CN116115649A - Application of bacillus subtilis JZXJ-7 in preparation of preparation for relieving vibrio parahaemolyticus infection - Google Patents

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of bacillus subtilis JZXJ-7 in preparation of a preparation for relieving vibrio parahaemolyticus infection. Based on the research of a vibrio parahaemolyticus infection mouse model, the bacillus subtilis JZXJ-7 enriches beneficial bacterial groups such as Acremonium, bifidobacterium, clostridium and the like by increasing the diversity of intestinal microorganisms of the mouse, remodels intestinal microecological stability, effectively resists the reduction of body mass, the increase of disease activity index and the colonization of intestinal pathogenic bacteria caused by the vibrio parahaemolyticus infection, and can relieve colon injury caused by Vp infection and maintain intestinal physiological functions. The preparation is expected to be a new strategy for preventing vibrio parahaemolyticus food poisoning and reducing antibiotic abuse, and has good application prospect.

Description

Application of Bacillus subtilis JZXJ-7 in the preparation of preparations for relieving Vibrio parahaemolyticus infection

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of bacillus subtilis JZXJ-7 in preparing preparations for relieving Vibrio parahaemolyticus infection.

Background technique

Vibrio parahaemolyticus (Vp) is a Gram-negative halophilic brevibacterium, which is widely distributed in the marine environment and is also commonly found in salted foods. According to the survey, the detection rate of Vp in aquatic products in my country is as high as 38.8%. Salmonella and Escherichia coli have become the primary pathogens threatening public health. In daily life, people eat seafood contaminated by Vp to cause food poisoning, which manifests as acute gastroenteritis symptoms such as abdominal pain, diarrhea and vomiting. Dehydration shock, systemic infection and even death may also occur in frail people. The traditional view is that the pathogenicity of Vp is related to the bacteria and virulent metabolites. The bacteria enter the body through the food chain and rely on the invasion and adhesion of the bacteria to destroy the intestinal wall barrier of the host. The heat-resistant direct hemolysin, Virulence factors such as urease and metalloprotease have cytotoxicity and aggravate the degree of intestinal infection. In addition, the team's previous research also found that Vp-induced intestinal flora disorder triggers an inflammatory response in the lamina propria and participates in the occurrence and development of intestinal inflammatory damage. Therefore, based on adjusting the intestinal microbial community structure, the method of reshaping the microecological stability may become a new strategy for the intervention of Vp food poisoning.

At present, the main therapeutic agents for Vp food poisoning are quinolones and ceftriaxone. Although antibiotics can kill pathogenic bacteria and remove virulence factors, frequent use will cause side effects such as drug-resistant bacteria, abnormal kidney function, and intestinal flora imbalance. Probiotics are defined by the World Health Organization as "live microorganisms that can have a beneficial effect on the health of consumers when ingested in appropriate quantities", and are widely used in food additions and dietary supplements. Studies have shown that probiotics have the functions of improving intestinal tissue morphology, inhibiting intestinal inflammation and optimizing the structure of flora, and are potential functional factors for alleviating foodborne pathogenic bacteria infection. Probiotic preparations such as Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus pentosus have become very common. However, studies on the probiotic efficacy of Bacillus subtilis have focused on nutrient absorption and improved lipid metabolism, and few studies have evaluated the effect of Bacillus subtilis on preventing pathogenic infection. In view of the unknown protective effect and mechanism of Bacillus subtilis on foodborne pathogenic bacteria infection, its application in the intervention of Vp infection is greatly limited.

Contents of the invention

In order to make up for the deficiencies in the prior art, the present invention provides the application of Bacillus subtilis JZXJ-7 in the preparation of preparations for relieving Vibrio parahaemolyticus infection.

Bacillus subtilis JZXJ-7 (Bacillus subtilis JZXJ-7, JZXJ-7) is a strain of probiotic isolated from Jinzhou shrimp paste by the team. The preservation number is GDMCC No: 62458. species collection center. Physiological characteristics and in vitro antibacterial experiments confirmed that JZXJ-7 can secrete bacteriocin and highly inhibit the growth of Vp, and is highly resistant to gastric acid and intestinal juice erosion (higher activity than common probiotics in the market), but this strain can damage Vp infection in vivo. palliative effect is unknown.

The purpose of the present invention is achieved through the following technical solutions:

Application of bacillus subtilis JZXJ-7 in preparation of preparation for treating/relief/improving Vibrio parahaemolyticus infection.

Preferably, the Vibrio parahaemolyticus infection has at least one of the following manifestations:

(1) blood in stool;

(2) Colonic pathological damage;

(3) Excessive intestinal inflammatory response;

(4) weakened antioxidant capacity;

(5) Increased intestinal wall permeability;

(6) Intestinal flora imbalance.

More preferably, the pathological damage of the nodules is specifically manifested as: loss of colonic mucosal structure, reduction of mucosal cells, shallower crypt depth, accompanied by hyperplastic connective tissue, and a large number of inflammatory cell infiltration in the lamina propria; decreased number of colonic goblet cells ; Colonic collagen deposition increased.

More preferably, the excessive intestinal inflammation refers to the up-regulation of interleukin-1β, interleukin-6 and tumor necrosis factor-α levels in colon tissue.

More preferably, said weakened antioxidant capacity refers to down-regulation of expression of catalase, superoxide dismutase and total antioxidant enzymes in colon tissue.

More preferably, the increase of intestinal wall permeability refers to the increase of serum lipopolysaccharide, diamine oxidase and D-lactic acid content.

More preferably, the dosage of the Bacillus subtilis JZXJ-7 is 10 ⁶ CFU/d-10 ⁸ CFU/d.

Compared with prior art, the beneficial effect of the present invention is:

The present invention found that Bacillus subtilis JZXJ-7 can relieve Vibrio parahaemolyticus infection, and intake of Bacillus subtilis JZXJ-7 can effectively resist weight loss, disease activity index increase and pathogenic bacteria colonization in mice caused by Vibrio parahaemolyticus infection At the same time, it can improve the pathological damage of the mouse colon, and effectively inhibit the intestinal physiological dysfunction caused by Vibrio parahaemolyticus infection; Bacillus subtilis JZXJ-7 enriches Akkermansia, Bi Beneficial flora such as Mycobacterium and Clostridium can alleviate the intestinal flora disorder caused by Vibrio parahaemolyticus infection and reshape the intestinal microecological stability. This preparation is expected to become a new strategy for preventing Vibrio parahaemolyticus food poisoning and reducing the abuse of antibiotics, and has a good application prospect.

Description of drawings

Figure 1 is an analysis diagram of the effect of Bacillus subtilis JZXJ-7 on the growth performance, disease index and intestinal bacterial load of Vibrio parahaemolyticus-infected mice.

Fig. 2 is an analysis diagram of the effect of Bacillus subtilis JZXJ-7 on colonic pathological damage in mice infected with Vibrio parahaemolyticus.

Fig. 3 is an analysis diagram of the effect of Bacillus subtilis JZXJ-7 on colonic inflammation, anti-oxidation and permeability of mice infected with Vibrio parahaemolyticus.

Fig. 4 is an analysis diagram of the effect of Bacillus subtilis JZXJ-7 on the colonic microbial α and β diversity of mice infected with Vibrio parahaemolyticus.

Fig. 5 is an analysis diagram of the effect of Bacillus subtilis JZXJ-7 on the phylum-level and genus-level flora structure of mice infected with Vibrio parahaemolyticus.

Fig. 6 is a graph of LEfSe analysis of the intestinal flora of Bacillus subtilis JZXJ-7-intervened mice.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The test methods used in the examples of the present invention, unless otherwise specified, are conventional methods; the materials, reagents, etc. used, unless otherwise specified, are commercially available reagents and materials.

experiment procedure

1. Materials and reagents

1.1. Materials

Experimental strains: Vibrio parahaemolyticus ATCC33847 (tdh ⁺ /trh ^- /tlh ⁺ ) is a typical virulent strain, Bacillus subtilis JZXJ-7 was screened from Jinzhou shrimp paste, and the strains were all preserved in Bulk Aquatic Product Storage and Safety of Bohai University Take control of the team.

Experimental animals: 28 6-week-old male specific pathogen-free C57BL/6J mice, body weight (17±2) g, purchased from Beijing Speifu Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0010), animal room temperature (20±2)°C, relative humidity (44±5)%, 12h light-dark cycle, temporary rearing for one week, no restriction on diet and drinking water.

1.2. Reagents

Phosphate buffered solution (phosphate buffered solution, PBS, pH=7.2), Soleibo (Beijing) Biotechnology Company; 4% paraformaldehyde tissue fixative, sodium chloride (NaCl), ether solution, lysate nutrient broth ( LB) medium, thiosulfate citrate bile salt sucrose agar (TCBS) medium, Beijing Luqiao Biotechnology Company; interleukin-1β, interleukin-6 and tumor necrosis factor-α ELISA kit, Wuhan enzyme immunoassay Biotechnology Co., Ltd; Hematoxylin Eosin Stain, Periodic Acid Schiff Stain, Masson Stain, Superoxide Dismutase, Catalase and Total Antioxidant Activity Kit, Lipopolysaccharide, Diamine Oxidase and D - Lactic acid kit, Nanjing Jiancheng Institute of Bioengineering.

1.3. Instrument

DL-CJ-2N ultra-clean bench, Suzhou Purification Company; SPX-25 biological constant temperature incubator, Shanghai Binglin Technology Co., Ltd.; 5804R high-speed desktop low-temperature centrifuge, Xiangyi (Hunan) Laboratory Instrument Development Company; ABI- stePone-Plus polymerase chain reaction instrument, Eppendorf, Germany; GI54DS vertical high temperature and high pressure steam sterilizer, Shandong Zhuolong Biotechnology Company; I-mark microplate reader, Bio-Rad, USA; HS-B7-B biological tissue Automatic embedding machine, HS-S7220-C paraffin embedding machine, KD-P tissue spreading machine, Hubei Beinuo Medical Technology Co., Ltd.; EcliPse-E100 optical microscope, Nikon Corporation, Japan.

1.4. Activation of experimental strains

Take ATCC33847 freeze-dried powder, streak continuously on LB agar medium, put it into 37°C incubator to activate for 24h; pick a single colony and inoculate it into LB liquid medium, and incubate at 37°C, 150r/min shaker for 12h; The resuscitated bacterial solution was transferred to fresh LB liquid medium containing 3% NaCl at a ratio of 1:100 for expansion. After growing to the logarithmic phase (OD _600nm = 0.8-1.0), the bacterial solution was transferred to 50 mL sterile centrifuged tube, centrifuged at 8,000r/min for 20min, discarded the supernatant, added an equal volume of sterile PBS solution to resuspend, centrifuged again, and repeated the above operation twice; finally, adjusted the concentration of ATCC33847 bacteria solution to 10 ⁶ CFU/ mL (the amount of bacteria is determined by TCBS plate counting), for later use.

Pick JZXJ-7 frozen at -80°C with an inoculation loop, streak on LB solid plate for activation, and culture at 30°C for 48 hours; pick a single colony with consistent growth shape and inoculate it in LB liquid medium, at 35°C, 180r/ Incubate on a shaker for 36 hours; collect the bacterial solution and centrifuge at 8,000r/min for 20 minutes to remove the supernatant to obtain the bacterial pellet; wash the bacterial pellet twice with sterile PBS solution, resuspend in fresh sterile saline, and use Measure the concentration of the bacterial solution at this time by the 10-fold dilution coating plate method, divide the bacterial solution into two parts according to the needs of animal experiments, adjust to 10 ⁷ and 10 ⁹ CFU/mL respectively, and store at 4°C for later use.

1.5. Animal experiments

Twenty-eight male C57BL/6J mice were randomly divided into 4 groups (n=7/group): control group (control, CK), model group (Vibrio parahaemolyticus, Vp), low dose intervention group (low dose Bacillus subtilis JZXJ- 7+Vp, L-JZXJ-7+Vp) and high dose intervention group (high dose Bacillus subtilis JZXJ-7+Vp, H-JZXJ-7+Vp). The mice in the intervention group were fed with 0.1 mL of JZXJ-7 bacterial solution containing 10 ⁷ and 10 ⁹ CFU/mL, respectively, for 14 days. On the 15th day, the mice in the Vp group and the two intervention groups were given 0.1 mL of ATCC33847 bacterial solution containing 10 ⁶ CFU/mL, and the mice in the CK group were given an equal volume of normal saline. After 7 days of exposure to infection, the experiment was terminated. After the mice were anesthetized with ether, the eyes were removed to collect blood, and the cervical vertebrae were dislocated to kill them. For the detection of flora, colon tissues with obvious lesions were collected, a part was fixed in 4% paraformaldehyde for pathological research, and the rest was frozen at -80°C for the determination of cytokines and antioxidant enzyme activities.

1.5.1. Evaluation of infection symptoms in mice

For the symptoms of Vp-induced infection in mice, the changes in body weight, disease activity index and Vp load in intestinal tissue were evaluated. During the exposure of the mice to Vp, the activity behavior, diet and water intake, mental state, feces properties and body weight of the mice in each group were observed and recorded. According to the evaluation criteria of the disease activity index, combined with changes in body weight, degree of loose stools and organ lesions and other indicators to score. In addition, take 0.5g of ileum, cecum or colon content and mix with 4.5mL sterile PBS solution, after 10-fold serial dilution, spread 3 serial appropriate dilutions on TCBS plates, incubate at 37°C for 24h, count the number of colonies, Expressed as lg (CFU/g), the plate count was performed in parallel with 3 experiments.

1.5.2. Analysis of colonic pathological damage

For Vp-induced colonic tissue lesions in mice, the number of intestinal epithelial goblet cells, degree of fibrosis and structural damage were evaluated. The fixed colon samples were dehydrated by alcohol gradient, cleared, soaked in wax, embedded, sectioned (thickness 5 μm), spread, patched, baked, conventional dewaxed and rehydrated, hematoxylin-eosin (htoxylin eosin, HE), Periodic acid-Schiff (PAS) and Masson (MASSON) staining, gradient dehydration, neutral gum mounting, using ordinary optical microscope (100×) blind reading, observation of colonic epithelial structure and inflammatory cells in the lamina propria Infiltration, the number of goblet cells and collagen deposition ratio were counted by Image J software, and 6 slices were randomly selected from CK group, Vp group, L-JZXJ-7+Vp group and H-JZXJ-7+Vp group according to the pathological scoring standard score.

1.5.3. Detection of colonic physiological function

For Vp-induced abnormal colon function in mice, the intestinal inflammatory response, antioxidant activity and intestinal wall permeability were evaluated. Interleukin (interleukin, IL)-1β (IL-1β), IL-6, tumor necrosis factor-α (tumournecrosis factor-α, TNF-α) kits were used to detect the levels of pro-inflammatory factors in the colon, catalase ( Catalase, CAT), superoxide dismutase (superoxide dismutase, SOD) and total antioxidant enzyme (Total antioxidanttenzyme, T-AOC) kits were used to detect the activity of antioxidant enzymes in the colon, lipopolysaccharide (lipopolysaccharide, LPS), diamine oxidation Enzyme (diamine oxidase, DAO) and D-lactic acid (D-lactic acid, D-LA) kits were used to detect the concentration of intestinal wall permeability markers in serum. The preparation and specific procedures of all samples were operated according to the instructions. The absorbance was read by the standard instrument, and a total of 9 indicators were measured in the colon tissue and serum, and the unit was pg/g, pg/mg or U/mg.

1.5.4. Detection of intestinal microbial community structure

16S rRNA sequencing was used to analyze Vp-induced intestinal bacterial disturbance in mice, which was completed by Hangzhou Guhe Biotechnology Company. CK group, Vp group and H-JZXJ-7+Vp group (n=6/group) were extracted using cetyltrimethylammonium bromide, a total of 18 samples of genomic DNA were detected by 1% agarose gel electrophoresis DNA purity and integrity; using purified genomic DNA as a template, select high-efficiency and high-fidelity enzymes for PCR to amplify the 16S rDNA V4 region; mix the PCR products at equal concentrations and use 2% agarose gel electrophoresis to detect; the constructed The library was sequenced on the Illumina HiSeq4000 platform. Use QIIME (V.1.7.0) to calculate the Alpha (α) diversity index, including Chao1, Shannon and Simpson. Based on bray curtis, use the R language package (V.2.15.3) for principal coordinate analysis (Principal Coordinate Analysis, PCoA); classify the sequence similarity ≥ 97% into an operational taxonomic unit (operational taxonomic unit, OTU), and draw Species composition map and clustering heat map at the phylum and genus levels to determine the species structure differences between the three groups; the sequencing results are uploaded to the online website, and linear discriminant analysis (linear discriminant analysis [LDA] size effect, LEfSe) is used to identify the differences between the groups Biomarker species.

Result analysis

1. Bacillus subtilis JZXJ-7 relieves symptoms of Vibrio parahaemolyticus infection

The body weight changes of the mice in each group are shown in Figure 1A. The body weight of the mice in the control (CK) group showed a steady upward trend, and the body weight of the mice in the model (Vp) group continued to decline after 2 days of exposure to Vibrio parahaemolyticus infection. JZXJ-7 intervention (L-JZXJ-7+Vp) failed to significantly inhibit weight loss, but slowed it down to a certain extent, high-dose JZXJ-7 (H-JZXJ-7+Vp) intervention reversed weight loss, the experimental At the end, it was consistent with the body weight change of the CK group, an increase of (6.88±0.41)%, and there was a statistical significance with the Vp group (P<0.05); and mild diarrhea and bloody stools, reflected in the disease activity index (DAI) score (Figure 1B), the DAI score of healthy mice in the CK group did not change, and the DAI score of the Vp group increased significantly, reaching 8.23±0.21 on the 7th day after exposure to infection , the DAI score of the L-JZXJ-7+Vp group (7.46±0.33) was not significantly different from that of the Vp group, while the DAI score of the H-JZXJ-7+Vp group (3.92±0.18) was significantly lower than that of the Vp group (P<0.05) ; The load of intestinal pathogenic bacteria is an important indicator for evaluating infection damage (Fig. 1C). Vibrio parahaemolyticus was not detected in the intestinal soluble matter of mice in CK group, Vp group, L-JZXJ-7+Vp group and H-JZXJ- The amount of Vibrio parahaemolyticus in the ileum and cecum of mice in the 7+Vp group was lower, and the amount of bacteria in the colon was the highest, respectively (3.52±0.13), (3.13±0.11) and (1.52±0.07) lg CFU/g, According to the difference analysis among the groups, the amount of Vibrio parahaemolyticus colonization in the ileum, cecum and colon of the mice in the H-JZXJ-7+Vp group was significantly lower than that in the Vp group (P<0.05). In summary, JZXJ-7 intake of 10 ⁸ CFU/d can effectively resist weight loss, increase of disease activity index and colonization of intestinal pathogens in mice caused by Vibrio parahaemolyticus infection.

2. Bacillus subtilis JZXJ-7 improves colonic pathological damage in mice

Infection by enteric pathogens begins in the epithelial tissue. HE staining of the mouse colon showed (Fig. 2A). The colonic crypts of the healthy mice in the CK group were clear and well-defined, the mucosal epithelium was complete, and there was no inflammatory cell infiltration. The cells decreased, the depth of crypts became shallower, accompanied by hyperplastic connective tissue, and a large number of inflammatory cell infiltration could be seen in the lamina propria. The infiltration was significantly reduced, the focal intestinal gland structure disappeared, and JZXJ-7 improved the invasion and damage of Vibrio parahaemolyticus to the colon; PAS staining showed (Figure 2B, 2D), which was comparable to the number of goblet cells in the colonic epithelium of the CK group, which was 28.93±1.41 The number of goblet cells in the Vp group was significantly reduced to 6.72±0.79 (P<0.01), and the high-dose JZXJ-7 intervention in the H-JZXJ-7+Vp group significantly alleviated the disappearance of goblet cells induced by Vibrio parahaemolyticus (23.95±1.12, P<0.05), while the L-JZXJ-7+Vp group had no significant improvement; MASSON staining showed (Fig. 2C, 2E), compared with the CK group mice intestinal tissue collagen deposition (6.03±1.64)% Compared with Vp group, Vibrio parahaemolyticus infection led to a significant increase in colonic collagen deposition, reaching (13.12±1.45)%. Collagen deposition in the mouse colon was reduced, and high-dose JZXJ-7 intervention significantly reduced the accumulation of extracellular matrix on the intestinal wall ([7.83±1.21]%), which was significantly different from the Vp group (P<0.05); The comprehensive score of physical science showed that (Fig. 2F), there were differences in the colonic pathological scores of the mice in each group, among which the scores of the Vp group and the L-JZXJ-7+Vp group were higher, being (8.26±0.98) and (7.33±1.02), respectively, There was no statistically significant difference between the two data, followed by the H-JZXJ-7+Vp group (2.89±1.21), and the CK group was the lowest (1.55±1.03). Compared with the Vp group, the high-dose JZXJ-7 intervention group effectively reduced Pathological score (P<0.05).

3. Bacillus subtilis JZXJ-7 enhances the physiological function of mouse colon

Intestinal pathological damage leads to intestinal physiological dysfunction. As shown in Figure 3A-3C, compared with the CK group, the contents of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the colon of mice in the Vp group They were increased by 3.55, 2.44 and 2.12 times respectively, indicating that the infection of Vibrio parahaemolyticus caused excessive inflammation in the colon of mice, dietary supplementation of JZXJ-7 could reduce the levels of three pro-inflammatory factors in the colon of mice, and the effect of high dose on inhibiting inflammation was better than that of low dose Dose, IL-1β, IL-6 and TNF-α levels in H-JZXJ-7+Vp group were only 0.32, 0.41 and 0.28 times of Vp group, the difference was statistically significant (P<0.05); The activity results of catalase (CAT), superoxide dismutase (SOD) and total antioxidant enzymes (T-AOC) in mouse colon (Fig. 3D-3F), the activity of CAT, SOD and T-AOC in colon of Vp group These values were significantly lower than those in the CK group (P<0.01), indicating that Vibrio parahaemolyticus infection caused the decline in intestinal antioxidant capacity, and JZXJ-7 intervention increased the activities of the three antioxidant enzymes in the colon to varying degrees, especially high-dose intervention (H The T-AOC activity of mice in -JZXJ-7+Vp) group was close to that of CK group (P>0.05). The detection results of intestinal wall permeability markers in serum showed (Fig. 3F-3H), the serum lipopolysaccharide (LPS), diamine oxidase (DAO) and D-lactic acid (D-LA) concentrations of mice in Vp group were significantly higher than CK group (P<0.01), respectively 1.78 times, 1.52 times, 1.54 times of CK group, supplemented with high-dose JZXJ-7 significantly reduced the concentration of LPS, DAO and D-LA in mouse serum, while low-dose intervention (L-JZXJ There was no statistical significance between -7+Vp) group and Vp group (P>0.05). In summary, there is a significant dose-dependent relationship between JZXJ-7 intervention and colonic excessive inflammation, anti-oxidation, and intestinal wall permeability, and mice presupplemented with high doses (10 ⁸ CFU/d) of JZXJ-7 can effectively inhibit Intestinal physiological dysfunction caused by Vibrio parahaemolyticus infection. Therefore, the follow-up study chose the high-dose intervention group to further explore the effect of JZXJ-7 on the structure of the intestinal microbial community in mice.

4. Bacillus subtilis JZXJ-7 remodels the homeostasis of intestinal flora in mice

4.1. Analysis of α and β diversity of intestinal flora

In the α-diversity index of intestinal flora, Chao1 reflects the richness of the flora, and Shannon and Simpson reflect the diversity of the flora. The larger the Chao1 index and the Shannon index, the smaller the Simpson index, indicating that the species in the sample is more abundant. The results of the study showed that compared with the CK group, the Chao1 index and Shannon index of the Vp group were extremely significantly decreased, and the Simpson index was extremely significantly increased (P<0.01), and the high-dose JZXJ-7 (H-J+V) intervention significantly Increasing the Chao1 index and Shannon index and decreasing the Simpson index (P<0.05) increased the α-diversity of intestinal flora (Fig. 4A-4C). β-diversity is used to evaluate the difference in microbial structure between groups. Principal coordinate analysis (PCoA) is used to visualize the difference. According to the degree of distribution between samples, the similarity of the bacterial community structure is judged. The closer the distance between the sample points, the difference in community composition is indicated. The smaller the , the more similar the sample community composition. As shown in Figure 4D, compared with the CK group, the PC1 and PC2 levels of the Vp group shifted to the lower right, indicating that Vibrio parahaemolyticus infection significantly changed the structure and composition of the intestinal flora of mice; compared with the Vp group, the H In the -J+V group, the level of PC1 and PC2 shifted to the upper left, suggesting that the structure of the intestinal flora of the mice after JZXJ-7 intervention was substantially improved, and it tended to change in the CK group. Through clustering, the OTUs are grouped according to similarity, as shown in Figure 4E, there are 444 OTUs unique to the CK group, 57 OTUs unique to the Vp group, and 85 OTUs unique to the H-J+V group; the CK group and the Vp group share 169 OTUs, 104 OTUs in H-J+V group and Vp group, 175 OTUs in CK group and H-J+V group, indicating that infection with Vibrio parahaemolyticus reduces the similarity with normal mouse flora, The species abundance was reduced, while pre-supplementation with JZXJ-7 increased the number of common OTUs between the H-J+V group and the CK group by 71, indicating that JZXJ-7 partially restored the intestinal species richness of Vibrio parahaemolyticus-infected mice.

4.2. Analysis of intestinal flora structure

The analysis of the taxonomy of the flora showed that there were significant differences in the microbial structure among the three groups, as shown in Figure 5A, at the phylum level, the relative abundance of species > 1% was defined as the dominant phylum, Bacteroidetes and Firmicutes accounted for the highest proportion in each group, and were the dominant species; compared with the CK group, the abundance of Bacteroidetes in the intestinal flora of mice in the Vp group was significantly reduced, Firmicutes and Proteobacteria The abundance of the phylum (Proteobacteria) was significantly increased (P<0.05); compared with the Vp group, the high-dose JZXJ-7 intervention (H-J+V) group significantly up-regulated the abundance of the Bacteroidetes phylum, and down-regulated the thick-walled phylum and Proteobacteria Phyla abundance (P<0.05). Among the non-dominant species, compared with the CK group, the relative abundance of Deferribacteres and Actinobacteriota in the Vp group increased; compared with the Vp group, after high-dose JZXJ-7 intervention Significantly down-regulate the abundance of Deferrobacteria and Actinomycetes, and increase the abundance of Verrucomicrobia. At the genus level (Fig. 5B), compared with the CK group, the abundance of Bacteroides, Lactobacillus and Ruminiclostridium in the Vp group was significantly reduced, and the abundance of Helicobacter, Odoribacter The abundance of Bacillus, Enterobacter, Vibrios and Tyzzerella was significantly increased; compared with the Vp group, the H-J+V group mice intestinal Bacteroides, Lactobacillus, The genera Bifidobacterium and Akkermansia were significantly up-regulated, and the relative abundances of Helicobacter pylori, Enterobacter and Vibrio were significantly down-regulated. Cluster analysis based on the representative population of mice (Figure 5C), the results showed that the H-J+V group and the CK group were clustered in the same group, and there was a significant difference from the CK group, which further verified that high-dose JZXJ-7 relieved side effects. The intestinal flora disturbance caused by Vibrio hemolyticus infection showed a microbial community structure close to that of the CK group.

4.3 Analysis of marker species of intestinal flora

The analysis of intestinal flora diversity and flora structure showed that high-dose JZXJ-7 intervention alleviated the dysbiosis of mice induced by Vibrio parahaemolyticus and reshaped the microecological balance. Therefore, in order to identify the signature microbial groups that resist Vibrio parahaemolyticus infection in the high-dose JZXJ-7 intervention group, the LefSe analysis of the mouse colonic flora and the LDA effect size discrimination (Figure 6A-6B), the results showed that 23 categories Groups significantly affected the biological characteristics of the microbiota during Vibrio parahaemolyticus infection, involving: 4 phyla, 5 classes, 4 families and 10 genera. Specifically, compared with the Vp group, JZXJ-7 intervention reduced the levels of Proteobacteria, Enterobacteriaceae, Streptococcus, Prevotella, Sutterella (Sutterella) and Vibrios (Vibrios) relative abundance, increased Verrucomicrobiota, Lachnospiraceae NK4A136 (Lachnospiraceae NK4A136), Clostridia UCG-014 (Clostridia UCG-014), double Relative abundance of Bifidobacterium, Akkermansia and Clostridium.

In the mouse model of Vibrio parahaemolyticus infection, the number of goblet cells and antioxidant enzyme activity in the mouse colon were significantly reduced, while the collagen deposition, inflammatory response and permeability of the colon were significantly increased, and the above damage can be effectively alleviated by supplementing JZXJ-7 , and this process is closely related to the adjustment of microbial community structure. Therefore, in order to clarify the role played by key microbiota, Spearman's correlation analysis was used to study the correlation between specific bacterial communities and infection indicators (Fig. 6C), the results showed that CAT, SOD, T-AOC activity and goblet cell The number of Clostridia UCG-014 (Clostridia UCG-014) (0.426≤r≤0.541), Akkermansia (Akkermansia) (0.612≤r≤0.794), Lachnospiraceae NK4A136 (Lachnospiraceae NK4A136) (0.449 ≤r≤0.613), Bifidobacterium (0.534≤r≤0.835), Verrucomicrobia (0.421≤r≤0.633), Clostridium (0.592≤r≤0.818) significantly Positive correlation (P<0.05), among which Akkermansia, Bifidobacterium and Clostridium had strong positive correlation (r≥0.75), and Proteobacteria (-0.294≤r≤- 0.387), Prevotella (-0.275≤r≤-0.397), Escherichia (-0.296≤r≤-0.401) and Vibrios (-0.328≤r≤ -0.423) was significantly negatively correlated (P<0.05), but the correlation coefficient was low; in addition, colon tissue collagen deposition, pro-inflammatory factor (IL-1β, IL-6 and TNF-α) levels, serum LPS, DAO and D- LA concentration was significantly correlated with Proteobacteria (0.585≤r≤0.763), Prevotella (0.579≤r≤0.793), Escherichia (0.592≤r≤0.831) and Vibrio (0.614≤r≤0.823) Positive correlation (P<0.05), and the correlation is strong (r≥0.75).

Apparently, the specific embodiments described above are only further detailed descriptions of the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit In the present invention, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (7)

1. Application of Bacillus subtilis JZXJ-7 in the preparation of preparations for treating/relieving/improving Vibrio parahaemolyticus infection. 2. The application according to claim 1, wherein the Vibrio parahaemolyticus infection has at least one of the following manifestations: (1) blood in stool; (2) Pathological damage to the colon; (3) Excessive intestinal inflammatory response; (4) weakened antioxidant capacity; (5) Increased intestinal wall permeability; (6) Intestinal flora imbalance. 3. The application according to claim 2, characterized in that the pathological damage of the nodules is specifically manifested as: loss of colonic mucosal structure, reduction of mucosal cells, shallower crypt depth, accompanied by hyperplastic connective tissue, and a large amount of lamina propria can be seen Inflammatory cell infiltration; decreased number of colonic goblet cells; increased colonic collagen deposition. 4. The application according to claim 2, characterized in that the excessive intestinal inflammatory response refers to the up-regulation of the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-α in colon tissue. 5. The application according to claim 2, characterized in that said weakened antioxidant capacity refers to the down-regulation of catalase, superoxide dismutase and total antioxidant enzyme expression in colon tissue. 6. The application according to claim 2, characterized in that, the increase in intestinal wall permeability refers to the increase in serum lipopolysaccharide, diamine oxidase and D-lactic acid content. 7. The application according to any one of claims 1 to 6, characterized in that the dosage of the Bacillus subtilis JZXJ-7 is 10 ⁶ CFU/d-10 ⁸ CFU/d.

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