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. 2024 May 31;23:101526. doi: 10.1016/j.fochx.2024.101526

Effects of Eurotium Cristatum on soybean (Glycine max L.) polyphenols and the inhibitory ability of soybean polyphenols on acetylcholinesterase under different conditions

Shuo-shuo Shi 1, Ting Hu 1,⁎
PMCID: PMC11200280  PMID: 38933989

Abstract

Most phenolic compounds in beans exist in complex, insoluble binding forms that bind to cell wall components via ether, ester, or glucoside bonds. In the process of solid-state fermentation, Eurotium Cristatum can produce many hydrolase enzymes, such as α-amylase, pectinase, cellulase and β-glucosidase, which can effectively hydrolyze ether, ester or glucoside bond, release bound polyphenols, and increase polyphenol content in soybeans. When the fermentation conditions of soybean were fermentation time 12 days, inoculation amount 15% and initial pH 2, the content of free polyphenols in fermented soybean was 2.79 mg GAE/g d.w, which was 4.98 times that of unfermented soybean. The contents of bound polyphenols and total phenols in fermented soybean were 0.62 mg GAE/g d.w and 3.41 mg GAE/g d.w, respectively, which were 2.38 times and 4.16 times of those in unfermented soybean. At the same time, the inhibitory effect of free polyphenols in fermented soybean on acetylcholinesterase reached 91.51%. Thus, our results demonstrated that solid state fermentation and Eurotium Cristatum can be used as an effective way to increase soybean polyphenol content and combat Alzheimer's disease.

Keywords: Soybeans, Eurotium Cristatum, Polyphenols, Acetylcholinesterase, Heatmap

Highlights

  • •

    Changes of free polyphenol and bound polyphenol contents in soybean before and after fermentation.

  • •

    The distribution of phenolic substances in soybean free polyphenols was analyzed by heatmap.

  • •

    Determination of inhibitory effect of soybean polyphenols on acetylcholinesterase before and after fermentation.

1. Introduction

A rapidly evolving food processing technique that has the potential to lower production costs, boost productivity, and lessen environmental pollution is the use of solid state fermentation technology to enhance the nutritional value of natural resources and bioactive ingredients (Singh, Chauhan, Kaur, & Pandey, 2020). Cellulase, protease, glycosylase, pectinase, and other hydrolase enzymes are released by microorganisms during fermentation. These hydrolase enzymes can break down plant cell walls (Leite, Salgado, Venâncio, Domínguez, & Belo, 2016; Leite, Silva, Salgado, & Belo, 2019; Nguyen, Wikee, & Lumyong, 2018) and release active ingredients by releasing the chemical bonds that hold them to cellulose, lignin, and other compounds.

As one of the most important crops, soybean is rich in primary and secondary metabolites, such as proteins, amino acids, carbohydrates, phenolic acids, isoflavones, saponins and other substances (Wang, Chen, & Zhang, 2014; Kim, Kim, & Yang, 2021). Studies have found that isoflavones, phenolic acids, saponins and other substances in soybean have strong biological activities such as antioxidant, anti-obesity, hypoglycemic, cholesterol-lowering and immunomodulatory (Zhang et al., 2003; Jayachandran & Xu, 2019; Ramdath, Padhi, Sarfaraz, Renwick, & Duncan, 2017), and pectin and dietary fiber in soybean epiderma have certain effects in preventing cardiovascular disease, coronary heart disease and improving intestinal osmotic pressure (Claudia, Elizabeth, & Rene´, B. Q., 2018; Yang et al., 2019). The bioavailability of soybean was improved after microbial fermentation. At the same time, under the action of microorganisms, bioactive ingredients are accumulated, thus improving the biological activity of soybeans and improving the quality of soybean products. In addition, anti-nutritional factors in soybeans, such as inositol hexaphosphate, trypsin inhibitors and lectins, are very low in fermented soybeans (Licandro et al., 2020; Anderson & Wolf, 1995).

Eurotium Cristatum is a kind of non-toxic and harmless probiotic bacteria with golden granules isolated from Fu brick tea, which gives Fu brick tea special aroma, color and taste. At the same time, studies have shown that E. cristatum has good effects in regulating blood sugar, lipid balance and cholesterol metabolism, enhancing immunity, alleviating obesity, regulating intestinal flora and inhibiting bacterial growth (Kang, Su, Duan, & Huang, 2019; Du, Wang, & Yang, 2019; Du, Li, Li, Zhu, & Wang, 2017). The biological activity of the fermented substance was significantly improved through the fermentation of E. cristatum. For example, the theabrownins of dark tea increased and the lipid-lowering activity of dark tea was significantly improved after it was fermented by E. cristatum (Xiao, Li, Wu, Zhong, & Gao, 2020). The contents of crude protein, amino acids, isoflavones, and total phenols in black beans fermented by E. cristatum have significantly increased, which also has great potential in oxidative disease, prevention of atherosclerosis, arthritis and other diseases (Xiao et al., 2021). After wheat bran fermentation, soluble dietary cellulose, total phenol, anthocyanin and phenylethanol were significantly increased. In addition, under the action of hydrolase enzyme secreted by E. cristatum, the content of ferulic acid was increased to 12.06 times, which further enhanced the antioxidant and anti-obesity effects of wheat bran (Lu, Jing, Li, Zhang, & Cao, 2022).

The purpose of this study was to investigate the effects of fermentation conditions on the content of soybean polyphenols and the changes of bioactivity of soybean polyphenols. The effects of fermentation time, inoculation amount and initial pH on the free and bound polyphenols of soybean were studied. The changes of main components in soybean during fermentation were analyzed, and the key factors that may affect the changes of lipid-lowering activity and anti-aging activity in soybean bioprocessing were speculated. This study provides a theoretical basis for the increase of polyphenol content in soybean substrate during solid state fermentation, and provides a new research direction for the development and utilization of soybean resources.

2. Materials and methods

2.1. Materials

Anhydrous sodium carbonate, gallic acid, anhydrous methanol, sodium hydroxide, anhydrous sodium sulfite, glucose, potassium chloride, potassium dihydrogen phosphate, fish peptone, ferrous sulfate heptahydrate, hydrochloric acid, potassium phosphate dibasic, Folin-Ciocalteu reagent, were obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Methanol, acetonitrile and formic acid are HPLC grade and purchased from Merck (USA).

AGAR powder, LP0021 yeast extract, acetylthiocholine iodide, acetylcholinesterase, 5,5′-Dithiobis(2-nitrobenzoic acid) were obtained from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China), Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China), OXOID Company of Britain (US), respectively. Soybeans purchased from Zaozhuang Guocheng Agricultural Products Co., Ltd. (Shandong, China). The strain E. cristatum J4 was isolated from the Fu-brick tea of Anhua (Hunan, China) for the first time in our research group. It was cultured on an AGAR slope every month for 6 days at 30 °C and stored at 4 °C.

2.2. Preparation of seed solution

1.20 g glucose, 0.15 g yeast extract, 0.6 g fish meal peptone, 0.3 g KH2PO4, 0.025 g KCl and 0.0005 g FeSO4∙7H2O were dissolved in a conical bottle containing 50 mL distilled water. The seed solution was sterilized at 121 °C for 20 min and placed at room temperature on the aseptic operating table. A piece of bacteria with diameter of 8 mm was extracted from the potato dextrose agar culture medium of E. cristatum cultured for 6 d using a sterile hole punch and inserted into the sterilized seed solution. Shaker incubator at 30 °C, 180 r/min for 2 d.

2.3. Extraction and content determination of polyphenols

The extraction of free polyphenols was performed as follows: 1.00 g of fermented soybean powder was added to a solution containing 20 mL of 80% methanol. The mixture was subjected to a water bath and maintained at a temperature of 40 °C for 1 h. Subsequently, centrifugation was carried out at a speed of 4000 r/min for 10 min, and the resulting supernatant was collected. To remove methanol, the supernatant underwent rotary evaporation at 40 °C (RE-2000 A rotary evaporator Yarong biochemical instrument factory, Shanghai,China) until its volume reached 20 mL, which was adjusted with distilled water. N-hexane (20 mL) is added to the mixture for degreasing, followed by separation from the organic phase. Further extraction with ethyl acetate (20 mL) was conducted twice, and the combined organic phases were collected. The obtained organic phase underwent evaporation at a temperature of 40 °C until complete evaporation of ethyl acetate occurred. It was then redissolved in a solution consisting of 5 mL of 80% methanol (v/v) solvent to obtain fermented soybean free polyphenol (FS-FP) and unfermented soybean free polyphenol (US-FP), respectively. Finally, these solutions were stored at −20 °C (Bei, Liu, Wang, Chen, & Wu, 2017).

Extraction of bound polyphenols was performed by adding 20 mL of a 4 M NaOH solution to the filter residue after the extraction of free polyphenols. The resulting mixture was hydrolyzed at room temperature for 4 h, followed by pH adjustment to 2.0 using 6 M HCl. After centrifugation and filtration, the filtrate was degreased with 20 mL of n-hexane, and the organic phase was separated. The aqueous phase was then subjected to two extractions with 20 mL of ethyl acetate, and the organic phases were combined. Subsequently, the obtained organic phase underwent evaporation at a temperature of 40 °C until complete evaporation of ethyl acetate occurred. It was then redissolved in a solution consisting of 5 mL of 80% methanol (v/v) solvent to obtain fermented soybean bound polyphenol (FS-BP) and unfermented soybean bound polyphenol (US-BP). The resulting solutions were stored in a − 20 °C refrigerator (Bei et al., 2017).

Determination of polyphenol content: The phenolic content of the samples was determined using the Folin-Ciocalteu method. A mixture of 1 mL polyphenol extract and 1 mL Folin-Cteu reagent was vigorously shaken and allowed to stand for 5 min. Subsequently, 6 mL deionized water and 2 mL of 15%(m/v) Na2CO3 solution were added sequentially. The reaction mixture was protected from light and incubated for 1 h, measure absorbance at 760 nm. The results were expressed as mg gallic acid equivalent (mg GAE/g d.w) (Bei et al., 2017). The obtained standard curve equation is y = 8.88× + 0.0401(R2 = 0.9996).

2.4. Single factor experiment on solid state fermentation conditions of E. cristatum

Effect of fermentation time on polyphenol content. 10.00 g soybeans were soaked in 25 mL pH 6 solution in a conical bottle overnight, and excess water was poured off. After sterilization at 121 °C for 20 min, the samples were placed on a sterile operating table and cooled to room temperature. Each sample was added with 10% seed solution (v/m) and fermented in an incubator at 30 °C and 60% humidity for 2, 4, 6, 8, 10, 12, 14 and 16 d, respectively.

Effect of inoculation amount on polyphenol content. 10.00 g soybeans were soaked in 25 mL pH 6 solution in a conical bottle overnight, and excess water was poured off. The samples were sterilized at 121 °C for 20 min, placed on a sterile operating table, and cooled to room temperature. Each sample was added with 5%, 10%, 15%, 20% and 25% seed liquid (v/m), respectively, and fermented in an incubator at 30 °C and 60% humidity for 8 d.

Effect of initial pH on polyphenol content. The pH of distilled water was adjusted by hydrochloric acid and sodium hydroxide solution to obtain pH 2, 4, 6, 8, 10 solutions. 10.00 g is soaked in 25 mL of different pH solution overnight, then excess water was poured away. After sterilization at 121 °C for 20 min, the samples were placed on a sterile operating table and cooled to room temperature. Each sample was added with 10% seed solution (v/m) and fermented in an incubator at 30 °C and 60% humidity for 8 d.

Fermented soybeans were dried in the oven at 45 °C to constant weight, and the fermented soybeans (FS) are crushed into powder for backup. The unfermented soybeans with the same treatment as the experimental group are fermented as the control group, named unfermented soybeans (US). fermented soybean free polyphenol (FS-FP), fermented soybean free polyphenol (US-FP), fermented soybean bound polyphenol (FS-BP) and unfermented soybean bound polyphenol (US-BP) were fermented.

2.5. Orthogonal optimization experiment

The optimal fermentation conditions were selected, and the L9 (33) orthogonal experiment was carried out according to orthogonal Table 1. The fermentation time (A), inoculation amount (B) and initial pH (C) were adjusted for the experiment, and the free polyphenol content was selected as the index to optimize the experiment.

Table 1.

Orthogonal optimization test factor level table.

Horizontal Factor
A fermentation time (d) B inoculation amount(%) C initial pH
1 8 10 2
2 10 15 4
3 12 20 6
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2.6. Analysis of free polyphenols by UPLC-MS/MS

Biological samples are freeze-dried by vacuum freeze-dryer (Scientz-100F). The freeze-dried sample was crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. Dissolve 50 mg of lyophilized powder with 1.2 mL 70% methanol solution, vortex 30 s every 30 min for 6 times in total. Following centrifugation at 12000g for 3 min, the extracts were filtrated (SCAA-104, 0.22 μm pore size; ANPEL, Shanghai, China) before UPLC-MS/MS analysis.

The detection method and data comparison results refer to the method of Chen et al., Fraga et al. (Chen et al., 2013; Fraga, Clowers, Moore, & Zink, 2010).

2.7. Soybean morphological change

The morphology of soybean before and after fermentation was observed. The soybean was crushed before and after fermentation and observed under electron microscope at different multiples.

2.8. Determination of acetylcholinesterase activity

At present, acetylcholinesterase is an attractive target for the treatment of Alzheimer's disease, and acetylcholinesterase inhibitors are the main approved drugs for the treatment of this neurodegenerative disease (Lee, Lee, & Ju, 2019).

In 96-well plates, 20 μL polyphenol samples and 120 μL 0.1 mol/L phosphate buffer (pH 8.0) were added, and 15 μL 0.5 U/mL acetylcholinesterase (phosphate buffer dissolved) was mixed. After the reaction at 25 °C for 10 min, 10 μL of 5 ‘5-dithiobibis (2-nitrobenzoic acid) (DTBN) at 10 mmol/L and 10 μL of 7.5 mmol/L acetylthiocholine iodide, were added and mixed evenly. The absorbance was measured at 405 nm after 30 min reaction at 37 °C. The calculation formula is as follows:

Inhibition rate%==1−A1−A2A0×100

where A1: the absorbance of the sample group; A2: the absorbance of the sample control group, buffer solution was used instead of enzyme solution; A0: refers to the absorbance of the blank group and replaces the sample with buffer solution.

2.9. Data analysis

All the experiments were used out in triplicate and the data were presented as the mean value standard deviation and treated with one-way analysis of variance (ANOVA). Significant differences (p < 0.05) between the means were determined using the Voeller-Duncan ‘s multiple range test which conducted by SPSS version 26 (SPSS Inc., Chicago, USA). Origin 2019b statistical software (OriginLab Co., USA) was carried out to prepare the data graphs. Orthogonal software assistant is used for orthogonal optimization analysis.

The types and relative contents of phenols in the samples were analyzed by using the compounds in the database, and two parallel experiments were conducted. The quantification of the compound is based on the MRM model to screen the precursor ion (Q1) of the target substance and remove the corresponding ion to remove interference. The chromatographic peaks were integrated and corrected with Multia Quant 3.0.2 edition, and the relative content of corresponding substances was calculated according to the peak area. Rstudio software was used to draw a heatmap of phenols.

3. Experimental results

3.1. Effect of fermentation time on polyphenol content

Fig. 1 shows the effects of different fermentation time on the contents of free polyphenols and bound polyphenols in soybeans. Fig. 1A shows the changes of free polyphenols and bound polyphenols in soybeans with the increase of fermentation time, and Fig. 1B shows the changes of the proportion of total phenols and two different forms of polyphenols in soybeans with the increase of fermentation time.The results are depicted in Fig. 1, with the increase of fermentation time, the free polyphenol content and total phenol content in soybean showed a trend of first increasing and then decreasing. The contents of free polyphenols, bound polyphenols and total phenols in unfermented soybeans were 0.56 mg GAE/g d.w, 0.26 mg GAE/g d.w and 0.82 mg GAE/g d.w, respectively. After 12 days of fermentation, the content of free polyphenols in soybean reached 1.79 mg GAE/g d.w, which was 3.20 times that of unfermented soybean, and the content of free polyphenols was significantly increased. After 16 days of fermentation, the content of bound polyphenols reached 0.85 mg GAE/g d.w, which was 3.30 times that of unfermented soybean. After 12 days of fermentation, the total phenol content reached 2.35 mg GAE/g d.w, which was 2.87 times that of unfermented soybean, and the free polyphenol content was relatively large. The above results indicated that fermentation could significantly increase the polyphenol content in soybean.

Fig. 1.

Fig. 1

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Effects of fermentation time on contents of free polyphenols, bound polyphenols and total phenols in soybean.

Note: Values in the figure are mean ± standard deviation (n = 3), and different letters in the same column indicate statistical differences between components, ANOVA, p < 0.05.

After 2 days of fermentation, the content of free polyphenols in soybean decreased slightly, while the content of bound polyphenols increased, which may be due to the fact that the growth of E. cristatum requires nutrients from soybean, and nutrients such as cellulose and protein combine with polyphenols (Dey, Chakraborty, Jain, Sharma, & Kuhad, 2016). The content of free polyphenols decreased slightly after 10 days of fermentation, which may be due to the fact that the bacteria reached a certain number at this time, the nutrient was insufficient, and the enzyme activity of the secreted hydrolase enzyme decreased, resulting in the reduction of the transformation and release of polyphenols.

Studies have shown that most phenolic compounds in legumes, grains and fruits bind to lignin, cellulose and polysaccharides in cell walls through ether, ester and glucoside bonds to form complex insoluble binding substances (Acosta-Estrada, Gutiérrez-Uribe, & Serna-Saldívar, 2014; Hur, Lee, Kim, Choi, & Kim, 2014). In the process of fermentation, E. cristatum produces hydrolase enzyme such as α-amylase, β-glucosidase, cellulase and protease. Under the action of hydrolase enzyme, the covalent bonds between binding phenols and proteins, starch, amines and other substances are hydrolyzed, resulting in free phenolic substances (Zou et al., 2019). At the same time, with the proliferation of E. cristatum and the secretion of hydrolase enzyme enzyme, the hydrolase enzyme enzyme can hydrolyze the protein, cellulose or starch in soybean into small molecular peptides, amino acids and oligosaccharides as energy, so that E. cristatum can grow well in soybean, thus improving the biosynthesis of phenolic compounds (Chen et al., 2020; Chen et al., 2020) and changing the content of free and bound phenols in soybean. Thus, the solid-state bioprocessed soybeans using E. cristatum enhances polyphenols, which may be applied in health care products and functional foods.

3.2. Effect of inoculation amount on polyphenol content

Fig. 2 shows the effects of different amount of inoculation on the contents of free polyphenols and bound polyphenols in fermented soybeans. Fig. 2A shows the changes of free polyphenols and bound polyphenols in soybeans with the increase of inoculation amount, and Fig. 2B shows the changes of the proportion of total phenols and two different forms of polyphenols in soybeans with the increase of inoculation amount.

Fig. 2.

Fig. 2

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Effects of the inoculum amount of E. cristatum on the contents of free polyphenols, bound polyphenols and total phenols in soybean.

Note: Values in the figure are mean ± standard deviation (n = 3), and different letters in the same column indicate statistical differences between components, ANOVA, p < 0.05.

As can be seen from the results in the Fig. 2, the free polyphenol content in soybean was 1.69 mg GAE/g d.w, which was 3.02 times that of unfermented soybean when the bacterial dosage was 10%. The content of bound polyphenols in soybean was 0.64 mg GAE/g d.w, which was 2.46 times that of unfermented soybean, and the total phenol content was 2.30 mg GAE/g d.w, which was 2.80 times that of unfermented soybean. The content of polyphenols in soybean was significantly changed with the change of the amount of E. cristatum. With the increase of the amount of E. cristatum, the activity of hydrolase enzyme was enhanced, and the polyphenols in soybean were easily dissociated. Cellulase produced by filamentous fungi breaks down plant cell walls, leading to the release of phenolic compounds.(Dey et al., 2016;) Moreover, protease can effectively transform bound polyphenols in protein into free phenols. Therefore, proteases may be one of the contributors to the enhancement of phenols during fermentation(Chen, Liu, et al., 2020; Chen, Wang, et al., 2020). At the same time, due to the growth of E. cristatum, the bacterial base increases, and the consumption of nutrients increases, which affects the biosynthesis of phenolic compounds.

3.3. Effect of initial pH on polyphenol content

The effects of different initial pH on the contents of free polyphenols and bound polyphenols in fermented soybeans, as Fig. 3 shows. Fig. 3A shows the changes in the contents of free polyphenols and bound polyphenols in fermented soybeans with the change of initial pH, and Fig. 3B shows the changes in the proportion of total phenols and two different forms of polyphenols in fermented soybeans with the increase of initial pH.

Fig. 3.

Fig. 3

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Effects of initial pH on the contents of free polyphenols, bound polyphenols and total phenols in soybean.

Note: Values in the figure are mean ± standard deviation (n = 3), and different letters in the same column indicate statistical differences between components, ANOVA, p < 0.05.

As can be seen from the Fig. 3, when the pH of the solution is 6, the free polyphenol content in fermented soybeans can reach 1.69 mg GAE/g d.w, the bound polyphenol content is 0.51 mg GAE/g d.w, and the total phenol content is 2.20 mg GAE/g d.w. They were 3.02, 1.96 and 2.68 times that of unfermented soybean. Studies have shown that the activity of E. cristatum is higher in acidic environment and it secretes more types of hydrolase enzyme, while the activity of E. cristatum is lower in alkaline condition, which affects the activity of hydrolase enzyme and thus the content of polyphenols (Jiang et al., 2022). In addition, different initial pH may also have certain effects on the structure and content of phenolic substances in fermented soybeans (Gulsunoglu-Konuskan, Karbancıoğlu-Güler, & Kılıç-Akyılmaz, 2021).

3.4. Experimental results of orthogonal optimization

According to the results of the previous three single factor experiments, the content of free polyphenols in soybeans after fermentation by E. cristatum accounted for a relatively large proportion, and was greatly affected by single factor conditions, and there were significant differences in the data within the groups. Therefore, in the orthogonal optimization experiment, the suitable fermentation conditions were selected, and the free polyphenol content was taken as the detection index according to the orthogonal Table 1, and the optimization experiment results were shown in Table 2.

Table 2.

Experimental results of orthogonal optimization.

Test No. A fermentation time(d) B inoculation amount(%) C initial pH Free polyphenols
(mg GAE/g d.w)
1 8 10 2 1.64
2 8 15 4 1.62
3 8 20 6 1.67
4 10 10 4 2.50
5 10 15 6 2.19
6 10 20 2 2.06
7 12 10 6 1.81
8 12 15 2 2.79
9 12 20 4 2.22
K1 1.643 1.983 2.163
K2 2.250 2.200 2.113
K3 2.273 1.983 1.890
Range 0.630 0.217 0.273
Factor A > C > B
Optimal A3B2C1
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The optimized range RA > RC > RB is obtained from Table 2, fermentation time (A), was the most influential factor on free polyphenols, followed by initial pH (C), followed by inoculation amount (B). According to K value (mean value), the optimal condition of soybean fermentation was determined as A3B2C1, which was consistent with the data obtained by orthogonal optimization experiment. Therefore, the optimal experimental conditions for soybean fermentation were fermentation time 12 days, bacterial dosage 15% and initial pH 2. Under these conditions, the content of free polyphenols in fermented soybean was 2.79 mg GAE/ G.D·W, which was 4.98 times that of unfermented soybean. The contents of bound polyphenols and total phenols in fermented soybean were 0.62 mg GAE/g d.w and 3.41 mg GAE/g d.w, respectively, which were 2.38 times and 4.16 times of those in unfermented soybean. The above experimental results showed that the polyphenol content in soybean could be significantly increased by the fermentation of E. cristatum.

3.5. Soybean morphology was observed by scanning electron microscopy

Scanning electron microscopy was used to observe the microscopic morphology of soybeans before and after fermentation and the attachment of E. cristatum. The experimental results are shown in Fig. 4. As can be seen in Fig. 4A, large pores appeared on soybeans after fermentation by E. cristatum. This phenomenon may be caused by the action of hydrolase enzyme secreted by E. cristatum on soybeans and the penetration of mycelium through soybean matrix. Fig. 4a, b, and c are electron microscopic images of unfermented soybeans at different magnifications. Pores on soybeans are small or non-porous, which may be related to mechanical failure. Fig. 4B and C can obviously observe the adhesion of E. cristatum. A large number of E. cristatum attached to soybean released a variety of hydrolase to hydrolyze cellulose, polysaccharide, protein and other macromolecular substances in soybean, further affecting the change of polyphenol content (Gebru & Sbhatu, 2020).

Fig. 4.

Fig. 4

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SEM image of fermented soybean and unfermented soybean.

A: Local map of 10.00 K× fermented soybean; B: Local map of 3.00 K× fermented soybean; C: Local map of 1.00 K× fermented soybean; a: Local map of 10.00 K× unfermented soybean; b: Local map of 3.00 K× unfermented soybean; c: Local map of 1.00 K× unfermented soybean.

3.6. UPLC-MS/MS analysis

According to the analysis of UPLC-MS/MS, as shown in Table 3 and Table 4, the free polyphenol in fermented soybean was mainly 6-Hydroxydaidzein, and its content increased significantly, which was similar to the results of Chen et al (Chen, Liu, et al., 2020; Chen, Wang, et al., 2020). Meanwhile, as shown in Fig. 5, the cluster heat map analysis of the two polyphenol samples showed significant differences in the polyphenol components of FS-FP and US-FP. Among them, flavonoids, tannins, lignans and coumarins in FS-FP had relatively high abundance, while the abundance of phenolic acids was lower than that of unfermented soybeans. This may be due to the combination of phenolic acids with cellulose, lignin, proteins, polysaccharides and other macromolecular substances in plants or conversion to other aromatic substances (Adaškevičiūtė, Kaškonienė, Barčauskaitė, Kaškonas, & Maruška, 2022; Chen, Wang, Yang, Wang, & Xu, 2023; Ma et al., 2022). As the main components of fermented soybean, flavonoids have potential research significance.

Table 3.

The relative content of the top 8 flavonoids in the FS-FP and UF-FP sample.

Sample Compounds Relative amount(106) Sample Compounds Relative amount(106)
FS-FP 6-Hydroxydaidzein 59.88 ± 0.62 UF-FP Sophoricoside 161.21 ± 12.4
Luteolin-4’-O-glucoside 39.07 ± 5.48 Genistein-7-O-galactoside 161.21 ± 12.4
Luteolin-3’-O-glucoside 34.61 ± 6.98 Apigenin-7-O-glucoside 157.28 ± 16.41
Kaempferol-3-O-neohesperidoside 44.95 ± 9.84 Galangin-7-O-glucoside 125.92 ± 20.98
7,4’-Dihydroxyflavone 38.19 ± 0.52 Daidzin 95.88 ± 6.04
6,7,8-Tetrahydroxy-5-methoxyflavone 38.07 ± 1.18 Apigenin-5-O-glucoside 106.58 ± 25.35
Cynaroside 33.84 ± 0.05 Daidzein-7-O-Glucoside-4’-O-Apioside 86.87 ± 1.04
Lonicerin 34.80 ± 1.44 Daidzein-7-O-apiosyl(1 → 6)glucoside 85.39 ± 7.85
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Table 4.

The relative content of the top 8 phenolic acids in the FS-FP and UF-FP sample.

Sample Compounds Relative amount(106) Sample Compounds Relative amount(106)
FS-FP Dimethyl phthalate 59.88 ± 0.62 UF-FP 6-O-Caffeoylarbutin 161.21 ± 12.4
Syringic acid 39.07 ± 5.48 5′-Glucosyloxyjasmanic acid 161.21 ± 12.4
Benzoylmalic acid 34.61 ± 6.98 Syringic acid 157.28 ± 16.41
Ferulic acid 44.95 ± 9.84 Hydrangeifolin I 125.92 ± 20.98
Vanillic acid 38.19 ± 0.52 Dimethyl phthalate 95.88 ± 6.04
2-(Formylamino)benzoic acid 38.07 ± 1.18 4-Nitrophenol 106.58 ± 25.35
3,4-Dimethoxyphenyl acetic acid 33.84 ± 0.05 Ferulic acid 86.87 ± 1.04
4-Nitrophenol 34.80 ± 1.44 Anthranilate-1-O-Sophoroside 85.39 ± 7.85
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Fig. 5.

Fig. 5

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Cluster heat map analysis of two polyphenol samples.

FS-FP: fermented soybean free polyphenol; US-FP: unfermented soybean free polyphenol.

3.7. Acetylcholinesterase inhibition effect

The inhibitory effects of four soybean polyphenols on acetylcholinesterase are shown in Fig. 6. The inhibitory rate of FS-FP (91.51 ± 0.43%) on acetylcholinesterase was higher than that of US-FP (45.69 ± 1.08%), and that of FS-BP (14.34 ± 0.36%) on acetylcholinesterase was higher than that of US-BP (4.67 ± 0.78%). The inhibitory effect of soybean polyphenols on acetylcholinesterase will be enhanced.

Fig. 6.

Fig. 6

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Inhibition effect of four polyphenol samples on acetylcholinesterase.

Note: Values in the figure are mean ± standard deviation (n = 3), and different letters in the same column indicate statistical differences between components, ANOVA, p < 0.05.

Other relevant studies have shown that the inhibitory effect of Perilla leaves on acetylcholinesterase was enhanced after fermentation with six kinds of probiotics, which may be mainly related to the changes of rutin, rorosemary acid, luteolin and flavonoids in the fermentation process (Wang et al., 2022). According to the results, the content, structure and types of polyphenols in the fermented soybean were changed. There was a significant increase in 6-Hydroxydaidzein, which may be the main reason for the decrease of acetylcholinesterase activity.

4. Conclusion

The experimental results show that when the fermentation conditions of soybean were fermentation time 12 days, inoculation amount 15% and initial pH 2, the content of free polyphenols in fermented soybean was 2.79 mg GAE/g d.w, which was 4.98 times that of unfermented soybean. The contents of bound polyphenols and total phenols in fermented soybean were 0.62 mg GAE/g d.w and 3.41 mg GAE/g d.w, respectively, which were 2.38 times and 4.16 times of those in unfermented soybean. At the same time, the inhibitory effect of free polyphenols in fermented soybean on acetylcholinesterase reached 91.51%. The contents and types of polyphenols in soybean were significantly increased by the fermentation of E.crustatum. By secreting a variety of hydrolase enzyme, the E.crustatum can metabolize and transform the bioactive ingredients in soybean and increase the abundance of various active ingredients. At the same time, after fermentation, the inhibition effect of free polyphenols on acetylcholinesterase was significantly improved compared with that without fermentation. This depends on the characteristics of the metabolites formed during the fermentation process and the type of conversion products that form polyphenol compounds, especially flavonoids. The results of this study are expected to be used in the production of new functional food or health care products. Meanwhile, further studies are needed to evaluate the safety of E.crustatum bioprocessed soybeans and their advantages in functional food development.

CRediT authorship contribution statement

Shuo-shuo Shi: Writing – review & editing, Writing – original draft, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation, Conceptualization. Ting Hu: Validation, Supervision.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.

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