CN116426406A

CN116426406A - Saliva combined lactobacillus DY802 and application thereof

Info

Publication number: CN116426406A
Application number: CN202211621413.2A
Authority: CN
Inventors: 丁轶; 范宏英; 俞露; 陈振辉; 罗紫倩; 古心怡
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2022-12-16
Filing date: 2022-12-16
Publication date: 2023-07-14
Anticipated expiration: 2042-12-16
Also published as: CN116426406B

Abstract

The invention provides a saliva combined lactobacillus DY802 and application thereof. The saliva combined lactobacillus DY802 is separated from fresh feces of healthy adults, has better acid resistance and bile salt resistance, and can reduce diarrhea and maintain intestinal canal steady state by producing a large amount of bacteriocin, thereby effectively preventing and treating chemotherapy-induced intestinal injury and effectively treating the problem of chemotherapy-induced intestinal injury of malignant tumor patients.

Description

Saliva combined lactobacillus DY802 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms and the technical field of medicines, and particularly relates to a saliva combined lactobacillus DY802 (Ligilactobacillus salivarius DY802, DY802 for short) and application thereof.

Background

Chemotherapy is one of the classical approaches to malignant tumor treatment that is concurrent with radiotherapy and surgery. Patients with malignant tumor can realize tumor shrinkage by chemotherapy and can also have different degrees of normal tissue damage. Adverse reactions, particularly those with diarrhea as a typical symptom, among which chemotherapy-induced intestinal injury occurs in 50% -80% of patients. Severe diarrhea can lead to not only intestinal flora disorders, but even death of the patient by induction of sepsis. The chemotherapy intestinal injury in the treatment process can cause the obvious reduction of the life quality of the patient, the poor treatment compliance, the prolongation of the whole treatment course is initiated, and finally the overall survival prognosis of the patient is influenced.

Currently, the treatment of chemotherapy-induced intestinal injury is mainly carried out as a supportive treatment and a drug treatment. Support treatment is mainly fluid infusion and rest. The medicine treatment is mainly carried out by loperamide, sulfasalazine, octreotide and the like. Although there is a well-established treatment regimen for chemotherapy diarrhea, 15% -30% of patients are ineffective in the above treatments, and refractory diarrhea occurs. Meanwhile, by using sulfasalazine as a typical sulfanilamide antibiotic, the disorder of intestinal flora cannot be improved, and even the steady state damage of intestinal micro-ecology can be aggravated, so that the wide application of the sulfasalazine is limited.

In recent years, a sterile mouse model suggests that mice are more tolerant to high doses of chemotherapeutic drugs in the absence of enteric bacteria; the mice experiments also suggest that the use of antibiotics in chemotherapy treatment will induce severe destruction of microbiota, exacerbating chemotherapy-induced diarrhea, while autologous fecal transplantation may alleviate diarrhea, and the laboratory data suggest that intestinal microbiota is involved in the development and progression of chemotherapy-induced diarrhea. Review of clinical data suggests that intestinal flora disorder, substantial reduction in flora species abundance and community diversity, etc. occur in colorectal cancer patients after chemotherapy with fluorouracil, irinotecan, etc., with the most significant reduction in the number of lactobacilli, bifidobacteria, bacteroides. The large clinical control test and meta-analysis show that the lactobacillus can remarkably prevent and treat diarrhea caused by chemotherapy of malignant tumor patients, and can reduce the use of other antidiarrheal medicines.

The lactobacillus salivarius (Ligilactobacillus salivarius) is a lactobacillus species and lactobacillus salivarius species, has no thixotropic enzymes, oxidases and motility, is a gram-positive probiotic which can grow in both aerobic and anaerobic environments, and is often present in the intestinal tracts of humans and animals. The saliva combined lactobacillus has been reported to have good application effects in aspects of host intestinal health, body immunity regulation and the like. And whether the saliva combined with lactobacillus can effectively play the role of the lactobacillus in maintaining intestinal health and the like or not depends on factors such as whether the strain has good tolerance capability to gastrointestinal tract environment, in vivo fixed value capability, whether characteristic bacteriocin can be produced, whether the effect of treating chemotherapeutic intestinal injury can be achieved after a certain dosage and time course and the like. Therefore, a strain with excellent comprehensive performance is screened out, and the strain can play an effective role in preventing and/or treating the chemotherapeutic intestinal injury, and is yet to be developed.

Disclosure of Invention

In order to solve the problems and the defects in the prior art, the invention provides a high-efficiency bacteriocin-producing saliva combined lactobacillus (Ligilactobacillus salivarius) DY802 and application thereof in preparing products for preventing or treating chemotherapeutic intestinal injury. The saliva combined lactobacillus DY802 can reduce diarrhea by producing a large amount of bacteriocin, maintain intestinal homeostasis, and effectively prevent and treat chemotherapy-induced intestinal injury, so as to solve the problem that the prior art cannot effectively treat the chemotherapy-induced intestinal injury of a malignant tumor patient.

According to a first aspect of the present invention, there is provided a Lactobacillus salivarius DY802, wherein the Lactobacillus salivarius DY802 is deposited with the microorganism strain collection in Guangdong province under the accession number GDMCC 62854. The storage time of the saliva combined lactobacillus DY802 is 2022, 10 and 09. The Guangdong province microorganism strain collection center is located at building 5 of Guangzhou Mitsui No. 100 institute of England.

The saliva combined lactobacillus DY802 is extracted and separated from fresh feces of healthy adults, has excellent gastrointestinal environment resistance and in vivo fixed value capability, and also has better bacteriocin production capability, so that the saliva combined lactobacillus DY802 can effectively regulate the gastrointestinal environment and maintain gastrointestinal stability, and has very remarkable curative effect in preventing and/or treating chemotherapy-induced intestinal injury.

According to a second aspect of the present invention, there is provided the use of lactobacillus salivarius DY802 in the manufacture of a medicament for the prevention and/or treatment of chemotherapeutic intestinal injury.

According to a third aspect of the present invention, there is provided the use of saliva in combination with lactobacillus DY802 for the preparation of a medicament for the prevention and/or treatment of digestive system diseases caused by chemotherapeutic intestinal injuries.

According to a fourth aspect of the present invention, there is provided a medicament for the prevention and/or treatment of chemotherapeutic intestinal injury, the medicament comprising lactobacillus salivarius DY802 and/or a culture of lactobacillus salivarius DY802.

According to a fifth aspect of the present invention, there is provided a pharmaceutical product for the prophylaxis and/or treatment of digestive system diseases, which comprises Lactobacillus salivarius DY802 and/or a culture of Lactobacillus salivarius DY802, and which digestive system diseases are caused by chemotherapeutic intestinal injury.

Preferably, the indication of the above-mentioned medicine includes at least one of abdominal pain, abdominal distension, nausea, vomiting, diarrhea, constipation.

Preferably, the above-mentioned pharmaceutical product further comprises a pharmaceutical adjuvant.

Preferably, the pharmaceutical adjuvant comprises at least one of a stabilizer, a wetting agent, an emulsifying agent, a binder, an isotonic agent.

Preferably, the pharmaceutical product is presented in at least one of the following forms: tablets, granules, powders, capsules, solutions, suspensions and freeze-dried preparations.

According to a sixth aspect of the present invention, there is provided a food product for regulating intestinal flora, the food product comprising lactobacillus salivarius DY802 and/or a culture of lactobacillus salivarius DY802. The food with the characteristics can regulate intestinal flora, maintain intestinal homeostasis, and particularly has obvious regulation effect on preventing and/or treating chemotherapy-induced intestinal injury or intestinal flora disorder caused by digestive system diseases caused by chemotherapy-induced intestinal injury.

According to a seventh aspect of the invention, there is provided a target nucleotide sequence capable of specifically recognizing Lactobacillus salivarius DY802, wherein the target nucleotide sequence is shown in SEQ ID No.2.

According to an eighth aspect of the present invention, there is provided a primer set specifically recognizing Lactobacillus salivarius DY802, characterized in that: the primer set comprises a first primer and a second primer; the first primer had a sequence of 5'-TTGGATTTAGTAAAAGTATCAAAGG-3' and the second primer had a sequence of 5'-TTTCATTGTAC ATCCCATTAGTG-3'.

According to a ninth aspect of the present invention, there is provided a method of identifying Lactobacillus salivarius DY802, comprising the steps of: s1, carrying out PCR (polymerase chain reaction) amplification on bacteria to be detected by adopting an amplification primer; s2, analyzing and identifying the amplification product in the S1; the amplification primer comprises a first primer and a second primer; the first primer had a sequence of 5'-TTGGATTTAGTAAAAGTATCAAAGG-3' and the second primer had a sequence of 5'-TTTCATTGTA CATCCCATTAGTG-3'.

Preferably, in the above method for identifying lactobacillus salivarius DY802, in S2, if the length of the amplified product is 120bp, the bacterium to be detected is judged to be lactobacillus salivarius DY802, and if the length of the amplified product is not 120bp, the bacterium to be detected is judged not to be lactobacillus salivarius DY802.

The saliva combined lactobacillus DY802 is extracted and separated from feces of healthy adults, ensures the safety and effectiveness of the saliva combined lactobacillus DY802 from bacterial sources, and has no obvious toxic or side effect on human bodies and high safety compared with the traditional medicine for treating the chemotherapeutic intestinal injury. The saliva combined lactobacillus DY802 of the invention has the specific effects of preventing and/or treating chemotherapy-induced intestinal injury as follows: (1) Saliva combined with lactobacillus DY802 can generate bacteriocin and other secondary metabolites, and can improve intestinal inflammation; (2) The saliva combined lactobacillus DY802 has high survival rate in the stomach and the intestines, can be implanted in the intestines, and can restore the stable state of intestinal flora; (3) Saliva combines lactobacillus DY802 to reduce intestinal damage under chemotherapy conditions and accelerate intestinal tissue repair. Therefore, the saliva combined with the lactobacillus DY802 has great application prospect in preparing medicines for preventing and/or treating chemotherapeutic intestinal injury.

Drawings

FIG. 1 shows colony morphology of Lactobacillus salivarius DY802 isolated in example 1.

FIG. 2 shows the gram staining results of Lactobacillus salivarius DY802 cells isolated in example 1.

FIG. 3 shows the 16S rDNA sequence alignment of Lactobacillus salivarius DY802 isolated in example 1.

FIG. 4 is the results of a biochemical test of the glycolysis of the isolated Lactobacillus salivarius DY802 in example 1; a is the test result of cellobiose, b is the test result of maltose, c is the test result of raffinose, d is the test result of mannitol, e is the test result of sucrose, f is the test result of salicin, g is the test result of sorbitol, h is the test result of esculin.

FIG. 5 is a genome circle map of the whole genome sequencing map of Lactobacillus salivarius DY802 in example 2.

FIG. 6 is an in vivo safety evaluation of mice with Lactobacillus salivarius DY802 in example 2; a represents the heart, b represents the lung, c represents the spleen, d represents the liver, and e represents the kidney.

FIG. 7 is bacteriocin excavation of Lactobacillus salivarius DY802 in example 5.

FIG. 8 is the amount of molded feces after treatment of a chemotherapeutic intestine-injured mouse with Lactobacillus salivarius DY802 in example 7.

FIG. 9 is the colon tissue length after treatment of chemotherapeutic intestine injured mice with saliva in combination with Lactobacillus DY802 in example 7.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.

Example 1

Isolation and identification of Lactobacillus salivarius DY 802:

the saliva of this example was extracted from Lactobacillus DY802 as follows: collecting fresh feces of healthy adults according to a solid-to-liquid ratio of 1:1000 (g/mL) ratio and PBS was added for dilution. mu.L of the sample was inoculated into MRS medium and cultured anaerobically at 37℃for 48 hours. And picking single colonies, and inoculating to an MRS liquid culture medium for enrichment culture. Wherein the formula of the MRS culture medium is as follows: 10g of peptone, 5g of yeast extract, 10g of beef extract, 20g of glucose, 5g of sodium acetate, 2g of diammonium citrate, 1mL of Tween 80, 0.58g of magnesium sulfate, 0.05g of manganese sulfate, 2g of dipotassium hydrogen phosphate, 15-17 g of agar and 1000mL of water, and regulating the pH to 6.12-6.2.

The morphological characteristics of the strain obtained by the culture were observed, the strain was subjected to gene sequence alignment, and the strain was identified by means of a sugar fermentation experiment, and the corresponding test steps and test results are shown below.

(1) Morphological characterization of Lactobacillus salivarius DY802

Colony characteristics: the strain is separated by five-area lines on an MRS plate, and is subjected to anaerobic culture for 48 hours at a constant temperature of 37 ℃ and grows well. Referring to FIG. 1, it can be seen that the colonies were convex, oval, smooth in surface, white with yellowish color and clean in edge.

Characteristics of the cells: after gram staining of the strain, the strain was observed with an oil microscope, and referring to fig. 2, it was seen that the gram staining was positive, and the strain was in the form of a thin and short rod without spores.

The strain provided by the invention is a probiotic separated from fresh feces of healthy adults, and ensures the safety and effectiveness of the strain from bacterial sources. In addition, after the strain is colonized and propagated in the human intestinal environment, the strain is only attached to intestinal epithelial cells of a host, can become a biological barrier of intestinal mucosa, and improves the barrier capacity of the intestinal mucosa of the host. Moreover, the strain can directly act on a human body in a live bacterial form, so that the safety of the strain is ensured.

(2) Gene sequence alignment of Lactobacillus salivarius DY802

In this example, the gene sequence alignment was performed on Lactobacillus salivarius DY802 by the following steps:

s1, extracting total DNA of a bacterial strain by using a bacterial genome DNA kit (TIANamp Bacteria DNA Kit), wherein the steps of the extraction method are carried out according to the instruction of the kit;

s2, performing PCR amplification on the extracted DNA by using universal primers of lactobacillus 16S rDNA;

s3, cutting gel after PCR electrophoresis, recovering target band gel, sequencing by Shanghai biological limited company, and obtaining a nucleotide sequence shown as SEQ ID No.1 after sequencing. In the NCBI database, the sequences of the Lactobacillus salivarius 16S rDNA genes were aligned using BLAST software tools.

The results obtained by the above test steps show that the homology of the obtained sequencing result with the 16S rDNA sequence of the Lactobacillus salivarius is 99%, so that the strain separated from fresh feces of healthy adults in the implementation can be identified as the Lactobacillus salivarius, and the strain is named as DY802, and the specific gene sequence comparison result of the DY802 is shown in FIG. 3.

In this example, the universal primer pair nucleotide sequence of the lactobacillus 16S rDNA used is as follows: a first primer 27f,5 '-agagttttgatcctggcttag-3'; a second primer 1492r,5'-GGTTACCTTGTTACGACTT-3'; the primers were synthesized by Beijing Optimus Co.

In this example, the total volume of the reaction system for PCR amplification was 20. Mu.L, 2. Mu.L of the template (DNA extracted from the strain), taKaRa Premix TaqTM. Mu.L, 1. Mu.L of each of the first primer and the second primer, and 6. Mu.L of double distilled water. In the negative control (control) reaction system, the template was replaced with double distilled water, and the rest components were the same. And the reaction conditions for PCR amplification in this example are as follows: 94 ℃ for 5min;94 ℃,60 s,60 ℃,60 s,72 ℃, 90s,30 cycles; 72 ℃ for 10min; preserving at 4 ℃.

(3) Identification of Lactobacillus salivarius DY802 by sugar fermentation experiments

The strain was subjected to a sugar fermentation experiment, which was performed according to the novel microbial trace biochemical series identification tube instructions, and the following biochemical metabolites of the isolated strain were detected: cellobiose, maltose, raffinose, mannitol, sucrose, salicin, sorbitol, esculin, and the results are shown in Table 1.

The results of the experiments in Table 1 were analyzed in combination with the Berger's bacteria identification manual data, and it was found that the physiological and biochemical characteristics of this strain were substantially consistent with those of the Lactobacillus salivarius standard strain ATCC11741, see FIG. 4.

TABLE 1 Biochemical identification results of Lactobacillus salivarius DY802

Note that: "+" is positive and "-" is negative.

The morphological characteristics, the gene sequence comparison and the test results of the sugar fermentation experiments of the strains show that the strain separated from the fresh feces of healthy adults can be identified as the lactobacillus salivarius, and the strain is preserved in the microorganism strain collection in Guangdong province at the 10 th month 09 of 2022, and is classified and named as: ligilactobacillus salivarius DY802 and accession number GDMCC 62854.

Example 2

Whole genome sequencing and safety evaluation of lactobacillus salivarius DY 802:

(1) Whole genome sequencing of Lactobacillus salivarius DY802

Whole genome sequencing was performed on lactobacillus salivarius DY802 as follows:

extracting genome DNA of the saliva combined lactobacillus DY802, and performing purity, concentration and integrity quality inspection by utilizing Nanodrop, qubit and 0.35% agarose gel electrophoresis; recovering large-fragment DNA by using a BluePIPP full-automatic nucleic acid recovery system; library construction using SQK-LSK109 ligation kit; sequencing on a machine; after the machine is started, the quality of the original data is controlled, and reads with low quality and too short length are filtered; genome assembly, namely performing de novo assembly on the filtered reads and performing error correction on the assembled draft genome; the resulting genome fractions were analyzed. Genome function annotations, including PHI-base, CARD, TCDB database annotations; genome analysis, genome map analysis.

The whole genome sequencing results of Lactobacillus salivarius DY802 are shown in FIG. 5, wherein Lactobacillus salivarius DY802 contains 1 circular genome and 2 circular plasmids, the genome size is 1.97Mb, the GC ratio is 33.03%, and the genome contains 1935 CDS regions, 7395bp repetitive sequences, 77 tRNA and 22 rRNA. And genome functional annotation indicates that the strain does not contain drug resistance genes and virulence genes obtained by horizontal gene transfer.

(2) Safety assessment of Lactobacillus salivarius DY802

(1) Antibiotic sensitivity

Sensitivity of Lactobacillus salivarius DY802 to 8 antibiotics was examined by a micro broth dilution method according to Probiotics general rule for food published by the society of food science and technology. The 8 antibiotics are: tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin, and gentamicin. Antibiotic susceptibility experiments were performed on Lactobacillus salivarius DY802 as follows:

s1, adjusting lactobacillus suspension grown to logarithmic phase to 1X 10 ⁹ CFU/mL, then adding antibiotic diluents with different concentrations (the concentration is 1-128 mg/mL), and carrying out anaerobic culture for 48 hours at 37 ℃;

s2.37 ℃ anaerobic culture for 48 hours, and then the minimum inhibitory concentration (Minimal Inhibit Concentration, MIC) of the strain on each antibiotic is read; strains are judged to be sensitive to the antibiotic (S), intermediate (I), resistant (R) and unwanted (non-required, n.r.) according to the bacterial resistance criteria provided by the general rule.

The safety evaluation results of the Lactobacillus salivarius DY802 are shown in Table 2, and as can be seen from Table 2, the MICs of the Lactobacillus salivarius DY802 for tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin and gentamicin are: 8mg/L, 64mg/L, 128mg/L, 4mg/L and 16mg/L, i.e., the bacterium is sensitive to 6 antibiotics prescribed in general rule.

TABLE 2 drug sensitivity of Lactobacillus salivarius DY802 to different antibiotics

(2) Animal safety assessment

Animal safety assessment experiments were performed with 5C 57BL/6 mice of 6 weeks of age. Animals were fed adaptively in the animal house for 5 days before the experiment, and then mice were subjected to salivary perfusion in combination with lactobacillus DY802, 0.2mL of DY802 bacteria solution with od=1 was perfused daily, and the total was perfused for 14 days. After the raising is finished, the experimental animal is sacrificed by neck breaking, and the organs are dissected and taken out by a scalpel for general analysis. The results show that the major organs of the mice, such as heart, liver, spleen, lung and kidney, have no abnormal structures, which suggests that DY802 is safe to apply in the mice, and reference can be made to FIG. 6.

The experimental animals and the experimental animal houses in the experiment meet the national regulations, and the standard compound feed is selected, so that the drinking water is not limited.

Example 3

Experiment of tolerance of Lactobacillus salivarius DY802 to artificial gastric juice:

an artificial gastric juice tolerance test was performed on lactobacillus salivarius DY802 as follows:

s1, respectively preparing artificial gastric juice with pH values of 2.0, 3.0 and 4.0;

s2, taking saliva and combining lactobacillus DY802, diluting the saliva and lactobacillus DY802 into 1X 10 by using sterile PBS ⁹ CFU/mL of bacterial suspension;

s3, absorbing 10 mu L of bacterial suspension in 99 mu L of artificial gastric juice with different pH values, respectively culturing in 96-well plates, carrying out anaerobic culture at 37 ℃ for 1-3 h, and measuring the number of living bacteria after initial and culture by using an enzyme-labeled instrument.

The results of the artificial gastric juice resistance test of Lactobacillus salivarius DY802 are shown in Table 3, and the data of Table 3 are analyzed, so that Lactobacillus salivarius DY802 shows a better artificial gastric juice resistance than Lactobacillus salivarius ATCC11741 (the artificial gastric juice resistance test method for testing Lactobacillus salivarius ATCC11741 is the same as that of Lactobacillus salivarius DY 802).

TABLE 3DY802 comparison of resistance to artificial gastric juice with ATCC11741

Example 4

Experiment of tolerance of Lactobacillus salivarius DY802 to artificial intestinal juice:

an artificial intestinal juice tolerance experiment was performed on lactobacillus salivarius DY802 as follows:

s1, preparing an artificial intestinal juice containing bile salt and having a pH value of 6.8;

s3, sucking 1 mu L of bacterial suspension into 99 mu L of artificial intestinal juice with different pH values, respectively culturing in 96-well plates, carrying out anaerobic culture at 37 ℃ for 1-3 h, and measuring the number of living bacteria after initial and culture by using an enzyme-labeled instrument.

The results of the artificial intestinal fluid resistance test of Lactobacillus salivarius DY802 are shown in Table 4, and the data of Table 4 are analyzed, so that Lactobacillus salivarius DY802 exhibits a better artificial intestinal fluid resistance than Lactobacillus salivarius ATCC11741 (the artificial intestinal fluid resistance test method for Lactobacillus salivarius ATCC11741 is the same as that of Lactobacillus salivarius DY 802).

TABLE 4DY802 comparison of Artificial intestinal fluid tolerance with ATCC11741

Example 5

Bacteriocin-producing ability of lactobacillus salivarius DY 802:

the synthetic gene cluster of potential ribosomally synthesized and post-translationally modified polypeptides (RiPPs) in the DY802 whole genome was mined using BAGEL 4. As a result, 4 potential hot spot areas (Areas of Interest, AOI) were found in the culture product of Lactobacillus salivarius DY802, including enterysin_ A, salivaricin _P_chain_b and nisin_F, as shown in Table 5 and FIG. 7. The results show that the saliva combined lactobacillus DY802 has stronger bacteriocin production capacity.

TABLE 5 prediction results of bacteriocins or RiPPPs of DY802

Example 6

Preparation of a medicament for the treatment/adjuvant treatment of chemotherapeutic intestinal injury:

the saliva is combined with lactobacillus DY802 as an active ingredient, and pharmaceutically acceptable auxiliary agents are added, so that the medicine for treating/assisting in treating the chemotherapeutic intestinal injury can be prepared. Pharmaceutically acceptable adjuvants in this embodiment, including at least one of stabilizer, wetting agent, emulsifier, binder, and isotonizing agent; and the prepared medicine is at least one of tablets, granules, powder, capsules, solutions, suspensions and freeze-dried preparations.

Example 7

Therapeutic effects of saliva against chemotherapeutic intestinal injury in combination with lactobacillus DY 802:

20C 57BL/6 mice of 6 weeks old were randomly divided into 4 groups after normal feeding for 7 days: control group (n=5), chemotherapy group (n=5), chemotherapy+atcc 11741 group (n=5), chemotherapy+dy 802 group (n=5).

Mice were dosed and analyzed for results as follows: on days 1-13, the mice in the Control group are subjected to PBS (phosphate buffered saline) 0.2mL gastric lavage, and the mice in the chemotherapy+ATCC 11741 group and the chemotherapy+DY802 group are respectively subjected to gastric lavage with 0.2mL of ATCC11741 and DY802 bacterial liquid with OD=1, and on days 8-10, the mice in the chemotherapy group, the chemotherapy+ATCC 11741 group and the chemotherapy+DY802 group are subjected to chemotherapy by intraperitoneal injection of 0.2mL of 50mg/Kg fluorouracil injection every day; collecting the total formed feces of each group of mice every day on days 13-15; on day 16, each index was anatomically examined.

The statistics of the number of the formed faeces of the mice are shown in figure 8, and the results show that the number of the formed faeces of a chemotherapy group is obviously reduced due to severe diarrhea; whereas the number of chemo + ATCC11741 and chemo + DY802 constitutive faeces was significantly greater than in the chemo-group, with the number of chemo + DY802 constitutive faeces being greater than in the chemo-ATCC 11741 group. In addition, the results of the statistical analysis of the colon length of each group of mice are shown in fig. 9, which shows that the colon length of the mice in the chemotherapy group is significantly shorter than that in the Control group, the colon length of the mice in the chemotherapy+atcc 11741 group and the colon length of the mice in the chemotherapy+dy 802 group is significantly longer than that in the chemotherapy group, and the colon length of the mice in the chemotherapy+dy 802 group is longer than that in the chemotherapy+atcc 11741 group. The statistics of the number of formed faeces and the colon length of the mice show that DY802 can remarkably treat the chemotherapeutic intestinal injury of the mice, and the improvement effect is superior to that of the standard strain ATCC11741. And the experimental results show that compared with the conventional drug therapy, the saliva combined lactobacillus DY802 can reduce adverse reaction caused by the drug and solve the problem that intestinal microecology cannot be improved.

Example 8

Specific molecular target excavation of lactobacillus salivarius DY 802:

specific molecular target mining was performed on lactobacillus salivarius DY802 as follows:

s1, downloading whole genome data of other 216 strains of Lactobacillus salivarius in NCBI database, and performing flood genome analysis on the 216 strains of Lactobacillus salivarius and DY802 by using Prokka (v 1.11) and Roary (v 3.11.2) software;

s2, after a core gene is obtained, identifying a gene with higher base substitution density by utilizing MEGA X (v10.2.2); obtaining a specific sequence of Lactobacillus salivarius DY802 different from other Lactobacillus salivarius;

s3, primer Premier 5 software is adopted to carry out Primer design on the specific sequence, and the specific molecular target sequence SEQ ID No.2 for identifying the bacteria is obtained.

Polymerase chain reaction (Polymerase Chain Reaction, PCR) was used to verify the effectiveness of saliva in combining specific molecular recognition target sequences of lactobacillus DY802. The detection template is the DNA of the strain, and the DNA extraction method refers to a DNA extraction kit of the genome of the rhizopus niveus. The amplification primers are as follows: 5'-TTGGATTTAGTAAAAGTATCAAAGG-3' and 5'-TTTCATTGTACATCCCATTA GTG-3'. The PCR reaction system and the PCR reaction conditions were as described in example 1. After the PCR is finished, the PCR is sent to a manufacturer company for sequencing, and the product length is analyzed to be 120bp, and is shown as SEQ ID No.2.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention, but these modifications or substitutions are all within the scope of the present invention.

Claims

1. A lactobacillus salivarius DY802, characterized in that: the Lactobacillus salivarius DY802 is deposited with the microorganism strain collection of Guangdong province under the accession number GDMCC 62854.

2. Use of a salivary combination lactobacillus DY802 as claimed in claim 1 for the preparation of a medicament for the prophylaxis and/or treatment of chemotherapeutic intestinal injury.

3. Use of a salivary combination lactobacillus DY802 as claimed in claim 1 for the manufacture of a medicament for the prophylaxis and/or treatment of digestive system diseases caused by chemotherapeutic intestinal injuries.

4. A medicament for preventing and/or treating chemotherapeutic intestinal injury, characterized in that: the pharmaceutical product comprises a lactobacillus salivarius DY802 as claimed in claim 1 and/or a culture of lactobacillus salivarius DY802.

5. A pharmaceutical product for preventing and/or treating digestive system diseases, characterized in that: the pharmaceutical product comprises the Lactobacillus salivarius DY802 as claimed in claim 1 and/or a culture of the Lactobacillus salivarius DY802, and the digestive system diseases are caused by chemotherapy-induced intestinal injury.

6. A pharmaceutical product for preventing and/or treating digestive system diseases according to claim 5, wherein: the indications of the medicine comprise at least one of abdominal pain, abdominal distention, nausea, vomiting, diarrhea and constipation. .

7. A food product with an adjustable intestinal flora, characterized in that: the food product comprises a lactobacillus salivarius DY802 as claimed in claim 1 and/or a culture of lactobacillus salivarius DY802.

8. A target nucleotide sequence specifically recognizing lactobacillus salivarius DY802 as claimed in claim 1, wherein: the target nucleotide sequence is shown as SEQ ID No.2.

9. A primer set specifically recognizing Lactobacillus salivarius DY802 as defined in claim 1, wherein: the primer set comprises a first primer and a second primer;

the sequence of the first primer is 5'-TTGGATTTAGTAAAAGTATCAAAGG-3', and the sequence of the second primer is 5'-TTTCATTGTACATCCCATTAGTG-3'.

10. A method of identifying lactobacillus salivarius DY802 as claimed in claim 1 comprising the steps of:

s1, carrying out PCR (polymerase chain reaction) amplification on bacteria to be detected by adopting an amplification primer;

s2, analyzing and identifying the amplification product in the S1;

the amplification primer comprises a first primer and a second primer;