The hemolytic activity of P. pentosaceus SNF15 was carried out as described by Rastogi et al. (14). Inoculate P. pentosaceus SNF15 activated on MRS agar into TSA (Qingdao hopebiol, China) blood agar containing 5% (v/v) off fiber sheep blood (Qingdao hopebiol, China) and cultured for 24 h at 37°C. Plates were then observed for hemolysis for β-hemolysis (clear halo around each colony), α-hemolysis (greenish halos around each colony), and γ-hemolysis (no halo around each colony). Staphylococcus aureus (CVCC186158, Guangzhou Legend Biotech Co., LTD) showing β-hemolysis was used as the positive control.

Antibiotic susceptibility testing of the isolated strain was determined by agar disk diffusion assay on MRS agar (Qingdao hopebiol, China), the results indicated as susceptible, S (inhibition zone ≥21 mm); intermediate, I (15 < inhibition zone <21 mm) and resistance R (inhibition zone ≤15 mm) according to Yang et al. (15). P. pentosaceus SNF15 was inoculated in MRS broth (Qingdao hopebiol, China) and cultured for 24 h at 37°C; the concentration of the isolated strain solution was adjusted to 1.5 × 10⁸ CFU/mL and spread on the surface of the MRS agar and allowed to dry. Antibiotic disks were placed on the MRS agar surface and cultured for 24 h at 37°C, and the inhibition zone around each disk was measured. The following antibiotic discs (Hangzhou Microbial Reagent Co., LTD, China) were used: penicillin G (10 units), ampicillin (10 μg), tetracycline (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), erythromycin (15 μg), clindamycin (2 μg), kanamycin (30 μg) and ciprofloxacin (30 μg).

Detect the growth performance and acid production capacity of P. pentosaceus SNF15 (16). The cultured solutions of isolated strains were added to 80 mL MRS broth (1% v/v) and hermetically incubated at 37°C. The absorbances at 600 nm and pH were measured once every 2 h and continued until 24 h of culturing. Acid production and growth curves were plotted with time as the horizontal coordinate and pH or OD_600nm as the vertical coordinate.

Using the agar well diffusion method, test the antimicrobial activity of the strain isolate (17). To explore the antagonistic effect of the supernatant of 24 h cultures of P. pentosaceus SNF15 on E. coli K99 (Isolated from the feces of diarrheic calves), P. pentosaceus SNF15 was inoculated into MRS Broth at a 1% dose and cultured at 37°C for 24 h. Then, the supernatant was obtained by centrifuging the culture solution at 12000 r/min for 20 min.

Briefly, nutrient agar (Qingdao hopebiol, China) was poured on the culture dish as the first layer, and the Oxford cup was gently placed on the agar after solidification. Then, E. coli K99 cultured overnight was diluted to 1 × 10⁶ CFU/ mL using normal saline. Then, the diluted E. coli K99 was mixed with soft Mueller-Hinton Agar (Qingdao hopebiol, China) and poured on nutrient agar as the second layer. After the second layer solidification, remove the Oxford cup. 200 μL of the prepared supernatant of the isolated strain was added into each Oxford cup hole and cultured at 37°C for 12 h. MRS broth as a negative control and tested in triplicate.

Analyze the main bacteriostatic substances of P. pentosaceus SNF15 (18). Use the same way as in 2.2.2. to get P. pentosaceus SNF15 supernatant. Catalase or protease K or NaOH (Beijing Solarbio Science & Technology Co., Ltd., China) was added to the supernatant of P. pentosaceus SNF15. The final concentrations were adjusted to 1 mg/mL for protease K, 5 mg/mL for catalase, and pH 7.0, respectively. The catalase group was placed in 37°C water bath for 1 h; the protease K group was placed in 37°C water bath for 3 h. The same method was used as 2.2.2 to determine the inhibitory effect of the treated supernatant on E. coli K99. The pH of the supernatant of P. pentosaceus SNF15 was adjusted to 7.0 using NaOH (Beijing Solarbio Science & Technology Co., Ltd., China). The original supernatant was used as a positive control to assess the impact of treatment on the antibacterial active substances in the supernatant.

Slightly modified according to Ye K’s method (19). The bile salt tolerance of P. pentosaceus SNF15 was assessed. To evaluate resistance to bile salt, a 5% inoculum size P. pentosaceus SNF15 solution, which had been cultured for 24 h, was inoculated into the MRS broth with 0.3% (w/v) bile salts, inoculated in common MRS broth as a control, and cultured at 37°C for 6 h.

Artificial gastroenteric fluid tolerance of P. pentosaceus SNF15 was assessed under gastrointestinal tract-like conditions. To prepare simulated gastric juice, MRS broth medium adjusted to pH 3.0 was added with 1 mg/mL pepsin (from Beijing Solarbio Science & Technology Co., Ltd., China) under sterile conditions. Similarly, to prepare artificial intestinal juice, 1 mg/mL of trypsin (1:250) (Beijing Solarbio Science & Technology Co., Ltd., China) was added to the sterile, pH 8.0 MRS broth. Inoculate into untreated MRS broth medium as a control. Fresh bacterial culture prepared in the same conditions mentioned above was cultured at 37°C for 4 h.

The absorbance at 600 nm was measured using a U-V spectrophotometer. The experiments were repeated thrice. The survival rate is calculated according to the following formula:

N0 is the absorbance at 600 nm of P. pentosaceus SNF15 cultured in processed MRS broth, and N1 is the absorbance at 600 nm of P. pentosaceus SNF15 cultured in common MRS broth.

Adhesion of P. pentosaceus SNF15 to the Caco-2 cell line in vitro was performed as reported (20). Caco-2 cells were maintained in a basal medium composed of DMEM medium (Wuhan Pricella Biotechnology Co., Ltd., China) supplemented with 20% FBS (Meilunbio^®, China) and 1%P/S (Beijing Solarbio Science & Technology Co., Ltd., China). Caco-2 cells were cultured at 37°C in an atmosphere containing 5% CO₂. 300,000 Caco-2 cells were inoculated on the 12-well plate, and the experiment was started after the cell density grew to more than 80%. Caco-2 cells were washed twice using PBS and then infected with 1 × 10⁸ CFU/mL P. pentosaceus SNF15 in basal medium without P/S and FBS. The experimental setup also included one control: a negative control without P. pentosaceus SNF15. After 2 h, PBS washed away non-adherent bacteria, and Caco-2 cells were digested with pancreatic enzymes and collected in sterile tubes. Cell counts of the negative control were performed under a microscope. Colony counting was performed by using the viable plate counting method.

The experiments were repeated thrice. The P. pentosaceus SNF15 adheres to the Caco-2 cell line was calculated using the following formula:

Where M1 is the number of adhesive strains (lgCFU/mL), and M0 is the cell count (lgCFU/mL) of the negative control.

In the current study, 30 half-male and half-female KM mice (5 weeks old, 30 ± 0.5 g initial body weight) purchased from SiPeiFu Biotechnology Co. LTD (Beijing, China) were raised in the Experimental Animal Center of Hebei Agricultural University under light-controlled (12-h light/dark cycle) and temperature-controlled (22 ± 1°C) conditions. The mice had free access to water and food.

Pediococcus pentosaceus SNF15 was statically cultured in MRS broth at 37°C for 24 h, and E. coli K99 was agitated cultured under 37°C 180 r/min for 12 h. Subsequently, P. pentosaceus SNF15 and E. coli K99 was harvested by centrifugation (4,000 r/min, 20 min) respectively. After washing twice using stroke-physiological saline solution, P. pentosaceus SNF15 was resuspended in an appropriate volume of stroke-physiological saline solution to a concentration of 1 × 10⁹ CFU/mL, and E. coli K99 was resuspended in an appropriate volume of stroke-physiological saline solution to a concentration of 1 × 10⁸ CFU/mL.

A total of 30 KM mice were randomly assigned to 5 groups, each containing 6 mice (n = 6): (I) CK group received 200 μL stroke-physiological saline solution for 14 days. (II) CN group received 200 μL E. coli K99 (1 × 10⁸ CFU/mL) for 1–7 days and stroke-physiological saline solution at 8–14 days. (III) CIP group received 200 μL E. coli K99 (1 × 10⁸ CFU/mL) at 1–7 days and 200 μL of 50 mg/kg Ciprofloxacin solution at 8–14 days. (IV) PPE group received 200 μL P. pentosaceus SNF15 solution (1 × 10⁹ CFU/mL) at 1–7 days and 200 μL E. coli K99 (1 × 10⁸ CFU/mL) at 8–14 days. (V) PTE group received 200 μL E. coli K99 (1 × 10⁸ CFU/mL) at 1–7 days and 200 μL P. pentosaceus SNF15 solution (1 × 10⁹ CFU/mL) at 8–14 days. All the administration methods were oral administration by gavage.

Based on Yang B et al. (21) develop fecal scoring criteria (Table 1) to evaluate the diameter of mice feces smudges.

The diarrhea rate of each group was calculated according to the formula. Body weight was recorded every 2 days, and fecal track scores were recorded every 3 days. On 15 days, after recording body weight, the mice were anesthetized and sacrificed by cervical dislocation. After blood was collected by eyeball enucleation, the weight of the liver, spleen, and small intestinal tissue was recorded, and then the jejunum tissues and cecal contents were collected. The diarrhea rate and weight change rate of mice were calculated according to the formula:

Where D1 and D0 are the diarrhea mice in this group and the total number of mice in this group.

Where W0 is the body weight of mice at 0 days and W1 is the body weight of mice at 2, 4, 6, 8, 10, 12, and 14 days, respectively.

The total RNA of jejunum tissue was extracted by using TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) and quantitatively analyzed using a NanoDrop D2000 instrument (Thermo Fisher Science, USA); the RNA was stored at −80°C. The mRNA was reverse-transcribed into cDNA using EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) kit. The reverse transcription reaction conditions were genomic DNA elimination at 42°C for 15 min and inactivation of the reverse transcriptase at 85°C for 5 s. qPCR assays were performed to assess the relative expression levels of the IL-6, IL-1β, and TNF-α. The expression level of GAPDH was used as the reference gene. The amplification conditions were pre-denaturation at 95°C for 1 min, followed by 40 cycles of PCR reaction with denaturation at 95°C for 5 s and annealing at 60°C for 15 s. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The specific primers for interleukin were IL-6 (5′-CTTCTTGGGACTGATGCTGGTGAC-3′; 5′-TCTGTTGGGAGTGGTATCCTCTGTG-3ʹ), IL-1β (5′-CCTGGGCTGTCCTGATGAGAG-3ʹ; 5ʹ-TCCACGGGAAAGACACAGGTA-3ʹ), TNF-α (5ʹ-GGACTAGCCAGGAGGGAGAACAG-3ʹ; 5ʹ-CAATGTGTCCGTCGTGGATCT-3ʹ), GAPDH (5ʹ-CAATGTGTCCGTCGTGGATCT-3ʹ; 5ʹ-GTCCTCAGTGTAGCCCAAGATG-3ʹ).

The jejunum samples were homogenized with pre-cooled PBS and were centrifuged at 12000 × g for 10 min at 4°C. Then, the collected supernatant was transferred to a sterile tube and used for ELISA analyses. Secretion levels of TNF-α, IL-6, and IL-1β in the jejunum samples homogenates were colorimetrically determined by enzyme-linked immunosorbent assay kits (mlbio, Shanghai, China) according to the manufacturer’s instructions.

Use standard saline buffer to gently rinse the contents of jejunum tissue samples and place them into a 4% (v/v) paraformaldehyde fixative for fixation. Subsequently, embed jejunum tissue in paraffin and cut into 5 μm sagittal sections. The sections were deparaffinized with xylene and stained with hematoxylin and eosin (H&E). Images of jejunum tissue were captured using a light microscope (NIKON, Tokyo, Japan) to observe pathological structural changes.

ZO-1, occludin, claudin-1, and MUC2 are crucial tight junction (TJ) proteins and mucin, contributing to intestinal physical barrier function (22). Immunofluorescence staining was performed on jejunum tissue. After antigen retrieval of jejunal sections, use 3% hydrogen peroxide to treat the sections, 0.3% Triton X-100 permeabilize and block with 3% BSA (Servicebio Technology, Wuhan, China) at room temperature for 30 min. Then, the sections were incubated overnight with primary antibodies directed against them. After incubation with the secondary antibody, stained sections in DAB (Servicebio Technology, Wuhan, China) and hematoxylin were observed under a microscope. Images were processed using ImageJ software.

Fresh content in the cecum was collected in a sterile tube. The contents were put in liquid nitrogen and then preserved at −80°C for the next step. The total DNA genome was extracted from colonic contents. PCR-amplified V3–V4 region of the 16S rRNA gene. Amplicons from all colon-content bacterial 16S rRNA samples were sent to Peisenor Biotechnology (Shanghai, China) for sequencing on an Illumina HiSeq 2500 platform. Data analysis was performed by using the Personalbio Cloud Platform.¹ Analysis of alpha diversity (α-diversity), beta diversity (β-diversity), rarefaction curve, and taxonomic composition was performed by using QIIME2 software (version 2019.4). β-diversity was displayed by principal coordinates analysis (PCoA) with the help of R software, QIIME2 software, and ape bag.

The positive control of S. aureus (CVCC186158) showed an apparent β-hemolytic ring. While the P. pentosaceus SNF15 has no hemolytic ring and is judged as γ-hemolysis, indicating that the P. pentosaceus SNF15 cannot dissolve erythrocytes (Figure 1B).

In the current study, P. pentosaceus SNF15 was assessed for antibiotic sensitivity properties. Antibiotic susceptibility tests showed that (Table 2) P. pentosaceus SNF15 was sensitive to penicillin G, ampicillin, tetracycline, ceftriaxone, chloramphenicol, doxycycline, erythromycin, and resistant to kanamycin and ciprofloxacin.

The growth activity of P. pentosaceus SNF15 in MRS broth is shown in Figure 1C. P. pentosaceus SNF15 began entering the logarithmic growth phase after culturing for 2 h and approximately reached the stationary phase after 12 h. The acid-producing characteristic of P. pentosaceus SNF15 in MRS broth is shown in Figure 1D. The pH of P. pentosaceus SNF15 culture solution decreased with the passage of culture time and tended to be stable at about 4.67 after 12 h of culture.

Tolerance to the gastrointestinal environment is a prerequisite as a probiotic that mainly colonizes the gut. The survivability of P. pentosaceus SNF15 to 0.3% bile salt and artificial gastroenteric fluid was monitored. The results demonstrated that (Table 3) the survival rate of P. pentosaceus SNF15 in 0.3% bile salt is 26.02%, and the survival rate in artificial gastric juice and artificial intestinal juice is 50.56 and 55.62%, respectively.

We performed adhesion tests to evaluate the ability of P. pentosaceus SNF15 to colonize the gastrointestinal tract. As shown in Table 3, the adhesion rate of P. pentosaceus SNF15 on Caco-2 cells is 13.93%.

The inhibition effect of the P. pentosaceus SNF15 was tested using the agar well diffusion method against E. coli K99. The results are shown in Table 4. P. pentosaceus SNF15 showed the antibacterial effect of inhibition on the E. coli K99, and the inhibitory diameter is 19.10 ± 0.436 mm. The effect of different treatments on the antibacterial activity of E. coli K99 of P. pentosaceus SNF15 supernatant is shown in Table 4. The control is supernatant without treatment. NaOH was added to the P. pentosaceus SNF15 supernatant to exclude the effect of organic acid and adjust the pH to 7.0. The result shows that it did not affect the antibacterial activity (p > 0.05). To exclude the effect of bacteriocin on antibacterial activity, the supernatant was treated with protease K. The result showed that the antibacterial activity of the treatment group was significantly decreased from that of the control group (p < 0.05). The supernatant was treated with catalase to eliminate the effect of hydrogen peroxide. As a result, the antibacterial activity of the treatment group was significantly decreased from that of the control group (p < 0.05). These results indicate that the main bacteriostatic substances of P. pentosaceus SNF15 include bacteriocin and hydrogen peroxide.

The basic information on the genome of P. pentosaceus SNF15 is shown in Table 5. The genome of P. pentosaceus SNF15 consists of 1,895,681 bp with 37.33% GC content. The genome of P. pentosaceus SNF15 consists of 1 chromosome (contig0001) and 2 plasmids (contig0002 and contig0003), and the chromosome and plasmid genome are circular. The length of the chromosome is 1,842,076 bp. The length of 2 plasmids is 31,328 and 22,277 bp, respectively. It contains 1,855 protein-coding sequences, 1,668,747 bp, accounting for 88.02% of the total length; the average gene length is 899 bp, the max gene length is 8,601 bp, and the min gene length is 90 bp. The total repetitive sequence length is 4,435 bp, and the repetitive sequence content is 0.23%. The genome contains 15 rRNA genes and 56 tRNA genes. The predicted coding genes were functionally annotated by aligning them with 5 databases: Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), evolutionary genealogy of genes Non-supervised Orthologous Groups (eggNOG), Carbohydrate-active enzymes (CAZy), and Comprehensive Antibiotic Research Database (CARD). Based on the protein sequence alignment, the annotated gene is compared with various databases to obtain the corresponding functional annotation information. Among them, 955 genes were annotated in the KEGG database, 1,466 genes were annotated in the GO database, 1,607 genes were annotated in the eggnog database, 57 genes related to carbohydrate activity were annotated by CAZy, and 1 drug resistance gene was annotated by CARD.

955 genes were annotated in the KEGG database, accounting for 51.48% of the total of P. pentosaceus SNF15 (Figure 2A). These genes were annotated with four categories (environmental information processing, genetic information processing, cellular Processes, and metabolism), including nineteen level 2 KEGG pathways and 76 “ko-” related reference pathways of metabolism annotated. Among the four categories of KEGG pathways, metabolism contains the most significant number of genes (558 genes), including level 2 pathways of 12 metabolism and 64”ko-” related pathways. In the level 2 pathways about metabolism, nucleotide metabolism (gene number: 80), carbohydrate metabolism (gene number: 121), and global and overview maps (gene number: 275) are the top three pathways. Carbohydrate metabolism mainly contains a total of 14 pathway information, including glycolysis/gluconeogenesis (ko00010), amino sugar and nucleotide sugar metabolism (ko00520), pyruvate metabolism (ko00620), pentose phosphate pathway (ko00030), and the number of related genes is 32, 31, 23, and 24, respectively. Nucleotide metabolism contains 2 pathways: purine metabolism (ko00230) has 56 genes annotated, and Pyrimidine metabolism (ko00240) has 42 genes annotated. Global and overview maps mainly consist of metabolic pathways, biosynthesis of secondary metabolites, and biosynthesis of antibiotics. It is worth noting that some pathways function as probiotic markers, such as terpenoid backbone biosynthesis, taurine and hypotaurine metabolism, and streptomycin biosynthesis (Table 6). These probiotic genes are an essential prerequisite for P. pentosaceus SNF15 to exert probiotic functions.

Annotation of coding genes against the GO database (Figure 2B). Gene functions could be classified into all three categories: biological process (BP), cellular component (CC), and molecular function (MF). 1,466 gene entries were annotated in the GO classification, accounting for 79.03% of all coding genes (1855) for 4,770 functions. The analysis revealed that 333 genes were enriched in CC terms, accounting for 22.71% of all GO coding genes. 445 and 443 genes were annotated, respectively, mainly focusing on cell and membrane. Others include extracellular region, nucleoid, macromolecular complex, organelle, etc. The analysis revealed that 938 genes were enriched in MF, accounting for 63.98% of all GO coding genes. 882 and 669 genes were annotated, respectively, mainly focusing on catalytic activity and binding. It also annotates nucleic acid binding transcription factor activity, signal transducer activity, transporter activity, etc. It is worth noting that 9 genes for antioxidant activity were annotated. The analysis revealed that 195 genes were enriched in BP terms, accounting for 13.30% of all GO coding genes. 771 genes were related to the metabolic process, and 641 were associated with the cellular process. The analysis also annotates single-organism processes, localization, biological regulation, response to stimulus, etc. It is worth noting that there are some functions, including reproduction, biological adhesion, and biological regulation, with 4 and 3 genes annotated, respectively.

Annotation of coding genes against the eggNOG database (Figure 2C) subsequently identified 1,607 genes, which accounted for 86.63% of the total of P. pentosaceus SNF15. The eggNOG annotation results indicated that the primary known genes of P. pentosaceus SNF15 are in carbohydrate transport and metabolism (149 genes, accounting for 9.12%), translation, ribosomal structure and biogenesis (140 genes, accounting for 8.57%), transcription (119 genes, accounting for 7.28%), and replication, recombination and repair (117 genes, accounting for 7.16%).

In the CAZy database, the 57 genes were annotated into 4 functional classifications (Figure 2D). Among those genes, 20 genes were annotated as Glycoside Hydrolases (GH), 20 genes were annotated as glycosyl transferases (GT), 9 genes were annotated as carbohydrate esterases (CE), and 8 genes were annotated as carbohydrate-binding modules (CBM), which account for 35.08, 35.08, 15.78, and 14.03%, respectively.

Compared with the CARD database (Table 7), as indicated by the results, the genome alignment of P. pentosaceus SNF15 identified 1 gene related to antibiotic resistance named OXA-224 (gene ID: GE000281), which causes penam; cephalosporin inactivation.

The experimental design in mice is shown in Figure 3A. Figure 3B shows the entire weight change of mice during the experiment. The body weight change of the CK group increased slowly during the trial for 1–14 days. The body weight change of the CN, CIP, and PTE groups was reduced rapidly during gavaged E. coli K99 for 1–7 days. The body weight change of the PPE group showed an increased trend during gavaged P. pentosaceus SNF15 for 1–7 days. At 8–14 days, the body weight change of the CN, CIP, and PTE groups slowly increased due to treatment with ciprofloxacin or P. pentosaceus SNF15, and the body weight growth trend for the CIP and PTE groups was better than the CN group. The body weight change of the PPE group showed a slowly decreasing trend but was still superior to the CN group.

Figures 3C–G show the fecal status score and diarrhea rate of all groups. The CK group had no diarrhea during the trial. The fecal status score and diarrhea rate of the CN, CIP, and PTE groups showed an increased trend. After treatment with ciprofloxacin or P. pentosaceus SNF15, both fecal status score and diarrhea rate significantly decreased. During only gavage for 1–7 days, the PPE group had no diarrhea, and some diarrhea appeared after gavage E. coli K99.

The organ index of mice was analyzed. Compared with the CK group, the liver shows considerable atrophy in the CN group (p < 0.05). The CIP, PPE, and PTE groups have no significant difference from the CN group (p > 0.05), and the liver index of PTE and PPE groups appears to be a rising trend and has no significant difference from the CK group (p > 0.05) (Figure 3H). Compared with the CK group, splenomegaly was found in the CN group (p < 0.05), and the CIP group of ciprofloxacin treatment, P. pentosaceus SNF15 prevention, and P. pentosaceus SNF15 treatment has no significant difference (p > 0.05) with the CK group (Figure 3I). There was no significant difference in intestinal coefficient (Figure 3J).

The results of HE staining of jejunal tissue are shown in Figure 4A. The intestinal villi of the CK group were clear and intact in structure, and the epithelial cells were arranged neatly. There was no edema within the lamina propria. Jejunal intestinal villi in the CN group were severely damaged, and there was prominent edema within the lamina propria of the intestinal villi. Compared with the CN group, the intervention of P. pentosaceus SNF15 and ciprofloxacin can significantly alleviate the intestinal damage caused by E. coli K99.

Figure 4 showed that, due to the stimulation of E. coli K99, compared with the CK group, claudin-1, ZO-1, occludin, and MUC2 in the CN group showed a notable decrease (p < 0.05). However, oral administration of P. pentosaceus SNF15 in advance significantly improved the expression of claudin-1, MUC2, occludin, and ZO-1 (p < 0.05), and the expression of claudin-1 was significantly increased compared with the CK group (p < 0.05). After infection with E. coli K99, the expression of P. pentosaceus SNF15, Claudin-1, MUC2, and ZO-1 in oral administration increased significantly compared with the CN group (p < 0.05), and the expression level of occludin did not change significantly (p > 0.05). Oral ciprofloxacin increased the expression of claudin-1, MUC2, and ZO-1 (p < 0.05), while the expression of occludin had no significant change (p > 0.05).

We detect expression mRNA levels of IL-6, IL-1β, and TNF-α in the jejunum to assess the anti-inflammatory properties. The expression mRNA levels of IL-6, IL-1β, and TNF-α in the intestines of mice infected with E. coli K99 were significantly up-regulated (Figures 5A–C). The mRNA expression levels of IL-6, IL-1β, and TNF-α in the CIP group were significantly reduced compared with the CN group (p < 0.05), and the mRNA expression levels of IL-6, and IL-1β had no significant difference with the CK group (p > 0.05). Compared with the CK group, the expression mRNA levels of TNF-α elevated significantly (p < 0.05). The mRNA expression levels of IL-6, IL-1β, and TNF-α in the PPE group were significantly decreased compared with the CN group (p < 0.05) and had no significant difference compared with the CK group (p > 0.05). The mRNA expression levels of IL-6 and IL-1β in the PTE group were significantly reduced compared with the CN group (p < 0.05), and TNF-α showed a downward trend but no significant difference (p > 0.05).

The secretion levels of IL-6, IL-1β, and TNF-α in jejunum tissues of each group were detected by ELISA (Figures 5D–F). The CN group has the same trend with expression mRNA levels, which is significantly decreased compared with the CK group (p < 0.05). The secretion levels of IL-6, IL-1β, and TNF-α in the CIP, PPE, and PTE groups were significantly reduced compared with the CN group (p < 0.05). The secretion levels of IL-6 and TNF-α in PPE group, secretion levels of IL-1β in CIP group had no significant difference with the CK group (p > 0.05).

In this research, 5 groups, including 15 samples, were subjected to high-throughput sequencing analysis. We obtain original data 1,001,103. After filigreeing and denouncing the original data, 879,706 effective-quality sequences were obtained from the 15 samples, and further, through merging and removal of chimeras, 524,424 high-quality sequences were obtained. The curves of good coverage index rarefaction in KM mice were flat and displayed saturated curves, demonstrating that the sequencing depth was sufficient in the current experiment (Figure 6A).