Preparation of rumen fluid inoculum and culture medium

Fresh rumen fluid was collected from sheep at Xuntian Husbandry Co., Ltd, Hebei, China. The rumen fluid was directly aspirated from thirty-five rumen-cannulated Dorper × Hu hybrid sheep, with an average age of 4.5 months and an average body weight of 38.32 ± 0.94 kg. The sheep were fed the base total mixed ration, based on rice straw, corn and cotton meal throughout the entire study period. For each round of strain screening, approximately 250 mL of rumen fluid from each of 3 sheep before the morning feeding, underwent filtration using 4 layers of sterile gauze. The filtered fluid was then mixed, pooled, and sealed within a preheated thermos flask. Afterward, collect the mixed rumen fluid and immediately transfer it to the laboratory, storing it at 39 °C as a backup. The culture medium was used in this study as follows (Table 1), and all the medium was autoclaved (121 °C, 15 min). It is worth noting that the medium was supplemented with FG after autoclaving.

Table 1 Preparation of medium componentsIsolation and purification of bacteria

All subsequent experimental steps were carried out in an anaerobic environment by YQX-II anaerobic incubator (Shanghai Haixiang Instrument Equipment Factory., Shanghai, China). The 5 mL rumen fluid sample was decimally diluted (10− 1, 10− 2) with the 39 °C saline solution, then the dilution solution was streaked with a sterilized wire loop onto the S-Screening medium plates [16] for 72 h in the AY6907 anaerobic jar (GeneScience, Wilmington, USA). The bacteria in a single colony arbitrarily selected from colonies on the S-Screening medium (solid medium with FG as the sole carbon source, shown in Table 1) plate were inoculated into L-LB medium (liquid medium based on standard LB broth) and cultured for 24 h. Subsequently, the enrichment cultures were decimally diluted (10− 2) with the 39 °C saline solution, and streaked onto the S-Screening medium plate for 72 h again. The process was reiterated until a consistent isolation of the purified single colony morphology was achieved, identifying it as the target strain [17]. The pure isolates were stored at -80 °C in 50% glycerol containing Man-Rogosa-Sharpe (MRS) medium until use.

Characterization and identification of the isolated bacteria

As the initial characterization of isolated bacteria, a small portion of the culture suspension was spotted on a sterile glass slide and stained by a bacterial Gram-staining kit, according to the manufacturer′s instruction [18]. The DNA was extracted using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech Co., Ltd, Shanghai, China). The DNA concentration and purity were checked by a Nanodrop 2000 (Thermo Scientific, Wilmington, USA), and then used as a template for polymerase chain reaction (PCR) amplification. The universal primer sequences were 27 F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492R: 5′-ACGGTTACCTTGTTACGACTT-3′, and the PCR reaction was conducted using the following conditions: The reaction procedure was pre-denaturation at 94 °C for 5 min; 94 °C denaturation for 30 s; annealing at 55 °C for 30 s; and 72 °C extension for 90 s. This was performed for 35 cycles. Finally, amplification was completed by incubation for 5 min at 72 °C [19]. The amplified products were sent to Sangon Biotech Co., Ltd, Shanghai, China. The nucleotide sequences were analyzed for sequence identity by BLAST in the GenBank of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). To obtain the GenBank accession numbers, the 16 S rRNA sequences of strains were uploaded into NCBI databases. The neighbor-joining method (1000 bootstrap value) of MEGA 6.0 software was used to construct a phylogenetic tree to visualize the phylogenetic relationships [20].

Determination of the growth curve of the target strain

Activated culture: the stored strain in glycerol at -80 °C was thawed and cultivated in S-MRS medium (solid medium based on standard MRS broth), followed by subculturing twice in L-MRS medium (liquid medium based on standard MRS broth). The 2 mL of activated culture was inoculated onto 200 mL L-MRS medium and cultured at 39 °C for 24 h (12 incubation time × 3 replicates). Every 2 h, the colony forming units in CFU/ml (determined after incubation by plating 100 µL bacteria solution onto S-MRS plates at 39 °C for 24 h), the OD600 value (determined by the UV-2000 model UV/visible spectrophotometer (UNIAC Instrument Co., Ltd., Shanghai, China)), and pH values (measured by the PHS-2 F model pH meter (OUSTOR Industrial Co., Ltd., Shanghai, China)) were obtained. Plate colony counts and plate photographs were taken using the Interscience Scan 4000 (France Interscience Co., Ltd., Cantal, France).

Detoxification for free gossypol and solid-state fermentation of cottonseed meal

The detoxification experiments were conducted with different free gossypol levels and carbon sources. Activated culture, l mL saline solution and 1 mL culture suspensions (around to 108 CFU/mL) were inoculated onto the 100 mL following fresh medium labeled, respectively: CONL (saline solution + L-MRS medium (2% (w/v) glucose) with 0.01% (w/v) free gossypol), GFFG (culture suspensions + L-MRS medium with glucose-free but 0.01% (w/v) free gossypol), HGFG (culture suspensions + L-MRS medium with half glucose (1% (w/v) glucose) and 0.01% (w/v) free gossypol), TGFG (culture suspensions + L-MRS medium (2% (w/v) glucose) with 0.01% (w/v) free gossypol), and incubated for 24 h at 39 °C. The bacterial solution samples were taken at 0, 6, 12, and 24 h to obtain the degradation efficiency of FG (DFG) and OD600 value, and calculated from the formula as follows:

Where CT is the FG concentration in the medium at time t = 6, 12, 24 h, and C0 is the FG concentration in the medium at time t = 0. The FG concentration was quantified by High Performance Liquid Chromatography (HPLC) with a Wufeng analytical instrument (Wufeng Co., Ltd., Shanghai, China) as the methods of Wang et al. [21].

To practically evaluate the degradation ability of FG in cottonseed meal (CSM) and fermentation performance of CSM for Lact. mucosae LLK-XR1, the experiment of solid-state fermentation from CSM was organized. The procured substrates of CSM were not ground to powder, it was performed with the ratio of material to water of 1:1 and the substrates were inoculated with 8% culture suspensions (around 108 CFU/mL). The trials were grouped into 4 blocks of 3 replicates, each labeled as CON (did not any treatment); SAF (saline solution + autoclaved substrates); CAF (culture suspensions + autoclaved substrates); CUF (culture suspensions + unautoclaved substrates). Then they were incubated for 5 d at 39 °C. After freeze-drying and crushing, the substrate samples were taken at D 3 and D 5 to obtain DFG.

Carbohydrate metabolism

Meanwhile, to clarify any uncertain enzymatic activity found in the selected potential probiotic Lact. mucosae LLK-XR 1, the API 50 CHL kit (BioMerieux, Marcy I’ Étoile, France) was used in this study, according to the manufacturer’s instructions [22]. This method uses the color change characteristics of the bromocresol violet indicator when exposed to acid and evaluates the metabolic ability of specific substrates.

Assessment of temperature tolerance

Activated culture, 10 mL activated cultures (108 CFU/mL) was centrifuged to collect stationary-phase cells (10,000 × g for 10 min at 4 °C), washed twice using sterile PBS (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and resuspended in PBS to initial volume. The bacteria suspension in 15 mL tubes was incubated for 120 min at 15, 25, 55, 39, and 65 °C. Suspension samples were collected at 0 and 120 min, followed by serial dilutions were prepared and plated onto S-MRS plates to obtain colony forming units. Tests were performed independently in triplicates to determine the Resistance index (RI), and calculated from the formula as follows [23].

$$\% RI = (\log }CFU}at}time = t/\log }CFU}at}time = o)*100$$

Assessment of low pH and bile tolerance

Activated culture, 1 mL activated cultures were incubated in 15 mL tubes of 9 mL modified L-MRS for 120 min at 39 °C according to [24]. The modified L-MRS medium was adjusted to pH 2.0, 3.0, 4.0, 5.0, 6.0, or supplemented with 0.2%, 0.3%, 0.4%, 0.5%, 0.6% (w/v) bovine bile salt (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the L-MRS medium at pH 6.7 or without bovine bile salt was served as a control. The steps to collect samples (only at 0 and 120 min) and obtain colony forming units, and calculated from the formula as follows:

$$\% RI = (\log }CFU}at}time = t/\log }CFU}at}time = o)*100$$

Assessment of antibiotic resistance

Antibiotic resistance of Lact. mucosae LLK-XR1 to the used antibiotics was evaluated by the Kirby-Bauer disc diffusion method. The following antibiotic discs (BKMAM Biotechnology Co., Ltd, Hunan, China) included: penicillin (10 µg), ampicillin (10 µg), ceftriaxone (30 µg), tetracycline (30 µg), erythromycin (15 µg), ciprofloxacin (5 µg), lincomycin (2 µg), compound sulfamethoxa (25 µg), chloramphenicol (30 µg), gentamycin (10 µg), amikacin (30 µg). Activated culture, the activated cultures with 0.5 McFarland turbid were homogeneously swabbed into S-MRS plates, followed by each of three antibiotic discs being dispensed on a plate. After anaerobic incubation for 48 h at 39 °C, inhibitory zones were recorded using by Interscience Scan 4000, and results were reflected as sensitive or resistant based on CSLI 2012 guidelines [25].

Assessment of antimicrobial activities

The most common pathogenic microbes used in this study, including Escherichia coli CICC 10,899, Staph. aureus CICC 21,600, and Salmonella. typhimurium CICC 21,483, were provided by the China Center of Industrial Culture Collection. Antipathogenic activities of cell-free supernatants of Lact. mucosae LLK-XR1 were assessed carried out as described by Jabbari et al. [26]. Activated culture, the activated cultures (108 CFU/mL) were centrifuged to collect the supernatant (10,000 × g for 10 min at 4 °C), then sterilized to remove stationary-phase cells using 0.22 μm cellulose acetate membrane filter (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Suspension of pathogenic bacteria cultured in L-LB was swabbed into S-LB medium (Solid medium based on standard LB broth), then plates (3 pathogenic bacteria × 3 replicates) were injected with 80 µL of cell-free supernatants into an 8 mm well, while the equal volume of sterile saline plate was served as the control. Following incubation for 24 h at 37 °C, plates were observed for the inhibitory zone. Referring to the description of a previous study, the inhibitory zone diameter is 7–9 mm indicated weak antimicrobial activities, 10–13 mm indicated intermediate antimicrobial activities, 14–17 mm indicated strong antimicrobial activities, and > 17 mm indicated very strong antimicrobial activities [26].

Assessment of cell surface properties

Assessment of cell surface properties was performed according to the procedure described by Muñoz-Provencio et al. [27]. Activated culture, 10 mL activated cultures (108 CFU/mL) were centrifuged (10,000 × g for 10 min at 4 °C), washed twice using sterile PBS (Phosphate buffer saline) and resuspended in PBS to initial volume as the final absorbance (A0) at 600 nm. Whereafter, 3 mL bacteria suspension was mixed with 1 ml of organic solvents: hexadecane, chloroform and ethyl acetate, respectively. The mixture was thoroughly vortexed for 1 min and incubation at 39 °C for 30 min, then absorbance (A1) at 600 nm of the aqueous layer was determined. E. coli CICC 10,899 and Salm. Typhimurium CICC 21,483 were used as the control. The percentage of affinity (% PA) was calculated from the formula as follows:

Assessment of auto-aggregation ability

Assessment of auto-aggregation was performed as described by Kos et al. [28]. Activated culture, the pre-treatment was conducted for obtaining bacterial suspension consistent with the previous description. The bacterial suspension was thoroughly vortexed for 1 min, and recorded the absorbance (A0) at 600 nm. Follow by incubation at 39 °C for 24 h, suspension samples were taken at different times (at 2, 6, 12, 24 h) to determine the absorbance (At). Percentage of auto-aggregation (% AA) was calculated from the formula as:

Assessment of hemolytic and catalase activity

Activated culture, the cultures were spotted onto blood agar plates (BKMAM Biotechnology Co., Ltd, Hunan, China) containing 5% (v/v) sterile defibrinated sheep blood agar, followed by incubation at 39 °C for 48 h. According to Cui et al. [29], the presence of greenish halos around the colonies indicates α-hemolytic, a clear halo indicates β-hemolytic, and no halo indicates γ-hemolytic (non-hemolytic). Staph. aureus CICC 21,600 was used as positive control for β-hemolytic.

The production of intracellular enzyme catalase was also crudely determined [22]. Activated culture, the activated cultures (around to 108 CFU/mL) were spotted on S-MRS plates. Followed by incubation at 39 °C for 24 h, the bacterial colony plates were flooded with 3 mL of 10% (v/v) hydrogen peroxide solution. The presence of gas bubbles on the colonies indicates a positive result.

Statistical analysis

All the experiments were carried out in triplicates, the experiment data are presented as mean ± standard deviation (SD). Statistical analyses were completed using the general linear model procedure of SAS [30]. The data, including the degradation efficiency of FG, OD 600 of LLK-XR1 under various carbon sources, and microbial adhesion, underwent one-way analysis of variance (ANOVA). The significant differences were analyzed by Duncan’s multiple comparison analysis. The replicate test served as the experimental unit. * P value < 0.05 and ** P value < 0.01 was considered statistically significant. The growth curve of Lact. mucosae LLK-XR1 are performed with Origin version 2021 (OriginLab, Massachusetts USA), and other data are performed with ChiPlot software (https://www.chiplot.online).