阅读说明：本技术 一种拮抗大肠杆菌o157：h7的戊糖片球菌及其应用 (An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof ) 是由 吴清平 李海新 李滢 谢新强 陈惠元 张菊梅 丁郁 王涓 陈谋通 薛亮 张淑红 于 2021-06-30 设计创作，主要内容包括：本发明公开了一种拮抗大肠杆菌O157：H7的戊糖片球菌及其应用。所述的戊糖片球菌为戊糖片球菌Pediococcus pentosaceus IM96,于2021年5月17日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO:61654。所述的菌株的活菌及发酵上清均具有良好抗菌活性,尤其对大肠杆菌O157：H7在体外、体内均有强的拮抗活性。此外该戊糖片球菌对胃肠液具有良好耐受性,安全性高,在体内能够缓解并改善由大肠杆菌O157：H7引起的肠道病理损伤、肠道炎症和肠道上皮屏障损伤。该戊糖片球菌将来可用于制备具有抗菌活性的药物、食品或食品添加剂、动物饲料和微生物制剂,具有良好的应用前景。(The invention discloses antagonistic Escherichia coli O157: h7 Pediococcus pentosaceus and its application. The Pediococcus pentosaceus is Pediococcus pentosaceus IM96, is preserved in Guangdong province microorganism strain preservation center at 17 th 5 th 2021, and has a preservation number of GDMCC NO: 61654. The viable bacteria and fermentation supernatant of the strain have good antibacterial activity, and particularly have antibacterial activity on Escherichia coli O157: h7 has strong antagonistic activity in vitro and in vivo. In addition, the pediococcus pentosaceus has good tolerance to gastrointestinal fluids and high safety, and can relieve and improve the gastrointestinal diseases caused by Escherichia coli O157: pathological damage to the gut, inflammation of the gut and damage to the epithelial barrier of the gut caused by H7. The pediococcus pentosaceus can be used for preparing medicaments, foods or food additives, animal feeds and microbial preparations with antibacterial activity in the future, and has good application prospect.)

1. Pediococcus pentosaceus IM96 with the preservation number: GDMCC No: 61654.

2. use of Pediococcus pentosaceus IM96 according to claim 1 for the manufacture of a medicament having antibacterial function.

3. The use according to claim 2, wherein the antibacterial agent is an agent capable of antagonizing the activity of O157: h7.

4. The use according to claim 3, wherein the drug with antibacterial function is a drug that relieves and reduces the activity of Escherichia coli O157: h7, and can ameliorate the inflammatory level of e.coli O157: h7 infection is a drug for pathological damage caused by small intestine.

5. Use of Pediococcus pentosaceus IM96 according to claim 1 for the production of a food product or food additive.

6. Use of Pediococcus pentosaceus IM96 according to claim 1 for the production of animal feed.

7. Use of pediococcus pentosaceus IM96 according to claim 1 for the preparation of a microbial preparation.

8. Use according to claim 7, wherein the microbial preparation is used as an antimicrobial in the field of pharmaceuticals, food additives or animal feed.

9. A pediococcus pentosaceus IM96 specific molecular target according to claim 1, characterized in that the nucleotide sequence is represented by seq ID No. 2.

10. A primer for identifying Pediococcus pentosaceus IM96 according to claim 1, comprising a forward primer: 5'-ATCGCCAAAGCCAAACGTC-3' and reverse primer: 5'-TTCCTCCTGTCAACTCATAGC-3' are provided.

The technical field is as follows:

the invention belongs to the technical field of microorganisms, and particularly relates to pediococcus pentosaceus with antibacterial activity and application thereof.

Background art:

enterohemorrhagic escherichia coli (EHEC) is a pathogenic subgroup of shiga toxin-producing escherichia coli (STEC) that can produce shiga toxin 1 and/or 2(Stx1 and Stx2), often containing adhesion genes (eae). Escherichia coli O157H 7 is the most common pathogenic serotype of enterohemorrhagic Escherichia coli. Enterohemorrhagic escherichia coli can cause systemic complications such as hemorrhagic diarrhea, hemorrhagic enteritis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Since enterohemorrhagic Escherichia coli was first discovered in 1982 in the United states, contamination of food with enterohemorrhagic Escherichia coli has occurred in many countries of the world, leading to many deaths. In 1996, a pandemic of the world's largest and large outbreak of E.coli O157 infection involving tens of thousands of people occurred in Japan. In germany, in 5 months 2011, a large-scale food poisoning event by EHEC O157 was outbreaked, resulting in infection of more than 4000 consumers, out of which 908 had hemolytic uremic symptoms and 40 died. Since the first report in 1986, China has discovered Escherichia coli EHEC O157 in many provinces and cities in sequence: sporadic cases of H7 infection. In addition, enterohemorrhagic escherichia coli is a major cause of morbidity and mortality in children under 5 years of age, particularly in developing countries. In conclusion, the food poisoning caused by enterohemorrhagic escherichia coli is always one of the major public health problems, and simultaneously causes great harm to the life and property safety of people. Currently for entero-hemorrhagic escherichia coli O157: the prevention and control of H7 is mainly by using antibiotics. However, due to the overuse and irregular use of antibiotics in recent years, many resistant microorganisms and super-resistant bacteria appear, which will bring serious threat to human health. Therefore, there is a need for a safe, stable, cost-effective antimicrobial agent that can reduce or even replace the use of antibiotics.

Lactic acid bacteria are recognized as food-grade safe microorganisms worldwide, and some strains have been listed as probiotics by FAO and WHO. A large number of researches show that the probiotic lactic acid bacteria have the functions of antagonizing pathogenic bacteria, regulating immunity, enhancing intestinal barrier and balancing intestinal flora. The lactobacillus can be directly used as a food additive to improve the health of human bodies, and the fermentation metabolite of the lactobacillus can be used as an antibacterial agent to inhibit the growth of a large number of food-borne pathogenic bacteria. Therefore, probiotics and their metabolites are of great interest as potential antibacterial agents in the treatment of diseases caused by pathogenic bacteria. Pediococcus pentosaceus (Pediococcus pentosaceus) belongs to the family Lactobacillaceae (Lactobacillus) and Pediococcus (Pediococcus). Pediococcus pentosaceus is a Generally Regarded As Safe (GRAS) species that has been included in the list of edible species by relevant national departments.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a novel polypeptide having antagonistic activity against Escherichia coli O157: h7 active Pediococcus pentosaceus IM96 and application thereof. The strain was deposited at the Guangdong province culture Collection (GDMCC) at 11/5/2021, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61654.

another object of the present invention is to provide a use of Pediococcus pentosaceus IM96 for producing a medicament having an antibacterial function.

Preferably, the drug having an antibacterial function is capable of antagonizing escherichia coli O157: H7.

it is further preferred to alleviate and reduce the risk of infection of the body with e.coli O157: the level of inflammation after H7, and can improve the activity of Escherichia coli O157: h7 infection is a drug for pathological damage caused by small intestine of infected people.

Another object of the present invention is to provide a use of Pediococcus pentosaceus IM96 for producing food or food additives.

Another object of the present invention is to provide the use of Pediococcus pentosaceus IM96 for the production of animal feed.

Another object of the present invention is to provide the use of Pediococcus pentosaceus IM96 for the preparation of a microbial preparation.

Preferably, the microbial preparation is used as an antibacterial agent in the fields of medicines, foods or food additives and animal feeds.

Another objective of the present invention is to provide a specific molecular target of pediococcus pentosaceus IM96 and primers for detecting the specific target, wherein the specific molecular target is shown in SEQ ID No.2, and the primers for detecting the specific target include a forward primer: 5'-ATCGCCAAAGCCAAACGTC-3' and reverse primer: 5'-TTCCTCCTGTCAACTCATAGC-3' are provided.

The invention has the beneficial effects that:

1. the Pediococcus pentosaceus IM96 provided by the invention is obtained by separating and purifying from Brassica napus pickled in inner Mongolian countryside, and the food source is safe and reliable. According to an antibiotic susceptibility test, Pediococcus pentosaceus IM96 is not resistant to 7 common antibiotics; meanwhile, the whole genome analysis finds that the strain has no virulence genes and drug resistance genes, so the safety is high.

2. Pediococcus pentosaceus IM96 has good Escherichia coli O157 antagonism: the activity of H7, the diameter of inhibition zone is as high as 24.67 mm.

3. Pediococcus pentosaceus IM96 has good gastrointestinal fluid resistance. The survival rate in gastric fluid was 80.31%, and in intestinal fluid was 80.21%.

4. Pediococcus pentosaceus IM96 can relieve and reduce the infection of organisms with Escherichia coli O157: the level of inflammation after H7, and can improve the activity of Escherichia coli O157: h7 infection causes pathological damage to the small intestine of infected persons.

Pediococcus pentosaceus IM96 was deposited at 11 th 5 th 2021 with the collection of microorganisms of the Guangdong province (GDMCC) at the address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61654.

drawings

FIG. 1 shows that Pediococcus pentosaceus IM96 antagonizes E.coli O157: graph of experimental results for H7;

FIG. 2 is a graph showing the results of the survival rate and weight change of mice;

FIG. 3 is a graph showing the results of the morphology of the small intestine of mice;

FIG. 4 is a graph showing the results of the mouse jejunal inflammation indicators;

FIG. 5 is a graph showing the results of the mouse jejunal intestinal epithelial barrier indicators;

FIG. 6 is a diagram of the validation of specific molecular targets of Pediococcus pentosaceus IM 96.

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

The present invention will be described in detail with reference to the following embodiments in order to better illustrate the objects, technical solutions and advantages of the present invention.

The media referred to in the examples below are as follows

MRS agar plate (g/L): 10.0g/L of peptone, 5.0g/L of beef extract, 4.0g/L of yeast extract powder, 20.0g/L of glucose and 801.0 ml/L, K of tween₂PO₄·3H₂02.0 g/L, sodium acetate 5.0g/L, and triammonium citrate 2.0g/L, MgSO g₄·7H₂0 0.2g/L、MnSO₄·4H₂00.05 g/L agar 20g/L solvent is water, and its preparation method comprises mixing the above components, and sterilizing. MRS liquid medium was without agar addition.

MRS broth (g/L): 10.0g/L of casein digest, 10.0g/L of beef extract, 4.0g/L of yeast extract powder, 2.0g/L of triammonium citrate and 5.0g/L, MgSO of sodium acetate₄·7H₂0 0.2g/L、MnSO₄·4H₂0 0.05g/L、K₂PO₄·3H₂02.0 g/L, glucose 20.0g/L, Tween 801.0 g/L, and water as solvent, and its preparation method comprises mixing the above components, and sterilizing.

Example 1 isolation and identification of lactic acid bacteria

1. Isolation of lactic acid bacteria

Collection of inner Mongolia autonomous region of ChinaTaking a Brassica napus sample from the Mandarin China, Colubel, as a sample, adding 0.1g of the Brassica napus sample into 5ml of MRS liquid culture medium in an aseptic environment, performing enrichment culture for 24h under an anaerobic condition at 37 ℃ after shaking and mixing uniformly, and sucking 0.5ml of bacterial liquid for gradient dilution. Adding physiological saline to obtain 10^-1To 10^-5Diluting the gradiental bacterial suspension, and respectively sucking 10^-3、10^-4、10^-5And (3) uniformly smearing three gradiental bacteria suspensions to an MRS agar culture medium by using a coating rod, and then culturing for 48 hours at 37 ℃ under an anaerobic condition. And selecting colonies with typical shapes on the plate to an MRS agar culture medium for streak purification, selecting a single colony after two purifications to inoculate into an MRS liquid culture medium, and culturing at 37 ℃ for 24 hours to collect thalli for extracting thalli DNA.

2. Identification of lactic acid bacteria

Bacterial DNA extraction was performed using a bacterial DNA extraction kit (Mabio, CHINA), followed by PCR amplification using 2 × PCR mix (Dongshengbio, CHINA). The PCR amplification primer adopts a 16SrRNA gene universal primer, and the sequence of an upstream primer is 27F: 5'-AGA GTTTGATCCTGGCTCAG-3', respectively; the sequence of the downstream primer is 1492R: 5'-CTACGGC TACCTTGTTACGA-3' are provided. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ 30s, 56 ℃ 30s and 72 ℃ 1min 30s, and annealing and extending at 72 ℃ for 10 min. The PCR products were recovered by gel cutting and subjected to one-generation sequencing (performed by Jinzhi Biotechnology, Inc., Suzhou). The sequence of the obtained 16SrRNA gene is shown as SEQ ID No. 1. This sequence was aligned with the NCBI database (https:// blast. NCBI. nlm. nih. gov), and as a result, it was suggested that the homology with Pediococcus pentosaceus was the highest, so this strain was named Pediococcus pentosaceus (Pediococcus pentosaceus) IM 96.

Pediococcus pentosaceus (Pediococcus pentosaceus) IM96 was deposited at 11/5/2021 with the GDMCC (GDMCC) in the guangdong province collection of microorganisms, address: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5, zip code: 510070, accession number: GDMCC No: 61654.

3. method for activating, culturing and preserving lactic acid bacteria

The pediococcus pentosaceus IM96 strain was removed from the freezer at-80 ℃ and activated twice in MRS agar medium. Selecting colonies, inoculating in MRS liquid culture medium, and anaerobically culturing at 37 deg.C for 24-48 h. Adding glycerol into the cultured bacterial liquid, and storing in a refrigerator at-80 deg.C.

EXAMPLE 2 evaluation of antibacterial ability of Pediococcus pentosaceus IM96

1. Preparation of lactic acid bacteria fermentation liquor

A single colony of Pediococcus pentosaceus IM96 was inoculated into MRS broth and cultured anaerobically at 37 ℃ for 48 hours. Centrifuging at 10000r × 4 deg.C for 10min to obtain fermentation supernatant. Filtering the fermentation supernatant by using a 0.22 mu m filter membrane to obtain cell-free supernatant fermented by Pediococcus pentosaceus IM96, and freezing the cell-free supernatant at-80 ℃ for later use.

2. Preparation of indicator bacterium

Escherichia coli O157: h7 ATCC43895, inoculating to LB liquid medium, culturing at 37 deg.C and 200r/min for 6H, adjusting concentration to 1 × 10⁸CFU/mL, antibacterial experiments were performed.

3. Pediococcus pentosaceus IM96 antibacterial ability determination

Taking 200 mu L of the prepared indicator bacterium liquid, uniformly coating the indicator bacterium liquid on a nutrient agar culture medium, placing sterile oxford cups on a flat plate by using sterilized tweezers, and adding 200 mu L of the prepared supernatant into each oxford cup. Meanwhile, MRS medium was used as a blank control group. The plate was placed in a refrigerator at 4 ℃ for uniform diffusion, and then cultured at 37 ℃ for 18 hours, and the diameter of the zone of inhibition was measured. As can be seen from FIG. 1, Pediococcus pentosaceus IM96 is responsible for Escherichia coli O157: h7 ATCC43895 has a zone of inhibition of 24.67 + -0.58 mm.

Example 3 evaluation of probiotic Properties of Pediococcus pentosaceus IM96

1. Resistance to Artificial gastric juice test of Pediococcus pentosaceus IM96

Tolerance to artificial gastric juice test: PBS (pH7.4) buffer solution was adjusted to pH 3.0 with 0.1mol/L HCl, pepsin (3g/L) was added, and after dissolution, the mixture was filtered through a 0.22 μm sterile filter membrane and ready to use. Activating the screened strain, inoculating the prepared bacterial liquid of Pediococcus pentosaceus IM96 in 10mL of MRS liquid with the inoculation amount of 2 percent of volume ratioStanding at 37 deg.C under anaerobic condition for 24 hr, centrifuging at 4000 Xg for 10min, collecting thallus, washing thallus with PBS (pH7.4) for 3 times, and adjusting bacterial liquid concentration to 10 with PBS⁹CFU/mL, spare. Adding 1mL of PBS resuspended lactobacillus bacterial liquid into 5mL of simulated gastric juice, adding 1.5mL of 0.5% (w/v) NaCl, mixing uniformly, quickly placing into a 37 ℃ incubator, culturing for 0h and 3h, diluting with sterile physiological saline according to a gradient of 1:10, sucking 100 mu L of diluent, uniformly coating the diluent in an MRS solid culture medium by using a disposable coating rod, culturing for 48h at 37 ℃, counting the viable lactobacillus, and calculating the survival rates of 0h and 3h of Pediococcus pentosaceus IM96 under the condition of pH 3.0 according to the following formula.

Survival rate (%). 3h survival number of strain/0 h survival number of strain × 100%

2. Tolerance to Artificial intestinal juice test of Pediococcus pentosaceus IM96

Tolerance to artificial intestinal juice experiment: PBS buffer was adjusted to pH 8.0 with 0.1mol/L NaOH, and 1mg/mL trypsin and 0.3% bovine bile salt were added. Adding 1mL of resuspended Pediococcus pentosaceus IM96 strain (bacterial suspension is prepared to be tolerant to artificial gastric juice) into 5mL of simulated intestinal fluid, quickly and uniformly mixing, putting the mixture into a 37 ℃ incubator, diluting the mixture by sterile physiological saline according to a gradient of 1:10 after 0h and 4h, sucking 100 mu L of diluent, uniformly coating the diluent in an MRS solid culture medium by using a disposable coating rod, culturing the mixture for 48h at 37 ℃, and counting viable bacteria of Pediococcus pentosaceus IM96, wherein the survival rate of different lactic acid bacteria strains for 4h is calculated by the following formula.

Survival rate (%). 4h survival number of strain/0 h survival number of strain × 100%

The result of an artificial gastrointestinal fluid simulation experiment on the screened strain shows that the gastric fluid resistant survival rate of Pediococcus pentosaceus IM96 is 80.31 +/-2.88%. The survival rate of the tolerant intestinal juice is 80.21 +/-3.46%.

3. Antibiogram of Pediococcus pentosaceus IM96

Salmonella typhimurium ATCC 14028, Enterobacter sakazakii ATCC 29544, Yersinia enterocolitica GDMCC60852, Pseudomonas aeruginosa ATCC 15442, and Bacillus sakazakii ATCC 29544,Respectively inoculating single colonies of Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19117, Bacillus cereus ATCC 14579 and Bacillus subtilis ATCC 6633 into LB liquid medium, culturing at 37 deg.C and 200r/min overnight, adjusting concentration to 1 × 10⁸CFU/mL. The antimicrobial spectrum of Pediococcus pentosaceus IM96 was determined by the Oxford cup agar diffusion method. As can be seen from the diameter of the inhibition zone in Table 1, Pediococcus pentosaceus IM96 has good antibacterial activity against the food-borne pathogenic bacteria, which indicates that Pediococcus pentosaceus IM96 is a potential probiotic with good application prospect and broad-spectrum antibacterial activity.

TABLE 1 bacteriostasis spectra of Pediococcus pentosaceus IM96

Example 4 Pediococcus pentosaceus IM96 genome and phenotypic safety evaluation thereof

1. Genome safety evaluation of Pediococcus pentosaceus IM96

Whole genome sequencing was performed on Pediococcus pentosaceus IM96 using the Illumina Nextseq 550 second generation sequencing platform. The extraction method of bacterial genome DNA is the same as that of the previous method. The second generation sequencing was performed by AMT Rapid DNA-Seq Kit for Illumina (CISTRO, CHINA) library and High Output v2.5 Kit (Illumina, USA).

The offline data is subjected to quality control by using Trimmomatic software (v0.39), and then is assembled by using SPAdes software (v 3.13.1). After assembly, the pediococcus pentosaceus genome was subjected to quality control evaluation using Quast software (v5.0.2), and for genomes that were qualified in quality control, the Prokka software (v1.13) was used for annotation. The comparison of VFDB (viral Factor database), ARG-Ant (inflammatory Resistance Gene-ANNOTAT) and CARD (the Comprehensive anti-inflammatory Research database) databases by Abricate software does not find that the bacteria contain virulence genes or drug Resistance genes.

2. Phenotypic safety evaluation of Pediococcus pentosaceus IM96

2.1 evaluation of the drug resistance phenotype of Pediococcus pentosaceus IM96

The sensitivity of Pediococcus pentosaceus IM96 to antibiotics was tested using the paper diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) standards. Seven antibiotics, namely ampicillin, erythromycin, clindamycin, tetracycline, chloramphenicol, imipenem and rifampin, were selected for the test, and the results are shown in Table 2 (note: R is drug-resistant and S is sensitive).

TABLE 2 susceptibility testing of antibiotics

As can be seen from Table 2, Pediococcus pentosaceus IM96 is sensitive to the antibiotics ampicillin, erythromycin, clindamycin, tetracycline, chloramphenicol, imipenem, and rifampicin.

2.2 evaluation of the hemolytic phenotype of Pediococcus pentosaceus IM96

mu.L of Pediococcus pentosaceus IM96 was spotted onto blood plates, respectively, using Staphylococcus aureus ATCC 25923 as control. The results were observed after incubation at 37 ℃ for 48 h. If a clear circular mark is observed around the colony, beta-hemolysis is observed, alpha-hemolysis is observed in brown or green, and gamma-hemolysis is not observed.

The results show that no phenomenon occurs around Pediococcus pentosaceus IM96, and the hemolytic type is gamma-hemolysis. The Pediococcus pentosaceus IM96 is therefore not hemolytic.

Example 5 Pediococcus pentosaceus IM96 relieves and improves mice from e.coli O157: pathological injury of intestinal tract, inflammation of intestinal tract and injury of epithelial barrier of intestinal tract caused by H7

1. Animal experiment and design

For the strain with antagonistic Escherichia coli O157: pediococcus pentosaceus IM96 with H7 activity, good probiotic properties and safety profileAnd (5) carrying out mouse experiment verification. A7 week old SPF rated C57BL/6 mouse from Beijing Huafukang Biotech GmbH. Mice were placed under controlled environmental conditions (temperature 23 ± 3 ℃, relative humidity 50% -60%, light/dark cycle 12/12h, free access to water and food during the experiment). Mice began adaptation 1 week prior to the experiment and were randomized into 4 groups. The mouse experiment was designed as follows: control group (Control group): intragastrically administering 0.1ml PBS every day, and intragastrically administering 0.1ml PBS 4h later; enterohemorrhagic escherichia coli O157: h7 infection group (EHEC group): intragastric administration of 1X 10 daily⁹cfu E.coli O157: h7 ATCC43895, gavage 0.1ml PBS 4H later; norfloxacin dried group (Norfloxacin group): intragastric administration of 1X 10 daily⁹cful E.coli O157: h7 ATCC43895, 5mg/kg of norfloxacin was gavaged after 4 hours; pediococcus pentosaceus IM96 dried group (IM96 group): intragastric administration of 1X 10 daily⁹cful E.coli O157: h7 ATCC43895, gavage 1X 10 after 4H⁸cfu Pediococcus pentosaceus IM 96. Gavage continued for 1 week.

2. Survival rate and weight change in mice

Experimental procedure measurements mice survival and weight changes were recorded and the results are shown in figure 2. The survival rate of the control group was 100%, whereas E.coli O157: the survival rate of the H7-infected group decreased to 80%. However, after Pediococcus pentosaceus IM96 dry prognosis, the survival rate of the IM96 dry prognosis group is increased to 100%, and the norfloxacin dry prognosis group has the same effect. Coli O157: the H7-infected group had significantly reduced body weight. Infection with escherichia coli O157 with significant remission of Pediococcus pentosaceus pentasaceus im96 and norfloxacin intervention: h7 mice lost weight. Statistical analysis was performed using software SPSS 26.0 using either t-test or one-way analysis of variance. Values are expressed as mean ± standard deviation, with p-values <0.05 considered statistically significant. In addition, p values are expressed as p <0.05, p <0.01, p < 0.001.

3. Morphological observations of jejunum and ileum tissues

At the end of the experiment, the mice were sampled and the jejunum and ileum of the mice were rapidly fixed in 10% neutral formalin. The tissue is dehydrated and transparent, embedded in paraffin and sliced to prepare tissue slices. Morphological observation of large and small intestine tissues was performed by H & E staining. The results are shown in FIG. 3.

As can be seen from FIG. 3, the villi in the small intestine of the control mice are completely structured and regularly arranged, and have no shedding phenomenon. The intestinal mucosa epithelium is intact, and the epithelial cell structure is normal without rupture. Coli O157: the H7 infected group has different erosion and shedding of small intestine villi, severe damage to intestinal mucosa, inflammatory cell infiltration and congestion in ileum. Compared with a model group, the norfloxacin and Pediococcus pentosaceus IM96 dry preparation group can relieve inflammatory infiltration of small intestine, relieve shedding of small intestine villi, relieve villi erosion and mucosal barrier damage, and protect small intestine villi structure and intestinal mucosal barrier.

4. Measurement of intestinal inflammation indicators

Preparing jejunum tissue homogenate, and detecting and analyzing the intestinal inflammation indexes and levels of the mice by using IL-1 beta, IL-6, TNF-alpha and IL-10 inflammatory factor Elisa kits. As can be seen from FIG. 4, in comparison with the control group, the ratio of Escherichia coli O157: h7 infection group was determined in e.coli O157: after H7 ATCC43895 is infected, the concentrations of proinflammatory factors IL-1 beta, IL-6 and TNF-alpha are obviously improved, and the successful modeling of the infection model is proved. Compared with the infected group, the proinflammatory factors IL-1 beta, IL-6 and TNF-alpha of the norfloxacin dried group and the Pediococcus pentosaceus IM96 dried group are obviously reduced. In addition, Pediococcus pentosaceus IM96 also significantly increased the production of the anti-inflammatory factor IL-10 compared to the infected group. In conclusion, Pediococcus pentosaceus IM96 has a good function of regulating intestinal inflammation. In particular, p <0.05, p <0.01, p < 0.001.

5. Intestinal epithelial barrier index measurement

Jejunal tissue homogenates were prepared and the content of MUC2, Occludin, ZO-1 in the jejunum of mice was measured for analysis of the epithelial barrier of the intestine of mice according to the protocol of the Elisa test kit. As can be seen from FIG. 5, in comparison with the control group, the ratio of Escherichia coli O157: h7 infection group was determined in e.coli O157: after H7 ATCC43895 infection, the concentration of MUC2, Occludin and ZO-1 in jejunum of mice is remarkably reduced, which indicates that the mice are infected by Escherichia coli O157: h7 disrupts the integrity of the intestinal barrier of mice. Compared with an infected group, the content of MUC2, Occludin and ZO-1 in the norfloxacin dried group and the Pediococcus pentosaceus IM96 dried group is remarkably increased. In conclusion, Pediococcus pentosaceus IM96 has a good function of repairing intestinal epithelial barriers. In particular, p <0.05, p <0.01, p < 0.001.

Example 6 excavation and validation of specific molecular targets of Pediococcus pentosaceus IM96

1. Excavation of specific molecular targets of Pediococcus pentosaceus IM96

Pangenomic analysis was performed on the genomes of Pediococcus pentosaceus IM96 and the other 68 strains of Pediococcus pentosaceus and the other 787 strains of Lactobacillus in the NCBI database using the Prokka (v1.11) and Roary (v3.11.2) software. After obtaining the core genome, genes containing a higher density of base substitutions were identified using Gubbins (v2.4.1) software. The Pediococcus pentosaceus IM96 obtained based on pan-genomic analysis has a specific sequence SEQ ID No.2 different from other Pediococcus pentosaceus and Lactobacillus. Designing a specific PCR amplification primer group according to a specific gene fragment sequence SEQ ID NO.2 of Pediococcus pentosaceus IM96 strain: a forward primer: 5'-ATCGCCAAAGCCAAACGTC-3' and reverse primer: 5'-TTCCTCCTGTCAACTCATAGC-3', primer set sequences are shown in Table 3 below.

TABLE 3 PCR primer set for detecting specific gene fragment sequence

2. Validity verification of identification target of Pediococcus pentosaceus IM96 specific molecule

The effectiveness of the specific molecular target sequence of Pediococcus pentosaceus IM96 was verified by Polymerase Chain Reaction (PCR) and agarose electrophoresis. The detection template is the DNA of bacteria, and the DNA extraction method is the same as the previous method.

The PCR reaction system is configured as follows:

the PCR reaction conditions were as follows:

after the PCR was completed, 5. mu.l of the PCR product was subjected to 1.2% agarose electrophoresis. If Pediococcus pentosaceus IM96 can generate a single specific band at 388bp, and other non-target bacteria can not generate a band at 388bp, the target pair has good efficiency of recognizing Pediococcus pentosaceus IM 96.

The result of PCR amplification product gel is shown in FIG. 6, where M is DL2000DNA standard marker, + is Pediococcus pentosaceus IM96, and C is negative control (the template of the control is DEPC water without genome). 1-230 are non-target pediococcus pentosaceus

As shown in FIG. 6, no specific amplification product was formed in any of the other Pediococcus pentosaceus and other species, except for the specific amplification product formed at 388bp after the primer amplification of the DNA of the target strain Pediococcus pentosaceus IM 96. The result proves that the PCR amplification primer is a specific molecular target of Pediococcus pentosaceus IM 96.

The strains used and the results of the tests are shown in table 4 below; in the table, "+" indicates positive and "-" indicates negative in the test result column.

TABLE 4 detection results of Pediococcus pentosaceus IM96 specific target

Although the preferred embodiments of the present invention have been disclosed, it should be understood that they are not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention should be subject to the content defined by the claims.

Sequence listing

<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)

GUANGDONG HUANKAI BIOTECHNOLOGY Co.,Ltd.

<120> an antagonistic Escherichia coli O157: h7 pediococcus pentosaceus and application thereof

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<213> Pediococcus pentosaceus IM96(Pediococcus pentosaceus)

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agacggctag ctcctaaaag gttaccccac cggctttggg tgttacaaac tctcatggtg 60

tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg atccgcgatt 120

actagcgatt ccgacttcgt gtaggcgagt tgcagcctac agtccgaact gagaatggtt 180

ttaagagatt agcttaacct cgcggtttcg cgactcgttg taccatccat tgtagcacgt 240

gtgtagccca ggtcataagg ggcatgatga tttgacgtcg tccccacctt cctccggttt 300

gtcaccggca gtctcactag agtgcccaac ttaatgctgg caactagtaa taagggttgc 360

gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac 420

ctgtcattct gtccccgaag ggaacctcta atctcttaga ctgtcagaag atgtcaagac 480

ctggtaaggt tcttcgcgta gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc 540

cccgtcaatt cttttgagtt tcaaccttgc ggtcgtactc cccaggcgga ttacttaatg 600

cgttagctgc agcactgaag ggcggaaacc ctccaacact tagtaatcat cgtttacggc 660

atggactacc agggtatcta atcctgttcg ctacccatgc tttcgagcct cagcgtcagt 720

tgcagaccag acagccgcct tcgccactgg tgttcttcca tatatctacg catttcaccg 780

ctacacatgg agttccactg tcctcttctg cactcaagtc tcccagtttc caatgcactt 840

cttcggttga gccgaaggct ttcacattag acttaaaaga ccgcctgcgc tcgctttacg 900

cccaataaat ccggataacg cttgccacct acgtattacc gcggctgctg gcacgtagtt 960

agccgtggct ttctggttaa ataccgtcac tgggtaaaca gttactctta cccacgttct 1020

tctttaacaa cagagcttta cgagccgaaa cccttcttca ctcacgcggc gttgctccat 1080

cagacttgcg tccattgtgg aagattccct actgctgcct cccgtaggag tctgggccgt 1140

gtctcagtcc caatgtggcc gattaccctc tcaggtcggc tacgtatcac tgccttggtg 1200

agcctttacc tcaccaacta gctaatacgc cgcgggtcca tccagaagtg atagcagagc 1260

catcttttaa aagaaaacca tgcggttttc tctgttatac ggtattagca tctgtttcca 1320

ggtgttatcc cctacttctg ggcaggttac ccacgtgtta ctcacccgtt cgccactcac 1380

ttcgtgttaa aatctcaatc agtacaagta cgtcataatc aattaacgga agttcg 1436

<210> 2

<211> 825

<212> DNA

<213> Pediococcus pentosaceus IM96(Pediococcus pentosaceus)

<400> 2

atgtttaaaa agtgtgaaat ttgtaataaa aagattggat tattttctaa accatttaca 60

actgatactg gtgcaaaagt atgtacctct tgtacatctt atgtagatcc aattttttta 120

actgaaaata ttacaacagc cgaatcaata gctttcgata tggataatga acttggaaat 180

tcttacttag caaaaattgg aaaagagtca ccttctgact tacgtaaaaa agaagaagag 240

gccgctaaac aagaactact tcaaaagcag aaagaagagg ctgaacgaca agaaatttta 300

aaacaagcac aagaaactat cgccaaagcc aaacgtcaaa aaatatatca ttttaaagtt 360

cgaggcgtat ctcactatga tttatcaaaa ttagttagtt atgcaaggaa aaatgactta 420

attttcccct atgatggttt cactgcatca gaaataaagg agatatgccc atacgaaaaa 480

gtttttgaaa cagacttagt gggaacatca ctaactatta gcttaattcc tgaacccgaa 540

aataaatatg atccaaacgc cataaaggta atagcaactc atgaagaaaa acaatttatg 600

ttaggttatg ttccaactga acatactaaa gaagttgcag atattatgca gaagcaaaat 660

gaaaacaaaa ttagaatgca catcagctat gagttgacag gaggaaaata caaaatagct 720

gaggaaaatt ttgatgattt ttcagatgat ccaaagctaa aaattcgtac tggaaaagtt 780

gaatatggtt ttaacataga aatccacgat atgaatattg attaa 825