CN115927106A

CN115927106A - Rhamnose cheese bacillus DY801 and application thereof

Info

Publication number: CN115927106A
Application number: CN202211621368.0A
Authority: CN
Inventors: 丁轶; 俞露; 张耀伟; 陈振辉; 王子欢; 文雅静
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: 2023-04-07
Anticipated expiration: 2042-12-16
Also published as: CN115927106B

Abstract

The invention provides a strain of Lactobacillus rhamnosus DY801 and application thereof. The Lactobacillus rhamnosus DY801 is preserved in Guangdong province microorganism culture collection center at 2022, 10 months and 9 days, and the preservation number is GDMCC 62853. The rhamnose cheese bacillus DY801 provided by the invention can improve the oxidative stress of intestinal tracts and the damage of small intestine villi under the chemoradiotherapy condition by generating a large amount of glutathione, reduce the inflammation index, can tolerate artificial gastric juice and intestinal juice, has no obvious toxic or side effect on human bodies, and is high in safety. The Lactobacillus rhamnosus DY801 or the culture thereof provided by the invention can be applied to preparation of medicines for preventing and/or treating the intestinal injury caused by radiotherapy and chemotherapy, can be used for preventing and/or treating digestive system diseases caused by the intestinal injury caused by radiotherapy and chemotherapy, and solves the problem that the intestinal injury caused by radiotherapy and chemotherapy of pelvic malignant tumors cannot be effectively treated in the prior art.

Description

Lactobacillus rhamnosus DY801 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a strain of Lactobacillus rhamnosus DY801 and application thereof.

Background

Radiotherapy and chemotherapy are one of the common clinical treatment methods for treating pelvic malignant tumor. After radiotherapy is carried out on patients with pelvic malignant tumor, about 75-81% of patients have radioactive intestinal injury; while chemotherapy, particularly with fluorouracil and irinotecan-containing chemotherapy regimens, is followed in approximately 50% to 80% of patients with chemotherapy-induced bowel damage. In particular intestinal flora translocation due to severe diarrhea can cause sepsis, with even a 5% mortality rate. The intestinal injury caused by radiotherapy and chemotherapy during the treatment process can cause the life quality of patients to be obviously reduced, the treatment compliance is poor, the whole treatment course is prolonged, and the overall survival prognosis of the patients is finally influenced.

Current treatments for chemoradiotherapy-induced intestinal injury are not optimistic. The radioactive intestinal injury treatment still remains in the stage of surgical excision of a seriously affected intestinal section and trial medicines, and the diagnosis and treatment consensus or guidance is very poor. While the treatment aiming at the chemotherapy-induced intestinal injury mainly carries out support treatment and drug treatment, wherein the drug treatment mainly comprises loperamide, sulfasalazine, octreotide and the like, but 9-30% of patients still have no effect on the drug treatment. However, even the drug therapy including non-steroidal anti-inflammatory drugs, steroid hormones and antibiotics can only improve local inflammation but cannot improve the intestinal flora disorder caused by the therapy, the overall therapeutic effect is poor, and the drugs can cause various adverse reactions such as nausea and vomiting, so that the wide application of the drugs is limited.

Lactobacillus rhamnosus (Lactobacillus rhamnosus) is a Lactobacillus species, lactobacillus rhamnosus species, an anaerobic, acid-resistant, non-spore-forming, gram-positive probiotic bacterium, present in the intestinal tracts of humans and animals. The Lactobacillus rhamnosus can survive and proliferate in gastric acid environment and in a culture medium containing bile, and can adhere to intestinal epithelial cells. In addition, the Lactobacillus rhamnosus can generate a biological membrane, promote the survival of intestinal crypts, reduce the apoptosis of intestinal epithelial cells and generate a series of soluble factors beneficial to intestinal tracts. The Lactobacillus rhamnosus can also inhibit some pathogens, such as Salmonella, escherichia coli, staphylococcus aureus with high pathogenicity, etc. The Lactobacillus rhamnosus also has a powerful immunoregulatory function, manifested by a reduction of monocyte activation and expression of inflammatory markers, and also an enhancement of macrophage function. Meanwhile, the Lactobacillus rhamnosus has the function of treating digestive system inflammations such as antibiotic-associated diarrhea, necrotizing enterocolitis of newborns, clostridium difficile infectious diarrhea and the like, and becomes one of the lactobacilli widely applied at home and abroad. However, the existing cheese bacillus rhamnosus has poor tolerance to gastric juice and intestinal juice of a human body and low glutathione content, so that the cheese bacillus rhamnosus effectively plays a role in the gastrointestinal tract of a host, and the treatment effect of the cheese bacillus rhamnosus is influenced. Therefore, it is of great importance to find a Lactobacillus rhamnosus strain that can tolerate gastric juice and intestinal juice of a host.

Disclosure of Invention

The invention provides a rhamnose cheese bacillus DY801 and application thereof, wherein the rhamnose cheese bacillus DY801 can improve the oxidative stress of intestinal tracts by generating a large amount of glutathione, has the characteristic of tolerance to gastric juice and intestinal juice of a human body, and can be used for preventing and/or treating chemoradiotherapy intestinal injury.

According to the first aspect of the invention, the invention provides a strain of lactobacillus rhamnosus DY801, which is deposited in Guangdong province microorganism

culture collection center

10, 9 and 2022 at the deposit address of Guangdong province microorganism research institute No. 100 Michelia Toolff, guangzhou, with the deposit number of GDMCC 62853.

The invention separates a new strain of Lactobacillus rhamnosus DY801 from fresh excrement of healthy adults in Guangzhou city, guangdong province. Compared with the existing rhamnose cheese bacillus, the rhamnose cheese bacillus DY801 provided by the invention is an excellent strain with a local source, and the specific embodiment is as follows: the strain can produce bacterial culture products such as glutathione and the like, and can improve oxidative stress and inflammation of intestinal tracts; the strain can tolerate artificial gastric juice and intestinal juice, has high survival rate in stomach and intestine, and can be planted in intestinal tract to recover the steady state of intestinal flora; in addition, the strain can reduce the damage of small intestinal villi under the conditions of radiotherapy and chemotherapy, reduce inflammation indexes and accelerate the repair of intestinal tissues. In addition, compared with the traditional medicine for treating the intestinal injury caused by radiotherapy and chemotherapy, the rhamnose cheese bacillus DY801 provided by the invention has no obvious toxic or side effect on human bodies and has high safety. In conclusion, the Lactobacillus rhamnosus DY801 provided by the invention has many excellent characteristics, can be used for effectively preventing and treating digestive system diseases caused by radiotherapy and chemotherapy intestinal injury, solves the problem that radiotherapy and chemotherapy intestinal injury after radiotherapy and chemotherapy of pelvic malignant tumors cannot be effectively treated in the prior art, and has great application prospects in preparation of medicines for preventing and/or treating radiotherapy and chemotherapy intestinal injury or foods and health-care products with auxiliary protection functions on radiation hazards.

According to a second aspect of the present invention, there is provided a use of the above-mentioned lactobacillus rhamnosus DY801 for the preparation of a medicament for the prevention and/or treatment of digestive system diseases.

Preferably, the digestive system disease includes at least one of abdominal pain, abdominal distension, nausea, vomiting, diarrhea, and constipation.

Preferably, the above-mentioned digestive system diseases are caused by intestinal injury.

Preferably, the intestinal injury is radiation intestinal injury or chemotherapy intestinal injury.

According to a third aspect of the present invention, there is provided a medicament for preventing and/or treating digestive system diseases, which comprises the above-mentioned cheesecaloides rhamnosus DY801 or the culture of the above-mentioned cheesecaloides DY801.

The rhamnose cheese bacillus DY801 or the culture thereof provided by the invention is applied to preparation of a medicine for preventing and/or treating digestive system diseases caused by radiotherapy and chemotherapy intestinal injuries, and the prepared medicine can effectively prevent and/or treat the radiotherapy and chemotherapy intestinal injuries, so that the problem that the radiotherapy and chemotherapy intestinal injuries after radiotherapy and chemotherapy of pelvic malignant tumors cannot be effectively treated in the prior art is solved.

Preferably, the above medicament further comprises pharmaceutically acceptable adjuvants: the pharmaceutically acceptable adjuvants include at least one of stabilizer, wetting agent, emulsifier, binder, and isotonic agent.

Preferably, the medicament is at least one of tablets, granules, powder, capsules, solutions, suspensions and freeze-drying agents.

According to a fourth aspect of the invention, the application of the rhamnose cheese bacillus DY801 in preparing food or health care products with auxiliary protection function on radiation hazard is provided.

According to a fifth aspect of the present invention, there is provided a food having an auxiliary protective function against radiation damage, which comprises the above-mentioned Lactobacillus rhamnosus DY801 or a culture of the above-mentioned Lactobacillus rhamnosus DY801.

The Lactobacillus rhamnosus DY801 or the culture thereof provided by the invention is applied to preparation of food or health-care products with auxiliary protection function on radiation hazard, and the prepared food or health-care products can play a certain auxiliary protection function on radiation hazard.

According to a sixth aspect of the present invention, there is provided a nucleotide sequence specifically recognizing the above-mentioned lactobacillus rhamnosus DY801, the nucleotide sequence being represented by SEQ ID:1 is shown.

The nucleotide sequence provided by the scheme can effectively distinguish the rhamnose cheese bacillus DY801 provided by the invention from other rhamnose cheese bacillus separation strains.

According to a seventh aspect of the present invention, there is provided a primer set specifically recognizing the above-mentioned lactobacillus rhamnosus DY801, the primer set comprising a primer set as set forth in SEQ ID:2 and SEQ ID: 3.

The primer group provided by the scheme can be used for carrying out PCR amplification on DNA of a bacterium to be detected, if a 431bp product is amplified, the bacterium to be detected is the rhamnose cheeseobacter DY801, and if the 431bp product is not amplified, the bacterium to be detected is not the rhamnose cheeseobacter DY801.

According to the eighth aspect of the invention, the primer group is used as a specific amplification primer, the genomic DNA of the to-be-detected rhamnose cheesecloth is used as a template for PCR amplification to obtain a PCR product, and the PCR product is identified by sequencing or electrophoresis.

Drawings

FIG. 1 is a colony morphology of Lactobacillus rhamnosus DY801 isolated and cultured in example 1.

FIG. 2 is a gram-stained bacterial body of Lactobacillus rhamnosus DY801 isolated and cultured in example 1.

FIG. 3 is a diagram showing the results of sequence alignment of the 16S rDNA gene of Lactobacillus rhamnosus DY801 in example 2.

FIG. 4 is a graph showing the results of glycolytic biochemical test in example 2 in which Lactobacillus rhamnosus DY801 was identified.

FIG. 5 is a genome-wide sequencing map and a genome circle map of Lactobacillus rhamnosus DY801 according to example 3.

FIG. 6 is a graph showing the results of the general evaluation of the safety evaluation of important organs in mice with Lactobacillus rhamnosus DY801 in example 4.

FIG. 7 is a graph showing the results of histopathological staining analysis of the important organs for the evaluation of safety of M.rhamnosus DY801 in mice in example 4.

FIG. 8 is a graph showing the results of measuring the glutathione content produced by Lactobacillus rhamnosus DY801 in example 7 by liquid chromatography-mass spectrometry.

FIG. 9 is a graph showing the survival rate of mice with chemotherapy-induced intestinal injury treated with Lactobacillus rhamnosus DY801 according to example 8.

FIG. 10 is an HE staining pattern (40X) of intestinal tissues after the treatment of chemoradiotherapy intestine-injured mice with Lactobacillus rhamnosus DY801 in example 8.

FIG. 11 is a graph showing the results of measurement of a blood inflammation index in a mouse after the treatment of the chemotherapy-and radiotherapy-induced intestinal injury mouse with the Lactobacillus rhamnosus DY801 according to example 8.

Detailed Description

Technical features in the technical solutions provided by the present invention are further clearly and completely described below with reference to specific embodiments, and it is obvious that the described embodiments are only a part of embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 isolation of Lactobacillus rhamnosus DY801

In the embodiment, a new lactobacillus rhamnosus strain is obtained by separating fresh excrement of healthy adults in Guangzhou city, guangdong province, is identified as lactobacillus rhamnosus DY801 by utilizing morphological characteristics, culture traits, physiological and biochemical characteristics and genetic characteristics 16SrDNA, and is preserved in the Guangdong province microorganism strain preservation center at 10.9.2022, the preservation address is Guangdong province microorganism research institute No. 100 Mzhongjilu, guangdong province, guangzhou city, the classification name is Lactcaseibacillus rhamnosus DY801, and the preservation number is GDMCC 62853.

In this example, after collecting fresh feces of healthy adults in Guangzhou city, guangdong province, the solid-to-liquid ratio was 1:1000 (g/mL) to fresh feces, PBS buffer was added to dilute the feces. And (3) inoculating 5 mu L of the diluted fresh excrement sample into an MRS culture medium, carrying out anaerobic culture at the constant temperature of 37 ℃ for 48 hours, and then picking out a single colony and inoculating the single colony into an MRS liquid culture medium for enrichment culture.

Wherein, the MRS culture medium formula involved in the culture process 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 adjusting the pH to 6.12-6.2.

The morphological characteristics of the isolated and cultured rhamnose cheese bacillus DY801 in this example are as follows:

(1) Characteristics of bacterial colony

In this example, isolated and cultured Lactobacillus rhamnosus DY801 was streaked on a plate and anaerobically cultured at a constant temperature of 37 ℃ for 72 hours, and the colony morphology is shown in FIG. 1. As can be seen from FIG. 1, the Lactobacillus rhamnosus DY801 has a good growth state, a convex colony, a round shape, a smooth and dense surface, a white color and regular edges.

(2) Characteristics of the cells

In this example, the morphology of isolated and cultured rhamnosus cheeseobacter DY801 was observed and stained by gram stain, and the results are shown in fig. 2. As can be seen from FIG. 2, the Lactobacillus rhamnosus DY801 has a long and thin rod shape, no movement, no spores, facultative anaerobe, and gram-positive staining.

Example 2 identification of Lactobacillus rhamnosus DY801

In this example, the bacterial total DNA of the Lactobacillus rhamnosus DY801 isolated and cultured in example 1 was extracted using a bacterial genomic DNA kit (TIANAmp Bacteria DNAkit), and the steps of the extraction method were performed according to the kit instructions. The extracted DNA was PCR amplified using universal primers for Lactobacillus 16S rDNA.

The nucleotide sequence of the 16S rDNA universal primer pair is as follows:

forward primer 27f:5 'AGAGAGTTTGATCCTGGCTCAG-3',

reverse primer 1492r:5 'GGTTACCTTGTTACGACTT-3';

the primers were synthesized by Beijing Optimalaceae biology Ltd.

The PCR amplification reaction system is 20 mu L, the template is 2 mu L, taKaRa Premix TaqTM is 10 mu L, the forward primer and the reverse primer are 1 mu L respectively, and double distilled water is 6 mu L. Meanwhile, a negative control (control) was set, and in the negative control reaction system, the template was replaced with double distilled water, and the other components were the same.

PCR amplification reaction conditions: 5min at 94 ℃; 60s at 94 ℃, 60s at 60 ℃, 90s at 72 ℃ and 30 cycles; 10min at 72 ℃; storing at 4 deg.C.

After PCR electrophoresis, the gel was cut, and the band gel of interest was recovered and sequenced by Shanghai Biotechnology Ltd (nucleotide sequence shown in SEQ ID: 4). In the NCBI database in the United states, the alignment of the sequence of the rDNA gene of Lactobacillus rhamnosus DY801 16S was performed using the BLAST software tool, and the results are shown in FIG. 3.

As can be seen from the sequencing results shown in FIG. 3, the homology between the rhamnose cheese bacillus DY801 provided by the invention and the 16S rDNA sequence of the rhamnose cheese bacillus reaches 99%, and the rhamnose cheese bacillus DY801 is identified to be the rhamnose cheese bacillus and named DY801, namely the rhamnose cheese bacillus DY801 obtained by separation and culture.

In addition, the present example further identifies lactobacillus rhamnosus DY801 by a sugar fermentation experiment:

the biochemical metabolites of the isolated candidate strain DY801 were detected according to the instructions of the novel microorganism micro-biochemical series assay tubes, and the results are shown in FIG. 4 and Table 1. As can be seen by combining the Bergey bacteria identification manual data and the data in FIG. 4 and Table 1, the physiological and biochemical characteristics of the strain are basically consistent with those of the Lactobacillus rhamnosus standard strain ATCC53103.

TABLE 1 Biochemical identification of Rhamniosus casei DY801

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

Example 3 Whole genome sequencing of Lactobacillus rhamnosus DY801

Genomic DNA of Lactobacillus rhamnosus DY801 isolated and cultured in example 1 was extracted, and quality control of purity, concentration and integrity was carried out by Nanodrop, qubit and 0.35% agarose gel electrophoresis. Meanwhile, a BluePippin full-automatic nucleic acid recovery system is used for recovering large-fragment DNA, an SQK-LSK109 connection kit is used for library construction, machine sequencing is carried out, original data after machine unloading are subjected to quality control, and low-quality and short-length reads are filtered; genome assembly is then performed, and de novo assembly of filtered reads is performed, and error correction is performed on the assembled draft genome. Then, genome component analysis and genome function annotation, including PHI-base, CARD, TCDB database annotation, are carried out. In addition, genome analysis and genome map analysis were also performed. The whole genome sequencing result of the Lactobacillus rhamnosus DY801 is shown in FIG. 5.

As can be seen from the genome sequencing and genome circled diagrams of FIG. 5, the genome size of M.rhamnosus DY801 is 3.01Mb, the GC ratio is 46.78%, and the genome contains 2873 CDS regions, 3626bp repeats, 60 tRNAs and 15 rRNAs. Genome function annotation suggests that lactobacillus rhamnosus DY801 contains 2 potential drug resistance genes poxtA and lmrB, but does not contain virulence genes obtained by horizontal gene transfer.

Example 4 evaluation of safety of Lactobacillus rhamnosus DY801

The Lactobacillus rhamnosus DY801 provided by the invention is a probiotic separated from fresh excrement of healthy adults, and the safety and the effectiveness of the Lactobacillus rhamnosus DY are ensured from a bacterial source. In addition, after the rhamnose cheese bacillus DY801 provided by the invention is colonized and propagated in human intestinal environment, only intestinal epithelial cells attached to a host can become a layer of biological barrier of intestinal mucosa, the barrier capability of the intestinal mucosa of the host is improved, and the rhamnose cheese bacillus DY801 can directly act on a human body in a viable bacteria form, so that the safety is ensured.

This example was conducted to examine the sensitivity of Lactobacillus rhamnosus DY801 to 8 antibiotics by a broth dilution method according to general guidelines for probiotic bacteria for food issued by the Chinese society for food science and technology. The 8 antibiotics were: tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin, and gentamicin. The suspension of lactobacilli grown to logarithmic growth phase was adjusted to 1X 10 ⁸ CFU/mL, then adding different concentrations of antibiotic diluent (concentration from 1-64 mg/mL), at 37 degrees C under anaerobic culture for 48 hours. After 48 hours, the Minimum Inhibitory Concentration (MIC) of the strain of lactobacillus rhamnosus DY801 for each antibiotic was read, and the strains were judged to be sensitive (S), intermediate (I), resistant (R) and not required (not required, n.r.) to the antibiotic according to the bacterial resistance standard provided by the general rule, and the results are shown in table 2.

TABLE 2 drug sensitivity of Lactobacillus rhamnosus DY801 to different antibiotics

As can be seen from Table 2, the MICs of the Lactobacillus rhamnosus DY801 for tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin and gentamicin are sequentially as follows: 8mg/L, 32mg/L, 64mg/L, 4mg/L, 64mg/L, 2mg/L, 4mg/L and 16mg/L, i.e.the bacteria are sensitive to 6 antibiotic bacteria as defined by general regulations.

In addition, in order to further evaluate the safety of the rhamnose cheese bacillus DY801, 3C 57BL/6 mice of 6-8 weeks old were selected for experiments. Animals were acclimatized for 5 days in the animal room prior to the experiment. The experimental animals and the experimental animal rooms conform to national regulations, standard compound feed is selected, and diet and drinking water are not limited. And (3) performing intragastric administration on the mice by using the rhamnose cheese bacillus DY801, wherein the intragastric absorbance OD =1 of DY801 bacterial liquid is 0.2mL every day. After the feeding is finished, the experimental animals are killed by breaking the neck, organs are dissected and taken out by adopting a scalpel, and important organs of the mice are observed, and the result is shown in fig. 6; histopathological staining analysis was performed simultaneously and the results are shown in FIG. 7.

As can be seen from FIG. 6, the general structures of the mouse vital organs, i.e., heart, liver, spleen, lung and kidney, were not abnormal; as can be seen from FIG. 7, no tissue damage was observed in the histopathological staining of mice. The results show that the Lactobacillus rhamnosus DY801 provided by the invention is safe to apply in mice.

Example 5 Lactobacillus rhamnosus DY801 has better resistance to artificial gastric juices

Respectively preparing artificial gastric juice with pH values of 2.0,3.0 and 4.0. The isolated and cultured rhamnose cheese bacillus DY801 obtained in example 1 was diluted to 10 degrees with sterile PBS buffer ⁹ CFU/mL of bacterial suspension. And (3) sucking 1 mu L of bacterial suspension into 99 mu L of artificial gastric juice with different pH values, culturing the bacterial suspension in a 96-well plate, carrying out anaerobic culture at 37 ℃ for 1-3 h, and measuring the number of the initial viable bacteria and the number of the cultured viable bacteria by using an enzyme-labeling instrument, wherein the results are shown in Table 3.

As can be seen from Table 3, compared with the standard strain ATCC53103 of the Lactobacillus rhamnosus, the Lactobacillus rhamnosus DY801 provided by the invention has better artificial gastric juice resistance.

TABLE 3DY801 comparison of Artificial gastric juice tolerance with ATCC53103

Example 6 Lactobacillus rhamnosus DY801 has better tolerance effect on artificial intestinal juice

Preparing the artificial intestinal juice with pH value of 6.8 and bile salt. The Lactobacillus rhamnosus DY801 isolated and cultured in example 1 was diluted to 10 with sterile PBS buffer ⁹ CFU/mL of bacterial suspension. 1 mul of the bacterial suspension was pipetted into 99 mul of artificial intestinal juice with different pH values and cultured in a 96-well plate, anaerobic culture was carried out at 37 ℃ for 1-3 h, and the number of viable bacteria was measured by a microplate reader, the results are shown in Table 4.

TABLE 4DY801 comparison of Artificial intestinal fluid resistance with ATCC53103

As can be seen from Table 4, the Lactobacillus rhamnosus DY801 exhibits better resistance to artificial intestinal juice than the Lactobacillus rhamnosus standard strain ATCC53103.

Example 7 Lactobacillus rhamnosus DY801 having a high glutathione producing ability

Adjusting absorbance to OD ₆₀₀ Adding 100 mu L of Lactobacillus rhamnosus DY801 bacterial liquid of =1 into 10mL of MRS broth containing L-tryptophan for culturing for 48 hours, taking 0.5mL of bacterial suspension into a 1.5mL centrifuge tube, adding 205 mu L of precipitator (acetonitrile: methanol = 1) containing 5 mu L of mixed internal standard, vortex mixing, standing on ice for 30 minutes, centrifuging at 12000rpm and 4 ℃ for 10 minutes, taking supernatant into a sample injection vial, using the supernatant as a sample to be detected, and performing mass spectrometry by upper liquid chromatography-mass spectrometry.

Preparing a blank following sample: mu.L of physiological saline IS taken, 5 mu.L of Internal Standard (IS), 100 mu.L of acetonitrile and 100 mu.L of methanol are added, vortex mixing IS carried out, the mixture IS kept still on ice for 30 minutes and IS centrifuged at 12000rpm and 4 ℃ for 10 minutes, and the supernatant IS taken out to be placed in a sample injection vial, and the mass spectrometry IS carried out as above.

Preparing a quality control sample: mu.L of physiological saline was taken, 5. Mu.L of Internal Standard (IS), 5. Mu.L of STD (standard), 100. Mu.L of acetonitrile and 100. Mu.L of methanol were added, vortexed, and allowed to stand on ice for 30 minutes, centrifuged at 12000rpm at 4 ℃ for 10 minutes, and the supernatant was taken out of a sample vial and subjected to mass spectrometry as described above.

The glutathione content in the supernatant is determined by liquid chromatography-mass spectrometry, that is, the glutathione content in the sample to be detected is detected, and the result is shown in fig. 8.

As can be seen from FIG. 8, compared with the standard strain ATCC53103 of the Lactobacillus rhamnosus DY801 provided by the invention, the Lactobacillus rhamnosus DY801 has better glutathione producing capability and higher total amount of glutathione.

Example 8 Lactobacillus rhamnosus DY801 effective in treating radiation and chemotherapy-induced intestinal injury by reducing mucosal congestion and edema

24 mice C57BL/6 with the age of 5-6 weeks are selected, and are randomly divided into 4 groups after being normally bred for 7 days: control group (n = 6), RCT + ATCC53103 group (n = 6), RCT + DY801 group (n = 6), where RCT indicates full abdominal irradiation.

On days 1-7, mice in the Control group were gavaged with 0.2mL of PBS buffer, and RCT + ATCC53103 and RCT + DY801 groups were gavaged with 0.2mL of ATCC53103 and DY801 bacteria solutions with absorbance OD = 1.

On day 8, after the RCT group, RCT + ATCC53103 group, and RCT + DY801 group mice were fasted and deprived of water for 2 hours, the mice were anesthetized, irradiated with 6MeV X-rays in a dose of 6Gy, followed by chemotherapy treatment by a single intraperitoneal injection of 0.2ml of 150mg/Kg fluorouracil injection, and deprived of fasting and deprived of water after completion of the injection.

On days 9-11, mice in the Control group were gavaged with 0.2mL of PBS buffer, and RCT + ATCC53103 and RCT + DY801 groups were gavaged with 0.2mL of ATCC53103 and DY801 bacteria solutions with absorbance OD = 1.

Mice were dissected on day 30 to test for each index.

The survival time of each group of mice was analyzed by Kaplan Meier survival, and the results are shown in FIG. 9. As can be seen from fig. 9, the mice in the Control group all survived, the mice in the RCT group all died before the end of the experiment, the mice in the RCT + ATCC53103 group and the mice in the RCT + DY801 group all survived when the experiment was terminated, and compared with the RCT + ATCC53103 group, the number of the mice in the RCT + DY801 group survived was more, and the survival time was longer, wherein the survival rate of the mice in the RCT + DY801 group reached 83.3%. The results show that the rhamnose cheeseobacter DY801 provided by the invention can obviously improve the survival in the bodies of the mice with the chemoradiotherapy injury, and the survival rate is higher than that of the standard strain ATCC53103.

The results of staining the groups of mice treated as described above with hematoxylin-eosin (HE staining) are shown in fig. 10. As can be seen from FIG. 10, pathological damage of intestinal tissues such as gland tissue destruction, mucosal hyperemia and edema, and short villi of small intestine appeared in the RCT group, while structural damage of glands in the RCT + DY801 group was reduced, and the mucosal hyperemia and edema condition was significantly improved. The results show that the rhamnose cheese bacillus DY801 provided by the invention can reduce mucosal hyperemia and edema so as to effectively treat the radiotherapy and chemotherapy intestinal injury.

In addition, this example also utilizes ELISA to detect inflammation markers IL-1 β, IL-6 and TNF- α in blood of each group of mice, and the results are shown in FIG. 11. As can be seen from FIG. 11, the contents of IL-1 beta, IL-6 and TNF-alpha in the blood of the RCT group mice are significantly higher than those of the Control group mice, and compared with the RCT group mice, the inflammation indexes of the RCT + ATCC53103 group mice and the RCT + DY801 group mice are significantly reduced, and the IL-1 beta inflammation index improvement effect of the RCT + DY801 group mice is better than that of the standard strain ATCC53103.

Example 9 specific molecular target excavation of Lactobacillus rhamnosus DY801

The complete genome data of the other 279 strains of Lactobacillus rhamnosus in the NCBI database were downloaded, and pan-genomic analysis was performed on the 279 strains of Lactobacillus rhamnosus described above and the Lactobacillus rhamnosus DY801 provided by the present invention using the software Prokka (v 1.11) and Roary (v 3.11.2). After obtaining the core gene, MEGA X (v10.2.2) was used to identify a gene having a high base substitution density. Specific sequences of the Lactobacillus rhamnosus DY801 different from other Lactobacillus rhamnosus are obtained based on the above analysis process. Primer Premier 5 software is adopted to carry out Primer design aiming at the specific sequence, and the specific molecular target sequence SEQ ID:1.

the effectiveness of the specific molecule recognition target sequence of the Lactobacillus rhamnosus DY801 is verified by adopting Polymerase Chain Reaction (PCR). The detection template is the DNA of bacteria, and the DNA extraction method refers to a Tiangen bacteria genome DNA extraction kit. The amplification primers are shown in Table 5. The PCR reaction system and the PCR reaction conditions in this example were the same as those of the OCR reaction system and the PCR reaction conditions in example 1. After the PCR is finished, the PCR product is sent to an industrial company for sequencing, and the length of the PCR product is 431bp and is analyzed as shown in SEQ ID:1 is shown.

TABLE 5 amplification primer sequences

Numbering	Primer sequence (5 '→ 3')
		SEQ ID：2	TGATACACTCTGCGACTT
SEQ ID：3	TCACATTAGCACCGACTA

In conclusion, the Lactobacillus rhamnosus DY801 provided by the invention can improve the oxidative stress of the intestinal tract and the damage of the villi of the small intestine under the condition of radiotherapy and chemotherapy by generating a large amount of glutathione, reduce the inflammation index, accelerate the repair of intestinal tract tissues, has the characteristics of tolerance to artificial gastric juice and intestinal juice, has high survival rate in the stomach and the intestine, and can fix the intestinal tract and restore the steady state of intestinal flora. In addition, compared with the traditional medicine for treating the radiotherapy and chemotherapy intestinal injury, the rhamnose cheese bacillus DY801 provided by the invention has no obvious toxic or side effect on human bodies, has high safety, and can reduce the adverse reaction caused by the medicine and overcome the problem of low treatment efficiency of the medicine. The Lactobacillus rhamnosus DY801 or the culture thereof provided by the invention is applied to preparation of medicines or foods for preventing and/or treating the radiotherapy and chemotherapy intestinal injury, can be used for preventing and/or treating digestive system diseases caused by the radiotherapy and chemotherapy intestinal injury, solves the problem that the radiotherapy and chemotherapy intestinal injury after radiotherapy and chemotherapy of pelvic malignant tumor cannot be effectively treated in the prior art, and has great application prospect in preparation of medicines or foods for preventing and/or treating the radiotherapy and chemotherapy intestinal injury.

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 various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The strain of the Lactobacillus rhamnosus DY801 is preserved in Guangdong province microorganism culture collection center at 10 months and 9 days of 2022, and the preservation number is GDMCC 62853.

2. Use of the Lactobacillus rhamnosus DY801 according to claim 1 for the preparation of a medicament or food for the prevention and/or treatment of digestive disorders.

3. The use of the Lactobacillus rhamnosus DY801 according to claim 1 for the preparation of a medicament or food for the prevention and/or treatment of digestive disorders, wherein: the digestive system disease is caused by intestinal injury.

4. The use of the Lactobacillus rhamnosus DY801 according to claim 1 for preparing a medicament or food for the prevention and/or treatment of digestive disorders, wherein: the intestinal injury is radiation intestinal injury or chemotherapy intestinal injury.

5. A medicament for preventing and/or treating digestive system diseases, characterized in that: the medicament comprises a culture of the Lactobacillus rhamnosus DY801 as defined in claim 1 or the Lactobacillus rhamnosus DY801 as defined in claim 1.

6. The use of the Lactobacillus rhamnosus DY801 according to claim 1 for the preparation of a food or health care product with auxiliary protection against radiation hazards.

7. A food with auxiliary protection function to radiation hazard is characterized in that: the food product comprises a culture of the Lactobacillus rhamnosus DY801 according to claim 1 or the Lactobacillus rhamnosus DY801 according to claim 1.

8. A nucleotide sequence specifically recognizing the Lactobacillus rhamnosus DY801 as claimed in claim 1, characterized in that: the nucleotide sequence is shown as SEQ ID:1 is shown.

9. A primer set specifically recognizing the Lactobacillus rhamnosus DY801 as set forth in claim 1, wherein the primer set comprises: the primer group comprises a primer set shown as SEQ ID:2 and SEQ ID: 3.

10. A method for identifying the lactobacillus rhamnosus DY801 as claimed in claim 1, which is characterized by comprising:

the primer group of claim 9 is used as a specific amplification primer, the genomic DNA of the Lactobacillus rhamnosus to be detected is used as a template for PCR amplification to obtain a PCR product, and the PCR product is identified by sequencing or electrophoresis.