JOURNAL OF FUNCTIONAL FOODS 1 (2 0 0 9) 1 1 9–12 7

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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
b
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

A R T I C L E I N F O A B S T R A C T

Article history: Soybean seeds with three different levels (low, intermediate and high) of isoflavones were
Received 14 July 2008 processed to soy flour and soy protein isolates (SPIs) and developed into functional soy
Accepted 29 August 2008 breads. The effect of factors involved in all steps of the process was investigated by tracking
Available online 11 October 2008 the composition and concentration of native forms of isoflavones. The total isoflavone con-
tents were 8033.3, 10570.1 and 15169.0 nmol/g DM (dry matter) in the three soybeans;
Keywords: 13201.5, 20034.4 and 26014.3 nmol/g DM in defatted soy flours; 9113.2, 13274.6 and
Soybean 17918.3 nmol/g DM in the SPI; 2782.7, 4081.4 and 5590.3 nmol/g DM in soy breads, respec-
Soy bread tively. The bread making processes did not affect the total isoflavone content, but changed
Isoflavones glucosides/acetylglucosides to aglycones. Malonylglucosides were stable prior to baking but
Food processing degraded to acetylglucosides and further to glucosides during baking. Our results provide
Functional food critical information for the production of functional soy breads that contain varying
Dough amounts of soy isoflavones.
Proofing Crown Copyright  2008 Published by Elsevier Ltd. All rights reserved.

1. Introduction malonylglycitin) and 600 -O-acetyl-7-O-b-D-glucosides (acetyl-

Interest in introducing soybean and soy products to the Wes-
tern 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-malo-
nyl-7-O-b-D-glucosides (malonylgenistin, malonyldaidzin and
genistin, acetyldaidzin and acetylglycitin) (Fig. 1). It has been
established that the bioavailability of isoflavones can be influ-
enced 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 gen-
istin during fermentation and that 600 -O-malonylgenistin
could be converted to 600 -O-acetylgenistin or genistin during
heating. For this reason, conversion among various isoflav-
ones 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  2008 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.jff.2008.09.013
120 JOURNAL OF FUNCTIONAL FOODS 1 ( 2 0 0 9 ) 1 1 9 –1 2 7

OH
OH

OH
O
HO O O O
HO

R2 R2
R1 O R1 O
OH OH

Aglycones 7-Ο-β-D-Glucosides
Daidzein R1=H R2=H Daidzin R1=H R2=H
Glycitein R1=H R2=OCH3 Glycitin R1=H R2=OCH3
Genistein R1=OH R2=H Genistin R1=OH R2=H

OH OH
OH OH

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

6"-O-Acetyl-7-O-β-D-Glucosides 6"-O-Malonyl-7-β-D-Glucosides
Acetyl Daidzin R1=H R2=H Malonyl Daidzin R1=H R2=H
Acetyl Glycitin R1=H R2=OCH3 Malonyl Glycitin R1=H R2=OCH3
Acetyl Genistin R1=OH R2=H 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 sig-
nificantly most likely due to the leaching of isoflavones into
water, and thermal degradation during cooking. They sug-
gested that a possible thermal degradation of isoflavones oc-
curred in addition to losses caused by leaching. Huang et al.
(2006) found that the isoflavone content of soy milk signifi-
cantly decreased during heat treatment. Significant losses of
isoflavones during production of soy beverage were also ob-
served by Jackson et al. (2002) during soaking and filtration.
Barbosa et al. (2006) showed that different processing param-
eters, such as ionic strength and pH, resulted in different iso-
flavone amounts and profile during soy protein isolate (SPI)
production and they attributed this to the endogenous b-glu-
cosidase 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 stabil-
ity 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 iso-
flavones when developing novel soy-containing functional
foods.
Bread made partially with soy represents a viable alterna-
tive 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 spe-
cific 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 isoflav-
ones 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 compo-
sition of individual and total isoflavones.
2. Materials and methods
2.1. 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, malonylgeni-
stin, malonylglycitin, acetyldaidzin, acetylgenistin and
 JOURNAL OF FUNCTIONAL FOODS 1 ( 20 0 9) 1 1 9–12 7 121

acetylglycitin were purchased from LC Laboratories (Woburn, bake lab of the Guelph Food Technology Centre in Guelph, ON,
MA, USA). Canada:

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 devel-
oped 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 temper-
ing. 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 defat-
ted 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 centrifu-
gation. The curd was neutralized with alkali to form a sodium
proteinate salt before drying. The SPI contained approxi-
mately 92% protein by dry weight.
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
(1) Dry yeast was added to a mechanical mixing bowl with
water and stirred gently until yeast dissolved. The mix-
ing 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 pre-
heated 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
20 C until subsequent analysis.
2.4. Extraction of isoflavones from soy samples
Soybean seeds with different levels of isoflavones (low, inter-
mediate 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 tem-
perature, 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. HPLC analysis

Table 1 – Soy bread formulation An HPLC system (Agilent Technology 1100 Series, Palo Alto,
Ingredient Amount (g) per loaf Relative %a CA, USA) equipped with a quaternary pump, an in-line degas-
ser, a thermostatic autosampler, and a diode array detector
Water 320 36.79
(DAD) was used for the identification and quantification of
Skim milk powder 28.8 3.31
Butter 4.68 0.54 various isoflavones in the samples. A Phenomenex Pheno-
Vegetable oil 3.68 0.42 sphere 5 ODS-2 column (150 · 4.6 mm, 5 lm) with a C18
Honey 56.16 6.46 guard column (Torrance, CA, USA) was used for the separa-
Salt 4.5 0.52 tion. The binary mobile phase consisted of acetonitrile (sol-
Yeast 7 0.80 vent A) and 2% acetic acid (solvent B), and a gradient
Whole wheat flour 310 35.64
program was used as follows: 0–40 min, 100–50% B; 40–
Soy flour 95 10.92
42 min, 50–20% B; 42–45 min, 20–100% B. The flow rate was
Soy protein isolate 30 3.45
Gluten flour 10 1.15 1.0 mL/min and the injection volume was 10 lL. The DAD col-
lected data from 190 to 900 nm, and absorbance at only
a Weight percentage of each ingredient.
260 nm was used to monitor and quantify isoflavones.
122 JOURNAL OF FUNCTIONAL FOODS 1 ( 2 0 0 9 ) 1 1 9 –1 2 7

2.6. Identification and quantification of isoflavones
The isoflavones in all samples were identified by a combina-
tion of the retention time in HPLC chromatograms, and UV
spectral pattern of isoflavone standards. Concentration of iso-
flavones, 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 Sta-
tistix software (V2.0, Analytical Software, Tallahassee, FL,
USA).
3. Results and discussion
3.1. Isoflavones in soybean seeds
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-
flavone composition and concentration. All three isoflavones,
daidzein, genistein and glycitein, and their respective b-glu-
cosides, 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 high-
er 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 differ-
ences were caused mainly by genetics.
3.2. Isoflavone change from soybean to soy flour
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, con-
centrations of the individual isoflavones (in different glyco-
sidic forms) and the total aglycone isoflavones were

mAU 10,11
3,4,5
7,8 12
1 2
5000
6 9
STD

SB
4000

D2P

D1P
3000

DAM

2000
DI

SPI
1000

SF

SS
0

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.
 JOURNAL OF FUNCTIONAL FOODS 1 ( 20 0 9) 1 1 9–12 7 123

Table 2 – Isoflavone content of soybean seeds, soy flour and soy protein isolate (nmol/g dry matter)A
Isoflavone SSB SFC SPID

Low isoflavone soybean (Cv. 2-1229)

Genistin 719.5 ± 69.5a 1959.1 ± 34.9b 1070.1 ± 39.6c
Malonylgenistin 1604.3 ± 88.9a 2044.5 ± 59.7b 1648.4 ± 8.2a
Acetylgenistin 49.1 ± 6.6a 1049.3 ± 14.0b 204.4 ± 0.3c
Genistein 116.1 ± 0.8a 445.8 ± 0.7b 2190.8 ± 38.9c
Total genistein 2489.0 ± 165.9a 5498.6 ± 38.1b 5113.6 ± 70.6c
Daidzin 841.1 ± 40.7a 2609.4 ± 102.0b 614.2 ± 23.2c
Malonyldaidzin 1563.2 ± 91.0a 2416.7 ± 89.2b 908.2 ± 7.0c
Acetyldaidzin NDa,E 1692.9 ± 79.2b 370.3 ± 1.0c
Daidzein 122.7 ± 2.1a 607.4 ± 6.5b 1998.4 ± 21.6c
Total daidzein 2527.1 ± 79.6a 7326.4 ± 62.9b 3891.2 ± 36.8c
Glycitin 100.9 ± 9.4a NDb NDb
Malonylglycitin 144.1 ± 6.2a 198.1 ± 3.5b 39.2 ± 1.0c
Acetylglycitin ND ND ND
Glycitein NDa 78.4 ± 8.7b 69.2 ± 0.2b
Total glycitein 245.1 ± 5.6ab 276.5 ± 4.6b 108.4 ± 0.9a

Total isoflavones 8033.3 ± 296.3a 13201.5 ± 215.3b 9113.2 ± 104.6c

Intermediate isoflavone soybean (Cv. 3-1543)

Genistin 936.7 ± 49.9a 3492.5 ± 73.2b 1780.3 ± 13.2c
Malonylgenistin 2125.1 ± 128.7a 3871.8 ± 11.9b 2130.7 ± 23.7a
Acetylgenistin 54.4 ± 2.6a 1102.9 ± 10.6b 262.2 ± 3.1c
Genistein 138.1 ± 17.9a 349.1 ± 4.0b 3250.9 ± 34.2c
Total genistein 3254.4 ± 82.1a 8816.4 ± 75.9b 7424.0 ± 57.8c
Daidzin 1067.4 ± 42.1a 4050.0 ± 78.9b 954.0 ± 9.2a
Malonyldaidzin 2102.7 ± 105.8a 4012.0 ± 33.1b 1128.1 ± 11.5c
Acetyldaidzin NDa 2022.5 ± 23.6b 469.8 ± 2.5c
Daidzein 137.9 ± 11.9a 399.3 ± 7.0b 2950.8 ± 22.1c
Total daidzein 3308.0 ± 129.1a 10483.8 ± 196.3b 5502.6 ± 27.0c
Glycitin 151.8 ± 16.9a NDb NDb
Malonylglycitin 198.1 ± 11.5a 291.1 ± 10.2b 56.1 ± 1.1c
Acetylglycitin ND ND ND
Glycitein NDa 39.7 ± 0.1b 123.9 ± 0.9c
Total glycitein 349.9 ± 16.9a 330.8 ± 5.2a 180.0 ± 4.7b

Total isoflavones 10570.1 ± 222.0a 20034.4 ± 351.6b 13274.6 ± 219.6c

High-isoflavone soybean (Cv. 3-1482)

Genistin 1514.6 ± 36.8a 5985.4 ± 13.1b 3215.5 ± 18.6c
Malonylgenistin 2844.1 ± 83.4a 7421.6 ± 109.1b 4286.2 ± 38.2c
Acetylgenistin 66.1 ± 6.2a 974.2 ± 8.3b 456.1 ± 1.7c
Genistein 230.2 ± 5.4a 547.4 ± 17.4b 4555.6 ± 20.3c
Total genistein 4655.0 ± 79.0a 14928.6 ± 147.8b 12513.5 ± 41.7c
Daidzin 1639.7 ± 73.8a 3928.6 ± 86.6b 926.2 ± 24.7c
Malonyldaidzin 2910.8 ± 95.1a 4454.8 ± 100.8b 1284.1 ± 11.6c
Acetyldaidzin NDa 1787.9 ± 38.3b 771.6 ± 15.2c
Daidzein 249.6 ± 12.7a 399.2 ± 12.2b 2241.8 ± 74.5c
Total daidzein 4800.1 ± 171.7a 10620.5 ± 117.9b 5223.7 ± 126.6a
Glycitin 206.5 ± 8.6a NDb NDb
Malonylglycitin 250.4 ± 8.4a 420.8 ± 8.0b 84.5 ± 0.4c
Acetylglycitin ND ND ND
Glycitein NDa 44.5 ± 3.1b 96.5 ± 0.4c
Total glycitein 456.9 ± 10.1a 465.2 ± 11.1a 181.1 ± 0.8b

Total isoflavones 15169.0 ± 140.2a 26014.3 ± 256.28b 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 (Table 2). The increase in aglycone isoflavones is likely due
compared to the soybean seeds, except for total glycitein to the defatting and dehulling processes. In addition, although
124 JOURNAL OF FUNCTIONAL FOODS 1 ( 2 0 0 9 ) 1 1 9 –1 2 7

the exact rate of increase in individual and total isoflavones 3.3. Isoflavone change from soy flour to soy protein isolate
varied among the three varieties studied, the relative isoflav-
one content, and thus the distinction among the low, inter- SPI was produced by extracting soy flour with water without
mediate and high-isoflavone contents, remained consistent pH adjustment, followed by precipitation, washing and dry-
from the soybean seeds to the soy flours. Interestingly, this ing. This process significantly decreased the b-glucosides,
process significantly affected the isoflavone profile as shown acetylglucosides and malonylglucosides, causing a significant
by the concentration of each isoflavone isomer in soy bean increase in the aglycones (Table 2) and hence isoflavone pro-
seeds and soy flour (Table 2) and the relative percentage of file changes, which is demonstrated by the change of the rel-
each isoflavone isomers (Fig. 3), particularly that of malonyl- ative percentage of the isoflavone isomers (Fig. 3). Total
and acetyldaidzin or malonyl- and acetylgenistin (Table 2 isoflavone content was significantly lower in the SPI than in
and Fig. 3). The acetyldaidzin or acetylgenistin levels were the soy flour, for all three soybean varieties. The reduction
either not detectable or were at very low concentrations in in total isoflavones could be caused by leaching during wash-
the soybean seeds; however, they were significantly increased ing (Grun et al., 2001; Mathias et al., 2006). Increased propor-
in the soy flours (Figs. 2 and 3). The emergence of acetyldaid- tion of aglycones could also be attributed to the hydrolysis
zin and acetylgenistin could be from the decarboxylation of of glucosides by endogenous b-glucosidase, the activity of
malonyldaidzin and malonylgenistin during the heating in which in soy flour has been studied in detail by Zhang et al.
this process, as the ratio between daidzin/malonlydaidzin in- (2004).
creased from an average of 0.54 to 0.99, and similarly from The magnitudes of decrease in individual and total isoflav-
0.47 to 0.89 for the genistin/malonylgenistin ratio. Both the one concentrations in the SPI (as compared to soy flour) were
decrease of the malonyl form and the production of isoflav- similar for all the three soybean varieties, providing further
one glucosides (daidzin and genistin) through deacylation, evidence of the maintenance of distinction in isoflavone con-
i.e. deacetylation and demalonylation process, can be attrib- tent from soy flour to SPI, as they were from soybean seeds to
uted to the increase of the ratios. Heating soy-enriched cook- soy flour. The average recovery of isoflavones in the SPI from
ies and soy milk and toasting soy flour have been reported to the soy flour was 67% (a percent ratio between the total iso-
cause conversion of isoflavone malonylglucosides to acetyl- flavone contents in the SPI and soy flour) for all three soybean
glucosides and glucosides (Coward et al., 1998; Murphy varieties, which is higher than those reported in the litera-
et al., 2002); however, this effect, as far as we are aware of, ture, e.g., Wang et al. (1998) reported a recovery rate of ca.
has not previously been observed in the processing of soy- 26% isoflavone from soy flour to SPI; Lin et al. (2006) showed
bean seeds to soy flour. a recovery rate of 27–40% from soy flour to SPI. The different

A 100 % of genistin B 100 % of daidzin C 100 % of glycitin

% of malonylgenistin % of malonyldaidzin % of malonylglycitin
% of Glycitein Isomers
% of Daidzein Isomers

80 80 80
% of Genistein Isomers

% of acetylgenistin % of acetyldaidzin % of acetylglycitin

% of genistein % of daidzein % of glycitein
60 60 60

40 40 40

20 20 20

0 0 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 100
% of genistin E 100
% of daidzin F 100
% of glycitin % of malonylglycitin
% of acetylglycitin % of glycitein
% of malonylgenistin % of malonyldaidzin
% of Genistein Isomers

80 80 80
% of Glycitein Isomers
% of Daidzezin Isomers

% of acetylgenistin % of acetyldaidzin
% of genistein % of daidzein
60 60 60

40 40 40

20 20 20

0 0 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

G 100
% of genistin H 100
% of daidzin I 100 % of glycitin
% of acetylglycitin
% of malonylglycitin
% of glycitein
% of malonylgenistin % of malonyldaidzin
% of Glycitein Isomers
% of Genistein Isomers

80 80 80
% of Daidzein Isomers

% of acetylgenistin % of acetyldaidzin
% of genistein % of daidzein
60 60 60

40 40 40

20 20 20

0 0 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

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.
 JOURNAL OF FUNCTIONAL FOODS 1 ( 20 0 9) 1 1 9–12 7 125

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 D1PD D2PE SBF

Low isoflavone soybean (Cv. 2-1229)

Genistin 446.5 ± 4.9a 407.3 ± 2.9b 371.2 ± 10.4c 294.0 ± 2.3d 368.0 ± 9.5c
Malonylgenistin 513.2 ± 7.6a 558.2 ± 5.5a 536.5 ± 11.8a 535.9 ± 10.6a 308.9 ± 7.8b
Acetylgenistin 192.4 ± 4.5a 62.7 ± 0.8b 37.6 ± 1.3c 30.4 ± 0.8d 110.9 ± 1.3e
Genistein 196.6 ± 2.9a 304.7 ± 3.0b 431.7 ± 8.7c 481.5 ± 3.9d 473.8 ± 4.9d
Total genistein 1299.1 ± 59.6a 1346.3 ± 35.2a 1377.0 ± 75.8a 1341.8 ± 45.1a 1261.7 ± 9.8a
Daidzin 484.4 ± 8.9a 403.4 ± 2.3b 355.2 ± 0.7c 276.2 ± 1.9d 482.8 ± 8.3ab
Malonyldaidzin 467.1 ± 10.2a 484.9 ± 3.4a 506.5 ± 10.9a 461.7 ± 6.4a 321.8 ± 6.4b
Acetyldaidzin 312.7 ± 5.9a 152.9 ± 4.2b 139.1 ± 8.1b 99.9 ± 5.2c 148.6 ± 2.2b
Daidzein 214.0 ± 5.7a 303.9 ± 4.2b 443.9 ± 3.5c 488.9 ± 8.3d 503.5 ± 9.5d
Total daidzein 1478.2 ± 24.6a 1331.8 ± 31.8a 1444.6 ± 44.8a 1326.8 ± 54.8a 1456.8 ± 37.2a
Glycitin NDG ND ND ND ND
Malonylglycitin 33.5 ± 0.9a 35.1 ± 0.3a 36.8 ± 0.7a 34.5 ± 0.2a 35.0 ± 1.0a
Acetylglycitin ND ND ND ND ND
Glycitein 31.4 ± 0.8a 32.3 ± 0.5a 31.1 ± 0.5a 32.5 ± 0.3a 29.9 ± 1.2a
Total glycitein 64.9 ± 1.2a 67.4 ± 0.6a 67.9 ± 0.3a 66.9 ± 0.4a 64.9 ± 1.6a

Total isoflavones 2866.8 ± 98.6a 2737.5 ± 84.3a 2895.5 ± 119.6a 2729.5 ± 78.3a 2782.7 ± 82.3a

Intermediate isoflavone soybean (Cv. 3-1543)

Genistin 683.1 ± 24.3a 584.6 ± 12.5b 507.6 ± 17.0c 439.4 ± 6.7d 548.4 ± 19.8bc
Malonylgenistin 765.6 ± 39.8a 759.4 ± 16.0a 719.0 ± 19.5a 731.4 ± 14.8a 574.0 ± 8.1b
Acetylgenistin 199.8 ± 8.5a 119.6 ± 1.0b 74.9 ± 2.3c 58.9 ± 0.6d 122.7 ± 4.3b
Genistein 231.8 ± 4.6a 465.4 ± 9.1b 627.7 ± 8.8c 698.6 ± 16.7d 697.5 ± 28.2d
Total genistein 1880.2 ± 75.6a 1929.0 ± 64.1a 1929.2 ± 28.0a 1928.3 ± 17.7a 1942.6 ± 15.9a
Daidzin 733.08 ± 22.4a 637.1 ± 17.3b 526.3 ± 16.8c 454.6 ± 8.7d 581.1 ± 22.9b
Malonyldaidzin 735.9 ± 25.6a 727.0 ± 27.4a 677.7 ± 10.8a 687.6±13.5a 558.4 ± 15.9b
Acetyldaidzin 365.7 ± 11.6a 200.3 ± 5.3b 151.0 ± 2.5c 139.1 ± 0.9d 179.8 ± 3.9bc
Daidzein 224.2 ± 8.9a 476.1 ± 4.4b 663.2 ± 10.2c 737.2 ± 17.8d 726.8 ± 28.2d
Total daidzein 2058.9 ± 98.6a 2040.6 ± 87.8a 2018.2 ± 79.3a 2018.6 ± 80.2a 2046.2 ± 55.1a
Glycitin ND ND ND ND ND
Malonylglycitin 52.0 ± 1.2a 54.4 ± 0.1a 51.8 ± 0.7a 52.8 ± 0.8a 51.2 ± 1.1a
Acetylglycitin ND ND ND ND ND
Glycitein 13.3 ± 0.8a 11.4 ± 0.2b 15.1 ± 3.0a 12.1 ± 0.9ab 14.4 ± 1.6a
Total glycitein 65.3 ± 1.2a 65.8±0.4a 66.9 ± 3.6a 64.9 ± 1.6a 65.6 ± 6.7a

Total isoflavones 4081.2 ± 145.8a 4045.4 ± 95.3a 4034.4 ± 78.5a 4041.8 ± 97.5a 4081.4 ± 56.3a

High-isoflavone soybean (Cv. 3-1482)

Genistin 1209.6 ± 28.3a 1044.1 ± 66.7b 842.6 ± 4.8c 716.7 ± 14.5d 1009.5 ± 62.6b
Malonylgenistin 1516.2 ± 17.9a 1457.7 ± 37.2a 1422.8 ± 73.4a 1427.3 ± 14.4a 1093.5 ± 36.0b
Acetylgenistin 193.2 ± 2.9a 132.5 ± 1.9b 82.7 ± 1.0c 64.9±1.8d 145.6 ± 3.2b
Genistein 343.1 ± 7.6a 726.1 ± 15.7b 999.3 ± 9.9c 1276.9 ± 53.0d 1185.1 ± 72.9d
Total genistein 3262.2 ± 26.8a 3360.3±85.1a 3347.5 ± 108.8a 3485.9 ± 74.2a 3433.8 ± 44.6a
Daidzin 729.3 ± 15.2a 578.0 ± 15.6b 456.3 ± 13.2c 368.0±4.4d 544.9 ± 15.1b
Malonyldaidzin 778.4 ± 14.8a 764.8 ± 15.3a 730.5 ± 15.6a 738.1 ± 13.9a 540.9 ± 14.4b
Acetyldaidzin 351.0 ± 5.69a 268.9 ± 4.4b 237.7 ± 8.9c 231.7 ± 8.3c 229.3 ± 4.4c
Daidzein 191.3 ± 4.59a 423.2 ± 11.4b 587.8 ± 4.8c 736.8 ± 12.2d 716.8 ± 16.0d
Total daidzein 2050.0 ± 58.6a 2034.8 ± 43.1a 2012.3 ± 65.4a 2074.6 ± 18.6a 2031.8 ± 26.8a
Glycitin ND ND ND ND ND
Malonylglycitin 77.3 ± 5.9a 78.3 ± 2.0a 75.6 ± 1.0a 76.6 ± 0.4a 74.3 ± 0.9a
Acetylglycitin ND ND ND ND ND
Glycitein 12.9 ± 0.5a 14.1 ± 0.4a 16.1 ± 1.0b 16.1 ± 0.6b 17.4 ± 2.5b
Total glycitein 90.3 ± 1.6a 92.4 ± 5.2a 91.7 ± 2.0a 92.7 ± 0.9a 91.7 ± 3.1a

Total isoflavones 5472.5 ± 94.5a 5491.5 ± 106.9a 5465.4 ± 98.6a 5681.2 ± 89.5a 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.
126 JOURNAL OF FUNCTIONAL FOODS
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 signifi-
cantly change through the bread production stages, although
there were significant changes in glycosylation patterns with
the individual isoflavones (Figs. 2 and 3). Conversion from glu-
cosides and acetylglucosides to aglycones took place at nearly
all stages, as was observed in increased aglycone (genistein,
daidzein and glycitein), and decreased glucoside and acetylg-
lucoside 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 concentra-
tions of malonylgenistin and malonlydaidzin were not signif-
icantly 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 proof-
ing 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 sus-
ceptibility of the malonylglucosides to enzyme hydrolysis is
the likely explanation.
Hydrolysis of glucoside isoflavones in soybean by endoge-
nous 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 isolat-
ing 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 pow-
der 6.83 U/g and wheat flour 4.14 U/g. Even though a new for-
mulation was used for the soy bread in this study, a
transformation in isoflavone form (i.e. hydrolysis of isoflav-
one glucosides and acetylglucosides) occurred as early as
the initial dough mixing period, and lasted through the proof-
ing stages, and this occurred regardless of soybean variety
(Table 3 and Fig. 3).
3.5. Isoflavone change during baking
Baking the proofed dough at 171 C for 37 min did not cause sig-
nificant loss of either total or aglycone isoflavones, but it did
significantly decrease malonylglucosides and increase b-glu-
cosides and acetylglucosides, regardless of soybean variety
(Table 3 and Fig. 3). This was again likely due to the decarboxyl-
ation 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
1 ( 2 0 0 9 ) 1 1 9 –1 2 7
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 acetylgluco-
sides 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-con-
taining bread ingredients (i.e. soy flour and SPI) were pro-
duced 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 inter-
conversion were examined at each step. While the isoflav-
one 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 proof-
ing stages and finally to the soy breads (Fig. 4). The correla-
tion 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 soy-
bean seeds to defatted soy flour due to the removal of oil,
although heating and endogenous enzyme caused intercon-
version 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
L
Total isoflavones (nmol/g DM)
25000
I
H
20000
15000
10000
5000
0
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, low-
isoflavone soybean variety (Cv. 2-1229); I, intermediate-
isoflavone 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.
 JOURNAL OF FUNCTIONAL FOODS
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 bak-
ing, 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 prod-
ucts such as bread. This understanding will help to maxi-
mize 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 Corpora-
tion for their assistance in production of the soy flour.
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