Luria Bertani (LB), Bacto Brain Heart Infusion (BHI), and DeMan, Rogosa and Sharpe (MRS) agar plates were used to isolate bacteria colonies. LB agar (pH 7.5) were prepared with 1.0 g of tryptone powder (Bio Basics Canada INC, Canada), 0.5 g of yeast extract (Bio Basics Canada INC, Canada), 1.0 g of NaCl (Fisher Scientific, USA) and 1.5 g of BactoAgar in 100 mL deionized water. BHI (Becton, Dickinson & Company, USA) and MRS (Oxoid, England) agar plates were prepared according to manufacturer’s procedures with the addition of 1% (w/v) BactoAgar.

Soy milk, carboxymethyl cellulose (CMC), starch agar plates were used to select colonies with proteolytic, cellulolytic and amylase activities, respectively. Soy milk agar was made by mixing 1 g of soy milk powder (Bulk Barn, Canada) and 2.5 g of BactoAgar (Becton, Dickinson & Company, USA) with 100 mL of water as described by Amoa-Awua et al. [31]. CMC agar plates were prepared by adding 1.0 g of CMC (Acros Organics, USA) and 1.5 g of BactoAgar to 100 mL of deionized water as previously described by Sazci et al. [32]. To prepare starch agar plates, 1 g of corn starch (Bulk Barn, Canada) and 1.5 g of BactoAgar were added to 100 mL of deionized. All plates were autoclaved for 25 min at 121 °C.

Five types of fermented soy products, including black fermented beans, red fermented beans, yellow fermented beans, soft fermented beans and Japanese Natto, were obtained from the local supermarket and used to isolate proteolytic bacteria. Approximately 1 cm3 of each source of fermented products was mixed with 500 μL of sterile water and serial dilutions were conducted to isolate approximately 100 clearly defined individual colonies of bacteria per plate. A hundred microliter of diluted samples from each of the five sources was plated onto the soymilk agar and incubated for 24 h in 37 °C. For each food source, colonies demonstrating the largest clear zone were selected and glycerol stocks were created for storage of isolates at − 80 °C. A total of six isolates were subjected to further screening for cellulase and amylase activity.

Contents from the proximal, middle and distal section of grass carp intestine were harvested to isolate bacteria with cellulolytic activity [30]. Diluted samples were first plated onto LB, BHI and MRS agar. Any colony present on the three plates was blotted onto the soymilk and CMC plates and incubated at 37 °C for 72 h. The clearance zone in the CMC agar plates was revealed by Congo Red staining as described [32, 33]. Colonies which were able to grow on the soymilk agar and exhibited the highest cellulase activity were selected and a total of three colonies were selected and further screened for enzymatic activities.

A single colony of each selected isolate was inoculated into 1 mL of BHI culture media and incubated for 18 h at 37 °C to extract crude enzymes [34]. The supernatant was harvested by centrifuging the liquid culture at 10,000 r/min for 15 min at 4 °C using an Eppendorf 5415D centrifuge. Twenty microliters of each crude enzyme extract were pipetted into the punctured hole on the soy milk, CMC and starch agar plates, respectively, and incubated at 37 °C for 72 h. Enzymatic activities were ranked by the clearance zone measurements taken from the edge of the colony (soy milk agar plate) or the edge of punctured hole (CMC and starch agar plates) to the outer edge of the clearance zone. The amylase clearance zone was revealed using an additional Gram’s Iodine Stain [31]. Each experiment was repeated three times using fresh inoculums each time. Commercial B. subtilis ATCC 6633 with protease [35], amylase [36] and cellulase activities [37] was used as the positive control and the blank culture media was used as the negative control.

All selected bacteria selected were characterized to the species or genus level by matrix assisted laser desorption ionization- time of flight mass spectrometry (MALDI-TOF MS). Isolates were individually streaked onto the BHI agar plate, and incubated for 18 h at 37 °C. Each fresh cultured bacterial colony was spotted onto the target plate (Bruker), air dried, and overlaid with 1 μL matrix solution mixed with an organic solvent solution made of 50% acetonitrile (Sigma Aldrich, Canada) and 2.5% trifluoroacetic acid (VWR, Canada). The target plate was then placed onto the MALDI-TOF MS instrument (Bruker) for analysis. Peptide mass fingerprint spectra was scored using MALDI-TOF MS software and database (MALDI Biotyper 3.0, Bruker). An isolate was automatically identified to the species level if the score was 2.0 to 3.0, or to genus level if the score was 1.7 to 1.999. No reliable identification was generated if the score was less than 1.7.

The 16S rRNA and gyrB genes were used to further identify the strain of bacteria isolates. Genomic DNA of each isolate was extracted using the PureLink Genomic DNA Mini Kit (Invitrogen, USA) according to manufacturers’ instructions. The partial 16S rRNA gene was amplified and sequenced using the same primer set of BSF8/20 and BSR534/18 [38] and the gyrB gene was amplified by the universal primer set of UP-1 and UP-2r and sequenced by the primer set of UP1S and UP2SR (Table 1) [39]. Before DNA sequencing using ABI Prism 3100 Automated Sequencer, the size of each PCR product was verified by agarose gel electrophoresis and the concentration of the PCR products was determined using a BioPhotometer Plus spectrometer (Eppendorf, USA) to ensure that the minimum concentration required. Bacterial strains were identified by blasting the sequencing results from both genes against nucleotide sequences in the GeneBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Soybean meal (48% crude protein content, Grand Valley Fortifiers, Canada) was sterilized by mild heating at 125 °C for 30 min to minimize lysine loss [40]. For each fermentation, bacteria inoculums of selected isolated were prepared fresh by incubating a single colony in the BHI culture media for 18 h at 37 °C to obtain a culture of 108 colonies forming unit (CFU)/mL. Solid state fermentation was set up by inoculating 2 × 108 CFU/mL isolated bacteria into the 2 g of soybean meal for each of the treatment group with a volume: weight ratio of 2:1. This mixture was set to ferment in an incubator at 42 °C for 48 h without any agitation. Each fermentation was performed in triplicate. Samples were collected at 24 and 48 h and stored at − 80 °C. Lyophilized samples were then ground into a fine powder for further analyses.

Total soluble proteins of the unfermented and fermented soybean meals were isolated as previously described by Hong et al. [20]. The quantification was carried out using the DC Assay Kit (Bio-Rad Laboratories, USA). Fractions of soluble protein extract were analyzed by 11% tris sodium–dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A volume of 15 μL of denatured total soluble protein sample was loaded to the gel, and subjected to electrophoresis for 100 min using 100 V. The gel was stained with Coomassie blue. Images were taken using ChemiDoc XRS+ (Bio-Rad Laboratories, USA) and analyzed with the Image Lab software.

To determine piglets’ immunoreactivity against an unfermented and fermented soybean meal, Western blots were performed. Total soluble protein of 50 μg each was loaded into an 11% SDS-PAGE as described above and then transferred to an Immobolin-P (Millipore, Billerica, USA) polyvinyl difluoride transfer membrane using the wet transfer method at 100 V for 100 min. The membrane was first blocked with buffer comprised of 5% skim milk, 0.1% Tween 20, and 1× TBS for 1 h before the primary antibody incubation. After several washes with washing buffer (0.1% Tween and TBS), the membrane was incubated with the primary antibody at 4 °C overnight. The primary antibody was prepared against soybean antigenic proteins glycinin and β-conglycinin following a published protocol [41]. Briefly, plasma was collected from 8-week-old piglets that were previously exposed to feed containing soybean meal for 21 d and developed an immune response to soybean allergens. The primary antibody was comprised of 20 μL of pooled pig serum and diluted to 1:500 with the blocking buffer. Rabbit anti-pig horseradish peroxidase-linked IgE was used as the secondary antibody (1: 100,000; Abcam). Proteins were detected by using Clarity Western Enhanced Chemiluminescence substrate (Bio-Rad Laboratories, USA) according to manufacturer’s instructions.

Lyophilized samples of unfermented and fermented soybean meal were subjected to the measurement of crude protein and amino acid profiles according to the corresponding AOAC official methods (moisture, method 934.01; crude protein, method 954.01; amino acids, method 994.12). Raffinose and stachyose were determined using high-performance liquid chromatography (HPLC, Shimadzu LC-15C, Japan) equipped with a differential detector (Shimadzu RID-10A, Japan) as described by Yao et al. [42]. Briefly, stachyose and raffinose were extracted by 70% (v/v) ethanol with the assistance of microwave. The supernatant was concentrated and reconstituted to 25 mL. A volume of 20 μL of each sample was injected into the HPLC system with 70% acetonitrile as the mobile phase. The temperature was set at 35 °C, and the flow rate was controlled at 1 mL/min. Serial diluted standards of stachyose (Dr. Ehrenstorfer, Germany) and raffinose (Dr. Ehrenstorfer, Germany) were used to determine the stachyose and raffinose concentrations.

All statistical analyses were performed using PRISM GraphPad Prism Version 5.03 (San Diego, CA). The data for bacteria screening were analyzed using one-way ANOVA with a Dunnet post-test to determine if any treatment group was significantly different from the positive control. Comparisons in the proximate analysis, amino acid profile and oligosaccharide concentrations of the unfermented and fermented samples were performed using one-way ANOVA followed by a Tukey post-test; observations were made in triplicate with one observation obtained daily. The assumption of no day effect was believed to be true. Data was expressed as mean ± SEM and considered to be significant if P