Novel green soybean shuidouchi fermented by Bacillus velezensis with multibioactivities

Abstract Soybeans are usually fermented by Bacillus subtilis to produce shuidouchi, which is a traditional fermentation soybean product in China. In the study, green soybeans were fermented by Bacillus velezensis to make a novel green soybean shuidouchi with multibioactivities. The processing conditions were optimized as follows: initial moisture content 75%, inoculum concentration 7 log CFU/g, and incubation time 24 h for prefermentation; water addition 50%, salt addition 6%, temperature 45°C, 3 days for postfermentation. The fermented green soybean shuidouchi (FGSS) showed 234.8 FU/g dry weight (DW) for the fibrinolytic activity and IC50 of 0.33 mg/ml for the anticoagulant activity. FGSS had higher contents of chemical components including 3.6 mg rutin (RE)/g DW of total flavonoids, 8.2 mg gallic acid (GAE)/g DW of total phenolics, 63.7 mg/g DW of reducing sugars, and 163.8 mg/g DW of peptides than the unfermented green soybean shuidouchi (UGSS). Moreover, it exhibited high antioxidant activities of 29.8, 85.1 μmol trolox equivalent (TE)/g DW, and 12.8 μmol Fe2+/g DW through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2’‐azino‐bis (3‐ethylbenzthiazoline‐6‐sulfonic acid) diammonium salt (ABTS), and ferric reducing antioxidant power (FRAP) experiments. Thus, a novel green soybean shuidouchi fermented by B. velezensis owing to multibioactivities can provide a theoretical basis for the further development of functional shuidouchi.

soybeans, which are further postfermented for 1 ~ 2 weeks (Chen, Liu, et al., 2019b). Shuidouchi contains many functional components including polypeptides, oligosaccharides, isoflavones, saponins, and other microbial secondary metabolites. The health benefits of shuidouchi includes protecting the intestinal tract and preventing colitis , preventing constipation in mice (Chen, Zhang, et al., 2019a), antioxidant activities (Xu et al., 2015), and antimutagenic and anticancer effects . Furthermore, shuidouchi has been found to possess a certain concentration of fibrinolytic enzyme, which may have a thrombolytic effect to prevent thrombus (Chen, Liu, et al., 2019b).
Bacillus species is the main microorganisms present in shuidouchi and plays an important role in the fermentation processes.
Both Bacillus subtilis and Bacillus amyloliquefaciens have been used as good starters to produce shuidouchi (Chen, Liu, et al., 2019b).
So far, soybean is the raw material of shuidouchi, and its variety affects both sensory characteristics and physicochemical properties of products (Luo et al., 2010). The contents of protein, moisture, and ash in green soybeans are similar to those of soybeans. However, the carbohydrate content of green soybeans is 4 times higher than that of soybeans, and the lipid content is very low. Green soybeans with the contents of high carbohydrate and low fat may be more suitable for the processing material of shuidouchi (Feng et al., 2015). Bacillus velezensis, as a novel species, was recently taxonomically classified as a closely related species B. amyloliquefaciens (Ye et al., 2018). So far, this species has been widely separated from diverse habitats, such as soil, water, and fermented foods. B. velezensis is potential starter culture of fermented foods . Recently, B. velezensis BS2 from a traditional Korean fermented seafood and B. velezensis SW5 from fish sauce showed fibrinolytic activity Yao et al., 2019). A novel fermented soybean food by various microbes (B. subtilis GD 1, B. subtilis N4, B. velezensis GZ1, L. bulgaricus, and H. anomala CICC 1728) showed peptide, total phenol, total flavonoid contents, suitable organic acid and biological amine contents, strong antioxidant and antifatigue activities (Cui et al., 2020). Liu et al. (2020) reported that B. velezensis DP-2 isolated from douchi was applied in soybean meal fermentation, it degrades soybean antigen proteins and improved crude protein, soluble protein and trichloroacetic acid-soluble protein to make it more suitable for animal feed. So far, no information is available regarding the soybean fermented food by single strain B. velezensis. There is no study on the green soybeans fermented by B. velezensis to make shuidouchi.
A strain showing high fibrinolytic activity, B. velezensis CAU263, was newly isolated from Chinese traditional fermented douchi. In this study, the strain was used to ferment green soybean shuidouchi (FGSS). The processing conditions were optimized, and the potential nutrients and multibioactivities of green soybean shuidouchi were further investigated. This is the first report on shuidouchi fermented by B. velezensis.

| Materials, microorganism, and reagents
Green soybeans were obtained from a local farm (Harbin, China).
Salt, paprika, and ginger powder were purchased from a supermarket (Beijing, China). B. velezensis CAU263 (CGMCC NO: 20318) isolated from Chinese traditional douchi was used in this study.
Ltd. Peptone and yeast extract were obtained from Oxoid Co. Ltd.
Rutin was gained from Shanghai Yuanye Biotechnology Co. Ltd.
Gallic acid was obtained from Shanghai Macklin Biochemical Co.
Ltd. L (+)-ascorbic acid (VC) was acquired from Fujifilm Wako Pure Chemical Corporation. Other chemical reagents were obtained from Beijing Chemical Works.

| Preparation of green soybean shuidouchi
Bacillus velezensis CAU263 was cultivated at 37°C for 8 h in LB medium (200 rpm) and used as a starter culture. The green soybeans (100 g dry weight) were first washed and soaked in distilled water (300 ml) for 10 ~ 18 h at 25°C. The soaked green soybeans were steamed at 121°C for 20 min and cooled to room temperature for prefermentation.
All samples were stored at −20°C after freeze-drying, pulverizing, and passing through a 20-mesh sieve.
The sensory properties of green soybean shuidouchi were evaluated (Lan et al., 2020). For all sensory evaluation, rating scores of 1 ~ 25 were used as follows: the scores of 21 ~ 25 were excellent, the scores of 16 ~ 20 were good, the scores of 11 ~ 15 were moderate, the scores of 6 ~ 10 were poor, and the scores of 1 ~ 5 were inferior.
The sensory evaluation score was the sum of color, appearance, flavor, and taste scores.
The sensory evaluation score and amino acid nitrogen content were used as evaluation indexes of green soybean shuidouchi to calculate their weighted average score as a comprehensive score for optimization in the following Equation (1). A higher comprehensive score indicated better quality .

| Extraction methods of green soybean shuidouchi
Water extract and ethanol extract were prepared. A 10-fold volume of distilled water was added to the shuidouchi freeze-dried powder and extracted using a shaking incubator at 200 rpm (25°C, 2 h). After centrifugation at 11,510 g for 10 min, the supernatant was collected and stored at 4°C for analysis. For ethanol extract, a 4-fold volume of 80% ethanol was added to the shuidouchi freeze-dried powder and extracted 20 min by using ultrasonic (480 W). After centrifugation at 1,840 g for 10 min, the supernatant was collected. Furthermore, a 3-fold volume of 80% ethanol was mixed with the sediment, and the previous extraction step was repeated two times. The supernatant was combined as ethanol extract and stored at 4°C for analysis.

| Determination of amino acid nitrogen
The amino acid nitrogen content was determined by the Chinese national standard (GB 5009.235-2016). In short, 5 g powder samples were added to 100 ml deionized water to homogenize, and the supernatant was obtained after centrifugation at 2,880 g for 10 min. The supernatant 10 ml was titrated to pH 8.2 with 50 mM NaOH. Acidity was calculated using the consumed volume of titrant. Amino was then fixed by adding excess formaldehyde to the solution. The samples were titrated to pH 9.2 with 50 mM NaOH afterward. The amino acid nitrogen content was determined using the consumed volume of titrant.

| Determination of peptides and ACE inhibiting activity
The content of peptides was estimated by the o-phthaldialdehyde (OPA) spectrophotometric assay. OPA solution contained 25 ml of 100 mM sodium tetrahydroborate, 10 ml of 5% sodium dodecyl sulfate (SDS), 1 ml of 40 mg/ml OPA (dissolved in methanol), and 0.1 ml of β-mercaptoethanol. The volume was adjusted to 50 ml with deionized water. The reaction was performed by adding 10 μl water extract solution to 400 μl OPA solution for 8 min at room temperature, and the absorbance was recorded at 340 nm. The content of peptides was represented as mg/g DW by using Gly-Leu as the standard.
Angiotensin I-converting enzyme inhibiting activity was determined by using the fluorescence-based method (Hong et al., 2016); the IC 50 value was used to express the result of ACE-inhibiting activity, which is defined as the green soybean shuidouchi concentration (mg/mL) required to inhibit 50% ACE activity.

| Determination of total phenolic contents (TPCs)
The total phenolic contents (TPCs) were measured using the modified Folin-Ciocalteu reagent method (Shi et al., 2020), namely, 80 μl water extract solution was loaded on a 96-well microplate, and 80 μl Folin-Ciocalteu reagent (50%) and 80 μl of 10% sodium carbonate solution were added and mixed well. After incubation in the dark at 30°C for 30 min, the absorbance at 760 nm was recorded. TPC samples were represented as mg GAE/g DW based on a standard curve of gallic acid (GAE).

| Determination of total flavonoid contents (TFCs)
The total flavonoid contents (TFCs) were estimated using the aluminum chloride colorimetric assay with slight modification . In short, ethanol extract solution (100 μl) was mixed with 6 μl of 5% NaNO 2 in a 96-well microplate and reacted for 6 min.
After 6 μl of 10% AlCl 3 was added to react for additional 6 min, the reaction was terminated by 80 μl of 4% NaOH. Finally, the total volume was adjusted to 200 μl with 50% ethanol. After 15 min, the absorbance at 510 nm was read. The calibration curve was made using rutin (RE) as a standard. TFC of samples were represented as mg RE/g DW.

| Determination of reducing sugars
The contents of reducing sugars were estimated using the dinitrosalicylic acid (DNS) method (Miller, 1959), namely, 3 ml water extract solution was added to a 10-mL test tube and mixed with 3 ml DNS reagent. The test tubes were immediately cooled to ambient temperature using distilled water after reaction at 100°C for 10 min and measured at 540 nm. The contents of reducing sugars were expressed as mg/g DW calculated by the calibration curve used glucose as a standard. (1)

| Determination of fibrinolytic activity
The fibrinolytic activity of water extract solution was estimated by Huy et al. (2016). In short, 0.4 ml of 0.72% fibrinogen solution was mixed with 1.4 ml of 50 mM phosphate buffer (pH 8.5) in test tubes and kept at 37°C for 5 min. The fibrin clot was formed by adding 0.1 ml of 20 U/ml thrombin solution and left at 37°C for 10 min. Then 0.1 ml water extract solution was added, and the reaction was conducted at 37°C for 60 min with stirring every 20 min. In the end, 2 ml of 0.2 mol trichloroacetic acid (TCA) was added to terminate the reaction. The absorbance was monitored at 275 nm after centrifugation at 11,510 g for 10 min. In the assay, one unit of fibrinolytic activity (FU) was defined as the amount of enzyme making 0.01 increase in absorbance in 1 min.

| Determination of anticoagulant activity
The anticoagulant activity was evaluated using the previous method with minor modification (Wei et al., 2011), namely, 50 mM Tris-HCl buffer (pH 7.2) was used as the solvent for fibrinogen and thrombin. Both 140 μl of 0.1% fibrinogen solution and 40 μl water extract solution were mixed in 96-well microplates. Then, the reaction was performed by adding 10 μl of 12 U/ml thrombin solution to the 96well plates for 10 min at 37°C and measured at 405 nm. The IC 50 value was used to express the result of anticoagulant activity which was defined as green soybean shuidouchi concentration (mg/ml) required to inhibit 50% anticoagulant activity.

| Determination of antioxidant activity
The assay of DPPH radical scavenging capacity was spectrophotometrically estimated by Shi et al. (2020)  FRAP activity was determined according to the previous report . The FRAP values were represented as μmol Fe 2+ per gram lyophilized powder of shuidouchi (μmol Fe 2+ /g DW).

| Statistical analysis
All results were performed in triplicate. Statistical analysis was carried out using SPSS 19.0 software (SPSS, USA) and expressed as mean value ± standard deviation. Statistical significance was determined at p < 0.05.

| Effects of prefermentation conditions on FGSS quality
The effects of prefermentation conditions on FGSS quality are shown in Figure 1. The sensory score and amino acid nitrogen con- respectively. Moreover, the comprehensive score was up to 90.1 (Table S1).
The quality of fermented soybean products during the processing conditions are usually related to many factors such as the fermentation microorganism, inoculum concentration, substrate moisture, fermentation temperature, and time (Shahzad et al., 2020).
In the production of shuidouchi, increment in the initial moisture content can alter the nutrient solubility and scavenge wastes of metabolites in the substrate which promote the growth of microorganisms (Rodriguez de Olmos & Selva Garro, 2020). Excessive moisture content causes reduction in oxygen transfer and heat dissipation. The inoculum concentration, fermentation temperature, and time were found to be important factors in the production of FGSS (Table S1). With cell growth, the fibrinolytic enzyme was biosynthesized, and the yield was growth-associated with B. subtilis (Wei et al., 2011). More bacteria may lead to a decrease in the amount of nutrition of per cell in a certain weight substrate, or even be damaged or dead because of oxygen deficiency and the accumulation of metabolic wastes (Feng et al., 2015). In addition, appropriate incubation temperature and time were needed for the growth and physiological processes of microorganisms. It has been reported that microorganisms may synthesize only a reduced number of proteins essential for growth and other physiological processes at high temperature. Longer incubation period and higher incubation temperature may also lead to the accumulation of toxic inhibitors and inhibition of metabolite production and enzyme activity (Rodriguez de Olmos & Selva Garro, 2020).

| Effects of postfermentation conditions on FGSS quality
The effects of postfermentation conditions on FGSS quality are shown in Figure 2. With the addition of water from 50% to 110%, the sensory score was continuously improved from 88.0 to 92.0  (Figures 1 and 2). The total fermentation time of FGSS was 4 days, obviously shorter than that of traditional douchi which usually ferments from several weeks to months. Addition of NaCl to fermented soybean products can improve the flavor and taste as well as inhibit harmful microorganisms for extending shelf life (Kim et al., 2020). The salt concentration in the FGSS product was almost 3% (Figure 2), which is lower than that of traditional fermented douchi. In the preparation of doenjang, higher concentration of NaCl caused reduction in isoflavone aglycones, especially daidzein and genistein which have greater anti-inflammatory and bioavailability effects than other forms of isoflavones . FGSS can be considered a low-salt fermented soybean food, which may fit the demand of public nutritional health.
The relationship between amino acid nitrogen content and the sensory of shuidouchi was also explored. Amino acid nitrogen is considered as the metabolite of microorganism of proteins that can reflect the degree of aging of douchi . As an important index of douchi products, its content is related to the flavor (Lan et al., 2020). The amino acid nitrogen content of FGSS was 0.51 g/100 g (Figure 2) which met the Sichuan provincial standard of douchi (DB 51 T391-2006) stating that the amount of amino acid nitrogen of shuidouchi should be ≥0.2 g/100 g. In the previous report, the mean amino acid nitrogen content of shuidouchi samples was 0.39 g/100 g (Chen, Liu, et al., 2019). Usually, traditional douchi is fermented by various microorganisms in the air. The natural fermentation process may cause some problems such as the long F I G U R E 1 Effects of prefermentation conditions including prefermentation time (a), inoculum concentration (b), and initial moisture content (c) on FGSS quality. The prefermentation time, inoculum concentration, and initial moisture content of FGSS were investigated by the single factor method. Data represent mean ± standard deviation (n = 3). Different letters in the same color indicate significantly different values (p < 0.05) period of fermentation, unstable quality, and potential safety risks (Tan et al., 2019). The use of pure starter (B. velezensis CAU263) in the study had been demonstrated to improve the quality of FGSS and shorten the fermentation process.

| Chemical components of FGSS
The chemical components of FGSS including the total flavonoids, total phenolics, reducing sugars, and peptides were analyzed (Table 1). Compared with the UGSS, TFC of FGSS was improved by 19.5% from 2.89 mg RE/g DW to 3.59 mg RE/g DW. TPCs and the content of reducing sugars in FGSS were 4.5 and 3.0 times higher than those of UGSS (1.82 → 8.16 mg GAE/g DW, 21.4 → 63.7 mg/g DW). Specifically, a significant increment was detected on the content of peptides, which increased from 13.9 mg/g DW in UGSS to

mg/g DW in FGSS.
Apart from the flavor improvements, the significant increment in chemical components in FGSS had been found. Total phenolic contents in FGSS were improved compared with UGSS (Table 1). The Abbreviations: DW, dry weight; FGSS, fermented green soybean shuidouchi; UGSS, unfermented green soybean shuidouchi.

TA B L E 1 Chemical components contents of UGSS and FGSS under the optimal conditions
correlation analysis indicated that polyphenols could be the major contributors of antioxidant activities (Khosravi & Razavi, 2021).
Recently, many researchers have reported on the enhanced antioxidant activities with the increased phenolic and flavonoids contents of fermented soybeans Shahzad et al., 2020).
Pigeon peas fermented by B. subtilis improved the total flavonoids (2.5 → 5.4 mg/g) and total phenolic contents (10.8 → 15.2 mg/g) during fermentation (Lee et al., 2015); 181.5% increment of the TPC in soybean fermented by B. amyloliquefaciens was reported in another study (Shahzad et al., 2020). Therefore, FGSS may be used as a good functional food source which is rich in chemical components.

| ACE inhibitory activity of FGSS
Angiotensin I-converting enzyme inhibitory activity of FGSS is shown in Figure 3.  (Li et al., 2016). Furthermore, ACE inhibitory activity of 76.5% was obtained after ripening for 2 weeks of douchi sample . Compared with these studies, FGSS may be considered as an outstanding source of ACE inhibitors based on the strong ACE inhibitory activity.

| Thrombolytic activities of FGSS
The anticoagulant activity of FGSS showed a dose-dependent manner ( Figure 4). FGSS exhibited the highest anticoagulant activity of 90.6%, with the IC 50 of 0.33 mg/ml at 0.78 mg/ml of FGSS, much higher than that of UGSS (35.1%), with the IC 50 of 1.14 mg/ml. Moreover, the fibrinolytic activity of FGSS was 234.8 FU/g DW, whereas the UGSS did not have fibrinolytic activity.
IC 50 of the anticoagulant activity in FGSS was 0.33 mg/ml, which is 3.5 times lower than that of UGSS (1.14 mg/ml) ( Figure 4).
Moreover, FGSS showed high fibrinolytic activity of 234.8 FU/g DW. The fibrinolytic activities of fermented soybean oil cake powder produced by four isolated strains B. amyloliquefaciens CB1, B. amyloliquefaciens CB2, B. amyloliquefaciens MB1, and B. amyloliquefaciens MB2 were in a range from 29.7 to 77.9 FU/g (Huy et al., 2016). The fibrinolytic activity of 48.8 FU/g was gained according to the fermentation of pigeon pea by B. subtilis 14,715 (Lee et al., 2015). Moreover, the fibrinolytic activity of chickpeas fermented by B. amyloliquefaciens was up to 39.28 FU/g (Wei et al., 2011). FGSS using B. velezensis CAU263 in this study had very high fibrinolytic activity. These suggest that FGSS has great potential to be developed as functional foods with high fibrinolytic activities.

| Antioxidant activities of FGSS
The antioxidant activities of FGSS are shown in Figure 5. Several studies had emphasized the importance of the fermentation process owing to the production of antioxidant compounds (Salim et al., 2017). The potential antioxidants in FGSS may be phenolics, flavonoids, and peptides, which explained the high antioxidant activities during fermentation. Similarly, fermented red beans showed an improvement of DPPH radical scavenging activity, total phenols, and total flavonoids during fermentation .
The fermented tofu could also promote the transformation of phenolics with low antioxidant activity into their corresponding forms with high antioxidant activity (Yin et al., 2020). The increased DPPH radical scavenging activity and FRAP values have a significant positive correlation between total phenols and total flavonoids in cheonggukjang (Piao & Eun, 2020). The microorganisms may play a vital role in the antioxidant activities of fermented foods. Mucor-and Aspergillusfermented douchi had higher DPPH value (2.7 ~ 23.1 μmol TE/g) than the bacteria-fermented Natto (2.6 ~ 4.5 μmol TE/g). FRAP assay showed significant difference between douchi (9.1 ~ 11.6 μmol Fe 2+ /g) and natto (4.7 ~ 6.1 μmol Fe 2+ /g) (Xu et al., 2015). In the current study, FGSS using B. velezensis CAU263 exhibited the high antioxidants.

| CON CLUS ION
A novel green soybean shuidouchi using B. velezensis CAU263 as a starter was developed. The optimal process conditions for green soybean shuidouchi improved the contents of total phenols, total flavonoids, peptides, and reducing sugars. The green soybean shuidouchi also had high fibrinolytic enzyme and antioxidant activities.
The study may be helpful for the development of shuidouchi with multibioactivities, which has the potential to improve human health.

CO N FLI C T S O F I NTE R E S T
The authors declare that there are no conflicts of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.