Food Sci. Biotechnol.
19(1): 107-113 (2010)
DOI 10.1007/s10068-010-0015-0
RESEARCH ARTICLE
Physicochemical and Sensory Characteristics of Soy Yogurt
Fermented with Bifidobacterium breve K-110, Streptococcus
thermophilus 3781, or Lactobacillus acidophilus Q509011
Seo Young Chang, Dong-Hyun Kim, and Myung Joo Han
Received: 29 July 2009 / Revised: 6 November 2009 / Accepted: 22 Novem 2009 / Published Online: 28 February 2010
© KoSFoST and Springer 2010
Abstract The physicochemical and sensory characteristics rings. Three main aglycone soysaponins have been identified
of soy yogurt fermented with Bifidobacterium breve K-
110, Streptococcus thermophilus 3781, or Lactobacillus
acidophilus Q509011 for 36 hr at 37oC were investigated
and the soyasaponin metabolites of soy yogurt were
measured. L. acidophilus reduced the pH of soy yogurt on
day 0 to optimum level. After 36 hr, the Brix dropped to
8.48-9.12oBx. The viable cell counts of soy yogurts on day
0 were 8.43 to 9.01 log CFU/mL. Among starters, B. breve
alone produced soyasapogenol B, although it was not
detected in non-fermented soybean. B. breve produced
poor sensory scores in all sensory aspects. By mixing B.
breve with S. thermophilus and L. acidophilus, the sensory
scores of B. breve could be improved. These findings
suggest that the mixed culture of S. thermopilus or L.
acidophilus with B. breve may be an ideal starter for the
preparation of soy yogurt.
Keywords: soy yogurt, Bifidobacterium breve K-110,
soyasaponin metabolite, sensory score, fermentation
Introduction
Soybeans and soy foods contain isoflavones, saponins,
phytic acids, phytosterols, trypsin inhibitors, and bioactive
peptides that have important biological activities (1).
Soyasaponins are triterpenoid glycosides with 4 common
Seo Young Chang, Myung Joo Han ( )
Department of Food and Nutrition, Kyung Hee University, Seoul 130-701,
Korea
Tel: +82-2-961-0553; Fax: +82-2-961-0538
E-mail: mjhan@khu.ac.kr
Dong-Hyun Kim
College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea
and labeled as soyasaponin A, B, and E (2). It is important
to understand the intestinal metabolism and absorption of
soyasaponins because humans obtain soyasaponins from
the diet. These components inevitably come into contact
with intestinal microflora in the alimentary tract and can be
metabolized by intestinal microflora (3). The metabolites
are then easily absorbed from the gastrointestinal tract
since most of the metabolites are nonpolar compared to the
parental components. These absorbed metabolites may
exhibit pharmacological activity (4). Saponins are assumed
to be poorly absorbed in the intestine. It is thought that the
sugar chains of the saponins have to be hydrolyzed by
bacterial enzymes in the lower intestine to liberate the
aglycones (5).
Ginseng saponins are deglycosylated by intestinal
bacteria such as Cubacterium sp., Streptococcus sp.,
Bifidobacterium sp., and Fucobacterium sp. to deglycosylated
metabolites via different types of ginsenosides (6). Human
bacteria collected from fecal material have been reported to
hydrolyze ginsenosides Rb1 and Rb2 to specific 20-β-O-
glycopyranosy-20(S)-protopanaxdiol, referred to as
compound K. Compound K transformed from ginseng
saponins is reported to have antitumor activity (8).
However, the biotransformation of soyasaponins by lactic
acid bacteria has not been studied.
Probiotics are defined as viable microorganisms that,
upon ingestion, have beneficial effects on the health of the
host by improving the properties of its indigenous
microflora (9). Traditionally, yogurt is manufactured using
Lactobacillus sp. and Streptococcus sp. such as L.
bulgaricus and S. thermophilus. While these lactic acid
bacteria provide some health benefits, they are neither
natural inhabitants of the intestine nor survive in the
gastrointestinal tract. However, Bifidobacterium sp., which
108
is a major microbe in human intestine, is considered a
probiotic for yogurt (9,10).
Fermented soy products have been perceived as healthy
foods and are therefore considered an important part of the
diet. Furthermore, the incorporation of probiotic bacteria as
dietary adjuncts has led to increased consumption of
probiotic products in Europe, the United States, and Asia
(12,13). Yogurt is a milk product fermented by L.
delbrueckii sp. bulgaricus and S. thermophilus. It originated
in the Balkans and the Middle East, is consumed
worldwide, and is considered beneficial for health (12).
Interest in the role of probiotics for human health goes
back at least as far as 1908 when Metchnikoff suggested
that man should consume milk fermented with lactobacilli
to prolong life (14,15).
Soybean has been used as a significant protein source in
the Korean diet due to its high yield ratio, inexpensive
price, excellent nutritive value, and assimilation ratio (16).
But, soybean has particular beany flavor from oxidation or
decomposition of organic compounds during processing,
and side effects such as flatulence occur with excessive
soybean consumption. It has been reported that fermenting
soybean with lactic acid bacteria generates fragrant
material and improves both the flavor and texture of
soybean products (17-20).
Therefore, this study investigated soyasaponin metabolites
in the soy yogurts fermented with lactic acid bacteria and
evaluated sensory evaluation of soybean yogurt to develop
the bioactive soy milk yogurt.
Materials and Methods
Materials and starter culture Soybean (Wonju, Korea)
was purchased in Hanaro Mart (Seoul, Korea). General
anaerobic medium (GAM) and blood liver (BL) medium
was purchased from Nissui Pharmaceutical Co., Ltd.
(Tokyo, Japan). Tryptic soy broth and de Man Rogosa
Sharpe (MRS) medium were purchased from Difco Co.
(Detroit, MI, USA). Anaero Pak Campylo was purchased
from Mitsubishi Gas Chemical Co. (Tokyo, Japan). Sheep
blood was purchased from Hanil Komed, Co., Ltd. (Seoul,
Korea).
Streptococcus thermophilus 3781 and Lactobacillus
acidophilus Q509011 were purchased from Korea
Research Institute of Bioscience and Biotechnology
(Daejeon, Korea). Bifidobacterium breve K-110 isolated
from human intestinal microflora, was used for starter
cultures (21). B. breve was cultured in GAM broth at 37oC
for 20 hr for yogurt starter. S. thermophilus and L.
acidophilus were cultured in tryptic soy broth at 37oC for
20 hr for yogurt starter.
S. Y. Chang et al.
Preparation of soy yogurt Soybeans were washed and
soaked in water for 24 hr at 4oC and then blended with a
Hallde cutter VCB-61 (Hallde Maskiner, Kista, Sweden).
The resulting slurry was filtered in 200 mesh sieve to
produce soy milk. The soymilk was adjusted to 10oBx, 5%
sugar was added, and the soymilk was heated for 5 min at
85oC and cooled to approximately 40oC. Then 2% of each
starter (approximately 107 cells) was added to the soy milk
and fermented for 36 hr at 37oC.
pH and titratable activity During the storage period,
soy yogurts were analyzed for pH and titratable acidity.
Ten mL of soy yogurt and 100 mL of deionized water were
mixed and the pH was then determined. Ten mL of soy
yogurt and 10 mL of deionized water were mixed, and 2-
3 drops of phenolphthalein were added. This mixture was
titrated to an end-point of pH 8.1 with 0.1 N NaOH. The
acidity of the soy yogurt was calculated as % lactic acid.
Brix During the storage period, the Brix values of the
soy yogurts were measured by refractometer (N-1 α; Atago,
Tokyo, Japan).
Viable cell count The viable cell counts in the soy
yogurts were analyzed during the 15-day storage period.
Lactobacilli MRS agar and BL medium were used for S.
thermophilus and L. acidophilus, and for B. breve,
respectively.
Identification of soyasaponin metabolites To extract
the soyasaponin fraction from soy yogurts, 1 g of soy milk
or each fermented soy yogurt was mixed with 10 mL of
methanol (MeOH) and centrifuged for 10 min at 2,000× g.
The supernatants were evaporated in a rotary evaporator
(Eyela Co., Tokyo, Japan), dissolved in 10 mL of MeOH
and then filtered with a 0.22-µM syringe filter (Type
Millex-HA; Millipore, Billerica, MA, USA). The filtrates
were analyzed by liquid chromatography-mass/mass
spectrometry (LC-MS/MS) to identity the soyasaponin
metabolites. LC-MS/MS analyses were performed on
Agilent G6410 Triple Quadrupole Mass Spectrometer with
an electro spray ionization (ESI) source, coupled with an
Agilent 1200 series high performance liquid chromatography
(HPLC) separations of the sample were performed on a
ZORBAX Eclipse XDB-C18 column (50 × 2.1 mm, i.d.,
1.8 µm, Agilent). For elution, a linear gradient was applied
from 20 to 95% B for 13 min (solvent A was 0.1%
aqueous formic acid; solvent B was acetonitrile). The
solvent flow rate was 0.3 mL/min. Mass spectra were
acquired in ESI mode using nitrogen gas at a temperature
of 350oC, flow rate 10 L/min, nebulizer pressure of 45 psi,
quadruple temperature of 30oC, and capillary voltage of
Characteristics of Fermented Soy Yogurt 109

4,000 V. Two µL samples were injected into the column
using an autosampler. Total ion chromatograms from 400
to 1,500 m/z in MS/MS positive and negative modes were
obtained.
Sensory evaluation After 36 hr of fermentation, soy
yogurts were evaluated at day 1 of storage at 4oC. The 18
university students were used for sensory panels. Seven-
point hedonic scales (1=dislike extremely, 7=like extremely)
for appearance, beany flavor, mouthfeel, bitterness,
sourness, and overall acceptability were used to evaluate
the soy yogurts.
Statistics analysisThe results obtained through this
experiment were analyzed with the statistical analysis system
(SAS) program. The significance of each sample was
established by one-way analysis of variance (ANOVA) and
verified by Duncan’s multiple range tests at p <0.05.
Results and Discussion
Characteristics of soy yogurt during After
storage
fermentation for 36 hr, the pH of the soy yogurt during 15-
day storage at 4oC is shown in Table 1. At day 0 of storage,
Table 1. Changes in pH of soy yogurt during 15-day of storage at 4 C
soy yogurt fermented by B. breve (4.72) and S. thermophilus
(4.66) showed higher pH than that fermented by L.
acidophilus (4.28). The most desirable yogurts in terms of
sensory characteristics have a pH value in the range of 4.0-
4.6 (22). At 36 hr of fermentation (day 0), L. acidophilus
dropped the pH of soy yogurt to an optimum level, but B.
breve and S. thermophilus produced slightly high pH.
Pyo and Song (23) reported that the pH of soy yogurt
fermented by a mixture of L. delbruecki sp. lactis and L.
plantarium at 35oC for 48 hr was 4.47. The pH of soy
yogurt fermented by B. breve (4.72 at day 0) or L.
acidophilus (4.29 at day 0) did not change during 15-day
storage (4.46, 4.39). The pH of soy yogurt fermented by S.
thermophilus decreased from 4.66 on day 0 to 4.47 on day
3 of storage and then did not change until day 15 of storage.
The titratable acidity of soy yogurt during 15-day
storage at 4oC is shown in Table 2. L. acidophilus (0.66%)
produced higher titratable acidity in soy yogurt on day 0
than B. breve (0.51%) and S. thermophilus (0.54%).
The Brix of soy yogurt during 15-day storage at 4oC are
shown in Table 3. To prepare soy yogurt, the soy milk was
adjusted to 10oBx, the Brix dropped to 8.48-9.12oBx after
36 hr of fermentation. The Brix of soy yogurt on day 0
were 9.12, 8.97, and 8.60oBx with B. breve, S. thermophilus,
and L. acidophilus, respectively. During 15-day storage,
o

Storage period (day)

Starter1)
0 3 6 9 12 15
BB A4.72±0.092) A4.74±0.06 A4.61±0.02 4.58±0.23 4.32±0.10 4.46±0.12
ST A4.66±0.11a. bB4.47±0.07b bB4.43±0.02b b4.39±0.05b b4.32±0.06b b4.36±0.07b
LA B4.29±0.040 C4.25±0.04 C4.23±0.06 4.21±0.18 4.16±0.07 4.39±0.07
1)BB, Bifidobacterium breve; ST, Streptococcus thermophilus; LA, Lactobacillus acidophilus
2)Means in a row (a,b) and in a column (A,B,C) followed by different superscripts are significantly different at p<0.05.

o
Table 2. Changes in titratable acidity of soy yogurt during 15-day of storage at 4 C

Storage period (day)

Starter1)
0 3 6 9 12 15
BB 0B0,51±0.03b2) B0.52±0.02b C0.58±0.11b 0.58±0.11b 0.76±0.04a B0.59±0.01b
ST B0.54±0.05b A0.63±0.03a B0.66±0.01a 0.66±0.01a 0.68±0.02a A0.66±0.01a
LA A0.66±0.04b A0.64±0.02b A0.72±0.03b 0.72±0.09b 0.74±0.05b A0.66±0.01b
1)BB, Bifidobacterium breve; ST, Streptococcus thermophilus; LA, Lactobacillus acidophilus
2)Means in a row (a,b) and in a column (A,B,C) followed by different superscripts are significantly different at p<0.05.

o
Table 3. Changes in Brix of soy yogurt during 15-day of storage at 4 C

Storage period (day)

Starter1)
0 3 6 9 12 15
BB 9.12±0.13a bA8.95±0.08ab2) AB8.74±0.20abb 8.85±0.07ab 8.79±0.13ab 8.60±0.28bb
ST 8.97±0.07a A8.93±0.07ab A8.85±0.07abc 8.75±0.21abc 8.45±0.21cb 8.55±0.21bc
LA 8.60±0.14b B8.55±0.07bb B8.40±0.28bb 8.35±0.49 8.15±0.07bb 8.00±0.56bb
1)BB, Bifidobacterium breve; ST, Streptococcus thermophilus; LA, Lactobacillus acidophilus
2)Means in a row (a,b) and in a column (A,B,C) followed by different superscripts are significantly different at p<0.05.
110
Table 4. Changes in viable cells of soy yogurt during 15-day of storage at 4oC
Starter1)
0 3
BB 8.75±0.69 8.93±0.66
ST 8.43±0.74 9.05±0.10
LA 8.96±0.30 9.10±0.08
BB, Bifidobacterium breve; ST, Streptococcus thermophilus; LA, Lactobacillus acidophilus
1)
the Brix of soy yogurt did not change significantly.
The viable cell count in soy yogurt during 15-day storage
at 4oC is shown in Table 4. The viable cell counts in soy
yogurt on day 0 were 8.75, 8.43, and 8.96 log CFU/mL
with B. breve, S. thermophilus, and L. acidophilus,
respectively. The viable cell counts in all soy yogurts were
maintained during 15-day storage at 4oC.
L. delbrueckii sp. bulgaricus and S. thermophilus are
traditionally used as yogurt starters, but these bacteria
neither naturally inhabit the intestine nor survive in the
gastrointestinal tract (24). Therefore, the use of probiotic
bacteria (lactobacilli, bifidobacteria, or these mixtures) in
fermented products has been increasing and the minimum
numbers of probiotic bacteria at consumption are 105-106
CFU/g (11,25). Donker et al. (13) mentioned that some
lactic acid bacteria grow slowly or poorly in soy milk.
However, in this study, the viable cell counts of soy yogurts
were more than 8 log CFU/g and probiotic bacteria such as
L. acidophilus, and B. breve had high numbers of viable
cells in soy yogurt. Farnworth et al. (26) reported that
probiotics generally do not grow rapidly in milk, but soy is
a good substrate for probiotics. Furthermore, the utilization
of prebiotics such as raffinose and stachyose could support
the growth of probiotic bacteria in soy yogurt. However,
the viable cell counts of this study showed that traditional
yogurt starters, as well as probiotics, grow well in soy
yogurt.
Identification of soyasaponin metabolites The soy-
Fig. 1. LC-MS/MS chromatograms of soybean juice (A) and soy yogurt (B).
S. Y. Chang et al.
Storage period (day)
6 9 12 15
9.09±0.43 9.29±0.02 9.30±0.04 9.13±0.13
9.05±0.08 9.21±0.04 9.20±0.06 8.88±0.30
9.22±0.21 9.30±0.15 9.44±0.19 9.12±0.04
asaponin fraction extracted from soybean showed 7 peaks
on LC-MS/MS analysis (Fig. 1). These peaks were
identified as soyasaponin Ab (I, m/z 1,435), soyasaponin
Af (II, m/z 1,273), soyasaponin Ah (III-a, m/z 1,243),
soyasaponin Ba (III-b, m/z 957), soyasaponin Bd (IV, m/z
941), soyasaponin Bc (V, m/z 911), soyasaponin Be (VI, m/
z 939), and soyasaponin βg (VII, m/z 1,067) by mass
spectra like those in the literature (1,2) (Fig. 2). The major
soyasaponins in soybean extract are soyasaponin Bd (IV,
m/z 941) and soyasaponin Bc (V, m/z 911). Among soy
yogurts fermented by various starters (B. breve, S.
thermophilus, and L. acidophilus), soyasapogenol B was
detected in soy yogurt fermented with B. breve alone, but
the soyasaponin metabolite(s), soyasapogenol, was not
detected soy yogurt fermented with S. thermophilus or L.
acidophilus. The productivity of a soyasaponin metabolite,
soyasapogenol B, in soy yogurt fermented with B. breve
was not significantly increased 1.06 fold during 15-day
storage at 4oC.
When the saponins such as ginsenoside Rb1, are orally
administered, their sugar chains are hydrolyzed by enzymes
in the intestinal microflora to liberate the aglycones that are
then absorbed into the blood and their pharmacological
activities are expressed (3). However, the hydrolysis of
their sugar chains between individuals may be significantly
different. Therefore, to continuously express the pharma-
cological effect of soyasaponins, the fermentation of soy-
asaponins by probiotics bifidobacteria or/and lactobacilli
are beneficial (27). Of probiotics tested, B. breve alone
Characteristics of Fermented Soy Yogurt 111

Fig. 2. Mass spectra of saponin of soybean juice and yogurt acquired in LC-MS/MS chromatograms of soybean juice and soy
yogurt. Acquired in ESI scan mode. Negative electrospray ion currents of soyasaponins (I to VIII in Fig. 1) in soy yogurt after−the
fermentation with B. breve K-110. The I to VIII refer to the assigned literature-reported compounds: MS spectra on the [M-H] of
soyasaponin Ab (I, m/z 1,435), soyasaponin Af (II, m/z 1,273), soyasaponin Ah (III-a, m/z 1,243), soyasaponin Ba (III-b, m/z 957),
soyasaponin Bd (IV, m/z 941), soyasaponin Bc (V, m/z 911), soyasaponin Be (VI, m/z 939), soyasaponin βg (VII, m/z 1,067), and
soyasapogenol B (VIII, m/z 441)

liberated the metabolite, soyasapogenol B, of soyasaponins.
It has been suggested that B. breve-fermented soy milk
yogurt produces soyasapogenol B easily absorbed from the
gastrointestinal tract and the absorbed metabolites may
exhibit pharmacological effect, such as anticarcinogenesis
(28,29).
Sensory evaluation of soybean yogurt To identify a
preferred soybean yogurt, the sensory scores of soy yogurts
on day 1 are given in Table 5. Scores for visual appearance,
beany flavor, mouthfeel, sourness, bitterness, and overall
acceptability of soy yogurts fermented by various starters
were significantly different. In the preliminary study, B.
breve and L. acidophilus fermented soy yogurts had poor
visual appearance, although S. thermophilus produced a
high appearance score. Therefore, we prepared soy yogurts
fermented with B. breve, S. thermophilus, and L. acidophilus
or these mixtures as starters and investigated their sensory
scores. S. thermophilus (5.56) produced a high appearance
score, followed by the mixture of B. breve, S. thermophilus,
and L. acidophilus (4.67), while the mixture of B. breve
and S. thermophilus (4.44), B. breve (3.77), the mixture of
B. breve and L. acidophilus (3.50), and L. acidophilus
(3.33) had poor visual appearance scores. Soy yogurt
fermented by B. breve (3.06) showed poor beany flavor
score. However, S. thermophilus (3.94), the mixture of B.
112 S. Y. Chang et al.

Table 5. Sensory scores of soy yogurt

Starter Visual Beany flavor Mouthfeel Sourness Bitterness Overall

appearance accetability
1)

BB 00 3.77±0.94
0 bcd2)
3.06±0.87 b
3.96±1.06
0 bc
3.17±0.79 d
3.94±1.16 b
3.61±0.61 b

ST 5.56±1.20 a
3.94±1.47 a
5.17±1.10 a 00
3.89±1.57 bcd
5.67±1.24 a
4.72±1.23 a

LA 3.33±1.14 d 0
3.50±1.38 ab
3.94±0.94
0 bc 0
4.72±0.75 ab 0
4.78±1.83 ab
3.28±1.41 b

BB+ST 0
4.44±1.62 bc
4.11±1.08 a
5.11±1.28 a 0
3.78±1.77 cd
5.56±1.42 a
4.56±1.50 a

BB+LA 0
3.50±1.58 cd
3.89±0.83 a
3.78±1.56 c 00
4.50±1.39 abc
4.44±1.95 b
3.22±1.63 b

BB+ST+LA 0
4.67±1.60 ab
4.27±1.07 a
4.72±1.07
0 ab
4.83±1.15 a
5.67±0.84 a
4.72±0.83 a

BB, Bifidobacterium breve; ST, Streptococcus thermophilus; LA, Lactobacillus acidophilus

1)

Means in a column followed by different superscripts are significantly different at p <0.05; 1=dislike extremely, 7=like extremely.
2)a-d

breve and L. acidophilus (3.89), the mixture of B. breve References

and S. thermophilus (4.11), and the mixture of B. breve, S.
thermophilus, and L. acidophilus (4.27) increased the
preference of beany flavor. Therefore, the beany flavor of
soy yogurt fermented by B. breve could be improved by
combining lactic acid bacteria. S. thermophilus (5.17) and
the mixture of B. breve and S. thermophilus (5.11) had high
mouthfeel scores. B. breve (3.96) alone had poor
mouthfeel, but could be improved by combination with S.
thermophilus (5.11). However, L. acidophilus (3.94) and
the mixture of B. breve and L. acidophilus (3.78) had poor
mouthfeel. B. breve (3.17), S. thermophilus (3.89), and the
mixture of B. breve and S. thermophilus (3.78) had low
preference in sourness. L. acidophilus (4.72), or combined
with B. breve (4.50) or with B. breve and S. thermophilus
(4.83) could improve sourness. B. breve (3.94), and the
mixture of B. breve and L. acidophilus (4.44) produced low
preference in bitterness. S. thermophilus (5.67), the mixture
of B. breve and S. thermophilus (5.56), and the mixture of
B. breve, S. thermophilus and L. acidophilus (5.67)
improved the preference of bitterness. B. breve (3.61), L.
acidophilus (3.28), and the mixture of B. breve and L.
acidophilus (3.22) showed low overall acceptability, but S.
thermophilus (4.72) and the mixtures with S. thermophilus
(B. breve, 4.56; B. breve and L. acidophilus, 4.72) had
good overall acceptability.
In conclusion, even though B. breve could hydrolyze
soyasaponin to liberate its aglycones such as soy-
asapogenol B, B. breve yielded poor sensory scores in all
sensory aspects of soy yogurt. But S. thermophilus
produced good sensory scores except sourness. The
fermentation of soy milk by the mixture B. breve with S.
thermophilus and L. acidophilus may improve the poor
sensory properties of B. breve. Therefore, the mixed culture
of B. breve, S. thermophilus, and L. acidophilus is a
probable soy yogurt starter that produces good quality
characteristics and liberates soyasaponin metabolites.
Acknowledgments This research was supported by a
research grant from Kyung Hee University (KHU-20080571),
Seoul, Korea.
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