Process Biochemistry 121 (2022) 286–297

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Fermentation performance, nutrient composition, and flavor volatiles in

soy milk after mixed culture fermentation
Xinhui Peng a, Yi Liao a, Kunyu Ren a, Yanwei Liu a, Mengmeng Wang a, Aihua Yu a, b, Tian Tian a,
Peilong Liao a, Zhaoxian Huang b, Huan Wang a, c, *, Lianzhou Jiang a, b, *
a
College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China
b
College of Food Science and Engineering, Hainan University, Haikou 570228, China
c
Key Laboratory of Soybean Biology of Chinese Education Ministry, Harbin 150030, China

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

Keywords: In this study, a mixed culture of lactic acid bacteria (LAB) and kombucha bacteria was used to ferment soy milk.
Soy milk The microbial composition and the rheological properties, isoflavones, vitamins, and volatile flavor substances
Mixed fermentation were quantitatively analyzed during fermentation. The fermentation process could significantly change the
Fermenting performance
viscoelastic rheological properties of soy milk. Most of the isoflavone glycosides were hydrolyzed to aglycones by
Nutrient composition
β-glucosidase produced during fermentation. The isoflavone content increased significantly as fermentation
Flavor compounds
progressed. B vitamins content increased significantly, riboflavin content increased from 117.44 to 162.58 µg/g
dry weight (dw), and cobalamin content, not detected before fermentation, increased to 106.73 µg/g dw. Mi­
crobial fermentation promoted protein aggregation and decreased the fat content of soy milk. After fermentation,
the content of characteristic flavor substances in soybean, such as hexanal, was significantly reduced, and some
new flavor compounds were generated from the fermentation-derived alcohols, esters, and acids. The combi­
nation of LAB and kombucha bacteria can enrich the taste and significantly enhance the nutritional properties
and antioxidant capacity of fermented soy milk and provides a new idea for the research and development of
fermented soy milk and its related plant-based fermented products.

1. Introduction
Soy milk is a plant-based nutritional and functional beverage that
contains protein with a high biological value similar to animal protein,
oligosaccharides, and high contents of essential fatty acids and
cholesterol-reducing soy isoflavones, among other beneficial constitu­
ents [1–3]. Soy milk also serves as a delivery medium for probiotics. The
fermentation of soy milk by probiotics, yeast, and other microorganisms
decreases the anti-nutritional factors in soybean, decomposes some
insoluble macromolecular compounds (such as the oligosaccharides
raffinose and stachyose) into small molecular compounds that can be
absorbed and utilized by the human body and can produce new nutrients
due to microbial autolysis, resulting in a more digestible and nutritious
health drink compared to unfermented soy milk [4]. Furthermore, the
aglycones, active phenolic substances, vitamins, and peptides produced
during soy milk fermentation play an important role in inhibiting tumor
cell growth and preventing obesity and osteoporosis [5].
Probiotic lactic acid bacteria (LAB) are an important component of
the human intestinal flora with a myriad of beneficial health effects [6],
such as inhibiting harmful bacteria in the intestinal tract and main­
taining the intestinal microecological balance [7,8]. LAB can also enrich
the nutritional value of food, improve the flavor and increase the added
value of products [9]. However, the taste of soy milk fermented by LAB
is undesirable to most consumers, and the low vitamin content of soy
products has become an important limiting factor to further promote
fermented soy milk.
When exploring the symbiotic relationship between probiotics and
base foods, it has always been a research focus to maintain the number
of probiotics in fermented products at a high level [10,11]. Kombucha is
a fermented functional beverage comprised of a complex bacterial sys­
tem of various microorganisms, mainly probiotics, such as yeast, acetic
acid bacteria (AAB), and a few LAB, and its characteristic
health-promoting properties have attracted much interest in recent
years [12]. It can produce a large number of vitamins during fermen­
tation, with antioxidant, lipid-lowering, and antibacterial effects. In the
current market, kombucha fungus has been used as the starting material

* Corresponding authors at: College of Food Science, Northeast Agricultural University, Harbin, Heilongjiang Province 150030, China.
E-mail addresses: whname@neau.edu.cn (H. Wang), jlzname@neau.edu.cn (L. Jiang).

https://doi.org/10.1016/j.procbio.2022.07.018
Received 9 April 2022; Received in revised form 26 June 2022; Accepted 17 July 2022
Available online 19 July 2022
1359-5113/© 2022 Elsevier Ltd. All rights reserved.
X. Peng et al.
for milk, cheese, and coffee fermentation to enhance the functional
characteristics of such foods [13]. However, there are few studies on soy
milk fermentation using kombucha starter, kombucha contains almost
no LAB, the fermentation time is too long, the fermentation state is
unstable, and in-depth mechanism research is needed.
In this study, a mixed starter culture of LAB and kombucha was used
to further improve the taste, flavor, and nutritional characteristics of
fermented soy milk. The microbial composition and the rheological
properties, resistance to oxidation and volatile flavor substances were
quantitatively analyzed during fermentation. The general law of mi­
crobial combined fermentation was explored, a novel functional soy
milk Beverage was developed and a new idea was provided for the
research and development of related products.
2. Materials and methods
2.1. Raw materials and instruments
Soybeans (Beidou 53, CNA20140575.5) were purchased from Bei­
dahuang Group Co. Ltd. (Jiamusi, Heilongjiang, China). The rotary
evaporator was from Aron Co. Ltd. (Pudong New Area, Shanghai,
China). HPLC analysis was performed using an Agilent 1260II Prime
high-performance liquid chromatograph (CA, USA). GC-MS was per­
formed using a Shimadzu GCMS-QP2020 chromatograph and a single-
quadrupole, four-stage mass spectrometer (Shimadzu Corp., Tokyo,
Japan). Absorbance values were determined using a Spectra MAX190
(Meigu Molecular Instruments Co. Ltd., Shanghai, China). MARS40
rotational rheometer was from Thermo Scientific (MA, USA). Confocal
laser microscope TCS SP8 was from Leica (Wetzlar, Germany). Kom­
bucha was purchased from Shaanxi Fuerbang Biotechnology Co. Ltd.
(Xi’an, Shaanxi, China). Four Lactobacillus bacteria (L. acidophilus,
L. bifidus, L. rhamnosus, and L. casei), used for matching the known lactic
acid species within the kombucha bacteria [14], were purchased from
Shandong Zhongke Jiayi Biological Engineering Co. Ltd. (Weifang,
Shandong, China).
2.2. Preparation of fermented soy milk
Soy milk was prepared by mixing 100 g of soybean with 800 mL of
water. Sucrose and FOS were added to soy milk to achieve a final mass
ratio of 4.8:4.8:100. After high-pressure homogenization (20 MPa, 3
min), the mixture was transferred to sterile bottles for sterilization in a
water bath (95 ℃, 15 min), then cooled to 37 ℃ and inoculated in an
aseptic environment at a mass ratio of soy milk to fermentation agent of
100:4.2. Fermentation was carried out in a temperature box at 32 ℃
with indirect shock ventilation every 2 h for 5 min to ensure the growth
of microorganisms. When the pH value of fermented soy milk
approached 4.5, the sealed samples were transferred to a refrigerator at
4 ℃ for 2 h for slow fermentation.
2.3. Determination of microbial content of fermented soy milk
The total number of LAB was enumerated on MRS medium (36 ℃ for
72 h) [15]. AAB and yeast counts were determined on potato glucose
agar medium and AAB culture medium, respectively, after incubation at
30 ℃ for 96 h [16,17]. For the three groups of fermented soy milk
samples, total DNA extraction was performed according to the DNA
extraction kit instructions for detecting the DNA concentration and
purity. Bacteria were PCR amplified with 338F (5′ -ACTCCTACGGGAG
GCAGCAG-3′ ) and 806R (5′ - GGACTACHVGGGTWTCTAAT-3′ ) primers,
fungi were amplified by PCR with primers using SSU0817F
(5′ -TTAGCATGGAATAATRRAATAGGA-3′ ) and SSU1196R
(5′ -TCTGGACCTGGTGAGTTTCC-3′ ) primers. Amplification was per­
formed under the following conditions: pre-denaturation at 95 ℃ for 3
min, followed by 35 cycles of pre-denaturation at 95 ℃ for 30 s,
annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 45 s; then, a final
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Process Biochemistry 121 (2022) 286–297
extension at 72 ℃ for 10 min based on the OTU species abundance
profiles after data homogenization [46].
2.4. Fermentation performance determination
2.4.1. Determination of pH and titratable acidity (TA) of fermented soy
milk
Changes in pH were monitored during soy milk fermentation using a
pH meter. TA was determined according to the AOAC method [18].
2.4.2. Determination of rheological properties of fermented soy milk
The rheological properties were determined by a low-amplitude
frequency sweep test conducted on a rotational rheometer fitted with
a stainless-steel flat probe (diameter of 40 mm, 1 mm gap) [19]. A
sequence of sweeps on samples was conducted at 25 ± 0.5 ℃, as follows:
(1) a strain sweep from 0 % to 50 % at a fixed frequency of 1 Hz, to
determine a constant strain of 0.5 %. (2) shear sweep from 0 to 500 1/s
and back to 0, the scanning time was 360 s (3) frequency sweep from 0.1
to 10 Hz at a fixed strain of 0.5 %. The protocol was repeated twice for
each sample.
2.4.3. Determination of basic components in fermented soy milk
The basic composition of fermented soy milk was analyzed by an
automatic milk composition analyzer. Before determination, the sample
was diluted 10 times with distilled water and homogenized using a high-
pressure homogenizer (20 MPa, 3 min).
2.4.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE)
SDS-PAGE was used to analyze the utilization of nitrogen sources in
soy milk by different LAB. After transferring 50 μL of the samples to 1.5
mL centrifuge tubes, 0.5 mL of sample treatment solution (20 % glycerol,
0.2 % SDS, 0.063 M Tris-HCl at pH 6.8) was added, followed by 0.36 g
urea, 20 μL of saturated bromophenol blue solution, 20 μL mercaptoe­
thanol, and sufficient distilled water to complete the total volume to 1
mL, then fully mixed to homogeneity. The mixture was left to stand at
room temperature for 12 h before electrophoresis [35]. After electro­
phoresis, the gel was scanned using a Canon scanner, and Scion Image
software was used to analyze the optical density of the protein bands on
the gel image.
2.4.5. Determination of microstructure of fermented soy milk
After a 10-fold dilution of the sample with distilled water, 1 mL was
placed into separate EP tubes. Nile red dye (40 μL, 1 mg/mL) and Nile
blue dye (40 μL, 10 mg/mL) were added, and then the samples were
incubated at room temperature for 30 min in the dark. The stained
sample was promptly placed on a slide and sealed with a cover slide.
Then, the microstructure was visualized by confocal laser scanning mi­
croscopy (TCS SP8, Leica Co. Ltd., Wetzlar, Germany) following the
standard method [20]. The excitation wavelengths were 488 and 633
nm for Nile red and Nile blue, respectively, and the samples were
captured with a 100 × oil mirror.
2.5. Analysis of β-glucosidase activity and nutrient composition
2.5.1. Determination of β-glucosidase activity in fermented soy milk
β-Glucosidase activity was determined by measuring the rate of hy­
drolysis of ρ-nitrophenyl β-D-glucopyranoside (pNPG) [21]. Briefly, 0.2
mL of 5 mM pNPG (dissolved in 0.1 M PBS pH 7.0) was added to 0.1 mL
of the soy milk sample, mixed slowly to homogeneity, and incubated at
37 ℃ for 30 min. The reaction was stopped by the addition of 0.4 mL of
0.5 M Na2CO3 solution (4 ℃) and centrifuged immediately (10,000 rpm,
20 min, 4 ℃). The absorbance value of the supernatant at 405 nm was
determined. In parallel, a blank control was prepared whereby 0.1 mL of
soy milk and 0.2 mL of pNPG solution were mixed evenly and immedi­
ately inactivated in a boiling water bath for 5 min, followed by the
X. Peng et al.
Fig. 1. Microbial growth: A) Dynamic growth curve; B) Fermentation endpoint content; C) Relative abundance of bacteria; D) Relative abundance of fungus. “J” is
co-fermentation by lactic acid bacteria (LAB) and kombucha bacteria at 1:1 mass ratio, “L” is LAB fermentation, “K” is kombucha bacteria fermentation.
addition of 5.0 mL 1 M Na2CO3 solution.
2.5.2. Determination of isoflavone content in fermented soy milk
To extract the isoflavones, 4 mL of the sample supernatant in a 10-mL
EP tube was mixed with 4 mL ethanol and HCl (final concentration of 1
M) and incubated at 80 ℃ for 60 min. After centrifugation at 10,000 rpm
for 10 min at high speed, 1 mL of the supernatant was filtered through a
0.22 µm membrane into an HPLC vial for subsequent analysis. The
chromatographic conditions were set according to Mi et al. [22].
Chromatographic column: C18 (250 mm × 4.6 mm, 5 µm); Mobile phase:
acetonitrile; Hydrophosphate solution (pH 3.0); Velocity of flow: 1.0
mL/min; UV detection wavelength: 260 nm; Sample size: 10 μL; Column
temperature: 30 ℃.
Standard curve equations for the four soy isoflavones:
Daidzein: Y=0⋅033X-0⋅269R2 R2=0⋅995
Genistein glycosides: Y=0⋅018X+0⋅225 R2=0⋅998
Isoflavone aglycones: Y=0⋅012X+2⋅488 R2=0⋅992
Genistein: Y=0⋅005X+2⋅573 R2=0⋅999
2.5.3. Determination of vitamin content in fermented soy milk
Samples were filtered through a 0.45-μm filter, and then 15 μL of the
filtered sample was injected into an HPLC equipped with a C18 column
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Process Biochemistry 121 (2022) 286–297
(250 mm × 4.6 mm, 5 mm). The vitamins were separated at a flow rate
of 1 mL/min and eluted by linear gradient elution with 0.01 % TFA
(solvent A) and methanol (solvent B). In the first 5 min [23], solvent A
was decreased from 95 % to 92 % and then linearly decreased to 5%.
After that, solvent A was gradually recovered to 95 %. Analysis was
performed at 245 and 280 nm. Calibration curves for pure compounds of
riboflavin, niacin, folic acid, and cobalamin were established. Results
were expressed as vitamin content (μg/mL) per milliliter of fermented
soy milk.
2.5.4. ABTS＋ and DPPH radical scavenging assay
To measure the ABTS＋ and DPPH antioxidant activity, the experi­
mental method of Yin et al. [24] and Jiang et al. [25] were modified.
According to the instructions of the total antioxidant capacity (T-AOC)
test kit (ABTS method), repeated the determination of each soybean
milk sample for 3 times with the microplate reader. Briefly, 5 mL of
sample was combined to homogeneity with the same volume of distilled
water and then centrifuged immediately (10,000 rpm, 8 ℃, 10 min). To
initiate the reaction, 1 mL of the supernatant was added to 1 mL of 0.2
mM DPPH radical ethanol solution (stored away from light) at room
temperature (20–25 ℃) and left to react for 30 min in a dark environ­
ment. Afterward, the sample was centrifuged (4000 rpm, 8 ℃, 10 min),
and the absorbance of the supernatant at 517 nm was determined. The
DPPH free radical clearance rate was calculated by the following
formula:
DPPHFreeradicalclearance(%) = [1 − (Ai − Aj)/Ac] × 100
X. Peng et al.
Fig. 2. Fermentation performance: A) pH value and titratable acidity (TA); B) Frequency sweep curve; C) Relationship between apparent viscosity and shear rate; D)
SDS-PAGE. “S” is unfermented raw soy milk, “J(1:1)” is co-fermentation by lactic acid bacteria (LAB) and kombucha bacteria at 1:1 mass ratio, “L” is LAB fermen­
tation, “K” is kombucha bacteria fermentation.
where Ai is the absorbance of 1 mL DPPH + 1 mL sample; Aj is the
absorbance of 1 mL distilled water + 1 mL sample; Ac is the absorbance
of 1 mL DPPH + l mL distilled water.
2.6. Determination of volatile flavor components in fermented soy milk
The soy milk sample (10.0 mL) was placed in a flask and equilibrated
at 45 ℃ for 25 min. After equilibration, the solid-phase microextraction
(SPME) fiber was inserted into the headspace of the flask for 40 min
(45 ℃). Then, it was rapidly inserted into the injector port of the GC-MS
for desorption (250 ℃, 1 min). The GC conditions were as follows:
column type, HP-5 capillary column (30 m × 0.25 mm, 0.25 µm);
injector temperature, 250 ℃; the GC oven was maintained at the initial
temperature of 35 ℃ for 3 min, then increased to 200 ℃ at 5 ℃/min,
followed by a further increase to 230 ℃ at 10 ℃/min and maintained at
230 ℃ for 10 min; carrier gas (He) flow rate, 0.80 mL/min; no shunt
injection [26]. The MS conditions were set as follows: electron ioniza­
tion source; ionization energy, 70 eV; ion source temperature, 200 ℃;
emission current, 200 μA; detection voltage, 350 kV; mass scanning
range 35–400 U. The relative content of each volatile flavor substance
was obtained by the peak area normalization method by computer
retrieval and identified by comparison with standard mass spectra
provided by the NIST mass spectral library.
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2.7. Statistical analysis
The data was repeated 3 times, and 3 parallel samples were selected
for each test to determine all indicators. Results were expressed as mean
± SD. Data, including Pearson’s correlation, were statistically analyzed
by ANOVA at a significance level of p < 0.05, using IBM SPSS 23.0 (IBM
Corp., Armonk, NY, USA). Illustrations were constructed using the Ori­
ginPro 2019b software (OriginLab, Northampton, MA, USA).
3. Results and discussion
3.1. Microbial content of fermented soy milk
As shown in Fig. 1A, when soy milk was fermented with LAB alone,
the strains grew well, increasing from 5.71 log to 8.85 log CFU/mL
within 12 h, proving that LAB could grow well in the soy milk envi­
ronment. When the mass ratio of LAB to kombucha bacteria was 2:1, the
growth of LAB in fermented soy milk occupies an absolute advantage
(Fig. 1B). The initial 3.31 log CFU/mL increased to 7.84 log CFU/mL
within 16 h. At this point, the LAB content of the system was as high as
48 %. In the mixed culture system of LAB and yeast, the growth of LAB
will be affected by yeast-derived metabolites, in particular, the fatty
acids [27]. At the same time, various compounds, such as
X. Peng et al. Process Biochemistry 121 (2022) 286–297

exopolysaccharides (EPS), biosurfactants, hydrogen peroxide (H2O2), Table 1

reuterin (3-hydroxypropionaldehyde), 4-hydroxyphenyllactic acid, cy­ Analysis of basic components of fermented soy milk.
clic dipeptides, and organic acids (e.g., lactic acid) produced by LAB will Basic soy milk Lactobacillus Kombucha Joint
inhibit the growth of yeast [28]. When the mass ratio of LAB to kom­ ingredients (g/ fermentation fermentation fermentation
bucha bacteria was 1:1 (Fig. 1A), there was a positive effect on the 100 g)
growth of LAB, yeast, and AAB in fermented soy milk, which increased Ash 1.30 1.59 ± 0.06a 1.48 ± 0.06a 1.54 ± 0.04a
from 2.63 log to 7.48 log CFU/mL, from 1.84 log to 6.68 log CFU/mL, ± 0.05a
and from 1.63 log to 6.83 log CFU/mL, respectively. The dominant Fat 2.41 1.72 ± 0.06b 1.79 ± 0.06a 1.75 ± 0.06a
± 0.09a
species was Acetobacter gluconicu (Gluconacetobacter), Lactobacillus Nonfat solids 15.94 17.24 ± 0.60a 16.55 ± 0.54a 16.82 ± 0.60a
rhamnosus and ZygoSaccharomyces bailii(Fig. 1C and Fig. 1D). At this ± 0.64b
time, the microbial population was split almost equally (32 %, 33 %, and Protein 3.36 4.43 ± 0.17a 4.47 ± 0.17a 4.44 ± 0.16a
36 %) among the microbial consortium, suggesting a symbiotic ± 0.10a
Lactose 8.77 10.28 ± 0.40a 9.65 ± 0.33a 9.89 ± 0.37a
fermentation of soy milk by bacteria and yeast. Yeasts hydrolyze sucrose
± 0.28a
into glucose and fructose and then use fructose to produce ethanol. AAB Total solids 17.98 21.74 ± 0.70a 20.88 ± 0.63b 21.34 ± 0.71a
metabolize ethanol and glucose into acetic acid and gluconic acid, ± 0.73b
respectively, and LAB assimilate metabolites produced by yeast, such as
proteins and vitamins, to achieve further growth [29]. Through quan­
titative comparative proteomics to predict the metabolic exchange in the protein molecules, forming colloidal particles and increasing the vis­
model system, it appeared that the LAB benefited from the presence of cosity of fermented soy milk [34]. When a protein gel is formed in the
S. cerevisiae due to the enhanced availability of amino acids [30]. At the presence of EPS-producing LAB, the EPS can modify the structure and
same time, it metabolized the arginine provided by the yeast to protect texture of the gel [35]. When the amount of EPS was increased, the
itself from pH stress while also enhancing the acid tolerance of molecular interaction made the shape of EPS irregular. At the same time,
S. cerevisiae by switching from acetate and lactate production to buta­ the association between the EPS and milk proteins caused internal
nediol production and by upregulating arginine deiminase [31]. friction and increased viscosity of the fermented soy milk. On the other
The results showed that under the condition of 32 ℃ when the mass hand, the protein and fat in soy milk are broken up by high-pressure
ratio of LAB to kombucha bacteria was 1:1, the microorganisms in fer­ homogenization, and the finer protein and fat particles are more
mented soy milk could survive well in the soy milk environment. evenly dispersed in the gel system, which is also conducive to the for­
Therefore, the follow-up experiments are mainly carried out at a mass mation of a more uniform and compact protein gel network structure.
ratio of LAB to kombucha bacteria of 1:1. Apparent viscosity is an important parameter affecting the sensory
quality of yogurt. As shown in Fig. 2C, the apparent viscosity of fer­
3.2. Fermentation performance mented soy milk of all three groups decreased with the increase in shear
rate and tended to be stable at a higher shear rate, indicative of a typical
3.2.1. pH and TA of fermented soy milk shear-thinning non-Newtonian fluid [36]. At low shear rates, the vis­
As shown in Fig. 2A, the initial pH and TA values of fermented soy cosity of soy milk fermented with LAB alone was the highest among the
milk prepared after inoculation with LAB only, kombucha bacteria only, three groups. This is because LAB produce EPS and participate in the gel
and mixed LAB and kombucha bacteria at 1:1 mass ratio were 6.78 and construction process. EPS and protein interact with each other, resulting
12.34◦ T, respectively. During the fermentation process, the pH values of in twining and overlapping of the molecular chains and hindering the
fermented soy milk of all groups showed a downward trend. In the flow of the solution. With the increase in shear rate, the network
mixed culture, the fermentation endpoint was reached at 30 h, and the structure of the fermented soy milk system was destroyed, the winding
pH and TA values of fermented soy milk were 4.31 and 78.33◦ T, chain segment was cut, and the shear resistance decreased, so the vis­
respectively. During fermentation, lactic acid produced by LAB meta­ cosity decreased. At high shear rates, the viscosity stabilizes at lower
bolism and acetic acid produced by AAB metabolism reduced the pH levels, and possible entanglement segments are completely cut and
value of fermented soy milk, which was consistent with the results of a flattened [37].
previous study on the combined fermentation of soy milk by LAB and
yeast [32]. The fermentation time with kombucha bacteria alone was 3.2.3. Basic components of fermented soy milk
the longest and reached the endpoint at 48 h, with pH and TA values of As shown in Table 1, at the end of fermentation, the fat content of
4.31 and 78.61◦ T, respectively. During the fermentation process, the TA fermented soy milk, irrespective of the starter culture, decreased
of soy milk gradually increased, resulting in a gradual decrease in the pH significantly, and the total solids increased significantly (p < 0.05). By
value. This increase in acidity was typically due to the formation of contrast, the ash, protein, and lactose contents showed no significant
organic acids [17]. changes. After inoculation of the starter culture, the protein in soy milk
would be consumed during the growth of the microorganisms. Mean­
3.2.2. Rheological properties of fermented soy milk while, the starter introduced a certain amount of protein, resulting in a
G′ and G" represent the elastic and viscous characteristics of the slight change in the protein content of the fermented soy milk compared
sample, respectively [33]. As shown in Fig. 2B, the G′ and G" values of with the unfermented soy milk. With the progress of fermentation, the
the three groups of fermented soy milk increased with the frequency fat content decreased significantly (p < 0.05), which may be due to the
(0.1–10 Hz), and G′ was higher than G" for all these samples, indicating production of lipase/esterase during the fermentation with LAB, which
that the elastic component was dominant and the samples showed can hydrolyze the fat into free fatty acids and mono-and diacylglycerols,
solid-like characteristics. soy milk treated with LAB alone had the and promote the synthesis of other flavor compounds, such as β-keto
highest G′ and G" values, followed by the mixed LAB and kombucha acids and acyl-CoA, initiated by the participation of the free fatty acids
bacteria (1:1 mass ratio) fermentation, which was closely followed by in further reactions [38]. It may also be because kombucha fermentation
the group fermented with kombucha bacteria alone. The results showed produces bile acid, which can lower the proportion of cholesterol by
that the fermentation process could significantly change the viscoelastic converting cholesterol to water-soluble derivatives. In addition, the
rheological properties of soy milk (p < 0.05). This result may be caused Kombucha fermentation process can also produce a substance that can
by, on the one hand, the continual decrease in the pH value due to the neutralize the cholesterol, glucuronic acid, which reduces the choles­
LAB-derived lactic acid and AAB-derived acetic acid during the terol content [39,40].
fermentation process, thus reducing the surface activity of soybean

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X. Peng et al. Process Biochemistry 121 (2022) 286–297

Fig. 3. Confocal laser microscopy images: A(a)1–4 are the images of unfermented bean milk, B(b)1–4 are the images of soy milk fermented by lactobacillus, C(c)1–4 are
the images of soy milk fermented by kombucha, D(d)1–4 are the images of soy milk fermented by joint strain; (A) 100 µm, (B), 50 µm.

291
X. Peng et al.
Fig. 4. Antioxidant capacity: A) β-glucosidase activity; B) DPPH free radical
scavenging activity; C) ABTS＋ radical scavenging activity. “J(1:1)” is co-
fermentation by lactic acid bacteria (LAB) and kombucha bacteria at 1:1
mass ratio, “L” is LAB fermentation, “K” is kombucha bacteria fermentation.
3.2.4. SDS-PAGE of fermented soy milk
Protein in soy milk is a source of nitrogen. According to their sedi­
mentation coefficient, soybean proteins can be divided into 2S, 7S, 11S,
and 15S. β-Glycinin, the main polypeptide of 7S, is composed of three
subunits: α’ (~72 kDa), α (~68 kDa) and β (~52 kDa), respectively.
11 S can be divided into acidic subunit A (~35 kDa) and alkaline sub­
unit B (~20 kDa). LAB and kombucha bacteria can ferment soy milk
protein to form peptides and amino acids. SDS-PAGE was used to
analyze the utilization of soy milk protein by the different starter cul­
tures. As shown in Fig. 2D, the band intensities were decreased by
fermentation. There was no significant difference between the band
intensities for the fermentation by LAB alone and kombucha bacteria
alone. Fermentation with LAB and kombucha bacteria was shown to
decompose protein most extensively among the starter cultures, result­
ing in the lowest band intensities. After fermentation, the asymmetry of
small molecule proteins increased, the protein molecules condensed,
and then the viscosity increased. Some of the protein decomposed into
free amino acids and served as the "nitrogen nutrient source" of micro­
organisms in soy milk [33]. Therefore, the cultures used in this
fermentation can utilize a variety of protein components in soy milk.
3.2.5. Microstructure of fermented soy milk
As shown in Fig. 3, in order to fully compare the macro distribution
and micro combination of protein and oil in fermented soy milk system,
two groups of micrographs with different magnification were obtained
(100 µm, 50 µm). All the fermented samples showed a porous network
structure, with extensive protein aggregation and visible fat globules.
Due to the high-pressure homogenization of soy milk, the oil and protein
particles in unfermented soy milk were small and dispersed. Lactic acid
produced during the fermentation process by LAB reduces the pH value
of soy milk and leads to protein aggregation and solidification [41]. The
large and concentrated protein aggregates increased the viscosity of
fermented soy milk. Correspondingly, the protein aggregation status of
soy milk fermented by kombucha bacteria alone was not as significant as
that by LAB alone, this may be explained by the fact that there were
relatively fewer LAB in kombucha bacteria, but the growth of AAB can
also reduce the pH of soy milk. On the one hand, due to the good growth
of yeast, AAB, and LAB, the pH value was greatly reduced by fermen­
tation with the kombucha consortium; on the other hand, more extra­
cellular polysaccharide was produced from the fermentation by LAB,
thus increasing the trend of protein aggregation. The results were
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Process Biochemistry 121 (2022) 286–297
Fig. 5. Correlation plot summarizing correlation coefficients between different
indicators. Red and blue reflect positive and negative correlations, respectively.
The depth of color represents the magnitude of the correlation coefficient.
consistent with the basic composition data and rheological analysis of
fermented soy milk.
3.3. β-Glucosidase activity and nutrient composition of fermented soy
milk
3.3.1. β-Glucosidase activity produced by bacteria in fermented soy milk
As shown in Fig. 4A, fermentation increased (p < 0.05) the initial
β-glucosidase activity of 2.42 mU/mL in soy milk to 58.88 mU/mL by
LAB alone, 65.94 mU/mL by kombucha bacteria alone, and 74.08 mU/
mL by the mixed culture. The change in β-glucosidase activity during
fermentation has been correlated to the growth of microorganisms [42].
β-Glucosidase reached maximal production in the early stage of the
stable growth period, but the activity decreased as the stable growth
period progressed, perhaps because of the downregulation of the
β-glucosidase gene at decreased pH values [43,44]. The mixed culture
fermentation promoted β-glucosidase activity, and similar results were
obtained in soy milk fermented with kefir [45]. In addition to some LAB,
including Lactobacillus acidophilus, L. rhamnosus, and L. plantarum, some
yeasts in the kombucha consortia also had glucosidase activity [46,47].
β-Glucosidase hydrolyzes the terminal β-D-glucoside bond of isoflavone
glycosides, converting them into aglycones [48]. The higher the activity
of glucosidase in fermented bean milk, the faster the strain converts
glycoside soybean isoflavone into side element soybean isoflavone, and
the stronger the antioxidant capacity of side element soybean isoflavone
is stronger, so the activity of fermented bean milk can determine the
potential antioxidant capacity of fermented bean milk. This was also
shown in the present experiment, the contents of β-glucosidase had a
good correlation with the ABTS＋ and DPPH radical scavenging rate
(Fig. 5).
3.3.2. Isoflavone content of fermented soy milk
During the mixed culture fermentation by LAB and kombucha bac­
teria, the isoflavone aglycones content increased from 53.87 to
358.13 µg/g dry weight (dw), and the genistein content increased from
59.14 to 422.08 µg/g dw (Table 2), being significantly higher than those
in soy milk fermented by LAB alone and kombucha bacteria alone
(p < 0.05). These results indicate that fermentation with LAB and
kombucha bacteria can improve the aglycone content in soy milk, which
corresponds to the increased β-glucosidase activity in the above results.
Tsangalis et al. [49] revealed that β-glucosidase-producing Bifidobacte­
rium animalis B-12 hydrolyzed isoflavone glycosides into bioactive and
bioavailable aglycones when cultured in soy milk prepared from soy
protein isolate. According to previous studies, soy milk fermented with
X. Peng et al. Process Biochemistry 121 (2022) 286–297

Table 2
Analysis of isoflavone content in fermented soy milk.
Time Daidzein (µg/g dw) Genistein glycosides (µg/g dw) Isoflavone aglycones (µg/g dw) Genistein (µg/g dw)
(h)
L K J L K J L K J L K J

0 220.15 220.15 220.15 337.34 337.34 337.34 53.87 53.87 53.87 59.14 59.14 59.14
± 7.74a ± 7.74a ± 7.74a ± 10.45a ± 11.45a ± 11.45a ± 1.86 h ± 1.86 f ± 1.86 f ± 2.83 h ± 2.83 h ± 2.26 h
6 216.58 217.27 215.65 328.46 335.51 323.84 65.47 67.78 64.35 61.26 64.22 68.16
± 9.16ab ± 9.22a ± 7.23ab ± 10.15ab ± 10.37a ± 11.43a ± 6.42 g ± 6.25gf ± 2.47 f ± 3.13 h ± 3.13 h ± 2.55 g
12 202.52 214.36 197.53 313.58 321.54 297.53 74.38 77.04 75.27 70.14 73.12 87.86
± 8.64b ± 8.47a ± 8.85b ± 10.85b ± 10.65ab ± 8.27b ± 5.71 g ± 5.72 g ± 2.53 f ± 3.32 g ± 3.87 g ± 3.15 f
18 166.15 193.16 155.76 287.38 303.43 256.25 93.73 92.39 107.35 82.67 86.15 113.32
± 8.14c ± 9.83b ± 6.54c ± 11.93c ± 10.52b ± 9.13c ± 6.04 f ± 6.92 f ± 4.63e ± 3.59 f ± 3.01 f ± 4.28e
24 137.27 175.26 126.23 252.17 272.38 203.62 139.47 131.48 234.16 108.24 110.24 165.21
± 5.58d ± 7.69b ± 4.86d ± 9.28d ± 9.34c ± 7.16d ± 4.38e ± 4.15e ± 8.59d ± 4.63e ± 4.34e ± 6.42d
30 121.39 132.51 92.29 227.63 222.74 138.44 158.43 167.43 272.54 144.84 149.83 240.64
± 4.36e ± 4.96c ± 4.31e ± 7.18e ± 7.53d ± 5.08e ± 6.33d ± 6.42d ± 11.27c ± 5.43d ± 5.38d ± 7.16c
36 112.73 100.95 53.65 189.35 175.87 63.28 190.59 204.53 311.22 185.89 199.53 336.74
± 4.28 f ± 4.43d ± 2.21 f ± 6.18 f ± 6.52e ± 1.57 f ± 8.31c ± 8.85c ± 14.39b ± 7.31c ± 7.15c ± 10.22b
42 99.62 67.38 14.42 157.34 127.05 3.67 244.66 264.52 335.26 249.15 269.35 406.15
± 3.18 g ± 3.38e ± 0.46 g ± 4.67 g ± 4.41 f ± 0.63 g ± 10.67b ± 10.67b ± 15.18ab ± 8.05b ± 8.16b ± 13.27a
48 87.08 61.04 ND 120.52 94.05 ND 283.41 298.27 358.13 302.47 313.42 422.08
± 2.28 h ± 1.21 f ± 4.76 h ± 3.32 g ± 9.23a ± 9.38a ± 14.77a ± 13.36a ± 12.42a ± 13.48a

L = Lactobacillus fermentation; K = kombucha fermentation; J = joint fermentation; ND = not detected in the detection range. Different letters in the same column
denote a significant difference (p < 0.05).

Table 3
Analysis of vitamin content in fermented soy milk.
Time Riboflavin (µg/g dw) Niacin (µg/g dw) Folic acid (µg/g dw) Cobalamin (µg/g dw)
(h)
L K J L K J L K J L K J

0 117.44 117.44 117.44 205.43 205.43 205.43 150.63 150.63 150.63 ND ND ND

± 5.26a ± 5.26d ± 5.26c ± 8.15a ± 8.15 g ± 8.15 g ± 7.27a ± 7.27 f ± 7.27 f
6 110.95 115.34 112.62 198.66 207.62 205.66 146.57 148.96 147.45 ND 5.25 5.37
± 5.35a ± 5.18d ± 5.28c ± 7.99a ± 7.95 g ± 7.32 g ± 7.81a ± 6.63 f ± 5.32 f ± 0.21 g ± 0.23 g
12 99.88 118.23 114.37 191.78 253.05 234.16 142.59 151.37 150.37 2.45 13.32 15.23
± 3.16b ± 5.06d ± 5.61c ± 6.96a ± 9.31 f ± 7.48 f ± 7.04a ± 5.12 f ± 6.68ef ± 0.08e ± 0.52 f ± 0.51 f
18 87.18 121.34 118.31 181.39 321.77 302.83 129.92 178.51 162.49 3.48 21.58 25.57
± 3.71c ± 4.24d ± 4.23c ± 7.23ab ± 10.26e ± 12.38e ± 4.39b ± 6.33e ± 7.26e ± 0.13d ± 1.24e ± 1.32e
24 75.53 147.76 129.77 168.84 485.71 423.94 117.04 254.79 232.48 5.57 41.93 52.63
± 2.18d ± 6.83c ± 3.86b ± 6.67b ± 15.38d ± 17.39d ± 3.28c ± 7.25d ± 8.29d ± 0.22c ± 2.41d ± 2.46d
30 67.36 168.98 137.05 143.27 603.15 536.55 108.78 365.73 335.83 8.44 68.22 75.21
± 2.33e ± 6.77b ± 6.73b ± 5.95c ± 27.39c ± 21.95c ± 3.32d ± 14.28c ± 11.34c ± 0.34b ± 2.35c ± 2.44c
36 63.66 189.33 148.27 129.31 721.07 645.37 102.82 454.47 416.44 9.11 82.58 90.34
± 1.96ef ± 8.21a ± 7.92ab ± 5.22d ± 26.23b ± 22.67b ± 4.75de ± 17.73b ± 14.42b ± 0.27ab ± 4.43b ± 4.28b
42 62.83 197.23 157.42 120.36 821.36 737.83 96.38 551.26 503.41 9.67 88.73 96.98
± 2.13 f ± 7.89a ± 7.41a ± 4.48de ± 28.44a ± 28.43a ± 7.26e ± 20.39a ± 19.08a ± 0.24a ± 4.28ab ± 3.26b
48 61.34 200.54 162.58 115.83 865.35 756.97 94.22 553.17 511.25 9.53 92.92 106.73
± 1.88 f ± 9.26a ± 7.36a ± 5.39e ± 29.30a ± 23.97a ± 3.94e ± 18.84a ± 21.34a ± 0.21a ± 3.47a ± 4.64a

L = Lactobacillus fermentation; K = kombucha fermentation; J = joint fermentation; ND = not detected in the detection range. Different letters in the same column
denote a significant difference (p < 0.05).

LAB and kefir can release corresponding phenolic substances and
convert isoflavone glycosides into their corresponding aglycone forms
[45,50]. Thus, the fermentation of soy milk can increase its aglycone
content and enhance its antioxidant effect. This was also shown in the
present experiment, the contents of β-glucosidase and soybean iso­
flavones had a good correlation (Fig. 5). Correspondingly, Marazza et al.
[51] found that the aglycone content increased with the increased DPPH
free radical scavenging rate in fermented soy milk. The results showed
that the mixed culture fermentation used in this study has a strong
ability to hydrolyze isoflavone glycosides and produce an ample quan­
tity of isoflavone aglycones.
3.3.3. Vitamin content of fermented soy milk
LAB will consume vitamins during fermentation, and almost no new
vitamins are synthesized, whereas kombucha bacteria will synthesize
some vitamins during soy milk fermentation [52]. As shown in Table 3,
there was a significant increase in the contents of all four vitamins
(riboflavin, niacin, folic acid, and cobalamin) measured during soy milk
fermentation (p < 0.05), except for the riboflavin, niacin, and folic acid
contents in soy milk fermented with LAB alone, which decreased.
Riboflavin is an integral cofactor of coenzymes involved in various
cellular metabolic activities. The mixed culture starter can produce a lot
of riboflavin during soy milk fermentation. However, riboflavin is a
prerequisite for the initial growth of a part of LAB, so part of the ribo­
flavin will be continuously consumed during the synthesis of riboflavin
[53]; therefore, a slight decrease in riboflavin content in the early
fermentation indicates that the production rate of riboflavin is insuffi­
cient to meet its rate of consumption. In addition, the contents of niacin
and folic acid increased from 205.43 and 150.63 µg/g dw before
fermentation to 865.35 and 553.13 µg/g dw, respectively, after
fermentation by the kombucha consortium alone. In the same group,
cobalamin was not detected before fermentation but increased to
92.92 µg/g dw after fermentation. The formation of cobalamin changes
the microenvironment of tryptophan residues in soybean protein and
increases hydrophobicity, which can alter the conformation of soybean
protein [54]. Study has shown that fermentation increases the content of
B vitamins in fermented dairy products, especially soy milk [55].

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Table 4
Analysis of volatile flavor components in fermented soy milk.
Number Category Compound Retention time Relative amount (% ± SD)
(min)
The original soy Lactobacillus Kombucha Joint
milk fermentation fermentation fermentation

1 Aldehydes 2-Octenal 15.65 1.98 ± 0.26 1.47 ± 0.42 0.27 ± 0.07 0.72 ± 0.21
2 trans-2,4-Heptadienal 13.48 6.47 ± 1.36 4.35 ± 0.74 1.48 ± 0.13 2.43 ± 0.88
3 Heptaldehyde 9.04 2.75 ± 0.61 1.36 ± 0.29 — —
4 n-Nonaldehyde 18.51 7.23 ± 2.23 2.38 ± 0.76 1.03 ± 0.33 1.36 ± 0.18
5 (E)-Hept-2-enal 28.43 0.17 ± 0.02 1.22 ± 0.27 — 0.32 ± 0.02
6 2,5-Dimethylbenzaldehyde 22.76 — — 15.32 ± 1.15 13.89 ± 1.82
7 Caproaldehyde 7.48 30.16 ± 10.56 16.42 ± 3.41 8.13 ± 1.85 8.74 ± 0.73
8 Benzaldehyde 11.69 1.37 ± 0.32 3.39 ± 0.45 1.02 ± 0.11 1.13 ± 0.17
9 2-Hexene aldehyde 4.08 1.46 ± 0.38 0.74 ± 0.02 — —
10 trans-2,4-Decadienal 29.73 15.28 ± 1.13 9.47 ± 0.89 8.49 ± 0.53 7.06 ± 1.40
11 Alcohols 1-Amyl alcohol 12.44 1.22 ± 0.51 1.31 ± 0.66 — —
12 n-Hexyl alcohol 16.34 — 0.36 ± 0.03 1.72 ± 0.52 2.58 ± 0.96
13 3-Octanol 17.85 0.68 ± 0.12 — — —
14 1-Octen-3-ol 14.82 13.66 ± 0.75 2.37 ± 0.28 8.42 ± 0.91 4.83 ± 0.76
15 1-Heptanol 13.92 — — 2.31 ± 1.05 1.88 ± 0.05
16 cis-3-Hexen-1-ol 19.67 — 1.04 ± 0.31 1.38 ± 0.29 1.53 ± 0.34
17 Geraniol 21.49 — — 8.46 ± 0.83 9.36 ± 1.07
18 Linalool 24.32 — — 20.32 ± 2.29 18.01 ± 1.39
19 1-Nonanol 20.37 — — — 0.78 ± 0.14
20 Esters Ethyl hexanoate 14.20 — 0.48 ± 0.14 1.19 ± 0.07 1.45 ± 0.57
21 Phthalic acid ester 15.10 0.33 ± 0.03 — 0.56 ± 0.06 0.46 ± 0.10
22 2-Hydroxy-benzoicacimethylester 8.38 1.25 ± 0.28 — 1.28 ± 0.36 1.41 ± 0.52
23 Butyl phthalate 33.57 — 2.92 ± 0.17 — 0.87 ± 0.05
24 Ketones Acetylacetone 5.08 — 3.31 ± 1.22 1.14 ± 0.58 1.44 ± 0.72
25 3,5-Octenaldehyde-2-ketone 12.86 — 4.29 ± 0.35 — 1.15 ± 0.08
26 Beta-ionone 6.63 — — 0.82 ± 0.02 0.71 ± 0.04
27 3-Octen-2-one 16.94 1.78 ± 0.77 1.38 ± 0.08 — —
28 2-Nonanone 7.12 — — 1.48 ± 0.11 0.59 ± 0.02
29 Butane-2,3-dione 2.35 — 4.35 ± 0.65 — —
30 5-Methyl-2-hexanone 10.87 1.72 ± 0.13 0.74 ± 0.46 0.49 ± 0.05 0.64 ± 0.03
31 Acids 2,4,4-Trimethylamyl ester Propionic 28.36 — — 1.37 ± 0.14 0.95 ± 0.08
acid
32 Caproic acid 23.52 — 15.47 ± 1.22 — 4.38 ± 0.35
33 octoic acid 26.57 — — 1.93 ± 0.72 1.86 ± 0.09
34 Nonanoic Acid 25.66 — — 0.72 ± 0.09 0.63 ± 0.16
35 Citric Acid 4.74 — — 1.33 ± 0.15 0.89 ± 0.06
36 Acetic Acid 2.97 0.31 ± 0.10 1.34 ± 0.16 2.49 ± 0.38 1.77 ± 0.21
37 Other n-Pentane 1.98 1.72 ± 0.19 1.32 ± 0.31 — —
38 Hexadiene-1,3 17.14 1.03 ± 0.07 — — —
39 Alanine 1.94 — 5.52 ± 1.71 — —
40 6-Methyl-3-octyne 34.28 — 3.68 ± 0.28 — 0.21 ± 0.03
41 4-Ethylphenol 3.64 — — 1.94 ± 0.37 1.72 ± 0.16
42 Octadecyl vinyl ether 28.45 0.14 ± 0.05 1.87 ± 0.44 — —
43 2-Pentylfuran 14.67 5.26 ± 0.30 3.42 ± 0.14 — 1.53 ± 0.36

"-": no detection within the detection range.

3.3.4. ABTS＋ and DPPH radical scavenging rate and correlation analysis
Different antioxidant compounds may exhibit different antioxidant
activities through different mechanisms. We applied two different
antioxidant evaluation assays of DPPH and ABTS＋ scavenging abilities
to analyze the antioxidant ability. As shown in Fig. 4B, unfermented soy
milk had a certain antioxidant capacity, displaying a DPPH free radical
clearance rate of 48.26 %. Among the fermented soy milk samples, those
fermented with the mixed starter culture had the highest DPPH anti­
oxidant activity (75.49 %), followed by 72.91 % for those fermented
with the kombucha consortium alone and 69.07 % for those fermented
with LAB alone. The ABTS＋ measurements in Fig. 4C showed similar
trends to DPPH. The contents of β-glucosidase and soybean isoflavones
had a good correlation with the ABTS＋ and DPPH radical scavenging
rate (Fig. 5). These results indicated that these compounds could
improve the antioxidant activity of fermented soy milk. Proteases, such
as peptidase and endopeptidase, secreted by microorganisms in kom­
bucha, can hydrolyze the peptide chain in plant proteins to form a large
number of low-molecular-weight active peptides. Peptides also play an
important role in the antioxidant activity of soybean products and have
a strong synergistic effect with antioxidants [56]. It was found that the
antioxidant activity of fermented soybean products was highly
correlated with the peptide content and protease activity [57]. Fer­
mented soy milk has a strong reducing activity and can react with free
radicals as an electron donor, thus interrupting the free radical chain
reaction and giving full play to the antioxidant effect.
3.4. Volatile flavor components in fermented soy milk
One of the main attributes influencing the acceptance of soy milk is
its unique beany flavor, also known as "grassy flavor" and "oxidized oil
flavor." It was found that some volatile compounds, including hexanal,
1-octene-3-ol, benzaldehyde, amyl alcohol, acetic acid, and n-nonal,
affected the flavor of soy milk [58]. As shown in Table 4 and Fig. 6, a
total of 43 volatile flavor compounds were detected in raw soy milk and
fermented soy milk. During the mixed culture fermentation, the LAB and
other microorganisms produced numerous alcohols (the end products of
amino acid catabolism). The contents of hexanal, 1-octene-3-ol, n-nonal,
and other compounds affecting the beany flavor decreased significantly
(p < 0.05), but some remained. In the process of soy milk fermentation,
hexanal and other volatile flavor substances can bind to the hydrophobic
regions of most proteins, and the binding constants increase with the
increase in the number of carbon chains, which may be the reason why

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X. Peng et al.
Fig. 6. Comparison and analysis of the composition of the flavor volatiles of soy milk fermented by different starter cultures.
some beany flavor remains [59]. In addition, organic acids produced
during the fermentation process, such as acetic acid and citric acid, can
impart soy milk with fresh acidity, and lactic acid produced by LAB can
neutralize the stimulation of acetic acid produced by AAB. Huang et al.
[60] found that compared to goat milk fermented with LAB, goat milk
co-fermented by LAB and yeast had several newly formed flavor com­
pounds, such as ethyl acetate, isoamyl acetate, 2-phenylethanol, and
methyl nonyl ketone, which masked the “goaty flavor” and imparted a
pleasant flavor. When Gerardi et al. [61] used yeast and LAB in the
fermentation of cherry fruit, the malic acid produced by substrate uti­
lization and the yeast was decarboxylated to lactic acid by LAB, effec­
tively reducing the acidity of fermentation. Kombucha fermentation can
generate a myriad of aromatic substances. Daenen et al. [47] screened
kombucha bacteria strains for β-glucosidase activity, which increases
the contents of linalool, methyl salicylate, (Z)− 3-hexen-1-ol, 1-octe­
ne-3-alcohol and several other volatile aromatic substances derived
from glycoside precursors. Gluconacetobacter intermedius promotes the
formation of aromatic substances, including acids, alcohols, and esters,
which can be used to produce traditional fermented foods in small
proportions [62,63].
4. Conclusion
In this study, a mixed starter culture of LAB and kombucha bacteria
was used to ferment soy milk, which enriched the species and content of
LAB in the kombucha consortium. Yeast, AAB, and LAB can grow well in
soy milk fermented by LAB and kombucha bacteria through shock
ventilation culture. The mixed culture fermentation can increase the
content of soybean isoflavones and vitamins in soy milk and signifi­
cantly enhance the nutritional characteristics of soy milk. Microscopic
and rheological results showed that the protein in soy milk could be
hydrolyzed to amino acids and peptides effectively during fermentation
by the mixed starter culture, which not only reducing the content of
peculiar flavor substances in soy milk but also endowing the fermented
soy milk with new characteristic aroma compounds. The improved
functional properties, basic composition, and antioxidant status can
improve the sensory properties and nutritional value of fermented soy
milk. This study can provide a new method for selecting a starter for soy
milk fermentation, enhancing the vitamin content of soy milk, and
improving the nutritional characteristics and flavor properties of soy
milk. Moreover, it can offer a theoretical basis for the research and
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Process Biochemistry 121 (2022) 286–297
development of fermented functional foods.
Funding
This work was financially supported by Excellent Youth Project of
Natural Science Foundation of Heilongjiang Province (YQ2021C023),
Funding for the Opening Project of Key Laboratory of Soybean Biology
of Chinese Education Ministry (NO. SBKF13).
Declarations of Competing Interest
The authors declare that they have no conflicts of interest.
References
[1] M.S.M. Wee, R. Yusoff, J.H. Chiang, Y. Xu, In vitro and in vivo studies on
intragastric soya protein-polysaccharide gels in a beverage matrix, Int. J. Food Sci.
Technol. 52 (2017) 1358–1366, https://doi.org/10.1111/ijfs.13415.
[2] J. Chen, C. Cui, H. Zhao, H. Wang, M. Zhao, W. Wang, K. Dong, The effect of high
solid concentrations on enzymatic hydrolysis of soya bean protein isolate and
antioxidant activity of the resulting hydrolysates, Int. J. Food Sci. Technol. 53
(2018) 954–961, https://doi.org/10.1111/ijfs.13668.
[3] L. Jiang, J. Hu, Y. Liu, Z. Jiang, Z. Wang, Z. Fan, Research progress in key
processing technology of soybean milk powder, Trans. Chin. Soc. Agric. Mach. 50
(2019) 1–11, https://doi.org/10.6041/j.issn.1000-1298.2019.06.001.
[4] L. Du, K.S. Ro, Y.J. Zhang, Y.J. Tang, W.B. Li, J.L. Xie, D.Z. Wei, Effects of
Lactiplantibacillus plantarum X7021 on physicochemical properties, purines,
isoflavones and volatile compounds of fermented soymilk, Process Biochem.
(2021), https://doi.org/10.1016/J.PROCBIO.2021.12.028.
[5] Z.H. Cao, J.M. Green-Johnson, N.D. Buckley, Q.Y. Lin, Bioactivity of soy-based
fermented foods: a review, Biotechnol. Adv. 37 (2019) 223–238, https://doi.org/
10.1016/j.biotechadv.2018.12.001.
[6] Q.X. Chen, S. Wang, J.Y. Guo, Q.G. Xie, E.E. Smith, Y. Song, B.L. Li, G.C. Huo, The
protective effects of Lactobacillus plantarum KLDS 1.0344 on LPS-induced
mammary gland inflammation in vitro and in vivo, Front. Immunol. 12 (2021),
770822, https://doi.org/10.3389/FIMMU.2021.770822.
[7] S. Ashman, H. Krishnamurthy, The gut microbiome, in: F. Yafi, N. Yafi (Eds.), Effect
of Lifestyle on Men’s Health, Academic Press, London, 2019, pp. 61–98, https://
doi.org/10.1016/B978-0-12-816665-9.00004-4.
[8] C. Xu, J. Ma, W. Wang, Z.J. Liu, L.Y. Gu, S.H. Qian, Z.M. Jiang, Preparation of
pectin-based nanofibers encapsulating Lactobacillus rhamnosus 1.0320 by
electrospinning, Food Hydrocolloid 124 (2022), 107216, https://doi.org/10.1016/
j.foodhyd.2021.107216.
[9] Y. Zhu, Y. Zhang, Y. Li, Understanding the industrial application potential of lactic
acid bacteria through genomics, Appl. Microbiol. Biotechnol. 83 (2009) 597–610,
https://doi.org/10.1007/s00253-009-2034-4.
[10] S. Hasani, I. Khodadadi, A. Heshmati, Viability of Lactobacillus acidophilus in rice
bran-enriched stirred yoghurt and the physicochemical and sensory characteristics
X. Peng et al.
of product during refrigerated storage, Int. J. Food Sci. Technol. 51 (2016)
2485–2492, https://doi.org/10.1111/ijfs.13230.
[11] M. Mousavi, A. Heshmati, A.D. Garmakhany, A. Vahidinia, M. Taheri, Optimization
of the viability of Lactobacillus acidophilus and physico-chemical, textural and
sensorial characteristics of flaxseed-enriched stirred probiotic yogurt by using
response surface methodology, LWT 102 (2019) 80–88, https://doi.org/10.1016/j.
lwt.2018.12.023.
[12] M. Coton, A. Pawtowski, B. Taminiau, G. Burgaud, F. Deniel, L. Coulloumme-
Labarthe, A. Fall, G. Daube, E. Coton, Unraveling microbial ecology of industrial-
scale Kombucha fermentations by metabarcoding and culture-based methods,
FEMS Microbiol. Ecol. 93 (2017), fix048, https://doi.org/10.1093/femsec/fix048.
[13] A. Lamia, B.A. Salwa, H. Moktar, Development of a beverage from red grape juice
fermented with the Kombucha consortium, Ann. Microbiol. 67 (1) (2017), https://
doi.org/10.1007/s13213-016-1242-2.
[14] M.L. Leal, L.V. Suarez, R. Jayabalan, J.H. Oros, A. Escalante-Aburto, A review on
health benefits of kombucha nutritional compounds and metabolites, CyTA-J. Food
16 (1) (2018) 390–399, https://doi.org/10.1080/19476337.2017.1410499.
[15] S. Jurate, K. Aiste, S. Dalia, Application of fermented soya as a bacterial starter for
production of fermented milk, Czech. J. Food Sci. 37 (6) (2019) 403–408, https://
doi.org/10.17221/194/2018-CJFS.
[16] K.V. Neera, H.V. Ramana, Batra, Occurrence of cellulose-producing
Gluconacetobacter spp. in fruit samples and Kombucha tea, and production of the
biopolymer, Appl. Biochem. Biotechnol. 176 (4) (2015) 1162–1173, https://doi.
org/10.1007/s12010-015-1637-8.
[17] N.K. Nguyen, P.B. Nguyen, H.T. Nguyen, P.H. Le, Screening the optimal ratio of
symbiosis between isolated yeast and acetic acid bacteria strain from traditional
kombucha for high-level production of glucuronic acid, LWT 64 (2) (2015)
1149–1155, https://doi.org/10.1016/j.lwt.2015.07.018.
[18] X. Xia, Y. Wang, X. Liu, Y. Li, J. Zhou, Soymilk residue (okara) as a natural
immobilization carrier for Lactobacillus plantarum cells enhances soymilk
fermentation, glucosidic isoflavone bioconversion, and cell survival under
simulated gastric and intestinal conditions, PeerJ 4 (2016), e2701, https://doi.org/
10.7717/peerj.2701.
[19] L. Li, B.Y. Liao, K. Thakur, J.G. Zhang, Z.J. Wei, The rheological behavior of
polysaccharides sequential extracted from Polygonatum cyrtonema Hua, Int. J.
Biol. Macromol. 109 (2017) 761–771, https://doi.org/10.1016/j.
ijbiomac.2017.11.063.
[20] E.P. Katherine, D. Ellen, H. Nasim, K. Ardjan, W.C. Philip, J.Z. Nicolaas, Confocal
imaging to reveal the microstructure of soybean processing materials, J. Food Eng.
147 (5) (2015) 8–13, https://doi.org/10.1016/j.jfoodeng.2014.09.022.
[21] Q. Bei, G. Chen, F. Lu, S. Wu, Z. Wu, Enzymatic action mechanism of phenolic
mobilization in oats (Avena sativa L) during solid-state fermentation with
Monascus anka, Food Chem. 245 (2018) 279–304, https://doi.org/10.1016/j.
foodchem.2017.10.086.
[22] Z.H. Mi, R. Hu, Y.F. Chen, The application of UPLC-MS on analysis of isoflavone in
fermented soybean milk, Food Sci. Technol. 43 (01) (2018) 302–305, https://doi.
org/10.13684/j.cnki.spkj.2018.01.055.
[23] P. Russo, V. Capozzi, M.P. Arena, G. Spadaccino, M.T. Dueñas, P. López, G. Spano,
Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-
enriched bread, Appl. Microbiol. Biot. 98 (8) (2014) 3691–3700, https://doi.org/
10.1007/s00253-013-5484-7.
[24] J.Y. Yin, S.P. Nie, C. Zhou, Y. Wan, M. Xie, Chemical characteristics and
antioxidant activities of polysaccharide purified from the seeds of Plantago asiatica
L, J. Sci. Food Agr. 90 (2) (2010) 210–217, https://doi.org/10.1002/jsfa.3793.
[25] Z.M. Jiang, S. Mu, C.L. Ma, Y. Liu, Y. Ma, M.H. Zhang, H.Y. Li, X.Q. Liu, J.C. Hou,
B. Tian, Consequencesof ball milling combined with high-pressure homogenization
on structure, physicochemical and rheological properties of citrus fiber, Food
Hydrocoll. 127 (2022), 107515, https://doi.org/10.1016/J.
FOODHYD.2022.107515.
[26] X. Shi, J. Li, S. Wang, L. Zhang, L. Qiu, T. Han, Q. Wang, C. Chang, S. Guo, Flavor
characteristic analysis of soymilk prepared by different soybean cultivars and
establishment of evaluation method of soybean cultivars suitable for soymilk
processing, Food Chem. 185 (2015) 422–429, https://doi.org/10.1016/j.
foodchem.2015.04.011.
[27] L. Bartle, K. Sumby, J. Sundstrom, The microbial challenge of winemaking: yeast-
bacteria compatibility, FEMS Yeast Res. 19 (4) (2019) foz040, https://doi.org/
10.1093/femsyr/foz040.
[28] B. Carbonetto, T. Nidelet, S. Guezenec, Interactions between Kazachstania humilis
yeast species and lactic acid bacteria in Sourdough, Microorganisms 8 (2) (2022)
240, https://doi.org/10.3390/microorganisms8020240.
[29] S.F. Meg, S.L. Fernando, R. Daniele, H. Cintia, G. Marcela, G. Sandra, I.I. Elza,
Evaluation of the isoflavone and total phenolic contents of kefir-fermented soymilk
storage and after the in vitro digestive system simulation, Food Chem. 229 (2017)
373–380, https://doi.org/10.1016/j.foodchem.2017.02.095.
[30] D. Xu, J. Behr, A.J. Geibler, J. Bechtnera, C. Ludwigb, R.F. Vogel, Label-free
quantitative proteomic analysis reveals the lifestyle of Lactobacillus hordei in the
presence of Sacchromyces cerevisiae, Int. J. Food Microbiol. 2 (294) (2019) 18–26,
https://doi.org/10.1016/j.ijfoodmicro.2019.01.010.
[31] J. Stadie, A. Gulitz, M.A. Ehrmann, R.F. Vogel, Metabolic activity and symbiotic
interactions of lactic acid bacteria and yeasts isolated from water kefir, Food
Microbiol. 35 (2) (2013) 92–98, https://doi.org/10.1016/j.fm.2013.03.009.
[32] A. Agarbati, M. Ciani, L. Canonico, E. Galli, F. Comitini, Exploitation of yeasts with
probiotic traits for kefir production: effectiveness of the microbial consortium,
Fermentation 8 (1) (2021) 9, https://doi.org/10.3390/FERMENTATION8010009.
[33] E. Kristo, C.G. Biliaderis, N. Tzanetakis, Modelling of rheological, microbiological
and acidification properties of a fermented milk product containing a probiotic
296
Process Biochemistry 121 (2022) 286–297
strain of Lactobacillus paracasei, Int. Dairy J. 13 (7) (2013) 517–528, https://doi.
org/10.1016/S0958-6946(03)00074-8.
[34] S. Jayarathna, H. Priyashantha, M. Johansson, J.K. Vidanarachchi, B.
C. Jayawardana, R. Liyanage, Probiotic enriched fermented soy-gel as a vegan
substitute for dairy yoghurt, J. Food Process Pres. 45 (1) (2020), https://doi.org/
10.1111/jfpp.15092.
[35] N. Yamada, Y. Kokean, K. Tsumura, Novel insights into the formation of soymilk
gel induced by ginger rhizome juice, Food Sci. Technol. Res. 25 (5) (2019)
751–754, https://doi.org/10.3136/fstr.25.751.
[36] X. Li, Y. Liu, C. Yi, Microstructure and rheological properties of mixtures of acid-
deami dated rice protein and dextran, J. Cereal Sci. 51 (1) (2017) 7–12, https://
doi.org/10.1016/j.jcs.2009.08.006.
[37] S.S. Singh, V.K. Aswal, H.B. Bohidar, Structural studies of agar-gelatin complex
coacervates by small angle neutron scattering, rheology and differential scanning
calorimetry, Int. J. Biol. Macromol. 41 (3) (2007) 301–307, https://doi.org/
10.1016/j.ijbiomac.2007.03.009.
[38] E. María, M.M. José, R. Blanca, M. Rosario, Characterization of a halotolerant
lipase from the lactic acid bacteria Lactobacillus plantarum useful in food
fermentations, LWT 60 (1) (2015) 246–252, https://doi.org/10.1016/j.
lwt.2014.05.063.
[39] C. Lee, J. Kim, S. Wang, S. Sung, N. Kim, H.H. Lee, Y.S. Seo, Y. Jung,
Hepatoprotective effect of Kombucha tea in rodent model of nonalcoholic fatty
liver disease/nonalcoholic steatohepatitis, Int. J. Mol. Sci. 20 (9) (2019) 2369,
https://doi.org/10.3390/ijms20092369.
[40] S.A. Villarreal-Soto, J. Bouajila, M. Pace, J. Leech, P.D. Cotter, J.P. Souchard,
S. Beaufort, Metabolome-microbiome signatures in the fermented beverage,
Kombucha, Int. J. Food Micro 333 (2020), 108778, https://doi.org/10.1016/j.
ijfoodmicro.2020.108778.
[41] D.W. Ningtyas, S. Hati, S. Prakash, Bioconversion and bioaccessibility of
isoflavones from sogurt during in vitro digestion, Food Chem. 343 (5) (2021),
128553, https://doi.org/10.1016/j.foodchem.2020.128553.
[42] O.N. Donkor, N.P. Shah, Production of β-glucosidase and hydrolysis of isoflavone
phytoestrogens by Lactobacillus acidophilus, Bifidobacterium lactis, and
Lactobacillus casei in soymilk, J. Food Sci. 73 (1) (2008) M15–M20, https://doi.
org/10.1111/j.1750-3841.2007.00547.x.
[43] G. Spano, A. Rinaldi, M. Ugliano, L. Moio, L. Beneduce, S. Massa, A β-glucosidase
gene isolated from wine Lactobacillus plantarum is regulated by abiotic stresses,
J. Appl. Microbiol 98 (4) (2005) 855–861, https://doi.org/10.1111/j.1365-
2672.2004.02521.x.
[44] M.S. Garro, L. Aguirre, G.S.D. Giori, Biological activity of Bifidobacterium longum
in response to environmental pH, Appl. Microbiol. Biot. 70 (5) (2006) 612–617,
https://doi.org/10.1007/s00253-005-0102-y.
[45] S.T.R. Baú, E.I. Garcia, Changes in soymilk during fermentation with kefir culture:
oligosaccharides hydrolysis and isoflavone aglycone production, Int. J. Food Sci.
Nutr. 66 (8) (2015) 845–850, https://doi.org/10.3109/09637486.2015.1095861.
[46] X.Y. Liu, M. Qian, Y.X. Shen, X. Qin, H. Huang, H. Yang, Y.L. He, W.D. Bai, An
high-throughput sequencing approach to the preliminary analysis of bacterial
communities associated with changes in amino acid nitrogen, organic acid and
reducing sugar contents during soy sauce fermentation, Food Chem. 349 (2021),
129131, https://doi.org/10.1016/J.FOODCHEM.2021.129131.
[47] L. Daenen, D. Saison, F. Sterckx, F.R. Delvaux, H. Verachtert, G. Derdelinckx,
Screening and evaluation of the glucoside hydrolase activity in Saccharomyces and
Brettanomyces brewing yeasts, J. Appl. Microbiol. 104 (2) (2008) 478–488,
https://doi.org/10.1111/j.1365-2672.2007.03566.x.
[48] Z. Yuksekdag, B. CinarAcar, B. Aslim, U. Tukenmez, β-Glucosidase activity and
bioconversion of isoflavone glycosides to aglycones by potential probiotic bacteria,
Int. J. Food Prop. 20 (sup3) (2018) S2878–S2886, https://doi.org/10.1080/
10942912.2017.1382506.
[49] D. Tsangalis, J. Ashton, A. Mcgill, N. Shah, Biotransformation of isoflavones by
bifidobacteria in fermented soymilk supplemented with D-glucose and L-cysteine,
J. Food Sci. 68 (2) (2003) 623–631, https://doi.org/10.1111/j.1365-2621.2003.
tb05721.x.
[50] B.H. Liu, X. Xiao, X.R. Zhou, X. Zhao, Effects of Lactobacillus plantarum CQPC01-
fermented soybean milk on activated carbon-induced constipation through its
antioxidant activity in mice, Food Sci. Nutr. 7 (6) (2019) 2068–2082, https://doi.
org/10.1002/fsn3.1048.
[51] J.A. Marazza, M.A. Nazareno, G.S. Giori, M.S. Garro, Enhancement of the
antioxidant capacity of soymilk by fermentation with Lactobacillus rhamnosus,
J. Funct. Foods 4 (3) (2012) 594–601, https://doi.org/10.1016/j.jff.2012.03.005.
[52] R. Jayabalan, R.V. Malbaša, E.S. Lončar, J.S. Vitas, M. Sathishkumar, A review on
kombucha tea-microbiology, composition, fermentation, beneficial effects,
toxicity, and tea fungus, Compr. Rev. Food Sci. Food Saf. 13 (4) (2020) 538–550,
https://doi.org/10.1111/1541-4337.12073.
[53] Kiran Thakur, Screening of riboflavin-producing lactobacilli by a polymerase chain
reaction-based approach and microbiological assay, J. Agr. Food Chem. 64 (9)
(2016) 1950–1956, https://doi.org/10.1021/acs.jafc.5b06165.
[54] Y. Li, M.D. Li, Z.J. Wang, L. Zhen, F. Teng, Spectroscopy analysis of interaction
between vitamin B12 and soybean protein isolate, Tra Chi Soc. Agr. Mac. 51 (1)
(2020) 341–348, https://doi.org/10.6041/j.issn.1000-1298.2020.01.037.
[55] Y.Y. Zhu, K. Thakur, J.Y. Feng, J.S. Cai, J.G. Zhang, F. Hu, P. Russo, G. Spano, Z.
J. Wei, Riboflavin-overproducing lactobacilli for the enrichment of fermented
soymilk: insights into improved nutritional and functional attributes, Appl.
Microbiol. Biot. 104 (13) (2020) 5759–5772, https://doi.org/10.1007/s00253-
020-10649-1.
[56] Q. Zhang, X. Tong, X. Sui, Z. Wang, B. Qi, Y. Li, L. Jiang, Antioxidant activity and
protective effects of Alcalase-hydrolyzed soybean hydrolysate in human intestinal
X. Peng et al.
epithelial Caco-2 cells, Food Res. Int. 2018 (6) (2018) 256–264, https://doi.org/
10.1016/j.foodres.2018.05.046.
[57] R.C. Cai, L. Li, M. Yang, Changes in bioactive compounds and their relationship to
antioxidant activity in white sufu during manufacturing, Int. J. Food Sci. Technol.
51 (7) (2016) 1721–1730, https://doi.org/10.1111/ijfs.13149.
[58] S. Kaneko, K. Kumazawa, O. Nishimura, Studies on the key aroma compounds in
soy milk made from three different soybean cultivars, J. Agr. Food Chem. 59 (22)
(2011), https://doi.org/10.1021/jf202942h (12204–11209).
[59] Kun Wang, Susan D. Arntfield, Binding of carbonyl flavours to canola, pea and
wheat proteins using GC/MS approach, Food Chem. 157 (2014) 364–372, https://
doi.org/10.1016/j.foodchem.2014.02.042.
[60] Z. Huang, L. Huang, G. Xing, X. Xu, C. Tu, M. Dong, Effect of co-fermentation with
lactic acid bacteria and K. marxianus on physicochemical and sensory properties of
goat milk, Foods 9 (3) (2020) 299, https://doi.org/10.3390/foods9030299.
297
Process Biochemistry 121 (2022) 286–297
[61] C. Gerardi, M. Tristezza, L. Giordano, Exploitation of Prunus mahaleb fruit by
fermentation with selected strains of Lactobacillus plantarum and Saccharomyces
cerevisiae, Food Microbiol. 84 (2019), 103262, https://doi.org/10.1016/j.
fm.2019.103262.
[62] Á. Gonzalez, N. Hierro, M. Poblet, N. Rozès, A. Mas, J.M. Guillamón, Application of
molecular methods for the differentiation of acetic acid bacteria in a red wine
fermentation, J. Appl. Microbiol. 96 (4) (2004) 853–860, https://doi.org/10.1111/
j.1365-2672.2004.02220.x.
[63] Y. Yamad, P. Yukphan, H.T.L. Vu, Y. Muramatsu, D. Ochaikul, Y. Nakagawa,
Subdivision of the genus Gluconacetobacter Yamada, Hoshino and Ishikawa 1998:
the proposal of Komagatabacter gen. nov., for strains accommodated to the
Gluconacetobacter xylinus group in the α-Proteobacteria, Ann. Microbiol. 62 (2)
(2012) 849–859, https://doi.org/10.1007/s13213-011-0288-4.