CN115975880B - Lactobacillus mucilaginosus CYQ09 and application thereof - Google Patents
Lactobacillus mucilaginosus CYQ09 and application thereof Download PDFInfo
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Abstract
The invention provides lactobacillus mucilaginosus CYQ09 and application thereof. The lactobacillus mucilaginosus CYQ09 is deposited with the microorganism strain collection of Guangdong province at 2022, 4 and 1, with the deposit number of GDMCC 62346. The fermented lactobacillus mucilaginosus CYQ09 provided by the invention can inhibit the inflammatory reaction induced by rays by producing a large amount of short chain fatty acid, 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 is high in safety. The fermented lactobacillus mucilaginosus CYQ09 or a culture thereof provided by the invention can be applied to the preparation of medicines for preventing and/or treating the radioactive intestinal injury, can be used for effectively preventing and/or treating digestive system diseases caused by the radioactive intestinal injury, and solves the problem that the radioactive intestinal injury after the radiotherapy of the malignant pelvic tumor cannot be effectively treated in the prior art.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus mucilaginosus CYQ09 and application thereof.
Background
Radiotherapy is one of the most effective means for treating pelvic malignant tumors, and about 35% -61% of patients with pelvic malignant tumors receive radiotherapy. Although radiotherapy may increase the local control rate of tumors, radiation damage to normal tissue is inevitable after radiation exposure. In addition, tissue not exposed to radiation conditions will also exhibit a similar damage profile to radiation exposure due to radiation-induced bystander effects. It is reported that about 75% -81% of patients undergo pelvic radiotherapy and then have radioactive intestinal injury, and the radioactive intestinal injury often leads to the reduction of life quality of the patients, poor treatment compliance, prolonged treatment course and influence on overall survival prognosis. Therefore, the method effectively relieves the damage of the radioactive intestines, thereby improving the life treatment and the overall survival prognosis of patients, and is a global tumor treatment requirement.
At present, only expert consensus exists in domestic treatment of radioactive intestinal injury, no guideline with higher evidence level can be referred, and drug treatment is commonly adopted clinically. However, the drug treatment based on non-steroidal anti-inflammatory drugs, steroid hormones and antibiotics can only improve local inflammation but can not improve intestinal flora disorder, and the overall treatment effect is poor; meanwhile, the medicines can cause various adverse reactions such as nausea, vomiting and the like, so that the wide application of the medicines is limited. Intestinal flora plays an important role in the occurrence and development of radiation intestinal lesions. The low dose radiation can cause the thinning of intestinal mucus layer and the increase of permeability; the radiation also directly causes anaerobic bacterial death through active oxygen, which causes flora disturbance, accelerates bacterial ectopic sites and aggravates local inflammation. In recent years, a plurality of scholars find that supplementing probiotics can relieve the radioactive intestinal injury through basic research, and a new strategy is provided for treating the acute radioactive intestinal injury.
The fermented lactobacillus mucilaginosus (Limosilactobacillus fermentum) is lactobacillus and lactobacillus mucilaginosus, is a gram positive bacterium without plasmids, has anaerobic acid resistance and does not produce spores, is mostly present in intestinal tracts of people and animals, and has the biological characteristics of acid resistance, bile salt resistance, various antibiotics resistance, short chain fatty acid production and the like. A large number of experiments show that the fermented lactobacillus mucilaginosus can be planted in the intestinal canal of a host for a long time and play an important role in regulating the intestinal flora. Meanwhile, the fermented lactobacillus mucilaginosus has the effects of treating inflammatory bowel disease, 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 range. However, the existing lactobacillus fermentum has poor tolerance to gastric juice and intestinal juice of human body, which results in limited function of lactobacillus fermentum in the gastrointestinal tract of host, and low content of short chain fatty acid, thereby affecting the therapeutic effect of lactobacillus fermentum. Therefore, it is of great importance to find a fermented lactobacillus mucilaginosus that is tolerant to gastric and intestinal fluids of the host.
Disclosure of Invention
The invention provides a lactobacillus mucilaginosus CYQ09 strain and application thereof, wherein the lactobacillus mucilaginosus strain can inhibit radiation-induced inflammatory reaction by generating a large amount of short chain fatty acid, has the characteristic of resisting gastric juice and intestinal juice of a human body, and can be used for preventing and treating radioactive intestinal injury.
According to a first aspect of the present invention there is provided a strain of Lactobacillus fermentum CYQ09 deposited at the microorganism strain collection, cantonese, calif. 1, 4, 2022, under the accession number GDMCC 62346 from the microorganism institute, cantonese, mitsui, guangdong.
The invention is characterized in that a novel fermented lactobacillus mucilaginosus strain is separated from fresh feces of healthy adults in Guangzhou city of Guangdong province, and is classified and named as Limosilactobacillus fermentum CYQ09. Compared with the existing fermented lactobacillus mucilaginosus, the fermented lactobacillus mucilaginosus CYQ09 provided by the invention is an excellent strain of local sources, and is specifically expressed as follows: the strain can produce bacterial culture products such as short chain fatty acid and the like, and can improve intestinal inflammation; the strain can resist artificial gastric juice and intestinal juice, has high survival rate in stomach and intestines, can be planted in intestinal tracts, and can restore stable 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 the radioactive intestinal injury, the lactobacillus mucilaginosus strain CYQ09 has no obvious toxic or side effect on human body and has high safety. In conclusion, the lactobacillus mucilaginosus CYQ09 has a plurality of 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 the radiotherapy of the malignant tumor of the pelvis cannot be effectively treated in the prior art, and has great application prospect in preparing 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 mucilaginosus CYQ09 as described above in the manufacture of a medicament for the prevention and/or treatment of digestive system diseases.
Preferably, the digestive system disease comprises at least one of abdominal pain, abdominal distension, nausea, vomiting, diarrhea, constipation.
Preferably, the 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 the prophylaxis and/or treatment of a digestive system disease, the medicament comprising lactobacillus fermentum CYQ09 as defined above or a culture of lactobacillus fermentum CYQ09 as defined above.
The fermented lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention is applied to the preparation of a medicament for preventing and/or treating digestive system diseases caused by radioactive intestinal injury, and the prepared medicament can effectively prevent and/or treat the radioactive intestinal injury, so that the problem that the radioactive intestinal injury after the radiotherapy of the malignant pelvic tumor cannot be effectively treated in the prior art is solved.
Preferably, the digestive system disease comprises at least one of abdominal pain, abdominal distension, nausea, vomiting, diarrhea, constipation.
Preferably, the 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: pharmaceutically acceptable adjuvants include at least one of stabilizers, wetting agents, emulsifiers, binders, isotonic agents.
Preferably, the medicine 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 the use of lactobacillus mucilaginosus CYQ09 as described above in the manufacture of a food or health care product having an auxiliary protective function against radiation hazards.
According to a fifth aspect of the present invention there is provided a food product with an auxiliary protective function against radiation hazards, the food product comprising lactobacillus fermentum CYQ09 as described above or a culture of lactobacillus fermentum CYQ09 as described above.
The lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention is applied to the preparation of foods or health care products with auxiliary protection function on radiation hazard, and the prepared foods or health care products can play a certain auxiliary protection function on the radiation hazard.
According to a sixth aspect of the present invention, there is provided a nucleotide sequence capable of specifically recognizing the above lactobacillus fermentum CYQ09, the nucleotide sequence being as shown in SEQ ID no: 1.
The nucleotide sequence provided by the scheme can effectively distinguish the lactobacillus fermentum CYQ09 provided by the invention from other lactobacillus fermentum isolates.
According to a seventh aspect of the present invention there is provided a primer set for specifically recognizing lactobacillus fermentum CYQ09 as defined above, the primer set comprising the sequence set forth in SEQ ID no: 2 and SEQ ID:3, and a nucleotide sequence shown in 3.
The primer group provided by the scheme can carry out PCR amplification on DNA of the bacteria to be detected, if 384bp products are amplified, the bacteria to be detected are the lactobacillus fermentum CYQ09, and if 384bp products are not amplified, the bacteria to be detected are not the lactobacillus fermentum CYQ09.
According to an eighth aspect of the present invention, there is provided a method for identifying the above-mentioned Lactobacillus fermentum CYQ09, wherein the above-mentioned primer set is used as a specific amplification primer, and the genomic DNA of Lactobacillus fermentum 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.
Drawings
FIG. 1 is a colony morphology of Lactobacillus fermentum CYQ09 isolated and cultured in example 1.
FIG. 2 is a graph showing the result of gram staining of cells of Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1.
FIG. 3 is a 16S rDNA phylogenetic tree constructed by the identification of Lactobacillus fermentum CYQ09 in example 2.
FIG. 4 is a graph showing the results of the glycolytic biochemical test for example 2 when Lactobacillus fermentum CYQ09 is identified.
FIG. 5 is a full genome sequencing map and a genome circle map of Lactobacillus fermentum CYQ09 of example 3.
FIG. 6 is a graph showing the results of evaluation of safety in mice of Lactobacillus fermentum CYQ09 in example 4.
FIG. 7 is a graph showing the results of detection of the content of short chain fatty acids produced by Lactobacillus mucilaginosus CYQ09 by gas chromatography-mass spectrometry in example 7.
FIG. 8 is a graph showing the results of an experiment for reducing radiation damage to small intestine organoids in vitro using Lactobacillus mucilaginosus CYQ09 of example 8.
FIG. 9 is a graph showing survival of mice treated with radiation intestinal injury by Lactobacillus mucilaginosus CYQ09 in example 9.
FIG. 10 is a HE staining pattern (100X) of intestinal tissue of mice treated with Lactobacillus mucilaginosus CYQ09 in example 9.
Detailed Description
The technical features of the technical solution provided in the present invention will be further clearly and completely described in connection with the detailed description below, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 isolation of Lactobacillus mucilaginosus CYQ09
In this example, a novel strain of Lactobacillus fermentum was isolated from fresh feces derived from healthy adults in Guangzhou, guangdong, and identified as Lactobacillus fermentum CYQ09 by using morphological, cultural, physiological and biochemical characteristics and genetic characteristics 16SrDNA, and deposited at the microorganism strain collection in Guangdong, 4 th month 1 of 2022, at a accession number of GDMCC 62346 under the classification designation Limosilactobacillus fermentum CYQ09, 100 Guangdong, first, and Guangzhou.
In this example, after collecting fresh feces from healthy adults in Guangzhou city in Guangdong, the solid-to-liquid ratio was 1:1000 (g/mL) ratio to fresh feces, PBS buffer was added for dilution. And 5 mu L of the diluted fresh fecal sample is inoculated into an MRS culture medium, anaerobic culture is carried out for 48 hours at the constant temperature of 37 ℃, and then single colony is selected and inoculated into the MRS liquid culture medium for enrichment culture.
Wherein, the formula of the MRS culture medium 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 regulating the pH to 6.12-6.2.
The morphological characteristics of lactobacillus mucilaginosus CYQ09 obtained after the separation and culture in this example are as follows:
(1) Colony characterization
In this example, lactobacillus mucilaginosus CYQ09 obtained by separation and culture was streaked on a plate, and cultured anaerobically at a constant temperature of 37℃for 48 hours, and the colony morphology is shown in FIG. 1. As shown in FIG. 1, the Lactobacillus mucilaginosus CYQ09 has good growth state, and the colony is milky white, round, convex and neat in edge.
(2) Characteristics of the cells
In this example, the bacterial form of Lactobacillus fermentum CYQ09 obtained by separation and culture was observed, and simultaneously stained by the gram stain method, and the results are shown in FIG. 2. As can be seen from FIG. 2, lactobacillus mucilaginosus CYQ09 has a short, rod-like shape, does not move, is spore-free, and is facultatively anaerobic and gram-positive.
EXAMPLE 2 identification of Lactobacillus mucilaginosus CYQ09
In this example, bacterial genomic DNA kit (TIANamp Bacteria DNA Kit) was used to extract the total bacterial DNA of Lactobacillus mucilaginosus CYQ09 isolated and cultured in example 1, 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 universal primer pair of the 16S rDNA is as follows:
forward primer 27f:5'-AGAGTTTGATCCTGGCTCAG-3' the number of the individual pieces of the plastic,
reverse primer 1492r:5'-GGTTACCTTGTTACGACTT-3';
primers were synthesized by Beijing Optimu Corp.
The PCR amplification reaction system was 20. Mu.L in total, 2. Mu.L for the template, taKaRa Premix TaqTM. Mu.L for the forward primer, 1. Mu.L for the reverse primer, and 6. Mu.L for the double distilled water. Meanwhile, a negative control (control) is arranged, and in a negative control reaction system, the template is replaced by double distilled water, and the rest components are the same.
PCR amplification reaction conditions: 94 ℃ for 5min;94 ℃ 60s,60 ℃ 60s,72 ℃ 90s,30 cycles; 72 ℃ for 10min; preserving at 4 ℃.
After PCR electrophoresis, the gel was cut, and the target band gel was recovered and sequenced by Shanghai Biotechnology Co., ltd (nucleotide sequence shown as SEQ ID: 4). In the NCBI database of America, the sequences of the Lactobacillus fermentum CYQ09 16S rDNA gene were aligned using BLAST software tools, and phylogenetic trees were constructed using MEGA X, and the results are shown in FIG. 3.
As can be seen from FIG. 3, the sequencing result of the fermented lactobacillus mucilaginosus CYQ09 provided by the invention has the homology of 99% with the 16S rDNA sequence of the fermented lactobacillus mucilaginosus, and the CYQ09 strain is identified as the fermented lactobacillus mucilaginosus and named as CYQ09, namely the fermented lactobacillus mucilaginosus CYQ09 obtained by separation and culture of the invention.
In addition, this example uses a sugar fermentation experiment to further identify lactobacillus mucilaginosus CYQ 09:
the biochemical metabolites of the isolated candidate strain CYQ09 were detected by the procedure described in the novel microorganism trace biochemical series identification tubes, and the results are shown in FIG. 4 and Table 1. In combination with the Berger's bacteria identification manual data, FIG. 4 and Table 1 show that the physiological and biochemical characteristics of this strain are substantially consistent with those of the Lactobacillus fermentum standard strain ATCC 14931.
TABLE 1 Biochemical identification of Lactobacillus mucilaginosus CYQ09
Note that: "+" is positive and "-" is negative.
EXAMPLE 3 Whole genome sequencing of Lactobacillus mucilaginosus CYQ09
Genomic DNA of Lactobacillus fermentum CYQ09 isolated and cultured in example 1 was extracted and subjected to purity, concentration and integrity quality control by agarose gel electrophoresis using Nanodrop, qubit and 0.35%. Then, a BluePIPP full-automatic nucleic acid recovery system is utilized to recover large-fragment DNA, a SQK-LSK109 connection kit is used for library construction, sequencing is carried out on the machine, quality control is carried out on the original data after the machine is started, and reads with low quality and too short length 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. Genomic component analysis was then performed, as well as genomic functional annotations, including PHI-base, CARD, TCDB database annotations. At the same time, genomic analysis and genomic profiling were also performed. The whole genome sequencing results of lactobacillus mucilaginosus CYQ09 are shown in fig. 5.
From the genome sequencing diagram and genome circle diagram of FIG. 5, it is seen that Lactobacillus fermentum CYQ09 has a genome size of 2.06Mb and a GC ratio of 51.83%, and its genome contains 2012 CDS regions, 2138bp repetitive sequences, 58 tRNA's and 15 rRNA. Genome functional annotation suggests that lactobacillus fermentum CYQ09 contains 1 potential drug resistance gene poxtA, but no level of gene transfer derived virulence genes.
Example 4 safety assessment of Lactobacillus mucilaginosus CYQ09
The lactobacillus mucilaginosus CYQ09 provided by the invention is a probiotic separated from fresh feces of healthy adults, and ensures the safety and effectiveness of the probiotic from bacterial sources. In addition, after the lactobacillus mucilaginosus CYQ09 provided by the invention is colonized and propagated in the human intestinal environment, the lactobacillus mucilaginosus CYQ09 is only attached to intestinal epithelial cells of a host, can become a biological barrier of intestinal mucosa, improves the barrier capacity of the intestinal mucosa of the host, can directly act on a human body in a bacterial form, and ensures the safety.
This example uses a micro broth dilution method to test the sensitivity of lactobacillus mucilaginosus CYQ09 to 8 antibiotics according to the "Probiotics general for food" issued by the society of food science and technology. The 8 antibiotics are: tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin, and gentamicin. The Lactobacillus suspension grown to logarithmic phase was adjusted to 1X 10 8 CFU/mL, antibiotic diluents (concentration from 1 to 256 mg/mL) were added at various concentrations, and anaerobic culture was performed at 37℃for 48 hours. After 48 hours the minimum inhibitory concentration (Minimal Inhibit Concentration, MIC) of lactobacillus fermentum CYQ09 strain against each antibiotic was read, and the strain was judged to be sensitive to the antibiotic (S), intermediated (I), resistant (R) and not needed (non required, n.r.) according to the bacterial resistance criteria provided in general, and the results are shown in table 2.
TABLE 2 drug sensitivity of Lactobacillus mucilaginosus CYQ09 to various antibiotics
As can be seen from table 2, the MIC of lactobacillus mucilaginosus CYQ09 against tetracycline, streptomycin, ciprofloxacin hydrochloride, clindamycin, vancomycin, chloramphenicol, ampicillin, and gentamicin were: 8mg/L, 32mg/L, 256mg/L, 4mg/L, 1mg/L and 16mg/L, i.e., the bacterium is sensitive to 6 antibiotics prescribed in general rule.
In addition, in order to further evaluate the safety of lactobacillus mucilaginosus CYQ09, 3C 57BL/6 mice of 6-8 weeks of age were selected for the experiment in this example. Animals were fed habitually in the animal house for 5 days prior to the experiment. The experimental animals and the experimental animal houses meet national regulations, standard compound feeds are selected, and the drinking water is not limited. Mice were subjected to gavage with lactobacillus mucilaginosus CYQ09 bacterial liquid, with daily gavage absorbance od=0.2 mL of CYQ09 bacterial liquid. After the raising, the experimental animals were sacrificed by neck breakage, the organs were removed by scalpel dissection, and the important organs of the mice were observed, and the results are shown in fig. 6.
As can be seen from FIG. 6, the important organs of the mice, such as kidney, liver, spleen, stomach, cecum and large intestine, are not abnormal, which indicates that the lactobacillus mucilaginosus CYQ09 provided by the invention is safe to apply in the mice.
Example 5 Lactobacillus mucilaginosus CYQ09 has a better tolerance to artificial gastric juice
Respectively preparing artificial gastric juice with pH value of 2.0,3.0,4.0. The Lactobacillus mucilaginosus CYQ09 obtained in the isolated culture of example 1 was diluted to 10 with a sterile PBS buffer 9 CFU/mL of bacterial suspension. 1. Mu.L of the bacterial suspension was aspirated into 99. Mu.L of artificial gastric juice of different pH values and cultured in 96-well plates, anaerobic cultured at 37℃for 1 to 3 hours, and the number of viable bacteria after initial and culture was measured using an ELISA reader, and the results are shown in Table 3.
As shown in Table 3, compared with the Lactobacillus fermentum standard strain ATCC14931, the Lactobacillus fermentum CYQ09 provided by the invention has better artificial gastric juice resistance.
Table 3 comparison of CYQ09 with ATCC14931 against Artificial gastric juice resistance
Example 6 Lactobacillus mucilaginosus CYQ09 has a better tolerability to artificial intestinal juice
Preparing artificial intestinal juice containing bile salt with pH value of 6.8. The Lactobacillus mucilaginosus CYQ09 obtained in the isolated culture of example 1 was diluted to 10 with a sterile PBS buffer 9 CFU/mL of bacterial suspension. 1. Mu.L of the bacterial suspension was aspirated and cultured in 96-well plates at 37℃in 99. Mu.L of artificial intestinal juice having different pH values, and the number of viable bacteria after initial and culture was measured by using an ELISA reader, and the results are shown in Table 4.
Table 4 comparison of CYQ09 with ATCC14931 resistant artificial intestinal juice
As can be seen from Table 4, lactobacillus mucilaginosus CYQ09 exhibits a better artificial intestinal juice resistance than the standard strain ATCC 14931.
Example 7 Lactobacillus mucilaginosus CYQ09 has a higher short-chain fatty acid production ability
The absorbance is taken to be adjusted to OD 600 100 μl of Lactobacillus mucilaginosus CYQ09 bacterial liquid with the concentration of=1 IS added into 8mL of MRS broth culture medium for culturing for 24 hours, 1mL of bacterial suspension IS taken out in a 1.5mL centrifuge tube, centrifugation IS carried out at 10000rpm/min for 2min, 100 μl of supernatant IS taken out, 5 μl of Internal Standard (IS) IS added, 5 μl of deionized water, 150 μl of methanol and 40 μl of 2.5% H 2 SO 4 0.05g of anhydrous sodium sulfate, vortexing for 1.5min, centrifuging (14000 rpm, room temperature, 5 min); taking the supernatant from a sample injection small bottle with a liner tube as a sample to be tested.
Preparing a follow-up blank sample: 100. Mu.L of physiological saline was taken and 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 were added 2 SO 4 0.05g of anhydrous sodium sulfate, vortexing for 1.5min, centrifuging (14000 rpm, room temperature, 5 min), and the steps are consistent with those of the sample to be tested.
Preparing a quality control sample: 100. Mu.L of physiological saline was taken, 5. Mu.L of Internal Standard (IS), 5. Mu.L of STD-4 (1000-fold diluted standard), 150. Mu.L of methanol, 40. Mu.L of 2.5% H were added 2 SO4,0.05g anhydrous sodium sulfate, vortex for 1.5min, centrifuge (14000 rpm, room temperature, 5 min), and the procedure is the same as the treatment of the sample to be tested.
The short chain fatty acid content in the supernatant, i.e. the short chain fatty acid content in the sample to be tested, was determined by gas chromatography-mass spectrometry, and the results are shown in fig. 7.
As can be seen from FIG. 7, the Lactobacillus fermentum CYQ09 provided by the invention has better capability of producing short-chain fatty acids compared with the Lactobacillus fermentum standard strain ATCC14931, and the total amount of the short-chain fatty acids is higher, especially the yields of acetic acid, propionic acid and butyric acid are higher than those of ATCC 149331.
EXAMPLE 8 fermentation of Lactobacillus mucilaginosus CYQ09 increases the tolerance of small intestine organoids to radiation injury
The small intestine crypt of the mouse is extracted and cultured to obtain the small intestine organoid, and the organoid is cultured on the 7 th day, and the following operation is carried out: control group at 37℃with 5% CO 2 Culturing normally under the condition; the organoids were irradiated with 6MeV X-rays at 6Gy and then with 5% CO at 37 ℃ 2 Culturing for 4 hours, and then replacing fresh culture medium; the +ATCC14931 group was irradiated with 6MeV X-ray 6Gy immediately after 50. Mu.L of the culture supernatant of the standard strain ATCC14931 was pipetted into the culture wells, and then with 5% CO at 37 ℃ 2 Culturing for 4 hours, and then replacing a fresh culture medium without bacterial culture supernatant for culturing for 24 hours; the +CYQ09 group is irradiated by sucking 50 mu L of culture supernatant of lactobacillus mucilaginosus CYQ09 into a culture hole, immediately irradiating with 6MeV X-ray 6Gy, and then irradiating with 5% CO at 37 DEG C 2 After 4 hours of incubation, the fresh medium without bacterial culture supernatant was replaced for 24 hours. The morphology of the small intestine organoids was observed after the completion of the culture, and the results are shown in FIG. 8.
As shown in FIG. 8, compared with the Lactobacillus fermentum standard strain ATCC14931, the organoids of Lactobacillus fermentum CYQ09 under the culture condition can maintain better forms, which indicates that the Lactobacillus fermentum CYQ09 has therapeutic effect on radiation injury.
Example 9 fermentation of Lactobacillus mucilaginosus CYQ09 reduces glandular lesions
24C 57BL/6 mice with the age of 4 weeks are selected and randomly divided into 4 groups after normal feeding for one week: control group (n=6), total abdominal irradiation group (n=6), total abdominal irradiation+atcc 14931 group (n=6), total abdominal irradiation+cyq09 group (n=6), and the following steps were performed:
on days 1-7, mice in the Control group were perfused with 0.2mL of PBS buffer, and groups of full abdominal irradiation +atcc14931 and full abdominal irradiation +cyq09 were perfused with 0.2mL of ATCC14931 and cyq09 bacteria solution with absorbance od=1, respectively;
on day 8, after each group of mice was fasted and water was prohibited for 2 hours, the mice were anesthetized, all except the control group, with full abdominal irradiation using 6MeV X-rays at a dose of 12Gy, and then the fasted and water prohibition was released;
on days 9-11, control mice were perfused with 0.2mL PBS buffer, and with 0.2mL od=1 ATCC14931 and CYQ09 bacteria respectively;
mice were dissected on day 32 to detect various indicators.
The survival time of each group of mice was analyzed by Kaplan Meier, and the results are shown in fig. 9. As can be seen from fig. 9, all the Control mice survived, all the mice in the full-abdomen-irradiated group had died before the end of the experiment, and all the mice in the full-abdomen-irradiated+atcc 14931 group and the full-abdomen-irradiated+cyq09 group survived at the end of the experiment, and the number of survived was greater in the full-abdomen-irradiated+cyq09 group and the survival time was longer than in the full-abdomen-irradiated+atcc 14931 group. The results show that the lactobacillus mucilaginosus CYQ09 provided by the invention can obviously improve survival in the bodies of radiation-damaged mice, and has better effect than that of the standard strain ATCC 14931.
The mice of each group treated as described above were stained with hematoxylin-eosin (HE staining), and the results are shown in fig. 10. As can be seen from fig. 10, the whole-abdomen irradiation group showed pathological damage to intestinal tissue such as gland tissue destruction and inflammatory infiltration, while the whole-abdomen irradiation +cyq09 group showed significantly reduced damage to gland structure and inflammatory cell infiltration. The results show that the lactobacillus mucilaginosus CYQ09 provided by the invention can reduce the radioactive intestinal injury.
EXAMPLE 10 specific molecular target excavation of Lactobacillus mucilaginosus CYQ09
The whole genome data of the other 155 strains of Lactobacillus fermentum in NCBI database is downloaded, and the Prokka (v 1.11) and Roary (v 3.11.2) software is used for performing the genome analysis on the 155 strains of Lactobacillus fermentum and the Lactobacillus fermentum CYQ09 provided by the invention. After the core gene was obtained, the gene having a high base substitution density was recognized by MEGA X (v10.2.2). The specific sequence of the lactobacillus fermentum CYQ09, which is different from other lactobacillus fermentum, is obtained based on the analysis flow. Primer Premier 5 software is adopted to carry out Primer design aiming at a specific sequence, so that a specific molecular target sequence SEQ ID capable of specifically recognizing the lactobacillus fermentum CYQ09 is obtained: 1.
the polymerase chain reaction (Polymerase Chain Reaction, PCR) is adopted to verify the effectiveness of the specific molecular recognition target sequence of the lactobacillus fermentum CYQ09. The detection template is bacterial DNA, the DNA extraction method refers to a Tiangen bacterial genome DNA extraction kit, and the amplification primer sequences are shown in Table 5. The PCR reaction system and the PCR reaction conditions in this example were the same as those in example 1. Sequencing by a manufacturer company after the PCR is finished, analyzing that the length of the product is 384bp, and the product is shown as SEQ ID:1.
TABLE 5 amplification primer sequences
| Numbering device | Primer sequence (5 '. Fwdarw.3') |
| SEQ ID：2 | AGTGAACCGCTGGAAAGA |
| SEQ ID：3 | TTGGGATGGTAACAGAAGG |
In conclusion, the lactobacillus mucilaginosus CYQ09 provided by the invention can inhibit the radiation-induced inflammatory reaction by producing a large amount of short chain fatty acid, reduce the damage of intestinal stem cells under the radiation condition, accelerate the repair of intestinal tissues, prolong the survival time of patients, and have the characteristics of tolerating gastric juice and intestinal juice of human bodies, has high survival rate in the stomach and intestines, can be planted in the intestines, and can restore the stable state of intestinal flora. In addition, compared with the traditional medicine for treating the radioactive intestinal injury, the lactobacillus mucilaginosus strain CYQ09 has no obvious toxic or side effect on human bodies, has high safety, can reduce adverse reaction caused by the medicine and overcomes the problem of low effective rate of medicine treatment. The fermented lactobacillus mucilaginosus CYQ09 or the culture thereof provided by the invention can be applied to the preparation of medicines or foods for preventing and/or treating the 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 after the radiotherapy of the malignant pelvic tumor cannot be effectively treated in the prior art, and has a huge application prospect in preparing the medicines or foods for preventing and/or treating the radioactive intestinal injury.
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 (6)
1. A strain of Lactobacillus mucilaginosus (Limosilactobacillus fermentum) CYQ09 was deposited at the microorganism strain collection, cantonese province, at 4/1/2022 under the accession number GDMCC 62346.
2. Use of lactobacillus mucilaginosus CYQ09 as claimed in claim 1 in the manufacture of a medicament for the prevention and/or treatment of radiation intestinal injury.
3. A medicament for the prevention and/or treatment of radiation intestinal injury, 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.
4. A nucleotide specifically recognizing lactobacillus fermentum CYQ09 as claimed in claim 1 wherein: the nucleotide sequence is shown as SEQ ID:1.
5. A primer set capable of specifically recognizing lactobacillus fermentum CYQ09 as claimed in claim 1 wherein: the primer group comprises a primer sequence shown in SEQ ID:2 and SEQ ID:3, and a nucleotide sequence shown in 3.
6. A method of identifying lactobacillus mucilaginosus CYQ09 as claimed in claim 1 wherein: PCR amplification is carried out by using the primer set as a specific amplification primer and genome DNA of the lactobacillus fermentum to be detected as a template to obtain a PCR product, and the PCR product is identified by sequencing or electrophoresis.
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| CN116751725B (en) * | 2023-07-27 | 2024-04-26 | 内蒙古农业大学 | Anti-phage fermented lactobacillus mucilaginosus and application thereof |
| CN117143767B (en) * | 2023-08-23 | 2024-06-11 | 浙江民生健康科技有限公司 | Breast milk-derived fermented lactobacillus mucilaginosus MSJK capable of regulating intestinal flora and application thereof |
| CN117801988B (en) * | 2023-12-15 | 2024-06-25 | 吉林省农业科学院(中国农业科技东北创新中心) | Lactobacillus mucilaginosus DP116 and application thereof in preparing products with functions of improving and protecting basement membrane |
| CN118028190B (en) * | 2024-04-15 | 2024-06-25 | 通化承诚药业有限公司 | Lactobacillus mucilaginosus BAS313 and application thereof in preparation of hypoglycemic products |
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