Alkaline protease (200,000 U/g) was obtained from Nanning Pangbo Biological Engineering Co., Ltd. (Nanning, China). High glucose Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). Cell counting kit 8 (CCK-8) was provided by Abbkine Scientific Co., Ltd. (Wuhan, China). The qRT-PCR kit was purchased from Takara Company (Dalian, China). H₂O₂ was purchased from Sigma-Aldrich (St. Louis, MO, USA). SOD, CAT and GSH assay kits were purchased from Nanjing Jian Cheng Bioengineering Institute (Nanjing, China). Goat anti-rabbit antibody such as β-actin, SOD1, heme oxygenase-1 (HO-1), zonulaoccluden-1 (ZO-1), claudin-1, keap1 and nuclear factor erythroid 2-related factor 2 (Nrf2) were purchased from Wuhan Service Bio-Technology Co., Ltd. (Wuhan, China).

About 25 g soybean meals were dispersed in distilled water in a ratio of 1:4 (w/v). The mixture was hydrolyzed using alkaline protease (10,000 U/g soybean meal) under the reactive conditions of 55°C, pH = 9 and 4.5 h. After pH was adjusted to 5.0, the mixture was placed in boiling water for 30 min to inactivate the enzymes, centrifuged at 5000 g for 10 min, and the supernatant was collected. Then, active carbon with a ratio of 1:10 (w/v) was added for decoloration and debittering. The supernatants was obtained using a membrane of 0.45 μm, then quickly freeze-dried and stored at 4°C for backup.

Identification of peptides was performed by an Easy-nLC 1,000 UHPLC system (Thermo Fisher Scientific, San Jose, CA, USA), online with an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Peptide separations were performed on Acclaim PepMap RSLC C18 threaded columns (Thermo Fisher Scientific, San Jose, CA, USA). The eluents of mobile phases A and B were 0.1% formic acid (FA) in water and 0.1% FA in acetonitrile, respectively. A gradient elution procedure was set: 0.0–3.0 min, 3% eluent B; 3.0–7.0 min, 3–8% eluent B; 7.0–46.0 min, 8–32% eluent B; 46.0–51.0 min, 32–44% eluent B. The polypeptide concentration was 10.0 mg/mL. The injection amount was 8.0 μL. The flow rate was 400 nL/min. The Orbitrap Fusion Mass Analyser collected all of the data throughout a 350–1,550 m/z scan range. Resequencing was used to analyze the spectra, and the UniProt database was used to look for matches in the MS/MS spectra.¹ Sequence comparison of reported peptides was performed using the BIOPEP database².

IPEC-J2 cells were grown in High-glucose Dulbecco’s Modified Eagle Medium (HGDMEM) supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum, respectively, at 37°C and 5% CO₂ in the incubator. For the experiments, IPEC-J2 cells were seeded into 6-well plates at a density of 2.5 × 10⁵ cells/mL for 2 mL or 96-well plates at a density of 1.0 × 10⁵ cells/mL for 100 μL. Cells were allowed to adhere for 24 h-36 h until they reached confluency before treatment. Cell viability was assessed using CCK-8 solution (1%, v/v). The protocol was as follows: Each well received a 10 μL CCK8 solution, and was incubated for 2 hours. An enzyme-linked immunosorbent assay (ELISA) was used to determine optical density (OD) at 490 nm.

To determine a model of cellular oxidative stress, after IPEC-J2 cells were adhere for 24 h, different concentrations (0.1, 0.5, 1, 2 or 4 mM) of H₂O₂ were added to the wells, then incubated for 1 h. The incubation time was selected according to the previous reports (Cai et al., 2013; Paszti-Gere et al., 2012). The optimal H₂O₂ concentration for decreasing cell viability was obtained for the following research. For the optimal SBP concentration, after IPEC-J2 cells reached confluency, different concentrations (0, 25, 50, 100, 200, 400, 800, 1,000, 2000 μg/mL) of SBP were added and went on incubating for 12 h, then incubated in H₂O₂ for 1 h. H₂O₂ was completely removed by washing with PBS twice. The viability of cell viability was measured with CCK8 to obtain the optimal SBP concentration for alleviating cell damage induced by H₂O₂.

The cell experiment was divided into 4 groups: (1) Control group: Incubated the cells with HGDMEM for 12 h, replaced the medium with fresh HGDMEM once, and continued to incubate for 1 h; (2) SBP group: Incubated the cells with HGDMEM supplemented with 100 μg /mL SBP for 12 h, replaced the above solution with fresh HGDMEM containing 100 μg /mL SBP, and continued to incubate for 1 h; (3) H₂O₂ treatment group: Incubated the cells with HGDMEM containing 1 mM H₂O₂ for 1 h; (4) SBP + H₂O₂ group: Incubated the cells with HGDMEM supplemented with 100 μg /mL SBP for 12 h, followed by 1 mM H₂O₂ treatment for 1 h.

After four treatments (Control, H2O2, SBP, SBP + H2O2), Total cellular RNA was completely extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the instructions. The cDNA in a 10 μL reaction system were synthesized by using PrimeScript RT reagent Kit (TakaRa, Dalian, China). Real-time PCR in a 10 μL reaction system were performed by using the SYBR Premix Ex Taq (TakaRa, Dalian, China), and the cycle conditions of each step were 95°C for 300 s, followed by 38 cycles of 95°C for 20 s, 60°C for 30 s and 72°C for 30 s. The primers used in this research are all listed in Table 1 (Xun et al., 2021). To normalize the levels of gene expression, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as a reference gene. The 2^-ΔΔCT method (Livak and Schmittgen, 2001) was utilized to analyze RT-PCR data and determine the relative fold changes of the target genes.

After four groups of cell treatments, total proteins from treated cells were extracted with RIPA Lysis buffer (Service biotechnology, Wuhan, China), and the extracted protein concentrations were accurately measured by BCA protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), equal amounts of proteins was isolated, then electro-transferred onto the methanol-activated polyvinylidene ifluoride (PVDF) membranes. The transferred membrane was placed in an incubation tank with TBST, quickly swabbed once, then added with 5% milk, closed in a decolourising shaker for 2 h at room temperature, and incubated in a shaker overnight at 4°C with the prepared primary antibodies. After being washed for three times with TBST, the treated membranes were incubated with the diluted secondary antibodies for 2 h. Enhanced chemiluminescence was used to observe the protein bands, and ImageJ (National Institutes of Health, Maryland, USA) software was used for analysis. All sample protein expressions were normalized using β-actin.

Antioxidant parameters including SOD, GSH-Px and CAT were analysed by xanthine oxidase-xanthine reaction, reduced glutathione and ammonium molybdate method, respectively. The determination of these antioxidants was carried out according to the instructions of the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

GraphPad Prism 7 (GraphPad Software, La Jolla, USA) was used to produce each graph. Statistical analysis was assessed using SPSS 20.0 one-way analysis of variance (ANOVA) (Sishu Software, Shanghai Co., Ltd. Shanghai, China). The replicates in each group were least 3 times. The Duncan test was used to perform multiple comparisons. The means ± standard deviations of the results were displayed, and differences were considered statistical significance at p < 0.05.

About 2,314 kinds of peptides were obtained in this study. The analysis of molecular weights for SBP showed that 500–1,500, 1,500–3,000, 3,000–5,000 and 5,000–10,000 Da accounted for 57.13, 38.63, 3.98 and 0.26%, respectively (Figure 1). It indicated that the molecular weights of 95.76% SBP were smaller than 3 kDa. About 15 identified soybean peptides and their potential bioactivity were listed in Table 2. By using the BIOPEP database and the known biological activity, the sequences or structures in the listed polypeptides can be identified as having a variety of activities, such as angiotensin-I-converting enzyme (ACE) inhibitory, antioxidant, and dipeptidyl peptidase (DPP) IV inhibitory activities.

Figure 2 revealed that the survivability of IPEC-J2 cells was significantly reduced after 1 h incubation when H₂O₂ concentration exceeded 1 mM (p < 0.05), therefore, 1 mM H₂O₂ was selected for the further research. Incubation time was selected as 1 h based to the previous report (Cai et al., 2013; Paszti-Gere et al., 2012).

Figure 3 showed that the cell viability had insignificant influence when SBP concentration was lower than 200 μg/mL during 12 h treatment (p > 0.05); however, it had insignificant influence when SBP concentration was lower than 80 μg/mL during 24 h treatment (p > 0.05), compared with the control group.

Under H₂O₂-induced condition, 100–800 μg/mL SBP could significantly alleviate H₂O₂-induced cell damages after 12 h or 24 h treatment (p < 0.05), compared with the single H₂O₂-induced group (Figure 4). Therefore, 100 μg/mL SBP was recommended for alleviating H₂O₂-induced cell damages in the further experiment.

Figure 5 showed that single H₂O₂ addition in IPEC-J2 cell incubation significantly reduced mRNA abundance as well as protein expression of ZO-1 (p < 0.05), while SBP addition significantly increased ZO-1 mRNA abundance. After the addition of SBP, Claudin-1 protein expression was significantly increased, and Claudin-1 and Occludin mRNA abundance were also increased, compared with single H₂O₂ group. It could be concluded that SBP pre-incubation increased intestinal compactness to safeguard intestinal cells from oxidative stress.

Figure 6 showed that H₂O₂ increased the mRNA abundance of Bax and cysteinyl aspartate-specific protease 3 (Caspase-3) (p < 0.05); instead, after SBP pre-incubation, they were down-regulated significantly (p < 0.05). In addition, H₂O₂ or SBP treatment had insignificant effect on B-cell lymphoma-2 (Bcl-2) mRNA abundance.

Figure 7 indicated that H₂O₂ significantly reduced the gene expressions of SOD1 (0.50 fold), CAT (0.78 fold), Nrf2 (0.78 fold) and HO-1 (0.74 fold) (p < 0.05), compared to the control group; however, SBP early addition before H₂O₂–induced cell incubation was able to upregulate the relative mRNA abundances of SOD1 (0.85 fold), CAT (1.15 fold), glutathione peroxidase1 (GPX1, 1.54 fold), Nrf2 (1.17 fold), HO-1 (1.05 fold) and quinone oxidoreductase1 (NQO1, 1.44 fold)(p < 0.05). Figure 7 also showed that H₂O₂ significantly declined the protein expressions of Nrf2, SOD1 and HO-1 (p < 0.05), however, SBP early addition during H₂O₂–induced cell incubation was able to increase the protein expressions of Nrf2, SOD1 and HO-1 (p < 0.05). Compared with the control group, the single SBP addition could upregulate the mRNA abundances of CAT (1.61 fold), GPX1 (1.45 fold), Nrf2 (1.21 fold) and NQO1 (1.92 fold) as well as protein expressions of Nrf2 and HO-1 (p < 0.05). Therefore, SBP was able to alleviate oxidative damage for H₂O₂-induced IPEC-J2 cells.

Table 3 showed that the enzyme vitality value of CAT, GSH-Px and SOD were significantly declined with the addition of H₂O₂ (p < 0.05); but SBP could significantly increase their enzyme activity (p < 0.05), indicating that SBP could increase antioxidant enzyme activity in H₂O₂-induced IPEC-J2 cells to alleviate H₂O₂-induced oxidative stress.