rui xin (Orcid ID: 0000-0002-7242-3235)

Does lactic fermentation influence soy yogurt protein digestibility– A

comparative study between soymilk and soy yogurt at different pH

Xin Rui, Qiuqin Zhang, Jin Huang, Wei Li, Xiaohong Chen, Mei Jiang, Mingsheng

Dong*

College of Food Science and Technology, Nanjing Agricultural University, Jiangsu

Province, P R China

*Corresponding author contact information:

Mingsheng Dong

College of Food Science and Technology

Nanjing Agricultural University

1 Weigang Road, Nanjing, Jiangsu, P.R. China

Tel: +86 25 84396989

Fax: +86 25 84399090

E-mail address: dongms@njau.edu.cn

This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jsfa.9256

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Abstract:

BACKGROUND: Lactic acid bacteria fermentation allows soymilk to form

yogurt-like product and accompanied with protein acidic coagulation. There is a

question if coagulation of soy protein during fermentation influences protein

digestibility when ingested. In the current study, soymilk (pH 6.3) and soy yogurt (SY)

at four varied pH (6.0, 5.7, 5.4, 5.1) were subjected to in vitro gastrointestinal

digestion (GIS) and comparison study was conducted.

RESULTS: Lactic fermentation allowed pH of soymilk gradually reduced to 5.1

in 330.0 min. Decline of pH made D[4,3] and D[v,90] increased from respectively

0.81 to 97 μm and 1.82 to 273 μm. Predominant proteins lost their solubility between

pH 6.0-5.7. Applying of GIS allowed SY samples especially SY-5.7, SY-5.4 and

SY-5.1 to show particles with predominant peak at around 10 μm and lower soluble

proteins compared to soymilk with reduction percentages of 18%, 28%, 43%.

Cleavage pattern of soy protein during GIS was barely affected by sample pH. But

less quantity of band at 33.9 kDa was found in SY-5.7, SY-5.4 and SY-5.1.

CONCLUSION: The results demonstrated lactic fermentation altered soy protein

digestibility. With the process of protein coagulation, SY-5.7, 5.4, and 5.1 had lower

bioaccessible protein contents compared to that of soymilk.

Key words: Soymilk; Lactic acid bacteria; pH; Protein digestibility

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1. Introduction

Proteins from plant sources are received increasing notice due to many

advantages and are considered environmental friendly and could be a complementary

to animal proteins. Soymilk is a traditional protein beverage made from crushed

soybeans after removal of insoluble residuals (okara).1 Aside from its benefits on

providing appreciable amounts of proteins, soymilk is free of cholesterol and lactose,

contains bioactive compounds such as isoflavone and saponin delivering potential

effects of attenuate risk of cardiovascular disease.2,3

Incorporation of lactic acid bacteria (LAB) into soymilk allows coagulation of

soy protein and made it a set-type yogurt like product.4,5 LAB during their growth

slowly release protons and allowed decline of pH which made soymilk proteins

gradually coagulated to form aggregates.6 Unlike casein gels, aggregates of soy

proteins are behaved particulate in nature other than network gels.7 This has made soy

protein gels varied texture and microstructure compared to that of casein gels.8 As

many studies have focused on LAB induced gelation process,6,9 fewer studies have

investigated if this particular structure influences digestibility of soy proteins.

Turgeon and his group in 2011 pointed out macronutrient like proteins needs to

be released from the food matrix to obtain real benefit of this compound.10 The

procedure of releasing nutrients from a complex food system to the intestinal lumen

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during digestion is defined as “bioaccessibility”11 and can be evaluated by in vitro

gastrointestinal digestion simulation (GIS).12 Many studies have suggested protein

bioaccessibility can be interfered by food matrix modification via different treatments

such as heating,13 fermentation,14,15 or addition of different additives.11 Therefore,

objective of this study is to find data that indicates that coagulation of soy protein

during fermentation influences protein digestibility when ingested. pH is an important

indicator during growth of LAB and also determines process of protein coagulation.

In the current study, soy yogurt was prepared at different pH levels and was

subsequently subjected to in vitro gastrointestinal digestion (GIS) and comparison

study was conducted in terms of particle size distribution, soluble protein content, and

electrophoretic profiles.

2. Materials and Methods

2.1. Materials

Whole soybean seeds were purchased from a local supermarket at Nanjing, P. R.

China and were stored at 4 °C until use. Molecular mass standard (20-150 kDa) was

purchased from Sangon Biotech (Shanghai, P. R. China). ±-amylase(from human

saliva, A1031), pepsin (from porcine gastric mucosa, P7125), pancreatin (from

porcine pancreas, P7545), and bile salts (from porcine, B8631) were obtained from

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Sigma-Aldrich Co. (St Louis, MO, USA). All other regents used were of analytical

grade.

Lactobacillus plantarum B1-6 was isolated in our lab from Krigiz boza, which is

a traditional type of fermented cereal based drink from Xinjiang province of China.

The strain has a gene accession number KM200717.

2.2. Inoculum preparation

The LAB culture were propagated twice in de Man-Rogosa and Sharp broth

(MRS, pH 6.2) at 37 °C for 24 h and they were subsequently inoculated into MRS

broth and cultured for another 16 h. The cells were and washed twice with sterile

physiological saline and harvested by centrifugation at 7500×g, 4 °C for 10 min

preparing for inoculation.

2.3. Preparation of soymilk and soy yogurt

Soybeans were selected, rinsed and soaked in distilled water for approximately 12

h and then drained and homogenized in 9-fold of distilled water to make slurry.

Filtration was then carried out using a 200-mesh screen cloth to remove the insoluble

soybean residue (okara). The filtrate, soymilk, was then heated at 108 oC for 15 min to

reduce the endogenous microbiota in the raw material. Part of the prepared soymilk

was left to cool down and inoculated with L. plantarum B1-6 at inoculation ratio of

3% (v/v) to make soy yogurt (SY). Fermentation temperature was set at 37 oC. An

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online pH meter (Heto PowerDry LL3000, Hamilton Bonaduz Co., Switzerland) was

used to monitor pH of the culture successively. Samples were transferred to

refrigerator (4 oC) immediately and as soon as possible subjected to the following

analysis.

2.4. Gelation experiments

A Physica MCR301 rheometer (Anton Paar, Austria) was used for the rheological

measurements. Soymilk samples, immediately after inoculation of L. plantarum B1-6,

was loaded between parallel plates (50 mm of diameter) with gap set at 1.0 mm. Edge

of samples was covered with silicon oil to prevent evaporation. Gelation process was

monitored at a strain of 1%, a frequency of 0.1 Hz and a constant temperature at

37 °C. Storage modulus (G2) and loss modulus (G22) were recorded every 2 min for 6 h.

Gelation time was defined at the point when G2e 1 Pa (log G2= 0).16 The corresponded

pH value was defined as gelation pH. According to the gelation pH, fermentation was

terminated at different times when pH of the culture dropped to 6.0, 5.7, 5.4 and 5.1

and samples collected were named respectively SY-6.0, SY-5.7, SY-5.4, and SY-5.1.

These samples, together with sample of soymilk were subjected to the following

study.

2.5. Viable cell counts

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 Viable lactic acid bacteria cell count was performed by enumeration on MRS

agar (pH 6.2±0.2, Oxoid-CM0361, Unipath, Basingstoke, UK) at 37 °C for 48 h. Cell

counts were expressed as log cfu ml-1. Three replicates were conducted.

2.6. Texture analysis

Texture analysis was performed according to a previous study with slight

modifications.17,18 Soy yogurts were evaluated by a TA.X2i texture analyzer

(Godalming, Surrey, UK). Samples were taken in cylindrical shape (1.5 cm

diameter×2.0 cm height) from the central part with a stainless steel cutter. A 5 kg load

cell was used and the test speed of 5.0 mm/s. The sample was compressed by a 5 cm

diameter cylinder probe to 35% deformation. The pre-test compression speed of the

probe was set as 1.0 mm/s. Hardness, springiness, cohesiveness, and gumminess were

calculated. Four replicates were conducted for each sample.

2.7. In vitro gastrointestinal digestion simulation (GIS)

Artificial saliva, gastric and duodenal juices were prepared to simulate human

buccal, gastric and duodenal digestion according to a previous published protocol.19

The ratio of food to digestive juices was set to be 2.5 (food) to 1.0 (saliva) to 1.5

(gastric juice) to 1.0 (bile) to 1.0 (pancreatic juice) based on volume units to mimic

human physiology conditions.19,20 Artificial saliva was prepared by dissolving

±-amylase (0.2 mg/mL, w/v) in phosphate buffer (20 mM, pH 7.0). Artificial gastric

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juice was prepared by dissolving pepsin (3.2 mg/mL, w/v) in 0.1 M HCl. Artificial

pancreatic juice and bile acids were prepared by dissolving pancreatin (0.4 mg/mL,

w/v) and bile salts (0.4 mg/mL, w/v) respectively in phosphate buffer (10 mM, pH

7.0). HCl or NaOH (4M) was used to adjust pH during digestion.

Buccal digestion simulation was initiated by firstly grinding the 25 mL of

soymilk (or SY samples) using a glass homogenizer which mimicked chewing

process and then 10 mL artificial saliva was added. The mixture was placed in a

shaking water bath (SWB series, Biobase, Shandong, China) at 55 rpm, 37 oC for 3

min for simulating of buccal digestion. Subsequently, the slurry was adjusted to pH

2.0 and artificial gastric juice (15 mL) was added. The mimic gastric digestion was

conducted at 55 rpm, 37 oC for 1 h. pH was then adjusted to 7.0 and proportional

artificial bile acids (10 mL) and pancreatic juice (10 mL) were added to simulate

duodenal digestion. The digestion was performed at 37 oC for 2 h at 150 rpm.

Aliquots were taken before digestion (P0), at the end of buccal digestion (P1), gastric

digestion (P2), and duodenal digestion (P3), respectively. All collected samples were

boiled for 5 min to terminate the enzymatic hydrolysis. Samples for particle size

distribution determination were taken immediately for the analyses, whereas samples

for evaluations of soluble protein content and electrophoresis were centrifuged at

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9800×g at 4 oC for 20 min to collect the supernatants and then frozen at -20 oC. GIS

was performed in triplicates for each sample.

2.8. Particle size distribution

Particle size distribution of soymilk and soy yogurt was measured by an

integrated laser light scattering instrument (Mastersizer 3000, Malvern Southborough,

MA, USA). The refractive index of the scatterers of the sample solution was set as

1.46 and a refractive index of the water dispersant was set as 1.333. Samples were

dropped into the chamber filled with water until an optimum dilution was reached.

The volume-weighted mean diameter D[4,3] was calculated to represent the size of

gel particles, whereas D[v,0.90] was also given to describe the diameter below which

90% of the volume of particles are found. The analysis was conducted in triplicates.

2.9. Soluble protein content

Total soluble protein content was determined according to the Bradford assay

using a previous protocol with bovine serum albumin as a standard.21 Triplicates were

carried out for each sample.

2.10. Electrophoresis

Aliquots taken at different digestion time points were subjected to sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which was carried

out under reducing conditions (presence of 5% ² -mercaptoethanol [² -ME]) using a

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12% polyacrylamide gel. SDS-PAGE was conducted on a Bio-Rad Miniprotein 3 unit

(Bio-Rad Laboratories, Inc., Hercules, CA, USA) with voltage 60 V for stacking gel,

and followed by 120 V for separating gel. A pre-stained molecular mass standard

(20-150 kDa, Sangon Biotech Co., Shanghai, China) was used. Gels were scanned

with Image Scanner III (GE Healthcare Biosciences, Uppsala, Sweden) and then

analyzed by using Quantity One software, version 4.6.2 (Bio-Rad Laboratories, Inc.,

Hercules, CA, USA).

2.11. Statistical analysis

Analysis of variance (ANOVA) and Duncan’s multiple comparison tests were

used to determine the significant differences between means (P<0.05) using IBM

SPSS Statistics (version 19, IBM Company, Chicago, IL, USA).

3. Results and discussion

3.1. Acidification induced gelation and growth of Lactobacillus plantarum B1-6

Soymilk has a starting pH of 6.30. Growing of Lactobacillus plantarum B1-6

allowed gradually decline of pH and increments in the storage modulus (G2) and loss

modulus (G22) during 6 h of fermentation (Fig 1A). G2 and G22 remained unchanged

during 0-140 min of fermentation and then increased rapidly from 140 min to 200 min.

Gelation time, which was assigned when the value of G2 was higher than 1 Pa (log

G2=0) based on the previous literature,16 was 163±6 min. The corresponded pH was

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5.86±0.02. Therefore, four pH levels were selected for in preparation of soy yogurts,

which were 6.0, 5.7, 5.4, and 5.1, to evaluate coagulation of soy protein during

fermentation influences protein digestibility when ingested. They were abbreviated as

SY-6.0, SY-5.7, SY-5.4, and SY-5.1 hereafter. It took 141.0±3.2 min, 204.0±1.2,

257.0±2.5 min and 330.0±4.1 min to form samples SY-6.0, SY-5.7, SY-5.4, and

SY-5.1, respectively. The growth of L. plantarum B1-6 as indicated by the viable

lactic bacteria cell counts is shown in Fig. 1B. The viable bacteria count of L.

plantarum B1-6 was observed 7.16±0.05, 7.52±0.06, 7.81±0.06, and 8.04±0.02

log cfu/ml in SY-6.0, SY-5.7, SY-5.4, and SY-5.1, respectively, indicating a steady

increment of L. plantarum B1-6 during fermentation.

3.2. Texture analysis

Texture properties including hardness, springiness, cohesiveness, and

gumminess were evaluated on soy yogurts collected at different terminal pH (Table 1).

SY-6.0 and SY-5.7 were either remained at emulsion state or had very weak texture

which were failed in deformation process, thus the results were not shown in the table.

SY-5.1 had higher values in hardness (90.50±3.81 g) and gumminess (54.06±2.71 g)

compared to that of SY-5.4. The latter represented 75.00±6.87 g and 47.41±4.34 g,

respectively. Springiness and cohesiveness values between the two gels appeared to

be similar. Increased hardness obtained from soy yogurt at lower pH indicated

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formation of stronger gel. SY-5.1 also showed greater gumminess value might be

related to a more compact structure. This was in agreement with previous results.22

3.3. Particle size distribution

Particle size distribution was followed for soymilk and SY samples during in

vitro gastrointestinal digestion simulation (GIS) (Fig. 2A-C). Soymilk, after buccal

digestion, showed a bimodel profile with size of particles peaked at approximately 0.5

μm and 2 μm (Fig. 2A). This was in agreement with results observed in a previous

study.6 The authors suggested the smaller particle population represented soymilk

protein particles whereas the larger ones were mostly related to fat globules.

Aggregation of those particles were observed along with process of lactic

fermentation and resulted in gradually increments in D[4,3] and D[v,90] from

respectively 0.81 to 97 μm and 1.82 to 273 μm (Fig. 2A). In sample SY-6.0, 0.1-1 μm

particle population was reduced and a new peak at approximately 6 μm has emerged,

indicating aggregation of protein particles. Particle distribution profile was greatly

modified in SY samples obtained at pH 5.7 or lower. SY-5.7, 5.4 and 5.1 gave

monodisperse distribution with particles peaked at around 12 μm, 20 μm, and 23 μm.

This has suggested a uniform structure has formed after mimic mastication for those

samples (Fig. 2A). Evolvement of larger size particles upon mastication in samples

with lower pH probably related to higher gel stiffness and more compact structure

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formed in those related samples. SY-5.1, in particular, afforded a population of

greater particle size at around 290 μm.

Gastric digestion has an significant impact on particle size distribution for all

investigated samples (Fig. 2B). D[4,3] and D[v,90] of SY-5.4 and SY-5.1 were

significantly reduced to 21-44% (D[4,3]) and 11-19% (D[v,90]) compared to values

obtained at previous stage, indicating dissociation of particles, whereas greater D[4,3]

and D[v,90] were obtained for soymilk and SY-6.0 digesta which suggestive of

particle aggregation. However, regardless of sample pH, soymilk and SY digesta

showed D[4,3] ranged from 21 to 27 μm and had very similar bimodal distribution

with a predominant population at around 10 μm and a minor fraction peaked at

approximately 100 μm. These changes were probably corresponded to conditions

applied in gastric digestion: including acidic pH (2.0), proteolytic hydrolysis, and

persistent mechanical stirring, which appeared to destabilize the matrix structure. It

seems in gastric environment particle size distribution was barely affected by

strengths of the gel. Similar profiles, namely, particles peaked at approximately 10

μm was observed for pasteurized human milk after gastric digestion.23

The subsequent duodenal digestion allowed soymilk digesta to evolve in both

smaller (0.01-1 μm) and larger (100-500 μm) particles, implied an extensive

destabilization of the emulsion. In this stage, soymilk digesta possessed the largest

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D[4,3] and D[v,90] values than that of all assayed SY samples, probably due to the

higher quantity of larger sizes particles (>100 μm) observed in soymilk digesta.

Larger particles (100-500 μm) might be associated with formation of aggregates. It is

interesting soymilk digesta formed higher quantity of large aggregates than all SY

samples, which might be due to changes of molecular interactions under duodenal

digestion condition. Previous studies demonstrated acid-induced soymilk gels were

mainly stabilized by van der Waals attraction, hydrogen bonding and hydrophobic

interations.9,24 Presence of these interactions between protein molecules probably

hindered extensive destabilization of the emulsion in this stage. Similar observation

was found at lower particle range (0.01-1 μm). SY-6.0 and SY-5.7 showed a broad

peak at lower particle sizes (0.01-1 μm) which was similar with soymilk digesta.

However, this peak was absent in sample SY-5.4 and SY-5.1. In addition, sample pH

also decided height of the peak at around 10 μm. The peak height, corresponding to

percentage(%) of particles increased from 5.2% to 10.0% along with decrease of

sample pH from 6.3 to 5.1. The results indicated dissociate/aggregation behaviors of

the particles were varied upon different sample pH, although all samples represented

similar profiles in previous gastric stage. It appeared matrix formed at gastric stage

was maintained higher integrity in this stage for SY samples especially for those with

lower pH (SY-5.4 and 5.1). Size of the particle may further influence susceptibility of

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soy proteins to enzymatic hydrolysis, then affect protein digestibility. One of our

earlier research supported this view.25

3.4 Soluble protein content

Soluble protein content in soymilk and SY samples at different gastrointestinal

phases are shown in Fig. 3. At P0 stage, soymilk presented the highest soluble protein

content (5.2 mg/mL) followed by SY-6.0 (3.9 mg/mL), whereas drastically lower

values (0.3-0.4 mg/mL) were observed in SY-5.7, 5.4, and 5.1 with no significant

differences between them (P>0.05). It seems most soymilk proteins loss their

solubility at pH between 5.7-6.0. Reduction of soluble protein during lactic

fermentation was observed in our previous study14 attributed to occurrence of acid

gelation.24,26 Protons released by hydrolytic action of lactic acid bacteria allowed

neutralization of the negative protein complexes formed from heated soymilk. A

previous study demonstrated acid gelation induced by fermentation of lactic acid

bacteria has a similar gelation mode to that of glucono-´ -lactone but showed a higher

gelation pH at 6.29.6 During the first phase of GIS digestion, mimic mastication (P1)

resulted in a similar result with that of P0 in all investigated samples expect SY-6.0,

which suggested mechanical chewing and incorporation of ±-amylase had little effect

on soluble protein content of SY. SY-6.0 gave a higher result at P1 stages and the

value was found to be no significant different compared to that of soymilk (P>0.05).

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In the following gastric digestion (P2), although soymilk and SY-6.0 digesta

maintained higher soluble protein contents than that of SY-5.7, 5.4 and 5.1,

differences between them were dramatically reduced and the latter three digesta

afforded climbing values. This was in accordance with the results found in particle

size distribution in which gastric phase allowed uniform distribution of particles in

different samples. In the subsequent duodenal (P3) stage, soymilk and SY-6.0 digesta

showed the highest soluble protein contents and lower soluble protein contents were

obtained in SY-5.7, 5.4, and 5.1 in descending order. SY-5.7, 5.4, and 5.1 showed

reductions of 18%, 28%, 43% respectively compared to the of soymilk (P<0.05).

SY-6.0 showed similar results (1% reduction) with soymilk digesta and no significant

difference (P>0.05) was observed between them.

Soluble protein content, expressed by measure of soluble proteins in the

supernatant of digestive mixture is an important index to evaluate bio-accessible

proteins.11,15 Proteins which were soluble at duodenal environment had higher

chances to be further absorbed whereas proteins retained in precipitate were probably

corresponding to greater protein aggregates which might be too large to cross gut

mucosa.27 Hence, soymilk, together with SY-6.0 seemed to elicit higher

bio-accessible proteins whereas lower bio-accessible proteins were found in SY-5.7,

5.4, and 5.1 in descending order. For SY samples, especially SY-5.7, 5.4, and 5.1,

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both gastric (P2) and duodenal (P3) stages have promoting effects in increasing of

soluble proteins. Protein originally stabilized by various forces at insoluble fraction

(P0) was seemed to be “released” to be soluble and potentially bio-accessible. Higher

the sample pH related to more a profound effect. With the decrement of sample pH,

less proteins were “released” during in vitro GIS to become soluble which might

correspond to higher integrity of the samples. It is interesting that lactic fermentation

of cow milk has a different manner when subject to gastrointestinal digestion. The

study showed yogurt possessed a higher soluble protein content than that of cow milk

after intestinal phase of GIS.15 This might be due to different gelation behavior of soy

and milk proteins.

3.5 Electrophoresis

Protein profile in soluble fraction of each sample was determined by sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reduced

condition (Fig. 4). Soymilk collected at P0 stage afforded several bands with MM

(molecular mass) ranged between 20-100 kDa. The predominant six bands, showed

with the number of 1-6, possessed molecular mass of 85.2 kDa, 76.6 kDa, 48.6 kDa,

40.0 kDa, 35.1 kDa and 20.1 kDa, respectively in an ascending order of the band

numbers. They were presumably corresponded to 7S globulin ±� subunit (band 1), 7S

globulin ± subunit (band 2), and 7S globulin ² subunit (band 3), 11S A3 subunit (band

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4), 11S acidic subunits (band 5), and 11S basic subunits (band 6) based on previous

studies.28,29 Profile was different in SY soluble fraction at P0 stage. Regardless of

terminal pH, all SY samples showed protein composition including five major bands

(7-11) with corresponding MM of 61.5 kDa, 55.4 kDa, 48.9 kDa, 40.0 kDa, and 25.7

kDa. It appeared 7S globulin ±� subunit, ± subunit and 11S acidic subunits were

absent in all investigated SY soluble fractions, whereas bands 9 and 10 might be

related to 7S globulin ² subunit and 11S A3 subunit based on similar MM with

respectively bands 3 and 4 but both were in presences of reduced contents. The absent

proteins might be distributed in the corresponding precipitates thus were not appeared

in the supernatant fraction. It can be presumed that lactic fermentation has triggered

different protein subunits into insoluble fraction at varied pH. A previous study

demonstrated addition of magnesium chloride (5 mM) coagulated most of soy

proteins including 7S subunits and 11S subunits and made them disappeared from the

supernatant fraction.27 The current findings indicated the coagulation behavior of

soymilk proteins during lactic fermentation might be different.

All SY samples except SY-6.0 showed similar electrophoretic profile before and

after buccal digestion (P1). Several additional proteins were seen on lane P1 (SY-6.0)

possessed identical MMs with band 1, 2, and 5. This has indicated 7S globulin ±�

subunit, 7S globulin ± subunit, and 11S acidic subunits were probably migrated to

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supernatant fraction of SY-6.0 after mechanical chewing and ±-amylase digestion.

This phenomenon was not seen in the SY-5.7, 5.4 and 5.1 (Fig. 4). No additional

bands were observed in the latter three samples but bands 7-11 were shown with

higher intensity. The results indicated SY samples starting from SY-5.7 presented

higher interacting forces between soymilk proteins thus hindered releasing of those

protein into soluble fraction. These results were in accordance with previous findings

in soluble protein content. Gastric digestion allowed disappearance of most major

fractions. Bands 1-6 were disappeared and seemed to be degraded to result in a few

degradation products, namely, bands 12-14 with identified MM of 33.9 kDa, 26.4

kDa, and 22.7 kDa. SY samples at P2 stage showed similar electrophoretic profile

with soymilk in terms of MMs of degradation products (as pointed by arrows). In the

subsequent duodenal digestion (P3) soymilk and SY-6.0 digesta showed similar

profile but with higher intensity in bands 12-14, indicating accumulation of these

products. For sample SY-5.7, 5.4 and 5.1, higher intensity was found in bands 13 and

14, whereas band 12 was observed with reduced intensity. The results indicated

sample pH has little effect on the MMs of cleavage products released during

enzymatic hydrolysis in gastric/duodenal phases. This is to say that LAB induced

protein coagulation process may not affect the cleavage pattern of soy protein during

in vitro GIS. But quantity of the products (bands 12-14) might be affected.

4. Conclusion

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 In the current study, soy yogurt prepared by L. plantarum B1-6 has a gelation

pH of 5.86±0.02. Soy yogurt (SY) was collected at four varied pH, namely, 6.0, 5.7,

5.4 and 5.1, were subjected to in vitro gastrointestinal digestion and their protein

digestibility was compared. The results suggested matrix of soy yogurts was greatly

interrupted by gastric (P2) and duodenal digestion (P3) and modifications in particle

size distribution, soluble protein content and electrophoretic patterns were observed.

At the end of duodenal digestion, SY samples were shown lower quantity in 100-500

μm particles (All SY samples), in absent of 0.01-1 μm particles (SY-5.4 and SY-5.1),

lower soluble protein content (SY-5.7, 5.4, 5.1), and fainter bands at 33.9 kDa which

probable a digestive product (SY-5.7, 5.4, 5.1) compared to that of soymilk digesta.

SY-6.0, after buccal digestion (P1), showed similar soluble protein content and

electrophoretic profile with soymilk in the corresponding digesta. Lower pH (5.1-5.4)

allowed the formation of a more compact structure and larger aggregates might

corresponded to the lower protein bioaccessiblity of soy yogurt. The results

demonstrated terminal pH might influence protein digestibility. Thus control of pH of

the final product will probably be an important index for maintain protein quality of

soy yogurt when ingested.

Acknowledgements:

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 This work was co-financed by National Natural Science Foundation of China

(No. 31501466), Natural Science Foundation of Jiangsu Province (BK20150660), and

the Fundamental Research Funds for the Central Universities (KJQN201647 &

KYZ201745). The author would also like to acknowledge the funding from the

Priority Academic Program Development of Jiangsu Higher Education Institutions

(PAPD) and Jiangsu Provincial Double-Creative Doctorate Plan.

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LIST OF TABLE

Table 1 Texture properties of soy yogurts (SY) at varied fermentation pH.

LIST OF FIGURES

Fig. 1 Microbial and acid gelation behavior of soymilk during the growth of L.

plantarum B1-6. (A) Changes in pH (△), Storage modulus G’ (■), and Loss

modulus G’’ (○); (B) Changes in viable lactic acid bacteria cell counts.

Fig. 2 Particle size distribution of soymilk and SY samples collected at varied phases

of in vitro GIS (A) P1: after simulated buccal digestion, (B) P2: after simulated

gastric digestion, (C) P3:after simulated duodenal digestion. Different patterns

represented the soymilk (blue closed circle), SY-6.0 (red closed square), SY-5.7

(green closed triangle), SY-5.4 (purple cross), SY-5.1 (blue open triangle). Data are

expressed as mean ± SD from triplicate experiments. Different letters within the same

column indicate significant difference (P <0.05)

Fig.3 Soluble protein contents of soymilk and SY samples at varied phases of in vitro

GIS (A) P0-before the GIS; P1-after simulated buccal digestion; P2-after simulated

gastric digestion; P3- after simulated duodenal digestion. The error bars represent the

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standard deviation of three replicates. Different letters within the same digestive phase

indicate significant difference (P <0.05)

Fig.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

profiles of proteins collected from supernatant at different phases of in vitro

gastrointestinal digestion simulation (GIS). P0-before GIS (P0); P1-after simulated

buccal digestion (P1); P2-after simulated gastric digestion (P2); P3-after simulated

duodenal digestion (P3). 1-14 represented predominant bands observed before and

after in vitro GIS.

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 Table 1 Texture properties of soy yogurts (SY) at varied fermentation pH1,2

Sample Hardness (g) Springiness (mm) Cohesiveness Gumminess (g)

b a a
SY-5.4 75.00±6.87 0.85±0.01 0.63±0.03 47.41±4.34 b

SY-5.1 90.50±3.81a 0.88±0.02a 0.60±0.03 a 54.06±2.71 a

1
The texture data for SY-5.7 and SY-6.0 were not available.
2 a-b
Values followed by different letters in the same column are significantly different (P<0.05).

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 A

Fig. 1

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 A

A D[4,3] D[v,0.90]
(μm) (μm)
Soymilk 0.81±0.02d 1.82±0.03c
SY 6.0 1.57±0.04d 5.00±0.10c
SY 5.7 20±3c 34±6c
SY 5.4 50±11b 119±36b
SY 5.1 97±29a 273±92a

B D[4,3] D[v,0.90]
(μm) (μm)
Soymilk 27±1a 58±8a
SY 6.0 24±1abc 44±6ab
SY 5.7 25±2ab 25±1c
SY 5.4 22±1bc 23±3c
SY 5.1 21±2c 32±13bc

C D[4,3] D[v,0.90]
(μm) (μm)
Soymilk 43±1a 164±3a
SY 6.0 29±2c 95±12b
SY 5.7 18±1e 23±1d
SY 5.4 33±1b 96±10b
SY 5.1 24±1d 38±2c

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 Fig. 2

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 Fig. 3

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 Fig. 4

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