CN115975880A - Lactobacillus mucilaginosus CYQ09 and application thereof - Google Patents
Lactobacillus mucilaginosus CYQ09 and application thereof Download PDFInfo
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Abstract
The invention provides a lactobacillus mucilaginosus strain CYQ09 and application thereof. The lactobacillus mucilaginosus CYQ09 is preserved in the Guangdong province microorganism culture collection center at 2022, 4 and 1, and the preservation number is GDMCC 62346. The lactobacillus mucilaginosus CYQ09 provided by the invention can inhibit the inflammatory reaction induced by rays by generating a large amount of short-chain fatty acids, has the characteristic of tolerating gastric juice and intestinal juice of a human body, has no obvious toxic or side effect on the human body, and has high safety. The lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention can be applied to the preparation of medicines for preventing and/or treating radioactive intestinal injury, can be used for effectively preventing and/or treating digestive system diseases caused by radioactive intestinal injury, and solves the problem that the radioactive intestinal injury after radiotherapy of pelvic malignant tumor can not be effectively treated in the prior art.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus mucilaginosus CYQ09 and application thereof.
Background
Radiotherapy is one of the most effective means for treating pelvic malignant tumor, and about 35-61% of patients with pelvic malignant tumor have received radiotherapy. While radiation therapy can improve the local control rate of tumors, radiation damage to normal tissues inevitably occurs after radiation exposure. Furthermore, tissue not exposed to radiation conditions will also exhibit damage similar to radiation exposure due to radiation-induced bystander effects. About 75-81% of patients are reported to have radioactive intestinal injury after pelvic radiotherapy, and the radioactive intestinal injury often causes the quality of life of the patients to be reduced, the treatment compliance is poor, the treatment course is prolonged, and the overall survival prognosis is influenced. Therefore, effective mitigation of radioactive bowel injury, thereby improving patient life treatment and overall survival prognosis, is a global tumor treatment need.
At present, only expert consensus exists in domestic treatment for radioactive intestinal injury, no guideline with higher evidence level is available for reference, and the medicine treatment is usually adopted clinically. However, the drug therapy including non-steroidal anti-inflammatory drugs, steroid hormones and antibiotics can only improve local inflammation but cannot improve intestinal flora disorder, and the overall therapeutic effect is poor; meanwhile, the above drugs can cause various adverse reactions such as nausea and vomiting, and thus limit their wide application. The intestinal flora plays an important role in the occurrence and development of radioactive intestinal injury. Low doses of radiation can cause thinning of intestinal mucus layers and increased permeability; radiation also causes anaerobic bacteria to die directly through reactive oxygen species, resulting in flora disturbance, accelerating bacterial translocation and exacerbating local inflammation. In recent years, a plurality of students find that the supplement of probiotics can relieve the radioactive intestinal injury through basic research, which provides a new strategy for treating acute radioactive intestinal injury.
The lactobacillus mucilaginosus (Lactobacillus fermentum) is lactobacillus and lactobacillus mucilaginosus, is a gram-positive bacterium without plasmid, anaerobe, acid resistance and spore production, mostly exists in human and animal intestinal tracts, and has the biological characteristics of acid resistance, bile salt resistance, multiple antibiotics resistance, short-chain fatty acid production and the like. A large number of experiments show that the lactobacillus mucilaginosus can be planted in host intestinal tracts for a long time and play an important role in regulating intestinal flora. Meanwhile, the lactobacillus mucilaginosus has the effects of treating inflammatory bowel diseases, relieving diarrhea, reducing obesity, regulating host immunity and the like, and becomes one of probiotics with a plurality of international literature records and wide application. However, the existing lactobacillus mucilaginosus has poor tolerance to gastric juice and intestinal juice of a human body, so that the lactobacillus mucilaginosus has limited effect in gastrointestinal tracts of hosts and low content of short-chain fatty acids, thereby influencing the treatment effect of the lactobacillus mucilaginosus. Therefore, it is of great importance to find a Lactobacillus mucilaginosus strain that is able to tolerate the gastric and intestinal juices of the host.
Disclosure of Invention
The lactobacillus mucilaginosus strain CYQ09 can inhibit inflammatory reaction induced by rays by producing a large amount of short-chain fatty acid, has the characteristic of tolerance to gastric juice and intestinal juice of a human body, and can be used for preventing and treating radioactive intestinal injury.
According to the first aspect of the invention, the lactobacillus mucilaginosus strain CYQ09 is preserved in Guangdong province microorganism strain preservation center at 1.4.2022, and the preservation address is Guangdong province microorganism research institute No. 100 Michelia furiosaefolia, guangzhou, and the preservation number is GDMCC 62346.
The invention separates a new strain of lactobacillus mucilaginosus strain from fresh excrement of healthy adults in Guangzhou city, guangdong province, and the strain is classified and named as Limosilactibacillus formem CYQ09. Compared with the existing lactobacillus mucilaginosus, the lactobacillus mucilaginosus CYQ09 provided by the invention is an excellent strain with native source, and is specifically embodied as follows: the strain can produce bacterial culture products such as short-chain fatty acid, and can improve intestinal inflammation; 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 intestinal stem cells under the radiation condition, accelerate the repair of intestinal tissues and further prolong the survival time of patients. In addition, compared with the traditional medicine for treating radioactive intestinal injury, the lactobacillus mucilaginosus strain CYQ09 has no obvious toxic or side effect on human bodies and has high safety. In conclusion, the lactobacillus fermentum CYQ09 provided by the invention has many excellent characteristics, can be used for effectively preventing and treating digestive system diseases caused by radioactive intestinal injury, solves the problem that the radioactive intestinal injury after radiotherapy of pelvic malignant tumor cannot be effectively treated in the prior art, and has great application prospects in preparation of medicines for preventing and/or treating the radioactive intestinal injury or foods and health-care products with auxiliary protection function on radiation hazard.
According to a second aspect of the present invention, there is provided the use of lactobacillus fermentum CYQ09 as described above, for the preparation of a medicament for the prevention and/or treatment of a disease of the digestive system.
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 a radioactive intestinal injury.
According to a third aspect of the present invention, there is provided a medicament for preventing and/or treating a disease of the digestive system, which comprises the above lactobacillus mucilaginosus CYQ09 or a culture of the above lactobacillus mucilaginosus CYQ09.
The lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention is applied to the preparation of the medicine for preventing and/or treating digestive system diseases caused by radioactive intestinal injury, and the prepared medicine can effectively prevent and/or treat the radioactive intestinal injury, thereby solving the problem that the radioactive intestinal injury after radiotherapy of pelvic malignant tumor can not be effectively treated in the prior art.
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 a radioactive intestinal injury.
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 present invention, there is provided a use of the lactobacillus mucilaginosus CYQ09 as described above in the preparation of a food or health care product with an auxiliary protection function against radiation hazards.
According to a fifth aspect of the present invention, there is provided a food product having an auxiliary protective function against radiation damage, which comprises the above lactobacillus mucilaginosus CYQ09 or a culture of the above lactobacillus mucilaginosus CYQ09.
The lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention is applied to the 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 lactobacillus mucilaginosus CYQ09 as set forth in SEQ ID:1 is shown.
The nucleotide sequence provided by the scheme can effectively distinguish the lactobacillus mucilaginosus CYQ09 provided by the invention from other lactobacillus mucilaginosus isolates.
According to a seventh aspect of the present invention, there is provided a primer set specifically recognizing lactobacillus mucilaginosus CYQ09 as described above, the primer set comprising a primer set shown as SEQ ID:2 and SEQ ID: 3.
The primer group provided by the scheme can carry out PCR reaction amplification on the DNA of the bacteria to be detected, if a 384bp product is amplified, the bacteria to be detected is the lactobacillus mucilaginosus CYQ09, and if the 384bp product is not amplified, the bacteria to be detected is not the lactobacillus mucilaginosus CYQ09.
According to the eighth aspect of the present invention, a method for identifying the lactobacillus mucilaginosus CYQ09 is provided, wherein the primer group is used as a specific amplification primer, the genomic DNA of the lactobacillus mucilaginosus to be detected is used as a template to perform PCR amplification, a PCR product is obtained, and the PCR product is identified by sequencing or electrophoresis.
Drawings
FIG. 1 is a colony morphology of Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1.
FIG. 2 is a gram-stained bacterial body of Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1.
FIG. 3 is a 16S rDNA phylogenetic tree constructed in example 2 when Lactobacillus fermentum CYQ09 was identified.
FIG. 4 is a graph showing the results of glycolysis biochemical tests performed in example 2 for the identification of Lactobacillus polymyxa CYQ09.
FIG. 5 is a whole genome sequencing map and genome circled map of Lactobacillus mucilaginosus CYQ09 of example 3.
FIG. 6 is a graph showing the results of evaluating the safety of Lactobacillus polymyxa CYQ09 in mice in example 4.
FIG. 7 is a graph showing the results of measuring the content of short-chain fatty acids produced by Lactobacillus mucilaginosus CYQ09 in example 7 by gas chromatography-mass spectrometry.
FIG. 8 is a graph showing the results of the experiment of Lactobacillus polymyxa CYQ09 in example 8 for reducing radiation damage to small intestine organoids in vitro.
FIG. 9 is a graph showing the survival rate of mice treated with radiation-induced intestinal injury by Lactobacillus polymyxa CYQ09 in example 9.
FIG. 10 is a HE staining pattern (100X) of intestinal tissue after treatment of radiation-induced intestinal injury mice with Lactobacillus mucilaginosus CYQ09 of example 9.
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 mucilaginosus CYQ09
In this example, a new lactobacillus mucilaginosus strain was isolated from fresh feces of healthy adults originated in Guangzhou, guangdong province, and was identified as lactobacillus mucilaginosus CYQ09 by using morphological characteristics, culture traits, physiological and biochemical characteristics, and genetic characteristics of 16SrDNA, and the lactobacillus mucilaginosus strain was deposited at the Guangdong province collection center of microbial cultures at 1.4.2022, with the deposition address of Guangdong province institute of microorganisms, michelia, mcA, guangzhou, and was classified and named as Limosilactibacillus fermentum CYQ09, with the deposition number of GDMCC 62346.
In this example, fresh feces of healthy adults from Guangzhou city, guangdong province were collected and mixed at a solid-to-liquid ratio of 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 Lactobacillus mucilaginosus CYQ09 obtained by the separation and the culture in the example are as follows:
(1) Characteristics of bacterial colony
In this example, the isolated and cultured Lactobacillus mucilaginosus CYQ09 was streaked on a plate and anaerobically cultured at 37 ℃ for 48 hours, and the colony morphology is shown in FIG. 1. As can be seen from FIG. 1, the Lactobacillus fermentum CYQ09 has good growth state, and the bacterial colony is milky white, round, convex and regular in edge.
(2) Characteristics of the thallus
In this example, the morphology of the isolated and cultured Lactobacillus fermentum CYQ09 was observed and stained by gram stain, and the results are shown in FIG. 2. As can be seen from FIG. 2, lactobacillus mucilaginosus CYQ09 is in the shape of a short rod, no movement, no spores, facultative anaerobic, gram-positive stain.
Example 2 identification of Lactobacillus mucilaginosus CYQ09
In this example, the total bacterial DNA of Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1 was extracted using a bacterial genomic DNA Kit (TIANAmp Bacteria DNA Kit), 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 'GGTTACCTTGTGACTT-doped 3';
the primers were synthesized by Beijing Optimalaceae biology Ltd.
The PCR amplification reaction system comprises 20 mu L of template, 2 mu L of TaKaRa Premix TaqTM 10 mu L of forward primer and reverse primer, 1 mu L of forward primer and 6 mu L of double distilled water. 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 ℃.
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 Lactobacillus mucilaginosus CYQ09 16S rDNA gene sequences were aligned using the BLAST software tool and phylogenetic trees were constructed using MEGA X, the results are shown in FIG. 3.
As can be seen from FIG. 3, the sequencing result of the Lactobacillus mucilaginosus CYQ09 provided by the invention has 99% homology with the 16S rDNA sequence of the Lactobacillus mucilaginosus, and the strain CYQ09 is identified to be the Lactobacillus mucilaginosus and named CYQ09, namely the Lactobacillus mucilaginosus CYQ09 obtained by separation and culture.
In addition, this example further identifies lactobacillus mucilaginosus CYQ09 by using sugar fermentation experiments:
the operation is carried out according to the specification of the novel microorganism micro-biochemical series identification tube, and the separated biochemical metabolites of the candidate strain CYQ09 are detected, and the results are shown in figure 4 and table 1. As can be seen by combining Bergey's Manual data of bacteria identification and FIG. 4 and Table 1, the physiological and biochemical characteristics of the strain are basically consistent with those of the Lactobacillus fermentum standard strain ATCC 14931.
TABLE 1 Biochemical identification results of Lactobacillus mucilaginosus CYQ09
Note: "+" is positive and "-" is negative.
Example 3 Whole genome sequencing of Lactobacillus mucilaginosus CYQ09
Genomic DNA of Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1 was extracted, and quality control of purity, concentration and integrity was performed by Nanodrop, qubit and 0.35% agarose gel electrophoresis. Then, 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, the original data after machine unloading is subjected to quality control, and low-quality and short-length reads are filtered; genome assembly is then performed, 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, were carried out. Meanwhile, genome analysis and genome map analysis are also performed. The whole genome sequencing results of Lactobacillus mucilaginosus CYQ09 are shown in FIG. 5.
From the genome sequencing and genome circled diagram of fig. 5, the genome size of lactobacillus mucilaginosus CYQ09 is 2.06mb, gc ratio is 51.83%, and the genome contains 2012 CDS regions, 2138bp repeats, 58 trnas and 15 rrnas. The genome functional annotation suggested that lactobacillus fermentum CYQ09 contained 1 potential drug resistance gene, poxtA, but no virulence genes obtained by horizontal gene transfer.
Example 4 safety assessment of Lactobacillus mucilaginosus CYQ09
The lactobacillus mucilaginosus CYQ09 provided by the invention is probiotics separated from fresh excrement of healthy adults, and the safety and the effectiveness of the lactobacillus mucilaginosus CYQ09 are ensured from the bacterial source. In addition, after the lactobacillus fermentum CYQ09 provided by the invention is colonized and propagated in the human intestinal environment, only intestinal epithelial cells attached to a host can become a layer of biological barrier of the intestinal mucosa, the barrier capability of the intestinal mucosa of the host is improved, and the lactobacillus fermentum CYQ09 can directly act on a human body in a viable bacteria manner, so that the safety is ensured.
In this example, the sensitivity of Lactobacillus mucilaginosus CYQ09 to 8 antibiotics was examined by broth microdilution according to the general Probiotics for food code published by the Chinese food science and technology society. The 8 antibiotics were: tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, and ammoniaBenzylpenicillin 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 (from 1-256 mg/mL), at 37 degrees C anaerobic culture for 48 hours. After 48 hours, the Minimum Inhibitory Concentration (MIC) of the lactobacillus mucilaginosus strain CYQ09 was read for each antibiotic, 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 susceptibility of Lactobacillus mucilaginosus CYQ09 to different antibiotics
As can be seen from Table 2, the MIC of Lactobacillus mucilaginosus CYQ09 for tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin and gentamicin are as follows: 8mg/L, 32mg/L, 256mg/L, 4mg/L, 1mg/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 Lactobacillus fermentum CYQ09, 3C 57BL/6 mice aged 6-8 weeks were selected for the experiment. Animals were acclimatized for 5 days in the animal house prior to the experiment. The experimental animals and the experimental animal rooms meet the national regulations, and standard compound feed is selected, so that the diet and drinking water are not limited. And (3) performing intragastric administration on the mice by using lactobacillus mucilaginosus CYQ09, wherein the intragastric absorbance OD =1 per day is 0.2mL of CYQ09 bacterial liquid. After the feeding, the experimental animals were sacrificed by cervical dislocation, and the organs were dissected and removed by a scalpel, and the important organs of the mice were observed, and the results are shown in fig. 6.
As can be seen from FIG. 6, no abnormality was found in the mouse vital organs, kidney, liver, spleen, stomach, cecum, and large intestine, which indicates that the Lactobacillus polymyxa CYQ09 provided by the present invention is safe for use in mice.
Example 5 Lactobacillus mucilaginosus CYQ09 has better tolerance to artificial gastric juice
Respectively preparing artificial gastric juice with pH values of 2.0,3.0 and 4.0. The Lactobacillus mucilaginosus CYQ09 isolated and cultured 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 viable bacteria after the culture by using an enzyme-labeling instrument, wherein the results are shown in table 3.
As can be seen from Table 3, compared with the Lactobacillus mucilaginosus standard strain ATCC14931, the Lactobacillus mucilaginosus CYQ09 provided by the invention has better artificial gastric juice resistance.
TABLE 3 comparison of the resistance of CYQ09 to ATCC14931 to artificial gastric juice
Example 6 Lactobacillus mucilaginosus CYQ09 has better tolerance effect on artificial intestinal juice
Preparing the artificial intestinal juice with pH value of 6.8 and bile salt. The Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1 was diluted to 10 degrees 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 4 comparison of CYQ09 and ATCC14931 Artificial gut fluid resistance
As can be seen from Table 4, lactobacillus fermentum CYQ09 exhibited better resistance to artificial intestinal juice than the standard strain of Lactobacillus fermentum ATCC 14931.
Example 7 Lactobacillus mucilaginosus CYQ09 has strong ability to produce short chain fatty acids
Adjusting absorbance to OD 600 =1 lactobacillus mucilaginosus CYQ09 bacteriumAdding 100 μ L of the solution into 8mL of MRS broth, culturing for 24 hr, centrifuging 1mL of the bacterial suspension in 1.5mL centrifuge tube at 10000rpm/min for 2min, collecting 100 μ L of the supernatant, adding 5 μ L of Internal Standard (IS), 5 μ L of deionized water, 150 μ L of methanol, 40 μ L of 2.5% H 2 SO 4 0.05g of anhydrous sodium sulfate, vortexed for 1.5min, and centrifuged (14000 rpm, room temperature, 5 min); taking the supernatant in a sample injection vial with a liner tube as a sample to be detected.
Preparing a follow blank sample: mu.L of physiological saline was taken and added with 5. Mu.L of Internal Standard (IS), 5. Mu.L of deionized water, 150. Mu.L of methanol, 40. Mu.L of 2.5% H 2 SO 4 0.05g of anhydrous sodium sulfate, vortexing for 1.5min, and centrifuging (14000 rpm, room temperature and 5 min), wherein the steps are consistent with the treatment of the sample to be detected.
Preparing a quality control sample: mu.L of physiological saline was taken and added with 5. Mu.L of Internal Standard (IS), 5. Mu.L of STD-4 (standard diluted 1000-fold), 150. Mu.L of methanol, 40. Mu.L of 2.5% H 2 SO4,0.05g of anhydrous sodium sulfate, vortexing for 1.5min, and centrifuging (14000 rpm, room temperature, 5 min), the steps being identical to those of the treatment of the sample to be tested.
The content of short-chain fatty acids in the supernatant was determined by gas chromatography-mass spectrometry, i.e., the content of short-chain fatty acids in the sample to be tested was detected, and the results are shown in fig. 7.
As can be seen from FIG. 7, compared with the Lactobacillus mucilaginosus standard strain ATCC14931, the Lactobacillus mucilaginosus CYQ09 provided by the invention has better capability of producing short-chain fatty acids, has higher total amount of short-chain fatty acids, and particularly has higher yield of acetic acid, propionic acid and butyric acid than ATCC 14931.
Example 8 Lactobacillus mucilaginosus CYQ09 increases the tolerance of small intestinal organoids to radiation damage
Extracting mouse small intestine crypt, culturing to obtain small intestine organoid, and culturing the organoid at the 7 th day as follows: control group at 37 ℃ C. And 5% CO 2 Normally culturing under the condition; the irradiation group was irradiated to organoids with 6MeV X-ray 6Gy, and then the CO was determined at 37 ℃ and 5% 2 Culturing for 4 hours, and then replacing a fresh culture medium; irradiation + ATCC14931 group 50. Mu.L of the culture supernatant of the standard strain ATCC14931 was pipetted into the culture well, immediately followed by 6MeV X-ray 6Gy irradiation, and then at 37 ℃、5％CO 2 Culturing for 4 hours, and then replacing a fresh culture medium without the bacterial culture supernatant to culture for 24 hours; irradiation + CYQ09 group 50. Mu.L of culture supernatant of Lactobacillus fermentum CYQ09 was aspirated into the culture well, immediately followed by 6MeV X-ray 6Gy irradiation, and then 5% CO at 37 ℃ C 2 The culture was continued for 4 hours, and then the culture medium containing fresh culture supernatant without bacteria was replaced for 24 hours. The morphology of the small intestine organoids after the completion of the culture was observed, and the results are shown in FIG. 8.
As can be seen from FIG. 8, compared with the Lactobacillus mucilaginosus standard strain ATCC14931, the organoid of the Lactobacillus mucilaginosus CYQ09 provided by the invention can maintain better shape under the culture condition, which shows that the Lactobacillus mucilaginosus CYQ09 has the treatment effect on radiation injury.
Example 9 Lactobacillus mucilaginosus CYQ09 fermented can reduce glandular injury
24 mice of 4 weeks old C57BL/6 were selected, and after one week of normal rearing, randomly divided into 4 groups: control group (n = 6), whole abdomen irradiation + ATCC14931 group (n = 6), whole abdomen irradiation + CYQ09 group (n = 6), and the treatment was performed according to the following procedure:
on days 1-7, mice in the Control group are subjected to intragastric lavage by adopting 0.2mL PBS buffer solution, and the whole abdominal irradiation + ATCC14931 group and the whole abdominal irradiation + CYQ09 group are subjected to intragastric lavage by respectively adopting 0.2mL ATCC14931 and CYQ09 bacterial solutions with absorbance OD = 1;
on the 8 th day, after each group of mice is fasted and water-deprived for 2 hours, anesthetizing the mice, performing full abdominal irradiation by using 6MeV X-rays except for a control group, wherein the dose is 12Gy, and then releasing the fasting and water-deprivation;
on days 9-11, mice in the Control group were subjected to intragastric lavage with 0.2mL of PBS buffer solution, and the whole abdominal irradiation + ATCC14931 group and the whole abdominal irradiation + CYQ09 group were subjected to intragastric lavage with 0.2mL of ATCC14931 and CYQ09 bacterial solutions with OD =1, respectively;
mice were dissected on day 32 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 whole abdominal irradiation group all died before the end of the experiment, the mice in the whole abdominal irradiation + ATCC14931 group and the whole abdominal irradiation + CYQ09 group all survived at the end of the experiment, and the mice in the whole abdominal irradiation + CYQ09 group survived more in number and survived longer than those in the whole abdominal irradiation + ATCC14931 group. The results show that the Lactobacillus fermentum CYQ09 provided by the invention can obviously improve the survival in the radioactive injury mice, and the effect is better than that of the standard strain ATCC 14931.
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 lesions of intestinal tissues such as gland tissue destruction and inflammatory infiltration appear in the whole abdominal irradiation group, while the gland structural lesions of the whole abdominal irradiation + CYQ09 group were reduced, and inflammatory cell infiltration was significantly reduced. The above results demonstrate that the Lactobacillus mucilaginosus CYQ09 provided by the invention can reduce the radioactive intestinal injury.
Example 10 specific molecular target excavation by Lactobacillus polymyxa CYQ09
The complete genome data of other 155 L.mucor fermentum strains in NCBI database are downloaded, and genome-wide analysis is carried out on the 155 L.mucor fermentum strains and the L.mucor fermentum CYQ09 provided by the invention by using Prokka (v 1.11) and Roary (v3.11.2) software. 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 lactobacillus mucilaginosus CYQ09 different from other lactobacillus mucilaginosus are obtained based on the 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 molecular recognition target sequence of the lactobacillus mucilaginosus CYQ09 provided by the invention is verified by Polymerase Chain Reaction (PCR). The detection template is the DNA of bacteria, the DNA extraction method refers to a Tiangen bacteria genome DNA extraction kit, and the amplification primer sequence is shown in table 5. The PCR reaction system and the PCR reaction conditions in this example were the same as those in the PCR 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 384bp and is analyzed as shown in SEQ ID:1 is shown.
TABLE 5 amplification primer sequences
| Numbering | Primer sequence (5 '→ 3') |
| SEQ ID：2 | AGTGAACCGCTGGAAAGA |
| SEQ ID：3 | TTGGGATGGTAACAGAAGG |
In conclusion, the lactobacillus mucilaginosus CYQ09 provided by the invention can inhibit inflammatory reaction induced by rays by generating a large amount of short-chain fatty acids, reduce the damage of intestinal stem cells under radiation conditions, accelerate intestinal tissue repair, prolong the survival time of patients, has the characteristics of tolerance to gastric juice and intestinal juice of human bodies, has high survival rate in the stomach and the intestine, can colonize the intestinal tract, and restore the intestinal flora homeostasis. In addition, compared with the traditional medicament for treating the radioactive intestinal injury, the lactobacillus fermentation strain CYQ09 has no obvious toxic or side effect on human bodies, has high safety, can reduce the adverse reaction caused by the medicament and solves the problem of low effective rate of medicament treatment. The lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention is applied to the preparation of medicines or foods for preventing and/or treating radioactive intestinal injury, can be used for effectively preventing and/or treating digestive system diseases caused by the radioactive intestinal injury, solves the problem that the radioactive intestinal injury occurring after radiotherapy of pelvic malignant tumor can not be effectively treated in the prior art, and has great application prospect in the preparation of medicines or foods for preventing and/or treating the radioactive 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 (10)
1. The lactobacillus mucilaginosus strain CYQ09 is preserved in Guangdong province microorganism strain preservation center at 2022, 4 and 1 days, and the preservation number is GDMCC 62346.
2. Use of lactobacillus fermentum CYQ09 according to claim 1 for the preparation of a medicament for the prevention and/or treatment of a disease of the digestive system.
3. Use of lactobacillus fermentum CYQ09 according to claim 2, for the preparation of a medicament for the prevention and/or treatment of diseases of the digestive system, wherein: the digestive system disease is caused by intestinal injury.
4. Use of lactobacillus fermentum CYQ09 according to claim 3, for the preparation of a medicament for the prevention and/or treatment of a disease of the digestive system, wherein: the intestinal injury is a radioactive intestinal injury.
5. A medicament for preventing and/or treating digestive system diseases, characterized in that: the medicament comprises lactobacillus mucilaginosus CYQ09 as claimed in claim 1 or a culture of lactobacillus mucilaginosus CYQ09 as claimed in claim 1.
6. Use of lactobacillus fermentum CYQ09 according to claim 1 for the preparation of a food or health product with auxiliary protection against radiation damage.
7. A food with auxiliary protection function to radiation hazard is characterized in that: the food product comprises lactobacillus mucilaginosus CYQ09 as described in claim 1 or a culture of lactobacillus mucilaginosus CYQ09 as described in claim 1.
8. A nucleotide sequence which specifically recognizes lactobacillus mucilaginosus CYQ09 as set forth in claim 1, wherein the nucleotide sequence comprises: the nucleotide sequence is shown as SEQ ID:1 is shown.
9. A primer set specifically recognizing lactobacillus mucilaginosus CYQ09 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 lactobacillus slime ferment CYQ09 as set forth in claim 1, wherein: the primer group of claim 9 is used as a specific amplification primer, the genomic DNA of the Lactobacillus mucilaginosus 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.
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