CN115820498B - Lactobacillus plantarum YJ2406 and application thereof - Google Patents
Lactobacillus plantarum YJ2406 and application thereof Download PDFInfo
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- CN115820498B CN115820498B CN202211573256.2A CN202211573256A CN115820498B CN 115820498 B CN115820498 B CN 115820498B CN 202211573256 A CN202211573256 A CN 202211573256A CN 115820498 B CN115820498 B CN 115820498B
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses lactobacillus plantarum (Lactobacillusplantarum) YJ2406 and application thereof. The lactobacillus plantarum YJ2406 provided by the invention has a preservation number of CGMCCNO.25907. The strain is separated from Guizhou dry pickled Chinese cabbage, grows well on an MRS culture medium, has no hemolysis phenomenon, is sensitive to various common antibiotics, does not carry antibiotic resistance genes, 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 strong capability of producing short chain fatty acid, has better in-vitro blood sugar and cholesterol reducing capability, has stronger inhibition capability to escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli, has better prevention and treatment effects to a mouse model induced by DSS, can obviously reduce inflammatory symptoms, can effectively regulate the intestinal flora structure of mice, and obviously increases the number of lactobacillus.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum YJ2406 and application thereof.
Background
Probiotics are a general term for microorganisms in the intestinal tract that promote the maintenance of a healthy state in the body and are beneficial to the body. The probiotics are various, mainly including: bifidobacterium species, bacillus species, lactobacillus species, and streptococcus species. Lactobacillus, streptococcus and bifidobacteria are also classified as lactic acid bacteria because they produce lactic acid, and bacillus, saccharomycete and propionic acid bacteria are classified as non-lactic acid producing types. Among them, the safety and probiotics of lactic acid bacteria are widely recognized, which can produce a large amount of lactic acid using fermentable carbohydrates, and the main forms include a rod shape, a sphere shape, a chain shape, or a barrier shape. Lactic acid bacteria in nature are various and widely distributed. The probiotics have application in the fields of medical treatment, livestock and food and the like due to the probiotic functions of the probiotics. Especially in disease control, probiotics have important roles including gastrointestinal disease treatment, neonatal disease treatment, immunity enhancement and the like. Inflammatory enteritis is a non-specific chronic intestinal disease that threatens the health of modern humans and animals. It is mainly induced by comprehensive factors such as genetics, environment and flora. Common clinical manifestations of the disease include abdominal pain and diarrhea, severe cases with bloody stool, and increased potential risk of canceration. Because of various induction reasons, the occurrence process is complex, and the prevention and the control often cannot be performed in time. As an adjunct to traditional drugs, probiotic preparations are becoming increasingly interesting in the prevention and treatment of inflammatory enteritis. At present, many new strains have been developed gradually.
Fermented foods are always a valuable source for developing novel probiotics, food-source lactobacillus not only has higher safety, but also has better properties than other sample sources, and traditional vegetable fermented products are naturally fermented by adhering microorganisms on the surfaces of vegetables. The lactic acid bacteria are used for fermentation to produce foods with special flavor and probiotics characteristics, and the bacterial activity directly affects the final quality and flavor of the fermented foods. Lactic acid bacteria play a key role in human and animal bodies, including participation in nutritional metabolism, microecological regulation, immune enhancement, intestinal barrier maintenance, and the like. Organic acid can be generated by lactic acid bacteria in growth metabolism to reduce the pH value of intestinal microecology, thereby inhibiting the growth of pathogenic bacteria. In addition, the produced bacteriocin and other antimicrobial active substances can inhibit the growth of saprophytic bacteria. Some lactic acid bacteria can bring health effects to a host by producing functional active factors, regulating intestinal flora, regulating the brain intestinal axis. Many genes directly involved in amino acids, vitamins and carbohydrates are lacking in the human and animal body, while intestinal microorganisms can complement them, assisting the host in completing a series of metabolic activities.
The screening process of probiotics is very strict, and the capabilities to be examined include safety, high acid tolerance, high bile salt tolerance, cell adhesion and the like. In addition to in vitro probiotic properties, the in vivo probiotic function of the strain in model animals is also a factor that must be examined. The fermented food is a valuable probiotic resource library, and a great deal of research has been conducted at present to show that excellent candidate strains of probiotics can be separated and screened from the fermented food.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum YJ2406 and provides an application of the lactobacillus plantarum YJ2406 in preparing anti-enteritis medicines, foods for regulating intestinal flora of human or animals and medicines for treating diseases caused by one or more of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the invention provides a lactobacillus plantarum YJ2406 which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC) at the 10 th and 13 th year of 2022, wherein the preservation number is CGMCC NO.25907.
Further, the lactobacillus plantarum YJ2406 is obtained by separating and screening pickled Chinese cabbage of eight villages defined in Ripingcounty of Guizhou province.
Furthermore, the DNA of the strain is extracted by adopting a root bacteria genome DNA extraction kit, the 16S rRNA amplification is finished by adopting a colony PCR technology, the PCR primers are the 16S rRNA universal primers 27F and 1492R, a target gene sequence consisting of 1027 base pairs (bp) is obtained, the nucleotide sequence is GGGGCGGTGGCGGGTGCTATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCTG., the gene sequence obtained by sequencing is input into an NCBI database for comparison, the similarity rate of the gene sequence to a standard strain (Lactobacillus plantarum strain JCM 1149) in Genebank reaches 99.52%, and the strain can be primarily identified as lactobacillus plantarum (Lactobacillus plantarum).
Furthermore, the lactobacillus plantarum YJ2406 grows well on an MRS agar culture medium, and the colony is milky white, smooth in surface, neat in edge and opaque, and is subjected to microscopic examination on the shape of the thallus, and gram staining is purple.
Further, the application of the lactobacillus plantarum YJ2406 in preparing anti-enteritis medicines is also provided.
Furthermore, the application of the lactobacillus plantarum YJ2406 in preparing medicines for treating diseases caused by one or more of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli is also provided.
Further, the application of the lactobacillus plantarum YJ2406 in preparing food for regulating intestinal flora of human or animals is also provided.
Furthermore, the lactobacillus plantarum YJ2406 has a certain tolerance to acid and bile salts and has a strong adhesion to human colon cancer cells HT-29.
Furthermore, the lactobacillus plantarum YJ2406 has better in-vitro blood sugar and cholesterol reducing capability, can effectively regulate the intestinal flora structure of mice, and remarkably increases the quantity of lactobacillus.
Furthermore, the lactobacillus plantarum YJ2406 has better prevention and treatment effects on a DSS mouse colonitis model, can relieve splenomegaly and colorectal atrophy phenomenon, and can reduce the level of various inflammatory factors
Compared with the prior art, the invention has the advantages that:
1. The lactobacillus plantarum YJ2406 provided by the invention is separated and screened from Guizhou pickled Chinese cabbage, 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.
2. Lactobacillus plantarum YJ2406 has strong antibacterial ability on common pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli and the like.
3. The lactobacillus plantarum YJ2406 has strong capability of producing short chain fatty acid, has better capability of reducing blood sugar and cholesterol in vitro, can effectively regulate the intestinal flora structure of mice, and obviously increases the quantity of lactobacillus.
4. Animal experiments show that the lactobacillus plantarum YJ2406 provided by the invention has better prevention and treatment effects on a DSS mouse colonitis model, and the inflammation index is obviously reduced. Therefore, the method is applied to the fields of functional foods and medicines, not only has practical production value, but also has very important significance for human and animal health
Drawings
FIG. 1 is a diagram showing the phylogenetic relationship between Lactobacillus plantarum YJ2406 and other strains;
FIG. 2 is a graph showing colony morphology and gram staining of Lactobacillus plantarum YJ2406 strain of the invention on MRS agar medium;
FIG. 3 is a graph showing the hemolytic activity of Lactobacillus plantarum YJ2406 strain;
FIG. 4 is a graph of total ion flow chromatograms of fermentation broth of Lactobacillus plantarum YJ2406 strain GC-MS;
FIG. 5 shows spleen index and colorectal length for a DSS-induced mouse colitis model;
FIG. 6 is a graph of structural analysis of intestinal flora of mice with DSS-induced colitis.
Detailed Description
The present invention will be further described below.
The invention provides a lactobacillus plantarum (Lactobacillus plantarum) YJ2406 which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO.25907 and the preservation address of the lactobacillus plantarum (Lactobacillus plantarum) in the year of 2022 and the month of 10 and 13: the korean district North Star, beijing city, part No.1, no. 3. The nucleotide sequence is:
GGGGCGGTGGCGGGTGCTATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCTG
The lactobacillus plantarum YJ2406 is obtained from pickled Chinese cabbage of eight villages defined in Li Ping county of Guizhou, and is frozen for later use after being separated and purified from the pickled Chinese cabbage sample.
Performing amplification culture on the frozen strain by using an MRS broth culture medium after activating and culturing, extracting strain DNA by using a root bacterium genome DNA extraction kit, and performing 16S rRNA amplification by using a colony PCR technology, wherein PCR primers are 16S rRNA universal primers 27F and 1492R; the 16s rRNA universal primers 27F and 1492R were synthesized by biological engineering (Shanghai) Inc., and the sequences were as follows:
27F：5-AGAGTTTGATCMTGGCTCAG-3；
1492R：5-GGTTACCTTGTTACGACTT-3。
The sequence is compared with BLAST in NCBI, and the similarity with the standard strain Lactobacillus plantarum strain JCM1149 reaches 99.52%, which can be preliminarily identified as lactobacillus plantarum and named as lactobacillus plantarum (Lactobacillus plantarum) YJ2406. The phylogenetic relationship of this strain with other strains is shown in FIG. 1.
The separation and purification method of the lactobacillus plantarum YJ2406 comprises the following steps: a small amount of pickled Chinese cabbage sample is taken in 50mL of MRS broth, fully and uniformly mixed by shaking, and placed in a shaking table at a constant temperature of 37 ℃ for 24h. 10-fold gradient dilution is adopted, the culture is spread on MRS agar culture medium, single colony is picked after 24h culture at 37 ℃, and the purification is carried out for 3 times continuously. The purified strain is inoculated into 600 mu L of MRS broth culture medium, shake-cultured for 18h at 37 ℃,400 mu L of sterile glycerol with the concentration of 50% is added, and the strain is frozen in an ultralow temperature refrigerator at-80 ℃ for standby.
The formula (per liter) and the preparation method of the MRS broth culture medium comprise the following steps: 10.0g of casein enzyme digest, 10.0g of beef extract powder, 4.0g of yeast extract powder, 2.0g of tri-ammonium citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 2.0g of dipotassium hydrogen phosphate, 20.0g of glucose, tween-80 and a final pH of about 5.7. When in use, 54.0g of the product is weighed, distilled water or deionized water 1L is added, the mixture is packaged in conical flasks, and the conical flasks are sterilized at 121 ℃ under high pressure for 15min.
Identification of lactobacillus plantarum YJ 2406: lactobacillus plantarum YJ2406 was inoculated on MRS agar medium, and after culturing at 37℃for 24 hours, the morphology of single colony was observed and recorded. Lactobacillus plantarum YJ2406 was gram stained using a kit method, and the stained bacterial morphology was observed and recorded under a microscope. As shown in FIG. 2-A and FIG. 2-B, lactobacillus plantarum YJ2406 grows well on MRS agar medium, and the colony form is milky white, round convex, flat in edge and smooth in surface; the microscopic examination shows that the thalli are rod-shaped and purple, and conform to the staining characteristics of gram-positive bacteria.
Example 1: antibiotic resistance and haemolysis of Lactobacillus plantarum YJ2406
1.1, Antibiotic resistance experiments:
The sensitivity of the strain to common antibiotics was tested by the paper sheet agar diffusion method. Lactobacillus plantarum YJ2406 strain 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 10min at room temperature, drug sensitive paper sheets are put in, after 24h of culture 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 3 times, and the test result refers to the American clinical laboratory standards committee (NCCLS) standard 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 Table 1, the Lactobacillus plantarum YJ2406 was sensitive (S) to the 6 antibiotics tested for tetracycline, ampicillin, ceftriaxone, clindamycin, clarithromycin and chloramphenicol, demonstrating the safety of the Lactobacillus plantarum YJ 2406.
TABLE 1 sensitivity of Lactobacillus plantarum YJ2406 Strain to 6 antibiotics
| Numbering device | Tetracycline | Ampicillin (Amoxicillin) | Ceftriaxone | Clindamycin | Clarithromycin | Chloramphenicol |
| YJ2406 | S | S | S | S | S | S |
1.2, Hemolysis experiment:
After resuscitating and activating the Lactobacillus plantarum YJ2406 strain for 3 generations, streaking and inoculating on a Columbia blood plate, culturing at 37 ℃ for 24 hours, and observing whether hemolysis rings appear around the colony to be detected. As shown in FIG. 3, the experimental results showed that no hemolytic loop appeared around the Lactobacillus plantarum YJ2406 colony, further demonstrating the safety of the strain.
Example 2: detection of primary fermentation characteristics of lactobacillus plantarum YJ2406
2.1, Evaluation of acid resistance and bile salt resistance:
The lactobacillus plantarum YJ2406 strain to be detected is revived and activated on an MRS agar plate for 3 generations, and the initial concentration of the bacterial liquid is regulated to be 1 multiplied by 106 6 CFU/mL. MRS broth medium of acidity (ph=3.0) and bile salt concentration (0.3%) was prepared using hydrochloric acid and porcine bile salt, respectively. 1mL of Lactobacillus plantarum YJ2406 strain solution of the strain to be tested is inoculated into a broth culture medium with pH=3.0, and is cultured for 18h at 37 ℃. 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 Lactobacillus plantarum YJ2406 strain to be tested was inoculated into MRS broth 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 the lactobacillus plantarum YJ2406 can still grow normal colonies on an MRS agar plate after being treated in an MRS broth culture medium with the pH value of=3.0 for 18 hours; salt tolerance test results show that the lactobacillus plantarum YJ2406 can still grow normal colonies on an MRS agar plate after being tolerant for 18 hours in an MRS broth culture medium with a bile salt concentration of 0.3%; the lactobacillus plantarum YJ2406 has stronger acid resistance and cholate resistance.
2.2 Cell adhesion test:
Lactobacillus plantarum YJ2406 is resuscitated and inoculated into MRS broth, and cultured 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. The cells were digested with 0.7mL of 0.25% trypsin-EDTA for 10min, and after the cells were completely detached, the digestion was terminated by adding 0.3mL of DMEM medium, and the medium after the completion of the adhesion test was collected for plate counting, and the result was used as the number of adhesion viable bacteria. And the standard strain LGG was used as a control. As shown in Table 2, the adhesion rate of Lactobacillus plantarum YJ2406 to human colon cancer cell HT-29 was 21.38% higher than that of the standard strain LGG. Wherein, the adhesion rate (%) = number of lactic acid bacteria at end period/initial lactic acid bacteria inoculation number×100%
TABLE 2 adhesion Rate of Lactobacillus plantarum YJ2406 to human colon cancer cells HT-29
2.3, Simulated gastric fluid, intestinal fluid tolerance experiments:
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. Resuscitating and activating Lactobacillus plantarum YJ2406 strain, regulating the concentration of bacterial liquid to 1X 10 8 CFU/mL, adding 1mL of bacterial liquid into 9mL of simulated artificial gastric fluid, performing 10-time gradient dilution, and sucking 20 mu L of bacterial liquid in a plate to obtain an initial viable count of the tolerating artificial gastric fluid; after the inoculated simulated gastric juice is cultured for 3 hours at 37 ℃, viable count is performed again to be used as the final viable count for tolerating the artificial gastric juice. Similarly, 1mL of lactobacillus plantarum YJ2406 fermentation broth with the concentration of 1X 10 8 CFU/mL is added into 9mL of simulated intestinal fluid, viable count is carried out, the viable count is counted again after culture for 6 hours at 37 ℃, and the survival rate is calculated. Survival = number of viable bacteria at end/number of initial viable bacteria x 100%. As shown in Table 3, lactobacillus plantarum YJ2406 strain has better tolerance to artificial simulated stomach and intestinal juice, the survival rate in artificial gastric juice after 3 hours is 136.0%, and the survival rate in artificial intestinal juice after 6 hours is 55.0%.
TABLE 3 tolerance of Lactobacillus plantarum YJ2406 Strain to artificial simulated gastric and intestinal fluids (%)
2.4, Measuring the content of short-chain fatty acid in lactobacillus plantarum YJ2406 fermentation liquor:
Preparation of fermentation liquor: after the lactobacillus plantarum YJ2406 preservation strain is activated and cultured for 24 hours, 4ul of bacterial liquid is sucked and added into a 4mLMRS broth culture medium, and the culture is carried out at 37 ℃ for 24 hours for standby, wherein the number of viable lactobacillus in the fermentation liquid is 1 multiplied by 10 9 CFU/ml.
Detection of short-chain fatty acids: the detection instrument was a gas chromatograph-mass spectrometer (GCMS-QP 2010 Plus) from Shimadzu corporation, and the chromatographic column was a fused silica capillary column (30 m. Times.0.25 mm. Times.0.25 um) Rtx-5 from RESTEK (Ruis Talct) corporation, U.S.A.. The GC temperature program was maintained at an initial temperature of 40℃for 5min, 5℃to 150℃per minute, 10℃to 280℃per minute, and 2min. The carrier gas is high purity helium (purity > 99.999%), flow rate: 1.0mL/min. MS conditions: the ionization mode is EI; the temperature is 200 ℃, the interface temperature is 220 ℃, and the mass scanning range m/z is 33-500. Taking 4mL of fermentation liquor, adding 10ul of 2-ethylbutyric acid internal standard solution with the concentration of 200ug/mL, sampling 1 mu L of sample in a mode of 1:3 of a split flow mode, setting the solvent delay time to 0.1min, and setting the temperature of a sample inlet to 270 ℃. The concentration of 5 short chain fatty acids (acetic acid, n-butyric acid, isobutyric acid, isovaleric acid, isocaproic acid) was calculated using the internal standard method. As shown in Table 4 and FIG. 4, the GC-MS detection result shows that the acetic acid content in the lactobacillus plantarum YJ2406 fermentation broth is the highest, which is 11.466ug/mL. As shown in FIG. 4, the composition further contains three kinds of butyric acid, i-butyric acid, 2-methylbutyric acid and i-valeric acid.
TABLE 4 Lactobacillus plantarum YJ2406 fermentation broth short chain fatty acid content (ug/mL)
| Strain number | Strain name | Acetic acid | N-butyric acid | Isobutyric acid | Isopentanoic acid | 2-Methylbutyric acid |
| YJ2406 | Lactobacillus plantarum | 11.466 | 0.075 | 0.119 | 0.358 | 0.107 |
Short chain fatty acids have an important role in maintaining the normal function of the large intestine and the morphology and function of colonic epithelial cells.
Example 3: evaluation of bacteriostatic Activity of Lactobacillus plantarum YJ2406 Strain
Escherichia coli (ESCHERICHIA COLI CMCCB 44102), staphylococcus aureus (Staphylococcus aureus CMCCB 50094), salmonella typhimurium (Salmonella typhimurium ATCC 14028), pseudomonas aeruginosa (Pseudomonas aeruginosa CMCCB 10104), campylobacter jejuni (Campylobacter jejuni ATCC 33291) and Campylobacter coli (Campylobacter coli ATCC 43478) were inoculated respectively to nutrient agar medium, resuscitated and activated 3 times. Sucking a proper amount of trypticase soy peptone liquid culture medium into a centrifuge tube, inoculating the activated pathogenic bacteria into the broth culture medium, and regulating the concentration of the bacterial liquid to be 1X 10 8 CFU/mL. 1mL of the mixture of the pathogenic bacteria and the broth is sucked up and 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 strain of bacteria, and three holes are formed as repetition. Resuscitating and activating the strain to be detected, and regulating the concentration of the cultured bacterial liquid to be 1 multiplied by 10 8 CFU/mL. And (3) sucking 50 mu L of bacteria liquid to be detected, adding the bacteria liquid to the hole of the pathogenic bacteria agar plate, and culturing for 24 hours at 37 ℃. After incubation, the diameter of the zone of inhibition around the perforation point was measured using a vernier caliper and recorded. The standard strain LGG was used as a control strain and the above experimental procedure was performed simultaneously with the strain to be tested. As shown in Table 5, the fermentation broth of Lactobacillus plantarum YJ2406 strain has stronger inhibitory activity on the growth of pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni, campylobacter coli, etc., and is superior to the antibacterial effect of LGG standard strain.
TABLE 5 evaluation of antibacterial Activity of Lactobacillus plantarum YJ2406 Strain (diameter: mm)
Example 4: test of the prevention action of Lactobacillus plantarum YJ2406 on the mouse colitis model
4.1, Laboratory animals and treatment groups
A total of 27 mice were randomly divided into 9 cages of 3 mice each. The animals were randomized into 3 groups of 3 cages each, including a placebo group (CK), a natural recovery group (DSS) and a YJ2406 treatment group, and experimental treatments are shown in table 6.
TABLE 6 animal experimental treatments
Wherein DSS is dextran sulfate sodium salt solution with concentration of 4% (w/v).
Preparation of bacterial suspension: the standard strain LGG and lactobacillus plantarum YJ2406 were resuscitated and activated for 3 passages. Centrifuging the bacterial liquid at 4 ℃ for 5min under the condition of 6000r/min, and discarding the 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.
4.2, 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 determination is to take out spleen by dissection immediately after the mice are sacrificed, wash with physiological saline, suck with filter paper and weigh. 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.
4.3 Experimental results
As shown in table 7, DSS treated groups had weight loss of 9.7% and 28.8% during the prophylaxis and treatment periods, respectively, while lactobacillus plantarum YJ2406 fed group was able to maintain weight substantially unchanged.
TABLE 7 weight variation of mice (Unit: g)
| Grouping | Onset of the prophylaxis period | End of the prevention period | End of treatment period |
| CK | 37.56 | 37.45 | 38.29 |
| DSS | 37.43 | 33.79 | 26.66 |
| YJ2406 | 35.99 | 35.28 | 35.62 |
As shown in Table 8, the four inflammatory factor indicators IL-1β, IL-6, IL-8 and TNF- α were substantially lower in both the prophylaxis and treatment phases than in the DSS group.
TABLE 8 measurement results of mouse serum inflammatory factors (pg/ml)
As shown in fig. 5 and table 9, lactobacillus plantarum YJ2406 fed group had lower spleen index and longer colorectal length. Feeding lactobacillus plantarum YJ2406 from multiple levels can alleviate inflammatory symptoms in mice.
Table 9 spleen index and colorectal length of mice
As shown by A, B, C, D in fig. 6, the intestinal flora 16S rRNA sequencing results showed that lactobacillus plantarum YJ2406 fed group had higher flora diversity and that the number of characteristic flora was higher than the control group and DSS treated group as shown by E in fig. 6. Further analysis found that the intestinal flora of YJ2406 contained large amounts of lactobacillus (Lactobacilus) compared to the control and DSS groups, which are globally recognized intestinal probiotics for animals.
In conclusion, lactobacillus plantarum YJ2406 strain has obvious relieving effect on the mouse colonitis induced by DSS.
Example 5: in vitro detection of hypoglycemic and cholesterol-lowering activity of lactobacillus plantarum YJ2406
5.1 Inhibition of alpha-glucosidase by Lactobacillus plantarum YJ2406
Alpha-glucosidase participates in the decomposition and utilization of carbohydrate, and research shows that inhibiting the activity of alpha-glucosidase can reduce the blood sugar level in human blood. After the lactobacillus plantarum YJ2406 preserved strain is resuscitated and cultured for 24 hours, inoculating the lactobacillus plantarum YJ2406 preserved strain into an MRS liquid culture medium according to the inoculum size of 2 percent, culturing the lactobacillus plantarum in the condition of 37 ℃ for 24 hours, centrifuging the bacterial liquid at 4 ℃ and 4000r/min for 15 minutes, and filtering the supernatant by a 0.22 mu m filter membrane to obtain a fermentation supernatant (CFS) for later use; 25 μl of the supernatant was taken, 50 μl of PNPG solution of 50 μl and 20mmol/L in PBS buffer (pH=6.8) was added, and the mixture was subjected to water bath at 37deg.C for 10min; adding 30 mu L of 20U/ml alpha-glucosidase solution, and continuing to react for 10min at 37 ℃; the reaction was terminated by adding 50. Mu.L of 1mol/L Na 2CO3 solution; in 96-well plates (365 μl), 3 replicates were set for each group; and measuring the absorbance at 405nm of the enzyme label instrument, and calculating to obtain the alpha-glucosidase inhibition rate (%). The calculation formula is as follows:
Wherein group A is alpha-glucosidase; group B is PBS buffer solution; c is a sample group to be detected and contains alpha-glucosidase and a sample to be detected; group D contained only the samples to be tested.
As shown in table 10, the lactobacillus plantarum YJ2406 fermentation supernatant has an activity of inhibiting alpha-glucosidase with an inhibition rate of 15.32% higher than that of the standard strain lactobacillus rhamnosus LGG, indicating that the lactobacillus plantarum YJ2406 preparation may have the potential to reduce blood glucose levels in animals and human blood.
TABLE 10 inhibition of alpha-glucosidase by Lactobacillus plantarum YJ2406 fermentation broth (%)
5.2 Degradation of cholesterol by Lactobacillus plantarum YJ2406
Studies show that some lactic acid bacteria can adsorb or absorb cholesterol, and the purpose of reducing the cholesterol in animals is achieved by discharging the lactic acid bacteria out of the body. Therefore, cholesterol degradation ability can be calculated by adding a certain amount of bile salt and cholesterol to a cholesterol-MRS medium (MRS-CHOL medium) and measuring a change in cholesterol concentration after culturing. The specific operation method is as follows:
5.2.1 preparation of cholesterol solution: cholesterol 0.06g, ox gall salt 0.12g, sucrose fatty acid ester 0.06g, glacial acetic acid 5ml and tween 0.6ml, and performing ultrasonic vibration until the cholesterol is completely dissolved, filtering with a 0.22 μm filter membrane under aseptic condition for later use;
5.2.2 preparation of cholesterol-MRS Medium (MRS-CHOL Medium): 12mL of 6mol/L NaOH solution of cholesterol solution is added into 300mL of MRS liquid culture medium;
5.2.3, inoculation culture: the lactobacillus plantarum YJ2406 strain is activated and cultured for 24 hours, and is inoculated into MRS-CHOL liquid culture medium with an inoculum size of 2 percent, and is cultured for 48 hours at 37 ℃, and the other part of the lactobacillus plantarum is not inoculated with a blank control.
5.2.4, Drawing of cholesterol standard curve:
Preparing mixed acid: mixing concentrated sulfuric acid and glacial acetic acid according to the proportion of 1:1, and shaking uniformly;
Phthalic aldehyde solution (1 mg/mL): 25ml of absolute ethyl alcohol and 25mg of phthalic aldehyde are mixed and dissolved into a brown volumetric flask, and the mixture is stored in a refrigerator;
Standard cholesterol working solution: 0.05g of cholesterol was fixed to a volume of 50mL using glacial acetic acid, and a cholesterol standard solution having a mass concentration of 1mg/mL was prepared; diluting with glacial acetic acid for 10 times to obtain standard cholesterol working solution;
As shown in Table 11, different amounts of samples were added, shaken well and then allowed to stand for 30min. Absorbance (OD value) was measured at a wavelength of 550nm, and a standard curve was drawn with cholesterol concentration as an abscissa and absorbance as an ordinate.
Table 11 amount of each reagent used (mL) in the calibration curve
| 0 | 1 | 2 | 3 | 4 | 5 | |
| Standard cholesterol working solution | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 |
| Glacial acetic acid | 0.5 | 0.4 | 0.3 | 0.2 | 0.1 | 0 |
| Phthalic dicarboxaldehyde | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 |
| Mixed acid | 4.3 | 4.3 | 4.3 | 4.3 | 4.3 | 4.3 |
5.2.5, Determination of cholesterol content:
Respectively taking bacterial suspension and blank control 500 mu L in a 5mL test tube, slowly adding 4.5mL absolute ethyl alcohol, standing for 10min, and centrifuging for 15min at 3000 r/min; adding 0.5ml of supernatant into a test tube, adding 0.2ml of 1mg/ml of phthalic aldehyde and 4.3ml of mixed acid, shaking uniformly, and standing for 30min; measuring absorbance at 550nm wavelength; calculating the cholesterol content in the sample from the standard curve;
wherein C0 is OD value of the blank control group; c1 is the OD value of the experimental group;
As shown in table 12, the degradation rate of the lactobacillus plantarum YJ2406 on cholesterol was 54.07%, which is superior to the standard strain lactobacillus rhamnosus-LGG, demonstrating that the lactobacillus plantarum YJ2406 has the potential to reduce cholesterol in animals and humans.
Table 12 degradation ability of Lactobacillus plantarum YJ2406 on cholesterol
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it will be apparent to those skilled in the art from this disclosure that various changes and modifications can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. A lactobacillus plantarum (Lactobacillusplantarum) YJ2406, characterized in that: the lactobacillus plantarum is preserved in China general microbiological culture Collection center (CGMCC) at the date of 10 and 13 of 2022, and the preservation number is CGMCC NO.25907.
2. A strain of lactobacillus plantarum YJ2406 according to claim 1, characterized in that: the lactobacillus plantarum YJ2406 grows well on an MRS agar culture medium, and is milky white in colony, smooth in surface, neat in edge, opaque, microscopic in shape of bacteria and purple in gram stain.
3. Use of a strain of lactobacillus plantarum YJ2406 according to claim 1, characterized in that: the application is the application in preparing anti-enteritis drugs.
4. Use of a strain of lactobacillus plantarum YJ2406 according to claim 3, characterized in that: the application is the application in preparing medicines for treating diseases caused by one or more of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and campylobacter coli.
5. Use of a strain of lactobacillus plantarum YJ2406 according to claim 1, characterized in that: the application is the application in preparing food for regulating intestinal flora of human or animals.
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