Microorganisms and basal substrate
B. velezensis 157 had been isolated from Eucommia ulmoides bark and was shown to exhibit various lignocellulolytic activities suited to SSF conversion of agro-industrial waste, which was collected in China Center for Type Culture Collection (M2017475). L. plantarum BLCC2-0015 (CTCC number M2015126) was obtained from Baolai-leelai Bio-tech Co., Ltd. (Taian, China) and is a widely accepted feed additive (originally isolated from pickles) that is used in mixed corn-soybean meal feed. In addition, Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were used as indicator organisms provided by the Institute of Animal Nutrition and Feed, Jilin Academy of Agricultural Sciences, China. Dried and corticate soybean meal (purchased from a local market in Gongzhuling, China) was sieved through a 40-mesh sieve prior to SSF.
Protein degradation capacity
The protein degradation capacity of B. velezensis 157 and L. plantarum BLCC2-0015 were measured using the agar well diffusion method. The crude supernatant of candidate strains were prepared according to the method(Wongputtisin et al. 2012a). Subsequently, the supernatants were determined using a soybean antigenic protein screening plate described by Liu. et al(Liu et al. 2007).
Preparation of fermented soybean meal
Two-stage fermentation of feed was performed using SBM as substrate, which was sterilized at 121 °C for 20 min. Prior to fermentation, B. velezensis 157 was cultured for 12 h in liquid LB medium at 37 °C. L. plantarum BLCC2-0015 was cultured for 16 h in liquid MRS medium, at 37 °C. The vegetative cells were resuspended in sterile 0.85% NaCl (108 CFU/mL) after washed thrice with sterile 0.85% NaCl.
The sterilized SBM (150 g) was conducted in a 500 mL Erlenmeyer flask with a sterile membrane, then sterile water and bacterial suspension in PBS to achieve a final moisture content of 40% in dry basis. During the first stage of fermentation, SBM was inoculated with B. velezensis 157 (8.0 log CFU/g) then fermentation was allowed to proceed for 24 h at 37 °C. For the second stage of fermentation, Erlenmeyer flask was sealed with a sterile rubber plug, the fermented mixture was inoculated with 8.0 log CFU/g of L. plantarum BLCC2-0015 then incubated for 48 h under anaerobic conditions at 37 °C. The addition of sterile 0.85% NaCl instead of inoculated bacteria were served as controls. All control and inoculated samples were tested in triplicate. 2 g of moist samples at 0 h, 24 h, 48 h and 72 h were collected for immediate determining for pH, microbial, enzyme activity, antimicrobial activity and lactic acid analyses, and the remaining samples were prevented continuous fermentation at 105 °C for 30 min(Shi et al. 2017a). Afterwards, all samples were dried for 24 h at 65 °C, cooled, ground, and subjected to SDS-PAGE, and physicochemical analysis.
Microorganisms and metabolites
The pH and microbiological counts were analyzed as described by Wang et. al(Jin et al. 2017). A lactic acid enzymology assay kit (Nanjing Jiancheng Technology Co., Ltd.) was used to determine lactic acid content using the manufacturer's protocol provided with the kit. Cellulase, xylanase, pectinase and β-mannase activities were analyzed by DNS method(Blibech et al. 2020; Parab et al. 2017; Salim et al. 2017). The activity of neutral protease was detected as mentioned by (Salim et al. 2017). Antimicrobial activity of FSBM was analyzed using an agar-well diffusion assay. The Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 were used as indicator organisms for antimicrobial test(Su et al. 2018). Moist samples after two-stage fermentation were diluted in 0.85% NaCl and transferred into a well in the MH agar containing corresponding pathogens at 37°C for 24 h. The control discs were impregnated with supernatant of uninoculated SBM.
Chemical analysis
Dried samples at 0 h, 24 h, 48 h, and 72 h were analyzed according to AOAC International Guidelines (2005) to determine contents of dry matter (DM), crude fibre (CF), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF), total phosphorus (P), Calcium (Ca), ash. The method reported by Ovissipour et al. was used to determine sample TCA-SP content(Ovissipour et al. 2013). An automated amino acid analyzer (LA8080; Hitachi, Tokyo, Japan) was used to determine amino acid profiles. Before analysis, the dried samples were hydrolyzed with 6 mol/L HCl at 110 °C for 24 h. Analyses of glycinin and β-conglycinin contents in uninoculated SBM and FSBM were conducted using an indirect competitive enzyme-linked immunosorbent assay (ELISA) kit as per kit instructions (Long zhou fang ke Bio Co., Ltd.).
SDS-PAGE
A Plant Protein Extraction Kit (Beijing Solarbio Science and Technology Co., Ltd.) was used to extract soluble proteins from uninoculated SBM and FSBM according to the protocol provided. Next, sample protein concentrations were determined using a Bio-Rad Protein Assay Kit (Bio-Rad, USA) and an SDS-PAGE system utilizing 12% polyacrylamide separating gels for 120 min at 65 mV to fractionate soluble proteins, as reported by (Shi et al. 2017a). Post-electrophoresis, gels were stained for 45 min with Coomassie Brilliant Blue R-250 (Bio-Rad, USA) then destained in 7% acetic acid.
Microscopic inspection
Physical property changes of samples were examined by SEM according to the protocol of the Electronic Microscopy Center of Xi'an Lianyi Sharing Information Technology Co. ltd. The microstructures of uninoculated SBM and FSBM were observed using a field-emission SEM (JSM-7900, JEOL, Japan) at ×1000, ×1,500, ×3,000 and ×5,000 magnifications.
Statistical analysis
Data were processed and evaluated by Student’s t tests and one-way analysis of variance (ANOVA) with Duncan’s multiple-range test using SPSS software (SPSS Inc., Chicago, IL, USA). A P-value ˂0.05 indicated a significant difference between groups, with each result expressed as mean ± standard deviation. Histograms and line graphs were drawn using GraphPad Prism 8.0 software.