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Tracking isoflavones: From soybean to soy flour, soy protein isolates to functional soy bread
Suqin Shaoa,b, Alison M. Duncanb, Raymond Yanga, Massimo F. Marconec, Istvan Rajcand, Rong Tsaoa,*
a

Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario, Canada N1G 5C9 Department of Human Health and Nutritional Sciences, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 c Department of Food Science, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1 d Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada N1G 2W1
b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 July 2008 Accepted 29 August 2008 Available online 11 October 2008 Keywords: Soybean Soy bread Isoflavones Food processing Functional food Dough Proofing

Soybean seeds with three different levels (low, intermediate and high) of isoflavones were processed to soy flour and soy protein isolates (SPIs) and developed into functional soy breads. The effect of factors involved in all steps of the process was investigated by tracking the composition and concentration of native forms of isoflavones. The total isoflavone contents were 8033.3, 10570.1 and 15169.0 nmol/g DM (dry matter) in the three soybeans; 13201.5, 20034.4 and 26014.3 nmol/g DM in defatted soy flours; 9113.2, 13274.6 and 17918.3 nmol/g DM in the SPI; 2782.7, 4081.4 and 5590.3 nmol/g DM in soy breads, respectively. The bread making processes did not affect the total isoflavone content, but changed glucosides/acetylglucosides to aglycones. Malonylglucosides were stable prior to baking but degraded to acetylglucosides and further to glucosides during baking. Our results provide critical information for the production of functional soy breads that contain varying amounts of soy isoflavones. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Interest in introducing soybean and soy products to the Western diet has grown rapidly in recent years due to the health promoting effects of soy protein and isoflavones. Soy and its constituent protein and isoflavones have been associated with reduced risk of cardiovascular disease (Rimbach et al., 2008), prostate (Nagata et al., 2007), breast (Steiner et al., 2007) and colon (MacDonald et al., 2005) cancers, and improved bone health (Barnes, 2003; Zhang et al., 2007). To date 12 isoflavones have been reported to be present in soybean, including three aglycones genistein, daidzein and glycitein, their respective 7-O-b-D-glucosides (genistin, daidzin and glycitin), 600 -O-malonyl-7-O-b-D-glucosides (malonylgenistin, malonyldaidzin and

malonylglycitin) and 600 -O-acetyl-7-O-b-D-glucosides (acetylgenistin, acetyldaidzin and acetylglycitin) (Fig. 1). It has been established that the bioavailability of isoflavones can be influenced by their chemical form in foods and the susceptibility to degradation during heating (Birt et al., 2001). Barnes et al. (1998) demonstrated that genistein could be formed from genistin during fermentation and that 600 -O-malonylgenistin could be converted to 600 -O-acetylgenistin or genistin during heating. For this reason, conversion among various isoflavones during processing of soybean products needs to be investigated. Although soy has been consumed in China for thousands of years, very little information is available regarding changes in both isoflavone profile and concentration during food

* Corresponding author: Fax: +1 519 829 2600. E-mail address: caor@agr.gc.ca (R. Tsao). 1756-4646/$ - see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jff.2008.09.013

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OH OH OH HO O HO R2 R1 O OH R2 R1 O OH O O O

Aglycones Daidzein R1=H R2=H Glycitein R1=H R2=OCH3 Genistein R1=OH R2=H
OH OH OH O O O O R2 R1 O OH O

7-Ο-β-D-Glucosides Daidzin R1=H R2=H Glycitin R1=H R2=OCH3 Genistin R1=OH R2=H
OH OH OH O O O O R2 O OH R1 O OH O

6"-O-Acetyl-7-O-β-D-Glucosides Acetyl Daidzin R1=H R2=H Acetyl Glycitin R1=H R2=OCH3 Acetyl Genistin R1=OH R2=H

6"-O-Malonyl-7-β-D-Glucosides

Malonyl Daidzin R1=H R2=H Malonyl Glycitin R1=H R2=OCH3 Malonyl Genistin R1=OH R2=H

Fig. 1 – Chemical structures of soy isoflavones.

processing. Grun et al. (2001) studied the profile of genistein, daidzein and their conjugates during thermal processing of tofu, and found that not only did the isoflavone profile change during the process, but the concentration also decreased significantly most likely due to the leaching of isoflavones into water, and thermal degradation during cooking. They suggested that a possible thermal degradation of isoflavones occurred in addition to losses caused by leaching. Huang et al. (2006) found that the isoflavone content of soy milk significantly decreased during heat treatment. Significant losses of isoflavones during production of soy beverage were also observed by Jackson et al. (2002) during soaking and filtration. Barbosa et al. (2006) showed that different processing parameters, such as ionic strength and pH, resulted in different isoflavone amounts and profile during soy protein isolate (SPI) production and they attributed this to the endogenous b-glucosidase activity. Currently, there has been a strong movement of public breeding programs in the United States and Canada to produce soybean for direct human consumption. However, the delivery of health benefits from soy will depend on the development of acceptable mainstream products. The realization of health benefits may also depend on the stability of the isoflavones during processing as well as on the specific profile of the isoflavone derivatives in the finished product. It is thus critical to understand the effect of processing on the abundance and distribution of the isoflavones when developing novel soy-containing functional foods.

Bread made partially with soy represents a viable alternative for incorporating soy into the Western diet. However, the effect of the soy bread production process on isoflavones is not clear. Soy bread production is a complex physicochemical process that involves major steps such as producing soy flour, SPI, dough mixing, yeast fermentation (proofing), and baking. During proofing, yeast (Saccharomyces cerevisiae) utilizes specific carbohydrates in bread ingredients to produce carbon dioxide and may alter the nutrient content and composition (Yoon et al., 2003). To the best of our knowledge, only one group has examined the changes in distribution of isoflavones during soy bread proofing and baking (Riedl et al., 2005; Zhang et al., 2003, 2004), and there has been no study evaluating isoflavone retention in all steps of the soy bread production process, particularly from the starting soybeans to soy flour and SPI. The objective of this study was therefore to investigate the effect of all factors involved in the soy bread production process by tracking the concentration and composition of individual and total isoflavones.

2.
2.1.

Materials and methods
Chemicals

All solvents were of HPLC grade from Caledon Laboratories (Georgetown, ON, Canada). Daidzein, genistein, glycitein, daidzin and genistin were purchased from Sigma–Aldrich (St. Louis, MO, USA); glycitin, malonyldaidzin, malonylgenistin, malonylglycitin, acetyldaidzin, acetylgenistin and

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acetylglycitin were purchased from LC Laboratories (Woburn, MA, USA).

bake lab of the Guelph Food Technology Centre in Guelph, ON, Canada: (1) Dry yeast was added to a mechanical mixing bowl with water and stirred gently until yeast dissolved. The mixing bowl was covered with cloth and the yeast was allowed to rise for approximately 10 min. (2) The mixture of the dry ingredients was added to the yeast solution using the dough hook at low speed, for approximately 6–8 min. (3) The dough was placed in a preheated proofing chamber at 90% relative humidity and 36–38 °C for 45 min to 1 h, until it doubled in size. (4) The dough was rolled for 1 min using a mechanical roller, formed into a traditional loaf shape in a loaf pan and then placed in the proofing chamber at the same humidity and temperature, and for the same length of time as stated above. (5) After the second proofing, the dough was put into a preheated oven (171 °C) and baked for 37 min. (6) The breads were cooled to room temperature, cut into 18 equal slices, slices 3, 10 and 16 were stored at À20 °C until subsequent analysis.

2.2.

Soybean, soy flour and soy protein isolate

Soybean seeds with three varieties containing different levels of isoflavones (Cv. 2-1229 (low), Cv. 3-1543 (intermediate), Cv. 3-1482 (high)) were grown and harvested in a winter nursery in Chile by Massait Agricultural Services on a contract with the soybean breeding program at the University of Guelph, Guelph, ON, Canada, in 2006. The three cultivars were developed from crosses involving high and low isoflavone parents by the University of Guelph’s soybean breeding program. Soy flour was produced by the POS Pilot Plant Corporation in Saskatoon, SK, Canada. The production of soy flour began with cleaning the soybean of any foreign materials using air classification and aspiration followed by drying and tempering. The dehulled beans were then subjected to humid heat to achieve a specific nitrogen solubility index (NSI) value of 80%. The full dehulled beans were passed through a series of flaking rollers until the desired thickness of 0.30 mm was achieved. The flakes were then extracted with hexane using a solvent extractor until the flakes contained <1% fat. The defatted soy flakes were then passed through a hammer mill followed by a US Standard No. 100 sieve (with at least 97% of the flour from the mill passed through the sieve). The defatted soy flour was then processed to SPI by the Solae Company (St. Louis, MO, USA). The soy flour was extracted with water without pH adjustment. The extract was clarified to remove the insoluble material and the supernatant was acidified to a pH range of 4–5 with HCl. The precipitated protein-curd (SPI) was collected and separated from the whey by centrifugation. The curd was neutralized with alkali to form a sodium proteinate salt before drying. The SPI contained approximately 92% protein by dry weight.

2.4.

Extraction of isoflavones from soy samples

2.3.

Soy bread

The ingredients used in the soy bread formulation are listed in Table 1. Soy flours each with a different level of isoflavones and their respective SPIs were used to prepare the soy bread. Three soy breads containing low, intermediate and high levels of isoflavones were made using the following procedure in the

Soybean seeds with different levels of isoflavones (low, intermediate and high) were ground into powder using a ball mill (Retsch MM2000, Schieritz, Hauenstein, Switzerland). Soy bread dough after mixing (at step 2), dough after the first proofing (step 3), dough after the second proofing (step 4), and the soy bread product were freeze-dried before they were ground into powder using a mortar and pestle. Soybean seed powder, soy flour, SPI, doughs after mixing and proofing stages, and the soy bread (0.5 g) were each extracted with 10 mL of 80% methanol for 12 h at room temperature on a VWR-Rocking platform Shaker (VWR, Batavia, IL, USA). The suspension was centrifuged at 3220g for 30 min at room temperature, the supernatant was then filtered through a 0.45 lm polyvinylidene difluoride (PVDF) filter (Whatman, Sanford, ME, USA) and stored at 4 °C before HPLC analysis within 24 h.

2.5.
Table 1 – Soy bread formulation Ingredient
Water Skim milk powder Butter Vegetable oil Honey Salt Yeast Whole wheat flour Soy flour Soy protein isolate Gluten flour

HPLC analysis

Amount (g) per loaf
320 28.8 4.68 3.68 56.16 4.5 7 310 95 30 10

Relative %a
36.79 3.31 0.54 0.42 6.46 0.52 0.80 35.64 10.92 3.45 1.15

a Weight percentage of each ingredient.

An HPLC system (Agilent Technology 1100 Series, Palo Alto, CA, USA) equipped with a quaternary pump, an in-line degasser, a thermostatic autosampler, and a diode array detector (DAD) was used for the identification and quantification of various isoflavones in the samples. A Phenomenex PhenosphereÒ 5 ODS-2 column (150 · 4.6 mm, 5 lm) with a C18 guard column (Torrance, CA, USA) was used for the separation. The binary mobile phase consisted of acetonitrile (solvent A) and 2% acetic acid (solvent B), and a gradient program was used as follows: 0–40 min, 100–50% B; 40– 42 min, 50–20% B; 42–45 min, 20–100% B. The flow rate was 1.0 mL/min and the injection volume was 10 lL. The DAD collected data from 190 to 900 nm, and absorbance at only 260 nm was used to monitor and quantify isoflavones.

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2.6.

Identification and quantification of isoflavones

The isoflavones in all samples were identified by a combination of the retention time in HPLC chromatograms, and UV spectral pattern of isoflavone standards. Concentration of isoflavones, including aglycones and their various glycosides, was calculated using the corresponding standard curves.

2.7.

Statistical analysis

All samples were extracted and analyzed in triplicate. Data were analyzed by standard analysis of variance (ANOVA) techniques, followed by a least significant difference (LSD) comparison of mean data values at a P-value <0.05, using StatistixÒ software (V2.0, Analytical Software, Tallahassee, FL, USA).

flavone composition and concentration. All three isoflavones, daidzein, genistein and glycitein, and their respective b-glucosides, malonylglucosides and acetylglucosides were found in all the soybean seeds examined (Fig. 2). The concentration of isoflavones in each cultivar was cultivar 2-1229, 8033.3 nmol/g dry matter, DM; cultivar 3-1543, 10570.1 nmol/ g DM; and cultivar 3-1482, 15169.0 nmol/g DM, with the three values significantly different from each other (Table 2). The soybean varieties used in this study were specifically selected to be of varying isoflavone contents, and they contained higher concentrations of total isoflavones than most soybeans used in other studies (Kim & Chung, 2007; Riedl et al., 2007; Toda et al., 2000). All three soybean varieties used in this study were grown under the same conditions thus the differences were caused mainly by genetics.

3.2.

Isoflavone change from soybean to soy flour

3.
3.1.

Results and discussion
Isoflavones in soybean seeds
The processing of the soybeans to soy flour, which included a series of moist heat treatments and a defatting step, caused significant changes in isoflavone content and profile (Table 2, Figs. 2 and 3). Within all three varieties of soybeans, concentrations of the individual isoflavones (in different glycosidic forms) and the total aglycone isoflavones were

Three varieties of soybean with genetically different levels of isoflavones (Cv. 2-1229; Cv. 3-1543; Cv. 3-1482) were used to study the effect of processing and soy bread making on iso-

mAU

3,4,5 1 2 6

10,11 7,8 9
STD

12

5000

SB
4000

D2P

D1P
3000

DAM
2000

DI

SPI
1000

SF

0

SS
5 10 15 20 25 30 35 40 min

Fig. 2 – Representative chromatograms of isoflavones at each soy bread production stage from soybean seeds to soy bread in high-isoflavone variety. SS, soybean seeds; SF, soy flour; SPI, soy protein isolate; DI, dry ingredients, mixed bread ingredient without water; DAM, dough after mixing; D1P, dough after first proofing; D2P, dough after second proofing; SB, soy bread; STD, isoflavone standards. Peak identities: 1, daidzin; 2, glycitin; 3, genistin; 4, malonlydaidzin; 5, malonylglycitin; 6, acetyldaidzin; 7, acetylglycitin; 8, malonlygenistin; 9, daidzein; 10, acetylgenistin; 11, glycitein; 12, genistein.

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Table 2 – Isoflavone content of soybean seeds, soy flour and soy protein isolate (nmol/g dry matter)A Isoflavone
Low isoflavone soybean (Cv. 2-1229) Genistin Malonylgenistin Acetylgenistin Genistein Total genistein Daidzin Malonyldaidzin Acetyldaidzin Daidzein Total daidzein Glycitin Malonylglycitin Acetylglycitin Glycitein Total glycitein Total isoflavones Intermediate isoflavone soybean (Cv. 3-1543) Genistin Malonylgenistin Acetylgenistin Genistein Total genistein Daidzin Malonyldaidzin Acetyldaidzin Daidzein Total daidzein Glycitin Malonylglycitin Acetylglycitin Glycitein Total glycitein Total isoflavones High-isoflavone soybean (Cv. 3-1482) Genistin Malonylgenistin Acetylgenistin Genistein Total genistein Daidzin Malonyldaidzin Acetyldaidzin Daidzein Total daidzein Glycitin Malonylglycitin Acetylglycitin Glycitein Total glycitein Total isoflavones

SSB
719.5 ± 69.5a 1604.3 ± 88.9a 49.1 ± 6.6a 116.1 ± 0.8a 2489.0 ± 165.9a 841.1 ± 40.7a 1563.2 ± 91.0a NDa,E 122.7 ± 2.1a 2527.1 ± 79.6a 100.9 ± 9.4a 144.1 ± 6.2a ND NDa 245.1 ± 5.6ab 8033.3 ± 296.3a

SFC
1959.1 ± 34.9b 2044.5 ± 59.7b 1049.3 ± 14.0b 445.8 ± 0.7b 5498.6 ± 38.1b 2609.4 ± 102.0b 2416.7 ± 89.2b 1692.9 ± 79.2b 607.4 ± 6.5b 7326.4 ± 62.9b NDb 198.1 ± 3.5b ND 78.4 ± 8.7b 276.5 ± 4.6b 13201.5 ± 215.3b

SPID
1070.1 ± 39.6c 1648.4 ± 8.2a 204.4 ± 0.3c 2190.8 ± 38.9c 5113.6 ± 70.6c 614.2 ± 23.2c 908.2 ± 7.0c 370.3 ± 1.0c 1998.4 ± 21.6c 3891.2 ± 36.8c NDb 39.2 ± 1.0c ND 69.2 ± 0.2b 108.4 ± 0.9a 9113.2 ± 104.6c

936.7 ± 49.9a 2125.1 ± 128.7a 54.4 ± 2.6a 138.1 ± 17.9a 3254.4 ± 82.1a 1067.4 ± 42.1a 2102.7 ± 105.8a NDa 137.9 ± 11.9a 3308.0 ± 129.1a 151.8 ± 16.9a 198.1 ± 11.5a ND NDa 349.9 ± 16.9a 10570.1 ± 222.0a

3492.5 ± 73.2b 3871.8 ± 11.9b 1102.9 ± 10.6b 349.1 ± 4.0b 8816.4 ± 75.9b 4050.0 ± 78.9b 4012.0 ± 33.1b 2022.5 ± 23.6b 399.3 ± 7.0b 10483.8 ± 196.3b NDb 291.1 ± 10.2b ND 39.7 ± 0.1b 330.8 ± 5.2a 20034.4 ± 351.6b

1780.3 ± 13.2c 2130.7 ± 23.7a 262.2 ± 3.1c 3250.9 ± 34.2c 7424.0 ± 57.8c 954.0 ± 9.2a 1128.1 ± 11.5c 469.8 ± 2.5c 2950.8 ± 22.1c 5502.6 ± 27.0c NDb 56.1 ± 1.1c ND 123.9 ± 0.9c 180.0 ± 4.7b 13274.6 ± 219.6c

1514.6 ± 36.8a 2844.1 ± 83.4a 66.1 ± 6.2a 230.2 ± 5.4a 4655.0 ± 79.0a 1639.7 ± 73.8a 2910.8 ± 95.1a NDa 249.6 ± 12.7a 4800.1 ± 171.7a 206.5 ± 8.6a 250.4 ± 8.4a ND NDa 456.9 ± 10.1a 15169.0 ± 140.2a

5985.4 ± 13.1b 7421.6 ± 109.1b 974.2 ± 8.3b 547.4 ± 17.4b 14928.6 ± 147.8b 3928.6 ± 86.6b 4454.8 ± 100.8b 1787.9 ± 38.3b 399.2 ± 12.2b 10620.5 ± 117.9b NDb 420.8 ± 8.0b ND 44.5 ± 3.1b 465.2 ± 11.1a 26014.3 ± 256.28b

3215.5 ± 18.6c 4286.2 ± 38.2c 456.1 ± 1.7c 4555.6 ± 20.3c 12513.5 ± 41.7c 926.2 ± 24.7c 1284.1 ± 11.6c 771.6 ± 15.2c 2241.8 ± 74.5c 5223.7 ± 126.6a NDb 84.5 ± 0.4c ND 96.5 ± 0.4c 181.1 ± 0.8b 17918.3 ± 252.0c

A Values are mean ± standard deviation (n = 3 independent runs). Within each row, values with different superscript letters are significantly different from each other by general ANOVA followed by an LSD comparison of means (P < 0.05). B SS, soybean seeds. C SF, soy flour. D SPI, soy protein isolate. E ND, not detected.

significantly greater in the soy flour (by a factor of 1.5–2) when compared to the soybean seeds, except for total glycitein

(Table 2). The increase in aglycone isoflavones is likely due to the defatting and dehulling processes. In addition, although

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the exact rate of increase in individual and total isoflavones varied among the three varieties studied, the relative isoflavone content, and thus the distinction among the low, intermediate and high-isoflavone contents, remained consistent from the soybean seeds to the soy flours. Interestingly, this process significantly affected the isoflavone profile as shown by the concentration of each isoflavone isomer in soy bean seeds and soy flour (Table 2) and the relative percentage of each isoflavone isomers (Fig. 3), particularly that of malonyland acetyldaidzin or malonyl- and acetylgenistin (Table 2 and Fig. 3). The acetyldaidzin or acetylgenistin levels were either not detectable or were at very low concentrations in the soybean seeds; however, they were significantly increased in the soy flours (Figs. 2 and 3). The emergence of acetyldaidzin and acetylgenistin could be from the decarboxylation of malonyldaidzin and malonylgenistin during the heating in this process, as the ratio between daidzin/malonlydaidzin increased from an average of 0.54 to 0.99, and similarly from 0.47 to 0.89 for the genistin/malonylgenistin ratio. Both the decrease of the malonyl form and the production of isoflavone glucosides (daidzin and genistin) through deacylation, i.e. deacetylation and demalonylation process, can be attributed to the increase of the ratios. Heating soy-enriched cookies and soy milk and toasting soy flour have been reported to cause conversion of isoflavone malonylglucosides to acetylglucosides and glucosides (Coward et al., 1998; Murphy et al., 2002); however, this effect, as far as we are aware of, has not previously been observed in the processing of soybean seeds to soy flour.

3.3.

Isoflavone change from soy flour to soy protein isolate

SPI was produced by extracting soy flour with water without pH adjustment, followed by precipitation, washing and drying. This process significantly decreased the b-glucosides, acetylglucosides and malonylglucosides, causing a significant increase in the aglycones (Table 2) and hence isoflavone profile changes, which is demonstrated by the change of the relative percentage of the isoflavone isomers (Fig. 3). Total isoflavone content was significantly lower in the SPI than in the soy flour, for all three soybean varieties. The reduction in total isoflavones could be caused by leaching during washing (Grun et al., 2001; Mathias et al., 2006). Increased proportion of aglycones could also be attributed to the hydrolysis of glucosides by endogenous b-glucosidase, the activity of which in soy flour has been studied in detail by Zhang et al. (2004). The magnitudes of decrease in individual and total isoflavone concentrations in the SPI (as compared to soy flour) were similar for all the three soybean varieties, providing further evidence of the maintenance of distinction in isoflavone content from soy flour to SPI, as they were from soybean seeds to soy flour. The average recovery of isoflavones in the SPI from the soy flour was 67% (a percent ratio between the total isoflavone contents in the SPI and soy flour) for all three soybean varieties, which is higher than those reported in the literature, e.g., Wang et al. (1998) reported a recovery rate of ca. 26% isoflavone from soy flour to SPI; Lin et al. (2006) showed a recovery rate of 27–40% from soy flour to SPI. The different

A 100
% of Genistein Isomers
80 60

% of genistin % of malonylgenistin % of acetylgenistin % of genistein

B 100
% of Daidzein Isomers
80 60 40 20 0

% of daidzin % of malonyldaidzin % of acetyldaidzin % of daidzein

C 100
% of Glycitein Isomers
80 60 40 20 0

% of glycitin % of malonylglycitin % of acetylglycitin % of glycitein

40 20

0 SS SF SPI DI DAM D1P D2P SB

SS

SF

SPI

DI

DAM

D1P

D2P

SB

SS

SF

SPI

DI

DAM

D1P

D2P

SB

D
% of Genistein Isomers

100
% of genistin % of malonylgenistin % of acetylgenistin

E
% of Daidzezin Isomers

100
% of daidzin % of malonyldaidzin % of acetyldaidzin

F
% of Glycitein Isomers

100

% of glycitin % of acetylglycitin

% of malonylglycitin % of glycitein

80 60

80 60

80 60

% of genistein

% of daidzein

40 20

40 20

40 20

0 SS SF SPI DI DAM D1P D2P SB

0 SS SF SPI DI DAM D1P D2P SB

0 SS SF SPI DI DAM D1P D2P SB

G
% of Genistein Isomers

100
% of genistin % of malonylgenistin

H
% of Daidzein Isomers

100
% of daidzin % of malonyldaidzin % of acetyldaidzin

I
% of Glycitein Isomers

100 80 60 40 20 0

% of glycitin % of acetylglycitin

% of malonylglycitin % of glycitein

80

80 60

% of acetylgenistin

60

% of genistein

% of daidzein

40

40 20

20

0 SS SF SPI DI DAM D1P D2P SB

0 SS SF SPI DI DAM D1P D2P SB

SS

SF

SPI

DI

DAM

D1P

D2P

SB

Fig. 3 – Percentage of isoflavone isomers at each soy bread production stage from soybean seeds to soy bread. A, B, and C, low-isoflavone variety (Cv. 2-1229); D, E, and F, intermediate-isoflavone variety (Cv. 3-1543); G, H, and I, high-isoflavone variety (Cv. 3-1482). SS, soybean seeds; SF, soy flour; SPI, soy protein isolate; DI, dry ingredients, mixed bread ingredient without water; DAM, dough after mixing; D1P, dough after first proofing; D2P, dough after second proofing; SB, soy bread.

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Table 3 – Isoflavone content at different stages of soy bread production from dry ingredient to soy bread (nmol/g dry matter)A Isoflavones DIB DAMC
407.3 ± 2.9b 558.2 ± 5.5a 62.7 ± 0.8b 304.7 ± 3.0b 1346.3 ± 35.2a 403.4 ± 2.3b 484.9 ± 3.4a 152.9 ± 4.2b 303.9 ± 4.2b 1331.8 ± 31.8a ND 35.1 ± 0.3a ND 32.3 ± 0.5a 67.4 ± 0.6a 2737.5 ± 84.3a

D1PD
371.2 ± 10.4c 536.5 ± 11.8a 37.6 ± 1.3c 431.7 ± 8.7c 1377.0 ± 75.8a 355.2 ± 0.7c 506.5 ± 10.9a 139.1 ± 8.1b 443.9 ± 3.5c 1444.6 ± 44.8a ND 36.8 ± 0.7a ND 31.1 ± 0.5a 67.9 ± 0.3a 2895.5 ± 119.6a

D2PE
294.0 ± 2.3d 535.9 ± 10.6a 30.4 ± 0.8d 481.5 ± 3.9d 1341.8 ± 45.1a 276.2 ± 1.9d 461.7 ± 6.4a 99.9 ± 5.2c 488.9 ± 8.3d 1326.8 ± 54.8a ND 34.5 ± 0.2a ND 32.5 ± 0.3a 66.9 ± 0.4a 2729.5 ± 78.3a

SBF
368.0 ± 9.5c 308.9 ± 7.8b 110.9 ± 1.3e 473.8 ± 4.9d 1261.7 ± 9.8a 482.8 ± 8.3ab 321.8 ± 6.4b 148.6 ± 2.2b 503.5 ± 9.5d 1456.8 ± 37.2a ND 35.0 ± 1.0a ND 29.9 ± 1.2a 64.9 ± 1.6a 2782.7 ± 82.3a

Low isoflavone soybean (Cv. 2-1229) Genistin 446.5 ± 4.9a Malonylgenistin 513.2 ± 7.6a Acetylgenistin 192.4 ± 4.5a Genistein 196.6 ± 2.9a Total genistein 1299.1 ± 59.6a Daidzin 484.4 ± 8.9a Malonyldaidzin 467.1 ± 10.2a Acetyldaidzin 312.7 ± 5.9a Daidzein 214.0 ± 5.7a Total daidzein 1478.2 ± 24.6a Glycitin NDG Malonylglycitin 33.5 ± 0.9a Acetylglycitin ND Glycitein 31.4 ± 0.8a Total glycitein 64.9 ± 1.2a Total isoflavones 2866.8 ± 98.6a

Intermediate isoflavone soybean (Cv. 3-1543) Genistin 683.1 ± 24.3a Malonylgenistin 765.6 ± 39.8a Acetylgenistin 199.8 ± 8.5a Genistein 231.8 ± 4.6a Total genistein 1880.2 ± 75.6a Daidzin 733.08 ± 22.4a Malonyldaidzin 735.9 ± 25.6a Acetyldaidzin 365.7 ± 11.6a Daidzein 224.2 ± 8.9a Total daidzein 2058.9 ± 98.6a Glycitin ND Malonylglycitin 52.0 ± 1.2a Acetylglycitin ND Glycitein 13.3 ± 0.8a Total glycitein 65.3 ± 1.2a Total isoflavones 4081.2 ± 145.8a

584.6 ± 12.5b 759.4 ± 16.0a 119.6 ± 1.0b 465.4 ± 9.1b 1929.0 ± 64.1a 637.1 ± 17.3b 727.0 ± 27.4a 200.3 ± 5.3b 476.1 ± 4.4b 2040.6 ± 87.8a ND 54.4 ± 0.1a ND 11.4 ± 0.2b 65.8±0.4a 4045.4 ± 95.3a

507.6 ± 17.0c 719.0 ± 19.5a 74.9 ± 2.3c 627.7 ± 8.8c 1929.2 ± 28.0a 526.3 ± 16.8c 677.7 ± 10.8a 151.0 ± 2.5c 663.2 ± 10.2c 2018.2 ± 79.3a ND 51.8 ± 0.7a ND 15.1 ± 3.0a 66.9 ± 3.6a 4034.4 ± 78.5a

439.4 ± 6.7d 731.4 ± 14.8a 58.9 ± 0.6d 698.6 ± 16.7d 1928.3 ± 17.7a 454.6 ± 8.7d 687.6±13.5a 139.1 ± 0.9d 737.2 ± 17.8d 2018.6 ± 80.2a ND 52.8 ± 0.8a ND 12.1 ± 0.9ab 64.9 ± 1.6a 4041.8 ± 97.5a

548.4 ± 19.8bc 574.0 ± 8.1b 122.7 ± 4.3b 697.5 ± 28.2d 1942.6 ± 15.9a 581.1 ± 22.9b 558.4 ± 15.9b 179.8 ± 3.9bc 726.8 ± 28.2d 2046.2 ± 55.1a ND 51.2 ± 1.1a ND 14.4 ± 1.6a 65.6 ± 6.7a 4081.4 ± 56.3a

High-isoflavone soybean (Cv. 3-1482) Genistin 1209.6 ± 28.3a Malonylgenistin 1516.2 ± 17.9a Acetylgenistin 193.2 ± 2.9a Genistein 343.1 ± 7.6a Total genistein 3262.2 ± 26.8a Daidzin 729.3 ± 15.2a Malonyldaidzin 778.4 ± 14.8a Acetyldaidzin 351.0 ± 5.69a Daidzein 191.3 ± 4.59a Total daidzein 2050.0 ± 58.6a Glycitin ND Malonylglycitin 77.3 ± 5.9a Acetylglycitin ND Glycitein 12.9 ± 0.5a Total glycitein 90.3 ± 1.6a Total isoflavones 5472.5 ± 94.5a

1044.1 ± 66.7b 1457.7 ± 37.2a 132.5 ± 1.9b 726.1 ± 15.7b 3360.3±85.1a 578.0 ± 15.6b 764.8 ± 15.3a 268.9 ± 4.4b 423.2 ± 11.4b 2034.8 ± 43.1a ND 78.3 ± 2.0a ND 14.1 ± 0.4a 92.4 ± 5.2a 5491.5 ± 106.9a

842.6 ± 4.8c 1422.8 ± 73.4a 82.7 ± 1.0c 999.3 ± 9.9c 3347.5 ± 108.8a 456.3 ± 13.2c 730.5 ± 15.6a 237.7 ± 8.9c 587.8 ± 4.8c 2012.3 ± 65.4a ND 75.6 ± 1.0a ND 16.1 ± 1.0b 91.7 ± 2.0a 5465.4 ± 98.6a

716.7 ± 14.5d 1427.3 ± 14.4a 64.9±1.8d 1276.9 ± 53.0d 3485.9 ± 74.2a 368.0±4.4d 738.1 ± 13.9a 231.7 ± 8.3c 736.8 ± 12.2d 2074.6 ± 18.6a ND 76.6 ± 0.4a ND 16.1 ± 0.6b 92.7 ± 0.9a 5681.2 ± 89.5a

1009.5 ± 62.6b 1093.5 ± 36.0b 145.6 ± 3.2b 1185.1 ± 72.9d 3433.8 ± 44.6a 544.9 ± 15.1b 540.9 ± 14.4b 229.3 ± 4.4c 716.8 ± 16.0d 2031.8 ± 26.8a ND 74.3 ± 0.9a ND 17.4 ± 2.5b 91.7 ± 3.1a 5590.3 ± 88.6a

A Each value is the mean and standard deviation of three independent runs. Within each row, values with different superscript letters are significantly different from each other by general ANOVA followed by an LSD comparison of means (P < 0.05). B DI, dry ingredient, mixed bread ingredient without water. C DAM, dough after mixing. D D1P, dough after first proofing. E D2P, dough after second proofing. F SB, soy bread. G ND, not detected.

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recovery was due to different parameters used by different groups during SPI production, e.g., soy flour was extracted at different pHs.

3.4. Isoflavone change during dough mixing and dough proofing
As summarized in Table 3, total aglycone and total isoflavone contents for genistein, daidzein and glycitein did not significantly change through the bread production stages, although there were significant changes in glycosylation patterns with the individual isoflavones (Figs. 2 and 3). Conversion from glucosides and acetylglucosides to aglycones took place at nearly all stages, as was observed in increased aglycone (genistein, daidzein and glycitein), and decreased glucoside and acetylglucoside concentrations from dough mixing through to the second proofing, regardless of soybean variety (Table 3 and Fig. 3), probably due to enzymatic hydrolysis. However, this was not observed for malonylglucosides in that concentrations of malonylgenistin and malonlydaidzin were not significantly affected by the bread making process in any of the soybean varieties (Table 3 and Fig. 3). This phenomenon was also observed by Zhang et al. (2004) in their soy dough proofing study. Although the exact mechanism behind the stability of the malonylglucosides is unknown, our earlier study on enzymatic hydrolysis of isoflavone glycosides did show a slower rate for malonylglucosides than for b-glucosides using isoflavone standards (Shao et al., unpublished). Lack of susceptibility of the malonylglucosides to enzyme hydrolysis is the likely explanation. Hydrolysis of glucoside isoflavones in soybean by endogenous b-glucosidases has been reported by several groups. Matsuura et al. (1989) first reported an increase of daidzein and genistein during soaking of soybean when producing soy milk, and later confirmed the enzymatic action by isolating b-glucosidases in soybeans (Matsuura & Obata, 1993). Zhang et al. (2004) studied the b-glucosidase activity in their soy bread ingredients, and found that b-glucosidase activity in bread yeast was 0.66 U/g, soy flour 10.7 U/g, soy milk powder 6.83 U/g and wheat flour 4.14 U/g. Even though a new formulation was used for the soy bread in this study, a transformation in isoflavone form (i.e. hydrolysis of isoflavone glucosides and acetylglucosides) occurred as early as the initial dough mixing period, and lasted through the proofing stages, and this occurred regardless of soybean variety (Table 3 and Fig. 3).

due to conversion of malonylconjugates. Moist heat increased the content of the b-glucoside conjugates, whereas dry heat caused an increase in the acetyl conjugates. Murphy et al. (2002) also indicated that isoflavones were not destroyed under normal thermal processing conditions but rather were subject to interconversions between different forms. They also found that in various soy foods, malonylglucosides and acetylglucosides can readily be converted to their respective, more stable non-conjugated b-glucoside.

4.

Conclusions

This study is the first of its kind in that all isoflavone-containing bread ingredients (i.e. soy flour and SPI) were produced from soybean varieties containing low, intermediate and high concentrations of isoflavones. This study is also unique because the isoflavones in all their native forms were followed from the soybean seeds through to the end soy bread product, and factors affecting their stability and interconversion were examined at each step. While the isoflavone content and composition were clearly affected by the different processing conditions during the development of soy bread, the effects were consistently observed in all three soy breads (Fig. 4). The distinction of low (L), intermediate (I) and high (H)-isoflavone content was maintained in all steps from soybean to soy flour to SPI to dough at different proofing stages and finally to the soy breads (Fig. 4). The correlation coefficients (R) for isoflavone content at all production steps between low and intermediate, intermediate and high, and high and low were 0.996, 0.998 and 0.999, respectively. In general, there was an isoflavone-enrichment effect from soybean seeds to defatted soy flour due to the removal of oil, although heating and endogenous enzyme caused interconversion of different forms of isoflavones during the process. Total isoflavones were decreased during the production of SPI from the soy flour, likely due to degradation under high pH, leaching and solvent extraction. The actual bread

30000 Total isoflavones (nmol/g DM) 25000 20000 15000 10000 5000 0
L I H

3.5.

Isoflavone change during baking

Baking the proofed dough at 171 °C for 37 min did not cause significant loss of either total or aglycone isoflavones, but it did significantly decrease malonylglucosides and increase b-glucosides and acetylglucosides, regardless of soybean variety (Table 3 and Fig. 3). This was again likely due to the decarboxylation and de-esterification of malonylglucosides under heating conditions. Several groups have reported changes caused by heating in conjugation profile of isoflavones in soy products. Coward et al. (1998) reported that baking and frying of SPI and textured vegetable proteins did not alter the total isoflavone contents but changed the profiles of individual isoflavones

SS

SF

SPI

DI

DAM

D1P

D2P

SB

Fig. 4 – Total isoflavone contents at each soy bread production stage from soybean seeds to soy breads. L, lowisoflavone soybean variety (Cv. 2-1229); I, intermediateisoflavone soybean variety (Cv. 3-1543); H, high-isoflavone soybean variety (Cv. 3-1482); SS, soybean seeds; SF, soy flour; SPI, soy protein isolate; DI, dry ingredients, all mixed bread ingredient without water; DAM, dough after mixing; D1P, dough after first proofing; D2P, dough after second proofing; SB, soy bread.

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making processes (from dry ingredients to the final bread) did not affect the total isoflavone content (Fig. 4), although further compositional changes, particularly the conversion of glucosides and acetylglucosides to aglycones before baking, occurred. Malonylglucosides were not affected during the dough mixing and proofing stages, but degradation of malonylglucosides to acetylglucosides (de-carboxylation) and further to glucosides (de-esterification) was observed during baking. Results from this study will expand our understanding of the factors that affect total and individual isoflavone contents in soy-containing functional food products such as bread. This understanding will help to maximize the retention of target bioactives, such as isoflavones, during functional food development.

Acknowledgements
This project was supported by the Food Research Program of the Ontario Ministry of Agriculture, Food and Rural Affairs (Project#026211) and the A-Base research of Agriculture and Agri-Food Canada. Support was also provided by the Solae Company who produced and donated the SPIs. The authors are grateful to the Guelph Food Technology Centre in Guelph, Ontario, for use of their bake lab and to Helle Thomsen for her help in baking the breads, and to the POS Pilot Plant Corporation for their assistance in production of the soy flour.
R E F E R E N C E S

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