Introducing Bacillus natto and Propionibacterium shermanii into soymilk fermentation: A promising strategy for quality improvement and bioactive peptide production during in vitro digestion

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Food Chemistry 455 (2024) 139585

Available online 5 June 2024

Introducing Bacillus natto and Propionibacterium shermanii into soymilk

fermentation: A promising strategy for quality improvement and bioactive

peptide production during in vitro digestion

Xiaohui Wu

, Honghong Liu

, Junqing Han

, Zhitong Zhou

, Jian Chen

Xiao Liu

Science Center for Future Foods, Jiangnan University, Wuxi 214122, China

Jiaxing Institute of Future Food, Jiaxing 314000, China

School of Biotechnology, Jiangnan University, Wuxi 214122, China

ARTICLE INFO

Keywords:

Bacillus natto

Propionibacterium shermanii

Fermented soymilk

In vitro digestion

Bioactive peptide

ABSTRACT

Herein, the texture properties, polyphenol contents, and in vitro protein digestion characteristics of soymilk

single- or co-fermented by non-typical milk fermenter Bacillus natto (B. natto), Propionibacterium freudenreichii

subsp. shermanii (P. shermanii), and traditional milk fermenter were evaluated. Co-fermenting procedure con-

taining B. natto or P. shermanii could raise the amounts of gallic acid, caffeic acid, and GABA when compared to

the unfermented soymilk. Co-fermented soymilk has higher in vitro protein digestibility and nutritional protein

quality. Through peptidomic analysis, the co-work of P. shermanii and Lactobacillus plantarum (L. plantarum) may

release the highest relative percentage of bioactive peptides, while the intervention of B. natto and Streptococcus

thermophilus (S. thermophilus) resulted in more differentiated peptides. The multi-functional bioactive peptides

were mainly released from glycine-rich protein, β-conglycinin alpha subunit 1, and ACB domain-containing

protein. These ndings indicated the potential usage of B. natto/S. thermophilus or P. shermanii/L. plantarum in

bio-enhanced soymilk fermentation.

1. Introduction

Growing environmental sustainability demand has fueled the rapid

development of plant-based dairy products, especially soymilk (Vogel-

sang-O’Dwyer, Zannini, & Arendt, 2021). Depending on the presence

and content of essential amino acids, soy proteins are of lower quality

and are less digestible than milk proteins. Besides, soy proteins usually

require more processing to become bioavailable (Guyomarc’h et al.,

2021). The application of microorganisms offers a reliable way of

nutritional enhancement of soymilk. Considering the potential syner-

gistic interactions within the microbial consortia, plenty of studies

emphasized the use of mixed-culture fermentation technologies to

enhance the sensory characteristics and nutritional content of soymilk,

which prompted the creation of soy-based dairy substitutes (like cheese

and yogurt) to satisfy the consumer demand (McClements, Newman, &

McClements, 2019). On this premise, the utilization of fermentation to

produce functional soymilk (owing anti-obesity, anti-inammatory,

immunomodulatory effects, and the potential to produce bioactive

peptides) is recognized as one of the most conspicuous future opportu-

nities for the invention of soymilk substances (Hasan et al., 2023; Mohd

Zaini et al., 2023).

Current research is focused on diversifying soymilk fermenters

beyond traditional lactic acid bacteria (LAB, such as L. plantarum and

S. thermophilus) to other potential probiotics. B. natto is generally used

as a starter for traditional natto fermentation, which is usually recog-

nized as a safe probiotic (Ruiz Sella, Bueno, de Oliveira, Karp, & Soccol,

2021). Furthermore, B. natto has been employed to develop functional

natto yogurt and ice cream with thrombolytic activity through the

fermentation of milk and soymilk (Nie et al., 2017; Zhang, Tong, Wang,

Lyu, & Yang, 2021). P. shermanii, known as a cheese-ripening adjunct

culture and generally regarded as safe (GRAS) bacterium, is highly

promising for bio-enhancing the nutritional prole of soy-derived

products (Assis, Matte, Aschidamini, Rodrigues, & Zachia Ayub,

2020). The real health benets and the potential bioactive functions

under real digestive situation of soymilk fermented by B. natto or

P. shermanii may be better illustrated by alterations in the type and

* Corresponding author at: Science Center for Future Foods, Jiangnan University, Wuxi 214122, China

E-mail address: liuxiao@jiangnan.edu.cn (X. Liu).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2024.139585

Received 20 February 2024; Received in revised form 26 April 2024; Accepted 6 May 2024

Food Chemistry 455 (2024) 139585

content of antioxidative bioactive peptides or polyphenols.

Research have been conducted intensively on the behavior of soy-

milk during simulated human gastrointestinal digestion (Wang, Ye,

Dave, & Singh, 2021; Wegrzyn, Acevedo-Fani, Loveday, & Singh, 2021).

To enhance the evaluation of the actual digesting qualities of fermented

soymilk, the in vitro digestion model offers a more practical, ethical, and

inexpensive approach to imitating the actual digestive procedure and

investigating the fundamental mechanisms. In contrast to conventional

static digestion research methods, the geometry, internal folding struc-

ture, and ecological environment of the human gastrointestinal

tract—including the dynamic secretion of gastrointestinal uid and

gastrointestinal peristalsis—can be replicated by some more complex

and representative dynamic digestive systems (Wang et al., 2019).

Among the many nutrients produced by fermentation, soy-derived

bioactive peptides (usually obtained from glycinin and β-conglycinin)

deserve special attention due to their high absorption rate, anti-cancer

activity, and other health-related activities (Li et al., 2022). Particular

bioactive properties differ depending on the specic microbial strains

and the arrangement and composition of the amino acids (Sanjukta &

Rai, 2016). Currently, these bioactive peptide sequences are usually

obtained through enzymatic hydrolysis, peptide separation, and LC-MS

identication (Singh et al., 2023). Besides, computer-based bioinfor-

matics simulation algorithms also provide a reasonable way to assess

and discover potentially bioactive peptides (Udenigwe, 2014). Notably,

given the critical role that food structure plays in the release of nutrients

(such as bioactive peptides and polyphenols) from the complex food

system into the intestinal, further research is suggested to reveal the real

in vitro digestive characteristics of soy yogurts fermented with various

bacteria strains (Chen et al., 2023; Turgeon & Rioux, 2011).

The aim of this study was to investigate whether the application of B.

natto and P. shermanii in soymilk fermentation could improve the

texture and nutritional properties of the resultant soymilk. Owing to the

ineffectiveness of B. natto and P. shermanii in producing acid, two

additional conventional LAB, L. plantarum, S. thermophilus, were added

to fermentation procedure to increase the acid-producing ability and to

produce gel-like food with texture characteristics of yogurt through

lactic fermentation (Peng & Guo, 2015). Specically, we compared the

physicochemical, microstructure, and textural characteristics of soymilk

fermented singly or in combination by four strains: L. plantarum,

S. thermophilus, P. shermanii, and B. natto. Furthermore, using the

healthy adult digestion model, the differences in the peptides released

from fermented soymilk following in vitro dynamic gastrointestinal

digestion identied by LC-MS/MS were also incorporated into this

study.

2. Materials and methods

2.1. Materials

Chemical standards for polyphenols (HPLC ≥99%) include proto-

catechuic acid, syringic acid, vanillic acid, p-hydroxybenzoic acid, gallic

acid, p-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid, and

GABA, which were purchased from Shanghai yuanye Bio-Technology

Co., Ltd. L-ascorbic acid and β‑sodium glycerophosphate were pur-

chased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd.).

The bacterial genome was extracted using the FastPure Bacteria DNA

Isolation Mini Kit DC112 (Vazyme Biotech Co.,Ltd.). Commercial soy

yogurt was purchased from a local supermarket. Rhodamine B,

-amylase (type IX-A), pepsin (P7000), and trypsin (P7545) were pur-

chased from Sigma Aldrich (St. Louis, MO, USA). The Pierce™ quanti-

tative colorimetric peptide assay and Pierce™ peptide desalting spin

column were obtained from Thermo Fisher Scientic. Milli-Q water was

contained in the mobile phase in mass spectrometric detection. Formic

acid and acetonitrile were of MS grade.

2.2. Soymilk sample preparation

Good-maturing soy particles were obtained from a local market

(Shiyue Daotian Co., Ltd., Heilongjiang province, China), chosen and

soaked overnight at room temperature in 10-fold volumes of distilled

water, then washed twice with clean water before soymilk preparation.

The coarse pulp (soy:water 1:8 (w/v)) was homogenized using a blender

(AUX HX-PB9311, China). The homogenate was rst ltered using a

120-mesh lter cloth and sterilized at 105 ◦C for 15 min (10% glucose

was added, w/v). Before bacterial fermentation, sterilized soymilk was

stored at 4 ◦C.

2.3. Cultivation

2.3.1. Pre-cultures

L. plantarum CICC 22696, S. thermophilus, P. shermanii CICC 10284,

and B. natto CICC 10454 were commercial strains or isolated in labo-

ratory. Strains were activated twice in MRS, GM17, RCM, and CM0002

medium for 24 h, respectively. In 12 mL tubes with 5 mL of the appro-

priate media, anaerobic strains were cultivated anaerobically. Pre-

cultures were propagated using conditions (temperature and media)

appropriate to each strain (Table S1).

2.3.2. Soymilk fermentation

Target products were obtained by inoculating of strains into steril-

ized soymilk (10

CFU/mL, 0.1% B. natto, 0.1% S. thermophilus, 0.01%

L. plantarum, and 0.1% P. shermanii, v/v). For groups using

S. thermophilus or P. shermanii, the inoculated groups were rst cultured

at 30 ◦C for 12 h in a Whitley A35 anaerobic workstation (Don Whitley

Scientic, England) under a strict anaerobic atmosphere, then used for

soymilk fermentation (37 ◦C, 10 h), and then preserved at 4 ◦C for 12 h.

The inoculated amount of bacterial culture was shown in Table 1. Un-

fermented soymilk was used as the control group.

2.3.3. Fermentation characteristics

The pH value, titratable acidity (TA), and viable counts were deter-

mined at 0, 2, 4, 6, 8 and 10 h during fermentation according to method

described before (Ge et al., 2022). The pH was measured with a digital

potentiometer (Mettler Toledo, Switzerland). TA was determined by

titration with NaOH using phenolphthalein as the indicator (Bai et al.,

2020). The total number of colonies of B. natto and P. shermanii was

counted using the plate pouring method. The corresponding media and

culture conditions are shown in Table S1.

2.4. Microstructure observations

The microstructure of fermented soymilk samples was observed

Table 1

Fermented soymilk groups and used strains.

Group Fermentation strain

A B. natto +S. thermophilus

B B. natto +L. plantarum

C B. natto +S. thermophilus +L. plantarum

D B. natto +S. thermophilus +L. plantarum +P. shermanii

E P. shermanii +S. thermophilus

F P. shermanii +L. plantarum

G P. shermanii +L. plantarum +S. thermophilus

H B. natto +P. shermanii +L. plantarum

B. natto B. natto

S. thermophilus S. thermophilus

L. plantarum L. plantarum

P. shermanii P. shermanii

Notes: The cell concentrations and proportions of the inoculum were: 10

CFU/

mL, 0.1% B. natto, 0.1% S. thermophilus, 0.01% L. plantarum, and 0.1%

P. shermanii, v/v. All strains were in the logarithmic phase of growth when

inoculated into soymilk.

X. Wu et al.

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Introducing Bacillus natto and Propionibacterium shermanii into soymilk fermentation: A promising strategy for quality improvement and bioactive peptide production during in vitro digestion

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