Feces were collected from a pig farm located in south Tibetan and were moved back to the lab as soon as possible. Then mixed with 70% glycerol as a 1:1 ratio to be fecal samples and stored at −80°C. When it is needed, 1 g of fecal samples was mixed with normal saline via vortex oscillations to get suspensions. Suspensions were diluted in a 10-fold gradient and the serial dilutions were streaked onto selective medium, anaerobic incubated (Anaerobic Incubator LAI-D2, Longyue, Shanghai, China) at 37°C till a single colony of appropriate size was grown. Picked single colony was inoculated on de Man, Rogosa, and Sharpe (MRS) solid medium plates in anaerobic conditions at 37°C to purify them and repeated this process three times for further purification. Purified strains were anaerobically incubated in MRS broth for 18 h. Then mixed bacterial suspension with 50% glycerol as 1:1 ratio and cryopreserved at −80°C for further identification.

The colonies were observed by size, shape, color, margins, and pellucidity. The isolates were done the Gram staining and were observed under an oil microscope (1,000 ×) to identify morphology for further classifying the strains. The utilization of starch by strains was detected by culturing in starch medium, and the starch in the medium was detected by iodine solution. Total DNA of purified strains was extracted using bacteria genomic DNA kit (CWBIO, Beijing, China). Molecular identification was done by PCR amplification using 27F and 1492R universal primers (Weisburg WG, 1991) with 1,500-bp product size. The forward primer is 5′-AGAGTTTGATCCTGGCTCAG-3′ and the reverse primer is 5′-GGTTACCTTGTTACGACTT-3′. PCR reactions were conducted in 50 μl reactions that include 1 μl of template DNA, 25 μl of 2.5 mM deoxynucleotide triphosphates (dNTPs), 22 μl of deionized water, 1 μl of 10 μM primer for three replications. For initial denaturation at 94°C for 4 min, then at 94°C for 45 s by 30 cycles, 55°C for 45 s, and 72°C for 1 min and an extension at 72°C for 10 min. The product of PCR was sequenced and blasted with the National Center for Biotechnology Information (NCBI) database. High homological Lactobacillus sequences of different species and different strains were selected to contrast with the sequences of the isolates to the construction of phylogenetic tree via Mega 7.0 software. The identified isolates were named L. amylovorus SLZX20-1 and kept in China General Microbiological Culture Collection Center (strain No. 20122).

Streaked and inoculated L. amylovorus SLZX20-1 onto a plate to activate the strain. In total, 1% of activated strains was anaerobically incubated in MRS broth at 37°C and tested OD600 nm value, pH, and counted viable bacteria numbers every 2 h. The L. amylovorus suspensions were diluted in a 10-fold gradient and streaked onto MRS medium, anaerobic incubated at 37°C to count viable bacteria numbers in plates, which colony numbers were between 30 and 300.

Tolerance to low pH and bile salts were assessed that 1% of activated strains was incubated with different pH (0, 2.5, 3.0, and 4.0) of sterile phosphate buffered saline (PBS) or with sterile PBS (Gibco, Brooklyn, NY, USA) containing different bile salts (Solarbio, Beijing, China) levels (0, 0.1, 0.2, and 0.3%). The mixture was incubated at 37°C for 0, 1, 2, 3, and 4 h to count viable bacteria numbers via the flat colony counting method. Taking the viable bacteria number of 0 h as the control, the survival rate of L. amylovorus SLZX20-1 in different pH conditions or different bile salt levels was calculated.

Antibiotic sensitivity was determined by drug-sensitive paper tablets (Hangzhou microbial reagent Co. LTD, China). Selected 1–2 common antibiotics from each class as a representative to comprehensively reflect the drug sensitivity of L. amylovorus SLZX20-1, such as amikacin, gentamicin, cefradine, carbenicillin, penicillin, norfloxacin, tetracycline, chloramphenicol, vancomycin, and erythromycin. In total, 1% of L. amylovorus SLZX20-1 was mixed in melted MRS solid medium till it solidified and dried for 3–5 min. The tablets were homogenized, spread in the plate, and gently compacted, with a spacing between each other not less than 24 mm. The distance from tablet center to the edge of plate was not less than 15 mm which prevents the crossing between each transparent circle. Plates were incubated in a 37°C anaerobic incubator for 36 h to observe the size of antibacterial circles. Every tablet was repeated in triplicate. The circle diameter <15 mm was resistant and 16–20 mm for medium sensitivity, the circle diameter of >20 mm was sensitive.

The determination of enzymatic spectrometry was done by using API-ZYM kit (Biomérieux, France). After 14 h cultures, L. amylovorus SLZX20-1 was centrifuged at 5,000 rpm for 10 min, removed the supernatant, resuspended the strains via normal saline, and adjusted their concentrations at 1 × 10⁹ colony forming unit (CFU)/ml. In total, 65 μl of bacteria suspension was added into strip wells and co-cultured in the anaerobic incubator at 37°C for 4 h. Then added a color developer into wells and recorded the color reaction results.

The supernatant of L. amylovorus SLZX20-1 was filtered and added into solid mediums containing ethyl4-hydroxy-3-methoxycinnamate to incubate at 37°C for 24 h for qualitative analysis of the feruloyl esterase production via the Oxford Cup method. Quantitative determination of feruloyl esterase activity was done by hyphenated to liquid chromatography (HPLC) (Agilent Technologies 1200 Series, Santa Clara, CA, USA) separation systems. The enzyme activity was defined that the required enzyme amount for degradation of ethyl4-hydroxy-3-methoxycinnamate per minute to generate 1 μmol ferulic acid is 1 U as the condition of 39°C, pH 7.0. After 36 h cultures, L. amylovorus SLZX20-1 was centrifuged at 5,000 rpm for 10 min, discarded the supernatant, washed strains, resuspended it with PBS, and then was broken by ultrasonic to obtain a crude solution of feruloyl esterase. In total, 400 μl crude solution of feruloyl esterase, 100 μl 10 mmol/l ethyl4-hydroxy-3-methoxycinnamate (solute with dimethyl sulfoxide), 500 μl sodium phosphate buffer of contained 2.5% (V/V) Triton-100 (pH 7.0) were co-incubator at 39°C for 45 min, then heated in 100°C for 10 min to terminate the reaction. Detected the production amount of ferulic acid via HPLC separation systems.

Antimicrobial activity was assessed against eight pathogens associated with Escherichia coli (E. coli) O157, K88, K99, 987P, Salmonella typhimurium (S. typhimurium) SL1344, Citrobacter rodentium (C. rodentium) DBS100, Salmonella pullorum (S. pullorum) CVCC1791, and Staphylococcus aureus (S. aureus) CVCC1882 (all strains were obtained from China Veterinary Culture Collection Center). Oxford Cup test was done for the detection of antimicrobial activity. Antibiotics (200 μl 0.1 mg/ml doxycycline hydrochloride) were used as a positive control, normal saline was used as a negative control. Briefly, L. amylovorus SLZX20-1 36 h cultures were centrifuged (5,000 × g for 10 min, 4°C), the supernatant and the strains were collected separately. The supernatant (filter by 0.22 μm filters), strains (wash three times and resuspend with sterile normal saline), and suspensions (L. amylovorus SLZX20-1 36 h cultures without centrifuging) were set to be three treatment groups. A drop of 200 μl antibiotics, normal saline, supernatant, strains, or suspensions was independently added into each hole of medium and co-cultured with different pathogens in the incubator for 12 h. Then the diameter of the inhibition zone was measured. Every group was repeated in triplicate.

Collected supernatant of L. amylovorus SLZX20-1 36 h cultures via centrifugation (5,000× g for 10 min, 4°C) and filtered it. In the exploration of antimicrobial substances, supernatant without any modification (pH is 4) was set as a control, adjusted pH of supernatant to 7, added protease k (1 mg/ml, adjusted pH to 8), trypsin (1 mg/ml, adjust pH to 3), or pepsin (1 mg/ml, adjust pH to 8.2) in the supernatant, which these treatments were constituted to be trial groups. Stood protease k, trypsin, and pepsin groups at 37°C for 2 h and then adjusted their pH to 7. A drop of 200 μl supernatant from every group was independently added into each hole of medium and co-cultured with different pathogens in the incubator at 37°C for 24 h. Then measured the diameter of the inhibition zone. Furthermore, the short-chain fatty acid (SCFA) production of the supernatant was analyzed by ion chromatography after a 400-fold dilution. SCFA was measured according to our previous studies (18, 19).

Recovery and passage of IPEC-J2 cells were referred to using the same method previous study (20). Before IPEC-J2 cells were fused to 90, 1% L. amylovorus SLZX20-1 was inoculated to MRS broth medium and grown for 12–14 h. Then, the mixture was centrifuged at 4°C 5,000 rpm for 10 min. The supernatant was discarded, and the strains were washed three times with PBS and resuspended by Dulbecco's Modified Eagle-F12 Ham Medium (DMEM/F12, Gibco, NY, USA) without serum and antibiotics and adjusted concentrations at 1 × 10⁷, 1 × 10⁸, or 1 × 10⁹ CFU/ml. Adhesion rate was determined by co-culture of L. amylovorus SLZX20-1 and IPEC-J2 cells. Different concentrations of L. amylovorus SLZX20-1 were added to six-well plates with grown and without non-adherent cells. They were incubated in an atmosphere of 5% CO₂ incubator at 37°C for 2 h. Then, discarded the supernatant and washed three times with PBS to fully remove the unadhered bacteria. In total, 1 ml of 0.1% Triton-100 (Sigma, Saint Louis, MO, USA) was added to each well, standing for 15 min to fully lyse the cells, free the bacteria, and then the bacterial fluid was moved into a centrifuge tube. The adhesion rate of the L. amylovorus SLZX20-1 was calculated via counting the numbers of live bacteria before and after adhesion by the flat colony counting method. Moreover, gram staining was used to observe the adhesion ability. Specifically, the process was similar to methods mentioned above except for the following points. Firstly, the IPEC-J2 cells were climbed on the glass slide. Secondly, after washing the unadhered bacteria, the cells and adhesive bacteria were fixed via formaldehyde for 30 min. Following, gram staining was done and the cell slides were put on the glass slides. Finally, adhesion condition of the co-culture of L. amylovorus SLZX20-1 and IPEC-J2 cells were examined under a microscope.

Separately cultured L. amylovorus SLZX20-1 and Enterotoxigenic Escherichia coli (ETEC) K88, collected strains and washed three times with PBS, resuspended by DMEM/F12, and adjusted concentrations of ETEC K88 at 1 × 10⁷ and 1 × 10⁸ CFU/ml. Competition, exclusion, and replacement trials were performed to test the antibacterial activity of L. amylovorus SLZX20-1. Co-culture of L. amylovorus SLZX20-1 and ETEC K88 onto IPEC-J2 cell plate for 2 h to test their competition. L. amylovorus SLZX20-1 was added onto the IPEC-J2 cell plate and incubated in an atmosphere of 5% CO₂ incubator at 37°C for 1 h. Then the supernatant was discarded and plates were washed three times with sterile PBS, subsequently, ETEC K88 was added to co-culture for 1 h again to test their repellency. The processes of the replacement trial were the same as the exclusion trial, but the difference between them lies in a different order of L. amylovorus SLZX20-1 addition. To be specific, ETEC K88 and IPEC-J2 cells were cocultured for 1 h, and L. amylovorus SLZX20-1 were add to the mixture for another 1 h. In three trials, all the controls were using DMEM/F12 to replace L. amylovorus addition. Finally, the inhibition rate of the L. amylovorus SLZX20-1 on ETEC was calculated via the count of the numbers of live ETEC K88 by the flat colony counting method. The formula of inhibition rate equal to the difference between the controls minus the experimental group was divided by the control group.

Intestinal porcine epithelial cells were added to six-well plates and grew till 80% of cells were confluent. After 14 h cultures, L. amylovorus SLZX20-1 were centrifuged, discarded the supernatant, washed strains with normal saline, resuspended by DMEM/F12 without serum, and set low, medium, and high-dose groups, in which bacteria suspension concentrations are 1 × 10⁷, 1 × 10⁸, and 1 × 10⁹ CFU/ml. In total, 2 ml different concentrations of L. amylovorus SLZX20-1 were added to six-well plates of IPEC-J2 cells, co-incubated in an atmosphere of 5% CO₂ incubator at 37°C for 6 h. RNA extraction was performed according to the instruction of cell RNA extraction kit (CWBIO, Beijing, China) and RNA reverse transcription-PCR (RT-PCR) was performed according to the instruction of RNA reverse transcription kit (Mei5 Biotechnology, Beijing, China) and done by qRT-PCR instrument (LightCycler 96, Roche, Shanghai, China). The primer sequence design of six HDPs is shown in Supplementary Table 1. The reaction system of real-time fluorescent quantitative PCR reactions was conducted in 10 μl reactions that include 2 μl of template DNA, 2 μl of real-time PCR super mix, 2 μl of deionized water, 0.5 μl of 10 μM forward primer, and 0.5 μl of 10 μM reverse primer. For initial denaturation at 95°C for 45 s, 95°C for 15 s then at 60°C for 15 s by 35 cycles, an extension at 72°C for 45 s.

All the procedures of this experiment were approved by the animal protection and utilization organization committee of China Agricultural University (CAU20171015-3). Sixty weaning C57BL/6 male mice were randomly divided into four groups, which are control group (CON, 150 μl normal saline), low dose group (LOW, 1 × 10⁷ CFU/ml), medium dose group (MID, 1 × 10⁸ CFU/ml), and high dose group (HIGH, 1 × 10⁹ CFU/ml) for intragastric administration of L. amylovorus SLZX20-1. Each group has three replicates, every replicate has five mice. Intragastric administration was performed every 2 days and the trial period is 14 d. Daily survival and abnormal status of mice were observed and recorded. Daily weight and feed intake were recorded too. Six mice were randomly selected from each group for sampling on day 14. Based on the daily weight changes and feed intake to calculate the average daily weight gain (ADG), average daily intake (ADFI), and feed to gain ratio (ratio of ADFI to ADG, F:G). Separated jejunum, ileum, cecum, colon, and collected chyme inside in 1.5 ml sterile centrifuge tube, put in liquid nitrogen immediately, and stored at −80°C for subsequent 16S rDNA sequencing.

After purification with AxyPrep DNA Purification kit (Axygen Biosciences, Union City, CA, USA), the PCR products were detected by agarose gel (2%) electrophoresis and were quantified using PicoGreen dsDNA Quantitation Reagent (Invitrogen, Waltham, MA, USA) on QuantiFluor-ST Fluorometer (Promega, Madison, WI, USA). After that, according to standard protocols, collected amplicons for paired-end sequencing (2 × 300 bp). This process did on the Illumina MiSeq platform (Allwegene, China). The raw data of this manuscript have been uploaded to NCBI SRA database, and the accession No. is PRJNA792839.

For raw FASTQ files analysis, the first step was demultiplexed and quality-filtered via QIIME (version 1.17). It follows with some basic principles: (i) sequencing reads were trimmed at the sites with an average quality score <20 over a 50 bp's sliding window and deleted trimmed reads <50 bp; (ii) the reads that contained mismatching barcode were deleted; and (iii) removed the paired reads with <10 bp overlapping.

UPARSE (version 7.1, http://drive5.com/uparse/) was used to gather OTUs with a 97% similarity. For chimeric sequences, using UCHIME identified and deleted. RDP Classifier (http://rdp.cme.msu.edu/) based on Silva (SSU115) 16S rRNA database was used to do the taxonomic analysis for each 16S rRNA gene sequence, and the confidence threshold is 70%. The diversity indexes, including Chao index, Shannon index, coverage indexes, and Metastats analysis, all were dependent on procedure Mothur v.1.21.1 to conduct. Primer 6 software (Primer-E Ltd., UK) was used for hierarchical clustering analysis. Software Venn diagrams for Venn figures and R tools for bacterial community figures. Using R tools to analyze the PCoA analysis. The significant differences between the two groups of microbial types were analyzed by linear discriminant analysis Effect Size (LEfSe) analysis.

The data and graphic analysis were performed by GraphPad prism 8.0. The results were shown on mean ± SEM. p < 0.05 was considered that differences are significant.

The morphology of colonies is shown in Figure 1A that diameter was about 1–2 mm with milky white color, opaque, smooth surface, and neat edges. Strains of L. amylovorus SLZX20-1 were proven to be the Gram-positive bacteria with rod-shaped, about 2–5 μm (Figure 1B). The amplified products size of 16S rDNA was about 1,500 bp and the sequencing length was 1,464 bp (Supplementary Figure 1). Sequencing results of 16S rDNA sequence were submitted to NCBI database for Basic Local Alignment Search Tool (BLAST) detection and were analyzed consanguinity via phylogenetic tree, which showed that the strains had 99.66% homology with L. amylovorus and genetically related with to L. amylovorus (Figure 1C). Further, L. amylovorus are major starch utilizers, we observed the utilization of starch by the isolated strain in starch medium (Supplementary Figure 2). Therefore, the strain was named as L. amylovorus SLZX20-1.

The growth stability period of L. amylovorus SLZX20-1 indicated that the strains grew rapidly (Figure 2A) and multiplied in nutrient-rich environments in a short time (Figure 2B). The number of live bacteria reached 1 × 10⁸ CFU/ml in 6 h and maintained this number till 24 h. When the strains entered the log growth period, the fluid pH of strains decreased quickly and slowed the decline rate bounded after 12 h with pH 3.93 at 24 h (Figure 2C), indicating the strong acid production capacity of L. amylovorus SLZX20-1. Meanwhile, L. amylovorus SLZX20-1 has shown strong acid tolerance ability at pH 4 and pH 3 with 70.55 and 57.13% survival rate for 2 h (Figure 2D). In addition it can tolerate lower concentration (0.1%) of bile salt environment instead of higher concentration (0.2%) of bile salt environment (Figure 2E). L. amylovorus SLZX20-1 was sensitive for cefradine, carboxypenicillins, tetracycline, chloramphenicol, vancomycin, erythromycin, and penicillin but insensitive to butamkana, norfloxacin, and gentamicin (Figure 2F). It can utilize fibrodilose, maltose, salicin, sucrose, raffinose, lactose, and synanthrin (shown in Supplementary Table 2). L. amylovorus SLZX20-1 has the stronger ability for enzyme production on leucine aromaminase, cystine amminoaraminase, acid phosphatase, Naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase, and β-glucosidase, while weaker on alkaline phosphatase, esterase, valine aromaminase and trypsin (Figure 2G; Supplementary Table 3). Meanwhile, L. amylovorus SLZX20-1 proved that the strains can secrete feruloyl esterase to degrade ethyl ferulate. In addition, the viability unit of feruloyl esterase was 105.7447 mU/mgprot via 72 h culture and liquid chromatography quantification analysis (Figure 2H).

The results of antimicrobial activity detection have shown that L. amylovorus SLZX20-1 can inhibit the growth of S. typhimurium SL1344, C. rodentium DBS100, S. pullorum CVCC1791, S. aureus CVCC1882, E. coli O157, E. coli K88, E. coli K99, and E. coli 987P, for which the inhibition ability for C. rodentium DBS100 was stronger than others (Figure 3A, Supplementary Figure 3, Table 1). C. rodentium DBS100 was further used to explore the antibacterial reagent in L. amylovorus SLZX20-1, the result showed that adjusted pH to neutral dismissing the antimicrobial activity of L. amylovorus SLZX20-1, which indicated that the inhibition effect was mainly exerted by the low pH of supernatant, due to its strong acid-producing capacity rather than proteins or peptides production from L. amylovorus SLZX20-1 (Figures 3B,C). The SCFAs in their supernatant were analyzed by ion chromatography. It was found that lactic acid and acetic acid were mainly produced. The yields of lactic acid and acetic acid were 14.62 and 3.51 g/l, respectively (Figure 3D).

L. amylovorus SLZX20-1 had certain adhesion for IPEC-J2 cells and this process was not easily affected by physical stimulation, such as washing and tinting (Figures 4A,B). Adhesion rates were 26.69, 20.21, and 4.38% by co-culturing with IPEC-J2 cells at low, medium, and high dose of L. amylovorus SLZX20-1. These results have shown that the adhesive rate of L. amylovorus SLZX20-1 was decreased following the increasing concentration of L. amylovorus SLZX20-1 per unit area of IPEC-J2 cells (Table 2). However, L. amylovorus SLZX20-1 proved that it did not inhibit E. coli K88 adhering to IPEC-J2 cells in every competition, exclusion, and replacement test (Supplementary Table 4). L. amylovorus SLZX20-1 was co-cultured with IPEC-J2 cells that significantly improved the expression level of antimicrobial peptides mRNA for NK-lysin, PEP2C, PG1-5, and PBD-1. Among that, high dose significantly improved NK-lysin, PEP2C expression (p < 0.05), medium dose significantly improved NK-lysin, PG1-5, PBD-1 expression (p < 0.05), but low dose significantly decreased PG1-5 expression (p < 0.05; Figure 4C).

Intragastric administration of different doses of L. amylovorus SLZX20-1 had no significant effect on the weight change of mice but showed a certain effect on promoting weight gain. There was no significant difference in the final body weight, ADG, and F:G ratio among the treatment groups (p > 0.05), but the ADF intake of the high group increased significantly (p < 0.05; Table 3).

The tissue morphology figures of the jejunum, ileum, and colon of mice are shown in Supplementary Figure 4. The results show that the tissue structure of each intestinal segment L. amylovorus SLZX20-1 mice with different doses by gavage was clear, and the intestinal villus of the small intestine were closely arranged. By measuring the villus height and recess depth of jejunum and ileum, it was found that different doses of L. amylovorus SLZX20-1 by gavage significantly reduced the recess depth of jejunum and ileum (p < 0.05). In addition, the villus height of mice in the low group was significantly reduced (p < 0.05), and the villus ratio of jejunum and ileum was significantly increased in the high dose group (p < 0.05; Table 4).

In order to evaluate the effect of the strain on the intestinal microbial composition of mice, the ileum samples were analyzed by 16S rDNA sequencing, it can be seen that there is no significant difference between the L. amylovorus SLZX20-1 group (1 × 10⁹ CFU/ml SLZX20-1) and the control group in terms of α-diversity (Figure 5A) and β-diversity (Figure 5B) (p > 0.05), but the samples in the SLZX20-1 group are more dispersed in terms of β-diversity. According to the common strains analyzed by the Venn diagram, there were 120 common strains between the SLZX20-1 group and the control group, and 356 differential strains in the SLZX20-1 group (Figure 5C).

The taxonomic distributions were further assessed to find the most abundant bacterial OTUs in each sample region. Based on the bacterial relative impairment of the top 11 phyla, whether in the control group or the SLZX20-1 group, Firmicutes were the most abundant flora in the ileum (99.43 and 97.60%, respectively), followed by Proteobacteria (0.44 and 1.31%, respectively). In addition, the abundance of Actinobacteria (0.66%), Bacteroidetes (0.36%), Verrucomicrobia (0.04%), and TM7 (0.02%) in SLZX20-1 group was higher than that in the control group (0.05, 0.07, 0.02, 0.03%, respectively; Figure 5D, Supplementary Table 5).

At the genus level, a total of 20 genera were identified, Lactobacillus was the most prevalent, and the relative abundance was 87.98 and 87.31% in the control group and the SLZX20-1 group, respectively. A decreased proportion of Candida_arthritis was observed in SLZX20-1 group compared with the control group (from 5.73 to 1.61%), and an increased abundance of Clostridium was observed in the SLZX20-1 group compared with the control group (from 0.62 to 2.15%). The abundance of Unspecified_Caulobacteraceae in the SLZX20-1 group (0.60%) was higher than the control group (0.18%) (Figure 5D, Supplementary Table 6). Furthermore, it was found that the average distribution index of 11 genera among the top 20 genera in the SLZX20-1 group was higher than the control group, except two genera in the ileum (the Unspecified_Coriobacteriaceae 0.05% and Prevotella 0.02%).

To identify the bacterial species' most characteristics of the SLZX20-1 group, LEfSe analysis of the taxa was conducted with linear discriminant analysis (LDA) scores >2 and p < 0.05. This approach revealed that 12 OTUs were differentially present between the SLZX20-1 group and the control group (Figure 5E, Supplementary Table 7) and further visualized the result of the Kruskal-Wallis rank-sum test with a LEfSe cladogram (Figure 5F).

The colon microbial composition analysis is further explored in Figure 6, the result showed that the treatment of L. amylovorus SLZX20-1 resulted in a slight increase in Chao1, Shannon, and Simpson index, but they did not reach the significant level (Figure 6A) (p > 0.05). In β-diversity analysis, the samples in the SLZX20-1 group were more dispersed, which was similar to the ileum (Figure 5B). Venn diagram showed that there are 787 common strains between the SLZX20-1 group and the control group in the colon, and 998 differential strains in the SLZX20-1 group (Figure 6C).

At the phylum level, a total of 10 phyla were identified, the most abundant phyla in the colon of the control group and SLZX20-1 group is Firmicutes (62.72 and 45.22%, respectively), followed by Bacteroidetes (29.50 and 36.05%). In addition, the abundance of Actinobacteria (9.21%), Verrucomicrobia (5.15%), Tenericutes (0.73%), and Deferribacteres (0.55%) in the SLZX20-1 group was higher than the control group (3.55, 0.01, 0.47, and 0.15% in the control group, respectively) (Figure 6D, Supplementary Table 8).

The genus level was further analyzed, and the results showed that a total of 20 genera were identified. The dominant genus in the control group was Lactobacillus (52.04%), but in SLZX20-1 group, the dominant genus was changed to Unspecified_S24_7 (27.03%) but not Lactobacillus (19.16%). In addition, the treatment of SLZX20-1 group increased the abundant of Unspecified_Clostridiales (6.13%), Akkermansia (5.15%), Unspecified_Lachnospiraceae (4.10%), Oscillospira (3.92%), Allobaculum (3.42%), and [Prevotella] (3.09%) than control group (3.04, 0.01, 1.74, 1.22, 0.02, and 0.36% in control group respectively). The abundance of Prevotella (0.51%) and Helicobacter (0.23%) was decreased in the SLZX20-1 group as compared with the control group (0.92 and 1.19% in the control respectively) (Figure 6D, Supplementary Table 9).

LEfSe analysis of the taxa showed that 12 OTUs were differentially present between the SLZX20-1 group and control group in the colon (Figure 6E, Supplementary Table 10), and the result of the Kruskal-Wallis rank-sum test was further visualized with a LEfSe cladogram (Figure 6F).