CN116426415B - Weissella sinica TE2103 and application thereof - Google Patents
Weissella sinica TE2103 and application thereof Download PDFInfo
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- CN116426415B CN116426415B CN202310222405.9A CN202310222405A CN116426415B CN 116426415 B CN116426415 B CN 116426415B CN 202310222405 A CN202310222405 A CN 202310222405A CN 116426415 B CN116426415 B CN 116426415B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses Weissella sinus (WEISSELLA CIBARIA) TE2103 and application thereof, relates to the technical field of microorganisms, and is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 12 months and 14 days in 2022, wherein the preservation address is North Star West No. 1,3 in the Chaoyang area of Beijing city, and the preservation number is CGMCC No.26188. The strain is separated from the intestinal tract of a Chinese toad, is sensitive to various common antibiotics, has better tolerance to acid, bile salt, artificial gastric juice and artificial intestinal juice, has stronger adhesion to human colon cancer cells HT-29, has stronger inhibition to various pathogenic bacteria such as escherichia coli, staphylococcus aureus, pasteurella multocida and the like, and has better prevention and treatment effects on a dextran sodium sulfate salt (DSS) induced mouse colonitis model.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Weissella shikimchi TE2103 and application thereof.
Background
Chinese toads (Bufo gargarizans) are widely distributed in China and have a large number, and are medicinal amphibians. The toad venom is prepared by collecting and processing white serum secreted by the posterior auricular glands and the cutaneous glands, and is a traditional rare Chinese medicinal material in China. The toad venom contains toad toxin, arginine and other substances, also contains steroid substances with heart strengthening effect, has the functions of heart strengthening and diuresis, detumescence and resuscitation, detoxification, anesthesia and pain relieving and the like, and has a certain anticancer effect. The toad skin is cuticle membrane naturally removed by toad, has the effects of clearing heat and detoxicating, detumescence and relieving pain, calming, promoting urination and the like, and has better curative effects on chronic liver diseases, various cancers, chronic tracheitis, ascites, sore and carbuncle treatment and the like. Studies have found that toads have abundant microbial species, including a large number of probiotics, on their body surfaces and in their intestinal tracts.
The antrum Weissella (WEISSELLA CIBARIA) is widely used in various natural fermentation foods, is a special-shaped lactic acid fermentation dominant bacterium in the early stage of naturally fermenting pickle, has the characteristics of fermentation complexity and product diversity, and can endow the pickle with better flavor. Meanwhile, the compound also has the probiotic characteristics of antioxidation, immunoregulation, extracellular polysaccharide production and the like. However, the existing antral Weissella has poor broad-spectrum antibacterial property, has high tolerance to acid and salt, is not reported, and often cannot meet the requirements of production and life.
Disclosure of Invention
The invention aims to provide Weissella antrum (WEISSELLA CIBARIA) TE2103 and application thereof, and aims to solve the problems that the existing Weissella antrum has poor broad-spectrum antibacterial property, is not clear in acid and salt tolerance, cannot meet the requirements of production and life and the like. The strain is separated from the intestinal tract of a Chinese toad, is sensitive to various common antibiotics, has better tolerance to acid, bile salt, artificial gastric juice and artificial intestinal juice, has stronger adhesion to human colon cancer cells HT-29, has stronger inhibition to various pathogenic bacteria such as escherichia coli, staphylococcus aureus, pasteurella multocida and the like, and has better prevention and treatment effects on a dextran sodium sulfate solution-induced mouse colonitis model.
In order to achieve the above purpose, the invention provides a Weissella shi TE2103, which is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 12 months and 14 days in 2022, wherein the preservation address is number 3 of West-road 1 in the Korean region North Star of Beijing city, and the preservation number is CGMCC No.26188.
The invention also provides a biocontrol microbial agent containing the Weissella food sinus TE 2103.
Preferably, the biocontrol agent is a bacterial suspension of Weissella food TE2103 with a bacterial suspension concentration of 1X 10 8 CFU/mL.
The biocontrol microbial inoculum provided by the invention can be used for biological control of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, listeria monocytogenes, staphylococcus hemolyticus, salmonella and/or pasteurella multocida.
The Weissella antrum TE2103 provided by the invention can be used for preparing medicines for treating diseases caused by escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, listeria monocytogenes, staphylococcus hemolyticus or pasteurella multocida.
The Weissella antrum TE2103 provided by the invention can be used for preparing anti-enteritis medicines.
The invention provides the Weissella antrum TE2103, solves the problems of poor broad-spectrum antibacterial property, low tolerance to acid and bile salts and the like of the prior Weissella antrum, and has the following advantages:
the Weissella antrum TE2103 provided by the invention is separated and screened from the intestinal tracts of Chinese toads, grows well on an MRS agar medium, has a certain tolerance to acid and bile salts, and has strong adhesion to human colon cancer cells HT-29.
The Weissella antrum TE2103 provided by the invention has stronger inhibition capability on common pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, listeria monocytogenes, staphylococcus hemolyticus, pasteurella multocida and the like.
The Weissella antrum TE2103 provided by the invention has good prevention and treatment effects on the mouse colonitis model induced by dextran sulfate sodium salt, and the inflammation index is obviously reduced.
Drawings
FIG. 1 is a phylogenetic relationship of Weissella food TE2103 with other strains according to the present invention.
FIG. 2 shows colony morphology and gram staining results of Weissella antrum TE2103 strain on MRS agar medium in the present invention.
FIG. 3 shows the results of the experiment of the hemolytic activity of the Weissella antrum TE2103 strain according to the present invention.
FIG. 4 shows the prophylactic and therapeutic effects of Weissella antrum TE2103 strain on DSS-induced colitis in mice according to the present invention.
FIG. 5 shows the results of colorectal length changes after DSS-induced colitis in mice treated with the Weissella antrum TE2103 strain of the present invention.
FIG. 6 shows the effect of Weissella antrum TE2103 on the structure of intestinal flora of mice with DSS-induced colitis according to the present invention.
FIG. 7 shows the effect of the strain Weissella antrum TE2103 on the intestinal flora diversity of mice with DSS-induced colitis according to the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but 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.
Description: the experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
The standard strain LGG in the invention is lactobacillus rhamnosus Lactobacillus rhamnosus ATCC 53103.
Experimental example 1 isolation, identification and safety evaluation of Strain
1. Isolation and identification of strains
Taking a small amount of content of intestinal tracts of Chinese toads, inoculating the content into a 50mLMRS broth culture medium, fully shaking and uniformly mixing, and placing the mixture into a shaking table at a constant temperature of 37 ℃ for culturing for 24 hours. 1mL of culture solution is sucked, 10-time gradient dilution is adopted, 20 mu L of each concentration gradient bacterial solution is taken and inoculated on MRS agar culture medium, single colony is picked up after 24h culture at 37 ℃, and purification and separation are carried out for 3 times. The purified strain is inoculated into 600 mu L of MRS broth culture medium, shake-cultured for 18h at 37 ℃, 400 mu L of sterilized glycerol with concentration of 50% (V/V) is added, and the strain is frozen in an ultralow temperature refrigerator at-80 ℃ for standby.
After the frozen strain is activated and cultured, the strain DNA is extracted by using an MRS broth culture medium, and the amplification of the 16S rRNA is completed by adopting a colony PCR technology by using the 16S rRNA universal primers 27F and 1492R with the following specific sequences (5 '. Fwdarw.3'):
27F(SEQ ID NO.1)：AGAGTTTGATCMTGGCTCAG，
1492R(SEQ ID NO.2)：GGTTACCTTGTTACGACTT。
Sequencing of the PCR product is completed by biological engineering (Shanghai) limited company, and the 16S rRNA sequence is shown as SEQ ID NO. 3. The sequence was aligned with BLAST in NCBI, and the similarity to standard strain WEISSELLA CIBARIA, 3385, was 98.32%, and could be initially identified as Weissella antrum, and designated as Weissella antrum (WEISSELLA CIBARIA) TE2103. The phylogenetic relationship of this strain with other strains is shown in FIG. 1. The strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with a preservation address of North Star Xilelu No. 1 and No.3 in the Korean region of Beijing as well as a preservation number of CGMCC No.26188 in 12 months and 14 days of 2022.
Antral Weissella TE2103 was inoculated on MRS agar medium, and after incubation at 37℃for 24 hours, the morphology of single colonies was observed and recorded. Gram staining was performed using a kit method, and the bacterial morphology after staining was observed and recorded under a microscope. The colony morphology and gram staining results of the Weissella antrum TE2103 strain on MRS agar medium are shown in FIG. 2, wherein A in the graph is a colony morphology graph, and B in the graph is a gram staining graph. It is known that Weissella antrum TE2103 grows well on MRS agar medium, the colony forms are milky white, round bulges, smooth edges and smooth surfaces, and the bacteria are rod-shaped and purple after microscopic examination, so that the bacterial strain meets the staining characteristics of gram-positive bacteria.
2. Detection of hemolytic and antibiotic resistance of Weissella antrum TE2103
The sensitivity of the strain to common antibiotics was tested by the paper sheet agar diffusion method. The strain of Weissella antrum TE2103 is activated and cultured, the concentration of bacterial liquid is regulated to 1X 10 6 CFU/mL, bacterial liquid is uniformly smeared on the surface of an MRS culture medium flat plate by using a sterile cotton swab, after the temperature is 10min, drug sensitive paper sheets are put in, after the temperature is cultured for 24 hours at 37 ℃, the diameter of a bacteriostasis ring around each drug sensitive paper sheet is measured by using a vernier caliper, each antibiotic is repeated for 3 times, and the test result refers to the American clinical laboratory standards Committee (NCCLS) to judge the drug sensitivity of the strain, and the results are expressed as sensitivity (S), intermediation (I) and drug resistance (R).
As shown in FIG. 3, the results of the experiment of the hemolytic activity of Weissella antrum TE2103 strain show that there was no hemolysis around the colony; the results of the statistics, presented in Table 1 below, demonstrate the safety of the Weissella antrum TE2103 strain, as sensitive (S) to the 6 antibiotics tested, tetracycline, ampicillin, ceftriaxone, clindamycin, clarithromycin and chloramphenicol.
TABLE 1 sensitivity of Weissella food TE2103 Strain to 6 antibiotics
Experimental example 2 detection of the Main fermentation characteristics of Weissella food TE2103
1. Evaluation of acid resistance and bile salt resistance
The strain to be tested, weissella antrum TE2103, is resuscitated and activated for 3 generations on an MRS agar plate, and the initial concentration of the bacterial liquid is regulated to be 1 multiplied by 10 6 CFU/mL. Hydrochloric acid and porcine bile salt were used to formulate MRS broth with acidity (ph=3.0) and bile salt concentration of 0.3%, respectively. 1mL of the strain to be tested, weissella antrum TE2103 broth was inoculated into a broth medium having a pH=3.0, and cultured at 37℃for 18 hours. After the completion of the culture, 20. Mu.L of the bacterial liquid was spread on MRS agar plate medium, and 3 replicates were set, and the culture was performed at 37℃for 18 hours. After incubation, the MRS plate surface was observed for colony growth. Similarly, 1mL of the strain to be tested, weissella antrum TE2103 bacterial liquid, was inoculated into MRS broth medium with a bile salt concentration of 0.3%, cultured at 37℃for 18 hours, plated, cultured at 37℃for 24 hours, and examined for colony growth.
Acid resistance test results show that normal colonies can still grow on an MRS agar plate after 18h treatment of the antral weissella t.e 2103 in an MRS broth medium with ph=3.0; salt tolerance test results show that the Weissella antrum TE2103 can still grow normal colonies on an MRS agar plate after being tolerant for 18 hours in an MRS broth culture medium with the bile salt concentration of 0.3%, and the Weissella antrum TE2103 has strong acid resistance and bile salt resistance.
2. Cell adhesion assay
Resuscitates the Weissella antraniliprole TE2103 and inoculates the resuscitated culture medium in MRS broth, and cultures the resuscitated culture medium at 37 ℃ for 24 hours. After the cultivation, the mixture is centrifuged for 10min at-4 ℃ and 5000r/min, and washed with sterile PBS buffer solution for a plurality of times. The bacterial suspension concentration is adjusted to 1X 10 6 CFU/mL for later use. Human colon cancer cells HT-29 were resuscitated, inoculated into six well cell culture dishes, supplemented with DMEM complete medium and incubated at 37℃in 5% CO 2, with medium replaced once for two days. When the cell attachment state reached 80%, digestion was performed using 0.25% pancreatin-EDTA, and subcultured. After the completion of the culture, the cells were counted by a cell counting plate, and the cell concentration was adjusted to 5X 10 6 cells/mL. 1mL of the cell suspension was added to one of the culture wells of a six-well cell culture dish and placed in an incubator for culture. Cells in the plates were grown to a monolayer, DMEM medium was discarded and each well was rinsed 3 times with sterile PBS. 1mL of the prepared bacterial suspension is added into a cell hole, the cell culture plate is slightly shaken, a small amount of bacterial liquid in the hole is sucked for plate counting, and the result is taken as the initial viable bacterial count in the bacterial suspension. The cell plates were incubated at 37℃for 2h, the medium was discarded and washed 3 times with sterile PBS buffer. Cells were digested with 0.7mL of 0.25% trypsin-EDTA for 10min, and after complete shedding of the cells, digestion was terminated by adding 0.3mL of DMEM broth, and the broth after the end of the adhesion experiment was collected for plate counting, and the result was used as the number of adhesion viable bacteria, and a standard strain LGG was used as a control, wherein the calculation formula of the adhesion rate was as follows.
Adhesion (%) = number of lactic acid bacteria at end period/number of initial lactic acid bacteria inoculation x 100%
The adhesion rate of Weissella antrum TE2103 to human colon cancer cells HT-29 is shown in Table 2 below. It was found that the adhesion rate of Weissella antrum TE2103 to human colon cancer cells HT-29 was 56.89% higher than that of the standard strain LGG.
TABLE 2 adhesion Rate of Weissella food TE2103 to human colon cancer cells HT-29
3. Simulated gastric juice and intestinal juice tolerance experiment
Simulated gastric and intestinal fluids were purchased from Shanghai leaf Biotechnology Inc. The artificial gastric juice simulated liquid comprises dilute hydrochloric acid, pepsin and sodium chloride, and the final pH=2.5; the artificial intestinal juice simulated fluid comprises potassium dihydrogen phosphate and trypsin, and the final pH=6.8. Resuscitates and activates the Weissella antrum TE2103 strain, adjusts the concentration of bacterial liquid to 1X 10 8 CFU/mL, takes 1mL bacterial liquid to be added into 9mL simulated artificial gastric juice, carries out 10-time gradient dilution, and absorbs 20 mu L of viable bacteria count of a plating plate to be used as an initial viable bacteria value of the tolerance artificial gastric juice; the simulated gastric juice after inoculation is cultured for 3 hours at 37 ℃, and then the plate is coated again to count the number of viable bacteria, and the number of viable bacteria is taken as the final viable bacteria value of the artificial gastric juice tolerance. Similarly, 1mL of fermentation broth of Weissella antrum TE2103 at a concentration of 1X 10 8 CFU/mL was added to 9mL of simulated intestinal fluid, viable count was performed, the viable count was counted again after culturing for 6 hours at 37℃and the survival rate was calculated. Survival = number of viable bacteria at end/number of initial viable bacteria 100%.
The tolerance of the Weissella antrum TE2103 strain to the artificial simulated gastric and intestinal fluids is shown in the following table 3, and the Weissella antrum TE2103 strain has better tolerance to the artificial simulated gastric and intestinal fluids, wherein the survival rate of the Weissella antrum TE2103 strain in the artificial gastric fluid after 3 hours is 136.0%, and the survival rate of the Weissella antrum TE2103 strain after 6 hours is 55.0%.
TABLE 3 tolerance of Weissella food TE2103 Strain to artificial simulated gastric and intestinal fluids (%)
Experimental example 3 evaluation of antibacterial Activity of Wessezia w.eati TE2103 Strain
Eight common pathogenic bacteria of escherichia coli (ESCHERICHIA COLI CMCCB 44102), staphylococcus aureus (Staphylococcus aureus CMCCB 50094), salmonella typhimurium (Salmonella typhimurium ATCC 14028), pseudomonas aeruginosa (Pseudomonas aeruginosa CMCCB 10104), listeria monocytogenes (Listeria monocytogenes ATCC 19115), salmonella haemolyticus (Staphylococcus haemolyticus ATCC 29970) Salmonella (Salmonella CMCC (B) 50094) and pasteurella multocida (Pasteurella multocidaATCC 51689) are respectively inoculated in a nutrient agar medium, recovered and activated for 3 times. And (3) inoculating the activated pathogenic bacteria into a broth culture medium for culture, and regulating the concentration of the bacterial liquid to be 1 multiplied by 10 8 CFU/mL. 1mL of fermentation liquor for sucking the pathogenic bacteria is added into 500mL of nutrient agar culture medium which is not solidified temporarily after sterilization (the temperature is cooled to about 40 ℃), and the mixture is fully mixed and split-packed into culture dishes according to the amount of 20mL per dish. After the culture medium is cooled and solidified, a puncher with the diameter of 6mm is used for punching holes on a flat plate, so that a pathogenic bacteria agar plate is manufactured, each plate corresponds to one pathogenic bacteria, and three holes are formed for repetition. Resuscitates and activates and cultures the Weissella antrum TE2103 strain, adjusts the concentration of the bacterial liquid to 1X 10 8 CFU/mL, sucks 50 mu L of the bacterial liquid into the hole of the pathogenic bacteria agar plate, cultures the bacterial liquid for 24 hours at 37 ℃, measures the diameter of a bacteriostasis ring around the punching point by using a vernier caliper, and takes a standard strain LGG as a control strain.
The evaluation results of the antibacterial activity of the Weissella antrum TE2103 strain are shown in the following table 4, and the fermentation broth of the Weissella antrum TE2103 strain has obvious inhibition effect on eight common pathogens tested, wherein the inhibition effect on staphylococcus aureus, salmonella typhimurium and listeria monocytogenes is superior to that of the LGG standard strain. The strain TE2103 can be used for preparing a biocontrol microbial inoculum, and can be used for biologically controlling the eight common pathogenic bacteria by regulating the concentration of bacterial suspension to be 1 multiplied by 10 8 CFU/mL.
Table 4 evaluation of antibacterial Activity of Weissella food TE2103 Strain (diameter: mm)
Experimental example 4 test of the preventive Effect of Weissella food TE2103 on the model of mouse colitis
1. Processing and grouping
A total of 27 mice were randomly divided into 9 cages of 3 mice each. The random groups were divided into 3 groups of 3 cages each, the groups including a placebo group (CK), a DSS model group (DSS), and a weissella food TE2103 treatment group. Experimental treatments are shown in table 5 below.
Dextran sodium sulfate salt solution (DSS): a4% (w/v) aqueous solution of DSS was prepared with dextran sulfate sodium salt.
Preparation of bacterial suspension: the standard strain LGG and weissella antrum TE2103 were resuscitated and activated for 3 passages. Centrifuging the bacterial liquid at-4deg.C and 6000r/min for 5min, and discarding supernatant. The bacterial cells were resuspended in sterile PBS buffer, the bacterial cell concentration was adjusted to 5X 10 9 CFU/mL and stored in a refrigerator for further use.
TABLE 5 animal experimental treatments
2. Experimental details
During the experiment, the activity status, the fecal status and the bloody stool were observed every day. At the end of the prophylaxis period (14 days) and treatment period (7 days), 3 mice were sacrificed randomly for serum inflammatory factor detection. Fresh blood samples were collected with a sterile centrifuge tube and centrifuged at 3000r/min for 10min to obtain serum. Serum inflammatory factors (IL-1. Beta., IL-6, IL-8 and TNF-. Alpha.) were measured using ELISA kit (Jiangsu Jingmei Biotech Co., ltd.) and the procedure was followed according to the kit instructions. Spleen index was measured by taking out the spleen, washing with physiological saline, sucking with filter paper, and weighing. Spleen index = spleen mass (g)/mouse body weight (g) 100%. At the end of the prevention period, fresh fecal samples were collected and sent to sequencing companies for sequencing of the hypervariable region of the 16S rRNA gene V3-V4, which was analyzed on the QIIME2 platform.
3. Results
The mice varied in body weight as seen in Table 6 below, and the DSS model group had 9.7% and 28.8% weight loss during the prophylaxis and treatment periods, respectively, while the Weissella food TE2103 treated group had a substantially constant body weight.
The results of serum inflammatory factor measurement of mice are shown in the following Table 7, and it is known that in the prevention period, four inflammatory factor indexes of IL-1β, IL-6, IL-8 and TNF- α of the Weissella food TE2103 treated group are higher than those of the blank control group, but lower than those of the DSS model group, so that the Weissella food TE2103 can relieve inflammation caused by DSS, and has an inflammation prevention effect; similarly, during the treatment period, the IL-1β, IL-6, IL-8 and TNF- α indicators of the TE2103 treated group were lower than those of the DSS model group, and the indicators were very similar to those of the blank control group, indicating that Weissella antracea TE2103 had therapeutic effects on DSS-induced enteritis.
TABLE 6 weight variation of mice (Unit: g)
TABLE 7 measurement results of mouse serum inflammatory factors (pg/mL)
The spleen index (the ratio of the spleen weight to the matrix weight of experimental animals; the weight after the spleen is taken and the residual blood is sucked by filter paper) of the feeding group of the Weissella antrum TE2103 is lower than that of the DSS model group, and the colorectal length is longer than that of the DSS model group, so that the feeding group of the Weissella TE2103 has the prevention and treatment effects on the mouse enteritis caused by the DSS, and the potential effect of the Weissella TE2103 in the prevention and treatment of the colonitis is suggested. The prophylactic and therapeutic effects of the Weissella antrum TE2103 strain on DSS-induced mouse colitis are shown in FIG. 4, wherein A in the figure is the spleen index at the end of the prevention period, and B in the figure is the spleen index at the end of the treatment period; the results of colorectal length changes after DSS-induced mouse colitis prevention and treatment by the weissella antrum TE2103 strain are shown in fig. 5, wherein a in the figure is the colorectal length at the end of the prevention period and B in the figure is the colorectal length at the end of the treatment period.
In combination with the sequencing result of the 16S rRNA of the intestinal flora, the effect of the weissella food-is TE2103 on the intestinal flora structure of the mice with DSS induced colitis is shown in fig. 6, wherein a in the figure is a weine (Venn) diagram with the flora structure, B in the figure is a figure with the number of operational classification units (OTUs) of the flora, and it is known that the number of operational classification units (OTUs) of the feeding group of the weissella food-is TE2103 is 554, which is higher than that of the blank control group and the DSS model group, which indicates that the feeding of the weissella food-is TE2103 can promote the increase of intestinal microorganism species. Further, according to the sequencing result of the intestinal flora 16S rRNA, the effect of feeding the antral Weissella strain TE2103 on three diversity indexes of the intestinal flora of mice with DSS induced colitis is shown in FIG. 7, including Sobs diversity index, shannon diversity index and Chao1 diversity index, wherein A is Sobs diversity index, B is Shannon diversity index and C is Chao1 diversity index. The results show that the three flora diversity indexes are higher than those of a DSS model group and even than that of a blank control group, so that the effect of adjusting the intestinal flora structure of mice by feeding the Weissella food is shown, the intestinal flora diversity is more abundant, and the Weissella food is shown that the Weissella food TE2103 can be used for preparing anti-enteritis medicines and has remarkable application value.
In conclusion, the antral Weissella TE2103 provided by the invention is sensitive to various common antibiotics, has better tolerance to acid, bile salt, artificial gastric juice and artificial intestinal juice, has stronger adhesion to human colon cancer cells HT-29, has stronger inhibition to various pathogenic bacteria such as escherichia coli, staphylococcus aureus, pasteurella multocida and the like, has better prevention and treatment effects on a dextran sodium sulfate salt (DSS) induced mouse colonitis model, and suggests that the strain has potential effects in preventing and treating colonitis.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (5)
1. The Weissella sinus (WEISSELLA CIBARIA) TE2103 is characterized in that the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 12 months and 14 days in 2022, the preservation address is North Star Xiyu No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.26188.
2. A biocontrol agent comprising the antral weissella TE2103 of claim 1.
3. The biocontrol microbial agent of claim 2, wherein said biocontrol microbial agent is a bacterial suspension of weissella antrum TE2103 having a bacterial suspension concentration of 1 x 10 8 CFU/mL.
4. Use of a biocontrol agent according to claim 2 or 3 for the biocontrol of non-disease diagnostic treatment of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, listeria monocytogenes, staphylococcus hemolyticus or/and pasteurella multocida.
5. Use of antral Weissella sp TE2103 as claimed in claim 1 for the preparation of an anti-colitis medicament.
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