Lactobacillus plantarum strain sourced from healthy people in cold region and application of lactobacillus plantarum strain

Abstract

The invention discloses a lactobacillus plantarum strain from healthy people in a cold region and application of the lactobacillus plantarum strain. The lactobacillus plantarum strain is named as PL-H1, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.22286. The lactobacillus plantarum PL-H1 screened by the invention can obviously relieve the disease index, intestinal canal pathological change, inflammatory response and the like of an inflammatory bowel disease (IBD) model mouse. Moreover, compared with a standard strain WCSF1, the strain has the high metabolism characteristic of tryptophan and the function of adjusting the metabolism of IBD host intestinal tryptophan, and the curative effect of preventing and treating IBD is more remarkable. In addition, the lactobacillus plantarum strain PL-H1 can also improve the liver lipid accumulation and NAFLD activity of MCD fed mice, improve the blood lipid level and liver injury of the MCD fed mice and prevent liver fibrosis of the MCD fed mice, can be used for preventing and treating fungal infection related diseases, and is good in safety.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus plantarum strain and its application, and particularly to a Lactobacillus plantarum strain derived from healthy people in cold regions and its application. The present invention belongs to the field of biotechnology. Background Art

[0002] Lactobacillus plantarum, a type of lactic acid bacteria, has an optimal growth temperature of 30-35°C, is anaerobic or facultatively anaerobic, with a rod-shaped morphology that can be straight or curved, occurring singly, in pairs, or in chains, and an optimal pH of around 6.5. It belongs to homofermentative lactic acid bacteria. Lactobacillus plantarum has many health benefits: ① It has a certain immunomodulatory effect; ② It inhibits pathogenic bacteria; ③ It reduces serum cholesterol levels and prevents cardiovascular diseases; ④ It maintains the balance of intestinal flora; ⑤ It promotes the absorption of nutrients; ⑥ It alleviates lactose intolerance; ⑦ It inhibits the formation of tumor cells, etc.

[0003] The incidence of inflammatory bowel disease (IBD, including ulcerative colitis UC and Crohn's disease CD) and vulvovaginal candidiasis and other diseases is on the rise globally, and its incidence is significantly higher in the north than in the south, which is related to latitude or temperature. Currently, the treatment methods for these diseases have limitations, such as large drug side effects and easy recurrence. Therefore, it is urgent to develop new treatment methods. Since probiotics can effectively colonize the colon for a long time and maintain intestinal health and play a role in treating IBD by producing short-chain fatty acids, improving the intestinal mucosal barrier, and reducing the production of pro-inflammatory factors. Currently, probiotic preparations have been applied to the treatment of inflammatory bowel disease. It has been reported in the literature that in the colon tissues of patients with inflammatory bowel disease and mice, the level of tryptophan in the host intestine decreases, and the content of indole substances decreases significantly, and its content is negatively correlated with the disease activity; furthermore, it causes abnormalities in the intestinal immune microenvironment, reduced secretion of mucosal protective substances, and increased production of pro-inflammatory factors. In addition, in the diseased tissues or secretions of diseases such as depression, there is often amino acid metabolism disorder, manifested as a significant decrease in amino acids such as tryptophan, which is related to the disease progression and severity, but the existing treatment methods are difficult to effectively regulate the amino acid metabolism imbalance. By orally supplementing tryptophan, due to the absorption in the upper digestive tract, the level of tryptophan reaching the colon is not sufficient to play a therapeutic role.

[0004] Tryptophan is an essential amino acid for the human body. Tryptophan and its metabolites have significant effects on some key physiological functions, including immune system stimulation and homeostasis, gastrointestinal motility, and even brain activity. Tryptophan can only be obtained from food intake. Under the metabolic action of intestinal microorganisms, it can be converted into various indole metabolites, such as indole acetic acid and indole acrylic acid, which can then promote the tight junctions between intestinal epithelia, repair the epithelial barrier, etc. to maintain intestinal health. Currently, strains such as Bifidobacterium and Escherichia coli that produce tryptophan manufactured through genetic engineering technology have shown effects in treating IBD.

[0005] Abnormal liver lipid metabolism refers to the pathological state of lipid metabolism imbalance in the liver, leading to excessive accumulation of fat in hepatocytes. This abnormality is mainly manifested as non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), hepatic fibrosis, and more severe diseases such as cirrhosis and primary hepatocellular carcinoma. These diseases together constitute a broad disease spectrum, namely non-alcoholic fatty liver disease (NAFLD). In 2020, researchers proposed to rename NAFLD as metabolic associated fatty liver disease (MAFLD) to emphasize the important role of metabolic disorders in its occurrence and development. Specifically, non-alcoholic fatty liver refers to the appearance of significant oil droplets or other forms of lipid accumulation when it exceeds 5% of the normal range. This excessive lipid not only causes the generation of toxic substances but may also lead to phenomena such as local inflammatory reactions and cell degeneration, such as ballooning degeneration, and these changes further progress to NASH. During this process, over time, the persistent inflammation and immune response can prompt a large number of dead hepatocytes to release signaling molecules, thereby activating adjacent stellate cells, transforming them into myofibroblasts, and migrating to the damaged area, triggering tissue repair mechanisms but also promoting the development of fibrosis. For patients with advanced fibrosis, obvious scar tissue has formed in their bodies, and this stage is called "cirrhosis". It is worth noting that among all patients diagnosed with NASH, 41% are accompanied by fibrosis to varying degrees, and 22% of patients with advanced fibrosis may further progress to irreversible cirrhosis. In addition, according to epidemiological data, approximately 2 - 3% of patients diagnosed with cirrhosis may evolve into primary HCC within three years. Therefore, early screening and intervention for these related diseases are of great significance for reducing their development risks and also pose new challenges to public health policies.

[0006] Fungal infections are diseases caused by pathogenic fungi invading the human body. According to the parts of the human body invaded by fungi, fungal infectious diseases can be divided into superficial mycosis, dermatomycosis, subcutaneous tissue mycosis, and systemic mycosis. The first two are collectively called superficial mycosis, and the latter two are also called deep mycosis. Probiotics have significant curative effects in the treatment of vaginal candidiasis, oral candidiasis, etc. Clinical studies have shown that probiotics can reduce the colonization of Candida on the human mucosal surface, relieve the symptoms and signs of fungal infections, improve the efficacy of traditional antifungal drugs, and play a role in the prevention and adjuvant treatment of candidiasis by maintaining the ecological balance of the mucosal flora. Summary of the Invention

[0007] The purpose of the present invention is to provide a Lactobacillus plantarum strain derived from healthy people in cold regions.

[0008] The second purpose of the present invention is to provide the application of the above-mentioned Lactobacillus plantarum strain.

[0009] In order to achieve the above purpose, the present invention adopts the following technical means:

[0010] Based on the epidemiological characteristics that the incidence of IBD is significantly lower in the north than in the south (about 1:3), the inventors of the present invention speculated that the normal population in the cold regions of the north (regions above 45 degrees north latitude) carried probiotic strains with the effect of treating IBD. Therefore, probiotic strains with the effect of treating IBD were isolated, screened, and identified from the vaginas of healthy women in Harbin, Heilongjiang Province, China.

[0011] A Lactobacillus plantarum strain derived from healthy people in cold regions isolated by the present invention is named PL-H1 and classified as Lactobacillus plantarum. The above-mentioned Lactobacillus plantarum strain is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the preservation address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 22286, and the preservation time is May 7, 2021.

[0012] Furthermore, the present invention also proposes the application of the above-mentioned Lactobacillus plantarum strain in the following aspects:

[0013] (1) Application in the preparation of drugs for preventing, relieving or treating inflammatory bowel disease and diseases related to tryptophan deficiency;

[0014] (2) Preparation for preventing, relieving or treating abnormal liver lipid metabolism and its related diseases;

[0015] (3) Application in the preparation of drugs for preventing, relieving or treating diseases caused by fungal infections.

[0016] Among them, preferably, the liver lipid metabolism disorder-related disease is non-alcoholic fatty liver.

[0017] Among them, preferably, the fungus is Candida albicans.

[0018] Among them, preferably, the disease caused by the fungal infection is vulvovaginal candidiasis.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. The Lactobacillus plantarum strain PL-H1 isolated in the present invention is derived from a healthy human body in a cold region, does not carry pathogenic genes, drug resistance genes, and genes interacting with the host, and has good safety;

[0021] 2. Compared with the Lactobacillus plantarum standard strain WCFS1, the Lactobacillus plantarum strain PL-H1 of the present invention can effectively alleviate the progression of inflammatory bowel disease (IBD), reduce the severity of the disease, decrease the disease activity index (DAI), and increase the body weight of the host and the length of the intestinal tract.

[0022] 3. The Lactobacillus plantarum strain PL-H1 of the present invention is characterized by a significantly increased ability to synthesize tryptophan and regulate the related pathways of tryptophan metabolites, can improve the metabolic process of tryptophan and its metabolites, regulate the imbalance of intestinal amino acid metabolism in IBD, and thus can be used to supplement and regulate the tryptophan metabolism disorder in related diseases such as depression, and alleviate the progression and severity of the disease.

[0023] 4. The Lactobacillus plantarum strain PL-H1 of the present invention can be applied to the prevention and treatment of related diseases such as non-alcoholic fatty liver. It can improve the liver lipid accumulation and NAFLD activity in mice fed with MCD, improve the blood lipid level and liver injury in mice fed with MCD, prevent the occurrence of liver fibrosis in mice fed with MCD, and improve the damaged intestinal barrier in mice fed with MCD by regulating tight junctions.

[0024] 5. Compared with the Lactobacillus plantarum standard strain WCFS1, the Lactobacillus plantarum strain PL-H1 of the present invention can effectively alleviate the progression of fungal infection and reduce the severity of the disease. Brief Description of the Drawings

[0025] Figure 1 It is the phylogenetic tree of the 16S rDNA nucleotide sequence of Lactobacillus plantarum PL-H1 and the strain identification result;

[0026] Figure 2 It is the prediction result of the genes related to the safety of Lactobacillus plantarum PL-H1;

[0027] Among them, (A) virulence gene; (B) drug resistance gene; (C) pathogen-host interaction gene;

[0028] Figure 3Comparison results of the efficacy of Lactobacillus plantarum PL-H1 and the standard strain WCFS1 on DSS-IBD mice;

[0029] Among them, (A) Results of changes in mouse body weight; (B) Results of changes in mouse spleen index; (C) DAI scores of each group; (D) Photos and statistical results of mouse colon length;

[0030] Figure 4 Mechanism analysis of the efficacy of Lactobacillus plantarum PL-H1 and the standard strain WCFS1 on DSS-IBD mice; Results;

[0031] Among them, (A) Results of HE staining and pathological scores of mouse colon; (B) Results of ZO-1 gene expression; (C) Results of IL-6 gene expression;

[0032] Figure 5 Analysis results of tryptophan transcription levels of Lactobacillus plantarum PL-H1 and the standard strain WCFS1;

[0033] Among them, (A) Expression results of phenylalanine, tyrosine, and tryptophan; (B) Expression level of tryptophan synthase;

[0034] Figure 6 Determination results of tryptophan and its metabolites in Lactobacillus plantarum PL-H1 strain and its supernatant;

[0035] Among them, (A) Relative abundances of tryptophan in Lactobacillus plantarum PL-H1 cells and supernatant; (B) Relative abundances of indole, indole lactic acid (ILA), indole-3-carboxaldehyde (IAId), indole pyruvic acid, tryptamine, indole-3-acetate, indole-3-acrylic acid (IA) in Lactobacillus plantarum PL-H1 cells and supernatant;

[0036] Figure 7 Lactobacillus plantarum PL-H1 improved liver lipid accumulation and NAFLD activity in MCD-fed mice;

[0037] Among them, (A) Representative images of H&E staining, 10X, scale bar 100 μm. (B) NAFLD activity score; (C) Representative images of Oil Red O staining, 10X, scale bar 100 μm; (D) Percentage of positive area of Oil Red O staining analyzed by Image J software; (E-H) mRNA expression of CD36, PPAR-r, FABP-1, and Fasn in mouse liver tissues detected by RT-qPCR; (I) Western blot analysis of PPAR-r / actin and FABP4 / actin in liver tissues; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA followed by Tukey's test;

[0038] Figure 8 Lactobacillus plantarum PL-H1 improves blood lipid levels and liver injury in MCD-fed mice;

[0039] Among them, (A) liver index, (B) serum cholesterol concentration (mmol / L), (C) serum triglyceride concentration (mmol / L), (D) serum HDL-C concentration (mmol / L), (E) serum LDL-C concentration (mmol / L), (F) serum total bile acid, (G) serum AST, (H) serum ALT. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA with Tukey's test;

[0040] Figure 9 Lactobacillus plantarum PL-H1 can prevent the occurrence of liver fibrosis in MCD-fed mice;

[0041] Among them, (A) representative images of Masson staining, 10X, scale bar 100 μm; (B) percentage of positive area of Masson staining analyzed using Image J software; (C) immunofluorescence staining of α-SMA (green) and DAPI (blue) in colon tissues. Scale bar, upper: 100 μm, lower: 20 μm; (D) area density of α-SMA immunofluorescent protein analyzed using Image J software; (E,F) mRNA expression levels of Col1a1 and Col3al in mouse liver tissues detected by RT-qPCR; (G) Western blot analysis of α-SMA / actin in liver tissues; *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA (ANOVA) with Tukey's test;

[0042] Figure 10 Lactobacillus plantarum PL-H1 improves liver immune-related indicators in MCD-fed mice;

[0043] Among them, (A) spleen index; (B) ELISA analysis of LPS in serum; (C-K) mRNA expressions of TGF-β, IL-10, IL-1β, IL-6, IL-17a, TLR4, F4 / 80, INOS, TNF-β, and IL-10 in mouse liver tissues detected by RT-qPCR; (L) Western blot analysis of IL-6 / actin, IL-17a / actin, IL-10 / actin, IL-22 / actin, iNOS / actin, CD163 / actin, PD-1 / actin, and PD-L1 / actin in liver tissues; (M) detection of PD-1 (green) and DAPI (blue) in colon tissues by immunofluorescence staining; scale bar, 100 μm; (N, Q) areas of PD-1 and PD-L1 immunofluorescent proteins; (O, R) mRNA expressions of PD-1 and PD-L1 in mouse liver tissues detected by RT-qPCR; *P < 0.05, **P < 0.01, ***P < 0.001, one-way analysis of variance (ANOVA) with Tukey's test;

[0044] Figure 11A-I and Figure 11J-N Lactobacillus plantarum PL-H1 improves the impaired intestinal barrier function in MCD-fed mice by regulating tight junctions;

[0045] Among them, (A) ELISA analysis of LPS in serum; (B, C) Colon length (cm); (D) Colon tissue stained with hematoxylin-eosin (H&E); 10X, 100 μm; The black arrow indicates the measured mucus layer; (E) Mucus layer thickness (μm); (F) Colon tissue stained with alcian blue-PAS; Scale bar, upper: 55 μm, lower: 30 μm; (G) Immunofluorescence staining of MUC-2 (green) and DAPI (blue) in colon tissue; Scale bar, upper: 100 μm, lower: 50 μm; (H) Detection of the mRNA expression level of MUC-2 in mouse colon tissue by RT-qPCR; (I) Area of MUC-2 immunofluorescent protein analyzed using Image J software; (J) Immunofluorescence staining of ZO-1 (green) and DAPI (blue) in colon tissue; Scale bar, upper: 100 μm, lower: 50 μm; (K) Area of ZO-1 immunofluorescent protein analyzed using Image J software; (L, M) Detection of the mRNA expression levels of Claudin1 and ZO-1 in mouse colon tissue by RT-qPCR; (N) Western blot analysis of Occludin / actin, E-cadherin / actin, and Claudin1 / actin in liver tissue; *P<0.05, **P<0.01, ***P<0.001, one-way analysis of variance (ANOVA) combined with Tukey's test;

[0046] Figure 12 Effect of Lactobacillus plantarum PL-H1 on the growth of Candida albicans;

[0047] Among them, (A) Colony-forming units of Candida albicans on YPD plates after treatment with Lactobacillus plantarum; Candida albicans control: 10 3 CFU / mL of Candida albicans cultured for 12 h, 24 h, and 48 h; 10 3 CFU / mL, 10 5 CFU / mL, and 10 8 CFU / mL of Lactobacillus plantarum: Different concentrations of Lactobacillus plantarum co-cultured with 10 3 CFU / mL of Candida albicans for 12 h, 24 h, and 48 h; (B) Candida albicans Log10CFU / mL (Tukey's test after analysis of variance: **P<0.01, ***P<0.001, ******P<0.0001); This figure shows the results obtained by averaging three independent replicate experiments, and the error bars represent the standard error (±SE);

[0048] Figure 13Comparison of the anti - Candida albicans effects of Lactobacillus plantarum and Lactobacillus delbrueckii subsp. bulgaricus; Log10 CFU / mL of Candida albicans with different concentrations of Lactobacillus plantarum and Lactobacillus delbrueckii subsp.; different times in the Lactobacillus bulgaricus treatment group (Tukey's test after analysis of variance: ***P < 0.001);

[0049] Figure 14 Growth curves of Lactobacillus plantarum PL - H1 and Lactobacillus delbrueckii subsp. bulgaricus in MRS broth and their co - culture with Candida albicans in co - culture broth;

[0050] Among them, (A) Candida albicans co - cultured with Lactobacillus plantarum PL - H1; (B) Candida albicans co - cultured with Lactobacillus delbrueckii subsp. bulgaricus; the curves represent the means of three independent experiments; error bars represent 95% CI;

[0051] Figure 15 Establishment of a murine model of vaginitis infected with Candida albicans and the therapeutic effect of Lactobacillus plantarum;

[0052] Among them, (A) Observation of fungi and epithelial morphology in the vaginal lavage fluid of VVC model mice and calcofluor white staining of vaginal fungi; (B) Quantification of fungi in the vaginal lavage fluid of VVC model mice; (C) Quantification of neutrophils in the vaginal lavage fluid of VVC model mice by Papanicolaou staining. Detailed implementation methods

[0053] The present invention will be further described below through examples, the purpose of which is only to better understand the research content of the present invention rather than to limit the protection scope of the present invention. Except for special cases described below, all the following examples are conventional experimental methods and operation steps in the art.

[0054] The formula of the strain isolation medium involved in the following examples is as follows: MRS medium (g / L): peptone 10 g / L, beef extract 8 g / L, yeast extract powder 4 g / L, glucose 20 g / L, K2PO4·3H2O 2 g / L, diammonium citrate 2 g / L, sodium acetate 5 g / L, MgSO4·7H2O 0.2 g / L, MnSO4 0.04 g / L, Tween 80 1 mL / L.

[0055] In the following examples, the PL - H1 strain involved is classified and named as Lactobacillus plantarum, and is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 22286.

[0056] The following method for preparing the bacterial suspension: inoculate the required strain into MRS liquid medium, culture it at 37°C for 24 h for activation, and activate it continuously twice to obtain the activation solution; inoculate the activation solution into MRS liquid medium at an inoculation amount of 2% (v / v), and culture it at 37°C for 30 h to obtain the bacterial solution; centrifuge the bacterial solution at 5000 rpm at 4°C for 10 min, filter it to obtain the bacterial cells, and resuspend the bacterial cells with PBS solution to obtain the bacterial suspension.

[0057] The test result data was statistically analyzed using ggplot2 in R language. *** represents p < 0.001, ** represents p < 0.01, * represents p < 0.05, and ns represents no significant difference.

[0058] Example 1: Isolation and identification of a Lactobacillus plantarum strain from healthy people in cold regions

[0059] (1) Source and isolation of the strain:

[0060] Based on the epidemiological characteristics that the incidence of IBD is significantly lower in the north than in the south (about 1:3), the inventor of the present invention speculated that normal people in the cold regions in the north (regions above 45 degrees north latitude) carry probiotic strains with the effect of treating IBD. Therefore, probiotic strains with the effect of treating IBD were isolated, screened and identified from the vaginas of healthy women in Harbin, Heilongjiang Province, China.

[0061] The experimental operations for isolation include: adding sterilized PBS buffer to the vaginal swabs of healthy women in a sterile container, fully shaking and then diluting and coating on the MRS solid medium plate, picking single colonies with different sizes and morphologies after culturing at 37°C under anaerobic conditions for 48 h and streaking on a fresh solid medium plate, then picking single colonies onto the slant, examining the colony morphology under the microscope after the slant culture is completed, and performing identification after examining that there are no contaminants under the microscope. Using the vaginal swabs of healthy women in cold regions as the isolation source, the obtained strains have higher safety and are more suitable for the production of health products.

[0062] (2) Strain identification: After culturing the above purified strains in the above MRS medium for 1 day, 1 mL of the bacterial solution was taken and centrifuged at 10,000 rpm for 1 min. The supernatant was discarded, and the cells were collected. DNA was extracted according to the operation steps of the MAGEN bacterial genomic DNA extraction kit. Using the extracted DNA as a template, the 16S rRNA was amplified by PCR (polymerase chain reaction) with the universal primers 337F (5’-GACTCCTACGGGAGGCWGCAG-3’) and 1492R (5’-TACGGCTACCTTGTTACGACTT-3’). The reaction system and program were configured according to the Phusion high-fidelity DNA polymerase instruction manual. Among them, the reaction system of PCR was 50 μL, and the annealing temperature was 55 °C. The obtained PCR product was sent to the Institute of Microbiology, Chinese Academy of Sciences in Beijing for sequencing.

[0063] The sequence of the 16S rRNA measured for the strain was proofread, and its sequence was as shown in SEQ ID NO.1. It was compared with the sequences of related species and genera in the GenBank database by BLAST of homologous sequences to determine the taxonomic status of the strain. Figure 1 After comparison, the strain was identified as Lactiplantibacillus plantarum, named PL-H1, and deposited in the China General Microbiological Culture Collection Center on September 8, 2023, with the deposit number CGMCC No. 22286.

[0064] (3) Detection of genes related to the safety of the strain

[0065] The genome sequencing of strain PL-H1 was performed on the BGI (MGISEQ-2000) platform. The raw image data obtained by BGI platform sequencing was converted into sequence data through Base Calling, and the results were stored in the FASTQ file format. Paired-end (PE) sequencing of the sample DNA was performed using BGI platform sequencing technology to construct a 280bp library. The SPAdes (version: v3.11.0) splicing software was used to splice the optimized sequences with multiple Kmer parameters to obtain the optimal assembly result, and finally a circular bacterial genome was obtained. Gene component prediction includes prediction of coding genes, non-coding RNAs, repetitive sequences, and prophages. RNAmmer (version: v1.2) was used to predict rRNA, and Prodigal (version: v2.6.3) was used to predict coding genes. The Pathogen-Host Interaction (PHI, http: / / www.phi-base.org / ) database was used to analyze pathogen-host interactions. The Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / main.htm) of pathogenic bacteria was used to predict virulence factors. The Comprehensive Antibiotic Research Database (CARD, https: / / card.mcmaster.ca / ) was used for genes related to antibiotic resistance. The results showed as Figure 2 , there were no virulence genes ( Figure 2 A), drug resistance genes ( Figure 2 B), or pathogen-host interaction genes ( Figure 2 C) in Lactobacillus plantarum PL-H1.

[0066] Example 2: Improvement ability of Lactobacillus plantarum PL-H1 on the symptoms of IBD model mice

[0067] (1) Experimental animals and grouping: A total of 12 2-month-old C57BL / 6 female mice (purchased from the Experimental Animal Department of Harbin Medical University), with a body weight of 16-18 g. After 1 week of adaptive feeding of the mice, the 12 mice were randomly divided into 4 groups (3 mice in each group): control group (no intervention), model group (DSS-IBD group), group of mice intervened with the commercially available Lactobacillus plantarum strain WCFS1 (WCFS1 group), and group of mice intervened with strain PL-H1 (PL-H1 group).

[0068] (2) Animal modeling and intervention methods: Control group (no intervention), the model group, WCFS1 group, and PL-H1 group established an IBD disease model by inducing colitis in mice with 3% DSS. During the whole experiment, the control group and the model group of rats were fed with normal feed + sterile water from the start to the end of the experiment, and each probiotic intervention group was fed with normal feed + purified water containing the bacterial suspension (the number of intervening strains was 2×10 8 CFU / mouse / day).

[0069] (3) Animal Observation Indicators and Analysis

[0070] (3.1) Mouse body weight detection: By accurately weighing the mouse's own weight, the quality change of the IBD mouse model is evaluated.

[0071] The results are as Figure 3 shown in Figure A. As can be seen from the figure, compared with the control group, the quality of the mice in the model group was significantly reduced. These changes reflect the physiological state of the IBD mouse model. After the intervention of each group of probiotics, especially the PL-H1 group, the weight of the mice increased significantly. The increase in the weight of the mice indicates that the probiotic intervention promoted the recovery or growth of the intestinal tissue of the IBD model, improved its structure and function, indicating that Lactobacillus plantarum PL-H1 can restore the healthy state of the digestive tract.

[0072] (3.2) Mouse spleen index detection: By dissecting and accurately weighing the mouse's own weight and spleen weight, the weight index of the mouse spleen is calculated, that is, the organ weight (mg) divided by the body weight (mg), to evaluate the quality change of the spleen.

[0073] The results are as Figure 3 shown in Figure B. As can be seen from the figure, compared with the control group, the spleen weight index of the mice in the model group increased significantly. The increase in the spleen index may indicate that the immune system of the mice is active or there is splenomegaly. These changes reflect the physiological state in which the immune system is damaged in the IBD mouse model. After the intervention of each group of probiotics, especially the PL-H1 group, the spleen index of the mice decreased significantly. The decrease in the spleen index of the mice indicates that the probiotic intervention promoted the recovery or growth of the spleen, improved its structure and function, indicating that Lactobacillus plantarum PL-H1 can restore the function of the immune system.

[0074] (3.3) Mouse disease activity index detection: The Disease Activity Index (DAI) is an important indicator for evaluating the severity of DSS-induced colitis in mice. By adding the scores of the three indicators of mouse body weight change, fecal occult blood or gross hematuria, and fecal morphology (diarrhea condition), the obtained score is the DAI.

[0075] As shown in Figure 3 Figure C, compared with the control group, the DAI score of the model group increased significantly. The significant increase in the DAI score indicates the severity of DSS-induced colitis, including the occurrence of inflammatory reactions and intestinal barrier damage. After the intervention of each group of probiotics, especially the PL-H1 group, the DAI score of the mice decreased significantly. This indicates that the probiotic intervention has a positive effect on restoring the healthy state of the digestive tract. The significant decrease in the DAI score reflects that Lactobacillus plantarum PL-H1 inhibits intestinal inflammatory reactions and repairs intestinal barrier damage by regulating the intestinal microbiota.

[0076] (3.4) Mouse colon length detection: By dissecting and precisely measuring the mouse colon length, the length changes of the mouse colon were evaluated.

[0077] The results are as Figure 3 shown in Figure D. It can be seen from the figure that compared with the control group, the colon length of the mice in the model group was significantly shortened. The shortening of the mouse colon length may indicate severe inflammation in the mouse intestine. These changes reflect the physiological state of the damaged immune system in the IBD mouse model. After the intervention of various probiotics, especially the PL-H1 group, the colon length of the mice increased significantly, indicating that the probiotic intervention promoted the recovery or growth of intestinal tissues, improved their structure and function, suggesting that the application of Lactobacillus plantarum PL-H1 can restore the function of intestinal tissues.

[0078] (3.5) Histopathological examination of mouse colon tissue: At the end of the experiment, the mice were dissected, and the colon tissues were taken. The colon tissues were cut open and longitudinally unfolded along the intestinal tract. After being rinsed clean, they were fixed in 4% paraformaldehyde solution. After dehydration, clearing, and wax infiltration, they were embedded. The embedded tissues were cut into continuous sections with a thickness of 4-5 μm. Histopathological scoring of inflammatory cell infiltration and tissue damage was performed by H&E staining (Hematoxylin and Eosin) to evaluate tissue structure, inflammatory cell infiltration, ulcer formation, crypt structure damage, etc.

[0079] As Figure 4 shown in Figure A, the pathological score of the mice in the model group increased significantly, and the morphological structure of the intestinal tract tissue changed, indicating the severity of DSS-induced colitis, including the occurrence of inflammatory reactions and intestinal barrier damage; among the mice in the probiotic intervention group, especially the mice in the PL-H1 group, the pathological score decreased significantly, indicating that the intervention of Lactobacillus plantarum PL-H1 can restore the function of the intestinal tissues in IBD mice.

[0080] (3.6) Detection of genes related to mouse colonic mucosal barrier and inflammation: Collect mouse colonic tissues, wash them with PBS to remove residues, then isolate the mucosal tissues and immediately place them in RNA protective solution and store at -80°C. When extracting RNA, Trizol reagent or commercial kits can be used, and DNase I is added during the extraction process to digest and remove genomic DNA contamination. The extracted RNA needs to be detected for purity (the ratio of A260 / A280 is between 1.8 - 2.0) by NanoDrop and confirmed for integrity (clear 28S and 18S bands) by agarose gel electrophoresis. Reverse transcribe the extracted RNA into cDNA using a high-efficiency reverse transcription kit. Random primers or oligo(dT) primers are recommended for the reaction. React at 42°C for 30 - 60 minutes, and then inactivate the enzyme at 85°C. Specific primers (such as ZO-1 mucosal barrier-related gene and IL-6 inflammation-related gene) need to be designed for qPCR detection, and GAPDH is recommended as the reference gene. The qPCR reaction system contains cDNA template, SYBR Green dye, and primers. The reaction conditions are usually set as pre-denaturation at 95°C for 2 minutes, followed by 40 cycles of amplification with 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. The 2 -ΔΔCt -method is used to calculate the relative expression level of the target gene.

[0081] As Figure 4 shown in B-C, ZO-1 in the model group decreased significantly ( Figure 4 B), and IL-6 increased significantly ( Figure 4 C), indicating the activation of the inflammatory response and the destruction of the tissue barrier in DSS-induced colitis. After intervention with various probiotics, especially in the PL-H1 group, the expression level of ZO-1 in mice increased significantly, and IL-6 decreased significantly, indicating that the intervention of Lactobacillus plantarum PL-H1 has a positive effect on restoring the integrity of intestinal tissues and reducing the inflammatory response in mice.

[0082] Example 3: Detection of the L-tryptophan-producing ability of Lactobacillus plantarum PL-H1

[0083] (1) Strain activation: Take the original glycerol tubes of PL-H1 and the standard strain WCFS1, thaw them at room temperature, aspirate 0.1 mL of the bacterial suspension and inoculate it onto MRS solid medium, spread it evenly, and place the petri dish in an incubator at 37°C for 48 h to obtain well-activated single colonies.

[0084] (2) Preparation of Lactobacillus cells and cell suspensions: Take the activated single colony, scrape a loop of the colony with an inoculation loop, and then inoculate it into a 250 mL Erlenmeyer flask containing 100 mL of MRS liquid medium. The pH of the MRS medium was adjusted to 7.0 ± 0.2 before sterilization, and the sterilization conditions were 121 °C for 30 min. Take 50 mL of the bacterial solution, centrifuge it at 3500 rpm / min for 15 min, and collect the supernatant and cells for transcriptome and metabolomics detection.

[0085] (3) Transcriptomics detection of the expression levels of tryptophan metabolism-related genes in PL-H1 cells: After RNA extraction, purification, and library construction of the cell samples, paired-end sequencing of the library was performed based on the Illumina HiSeq sequencing platform. The raw data was saved in FASTQ format, and FastQC was used to perform quality detection and data filtering on the downloaded data. The reference genome index was established through Bowtie2 (http: / / bowtie-bio.sourceforge.net / index.shtml), and the high-quality sequencing data after quality control analysis was aligned to the reference genome of Lactobacillus plantarum (GenBank accession number: GCA_000203855.ASM20385v3). Quantitative analysis of gene expression levels in each sample was performed through the HTSeq software (http: / / www-huber.embl.de / users / anders / HTSeq), and then differential analysis of gene expression was performed using DESeq (V1.18.0). The conditions for screening differentially expressed genes were: Log2|fold change| of expression fold difference > 1 and significant P < 0.05. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the number of differentially expressed genes included in different levels of each KEGG pathway was counted, and then the main metabolic pathways and signaling pathways involved in the differentially expressed genes were determined through GSEA;

[0086] The results are as Figure 5 shown. The GSEA analysis results of the strain's metabolic pathway showed that the phenylalanine-tyrosine-tryptophan biosynthesis pathway of Lactobacillus plantarum PL-H1 was significantly upregulated ( Figure 5 A); the expression level of the tryptophan synthase of the strain showed that the expression level of the tryptophan synthase of Lactobacillus plantarum PL-H1 was significantly higher than that of the standard strain WCFS1 ( Figure 5 B).

[0087] (4) Metabolomics detection of tryptophan content in PL-H1 bacteria and supernatant: Sample preparation and analysis conditions for liquid chromatography-mass spectrometry. Thaw the freeze-dried samples and the supernatant. The bacteria were subjected to repeated freeze-thaw cycles to obtain bacterial lysates. For extraction, an extraction agent (150 μL; acetonitrile: methanol = 1:4, V / V) containing the internal standard 2-chloro-L-phenylalanine was mixed with 50 μL of the sample. The mixture was vortexed for 3 minutes and then centrifuged at 12,000×g for 10 minutes at 4 °C. The supernatant was collected, stored at 20 °C for 30 minutes, and then subjected to another centrifugation at 12,000×g for 3 minutes (4 °C). Then the supernatant was collected, filtered through a 0.22 μm pore-size membrane on a laminar flow bench, and transferred to a new sample vial for subsequent analysis (Yu et al., 2021). The chromatographic column was a Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm). The mobile phase consisted of different proportions of acetonitrile and 0.1% formic acid (SigmaAldrich, St. Louis, MO, USA) and ultrapure water and 0.1% formic acid. The elution gradient started from 5% acetonitrile, held for 11 min, and then linearly increased to 90% acetonitrile. The mobile phase was held at 90% acetonitrile for 1 min and then returned to 5%. The column was equilibrated with 5% acetonitrile for 1.9 minutes before the next injection. The column temperature was 40 °C, the column temperature was stable, and the repeatability between batches was good.

[0088] The results are as Figure 6 shown, the tryptophan metabolism ability in PL-H1 bacteria and supernatant increased, and the tryptophan content in the supernatant was higher than that in the bacteria ( Figure 6 A); moreover, the metabolites of the tryptophan indole pathway in PL-H1 bacteria increased. Among them, the contents of indole lactic acid (ILA), indole-3-acetaldehyde (IAId), and indole pyruvic acid were less than those in the supernatant, but tryptamine, indole-3-acetic acid, and indole-3-acrylic acid (IA) were only present in the bacteria ( Figure 6 B).

[0089] Example 4: Application of Lactobacillus plantarum PL-H1 in preventing, alleviating or treating liver lipid metabolism disorders and their related diseases (such as non-alcoholic fatty liver, etc.)

[0090] Experimental method:

[0091] 1. Animals and model making method:

[0092] Experimental animals: A total of 18 female C57BL / 6 mice at 2 months of age (purchased from the Experimental Animal Department of Harbin Medical University), with a body weight of 16 - 18 g. Model making method: All mice were randomly divided into 3 groups, including the Control group, the MCD group, and the MCD + PL-H1 group. Mice in the Control group were given a control diet, mice in the MCD group were given an MCD diet (methionine- and choline-deficient feed), and mice in the MCD + PL-H1 group were given an MCD diet and intragastrically administered 200 μl of PL-H1 bacterial solution per mouse per day. The modeling period was 4 weeks.

[0093] 2. Lactobacillus plantarum PL-H1 and its feeding:

[0094] Preparation of bacterial medium, MRS agar medium: Add 13.06 g of MRS broth and 3.5 g of agar to 250 ml of sterile deionized water, mix well, adjust the pH value to 6.5 ± 0.2, sterilize at 121 °C under high pressure for 15 minutes, pour into a bacterial culture dish, and store at 4 °C after cooling.

[0095] MRS broth medium: Add 13.06 g of MRS broth to 250 ml of sterile deionized water, mix well, adjust the pH value to 5.7 ± 0.2, sterilize at 118 °C under high pressure for 15 minutes, and store at 4 °C;

[0096] Inoculation and culture of bacteria. Resuscitate the Lactobacillus plantarum PL-H1 bacterial solution stored at -80 °C, inoculate PL-H1 on MRS agar medium using the plate three-segment streaking method, anaerobically culture at 37 °C for 24 - 48 hours, pick a single colony and add it to MRS broth medium, and culture on a shaker at 37 °C. Measure the OD value of the bacterial solution at a wavelength of 600 nm every 1 hour to obtain the growth curve of PL-H1. According to the McFarland turbidity standard, when OD600 = 1.0, the bacterial concentration is approximately 1×10 9 CFU / ml. Take 1 ml of the bacterial solution and centrifuge for 15 minutes at 10,000 revolutions per minute, wash 3 times with sterile PBS, and resuspend in 200 μl of sterile PBS to obtain the PL-H1 bacterial solution.

[0097] 3. Histological observation of fatty liver:

[0098] 3.1 H&E staining:

[0099] (1) Dewaxing: Preheat the paraffin section at 68 °C for 10 minutes, immerse it in xylene for 10 minutes 3 times in sequence, absolute ethanol for 10 minutes, gradient ethanol (95%, 90%, 85%, 80%, 75%) for 5 minutes each, and wash with distilled water for 1 minute.

[0100] (2) Hematoxylin staining: Immerse the section in hematoxylin staining solution for 10 minutes and rinse with running water for 10 minutes.

[0101] (3) Eosin staining: Immerse the section in eosin staining solution for 10 minutes.

[0102] (4) Dehydration: Immerse the section in gradient ethanol (75%, 80%, 85%, 90%, 95%) for 30 seconds each in sequence, anhydrous ethanol for 5 minutes twice, and xylene for 5 minutes twice.

[0103] (5) Sealing the slide: Seal the slide with neutral resin, bake the slide at 60 °C for 1 day, and observe and take pictures using a stereomicroscope.

[0104] 3.2 Oil Red O staining

[0105] (1) Fixation: For frozen sections, it is recommended that the thickness be 10 μm. Do not fix or fix with 10% formalin for 30 minutes, and wash twice with distilled water for 3 minutes each time.

[0106] (2) Immerse the section in 60% isopropanol for 20 - 30 s for washing.

[0107] (3) Oil Red O staining: Immerse the section in the modified Oil Red O staining solution (cover with a lid), and stain in a sealed state for 10 - 15 minutes.

[0108] (4) Differentiation: Immerse in 60% isopropanol for a short wash for 5 - 10 s to remove the staining solution, and wash with distilled water for 1 minute.

[0109] (5) Hematoxylin staining: Drop Mayer hematoxylin staining solution to counterstain the nucleus for 1 - 2 minutes.

[0110] (6) Blueing: Rinse with tap water for 10 minutes or with dilute lithium carbonate solution for 3 minutes for blueing. Slightly wash in distilled water, and blot the surrounding moisture with filter paper.

[0111] (7) Sealing the slide: Seal the slide with glycerol gelatin, and observe and take pictures using a stereomicroscope.

[0112] 3.3 Masson staining

[0113] (1) Deparaffinization: Preheat the paraffin section at 68 °C for 10 minutes, immerse it in xylene for 10 minutes three times in sequence, anhydrous ethanol for 10 minutes, gradient ethanol (95%, 90%, 85%, 80%, 75%) for 5 minutes each, and wash with distilled water for 1 minute.

[0114] (2) Hematoxylin staining: Drop iron hematoxylin staining solution to cover and stain the section for 5 - 10 minutes. Wash away the excess staining solution with distilled water, drop acidic differentiation solution for differentiation for 5 - 15 s, and wash with distilled water for 30 s.

[0115] (3) Drop Masson bluing solution for bluing for 3 - 5 minutes, and wash with distilled water for 30 s.

[0116] (4) Add ponceau magenta staining solution and stain for 5 - 10 min, then add weak acid working solution and wash for 30 s.

[0117] (5) Add phosphomolybdic acid solution and treat for 1 - 2 min, then add weak acid working solution and wash for 30 s.

[0118] (6) Add aniline blue staining solution and stain for 1 - 2 min, then add weak acid working solution and wash for 30 s.

[0119] (7) Dehydrate rapidly with 95% ethanol for 2 - 3 s, dehydrate with absolute ethanol twice, 5 - 10 s each time.

[0120] (8) Clear with xylene twice, 1 - 2 min each time, seal with neutral gum, bake the slides at 60 °C for 1 day, observe and photograph using a stereomicroscope.

[0121] 3.4 Immunofluorescence staining

[0122] (1) Deparaffinization: Preheat the paraffin sections at 60 °C for 10 minutes, then immerse them successively in xylene at 60 °C for 10 minutes, xylene at room temperature for 10 minutes, and gradient ethanol (100%, 95%, 70%, 50%, 30%) for 5 minutes each, and wash with deionized water for 30 s twice.

[0123] (2) Antigen retrieval: Immerse the sections in citrate buffer at 98 °C for 30 minutes, cool naturally to room temperature, wash with PBS for 3 minutes three times, and circle the tissue with an immunohistochemistry pen.

[0124] (3) Blocking: Drop 5% fetal bovine serum on the tissue and incubate in a humidified box at room temperature in the dark for 30 minutes.

[0125] (4) Primary antibody incubation: Drop the diluted primary antibody on the tissue and incubate in a humidified box at 4 °C overnight.

[0126] (5) Secondary antibody incubation: After washing with PBS for 5 minutes three times, drop the diluted secondary antibody on the tissue and incubate in a humidified box at room temperature in the dark for 1.5 hours.

[0127] (6) DAPI staining: After washing with PBS for 5 minutes three times, drop DAPI staining solution on the tissue and incubate in the dark for 3 minutes.

[0128] (7) Mounting: Wash with PBS for 5 minutes three times, wait for the tissue to dry, then drop anti - quenching mounting medium to mount the slides, and observe and photograph using a confocal microscope or fluorescence microscope.

[0129] 4. Analysis and detection of biochemical indicators in the serum of fatty hepatitis:

[0130] For the determination of total triglyceride (TG), total cholesterol (TCHO), total bile acid (TBA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in serum, the operations were performed according to the instructions of the kits provided by Nanjing Jiancheng Bioengineering Institute.

[0131] 5. Gene detection (PCR) and protein detection (WB) in liver and colon tissues:

[0132] 5.1 Extraction of total RNA from liver and colon tissues:

[0133] (1) Take 0.05 - 0.1 g of frozen intestinal tissue and place it in a 1.5 ml EP tube. First, add 150 μl of Trizol, cut the tissue into pieces, and then add 350 μl of Trizol and grind thoroughly.

[0134] (2) Add 200 μl of chloroform, shake well for 15 seconds, and let it stand at room temperature for 10 minutes.

[0135] (3) Centrifuge at 12,000 r / min at 4°C for 10 minutes, and take the colorless supernatant.

[0136] (4) Add 400 μl of isopropanol, invert and mix well, and let it stand at room temperature for 10 minutes.

[0137] (5) Centrifuge at 12,000 r / min at 4°C for 10 minutes, discard the supernatant (pour it directly, do not discard the precipitate).

[0138] (6) Add 1 ml of 75% ethanol (function: wash), vortex mix or pipette up and down.

[0139] (7) Centrifuge at 12,000 r / min at 4°C for 3 minutes, discard the supernatant, and let it stand at room temperature to dry for 15 - 20 minutes.

[0140] (8) Add 30 μl of RNase-Free Water to dissolve the RNA and store it at -80°C.

[0141] 5.2 Reverse transcription:

[0142] Using the total RNA from intestinal tissue as a template, a reverse transcription kit was used. According to the instructions, the components required for the reverse transcription reaction were added, centrifuged and mixed well, and then loaded onto the machine; VRNA = 1000 (ng) / Concentration RNA (ng / μl), unit: μl.

[0143] 5.3 Real-Time PCR:

[0144] Using the cDNA of liver and colon tissues as templates, add the components required for PCR according to the instructions of the PCR kit, centrifuge and mix well, and perform amplification detection in a Real-Time PCR instrument.

[0145] 5.4 Western Blot (WB)

[0146] (1) Preparation of tissue protein samples: Longitudinally cut the liver and colon tissues of mice, wash the intestinal contents with pre-cooled sterile PBS, take 0.5 cm and put it into a premixed solution of 300 μl RIPA, PMSF and phosphatase inhibitor (60:1:2). After cutting and grinding the tissue, sonicate for 5 seconds and pause for 15 seconds (150 w, 20 kHz) and repeat 4 times. After standing on ice for 30 minutes, centrifuge at 12,000 rpm for 15 minutes and take the supernatant as the tissue protein sample, and detect the protein concentration of the sample using the BCA method. Dilute the sample according to the protein concentration of 50 μg / 10 μl, add protein loading buffer in a ratio of 4:1, mix well and denature at 100 °C for 10 minutes.

[0147] (2) Preparation of SDS-PAGE gel (unit: ml): Prepare the separating gel and stacking gel according to the following ratio, insert the comb and wait for natural solidification.

[0148]

[0149] (3) Electrophoresis: Place the gel in the electrophoresis tank and pour in the rapid electrophoresis solution, carefully pull out the comb, slowly add 10 μl of protein sample and protein Marker into the gel wells, set the electrophoresis instrument to constant voltage 200 mV for 30 minutes, and stop electrophoresis before the protein reaches the bottom.

[0150] (4) Transfer membrane: Take out the electrophoresed gel and place it on a moist sponge and filter paper, cover a properly sized methanol-activated PVDF membrane, put it into the transfer membrane clip and insert it into the transfer membrane tank filled with pre-cooled rapid transfer membrane solution according to the electrode direction, set the electrophoresis instrument to constant current 400 mA for 30 minutes.

[0151] (5) Blocking and incubation with primary antibody: Take out the PVDF membrane containing protein and put it into the rapid blocking solution, incubate on a shaker at room temperature for 30 minutes. After washing with TBST, put it into the diluted primary antibody and incubate on a shaker at 4 °C overnight.

[0152] (6) Incubation with secondary antibody and development: Wash the PVDF membrane in TBST for 10 minutes 3 times, add the diluted secondary antibody, and incubate at room temperature for 1.5 hours. After washing 3 times with TBST, add the chemiluminescent developing solution, develop and take pictures in a multifunctional imager, and use ImageJ to calculate the band density.

[0153] 6. Mucosal barrier function of mouse intestinal tissue:

[0154] 6.1 Alcian blue-PAS staining

[0155] (1) Deparaffinization: Preheat the paraffin section at 68 °C for 10 minutes, then immerse it in xylene for 10 minutes three times successively, in absolute ethanol for 10 minutes, in gradient ethanol (95%, 90%, 85%, 80%, 75%) for 5 minutes each, and wash it with distilled water for 1 minute.

[0156] (2) Alcian blue staining: Immerse the section in 3% acetic acid solution for 2 minutes, in Alcian blue staining solution for 10 minutes, rinse it with running water for 2 minutes, and wash it with distilled water for 30 seconds twice.

[0157] (3) Schiff staining: Immerse the section in periodic acid for 5 minutes, wash it with distilled water for 30 seconds twice, immerse it in Schiff solution for 10 minutes, and rinse it with running water for 2 minutes.

[0158] (4) Hematoxylin staining: Immerse the section in hematoxylin staining solution for 8 minutes, and rinse it with running water for 10 minutes.

[0159] (5) Dehydration: Immerse the section in gradient ethanol (75%, 80%, 85%, 90%, 95%) for 5 minutes each, in absolute ethanol for 5 minutes twice, and in xylene for 5 minutes twice.

[0160] (6) Sealing: Seal the section with neutral resin, bake the section at 60 °C for 1 day, and observe and photograph it using a stereomicroscope.

[0161] 6.2 ELISA

[0162] Operate according to the instruction manual of Jianglai ELISA kit.

[0163] (1) Serum preparation: Let the whole blood sample of mice stand at room temperature for 30 minutes, centrifuge it at 1000×g for 20 minutes to obtain the supernatant, and get the serum sample.

[0164] (2) Biotinylated antibody labeling: Add 100 μl of the sample, standard, and universal diluent (blank control) to the wells to be tested coated with antibodies respectively, incubate at 37 °C for 60 minutes. Discard the liquid, add 100 μl of biotinylated antibody working solution to each well, incubate at 37 °C for 60 minutes. Discard the liquid, add 300 μl of 1× washing solution to each well, and wash the plate 3 times.

[0165] (3) HRP enzyme binding: Add 100 μl of enzyme conjugate working solution to each well, incubate at 37 °C for 30 minutes, and wash the plate 5 times.

[0166] (4) TMB substrate color development: Add 90 μl of substrate to each well, cover it with a sealing film, incubate at 37 °C for 30 minutes, add 50 μl of stop solution, and immediately measure the OD value of each well at a wavelength of 450 nm.

[0167] (5) Data analysis: After subtracting the OD value of the blank well from the OD value of the sample well, the concentration of the protein to be measured in the sample is obtained from the standard curve drawn by subtracting the OD value of the blank well from the OD value of the standard well.

[0168] Results:

[0169] 1. The results are as Figure 7 shown in A-I. Lactobacillus plantarum PL-H1 improves liver lipid accumulation and non-alcoholic fatty liver disease (NAFLD) activity in mice fed a methionine-choline-deficient (MCD) diet.

[0170] 2. The results are as Figure 8 shown in A-H. Lactobacillus plantarum PL-H1 improves liver damage and serum lipid distribution in mice fed an MCD diet.

[0171] 3. The results are as Figure 9 shown in A-G. Lactobacillus plantarum PL-H1 can prevent the occurrence of liver fibrosis in mice fed an MCD diet.

[0172] 4. The results are as Figure 10 shown in A-R. Lactobacillus plantarum PL-H1 improves liver immune-related indexes in mice fed an MCD diet.

[0173] 5. The results are as Figure 11A-I and Figure 11J-N shown. Lactobacillus plantarum PL-H1 improves the impaired intestinal barrier function in mice fed an MCD diet by regulating tight junctions.

[0174] Example 5: Experiment on the anti-Candida albicans effect of Lactobacillus plantarum PL_H1

[0175] 1. Experimental method:

[0176] 1.1 Strains, culture media and culture conditions

[0177] (1) Lactobacillus: Take out 50 μl of the frozen stock solution of Lactobacillus plantarum PL_H1 or Lactobacillus delbrueckii subsp. bulgaricus stored in a -80 °C refrigerator, and inoculate it into 5 mL of MRS medium. Culture it in a constant temperature incubator at 37 °C and 170 rpm for 24 hours. After the culture is completed, activate it again according to the above operation to keep Lactobacillus plantarum PL_H1 and Lactobacillus delbrueckii subsp. bulgaricus in good growth state.

[0178] (2) Candida albicans: Take out the Candida albicans cryopreservation solution stored in the -80°C refrigerator. Use a sterilized inoculation loop to pick up an appropriate amount of Candida albicans and inoculate it in yeast extract peptone dextrose agar medium. Incubate it statically in a 37°C constant temperature incubator for 24 - 48 hours. After the cultivation is completed, activate it again according to the above operation to keep Candida albicans in a good growth state.

[0179] 1.2 Counting of Lactobacillus and Candida albicans

[0180] In this study, Lactobacillus plantarum PL_H1 and Lactobacillus delbrueckii subsp. bulgaricus were respectively selected for co-culture with Candida albicans. Before co-culture, the counting plate method was used to directly count Lactobacillus and Candida albicans through an optical microscope. After co-culture, Candida albicans was inoculated in yeast extract peptone dextrose agar medium for 24 - 48 hours, and then the colony counting was carried out by the dilution plate coating method.

[0181] (1) Counting plate method: Lactobacillus was diluted with MRS liquid medium and Candida albicans was diluted with YPD medium by 10-fold gradients respectively. The dilution multiples were 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 in turn. Use a pipette to suck a certain amount of the diluted bacterial suspension and add it to the counting pool of the counting plate. The volume of the counting pool is 0.1 mm3. Calculate the concentration of the bacterial suspension by counting the number of Lactobacillus or Candida albicans under the microscope, and then use it for the co-culture experiment. Calculate the amount of bacteria through the following formula:

[0182] Colony number per milliliter = (total number of bacteria in the counting pool × dilution multiple) / volume of the counting pool

[0183] (2) Dilution plate coating method: Candida albicans and Lactobacillus in the control group and co-culture group were diluted with PBS buffer by 10-fold gradients. The dilution multiples were 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 in turn. Use a pipette to suck a certain amount of the diluted bacterial suspension and put it into a sterile petri dish in turn. Spread the bacterial suspension in the sterile petri dish evenly. Three duplicate plates of the same kind will be set for each different dilution degree. Place the plates in a 37°C constant temperature incubator for cultivation. Take them out after 24 - 48 hours and count the number of colonies on each plate. Each single colony represents a fungus in the original sample. Count Candida albicans and Lactobacillus through the following formula:

[0184] Colony forming units per milliliter (CFU / mL) = (number of colonies on the plate × dilution multiple) / sampling volume of the bacterial suspension

[0185] 1.3 Analysis of the effect of Candida albicans on the growth of Lactobacillus by liquid co-culture method

[0186] Candida albicans was cultured on yeast extract peptone dextrose agar medium, then scraped off from the medium with an inoculation loop, and resuspended to 10 8 CFU / mL in YPD liquid medium by the counting plate counting method; two Lactobacillus strains were cultured in MRS liquid medium and resuspended to 10 8 CFU / mL respectively in MRS liquid medium by the counting plate counting method;

[0187] Control group: Take a 15 mL centrifuge tube and add 1 mL of 10 8 CFU / mL Candida albicans, 2.5 mL of MRS liquid medium, and 1.5 mL of YPD liquid medium into it;

[0188] 10 8 CFU / mL Lactobacillus plantarum group: Take a 15 mL centrifuge tube and add 1 mL of 10 8 CFU / mL Lactobacillus plantarum PL_H1, 1 mL of 10 8 CFU / mL Candida albicans, 1.5 mL of MRS liquid medium, and 1.5 mL of YPD liquid medium into it;

[0189] 10 8 CFU / mL Lactobacillus delbrueckii subsp. bulgaricus group: Take a 15 mL centrifuge tube and add 1 mL of 10 8 CFU / mL Lactobacillus delbrueckii subsp. bulgaricus, 1 mL of 10 8 CFU / mL Candida albicans, 1.5 mL of MRS liquid medium, and 1.5 mL of YPD liquid medium into it;

[0190] Prepare 3 replicates of each of the above groups and incubate them at 37°C. Take one sample at 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, and 72 hours of incubation. Inoculate the control group and the Lactobacillus plantarum and Lactobacillus delbrueckii subsp. bulgaricus after co-culture onto MRS agar medium by the dilution plate coating method, and perform colony counting and plot the growth curves of the two Lactobacillus strains after incubation at 37°C for 24 - 48 hours.

[0191] 1.4 Analysis of the effect of Lactobacillus on the growth of Candida albicans by liquid co-culture method

[0192] (1) Candida albicans was cultured on yeast extract peptone dextrose agar medium, then scraped off from the medium with an inoculation loop, and resuspended to 10 3 CFU / mL in YPD liquid medium by the counting plate counting method; two Lactobacillus strains were cultured in MRS liquid medium and resuspended to 10 3 CFU / mL and 10 5CFU / mL, 10 8 CFU / mL;

[0193] (2) Control group: Take a 15 mL centrifuge tube and add 10 3 CFU / mL of Candida albicans 1 ml, MRS liquid medium 2.5 ml, and YPD liquid medium 1.5 ml;

[0194] (3) 10 3 CFU / mL Lactobacillus plantarum group: Take a 15 mL centrifuge tube and add 10 3 CFU / mL of Lactobacillus plantarum 1 ml, 10 3 CFU / mL of Candida albicans 1 ml, MRS liquid medium 1.5 ml, and YPD liquid medium 1.5 ml;

[0195] (4) 10 5 CFU / mL Lactobacillus plantarum group: Take a 15 mL centrifuge tube and add 10 5 CFU / mL of Lactobacillus plantarum PL_H1 1 ml, 10 3 CFU / mL of Candida albicans 1 ml, MRS liquid medium 1.5 ml, and YPD liquid medium 1.5 ml;

[0196] (5) 10 8 CFU / mL Lactobacillus plantarum group: Take a 15 mL centrifuge tube and add 10 8 CFU / mL of Lactobacillus plantarum 1 ml, 10 3 CFU / mL of Candida albicans 1 ml, MRS liquid medium 1.5 ml, and YPD liquid medium 1.5 ml;

[0197] (6) 10 3 CFU / mL Lactobacillus delbrueckii subsp. bulgaricus group: Take a 15 mL centrifuge tube and add 10 3 CFU / mL of Lactobacillus delbrueckii 1 ml, 10 3 CFU / mL of Candida albicans 1 ml, MRS liquid medium 1.5 ml, and YPD liquid medium 1.5 ml;

[0198] (7) 10 5 CFU / mL Lactobacillus delbrueckii subsp. bulgaricus group: Take a 15 mL centrifuge tube and add 105 CFU / mL of Lactobacillus delbrueckii 1 ml, 10 3 CFU / mL of Candida albicans 1 ml, MRS liquid medium 1.5 ml, and YPD liquid medium 1.5 ml;

[0199] (8) 10 8CFU / mL Lactobacillus delbrueckii subsp. bulgaricus group: Take a 15 mL centrifuge tube and add 10 8 CFU / mL of Lactobacillus delbrueckii 1 mL, 10 3 CFU / mL of Candida albicans 1 mL, 1.5 mL of MRS liquid medium, 1.5 mL of YPD liquid medium;

[0200] (9) Prepare 3 replicates of each group and incubate them at 37°C. Take one sample each at 12 hours, 24 hours, and 48 hours of incubation. Inoculate the control group and the Candida albicans after co-culture onto yeast extract peptone dextrose agar medium by the dilution plate coating method, and perform statistical analysis after incubating at 37°C for 24 - 48 hours. Count the colonies on the YPD medium supplemented with 1.5 mg / mL chloramphenicol to eliminate the influence of lactobacilli on the counting.

[0201] Results:

[0202] 1. Lactobacillus plantarum PL-H1 can inhibit the growth of Candida albicans

[0203] In this study, 10 3 CFU / mL, 10 5 CFU / mL, 10 8 CFU / mL of Lactobacillus plantarum and 10 3 CFU / mL of Candida albicans were co-cultured at 37°C for 12 hours, 24 hours, and 48 hours. A control group of Candida albicans was inoculated into a mixed medium of YPD and MRS, with the environmental conditions the same as those of the co-culture group. Subsequently, the Candida albicans in the treatment group and the control group were inoculated onto YPD agar medium containing 1 mg / mL chloramphenicol by the dilution plate coating method, and colony counting was performed after incubating at 37°C for 24 - 48 hours.

[0204] The results are as Figure 12 shown. The colony counting results indicate that after co-culturing Lactobacillus plantarum PL-H1 with Candida albicans for 12 hours, compared with the 10 3 CFU / mL Candida albicans control group, 10 5 CFU / mL of Lactobacillus plantarum PL-H1 (P < 0.01), 10 8 CFU / mL of Lactobacillus plantarum PL-H1 (P < 0.0001) had antibacterial effects on 10 3 CFU / mL of Candida albicans. After co-culturing for 24 hours, compared with the 10 3 CFU / mL Candida albicans control group, 10 3 CFU / mL of Lactobacillus plantarum PL-H1 (P < 0.001), 10 5 CFU / mL of Lactobacillus plantarum PL-H1 (P < 0.0001), 108 10 CFU / mL Lactobacillus plantarum PL-H1 (P < 0.0001) has antibacterial activity against 10 3 CFU / mL Candida albicans. After 48 hours of co-culture, compared with the control group of 10 3 CFU / mL Candida albicans, 10 3 CFU / mL Lactobacillus plantarum PL-H1 (P < 0.001), 10 5 CFU / mL Lactobacillus plantarum PL-H1 (P < 0.0001), 10 8 CFU / mL Lactobacillus plantarum PL-H1 (P < 0.0001) has antibacterial activity against 10 3 CFU / mL Candida albicans. The above results suggest that Lactobacillus plantarum PL-H1 has the effect of inhibiting the growth of Candida albicans and shows time-dose dependence.

[0205] 2. Comparison of the inhibitory effects of Lactobacillus plantarum PL-H1 and Lactobacillus delbrueckii subsp. bulgaricus on the growth of Candida albicans

[0206] The antibacterial activities of 10 8 CFU / mL Lactobacillus plantarum PL-H1 and Lactobacillus delbrueckii subsp. bulgaricus at 12 hours, 24 hours, and 48 hours were compared by plotting a line graph. The results are as Figure 13 shown. It was observed that the inhibitory effect of 10 8 CFU / mL Lactobacillus plantarum PL-H1 on Candida albicans at 24 hours (P < 0.001) and 48 hours (P < 0.001) was stronger than that of 10 8 CFU / mL Lactobacillus delbrueckii subsp. bulgaricus. It is suggested that the inhibitory effect of Lactobacillus plantarum PL-H1 on the growth of Candida albicans is stronger than that of Lactobacillus delbrueckii subsp. bulgaricus when the co-culture time reaches 24 hours and 48 hours.

[0207] 3. Candida albicans does not affect the growth results of Lactobacillus plantarum PL-H1 and Lactobacillus delbrueckii subsp. bulgaricus

[0208] To clarify the effect of co-culture with Candida albicans on the growth of Lactobacillus plantarum and Lactobacillus delbrueckii subsp. bulgaricus, 10 8 CFU / mL of Lactobacillus plantarum and 10 8 CFU / mL of Lactobacillus delbrueckii subsp. bulgaricus were co-cultured with 10 8 CFU / mL Candida albicans. The colony counts of the two strains of lactobacilli were performed by the dilution plate coating method on MRS agar medium at 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, and 72 hours, respectively, and a line graph was plotted for comparison.

[0209] The results are asFigure 14 As shown, the results showed that Lactobacillus plantarum PL-H1 and Lactobacillus delbrueckii subsp. bulgaricus showed an increasing trend between 12 hours and 60 hours, and the colony count decreased between 60 hours and 72 hours due to factors such as cell death. Compared with the control group, the growth rates of Lactobacillus plantarum PL-H1 and Lactobacillus delbrueckii were not affected by co-culture with Candida albicans (P>0.05).

[0210] 4. Efficacy of Lactobacillus plantarum PL-H1 in treating murine vulvovaginal candidiasis (VVC) caused by Candida albicans

[0211] Based on the in vitro experiment of Lactobacillus plantarum PL-H1 against Candida albicans, a murine VVC model was established, and Lactobacillus plantarum PL-H1 (10 9 CFU / ml, for 1 week) was administered vaginally for experimental treatment to evaluate the efficacy of Lactobacillus plantarum in treating VVC. The results were as Figure 15 shown. The results showed that fungal hyphae could be detected in the vaginal lavage fluid of VVC model mice, and the fungal colony count increased compared with the control group (p<0.01) ( Figure 15 A); after treatment with Lactobacillus plantarum, changes such as a decrease in the number of neutrophils and a decrease in the fungal colony count were observed in the vaginal lavage fluid of VVC mice (p<0.01) ( Figure 15 B). The results suggest that Lactobacillus plantarum PL-H1 can significantly inhibit fungal proliferation and host inflammatory responses in the VVC vagina, playing a role in treating VVC.

Claims

1. A Lactobacillus plantarum strain isolated from healthy people in cold regions, named PL-H1, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 22286.

2. Use of the Lactobacillus plantarum strain according to claim 1 in the following aspects: (1) Use in the preparation of a drug for preventing, alleviating or treating inflammatory bowel disease and diseases related to tryptophan deficiency; (2) Use in the preparation of a drug for preventing, alleviating or treating abnormal liver lipid metabolism and its related diseases; (3) Use in the preparation of a drug for preventing, alleviating or treating diseases caused by fungal infections.

3. The application according to claim 2, characterized in that, The liver lipid metabolism disorder-related disease is non-alcoholic fatty liver.

4. The application according to claim 2, characterized in that, The fungus is Candida albicans.

5. The application according to claim 2, wherein The disease caused by fungal infection is vulvovaginal candidiasis.

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