supplementation of almond powder into the soy bread product successfully resulted in double the amount of isoflavone aglycones (~30%) (Lodi 2006). Almonds, however, are considered a major allergen and their inclusion into food products requires declaration as such. Additionally, rigorous cleaning of manufacturing equipment is required to remove allergen contaminants between product lines, and can be a costly, labor-intensive, and time-consuming process for commercial bakeries.

The fermentation of soy ingredients has shown to significantly increase the amount of bioavailable isoflavone aglycones in various soyfoods, and is frequently attributed to β-glucosidase activity of the fermenting microorganisms

(Coward and others 1993; Pyo and others 2005; Chien and others 2006). Lactic acid bacteria, particularly Lactobacillus spp., are the fermenting microorganisms

most frequently associated with sourdough fermentation, and several strains have been shown to exhibit β-glucosidase activity (Sestelo and others 2004; De

Angelis, Gallo, and others 2005; Pyo and others 2005; Di Cagno and others

2010; Michlmayr and others 2010). Additionally, sourdough fermentation has been shown to benefit bread quality by flavor and aroma enhancement, improvement of gas retention and loaf volume, increased mineral bioavailability, and shelf-life extension. Given this, the overall objective of this study was to utilize sourdough fermentation to match or enhance the conversion of isoflavone

β-glucosides to aglycones observed upon the addition of almond powder, without negatively impacting the physical and thermal properties, as well as overall acceptability, of the regular (yeast-leavened) soy bread product. The specific objectives of this study include:

Aim 1: To quantitatively and qualitatively assess the isoflavone profile of the sourdough throughout the fermentation process, and in fresh and stored sourdough soy bread and regular soy bread by RP-HPLC analysis.

• Hypotheses: Sourdough fermentation will result in an increasing amount of

isoflavone aglycones and corresponding decrease in isoflavone simple β-

glucosides with fermentation time, and the isoflavone profile of regular and

sourdough soy breads will remain stable over storage.

Aim 2: To characterize and compare the physical and thermal properties of sourdough and yeast-leavened soy dough, as well as fresh and stored sourdough soy bread and regular soy bread.

• Hypotheses: Sourdough fermentation will not significantly alter the

physical and thermal properties of soy dough and fresh bread, however,

will result in a more shelf-stable product.

Aim 3: To determine the overall acceptability of sourdough soy bread and regular soy bread, and preference between the two, by sensory analysis.

• Hypotheses: The sourdough soy bread will have equal or greater overall

acceptability compared to the regular soy bread, and will be equally

preferred, or preferred more than the regular soy bread.

CHAPTER 3: LITERATURE REVIEW

3.1 Soy Isoflavones

3.1.1 Health benefits

The soy isoflavones daidzein, genistein, and glycitein (Figure 3.1) are non-nutritive phytochemicals that mimic the action of human 17β-estradiol, and their consumption has been correlated with risk reduction for various diseases and illnesses, including various cancers, heart disease, osteoporosis, and menopausal symptoms (Pyo and others 2005). These non-steroidal dietary estrogens preferentially bind the estrogen receptor (ER) subtype β, in contrast to

17β-estradiol, which binds both α and β ER subtypes with high affinity (De

Angelis, Stossi and others 2005). In addition to the varying ligand affinities of the

ER subtypes, differences in their tissue distribution patterns exist (Figure 3.2), providing additional information on the distinct functionality of phytoestrogens compared to steroidal 17β-estradiol. For example, ERβ is highly expressed in the prostate epithelium, ovarian follicles, lung, urogenital tract and intestinal epithelium, while ERα expression is either extremely low or not measurable in these tissues (Weihua and others 2003).

Figure 3.1. Chemical structures for 17-β-estradiol and the isoflavone aglycones, daidzein, genistein, and glycitein (modified from Song and others 1999).

Figure 3.2. Estrogen receptor (ER) subtype (α and/or β) distribution in males and females (Setchell and Cassidy 1999).

Isoflavones also possess a number of non-estrogenic activities, which may further contribute to their numerous health-promoting qualities. Soy isoflavones act as antioxidants due to their phenolic structure and hydroxyl groups that are capable of stabilizing radical oxygen species, thereby reducing oxidative DNA damage. Genistein has, also, been shown to be an inhibitor of protein tyrosine kinase, an inhibitor of prostaglandin synthesis, a regulator of cell proliferation by influencing various growth factors, and acts as a suppressor of metastasis and angiogenesis (Setchell 1998; Swami and others 2009).

Combined, these estrogenic and non-hormonal functionalities make soy isoflavones probable modulators of cancer cell initiation and proliferation in various tissues, such as human prostate cells.

Based on previous findings that genistein inhibits prostate cancer cell growth in culture, and abolishes oxidative stress-induced activation of NF-κB in prostate cancer cell lines, Hussain and others (2003) investigated the in vivo effects of genistein in prostate cancer patients with rising serum prostate-specific antigen (PSA) levels. The 41 study participants consumed 100 mg of soy isoflavone twice daily for a minimum of 3 months and a maximum of 6 months, with the median of supplementation being 5.5 months. Thirty-nine patients were assessed for response and, although there were no sustained decreases in PSA, there was a decrease in the rate of rise of serum PSA (p = 0.01). These observations provided in vivo evidence that the consumption of soy isoflavones may be beneficial for prostate cancer patients.

3.1.2 Bioavailability

Soy isoflavones may exist in nature as aglycones (daidzein, genistein, and glycitein), however, they are predominately found as β-glucosides (daidzin, genistin, and glycitin), in which a glucose moiety is attached to the aglycone structure at position 7 of the A ring (Figure 3.3). The simple β-glucosides may have a malonyl or acetyl group esterified at the 6”-O- of the glucose moiety, forming the higher molecular weight 6”-O-malonylglucosides and 6”-O- acetylglucosides, respectively (Mathias and others 2006). Interconversion between the various isoflavone forms may occur as a result of chemical or enzymatic hydrolysis and, therefore, varying isoflavone profiles are observed in soyfoods that are subjected to different processing techniques. Additionally, the estrogenic and non-hormonal, health-promoting functionalities of soy isoflavones have been attributed to the aglycone forms, primarily genistein and daidzein

(Setchell and others 2002). Given this, numerous studies have been conducted to determine the exact mechanism by which isoflavones are absorbed and reach systemic circulation, which are the necessary prerequisites for tissue distribution and exertion of their physiological effects (Larkin and others 2008).

Figure 3.3. Chemical structure of the isoflavone glucosides (modified from Song and others 1998).

Isoflavone glucosides exhibit lower partition coefficients (indicating a greater degree of hydrophilicity) and have higher molecular weights than their aglycone counterparts; therefore, isoflavone glucosides have a decreased likelihood for passive diffusion across biological membranes and exhibit poor

absorption in the small intestine compared to their aglycone forms (Xu and others

1995; Scalbert and Williamson 2000). Setchell and others (2002) investigated whether intact isoflavone glucosides could be absorbed from the intestinal tract and detected in the peripheral circulation in vivo. In this study, healthy adults ingested either 50 mg of either daidzin or genistin, or 250 mL soymilk consisting of mostly isoflavone glucosides, with plasma being collected at regular intervals before and after ingestion for analysis by electrospray ionization mass spectrometry and gas chromatography-mass spectrometry. Plasma collected 1,

2, and 8 h after ingestion of pure isoflavone glucosides or soymilk predominately containing isoflavone glucosides failed to show measureable traces of either daidzin or genistin, therefore, it was concluded that isoflavone glucosides could not be absorbed intact across the intestinal epithelium of healthy adults.

Izumi and others (2000) conducted a similar study in which the absorption of soy isoflavones was investigated by evaluating total isoflavone concentrations in plasma after ingestion of low (0.11 mmol) and high (1.7 mmol) dosages of either aglycones or glucosides. Following low dose intake, subjects displayed the highest concentration of isoflavones in plasma at 2 h and 4 h post ingestion of isoflavone aglycones and glucosides, respectively. This finding provides evidence for a faster absorption of isoflavone aglycones compared to glucosides.

Additionally, following low dose intake of aglycones, the highest plasma concentration was twice of that for the low dose intake of isoflavone glucosides, suggesting that aglycones are absorbed in greater amounts, in addition to being absorbed faster, when compared to isoflavone glucosides. This finding was even

more pronounced following intake of the high dosage, after which the highest isoflavone concentration in plasma was 5 times higher after aglycone intake compared to glucoside intake.

Glycosidic hydrolysis of isoflavone glucosides, the necessary prerequisite for passive diffusion of isoflavone aglycones into the intestinal epithelium, occurs via the ubiquitous enzyme β-glucosidase, which cleaves β 1,4-glycosidic linkages. β-glucosidase activity may be expressed by the food (if endogenous to the food or added during processing), in the cells of the intestinal epithelium, or by colonic bacteria (Scalbert and Williamson 2000). The mammalian β- glucosidases that have been identified, include a cytosolic β-glucosidase enzyme present in the small intestine, liver, and kidney, as well as a membrane-bound lactase phlorizin hydrolase (LPH) enzyme, which is present on the luminal side of the brush border of the small intestine and capable of hydrolyzing isoflavone glucosides to their more hydrophobic, aglycone forms (Day and others 2000;

Larkin and others 2008). Isoflavone glucosides that are not deglucosylated in the small intestine will pass to the colon, where bacterial β-glucosidases may hydrolyze the isoflavone glucosides to their aglycone forms. Following initial absorption, soy isoflavones undergo enterohepatic circulation where liver glucuronosyl-transferases and sulfotransferases produce glucuronide and sulphate conjugates, respectively, that can be transported to tissue via the systemic circulation for exertion of their physiological effects (Figure 3.4) (Xu and others 1995; Larkin and others 2008).

Figure 3.4. Diagram depicting isoflavone metabolism and enteric recycling (Larkin and others 2008).

3.1.3 Effects of processing on the soy isoflavone profile

In unprocessed soybeans, isoflavones predominately occur as high molecular weight malonylglucosides (Coward and others 1998); however, interconversion between the various isoflavone forms may result from chemical and/or heat induced decarboxylation or enzymatic hydrolysis of the glucose moiety (Figure 3.5). Given this, varying isoflavone profiles are observed in soyfoods that are subjected to different processing techniques. Coward and others (1993) investigated the isoflavone content of different soy food products, and noted total isoflavone concentrations ranging from 0.1-3.0 mg/g and varying isoflavone profiles. In fermented soy products, such as miso and tempeh, the total isoflavone concentration was similar to that of non-fermented foods, however, the isoflavones were primarily in their aglycone form. This was not the case for non-fermented foods, which almost exclusively contained β-glucoside conjugates. Of the acetyl- and malonyl- glucoside conjugates, 6”-O- malonyldaidzin and 6”-O-malonylgenistin were more prominently found. When stimulated by increased temperatures, however, the malonyl conjugates are subject to decarboxylation to their corresponding 6”-O-acetylglucosidic forms.

This was noted in the isoflavone profile of toasted soy flakes. Wang and Murphy

(1996) similarly found a decrease in malonyl conjugates, and a concomitant increase in the respective acetyl conjugates, during the soaking and cooking of tempeh, tofu and soymilk. After the fermentation of tempeh, a significant increase in the aglycone concentration occurred, which the author attributed to fungal enzymatic activity.

Figure 3.5. Interconversion of the isoflavone forms, using daidzin as an example (Riedl and others 2005).

More recently, Mathias and others (2006) investigated the effects of heat and pH on the conjugated forms of genistin and daidzin. The conjugated glucosides were exposed to temperatures of 25, 80 and 100 °C and to acidic (pH

= 2), neutral (pH = 7) and alkaline (pH = 10) conditions. The variance in decarboxylation and degradation rates of the conjugated glucosides was attributed to the chemical structures of the isomers. Malonyl conjugates, which have an additional carboxyl group as compared to acetyl conjugates, were shown to be considerably more stable, especially under acidic conditions. 19

Regardless of pH, however, the stability of malonyldaidzin decreased as temperature increased. The highest degree of interconversion (65%) of malonyldaidzin occurred at conditions of pH 10 and 100 °C. Acetyldaidzin varied from this slightly, having the greatest amount of interconversion at pH 10 and 80

°C. The conjugated forms of genistin showed a similar trend. Malonylgenistin and acetylgenistin were least stable at pH 10 with increasing interconversions as temperature increased. Genistin conjugates also displayed considerable interconversion in acidic conditions at 100 °C, opposed to daidzin conjugates that were significantly more stable at pH 2.

Pyo and others (2005) observed the effects of fermentation on the isoflavone profile of soymilk. Soymilk was incubated for 48 h at 37 °C with the following 4 strains of lactic acid bacteria (LAB): Lactobacillus plantarum KFRI

00144, Lactobacillus delbrueckii subsp. lactis KFRI 01181, Bifidobacterium breve

K-101, and Bifidobacterium thermophilum KFRI 00748. A control was also incubated under the same conditions without the 4 LAB strains present. After fermentation, the control had a total isoflavone concentration (genistein, daidzein, genistin and daidzin) of 150.3 µg/mL, with glucosides comprising 86% of the total. In the sample incubated with the 4 strains of LAB, however, the total concentration of isoflavones was 160.7 - 167.2 µg/mL with the aglycones, genistein and daidzein) comprising 100% of the total. This conversion correlated to the β-glucosidase activity of the LAB, responsible for the hydrolysis of the β- glucosidic bonds.

A similar study was conducted by Chien and others (2006), in which the isoflavone profile of soymilk was observed after a 24 h fermentation period with

Streptococcus thermophilus BCRC 14085, Lactobacillus acidophilus BCRC

14079, Bifidobacterium infantis BCRC 14633 and B. longum B6, both individually and in combination. Results showed a lower total isoflavone content in the fermented soymilk (81.94–86.61 mg/mL) as compared with the non-fermented soymilk (87.61 mg/mL). Regardless of the starter organism(s) used, however, there was a significant decrease in the concentrations of β-glucosides, acetylglucosides, and malonylglucosides, with a correlating increase in aglycones. Lactobacillus acidophilus BCRC 14079 showed the highest β- glucosidase activity and correlating increase in aglycones, with daidzein, genistein, and glycitein increasing from 14.24%, 6.89% and 2.45%, to 36.20%,

28.80% and 12.44%, respectively, after 24 h of fermentation.

3.1.4 Soy isoflavone HPLC analysis

In order for soy isoflavone profiles to be assessed, reliant extraction, identification and quantification methods must be employed. Wilkinson and others (2002) reviewed over 90 reports for the methodologies used and their efficacy. The most common extraction solvents used were 80% methanol at room temperature or 4°C, and mixtures of acetonitrile-hydrochloric acid (0.1 M) and water. The main analytical techniques employed involved high-performance liquid chromatography (HPLC) with UV (ultraviolet light) or diode array detection

(DAD), in conjunction with reversed-phase C18 stationary phases with gradient elution. Internal standards were frequently used to evaluate analyte loss

occurring during the extraction process. The reported recoveries were generally greater than 90%, however, several reports indicate that the recovery of internal standards can vary substantially with the soy food matrix analyzed. Some of the compounds utilized as internal standards include apigenin, flavone, and trihydroxydeoxybenzoin (THB).

3.2 Sourdough fermentation

3.2.1 Microbiotic associations

Sourdough is comprised of various LAB that are often associated with yeasts in an approximate ratio of 100:1, as well as flour and water (Corsetti and

Settanni 2007). The composition of the microbiotic population may vary tremendously, depending on the fermentation environment and implemented controls of the ecosystem. The lactic acid bacteria most frequently isolated in sourdough fermentation, however, include the species Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus brevis. The primary yeasts isolated in sourdough are from the species Saccharomyces cerevisiae,

Saccharomyces exiguous, Candida krusei, Pichia norvegensis, and Hansenula anomala (Gobbetti 1998). The exact composition of the microbial population is critical due to the variance in metabolic processes across strains and is, therefore, capable of influencing the product’s organoleptic, structural, shelf-life, and nutritional properties.

Characterization of the LAB in traditional sourdough products has led to the identification of the common association between hetero- and homofermentative lactic acid bacteria (De Vuyst and Vancanneyt 2007). The

frequent and stable partnership between obligately heterofermentative LAB (such as Lactobacillus sanfranciscensis and Lactobacillus brevis) and facultatively heterofermentative LAB (such as Lactobacillus plantarum) is frequently attributed to differing carbohydrate metabolisms, which decreases competition for preferred carbon sources. For example, of the four flour carbohydrates available for metabolic utilization (maltose, sucrose, fructose, and glucose), Lactobacillus sanfranciscensis and Lactobacillus brevis preferentially metabolize maltose, while Lactobacillus plantarum preferentially ferments glucose, fructose, and maltose, with the latter being subject to carbon catabolic suppression (De Vuyst and others 2009).

During hexose metabolism by the obligately heterofermentative

Lactobacillus sanfranciscensis and Lactobacillus brevis (Figure 3.6), maltose crosses the cell membrane via a maltose/H+ symporter and is cleaved by maltose phosphorylase to glucose-1-phosphate and glucose. Glucose-1- phosphate is further metabolized via the pentose-phosphate shunt for energy production, and glucose is excreted outside of the cell, thereby, providing an energy source for other microorganisms such as Lactobacillus plantarum.

Furthermore, fructose can be utilized as an electron acceptor, resulting in the formation of acetate and the synthesis of an additional ATP (Gänzle and others

2007).

Figure 3.6. Carbohydrate metabolism of the obligately heterofermentative Lactobacillus sanfranciscensis (Gänzle and others 2007).

The facultatively heterofermentative Lactobacillus plantarum metabolizes hexoses via the Emden-Meyerhoff pathway, in contrast to the pentose-phosphate shunt utilized by obligately heterofermentive LAB. During this process, maltose and fructose are only consumed after the depletion of glucose, making

Lactobacillus plantarum an ideal compliment to the obligately heterofermentative

Lactobacillus brevis and Lactobacillus sanfranciscensis. The main end-product of the Emden-Meyerhoff pathway is lactate. Obligately heterofermentative LAB produce lactate, ethanol, and carbon dioxide, unless electron acceptors, such as fructose, are available to divert the formation of ethanol to acetate production

(Gänzle and others 2007). The ideal balance of lactic and acetic acids produced from these combined pathways results in favorable sensory qualities of sourdough bread.

3.2.2 Flavor and aroma development

Sourdough bread is commonly recognized by its distinct flavor and aroma, which is attributed to compounds that are produced during the fermentation and baking processes. The fermentation quotient (FQ) describes the ratio of lactic acid to acetic acid, and can highly influence the organoleptic properties of the bread (Röcken and others 1992). Depending on the carbohydrate catabolic pathways of the microorganisms present and the substrates made available to them, the FQ may vary appreciably. Research by Gobbetti and others (2005) attributes this variance to the availability of external electron acceptors, such as citrate, malate, fructose, and oxygen. In the carbohydrate catabolism of

Lactobacillus sanfranciscensis, acetate kinase has the potential to generate additional energy when fructose is available as an electron acceptor. This reaction diverts the production of ethanol to acetic acid, therefore, decreasing the fermentation quotient and enhancing the flavor and microbial stability of the bread.

There are several other compounds responsible for the distinct flavor and aroma of sourdough bread. Lactic acid bacteria present in the dough metabolize amino acids that may yield aldehydes, ketoacids, ammonia, amines and alcohols

(Carnevali and others 2007). Glutamine is metabolized to glutamate by LAB, and is accumulated in the dough during fermentation, further contributing flavor

components (Moroni and others 2009). Several Lactobacillus species produce increased amounts of ornithine that is further metabolized to 2-acetyl-1pyrroline, the compound responsible for the roasty note of wheat bread crust. Lipid oxidation during wheat and rye flour storage and dough mixing produces a number of aroma compounds, such as (E)-2-nonenal and other aldehydes that are responsible for a “fatty”, “metallic”, or “green” flavor. Fermentation by LAB, however, further metabolizes (E)-2-nonenal to its corresponding alcohol that imparts a much stronger aroma (Gänzle and others 2007).

The sensory attributes of sourdough bread can vary greatly depending on the fermenting microorganisms present, the substrates made available to them, and the implemented fermentation conditions. Given this, sensory studies involving sourdough bread typically utilize highly trained panelist for characterization and differentiation of sourdough products by descriptive analysis and for the identification of aroma volatiles (Hansen and Hansen 1996; Lotong and others 2000; Kirchhoff and Schieberle 2001; Meignen and others 2001).

Hansen and Hansen (1996) found that bread produced with 15% sourdough fermented with Lactobacillus plantarum was perceived as “very sour”, “metallic”, and imparting a “bitter aftertaste”. When supplemented with Saccharomyces cerevisiae, however, the bread was described as having a more “aromatic wheat bread flavor”. Sensory analysis of sourdough wheat bread by Meignen and others (2001), however, resulted in high ratings of descriptors such as “acid”,

“bitter”, and “vinegar taste” for sourdough fermented with a mixed culture of

Lactobacillus brevis and Saccharomyces cerevisiae.

3.2.3 Nutritional benefits

Sourdough LAB may, also, hold promise for individuals who suffer from an autoimmune response to gluten metabolites. Wheat, rye, and barley contain gliadins that are metabolized, via proteolytic digestion, to polypeptides rich in proline and glutamine. While intestinal peptidases are present to further digest the proline and glutamine rich polypeptides, these antigenic determinants resist further degradation and, in individuals with Celiac Sprue, may initiate a T-cell mediated response (Gobbetti and others 2005). Select sourdough LAB, however, are capable of hydrolyzing these metabolites, and during prolonged fermentation (12-24 h) can entirely eradicate the toxic compounds (Moroni and others 2009).

Sourdough fermentation has potential for many other nutritional applications. Several strains of sourdough LAB are capable of producing fructan exopolysaccharides, including the prebiotic fructooligosaccharides and inulin that aid in the propagation of probiotic bacteria (Gänzle and others 2007). Phytic acid, the major storage form of phosphorus in grains, decreases the nutritional quality of bakery products due to its chelating effect on cations (Gobbetti and others 2005). The resulting decrease in bioavailability of these minerals, however, may be counteracted by the phytase activity of many strains of sourdough LAB. Lactobacillus sanfranciscensis CB1 has been characterized for its high phytase activity, and when present in sourdough fermentation, may reduce phytic acid levels over 50%. Additionally, the pH of sourdough fermentation is optimal for endogenous phytase activity of wheat and rye flours,

further contributing to the solubility and bioavailability of the minerals present

(Moroni and others 2009).

3.2.4 Shelf-life extension

To apply the benefits of sourdough fermentation to current industry, the product must have a controlled and sustainable microbial composition, as well as a shelf-life that satisfies current standards. Sourdough LAB fulfill this criteria through several mechanisms. The production of antimicrobial compounds plays a key role in the preserving qualities of sourdough. Organic acids, ethanol, hydrogen peroxide, phenyllactic acid, bacteriocins, and fungicins are a few of many antimicrobial compounds produced by sourdough LAB. Lactobacillus reuteri LTH2584 produces the antibiotic reutericyclin, inhibiting growth of sensitive bacteria while promoting its own continuous presence and a constant microbial composition (Gobbetti and others 2005). Lactobacillus plantarum conserves its substrate mass by producing the anti-fungal compounds phenyllactic acid and 4-hydroxyphenyllactic acid. These compounds work synergistically with the common industrial additive, calcium propionate, in wheat sourdoughs, and allow for a reduction of calcium propionate by approximately

30% (Moroni and others 2009). Furthermore, retarding starch retrogradation, a key component of bread staling, may extend the shelf-life. Both the acidification of sourdough and the proteolytic activity of sourdough LAB decrease starch retrogradation, producing a more palatable product. Amylolytic activity, although uncommon in sourdough LAB, produces the same effect (Moroni and others

2009).

3.2.5 Beta-glucosidase activity

Lactic acid bacteria are often present in fermented food products and several strains have been identified as exhibiting β-glucosidase activity.

Research has been directed towards mapping the enzymatic activity of β- glucosidase LAB for improved understanding of their contribution to the human diet. The hydrolysis of β 1,4-glycosidic bonds increases the bioavailability of many non-digestible food components, and may be applied to manufacturing processes for the nutritional enhancement of food products.

Lactobacillus sanfranciscensis CB1, responsible for the distinct flavor and aroma of San Francisco sourdough bread, displays β-glucosidase activity unique to its strain. As described by De Angelis, Gallo, and others (2005), the hexameric, 288 kDa enzyme functioned optimally at pH 7.5 and 40 °C, and was predominately activated when starved of its preferred carbon source, maltose.

Under these conditions, Lactobacillus sanfranciscensis used cellobiose, methyl-

β-glucoside, arbutin, amygdalin, and salicin as carbon sources. With glucose or maltose made available at concentrations of 0.5%, β-glucosidase activity was exhibited; however, when replaced with cellobiose as subtrate, expression increased 5 fold. This metabolic flux demonstrates the importance of substrate and microbiota interaction and compatibility.

Sestelo and others (2004) reported characteristics on the β-glucosidase of

Lactobacillus plantarum USC1 isolated from wine. The extracellular enzyme was estimated to have a molecular mass of 40 kDA and displayed broad substrate specificity. The enzyme hydrolyzed glucose dimmers linked by (β-1,4), (β-1,3),

(β-1,2), and (β-1,6) bonds, with a respective hydrolysis rate order, and was capable of hydrolyzing both β-diglucosides and aryl-β-glucosides. The enzyme was not affected by the presence of oxygen, however, repression did occur when glucose levels exceeded 0.2% w/v. A sharp-optimum pH curve was observed, with optimal activity at pH 5.0 and activity ranging between pH 4.5 and 8.0. With ideal environmental paramenters set (pH 5.5 and 45 °C), the enzyme displayed

Km and Vmax values for the synthetic substrate, 4-nitrophenyl-β-D- glucopyranoside (pNPG), of 1.82 mM and 4.89 nmol/ml/min, respectively. These results were comparable to characteristics found for other bacterial β- glucosidases. Grape wine typically ranges between pH 3.0 – 3.5, therefore decreasing the enzymatic activity to approximately 33%. Sourdough, however, may be a more suitable medium for optimal Lactobacillus plantarum USC1 β- glucosidase activity. The normal pH of sourdough ranges between pH 4.0 – 4.5

(Gobbetti 1998), and this less acidic environment would result in a sharp increase in β-glucosidase activity according to the findings of Sestelo and others

(2004).

Lactobacillus brevis SK3 isolated from wine starter culture also displayed

β-glucosidase activity, as described by Michlmayr and others (2010). The β- glucosidase of Lactobacillus brevis exhibited characteristics vastly different from that of Lactobacillus plantarum. The intracellular enzyme was shown to be a homotetramer with a molecular weight of 330 kDa, and performed optimally at pH

5.5 and 45 °C. The Km for pNPG under optimal conditions was significantly lower than that reported for the β-glucosidase of Lactobacillus plantarum (0.22 mM

compared to 1.82 mM). The β-glucosidase of Lactobacillus brevis, however, displayed limited inhibition by glucose, thereby, making the enzyme more suitable for fermentations with a high carbon source concentration.

Pyo and others (2005) investigated the β-glucosidases of LAB isolated from soymilk. Of the 31 strains isolated, the 4 strains that displayed greater β- glucosidase activity were reviewed and include: Lactobacillus plantarum KFRI

00144, Lactobacillus delbrueckii subsp. lactis KFRI 01181, Bifidobacterium breve

K-101, and Bifidobacterium thermophilum KFRI 00748. Of these strains,

Lactobacillus plantarum KFRI 00144 showed the highest activity (24.7 mU/mL).

β-glucosidase activity and glucoside hydrolysis of the 4 strains was observed during a 48 h incubation period at 37 °C in soymilk. A control (soymilk with no bacterial strains present) was also incubated under the same conditions. After

48 h, the total concentration of the isoflavones genistin, daidzin, genistein, and daidzein, was 150.3 µg/mL in the control sample, with glucosides comprising

86% of the total concentration. The soymilk fermented with the 4 bacterial strains, however, had a total concentration of 160.7 - 167.2 µg/mL, with the bioavailable aglycones comprising 100% of the total isoflavone concentration

(Pyo and others 2005).

3.2.6 Structural and textural changes

Sourdough fermentation, also, exhibits influence on the structural components of sourdough bread products. Sourdough LAB produce strain- specific exopolysaccharides (EPS), which improve dough rheology, gas retention, bread volume, and crumb firmness. Mouthfeel and textural quality are

also improved with the synthesis of EPS (Lacaze and others 2007).

Exopolysaccharides of interest for sourdough application are homopolysaccharides synthesized from sucrose. Extracellular glucosyltransferases and fructosyltransferases catalyze the transfer of glucose or fructose, respectively, to an acceptor polymer chain (Gänzle and others 2007).

These polymers vary in length, branching, and linking in a LAB strain-specific manner.

Dextran, an exopolysaccharide comprised of repeating (1-6) linked alpha-

D-glucopyranosyl residues, is noted for its hydrocolloid characteristics. Although the use of dextran is not widespread commercially, interest has been elicited for use as a stabilizing, gelling, or bodying agent. With regards to the production of baked goods, dextran has been shown to interact with the gluten network of wheat and rye, improving gas retention and loaf volume (Lacaze and others

2007).

LAB may, additionally, influence the gluten network of wheat sourdoughs by increasing the availability of low-molecular weight thiol compounds.

Lactobacillus sanfranciscensis exhibits activity of an R-specific alcohol dehydrogenase that catalyzes the reduction of hexanal, further leading to increased acetate concentrations. Gänzle and others (2007) hypothesized a linkage between this increased production of acetate and the reduction of oxidized glutathione, the compound responsible for gluten cross-linking. By favoring the formation of reduced glutathione, Lactobacillus sanfranciscensis

mimics the function of dough mixing, leading to further inflation of gluten proteins

(Gobbetti and others 2005).

Dough extensibility is frequently assessed to measure large deformations that correlate to baking performance, such as loaf volume, as well as glutenin composition of doughs and the effect of mixing time on dough extensibility

(Anderssen and others 2004; Abang Zaidel and others 2008). Anderssen and others (2004) described the 5 possible viscoelastic stages observed upon uniaxial dough extension (Figure 3.7). The maximum resistance (Rmax), or maximum load/force (N), occurs after maximum alignment of dough macromolecules, which is an almost instantaneous occurrence in doughs with a strong gluten network, and slower response in doughs with a less developed gluten network (displaying a more pronounced equilibrium response). Studies using the extensograph have demonstrated decreased extensibility of doughs upon the addition of acid and in the presence of salt (Tsen 1966 and Tanaka and others 1967), which is believed to be due to the increased solubility of gluten at acidic pH values (Takeda and others 2001). The increased solubility of gluten has been hypothesized to increase access of endogenous and microbial proteolytic enzymes to the gluten network, allowing for the partial hydrolysis of low molecular weight glutenins during sourdough fermentation (Thiele and others

2002; Gänzle and others 2008).

Figure 3.7. Extensogram displaying the 5 viscoelastic stages of uniaxial dough extension (Anderssen and others 2004).

3.3 Thermal analysis of bread

Thermal analysis techniques, including thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), are frequently used to study the moisture distribution, thermal transitions, and overall staling process of baked goods (Davidou and others 1996; Baik and Chinachoti 2000, 2001; Vittadini and

Vodovotz 2003; Lodi and Vodovotz 2008). Thermogravimetric analysis allows for moisture content determination by applying a linear temperature ramp and observing the weight loss of the sample. When used in conjunction with DSC,

“freezable” (FW) and “unfreezable” water (UFW) populations can be determined by the observation of an endothermic ice melt peak near 0 °C, representative of

the FW population. Moisture migration has been shown to be a key event of bread staling, with FW populations decreasing with storage time, and an overall migration of water from the bread crumb to crust (Baik and Chinachoti 2000;

Vittadini and Vodovotz 2003). The addition of soy flour to wheat bread, however, has been shown to result in a more homogeneous water distribution throughout the bread crumb, as well as retard moisture redistribution during storage, thereby, slowing the staling rate of bread (Lodi and others 2007; Lodi and

Vodovotz 2008).

In addition to the moisture migration events that characterize bread staling, the recrystallization of amylopectin has, also, been shown to be a key event of the staling process. Differential scanning calorimetry has frequently been used to study the retrogradation of starch, with amylopectin melts generally occurring between 40 and 60 °C, and melt enthalpy increasing with storage time

(Baik and Chinachoti 2000; Vittadini and Vodovotz 2003; Lodi and Vodovotz

2008). Vittadini and Vodovotz (2003) investigated the effect of soy flour supplementation to wheat breads on the rate of amylopectin recrystallization, and noted a significant decrease in amylopectin recrystallization in breads supplemented with soy flour that could not alone be explained by decreased amylopectin concentrations. For example, a bread in which 40% of the flour weight was replaced with soy flour was expected to have a 40% reduction in amylopectin recrystallization due to a 40% decrease in amylopectin concentration; however, a 76% reduction in amylopectin recrystallization was observed in the soy bread containing 40% soy flour on the second day of storage

when compared to the control (wheat bread). Baik and Chinachoti (2001) had previously suggested a correlation between lower amylopectin recrystallization rates and decreased moisture migration from bread crumb to crust. Given this, the decreased rate of amylopectin recrystallization in soy bread observed by

Vittadini and Vodovotz (2003) may be attributed to the higher water-holding capacity of soy flour compared to wheat flour and, therefore, decreased moisture migration that is observed with soy-supplemented wheat breads (Lodi and others

2007).

In addition to the endothermic peaks representing FW populations and amylopectin crystals that are observable by DSC analysis, a third endothermic peak occurring between 100 and 130 °C is frequently observed in bread, and is attributed to the melting of amylose-lipid complexes. Czuchajowska and

Pomeranz (1989) hypothesized that these complexes are formed during and/or immediately after baking, and additional studies have shown that amylose-lipid complexes remain stable with storage time as shown by DSC analysis (Davidou and others 1996; Lodi and Vodovotz 2008). Furthermore, the complexation of lipid with amylose is believed to result in a decrease in starch retrogradation, as amylose complexed with lipid would no longer be available to co-crystallize with amylopectin molecules. This was shown by Davidou and others (1996), however, lipid complexation with amylose only appeared to inhibit starch retrogradation after extended storage.

CHAPTER 4: MATERIALS AND METHODS

4.1 Dough formulation and bread production

The formulations of ingredients (%) for regular soy bread (R-SB) and sourdough soy bread (SD-SB), are shown in Table 4.1, and the ingredient manufacturers are given in Table 4.2.

Table 4.1. Ingredients and formulations (%) for regular soy bread (R-SB) and sourdough soy bread (SD-SB). Formulation (%) Ingredient R-SB SD-SB Water 36.6 26.2 Bread flour 40.7 30.2 Soy flour 12.2 12.3 Soymilk powder 4.1 4.1 Sugar 2.0 2.0 Shortening 2.0 2.0 Wheat gluten 1.0 1.0 Salt 0.8 0.8 Dough conditioner 0.1 0.1 Yeast 0.4 0.0 Preferment 0.0 21.2

Table 4.2. Soy bread ingredients and their corresponding manufacturers. Ingredient Manufacturer Bouncer High Gluten Flour Bay State Milling Co., Quincy, MA, U.S.A. Baker’s Soy Flour ADM, Decatur, IL, U.S.A. Benesoy™ Soymilk Powder Devansoy, Carroll, IA, U.S.A. Granulated Sugar Gordon Food Service, Grand Rapids, MI, U.S.A. Crisco® All-Vegetable J.M. Smuckers, Orrville, OH, U.S.A. Shortening Vital Wheat Gluten Bob’s Red Mill, Milwaukie, OR, U.S.A. Iodized Salt U.S. Foodservice, Columbia, MD, U.S.A. Dough Conditioner Caravan Ingredients, Lenexa, KS, U.S.A. Instaferm RED Instant Dry Lallemand, Montréal, QC, Canada Bakers Yeast Florapan LA4 Lallemand, Montréal, QC, Canada

4.1.1 Regular soy bread

Regular soy bread was prepared by a sponge-dough method (U.S. Patent

Number: 7,592,028), in which a wheat-based sponge was proofed at room temperature (~20 °C) for 2 h, prior to the addition of the remaining ingredients

(dough). The ingredients were mixed with an electric mixer for approximately 5 min, or until fully hydrated. The dough was then hand kneaded and formed into a loaf before being proofed (CM2000 combination module, InterMetro Industries

Corp., Wilkes-Barre, PA, U.S.A.) at 40 °C and 95% relative humidity (RH) for 1 h.

Analysis of dough took place after the final proofing period. The proofed dough was then baked for 50 min at 152 °C (Jet air oven, JA14, Doyon, Linière,

Québec, Canada), cooled at room temperature for 3 h, sliced (Doyon SM302 bread slicer, Linière, Québec, Canada), and sealed in polyethylene bags prior to analysis of fresh bread samples. Stored bread was stored, unsliced, in polyethylene bags for 10 days at room temperature (~20 °C) prior to slicing and analysis.

4.1.2 Sourdough soy bread

A sourdough starter culture (preferment) was generated 5 days prior to sourdough soy bread production, or until the preferment displayed optimal gas production (Figure 4.1) and reached a pH of approximately 3.5. Florapan LA4

(Lallemand, Montréal, QC, Canada), containing Lactobacillus brevis,

Lactobacillus plantarum, and Saccharomyces cerevisiae, was incorporated into the preferment mixture at 0.1% preferment flour weight, and was propagated daily by pouring off approximately 40% of the preferment mixture before incorporating bread flour and water in a 1:1 ratio. The resulting dough yield of the mixture was 200 (Equation 4.1), therefore, the preferment was considered a liquid sourdough (Decock and Cappelle 2005).

Equation 4.1

Dough yield (DY) = (Flour weight + Water weight) X 100

Flour weight

Figure 4.1. Image of optimally active, liquid sourdough preferment.

The sourdough soy bread was generated using a modified formulation of the regular soy bread. The preferment was incorporated into the dough at 21.2% w/w, and the ingredients were mixed for approximately 5 min, or until fully hydrated. The dough was kneaded and loaves formed by hand, prior to a 9 h proofing period at 30 °C (Isotemp® Oven 200 Series, Model 215F, Fisher

Scientific, Fair Lawn, NJ, U.S.A.). Dough analyses took place immediately following the 9 h proofing period. For bread production, fermented dough was baked for 50 min at 152 °C (Jet air oven, JA14, Doyon, Linière, Québec,

Canada), cooled at room temperature for 3 h, sliced (Doyon SM302 bread slicer,

Linière, Québec, Canada), and sealed in polyethylene bags prior to analysis of fresh bread samples. Stored bread was stored, unsliced, in polyethylene bags for 10 days at room temperature (~20 °C) prior to slicing and analysis.

4.1.3 Dough formulations for HPLC analysis

The isoflavone profiles of 4 dough formulations were analyzed by RP-

HPLC in order to elucidate the mechanism for isoflavone conversion occurring during sourdough fermentation. The formulations of the 4 doughs are given in

Table 4.3, and the ingredients used are the same as those listed in Table 4.2.

Table 4.3. Formulation (%) of ingredients for control soy dough (C-SD), yeast soy dough (Y-SD), lactic acid bacteria soy dough (LAB-SD), and sourdough soy dough (SD-SD). Formulation (%) Ingredient C-SD Y-SD LAB-SD SD-SD Water 36.8 36.6 26.2 26.2 Bread flour 40.9 40.7 30.2 30.2 Soy flour 12.3 12.2 12.3 12.3 Soymilk powder 4.1 4.1 4.1 4.1 Sugar 2.0 2.0 2.0 2.0 Shortening 2.0 2.0 2.0 2.0 Wheat gluten 1.0 1.0 1.0 1.0 Salt 0.8 0.8 0.8 0.8 Dough conditioner 0.1 0.1 0.1 0.1 Yeast 0.0 0.4 0.0 0.0 Preferment 0.0 0.0 21.2 21.2

Two of the 4 dough types were prepared by inclusion of a preferment, and include soy sourdough (SD-SD) and lactic acid bacteria soy dough (LAB-SD).

The remaining 2 dough types were prepared by a straight-dough method, and include yeast-leavened soy dough (Y-SD) and control soy dough (C-SD). These doughs were produced in order to isolate the potential sources of β-glucosidase present in the sourdough soy bread, which include Saccharomyces cerevisiae

(baker’s yeast), lactic acid bacteria (Lactobacillus brevis and Lactobacillus plantarum), and the endogenous β-glucosidase activity of the soy and wheat ingredients (soy flour, soy milk powder, wheat flour) (Zhang and others 2004;

Riedl and others 2005).

A control soy dough (C-SD) formulation was generated, in which no fermenting microorganisms were present, and a lactic acid bacteria soy dough

(LAB-SD) was formulated, in which Lactobacillus brevis (Florapan L62,

Lallemand, Montréal, QC, Canada) and Lactobacillus plantarum (Florapan L62,

Lallemand, Montréal, QC, Canada) were the only fermenting microorganisms present. The isoflavone profile of these 2 doughs, as well as yeast-leavened soy dough (Y-SD; fermented by Saccharomyces cerevisiae) and soy sourdough (SD-

SD; fermented by Lactobacillus brevis, Lactobacillus plantarum, and

Saccharomyces cerevisiae), were evaluated at the beginning and end of a prolonged proofing period (9 h) at 30 °C (Isotemp® Oven 200 Series, Model

215F, Fisher Scientific, Fair Lawn, NJ, U.S.A.) by RP-HPLC. Analyses of variance (ANOVA) and Tukey’s HSD tests were conducted to analyze differences between sample means at the 95% confidence interval. In addition to

the isoflavone characterization of these 4 doughs, the isoflavone profile of the soy mix (1:3 w/w/ ratio of soy milk powder to soy flour) was determined in order to observe the effect of dough formation on the isoflavone profile of the initial ingredients.

4.2 Isoflavone extraction and analysis

4.2.1 Isoflavone extraction

Isoflavones from the bread crumb and dough were extracted from their matrices for qualitative and quantitative analysis via RP-HPLC. Following the 3 h cooling period, 0.5 g of bread crumb was homogenized in 5 mL of acetonitrile in water (60%, v/v) using a PT 3100 Polytron homogenizer (Kinematica, Inc.,

Bohemia, NY, U.S.A.). Homogenized samples were vortexed and sonicated for

15 min (Mechanical Ultrasonic Cleaner FS30H, Fisher Scientific, Fair Lawn, NJ,

U.S.A.), before centrifuging at 3000 rpm for 30 min (IEC HN-SII Centrifuge,

Damon/IEC Division, Needhamhts, MA, U.S.A.). The resulting supernatant was collected and extraction process repeated twice, for a collective pool of 15 mL of extract. From the pooled extract, 2 mL was transferred to a separate vial, the solvent evaporated under 5.0 grade nitrogen to prevent isoflavone interconversion, and dried extracts stored at or below -20 °C to prevent degradative loss of isoflavones. Isoflavones were extracted from dough at various time intervals during the 9 h proofing period (0, 2, 4, 6, and 9 h). To account for the higher moisture content of the dough samples, extracts were obtained from 1 g samples and extracted using pure acetonitrile.

4.2.2 RP-HPLC isoflavone analysis

Isoflavone extracts were redissolved in 2 mL of methanol in water (80%, v/v) by sonication, and filtered through a 0.20 µm syringe filter prior to analysis by

RP-HPLC using a Waters Symmetry® C18 column, with dimensions of 4.6 x 75 mm and 3.5 µm particle size (Waters Corporation, Milford, MA, U.S.A.).

Qualitative analysis was performed using an Agilent 1100 Series HPLC equipped with a multiple wavelength detector monitoring at 260 nm, manual injector with a

20 µL injection loop, and ChemStation software (Rev.A.08.03) for analysis

(Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). Quantitative analysis of 10

µL sample injections was performed (with the help of Jennifer Ahn-Jarvis) using a

Waters 2695 Alliance® HPLC equipped with a Waters 996 photodiode array

(PDA) detector and Empower 2 software (Waters Corporation, Milford, MA,

U.S.A.). Table 4.4 displays the mobile phase elution gradient used on both

HPLC systems. Solvent A consisted of acetic acid in water (1% v/v) and solvent

B was pure acetonitrile. Pump flow rates were set at 1.0 mL/min and column ovens maintained at 35 °C.

Table 4.4. Mobile phase elution gradient for isoflavone analysis by RP-HPLC. Time (min) Solvent A (%) Solvent B (%) 1 90 10 23 65 35 26 25 75 29 90 10 35 90 10

4.2.3 Isoflavone quantification

Stock standard solutions were prepared by dissolving crystalline standards (LC Laboratories, Woburn, MA, U.S.A.) in methanol via sonication, to a final concentration of 1 mg/mL. To optimize solubility, dimethyl sulfoxide

(DMSO) was added to daidzin, genistin, and glycitin standard solutions (5% v/v), and glycitein standard solution (30% v/v). Static UV spectrophotometer readings were used to determine standard concentrations using extinction coefficients reported by Murphy and others (2002). Working standard solutions were prepared by creating an equimolar mixture of stock standard solutions, and serially diluting by transferring 1 part of the equimolar solution into 2 parts methanol. In this manner, 5 serially-diluted solutions were prepared and used to generate standard curves for qualitative analysis of isoflavone extracts.

Appendix A displays the standard curves for isoflavone aglycones and simple β- glucosides acquired with the Agilent HPLC system.

4.3 Physical properties

4.3.1 Dough extension

The Instron Universal Testing Machine 5542 (Instron Corp., Norwood, MA,

U.S.A.) was used to test uniaxial dough extensibility. Approximately 26 g of proofed dough was flattened to 5 mm sheets using a Culver press (Fred S.

Culver, Inc., Summit, NJ, U.S.A.). Dough was covered with plastic wrap, placed between metal plates, and ~4000 lbs/in2 was applied over a 2 h period for gluten relaxation (Morgenstern and Holsti 1996). The top layer of plastic wrap was

removed and the dough allowed to rest for 10 minutes to prevent stickiness and facilitate cutting. Immediately prior to testing, a 3.0 x 0.5 cm strip was prepared, placed on the Instron stage, and secured by clamps. A dough hook attachment pulled the dough at a crosshead speed of 150 mm/min, and the test was stopped upon dough rupture. Bluehill® 2 software version 2.17 (Instron Corp., Norwood,

MA, U.S.A.) recorded maximum force (N) and extension at maximum load (mm), as displayed in the resulting extensograms. At least 6 intra-dough replicates were obtained for 3 dough batches, therefore, a minimum of 18 replicates were obtained for each dough type.

4.3.2 Bread crumb hardness

Bread crumb hardness was determined using the Instron Universal

Testing Machine (Instron Corp., Norwood, MA, U.S.A.) and the AACC method

74-09 (AACC 2000). Samples were cut to dimensions of 25 x 25 x 25 mm3 using an electronic carving knife to minimize compressive deformation prior to testing.

A uniaxial compression (applied with a 35 mm diameter plunger) with crosshead speed of 100mm/min was utilized to mimic mastication, with crumb hardness corresponding to the force (N) required for 40% compression. The instrument software (Bluehill® 2 version 2.17, Instron Corp., Norwood, MA, U.S.A.) automatically recorded the compressive load (N) and plunger extension (mm) for the generation of compression curves. At least 6 intra-loaf replicates were obtained for a minimum of 2 loaves, therefore, at least 12 replicates were obtained for each bread type and given storage condition (fresh or stored).

4.3.3 Specific loaf volume

The specific loaf volume (cm3/g) was determined using the AACC method

10-05 (rapeseed displacement) and the loaf mass (g) (AACC 2000).

Measurements of loaf mass were taken immediately following the 3 h cooling period to reduce variability resulting from evaporative losses following baking. A minimum of 3 replicates were obtained for each bread type.

4.3.4 Crust color

The crust color of sourdough soy bread and regular soy bread was determined using a Chroma Meter CR-300 (Konica Minolta Sensing Americas,

Inc., Ramsey, NJ, U.S.A.) using the Commission Internationale d’Eclairage color model (Clydesdale 1978). Lightness (L), ranging from 0 (black) to 100 (white), and the chromatic components, a* (ranging from red to green) and b* (ranging from yellow to blue), which range from -120 to +120, are depicted in Figure 4.2.

At least 3 replicates were obtained for each loaf, and 4 loaves analyzed for each bread type, therefore, a total of 12 replicates were obtained for both SD-SB and

R-SB.

Figure 4.2. Commission Internationale d’Eclairage color model depicting values of lightness (L), and chromatic components, a* and b* (Clydesdale 1978).

4.4 Thermal analysis

4.4.1 Thermogravimetric analysis (TGA)

Thermogravimetric measurements of dough and bread crumb samples were obtained with a Thermogravimetric Analyzer Q5000 (TA Instruments, New

Castle, DE, U.S.A.). Samples weighing between 15-20 mg were placed on a tared, stainless steel pan (PerkinElmer Life and Analytical Sciences, Inc., Boston,

MA, U.S.A.), and immediately placed on the instrument platform for analysis.

Samples were heated within the instrument chamber using a linear ramp of 5

°C/min, from room temperature (approximately 20 °C) to 180 °C. The thermograms, displaying the sample weight as a function of temperature and the derivative of weight loss, were analyzed using Universal Analysis™ 2000

software (TA Instruments, New Castle, DE, U.S.A.). Moisture content of samples was determined from the initial and final weights of the sample (Equation 4.2), under the assumption that resulting weight losses were solely attributed to the evaporation of water and other volatile substances (Fessas and Schiraldi 2001).

Equation 4.2

Moisture content (%) = initial sample weight (g) – final sample weight (g) X 100

initial sample weight (g)

4.4.2 Differential scanning calorimetry (DSC)

Calorimetric measurements were taken using a Differential Scanning

Calorimeter Q100 (TA Instruments, New Castle, DE, U.S.A.) for the determination of FW and UFW populations, and for measuring thermal transitions associated with starch retrogradation. Bread crumb samples (~10 mg) and references (empty) were prepared in stainless steel pans, hermetically sealed with O-rings (PerkinElmer Life and Analytical Sciences, Inc., Boston, MA,

U.S.A.), and placed onto separate thermocouples within the DSC chamber.

Chamber heating and cooling was performed at a linear rate of 5 °C/min. The experimental procedure included cooling from room temperature (~20 °C) to -50

°C, a 3 min isothermal period, and heating to 150 °C.

Thermograms displayed the differential heat flow (W/g) between reference and sample pans versus temperature (°C), and were analyzed using Universal

Analysis™ 2000 software (TA Instruments, New Castle, DE, U.S.A.). Thermal

transitions of interest were analyzed by peak integration for determination of their associated enthalpies (J/g). Endothermic peaks occurring near 0 °C were attributed to ice melting (Reid and others 1993; Vittadini and Vodovotz 2003), allowing for the determination of the percent FW present in the sample

(Equation 4.3). Endothermic peaks occurring between 40 and 60 °C were attributed to amylopectin melting, and endothermic peaks observed between 100 and 130 °C were associated with melting of amylose-lipid complexes (Lodi and

Vodovotz 2008).

Equation 4.3

Percent “freezable” water (%FW) = Enthalpy at 0 °C (J/g) X 100

Latent heat of fusion of water (333.2 J/g)

4.5 Sensory analysis

Overall liking of regular soy bread and sourdough soy bread, and preference between the 2, was evaluated amongst 60 adult (18 years of age or older) participants using a 9-point hedonic scale and paired comparison

(preference) test, respectively. The study was approved for exemption from The

Ohio State University institutional review board (IRB) under category 6. The letter of approval is shown in Appendix B. A sample size of 60 was determined to be appropriate for analysis based on previously published sample sizes for 9- point hedonic and paired comparison (preference) testing (Carpenter and others

2000; Afolabi and others 2001; Rose and others 2011). Participants were

recruited via Ohio State University email list servers (Appendix C), with individuals with known allergies to wheat and soy and pregnant women being discouraged from participating.

Regular soy bread and sourdough soy bread samples were baked and prepared the day prior to testing, and were labeled with randomized 3-digit codes. All sample sets were counterbalanced and randomized in their presentation. Prior to test administration, participants were asked to review, sign, and date an informed consent form (Appendix D). An electronic questionnaire

(Appendix E) was used to collect responses with Compusense® version 5.2 software (Compusense, Inc., Guelph, ON, Canada), and a 2-sample t-test was performed to determine statistical significance. Preference testing was conducted via ballot entry (Appendix F), and statistical significance determined at the 95% confidence interval.

CHAPTER 5: RESULTS AND DISCUSSION

5.1 Aim 1: Quantitative and qualitative assessment of the isoflavone

profile of fermenting sourdough, and fresh and stored sourdough

soy bread and regular soy bread by RP-HPLC

5.1.1 Isoflavone profile of fermenting soy dough

The isoflavone profiles of 4 dough formulations were evaluated at the beginning and end of a 9 h proofing period at 30 ºC, with the aim of elucidating the mechanism of isoflavone interconversion observed during extended 24 h fermentation of SD-SD (Figure 5.1). The dough types analyzed varied in their potential sources for β-glucosidase activity, which include the wheat flour and soy ingredients (soy flour and soy milk powder), Saccharomyces cerevisiae, and the

LAB, Lactobacillus brevis and Lactobacillus plantarum (Zhang and others 2004;

Riedl and others 2005).

Figure 5.1. Overlay of chromatograms displaying the isoflavone profile of SD-SD at 0, 9, and 24 h of fermentation at 30 °C. Dashed lines highlight the conversion of the isoflavone simple β-glucosides, daidzin and genistin, to their corresponding aglycone forms (courtesy of Jennifer Ahn-Jarvis).

The control dough type (C-SD) contained no fermenting microorganisms and, therefore, the wheat and soy ingredients were the only potential sources of

β-glucosidase activity available for deglucosylation of isoflavone β-glucosides.

The yeast dough type (Y-SD) contained Saccharomyces cerevisiae in addition to the endogenous β-glucosidase activity of wheat and soy ingredients, whereas the lactic acid bacteria dough type (LAB-SD) contained Lactobacillus brevis and

Lactobacillus plantarum in addition to wheat and soy ingredients. Lastly, the soy sourdough (SD-SD) contained all of the above-mentioned potential sources of β- glucosidase activity.

In addition to the analyses of these 4 dough types, the isoflavone profile of the initial soy mix (1:3 w/w ratio of soy milk powder to soy flour) was evaluated in order to determine the effect of dough formation on the isoflavone profile of the initial soy ingredients. Table 5.1 displays the isoflavone profile of the initial soy mix (SM), which contained predominantly malonyl-glucosides (66.9%) and simple

β-glucosides (27.1%). This observation is in accordance with previous findings for soy flour, which was determined to contain primarily malonyl-glucosides

(~80%) and simple β-glucosides (~15%), with very small amounts of acetylglucosides and aglycones (Murphy and others 2002).

Table 5.1. Isoflavone profile (aglycones and various conjugates given as % of total isoflavone concentration) for SM. SM Aglycone (%) 1.69 ± 0.0794

Simple-glucoside (%) 27.1 ± 1.42

Acetyl-glucoside (%) 4.29 ± 0.130

Malonyl-glucoside (%) 66.9 ± 2.33

The observed isoflavone profiles and pH of the 4 dough types at the beginning (T0) and end (T9) of fermentation are given in Table 5.2, and are

displayed graphically in Figure 5.2. The initial isoflavone profiles (T0) of the dough types varied significantly from the SM isoflavone profile (p < 0.05), suggesting that there is significant transformation of the isoflavone profile upon inclusion of the soy ingredients into the dough. Most notable is the significantly larger malonyl-conjugate pool of the SM compared to the various dough types.

Malonyl-conjugates have been shown to be highly unstable under conditions of moist heat, and will decarboxylate to β-glucosides in room temperature solution at a rate of 0.2-0.3 mol% per h (Murphy and others 2002). Given this, it is not surprising that the addition of water and the application of heat via dough mixing and kneading would result in significantly smaller malonyl-conjugate pools in the various doughs compared to the SM.

Table 5.2. Isoflavone profiles (aglycones and various conjugates given as % of total isoflavone concentration) and dough pH for C-SD, Y-SD, LAB-SD, and SD-SD at the beginning (T0) and end (T9) of fermentation at 30 ºC. C-SD Y-SD LAB-SD SD-SD

Aglycone (%) 7.72 ± 0.694a 11.8 ± 0.949b 6.95 ± 0.490a 5.37 ± 0.700c

Simple-glucoside (%) 48.3 ± 1.20a 51.8 ± 1.46b 55.1 ± 2.49c 29.8 ± 1.68d

Acetyl-glucoside (%) 15.8 ± 0.284a 14.8 ± 1.04b 15.5 ± 0.792ab 9.32 ± 0.666c

Malonyl-glucoside (%) 28.2 ± 1.10a 21.6 ± 0.955b 22.5 ± 1.13bc 55.5 ± 2.55d

a b c c

56 Dough pH 6.35 ± 0.0643 6.07 ± 0.0503 5.77 ± 0.0781 5.71 ± 0.0985

Aglycone 55.8 ± 3.53a 64.1 ± 4.43b 67.6 ± 2.76c 56.1 ± 4.41a

Simple-glucoside (%) 9.03 ± 0.334a 18.1 ± 0.666b 3.60 ± 0.128c 12.4 ± 0.901d

Acetyl-glucoside (%) 2.75 ± 0.380a 7.54 ± 0.725b 4.95 ± 0.0426c 4.62 ± 0.304c

Malonyl-glucoside (%) 32.5 ± 1.33a 10.2 ± 1.18b 23.9 ± 0.860c 26.9 ± 1.94d

Dough pH 6.27 ± 0.0351a 5.76 ± 0.0173b 4.68 ± 0.241c 4.22 ± 0.136d

Figure 5.2. Isoflavone profiles (aglycones and various glucosides given as % of total isoflavone concentration) for C-SD, Y-SD, LAB-SD, and SD-SD at the beginning (T0) and end (T9) of fermentation at 30 ºC.

In addition to the significantly different isoflavone profile observed between

SM and dough types, the initial isoflavone profiles (T0) of the doughs varied considerably from one another. This finding suggests that differences in the dough preparation methods and ingredients can exert significant effects on the immediate isoflavone composition of the resulting doughs. This observation was most prominently displayed in SD-SD at T0, which varied significantly from the 3 other dough types at T0 in all isoflavone forms (p < 0.05). SD-SD, however,

most closely resembled SM in its isoflavone profile, displaying a significantly greater malonyl-conjugate pool (p < 0.05) and significantly smaller simple β- glucoside pool (p < 0.05) compared to the other dough types. Malonyl- glucosides have been shown to be least stable under alkaline conditions (pH 10) and considerably more stable under acidic conditions (Murphy and others 2002;

Mathias and others 2006); therefore, the lower dough pH of SD-SD at T0 may be partially responsible for the larger malonyl-conjugate pool observed.

Of the dough types analyzed, LAB-SD resulted in the greatest proportional increase in isoflavone aglycones and, correspondingly, the greatest proportional decrease in isoflavone simple β-glucosides. This observation may be attributed to potential β-glucosidase activity of the particular Lactobacillus brevis and

Lactobacillus plantarum strains present in the preferment. Several strains within each of these species have been identified as possessing β-glucosidase activity

(Sestelo and others 2004; Pyo and others 2005; Di Cagno and others 2010;

Michlmayr and others 2010), with Lactobacillus plantarum strains frequently displaying the highest activity of LAB species analyzed (Di Cagno and others

2010).

Y-SD and SD-SD displayed considerable decreases in malonyl-conjugate pools during the fermentation period compared to C-SD and LAB-SD, which displayed marginal increases in their malonyl-glucoside pools. Y-SD and SD-SD, also, exhibited significant decreases (p < 0.05) in total isoflavone concentration within the first 6 h of fermentation, which steadily increased during extended fermentation to 24 h (Figure 5.3). Y-SD and SD-SD displayed total isoflavone

concentrations of 871 and 856 nmol/g at T0, which decreased to 542 and 537 nmol/g at T9, respectively. These trends did not correlate to dough pH over fermentation time, therefore, these observations may have resulted from the metabolic activity of Saccharomyces cerevisiae and/or differing extraction efficiency of the various isoflavone forms from the given dough types over fermentation. Figure 5.4 displays the isoflavone concentrations of the various isoflavone forms of SD-SD (nmol/g of dough) over 24 h of extended fermentation at 30 °C, and clearly displays the decreasing malonyl-conjugate content observed at the beginning of fermentation in these dough types.

Figure 5.3. Total isoflavone concentration (nmol/g of dough) for SD-SD and Y- SD over 24 h of fermentation at 30 °C.

Figure 5.4. Isoflavone concentrations (nmol/g) of aglycones and various conjugates of SD-SD over 24 h of fermentation at 30 °C.

5.1.2 Isoflavone analysis of fresh and stored R-SB and SD-SB

The isoflavones of R-SB and SD-SB were quantified via RP-HPLC and are reported as collective aglycones, simple β-glucosides, and acetyl- and malonyl-glucosides (nmol/g) in Table 5.3. The total isoflavone concentration of the SD-SB was significantly (p < 0.05) lower than that of R-SB (390 and 478 nmol/g, respectively). This observation paralleled the decreased concentrations observed in SD-SD upon extended fermentation. R-SB required a shorter fermentation time (1 h at 40 °C) and, therefore, the higher total isoflavone

concentration compared to SD-SB is expected based on the data observed with

Y-SB and SD-SB. Regardless of the lower total concentration of isoflavones observed in SD-SB, however, a significantly higher concentration (p < 0.01) of isoflavone aglycones was observed in SD-SB relative to R-SB (125 and 80.3 nmol/g, respectively). Additionally, R-SB contained significantly higher concentrations of simple β-glucosides and acetyl-glucosides compared to SD-SB

(p < 0.001). Malonyl-glucosides, however, did not vary significantly between the

2 bread types, with R-SB and SD-SB containing 170 and 162 nmol/g, respectively.

Table 5.3. Isoflavone composition (aglycones and various glucosides given in nmol/g) of R-SB and SD-SB, with significance established at p < 0.05. R-SB SD-SB

Aglycone (nmol/g) 80.3 ± 10.3a 125 ± 4.10b

Simple β-glucoside (nmol/g) 172 ± 0.969a 77.2 ± 2.82b

Acetyl-glucoside (nmol/g) 55.4 ± 2.69a 25.7 ± 2.06b

Malonyl-glucoside (nmol/g) 170 ± 7.80a 162 ± 7.43a

Total (nmol/g) 478 ± 21.7a 390 ± 14.2b

Figure 5.5 displays the isoflavone profiles of these 2 breads as collective aglycones, simple β-glucosides, acetyl-glucosides, and malonyl-glucosides. The proportion of aglycones in SD-SB was almost double of that in R-SB (32% and

17%, respectively) and correspondingly, the simple β-glucoside pool of SD-SB 61

was significantly smaller than that of R-SB (p < 0.001). The proportions of acetyl- and malonyl-glucosides, also, varied significantly between bread types (p <

0.001), although the malonyl-conjugate concentration present in the 2 breads did not vary significantly. The malonyl-glucosides represented one of the largest pools of isoflavones in both bread types, suggesting preference for this isoflavone form in both dough and bread systems. Furthermore, the isoflavone profiles of R-SB and SD-SB were reevaluated after 10 days of room temperature storage, and were shown to remain stable with storage time (Figure 5.6). This finding suggests that the isoflavone profile achieved in a dough system can be stabilized upon baking, and remain shelf-stable for sustained delivery of the achieved isoflavone profile to the target population.

Figure 5.5. Isoflavone profiles (aglycones and various glucosides given as % of total isoflavone concentration) for R-SB and SD-SB.

Figure 5.6. Overlay of chromatograms (obtained from Agilent HPLC) displaying the isoflavone profile of fresh and stored SD-SB.

5.2 Aim 2: Physical and thermal properties of sourdough and yeast-

leavened dough, and fresh and stored sourdough soy bread and

regular soy bread

5.2.1 Dough extensibility

Dough extensibility measures were obtained to assess the effect of sourdough fermentation on the gluten performance and overall extensibility of soy-supplemented wheat dough, as compared to yeast-leavened, soy- supplemented wheat dough. Figure 5.7 displays the resulting extensograms for soy-supplemented wheat doughs obtained by (A) sourdough fermentation and

(B) fermentation with traditional baker’s yeast, with each line representing 1 of 7 replicates for the given dough batch. Both dough types displayed very minor equilibrium responses, suggesting the presence of strong, well-developed gluten networks (Anderssen and others 2004). Table 5.4 displays the maximum load

(N) and the extension achieved at maximum load (mm) for the soy-supplemented wheat dough fermented with traditional baker’s yeast (R-SD), and for the soy- supplemented wheat dough obtained by sourdough fermentation (SD-SD). The

R-SD displayed a significantly greater maximum load and extension at maximum load compared to SD-SD (p < 0.001). This data suggests a greater polymer network and, therefore, greater dough strength, in the R-SD compared to the SD-

SD (Abang Zaidel and others 2008).

Figure 5.7. Extensograms displaying the load (N) versus hook extension (mm) for soy-supplemented wheat dough obtained by (A) sourdough fermentation and (B) fermentation with traditional baker’s yeast.

Table 5.4. Maximum load (N) and extension at maximum load (mm) for R-SD and SD-SD. R-SD SD-SD

Maximum Load (N) 0.607 ± 0.111a 0.413 ± 0.131b

Extension at Maximum Load (mm) 29.7 ± 4.76a 17.1 ± 2.45b

These observations are in accordance with similar studies in which the effects of sourdough fermentation or acidification on wheat dough extensibility was investigated, with these observations generally being attributed to microbial proteolysis and dough acidification. In dough systems with a pH below 4.0, there is a substantial net positive charge and increased electrostatic repulsion that results in the reduction of intra- and intermolecular disulfide bonds, thereby, increasing gluten solubility and increasing access by endogenous and microbial proteolytic enzymes (Thiele and others 2002; Schober and others 2003; Arendt and others 2007). Low molecular weight glutenins are, thereby, partially hydrolyzed during sourdough fermentation, with the gliadin fraction of gluten being degraded to a lesser extent (Gänzle and others 2008). The result of this proteolytic activity is an improvement in bread flavor due to the accumulation of amino acids that acts as precursors to flavor volatiles, as well as a large reduction in dough elasticity (Clarke and others 2004; Arendt and others 2007).

The given microbial activity of the dough, presumed proteolytic activity, and relatively low pH (4.22 ± 0.0985) are, therefore, the likely causative agents for the observed effects of sourdough fermentation on dough extensibility.

5.2.2 Specific loaf volume

The specific loaf volume (cm3/g) of regular soy bread (R-SB) and sourdough soy bread (SD-SB) are presented in Table 5.5. Loaf volume was determined by rapeseed displacement, and loaf weights were measured using an analytical scale after 3 h of cooling. The specific loaf volumes of R-SB and SD-

SB were 2.32 and 2.21, respectively, and were not statistically different.

Sourdough fermentation has been reported to have both positive and negative effects on dough structure and texture and, therefore, various effects on loaf volume have been observed with sourdough fermentation.

Table 5.5. Specific loaf volumes (cm3/g) for R-SB and SD-SB, with significance established at p < 0.05. R-SB SD-SB

Specific loaf volume (cm3/g) 2.31 ± 0.0966a 2.21 ± 0.0695a

In substantially acidic sourdough systems (pH < 4), the reduction of intra- and intermolecular disulphide bonds is encouraged resulting in the solubilization of gluten proteins and proteolytic degradation of the gluten network by endogenous and microbial proteases (Thiele and others 2002; Schober and others 2003; Clarke and others 2004; Arendt and others 2007). Degradation of the gluten network, in turn, results in decreased dough elasticity, decreased gas retention, and reduced loaf volume. Several strains of sourdough lactic acid bacteria (LAB), however, have been shown to produce exopolysaccharides

(EPS), such as fructan and glucan. These homopolysaccharides (HoPS) act as bodying agents, and may replace hydrocolloid additives currently used in industry

(Tieking and others 2003; Arendt and others 2007; Corsetti and Settanni 2007).

Given both the positive and negative effects of sourdough fermentation on the structural stability of dough and the resulting baked product, it is not surprising that sourdough fermentation did not exert significant effects on the specific loaf volume of the soy-supplemented wheat dough. While the substantially acidic dough (pH 4.22 ± 0.0985) may have resulted in considerable gluten degradation (as implied by its reduced dough extensibility), the potential for LAB present in the sourdough culture to produce HoPS may have negated these deleterious effects with regards to loaf volume.

5.2.3 Crust color

The crust color of R-SB and SD-SB was determined using a handheld colorimeter, which reported values of lightness (L) and the chromatic components, a* and b*, depicted in Figure 4.2. The crust of SD-SB was found to be significantly lighter than that of R-SB (p < 0.001), exhibiting L values of 36.7 and 33.5, respectively (Table 5.6). Maillard browning is a nonenzymatic reaction between amino acids and reducing sugars that results in the formation of brown pigments, and is the reaction responsible for the darker coloration of bread crust compared to bread crumb. Maillard browning is largely influenced by pH, with an optimum Maillard browning rate occurring at pH 10 and decreasing with increased acidity (Wolfram and others 1974; Ashoor and Zent 1984; Renn and

Sathe 1997). Therefore, the lighter crust color of the sourdough bread was

expected due to the significantly more acidic dough (pH 4.22) compared to the regular soy dough (pH 6.07) (p < 0.001).

Table 5.6. Values of lightness (L) and the chromatic components a* and b* for the crust of SD-SB and R-SB, as well as the corresponding dough pH prior to baking. SD-SB R-SB

Lightness (L) 36.7 ± 1.65a 33.5 ± 1.03b

Chromatic component a* +15.4 ± 1.18a +14.9 ± 0.946b

Chromatic component b* +21.9 ± 1.05a +16.7 ± 1.79b

Dough pH 4.22 ± 0.136a 6.07 ± 0.0503b

In addition to crust lightness, the chromatic components a* (ranging from red to green) and b* (ranging from yellow to blue) were also recorded. There was no significant difference in the chromatic component a*, with the SD-SB and the R-SB crusts displaying values of +15.4 and +14.9, respectively, indicating a slight red coloration to the crust. A significant difference in the chromatic component b* was observed however (p < 0.001), with SD-SB crust displaying a more yellow hue compared to that of R-SB (+21.9 and +16.7, respectively). The soy ingredients used in this study, also, display a natural yellow coloration; therefore, the more yellow crust of SD-SB may have been due to decreased masking of this natural hue by brown pigments formed during the Maillard

browning process. Images displaying the crust colors of SD-SB and R-SB are depicted in Figure 5.8.

Figure 5.8. Images displaying the crust color of SD-SB and R-SB.

5.2.4 Bread crumb hardness

Crumb hardness, or the force required to compress a 25 cm3 bread crumb sample to 40% it’s original height, was determined for fresh and stored R-SB and

SD-SB (Table 5.7). The fresh SD-SB bread crumb was significantly firmer than that of fresh R-SB (p < 0.05), exhibiting crumb hardness values of 17.0 and 13.54

N, respectively. This observation may have, in part, resulted from the decreased

elasticity of the dough and, therefore, decreased gas retention and resulting specific loaf volume (Arendt and others 2007). Because the specific loaf volume provides an inverse measure of density, a denser loaf would require more force to compress compared to a more porous, less dense structure.

Table 5.7. Bread crumb hardness (N) for fresh (day 1) and stored (day 10) SD- SB and R-SB, with significance established at p < 0.05. Fresh Stored

SD-SB R-SB SD-SB R-SB

Hardness (N) 17.0 ± 1.81a 13.5 ± 1.60b 22.4 ± 3.41c 15.5 ± 1.86ab

Bread crumb hardness after 10 days of room temperature storage was, also, evaluated for SD-SB and R-SB. One of the 3 R-SB loaves to be evaluated, however, displayed mold growth on the 7th day of storage and, as a result, was not evaluated for crumb hardness (Figure 5.9). In addition to the fungal growth observed with R-SB, the remaining R-SB loaves displayed a loss of crumb structure upon slicing (Figure 5.10), and evidence (exopolysaccharide production and uncharacteristic aroma volatiles) of bacterial growth and fermentation.

Figure 5.9. Image displaying mold growth on RSB after 10 days of storage.

Figure 5.10. Images of slices of (A) fresh R-SB, (B) stored R-SB, (C) fresh SD- SB, and (D) stored SD-SB.

These observations were evident in the crumb hardness of the stored R-

SB (15.5 N), which was not significantly different from the crumb of fresh R-SB

(13.5 N). The SD-SB, although microbially stable with storage time, did display a significant increase in bread crumb hardness (p < 0.05). Following storage, 22.4

N were required for 40% bread crumb compression of stored SD-SB, compared to 17.0 N required for compression of the fresh SD-SB crumb (Figure 5.11).

Figure 5.11. Compression curves displaying the compressive load (N) versus time (s) for (A) fresh SD-SB and (B) stored SD-SB.

5.2.5 Water properties of dough

Thermogravimetric analysis (TGA) and differential scanning calorimetry

(DSC) were utilized to determine the moisture content and distinguish between

FW and UFW populations of R-SD and SD-SD. Both dough types displayed similar moisture contents (%) as determined by TGA (Table 5.8). R-SD and SD-

SD exhibited weight losses of 42.47% and 42.91%, respectively, when a linear heating ramp was applied. Figure 5.12 displays a typical thermogram obtained

for SD-SD, depicting sample weight loss (%) with increasing temperature, as well as the derivative of weight loss (%/ºC). Two derivative peaks were evident in dough TGA thermograms, occurring at approximately 43 °C and 105 °C.

Simmons and others (2012) observed a similar trend, noting TGA derivative peaks near 55 °C and 112 °C in soy dough. Wheat dough analyzed, however, only exhibited a derivative peak at 112 °C, therefore, the derivative peak occurring at 55 °C in soy dough was attributed to the evaporation of water from soy protein.

Table 5.8. Moisture content (%) and water distribution properties for R-SD and SD-SD, as determined by TGA and DSC. R-SD SD-SD

Moisture content (%) 42.5 ± 0.423a 42.9 ± 0.936a

Ice melt enthalpy (J/g) 88.8 ± 2.57a 94.9 ± 3.06b

Ice melt peak temperature (ºC) -1.70 ± 0.683a -3.91 ± 0.728b

FW (%) 26.7 ± 0.773a 28.5 ± 0.918b

UFW (%) 15.8 ± 0.975a 14.42 ± 1.34b

Figure 5.12. Typical TGA thermogram obtained for SD-SD, displaying sample weight loss (%) and the derivative of weight loss (%/ºC) with increasing temperature (ºC).

FW and UFW populations were distinguished by DSC. Endothermic peaks occurring near 0 ºC were attributed to ice melting (Reid and others 1993;

Vittadini and Vodovotz 2003) and, therefore, represented the FW present in the sample. SD-SD displayed a significantly larger endothermic peak occurring at this temperature (p < 0.001) and, therefore, exhibited a larger FW population compared to R-SD (p < 0.001). This endothermic peak (shown in Figure 5.13), also, occurred at lower temperatures in SD-SD compared to R-SD (-4.12 and -

1.75 ºC, respectively) (p < 0.001), suggesting that the FW population of SD-SD was more tightly bound to dough macromolecules.

Figure 5.13. Thermogram displaying the endothermic peak associated with ice melting for R-SD and SD-SD.

5.2.6 Thermal analysis of fresh and stored bread

TGA and DSC were, also, utilized to determine the moisture content (%) and water populations of fresh and stored R-SB and SD-SB. Additionally, DSC thermograms of fresh and stored bread samples were analyzed for transitions associated with bread crumb staling. These transitions include the melting of amylopectin crystals occurring between 40 and 60 ºC, and the melting of amylose-lipid complexes between 100 and 130 ºC (Lodi and Vodovotz 2008).

The moisture properties and thermal transitions of interest were fairly similar

between fresh R-SB and SD-SB, and exhibited similar trends upon storage

(Table 5.9). Exceptions to this generalization were the significantly lower peak temperature associated with ice melting in fresh SD-SB compared to fresh R-SB

(p < 0.05), and significantly lower enthalpy associated with melting of amylose- lipid complexes in fresh and stored SD-SB compared to fresh and stored R-SB (p

< 0.05).

Table 5.9. Moisture and thermal properties for fresh and stored R-SB and SD-SB, with significance established at p < 0.05. R-SB SD-SB

Fresh Stored Fresh Stored

Moisture content (%) 42.3 ± 0.551a 41.7 ± 0.589a 42.0 ± 0.982a 42.0 ± 0.517a

Ice melt enthalpy (J/g) 82.4 ± 3.82a 70.6 ± 4.53b 83.6 ± 2.12a 75.3 ± 4.67b

Ice melt peak temperature (ºC) -2.57 ± 0.374a -3.62 ± 0.531bc -3.45 ± 0.409b -4.14 ± 0.321c

FW (%) 24.7 ± 1.15a 21.2 ± 1.36b 25.1 ± 0.637a 22.6 ± 1.40b

a b a b

79 UFW (%) 17.6 ± 0.985 20.5 ± 1.88 16.9 ± 1.11 19.4 ± 1.71

Amylopectin melt enthalpy (J/g) 0.375 ± 0.131a 1.51 ± 0.233b 0.402 ± 0.139a 1.54 ± 0.164b

Amylose-lipid melt enthalpy (J/g) 1.39 ± 0.150a 1.09 ± 0.115b 0.682 ± 0.196c 0.706 ± 0.0671c

Amylose-lipid complex formation is hypothesized to occur during and/or immediately after baking (Czuchajowska and Pomeranz 1989), and to remain stable with storage time (Davidou and others 1996; Lodi and Vodovotz 2008).

Given that the initial ingredient composition of SD-SD was minimally modified from that of R-SD, the discrepancy in enthalpies associated with the melting of amylose-lipid complexes of SD-SB and R-SB is likely attributable to the sourdough fermentation process. The decreased concentration of amylose-lipid complexes in SD-SB, as indicated by DSC, may have occurred as a result of amylolytic activity of the LAB present in the sourdough starter culture. While amylolytic activity of LAB is strain specific, several strains of Lactobacillus plantarum have been shown to exhibit this activity (Giraud and others 1991;

Corsetti and others 1998); therefore, Lactobacilus plantarum present in the sourdough starter culture may have resulted in a smaller amylose pool available for complexation with lipids.

The complexation of lipid with amylose is believed to result in a decrease in starch retrogradation, however, this has only been shown to be effective upon extended storage (Davidou and others 1996). This finding is further supported by the similar trends observed for amylopectin recrystallization occurring over short-term (10-day) storage in 2 breads with significantly different concentrations of amylose-lipid complexes (R-SB and SD-SB). Figure 5.14 displays the endothermic peak associated with melting of amylose-lipid complexes in fresh R-

SB and SD-SB. Figure 5.15 displays the transition associated with the melting of amylopectin in fresh and stored SD-SB.

Figure 5.14. Thermogram displaying the endothermic peak associated with melting of amylose-lipid complexes in fresh SD-SB and R-SB.

Figure 5.15. DSC thermogram displaying the transition associated with the melting of amylopectin crystals in fresh and stored SD-SB.

The moisture contents (%) of R-SB and SD-SB were determined to be

42.3% and 42.0%, respectively. These values were only slightly lower than those observed in R-SD and SD-SD samples, which may be attributed to excessive evaporative losses occurring in the dough samples prior to analysis.

Fresh bread samples displayed significantly lower FW populations (24.7% and

25.1% for R-SB and SD-SB, respectively), however, compared to R-SD and SD-

SD (26.7% and 28.5%, respectively)(p < 0.05). This data suggests that substantial evaporative moisture loss did occur during the baking process and, therefore, supports the logic that the moisture content of dough samples was not

accurately measured by TGA. Figure 5.16 displays the moisture contents and

FW and UFW populations of SD-SD and fresh and stored SD-SB.

Figure 5.16. Moisture contents and FW and UFW populations (% of total sample weight) for SD-SD and fresh and stored SD-SB.

The moisture content of R-SB and SD-SB crumb remained stable over 10 days of storage, exhibiting moisture contents of 41.7% and 42.0%, respectively.

Moisture migration from bread crumb to crust is a critical event associated with the staling of wheat bread (Baik and Chinachoti 2001). The increased water- holding capacity of soy flour, however, has been shown to decrease the amount of moisture redistribution occurring with bread storage (Vittadini and Vodovotz

2003; Lodi and others 2007). Vittadini and Vodovotz (2003) observed moisture loss proportional to the amount of soy flour included in the bread formulation,

with moisture loss in soy breads ranging from 1.7% to 3.1% over 7 days of

storage. This study did not specify whether bread samples were stored intact or

sliced, however. Given this, it is plausible that intact soy bread containing

approximately 26% soy ingredients (dry basis, w/w) would exhibit a stable bread

crumb moisture content over 10 days of storage.

5.3 Aim 3: Overall acceptability of sourdough soy bread and regular soy

bread, and preference between the two, as determined by sensory

analysis

The overall liking of R-SB and SD-SB was determined by sensory analysis

using a 9-point hedonic scale, with a rating of 1 corresponding to “dislike

extremely, a rating of 9 corresponding to “like extremely”, and a rating of 5

corresponding to “neither like nor dislike”. Preference for either R-SB or SD-SB

was determined by a paired comparison, forced choice preference test via ballot

entry (Appendix F), whereas overall liking for samples was determined via an

electronic questionnaire (Appendix E). Overall liking of SD-SB (4.82) was found

to be significantly less (p < 0.001) than that of R-SB (6.93), and consequently, 47

out of 55 participants indicated preference for the R-SB (p < 0.001) (Stone and

others 2012).

Sensory studies are frequently utilized to describe the flavor and aroma

components of sourdough bread (Hansen and Hansen 1996; Kirchhoff and

Schieberle 2001; Meignen and others 2001); however, few studies have been

conducted that directly compare the overall liking of sourdough bread compared

to yeast-leavened bread. The sensory attributes of sourdough bread greatly

depend upon the fermenting organisms present and the implemented fermentation conditions. Hansen and Hansen (1996) found that bread produced with 15% sourdough fermented with Lactobacillus plantarum was perceived as

“very sour”, “metallic”, and imparting a “bitter aftertaste”. Similarly, sensory analysis of sourdough wheat bread by Meignen and others (2001) resulted in high ratings of descriptors such as “acid”, “bitter”, and “vinegar taste” for sourdough fermented with a mixed culture of Lactobacillus brevis and

Saccharomyces cerevisiae. Sensory analysis of SD-SB produced similar descriptors, including “very acidic”, “very sour”, “vinegar”, and “bitter aftertaste”, which most likely attributed to the lower score for overall liking obtained for this sample.

Furthermore, participants were not informed of the varieties of bread that they would be sampling. Only 8 individuals indicated sourdough as being their preferred variety of bread, whereas 11 individuals preferred “white” bread and 35 individuals indicated “whole wheat” as their preferred bread variety when polled via an electronic questionnaire (Appendix E). The indicated preference for yeast-leavened breads amongst participants may have been a source of bias, and resulted in the overall preference for R-SB. Given this, participant recruitment based on an equally-distributed preference for sourdough and yeast- leavened bread varieties may have been a more appropriate means for discerning accurate measures of overall liking for the 2 bread varieties, and for determining preference between the two.

CHAPTER 6: CONCLUSIONS

6.1 Aim 1: Quantitative and qualitative assessment of the isoflavone

profile of fermenting sourdough, and fresh and stored sourdough

soy bread and regular soy bread by RP-HPLC

The isoflavone profiles of 4 dough formulations were evaluated at the beginning and end of a 9 h proofing period in order to elucidate the mechanism of isoflavone conversion observed during fermentation of SD-SD. The isoflavone profile of the initial soy ingredients (SM) was determined to be comprised of primarily malonyl-glucosides (70%) and simple β-glucosides (27%). Analysis of the isoflavone profile of the 4 dough types prior to fermentation (T0) indicated the substantial effect of dough assembly on the isoflavone profile. All 4 dough types exhibited substantially lower malonyl-conjugate pools and, correspondingly, larger simple β-glucoside pools, most likely due to the addition of water and application of heat during dough mixing and kneading. This observation was less pronounced in SD-SD, whose isoflavone profile most closely resembled that of the initial soy ingredients. The larger malonyl-conjugate pool of SD-SD may have been partially attributed to the increased stability of this isoflavone form under acidic conditions.

At the end of the 9 h proofing period, the dough fermented by the LAB,

Lactobacillus brevis and Lactobacillus plantarum (LAB-SD), exhibited the 86

greatest proportional increase in isoflavone aglycones and, correspondingly, the greatest proportional decrease in isoflavone simple β-glucosides. This observation may be attributed to the potential β-glucosidase activity of the specific strains used in production of the preferment. Furthermore, this finding suggests that the presence of Saccharomyces cerevisiae present in the sourdough starter culture may hinder the deglucosylation of isoflavone simple β- glucosides to aglycones, since this conversion was less pronounced in SD-SD.

Regardless, sourdough fermentation of soy dough successfully resulted in a comparable proportional increase in isoflavone aglycones observed in soy dough upon the addition of almond powder (5% w/w/), with R-SB and SD-SB containing approximately 17% and 32% of isoflavones in the aglycone form, respectively. Furthermore, the isoflavone profiles observed in R-SB and SD-SB remained unchanged following 10 days of room temperature storage. These findings suggest that sourdough fermentation results in a soy bread variety that may be used in clinical trials for the sustained delivery of twice the amount of isoflavone aglycones achieved in the regular soy bread variety, without the addition of major allergens.

6.2 Aim 2: Physical and thermal properties of sourdough and yeast-

leavened dough, and fresh and stored sourdough soy bread and

regular soy bread

The physical and thermal properties of sourdough and yeast-leavened soy dough, and fresh and stored sourdough and regular soy bread were evaluated to determine the effects of sourdough fermentation on the loaf quality of a U.S.

patented, high soy protein-containing bread. Both soy sourdough (SD-SD) and regular soy dough (R-SD) displayed moisture contents of approximately 43%, however, SD-SD exhibited a significantly larger FW population compared to R-

SD. Soy sourdough was determined to be significantly less extensible than R-

SD, which may have resulted in decreased gas retention of the dough and, in- turn, a smaller specific loaf volume and increased crumb hardness of the sourdough soy bread (SD-SB) compared to the regular soy bread (R-SB).

Sourdough fermentation did result in a significantly lighter crust in SD-SB compared to R-SB, however, which was attributed to the significantly lower pH of

SD-SD compared to R-SD. Furthermore, SD-SB appeared to be substantially more shelf-stable than R-SB. After 7 days of room temperature storage, 1 of 3 loaves of R-SB displayed substantial mold growth, with the other 2 loaves exhibiting signs of bacterial growth upon analysis at day 10 (pungent off-odors, exopolysaccharide production, and loss of crumb structure). Despite the difference observed with the microbial stability of the 2 breads, both SD-SB and

R-SB exhibited similar trends in starch retrogradation upon storage.

6.3 Aim 3: Overall acceptability of sourdough soy bread and regular soy

bread, and preference between the two, as determined by sensory

analysis

Sensory analysis was conducted to determine the sensory acceptability of

SD-SB compared to R-SB. Overall liking of SD-SB and R-SB was determined on a 9-point hedonic scale, and preference between the 2 breads was determined by a paired comparison, forced choice preference test. The overall liking of R-SB

was significantly higher than that of SD-SB and, consequently, R-SB was the preferred bread amongst the majority of participants. Participants described SD-

SB as being “very acidic” and “very sour”, which may have been a reflection of the overall participant preference for whole wheat and white bread varieties.

Participant recruitment based on an equally-distributed preference for sourdough and yeast-leavened bread varieties may be a more appropriate means for discerning accurate measures of liking and preference between 2 distinctly different bread varieties in future studies.

Future work

Several strains of lactic acid bacteria have been shown to exhibit β- glucosidase activity, with the activity varying tremendously across strains.

Therefore, there is potential to further optimize the deglucosylation of isoflavone simple β-glucosides by sourdough fermentation with lactic acid bacteria with high expression of β-glucosidase activity.

Furthermore, the sensory attributes and overall loaf quality of sourdough bread can vary substantially based on the fermenting microorganisms present, the substrates made available to them, and the fermentation conditions employed. Therefore, the sensory attributes of this product could be improved by altering one or more of these factors.

Lastly, appropriate solvents to optimize the extraction efficiency of isoflavones over extended fermentation should be investigated.

REFERENCES

AACC. 2000. Approved methods of the American Association of Cereal Chemists. 10th ed. St. Paul, MN: American Association of Cereal Chemists. 1200 p.

Abang Zaidel DN, Chin NL, Abdul Rahman R, Karim R. 2008. Rheological characterization of gluten from extensibility measurement. J Food Eng 86:549- 556.

Afolabi WAO, Oguntona CRB, Fakunmoju BB. 2001. Acceptability and chemical composition of bread from beniseed composite flour. Nutrition Food Sci 31(6):310-313.

Anderssen RS, Bekes F, Gras PW, Nikolov A, Wood JT. 2004. Wheat-flour dough extensibility as a discriminator for wheat varieties. J Cereal Sci 39:195- 203.

Arendt EK, Ryan LAM, Dal Bello F. 2007. Impact of sourdough on the texture of bread. Food Microbiol 24:165-174.

Ashoor SH, Zent JB. 1984. Maillard Browning of Common Amino Acids and Sugars. J Food Sci 49:1206-1207.

Baik M, Chinachoti P. 2000. Moisture Redistribution and Phase Transitions During Bread Staling. Cereal Chem 77(4):484-488.

Baik M, Chinachoti P. 2001. Effects of Glycerol and Moisture Gradient on Thermomechanical Properties of White Bread. J Agr Food Chem 49:4031-4038.

Carnevali P, Ciati R, Leporati A, Paese M. 2007. Liquid sourdough fermentation: Industrial application perspectives. Food Microbiol 24(2):150-154.

Carpenter RP, Lyon DH, Hasdell TA [Ed]. 2000. Guidelines for Sensory Analysis in Food Product Development and Quality Control. 2nd ed. Gaithersburg, MD: Aspen Publishers, Inc. 210 p.

Chien H, Huang H, Chou C. 2006. Transformation of isoflavone phytoestrogens during the fermentation of soymilk with lactic acid bacteria and bifidobacteria. Food Microbiol 23:772-778.

Clarke CI, Schober TJ, Dockery P, O’Sullivan K, Arendt EK. 2004. Wheat Sourdough Fermentation: Effects of Time and Acidification on Fundamental Rheological Properties. Cereal Chem 81(3):409-417.

Clydesdale FM. 1978. Colorimetry: methodology and applications. CRC Cr Rev Food Sci 10(3):243-301.

Corsetti A, Gobbetti M, Balestrieri F, Paoletti F, Russi L, Rossi J. 1998. Sourdough Lactic Acid Bacteria Effects Bread Firmness and Staling. J Food Sci 63(2):347-351.

Corsetti A, Settanni L. 2007. Lactobacilli in sourdough fermentation. Food Res Int 40:539-558.

Coward L, Barnes N, Setchell K, Barnes S. 1993. Genistein, Daidzein, and Their β-Glycoside Conjugates: Antitumor Isoflavones in Soybean Foods from American and Asian Diets. J Agr Food Chem 41(11):1961-1967.

Coward L, Smith M, Kirk M, Barnes S. 1998. Chemical modification of isoflavones in soyfoods during cooking and processing. Am J Clin Nutr 68(suppl):1486S-1491S.

Crowley P, Schober TJ, Clarke CI, Arendt EK. 2002. The effect of storage time on textural and crumb grain characteristics of sourdough wheat bread. Eur Food Res Technol 214:489-496.

Czuchajowska Z, Pomeranz Y. 1989. Differential scanning calorimetry, water activity, and moisture contents in crumb center and near crust zones of bread during storage. Cereal Chem 66:305-309.

Davidou S, Meste ML, Debever E, Bekaert D. 1996. A contribution to the study of staling of white bread: effect of water and hydrocolloid. Food Hydrocolloid 10(4):375-383.

Day AJ, Cañada FJ, Díaz JC, Kroon PA, Mclauchlan R, Faulds CB, Plumb GW, Morgan MRA, Williamson G. 2000. Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase. FEBS Lett 468:166-170.

De Angelis M, Gallo G, Settanni L, Corbo MR, McSweene PLH, Gobbetti M. 2005. Purification and Characterization of an Intracellular Family 3 β-Glucosidase from Lactobacillus sanfranciscensis CB1. Ital J Food Sci 17:131-142.

De Angelis M, Stossi F, Waibel M, Katzenellenbogen BS, Katzenellenbogen JA. 2005. Isocoumarins as estrogen receptor beta selective ligands: Isomers of

isoflavone phytoestrogens and their metabolites. Bioorgan Med Chem 13:6529- 6542.

Decock P, Cappelle S. 2005. Bread technology and sourdough technology. Trends Food Sci Tech 16:113-120.

De Vuyst L, Vancanneyt M. 2007. Biodiversity and identification of sourdough lactic acid bacteria. Food Microbiol 24:120-127.

De Vuyst L, Vrancken G, Ravyts F, Rimaux T, Weckx S. 2009. Biodiversity, ecological determinants, and metabolic exploitation of sourdough microbiota. Food Microbiol 26:666-675.

Di Cagno R, Mazzacane F, Rizzello CG, Vincentini O, Silano M, Giuliani G, De Angelis M, Gobbetti M. 2010. Synthesis of Isoflavone Aglycones and Equol in Soy Milks Fermented by Food-Related Lactic Acid Bacteria and Their Effect on Human Intestinal Caco-2 Cells. J Agr Food Chem 58:10338-10346.

Erdman JW Jr, Fordyce EJ. 1989. Soy products and the human diet. Am J Clin Nutr 49:725-737.

Fessas D, Schiraldi A. 2001. Water properties in wheat flour dough I: Classical thermogravimetry approach. Food Chem 72:237-244.

Food and Drug Administration. 1999. Food labeling: health claims; soy protein and coronary heart disease; final rule. Fed Regist 64:57699-57733.

Friedman M, Brandon DL. 2001. Nutritional and Health Benefits of Soy Proteins. J Agr Food Chem 49(3):1069-1086.

Gänzle MG, Loponen J, Gobbetti M. 2008. Proteolysis in sourdough fermentations: mechanisms and potential for improved bread quality. Trends Food Sci Tech 19:513-521.

Gänzle MG, Vermeulen N, Vogel RF. 2007. Carbohydrate, peptide and lipid metabolism of lactic acid bacteria in sourdough. Food Microbiol 24:128-138.

Giraud E, Brauman A, Keleke S, Lelong B, Raimbault M. 1991. Isolation and physiological study of an amylolytic strain of Lactobacillus plantarum. Appl Microbiol Biot 36:379-383.

Gobbetti M. 1998. The sourdough microflora: Interactions of lactic acid bacteria and yeasts. Trends Food Sci Tech 9:267-274.

Gobbetti M, De Angelis M, Corsetti A, Di Cagno R. 2005. Biochemistry and physiology of sourdough lactic acid bacteria. Trends Food Sci Tech 16:57-69.

Hansen Å, Hansen B. 1996. Flavour of sourdough wheat bread crumb. Z Lebensm Unters Forsch 202:244-249.

Hussain M, Banerjee M, Sarkar FH, Djuric Z, Pollak MN, Doerge D, Fontana J, Chinni S, Davis J, Forman J, Wood DP, Kucuk O. 2003. Soy Isoflavones in the Treatment of Prostate Cancer. Nutr Cancer 47(2):111-117.

Izumi T, Piskula MK, Osawa S, Obata A, Tobe K, Saito M, Kataoka S, Kubota Y, Kikuchi M. 2000. Soy Isoflavone Aglycones Are Absorbed Faster and in Higher Amounts than Their Glucosides in Humans. J Nutr 130:1695-1699.

Kirchhoff E, Schieberle P. 2001. Determination of Key Aroma Compounds in the Crumb of a Three-Stage Sourdough Rye Bread by Stable Isotope Dilution Assays and Sensory Studies. J Agr Food Chem 49:4304-4311.

Lacaze G, Wick M, Cappelle S. 2007. Emerging fermentation technologies: Development of novel sourdoughs. Food Microbiol 24:155-160.

Larkin T, Price WE, Astheimer L. 2008. The Key Importance of Soy Isoflavone Bioavailability to Understanding Health Benefits. CRC CR Rev Food Sci 48:538- 552.

Lee J, Renita M, Fioritto RJ, Martin SKS, Schwartz SJ, Vodovotz Y. 2004. Isoflavone Characterization and Antioxidant Activity of Ohio Soybeans. J Agr Food Chem 52:2647-2651.

Lodi A. 2006. Physico-chemical and Molecular Characterization of Soy Bread Containing Almond. [DPhil dissertation]. Columbus, OH: The Ohio State University. 194 p.

Lodi A, Abduljalil AM, Vodovotz Y. 2007. Characterization of water distribution in bread during storage using magnetic resonance imaging. Magn Reson Imaging 25:1449-1458.

Lodi A, Vodovotz Y. 2008. Physical properties and water state changes during storage in soy bread with and without almond. Food Chem 110:554-561.

Lotong V, Chambers E, Chambers DH. 2000. Determination of the sensory attributes of wheat sourdough bread. J Sens Stud 15:309-326.

Mathias K, Ismail B, Corvalan C, Hayes K. 2006. Heat and pH Effects on the Conjugated Forms of Genistin and Daidzin Isoflavones. J Agr Food Chem 54:7495-7502.

Meignen B, Onno B, Gélinas P, Infantes M, Guilois S, Cahagnier B. 2001. Optimization of sourdough fermentation with Lactobacillus brevis and baker’s yeast. Food Microbiol 18:239-245.

Michlmayr H, Schümann C, Barreira Braz de Silva NM, Kulbe KD, Del Hierro AM. 2010. Isolation and basic characterization of a β-glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture. J Appl Microbiol 108:550-559.

Morgenstern MP, Newberry MP, Holst SE. 1996. Extensional Properties of Dough Sheets. Cereal Chem 73(4):478-482.

Moroni AV, Dal Bello F, Arendt EK. 2009. Sourdough in gluten-free bread- making: An ancient technology to solve a novel issue? Food Microbiol 26:676- 684.

Murphy PA, Barua K, Hauck CC. 2002. Solvent extraction selection in the determination of isoflavones in soy foods. J Chromatogr B 777:129-138.

Pyo Y, Lee T, Lee Y. 2005. Enrichment of bioactive isoflavones in soymilk fermented with β-glucosidase-producing lactic acid bacteria. Food Res Int 38:551-559.

Reid DS, Hsu J, Kerr W. 1993. Calorimetry. In: Blanshard JMV, Lillford PJ, editors. The glassy state in foods. Nottingham, U.K.: Nottingham University Press. p 123-132.

Renn PT, Sathe SK. 1997. Effects of pH, Temperature, and Reactant Molar Ratio on L-Leucine and D-Glucose Maillard Browning Reaction in an Aqueous System. J Agr Food Chem 45:3782-3787.

Riedl KM, Zhang YC, Schwartz SJ, Vodovotz Y. 2005. Optimizing Dough Proofing Conditions To Enhance Isoflavone Aglycones in Soy Bread. J Agr Food Chem 53:8253-8258.

Röcken W, Rick M, Reinkemeijer M. 1992. Controlled production of acetic acid in wheat sour doughs. Z Lebensm Unters Forsch 195:259-263.

Rose DJ, Ogden LV, Dunn ML, Jamison RG, Lloyd MA, Pike OA. 2011. Quality and Sensory Characteristics of Hard Red Wheat after Residential Storage for up to 32 Y. J Food Sci 76(1):S8-S13.

Scalbert A, Williamson G. 2000. Dietary Intake and Bioavailability of Polyphenols. J Nutr 130:2073S-2085S.

Schober TJ, Dockery P, Arendt EK. 2003. Model studies for wheat sourdough systems using gluten, lactate buffer and sodium chloride. Eur Food Res Technol 217:235-243.

Sestelo ABF, Poza M, Villa TG. 2004. β-Glucosidase activity in a Lactobacillus plantarum wine strain. World J Microb Biot 20:633-637.

Setchell KDR. 1998. Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. Am J Clin Nutr 68(suppl):1333S-46S.

Setchell KDR, Cassidy A. 1999. Dietary Isoflavones: Biological Effects and Relevance to Human Health. J Nutr 129:758S-767S.

Setchell KDR, Brown NM, Zimmer-Nechemias L, Brashear WT, Wolfe BE, Kirschner AS, Heubi JE. 2002. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability. Am J Clin Nutr 76:447-453.

Simmons AL, Smith KB, Vodovotz Y. 2012. Soy ingredients stabilize bread dough during frozen storage. J Cereal Sci (Forthcoming).

Song TT, Barua K, Buseman G, Murphy PA. 1998. Soy isoflavone analysis: quality control and a new internal standard. Am J Clin Nutr 68(suppl):1474S- 1479S.

Song TT, Hendrich S, Murphy, PA. 1999. Estrogenic Activity of Glycitein, a Soy Isoflavone. J Agr Food Chem 47:1607-1610.

Stone H, Bleibaum R, Thomas HA, editors. 2012. Sensory Evaluation Practices. 4th ed. San Diego, CA: Academic Press. 446 p.

Swami S, Krishnan AV, Moreno J, Bhattacharyya RS, Gardner C, Brooks JD, Peehl DM, Feldman D. 2009. Inhibition of prostaglandin synthesis and actions by genistein in human prostate cancer cells and by soy isoflavones in prostate cancer patients. Int J Cancer 124:2050-2059.

Takeda K, Matsumura Y, Shimizu M. 2001. Emulsifying and Surface Properties of Wheat Gluten under Acidic Conditions. J Food Sci 66(3):393-399.

Tanaka K, Furukawa K, Matsumoto H. 1967. The effect of acid and salt on the farinogram and extensogram of dough. Cereal Chem 44:675-680.

Thiele C, Gänzle MG, Vogel RF. 2002. Contribution of Sourdough Lactobacilli, Yeast, and Cereal Enzymes to the Generation of Amino Acids in Dough Relevant for Bread Flavor. Cereal Chem 79(1):45-51.

Tieking M, Korakli M, Ehrmann MA, Gänzle MG, Vogel RF. 2003. In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria. Appl Environ Microb 69(2):945-952.

Tsen, CC. 1966. A note on effects of pH on sulfhydryl groups and rheological properties of dough and its implication with the sulfhydryl-disulfide interchange. Cereal Chem 43:456-460.

Vittadini E, Vodovotz Y. 2003. Changes in the Physicochemical Properties of Wheat- and Soy-containing Breads During Storage as Studied by Thermal Analyses. J Food Sci 68:2022-2027.

Wang H, Murphy P. 1996. Mass Balance Study of Isoflavones during Soybean Processing. J Agr Food Chem 44:2377-2383.

Weihua Z, Andersson S, Cheng G, Simpson ER, Warner M, Gustafsson J. 2003. Update on estrogen signaling. FEBS Lett 546:17-24.

Wilkinson AP, Wähälä K, Williamson G. 2002. Identification and quantification of polyphenol phytoestrogens in foods and human biological fluids. J Chromatogr B 777:93-109.

Wolfrom ML, Kashimura N, Horton D. 1974. Factors Affecting the Maillard Browning Reaction between Sugars and Amino Acids. Studies on the Nonenzymatic Browning of Dehydrated Orange Juice. J Agr Food Chem 22(5):796-800.

Xu X, Harris KS, Wang H, Murphy PA, Hendrich S. 1995. Bioavailability of Soybean Isoflavones Depends upon Gut Microflora in Women. J Nutr 125:2307- 2315.

Zhang YC, Lee JH, Vodovotz Y, Schwartz SJ. 2004. Changes in Distribution of Isoflavones and β-glucosidase Activity During Soy Bread Proofing and Baking. Cereal Chem 81(6):741-745.

APPENDIX A:

ISOFLAVONE AGLYCONE AND SIMPLE β-GLUCOSIDE STANDARD

CURVES ACQUIRED WITH AGILENT HPLC SYSTEM

Daidzein 12000 y = 2.0004E+06x 10000 R² = 9.9915E-01

8000

6000

4000 Peak Area (260 nm)

2000

0 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 6.00E-03 umol Injected

Daidzin 7000 y = 1.0108E+06x 6000 R² = 9.9987E-01

5000

4000

3000

2000 Peak Area (260 nm)

1000

0 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 6.00E-03 7.00E-03 umol Injected

Genistein 20000 18000 y = 2.5485E+06x 16000 R² = 9.9947E-01 14000 12000 10000 8000 6000 Peak Area (260 nm) 4000 2000 0 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 6.00E-03 7.00E-03 umol Injected

Genistin 8000 y = 1.1892E+06x 7000 R² = 9.9983E-01

6000

5000

4000

3000

Peak Area (260 nm) 2000

1000

0 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 6.00E-03 7.00E-03 umol Injected

6000 Glycitein

5000 y = 1.1613E+06x R² = 9.7685E-01

4000

3000

2000 Peak Area (260 nm)

1000

0 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 umol Injected

Glycitin 3500 y = 8.2671E+05x 3000 R² = 9.9976E-01

2500

2000

1500

Peak Area (260 nm) 1000

500

0 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 umol Injected

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APPENDIX B:

APPROVAL LETTER FOR EXEMPTION FROM IRB REVIEW

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APPENDIX C:

SENSORY ANALYSIS RECRUITMENT LETTER

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From: Yael Vodovotz ([email protected]) To: Graduate Listserve Undergrad Listserve Cc: [email protected] Subject: Help needed in evaluating two soy bread varieties!

The Sensory Analysis Laboratory, Food Science & Technology Date: June 4th, 2012 Time: 10:00 AM – 4:00 PM Place: Parker Food Science & Technology Building, Room 122 Sensory Laboratory, 2015 Fyffe Ct., Columbus OH (Building 64)

Your help is needed in evaluating soy bread! In this study, you will be evaluating two varieties of soy bread for overall acceptability, and indicating your preference between the two. A brief demographic survey will conclude the study. This study should not take more than 5 minutes of your time!

• You must be at least 18 years of age to participate. • Individuals who are allergic to wheat and/or soy are discouraged from participating Answers will be submitted both electronically and by ballot submission. Your participation is completely voluntary and you may choose to leave the study at any time. All responses will remain confidential and will not be linked to your identity. Questions regarding this study may be directed to Gabrielle Yezbick ([email protected]).

Thank you!

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APPENDIX D:

SENSORY ANALYSIS INFORMED CONSENT FORM

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The Ohio State University Consent to Participate in Research

Study Title: Overall Acceptability of Soy Bread and Sourdough Soy Bread Researcher: Dr. Yael Vodovotz

This is a consent form for research participation. It contains important information about this study and what to expect if you decide to participate.

Your participation is voluntary. Please consider the information carefully. Feel free to ask questions before making your decision whether or not to participate. If you decide to participate, you will be asked to sign this form and will receive a copy of the form.

Purpose: The purpose of this study is to determine the consumer acceptability of a soy bread and a sourdough soy bread, and to determine consumer preference between the two.

Procedures/Tasks: You will be given two soy bread samples (one sourdough and one regular) and asked to evaluate the two based on your overall liking. You will, then, be asked which sample you prefer. Following this, you will be asked to complete a brief demographic survey.

Duration: The study will last approximately 5 minutes. You may leave the study at any time. If you decide to stop participating in the study, there will be no penalty to you. Your decision will not affect your future relationship with The Ohio State University.

Risks and Benefits: The risks associated with this protocol are expected to be minimal unless you have a known allergy to wheat and/or soy. If you have these allergies, you are discouraged from participating in this study. Moreover, if you have not eaten any foods containing these items (wheat and soy) before this study, you will be discouraged from participating, but not disallowed.

Confidentiality: Efforts will be made to keep your study-related information confidential. However, there may be circumstances where this information must be released. For example, personal information regarding your participation in this study may

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be disclosed if required by state law. Also, your records may be reviewed by the following groups (as applicable to the research): • Office for Human Research Protections or other federal, state, or international regulatory agencies; • The Ohio State University Institutional Review Board or Office of Responsible Research Practices; • The sponsor, if any, or agency (including the Food and Drug Administration for FDA-regulated research) supporting this study.

Participant Rights: You may refuse to participate in this study without penalty or loss of benefits to which you are otherwise entitled. If you are a student or employee at Ohio State, your decision will not affect your grades or employment status.

If you choose to participate in the study, you may discontinue participation at any time without penalty or loss of benefits. By signing this form, you do not give up any personal legal rights you may have as a participant in this study.

Contacts and Questions: For questions, concerns, or complaints about the study, you may contact Gabrielle Yezbick, the study coordinator, by email ([email protected]) or by telephone (614-247-7686).

For questions about your rights as a participant in this study or to discuss other study-related concerns or complaints with someone who is not part of the research team, you may contact Ms. Sandra Meadows in the Office of Responsible Research Practices at 1-800-678-6251.

If you are injured as a result of participating in this study or for questions about a study-related injury, you may contact Yael Vodovotz, the principal investigator, by email ([email protected]) or by telephone (614-247-7696).

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Signing the consent form I have read (or someone has read to me) this form and I am aware that I am being asked to participate in a research study. I have had the opportunity to ask questions and have had them answered to my satisfaction. I voluntarily agree to participate in this study.

I am not giving up any legal rights by signing this form. I will be given a copy of this form.

______Printed name of subject Signature of subject

______AM/PM Date and time

______Printed name of person authorized to Signature of person authorized to consent consent for subject (when applicable) for subject (when applicable)

______AM/PM Relationship to the subject Date and time

Investigator/Research Staff I have explained the research to the participant or his/her representative before requesting the signature(s) above. There are no blanks in this document. A copy of this form has been given to the participant or his/her representative.

______Printed name of person obtaining consent Signature of person obtaining consent

______AM/PM Date and time

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APPENDIX E:

SENSORY ANALYSIS ELECTRONIC QUESTIONNAIRE

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APPENDIX F:

SENSORY ANALYSIS PREFERENCE TEST BALLOT

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Soy Bread Preference Test

Of the two samples you just evaluated, please indicate which sample you preferred by checking the appropriate box below.

Tray #:______

Sample 660 Sample 568

Comments:ää

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