Materials
Anhydrous sodium carbonate, L (+) -ascorbic acid, gallic acid, soluble starch, anhydrous ethanol, potassium persulfate, phenol, potassium tartrate, sodium hydroxide, anhydrous sodium sulfite, glucose, potassium chloride, potassium dihydrogen phosphate, fish peptone, ferrous sulfate heptahydrate, hydrochloric acid, 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). 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), were obtained from Shanghai yuanye Bio-Technology Co., Ltd(Shanghai, China).
AGAR powder, α-amylase (pig pancreas), 3, 5-dinitrosalicylic acid, pepsin, trypsin, corn starch, wheat starch, LP0021 yeast extract, were obtained from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China), Shanghai Macklin Biochemical Technology Co., Ltd (Shanghai, China), OXOID Company of Britain (US). 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 ℃ and stored at 4 ℃.
Preparation of seed solution
1.20 g glucose, 0.15 g yeast extract, 0.6 g fish meal peptone, 0.3 g KH2PO4, 0.025g KCl and 0.0005g FeSO4∙7H2O were dissolved in a conical bottle containing 50 mL distilled water. The seed solution was sterilized at 121℃ for 20min and placed at room temperature on the aseptic operating table. A piece of bacteria with diameter of 8 mm was extracted from the PDA 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℃, 180 r/min for 2 d.
Solid-state fermentation preparation
10.00 g of soybeans were soaked in 25 ml of hydrochloric acid solution pH 2. Soak overnight and pour off excess water. After sterilization for 20 min at 121℃, the samples were placed on aseptic operation table, cooled to room temperature, inoculated with bacteria by 15% (m/v), fermented for 12 days, dried at 45℃ for reserve.
Extraction and content determination of polyphenols
Extraction of free polyphenols. 1.00 g of fermented soybean powder was added to 20 ml of 80% methanol solution, water bath at 40 KHz and 40℃ for 1 h, then centrifuge at 4000 r/min for 10 min, and take the supernatant. In the supernatant, the methanol was evaporated by rotation at 40℃, and the volume was fixed to 20 mL with distilled water. Add 20 mL n-hexane for degreasing, leave out the organic phase, extract twice with 20 mL ethyl acetate, combine the organic phase, collect the water phase for use. The organic phase was fermented at 40℃, ethyl acetate was completely evaporated and redissolved with 5 mL 80% methanol (v/v) to obtain fermented soybean free polyphenol (FS-FP), and unfermented soybean free polyphenol (US-FP), respectively, The solution is stored at -20℃ (Bei et al., 2017).
Extraction of bound polyphenols. 20 mL 4 M NaOH solution was added into the filtrate after the extraction of free polyphenols. After hydrolysis at room temperature for 4 h, the filtrate was adjusted to a pH value of 2.0 with 6 M HCl. The filtrate after centrifugation and filtration was degreased with 20 mL n-hexane, and the organic phase was left out. Aqueous phase was extracted twice with 20 mL ethyl acetate and organic phase was incorporated. The organic phase was fermented at 40℃, ethyl acetate was completely evaporated and redissolved with 5 mL 80% methanol (v/v) to obtain fermented soybean bound polyphenol (FS-BP), and unfermented soybean bound polyphenol (US-BP), the solution was stored in -20℃ fridge (Bei et al., 2017).
Determination of polyphenol content. Folin-Ciocalteu method was applied to determine the phenolics content of the studied samples. 1 mL of Folinol-Ciocalteu reagent was added into 1 mL of polyphenol extract, shake well and let stand for 5 min, 6 mL deionized water and 2 mL of 15% (m/v) Na2CO3 are added in sequence, react for 1h under light protection, measure absorbance at 760 nm, and express it as mg gallic acid equivalent (mg GAE/g d.w) (Bei et al., 2018). The obtained standard curve equation is y = 8.88x + 0.0401(R2 = 0.9996).
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 seconds every 30 minutes for 6 times in total. Following centrifugation at 12000 rpm for 3 min, the extracts were filtrated (SCAA-104, 0.22 µm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before UPLC-MS/MS analysis.
The sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/; MS, Applied Biosystems 6500 Q TRAP, https://sciex.com.cn/). The analytical conditions were as follows, UPLC: column, Agilent SB-C18 (1.8 µm, 2.1 mm ×100 mm); The mobile phase was consisted of solvent A, pure water with 0.1% formic acid, and solvent B, acetonitrile with 0.1% formic acid. Sample measurements were performed with a gradient program that employed the starting conditions of 95% A, 5% B. Within 9 min, a linear gradient to 5% A, 95% B was programmed, and a composition of 5% A, 95% B was kept for 1 min. Subsequently, a composition of 95% A, 5.0% B was adjusted within 1.1 min and kept for 2.9 min. The flow velocity was set as 0.35 mL per minute; The column oven was set to 40°C; The injection volume was 2 µL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.
The ESI source operation parameters were as follows: source temperature 500°C; ion spray voltage (IS) 5500 V (positive ion mode)/-4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), curtain gas (CUR) were set at 50, 60, and 25 psi, respectively; the collision-activated dissociation (CAD) was high. QQQ scans were acquired as MRM experiments with collision gas (nitrogen) set to medium. DP (declustering potential) and CE (collision energy) for individual MRM transitions was done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.
DPPH radical scavenging activity assay
Scavenging activity against DPPH radical was performed according to Saelee et al. (Saelee et al., 2020) reported. 0.175 mmol/L DPPH solution was configured. The 1.0 mL of soybean polyphenol sample solution before and after fermentation was removed into the colorimetric tube, and 2 mL of DPPH solution was added to mix evenly. After reaction for 30 min under the condition of avoiding light, the absorbance was determined at the wavelength of 517 nm. Ascorbic acid was used as positive control and the experiment was repeated for 3 times.
Determination of ABTS free radical (ABTS∙+)scavenging activity
Scavenging activity against ABTS∙+ was evaluated in light of the approach of Re et al. (Re et al., 1999) reported. The absorbance was measured at 734 nm after absorbing 20 µL polyphenol sample solution diluted 5 times and mixing it with 180 µL ABTS∙+ working solution. The absorbance was measured at 734 nm after reaction for 10 min in the dark. Ascorbic acid was used as positive control and the experiment was repeated for 3 times.
Determination of α-amylase inhibition rate
Scavenging inhibitory effect on α-amylase of the approach of Li et al. (Li et al., 2018) reported. The α-amylase was dissolved in a phosphoric acid buffer at pH 6.9 at 25 mmol/L to produce 1U/mL α-amylase solution. 0.5mL soybean polyphenol extract before and after fermentation was mixed with 0.5mL 1U/mL α-amylase solution in 37℃ water bath for 10 min. 1% soluble starch solution (1 mL) was added in 37℃ water bath for mixing reaction for 10 min, 1 mL dinitrosalicylic acid solution was added to terminate the reaction, and boil water bath for 10 min, ice water cooling to room temperature, and set volume to 10 mL. The absorbance of the reaction solution was measured at 540 nm.
Effects of different soybean polyphenols on secondary structure of α-amylase
1 U/mL α-amylase, 2 mL mixture of soybean polyphenols and 1 U/mL α-amylase were scanned by a circular dichrometer in the far ultraviolet region (190 nm to 260 nm). After reacting at 37℃ for 10 min, scanning was performed at 37℃ with a scanning speed of 60 nm/min, response time of 1 s and slit width of 1 nm. Baseline scanning was started with 25 mmol/L PBS buffer solution.
Changes in glycemic index
In vitro starch digestibility of the FP in soybean was measured using the method of Uğur et al (Uğur et al., 2022). Prepare 100.00 mg starch (corn, wheat, rice) dissolved in 50 mL deionized water, after dispersed, boiling water bath for 10 min, cooling to 37 ℃, keep the temperature constant for use.
5 mL FP sample was added into the starch solution, 2 mL α-amylase solution (7 U/mL, prepared with PBS solution with pH 7) was added, and the pH was adjusted to 2 by shaking at 37℃ for 2 min. After 2 mL pepsin solution (1 mg/mL, prepared with 0.02 M HCl) was added, it was oscillated at 37℃ for 60min. At the end of the reaction, the pH was adjusted to neutral and 2 mL alpha-amylase solution and 1 mL trypsin solution (1 mg/mL) were added successively. Samples were taken at 10, 20, 30, 60, 90, 120, 180, 240 min, boiling water bath for 5 min to inactivate enzyme, and centrifugation, supernatant was taken, and glucose content was determined. 1 mg/mL glucose was used as the standard substance and deionized water was used as the control group. The hydrolysis rate of starch was calculated by the following formula.
GI calculation
The GI value is calculated using the method of Isabel et al (Isabel et al., 1997) .and Piecyk et al (Piecyk et al., 2019).
Ct: t time is the hydrolysis rate of starch; C∞: starch hydrolysis rate at stop reaction; k: digestive kinetics constant; t: Time.
Data analysis
All the experiments were carried out in triplicate and the data were presented as the mean value standard deviation and conducted 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.
The types and relative contents of phenols in the samples were analyzed by using the compounds in the database, and three 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.