A strain of Bifidobacterium longum GY2 with anti-hepatitis B virus function and its application

Abstract

The present invention discloses a strain of Bifidobacterium longum GY2 with anti-hepatitis B virus function and its application. The preservation number of the Bifidobacterium longum GY2 is: GDMCC No: 64873. The Bifidobacterium longum GY2 provided by the present invention can inhibit the replication of HBV DNA and the production of HBeAg and HBsAg both in vivo and in vitro, and can delay the occurrence of tumors in nude mice bearing tumors, and has a certain gastrointestinal fluid tolerance ability, can colonize in the intestine, effectively antagonize multiple pathogenic bacteria, is sensitive to multiple antibiotics, has no hemolytic activity, has good probiotic properties and safety, and has significant functions in vivo and in vitro, which has great application potential and utilization value in the development of functional foods or medicines for preventing and treating HBV infection and related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a lactic acid bacterium with anti-hepatitis B virus (HBV) function and application thereof. Background Art

[0002] In recent decades, increasing evidence has shown that hepatitis B virus (HBV) infection can lead to serious liver diseases such as liver failure, cirrhosis, hepatocellular carcinoma (HCC) or death.

[0003] Due to the lack of drugs with sustained virological response, the number of hepatitis B patients is increasing every year. At present, the treatment for HBV infection is mainly chemical antiviral treatment, among which interferon (IFN) and nucleoside analogues (NAS) are the main ones. IFN is a soluble glycoprotein with immunomodulatory function produced by various cells. It is an important antiviral cytokine and is divided into IFN-α and pegylated IFN-α. In clinical practice, IFN is the first choice of anti-HBV drug. Its advantages are fixed course of treatment, low resistance, high serum antigen conversion rate, and dual effects of anti-HBV and immune regulation. However, its high price, poor tolerance, long-term injection and easy to cause serious adverse reactions such as liver damage limit its wide application in clinical practice. In addition, nucleoside drugs are the most widely used anti-HBV drugs on the market. Clinically used anti-HBV nucleoside analogs include tenofovir alafenamide fumarate, adefovir, lamivudine (LAM), entecavir and telbivudine, etc. They have become a hot topic in the research of antiviral drugs in recent years due to their safety, effectiveness and ease of use. However, nucleoside analogs can only prevent viral replication, but cannot completely eliminate HBV and have no ability to remove HBV DNA. They are also prone to drug resistance and the therapeutic effect is not outstanding. Generally, the price of anti-HBV drugs is high, and patients need to take them for many years or even for life. The compliance of continuous anti-HBV medication is not high and they need to bear the inevitable side effects caused by the drugs. In addition, HBV patients have special immune functions and immune tolerance to HBV DNA, and their immune coordination functions have significant individual differences. Immunotherapy cannot completely eliminate HBV in the body. Therefore, it is very necessary and urgent to develop a new drug that has anti-HBV effect and is safe and has no side effects.

[0004] At present, the search for safe and effective functional lactic acid bacteria is gaining increasing attention and becoming one of the research hotspots. Lactic acid bacteria are a type of food-grade microorganisms that are generally recognized as safe (GRAS) with a long history of safe consumption. They have anti-inflammatory, antioxidant, immune regulation, antiviral and anti-tumor functions. Since the liver is directly connected to the intestine through the portal vein circulation, the pathogenesis of liver-related diseases is related to negative changes in the gut microbiota (GM). This connection study shows that lactic acid bacteria can restore the complexity and colonization resistance of intestinal bacteria, and reduce the risk of liver disease through multiple mechanisms such as regulating host GM, preventing disorder-related endotoxemia, maintaining intestinal epithelial barrier function, and inhibiting the entry of intestinal bacteria and pathogen-associated molecular patterns (PAMPs) into the systemic circulation. In addition, studies have found that lactic acid bacteria relieve liver oxidative stress by increasing the expression of antioxidant enzymes, promoting the growth of beneficial microorganisms, and producing anti-inflammatory metabolites; and upregulating the expression of tumor suppressor genes, inhibiting the expression of oncogenes in liver cancer; or converting non-nutritional dietary components such as proanthocyanidins into metabolites with anti-cancer effects. In addition, lactic acid bacteria can not only produce short-chain fatty acids (SCFA), but also promote the growth of SCFA-producing bacteria in GM. SCFA is known to regulate anti-inflammatory responses, regulate cell differentiation and proliferation; and protect intestinal epithelial function to prevent bacterial endotoxemia by limiting the entry of intestinal bacteria and their metabolites into the liver, such as increasing goblet cell mucus secretion and the ability to release antimicrobial peptides to protect the intestinal epithelium from pathogens. By feeding SCFA HBx Studies in transgenic (HBxTg) mice have demonstrated that SCFAs promote the expression of the tumor suppressor, anergy homeobox 2, and also reduce HBx The cell viability of the transfected cell line was significantly reduced, and the viability of human primary hepatocytes was not affected, which effectively delayed the progression of chronic liver disease. In addition, some scholars found that Bifidobacterium adolescentis ( Bifidobacterium adolescentis) The cell extract of SPM0212 reduced the extracellular HBsAg level by 50% in a dose-dependent manner, and its gene expression was also inhibited by 40%. At the same time, the expression level of IFN-induced anti-myxovirus A (Myxovirus resistance A) was significantly increased. With the deepening of research on lactic acid bacteria, many countries around the world have widely used it in many fields such as medicine, food, daily necessities, and animal husbandry, and its related products are even more dazzling. However, the antiviral research and application of lactic acid bacteria started late, especially in my country, and there is still a blank in the market for antiviral lactic acid bacteria products. Therefore, it is of great significance to screen functional lactic acid bacteria with antiviral effects on HBV and conduct in-depth research on anti-HBV mechanisms and related product development. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Bifidobacterium longum ( Bifidobacterium longum ) GY2, whose deposit number is: GDMCC No: 64873. The lactic acid bacteria GY2 of the present invention has a good effect of inhibiting HBV DNA replication in animals and has great potential in anti-HBV in humans.

[0006] The second object of the present invention is to provide the use of Bifidobacterium longum GY2 in the preparation of products against HBV or preventing and controlling pathogenic bacteria.

[0007] Preferably, the pathogenic bacteria include enterohemorrhagic Escherichia coli ( Enterohemorrhagic Escherichia coli )O157:H7, Yersinia enterocolitica ( Yersinia enterocolitica ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Escherichia coli ( Escherichia coli ), Salmonella typhimurium ( Salmonella Typhimurium )、Bacillus subtilis( Bacillus subtilis ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ) and Listeria monocytogenes ( Listeria monocytogenes ).

[0008] Preferably, the product comprises a medicine, a health product, a food or a dietary supplement.

[0009] The third object of the present invention is to provide a product for fighting HBV or preventing and controlling pathogenic bacteria, which contains Bifidobacterium longum GY2 or its fermentation culture or a processed product of the fermentation culture as an active ingredient.

[0010] Preferably, the processed product of the fermentation culture includes a concentrated solution of the fermentation culture or a dried product thereof, a filtrate of the fermentation culture, or a lysate of the fermentation culture.

[0011] The fourth object of the present invention is to provide a specific molecular target for detecting the Bifidobacterium longum GY2 according to claim 1, wherein the molecular target has a nucleotide sequence as shown in SEQ ID NO.1.

[0012] The fifth object of the present invention is to provide a primer set for the above-mentioned molecular target, which primer set includes a primer pair with nucleotide sequences as shown in SEQ ID NO.2-3 and a primer pair as shown in SEQ ID NO.4-5.

[0013] The sixth object of the present invention is to provide the use of the above-mentioned molecular target and primer set in detecting Bifidobacterium longum GY2.

[0014] The seventh object of the present invention is to provide a method for detecting Bifidobacterium longum GY2, comprising the following steps: extracting DNA of the sample microorganism to be tested, performing PCR amplification on it using the above-mentioned primer set, and subjecting the amplified product to gel electrophoresis. If two bands of 246 bp and 274 bp are obtained at the same time, it is determined that Bifidobacterium longum GY2 is detected in the sample; if two bands of 246 bp and 274 bp are not obtained at the same time, it is determined that Bifidobacterium longum GY2 is not detected in the sample.

[0015] Beneficial effects of the present invention:

[0016] The Bifidobacterium longum GY2 provided by the present invention is isolated from fecal samples of healthy people. It can inhibit the replication of HBV DNA and the production of HBeAg and HBsAg in vitro; at the same time, it has good safety performance; it can inhibit the replication of HBV DNA and the production of HBeAg and HBsAg in vivo, and can also delay the occurrence of tumors in tumor-bearing nude mice. The Bifidobacterium longum GY2 screened and provided by the present invention has both good probiotic properties and safety of probiotics, and has significant functions in vivo and in vitro, which has great application potential and utilization value in the development of functional foods or medicines for the prevention and treatment of HBV infection and related diseases.

[0017] Bifidobacterium longum ( Bifidobacterium longum ) GY2 was deposited on July 16, 2024 in the Guangdong Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 64873. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Phylogenetic tree of 16S rRNA gene of Bifidobacterium longum GY2.

[0019] Figure 2 :In vitro experimental diagram of Bifidobacterium longum GY2. (A) Cell viability; (B) Cell number; (C) Relative expression level of extracellular HBV DNA; (D) HBV DNA inhibition rate; (E) Extracellular HBeAg inhibition rate; (F) Extracellular HBsAg inhibition rate. Among them, the difference compared with the Control group, * indicates P< 0.01, ** indicates P< 0.001,*** indicates P< 0.0001.

[0020] Figure 3 : In vitro probiotic properties of Bifidobacterium longum GY2. (A) Tolerance to gastric and intestinal fluids; (B) Hydrophobicity; (C) Aggregation.

[0021] Figure 4 : Hemolytic activity of Bifidobacterium longum GY2.

[0022] Figure 5 : Experiment of subcutaneous ectopic implantation of Bifidobacterium longum GY2. (A) Changes in body weight of nude mice; (B) Body weight of nude mice at 37 days; (C) Tumor weight; (D) Changes in tumor volume of nude mice; (E) Tumor volume of nude mice at 37 days; (F) Tumor inhibition rate; (G) Nude mice. * indicates P <0.05, ** indicates P <0.01, *** indicates P <0.0001.

[0023] Figure 6 :In vivo evaluation of the anti-HBV effect of Bifidobacterium longum GY2. (A) HBV DNA expression level in nude mouse serum; (B) HBeAg expression level in nude mouse serum; (C) HBsAg expression level in nude mouse blood. Among them, * indicates P< 0.05, ** indicates P< 0.01,*** indicates P< 0.0001.

[0024] Figure 7 : Serum cytokine determination. (A) Serum IL-6 expression level; (B) Serum IL-10 expression level; (C) Serum AFP expression level. Among them, * indicates P< 0.05, ** indicates P< 0.01,*** indicates P< 0.0001. DETAILED DESCRIPTION

[0025] In order to more clearly describe the technical solution of the present invention, it is further described below in conjunction with specific embodiments, but it cannot be used to limit the present invention. This is only a partial embodiment of the present invention.

[0026] Example 1 Isolation and Identification of Lactic Acid Bacteria

[0027] Clinical feces of healthy people were collected as samples. In a sterile environment, 1.0 g ± 0.1 g of the sample was weighed and added to 10 mL of sterile mMRS liquid culture medium. After homogenization, it was enriched and cultured under anaerobic conditions at 37°C for 12 h. 1.0 mL of the enrichment solution was taken and added to 9.0 mL of sterile saline for gradient dilution to prepare 10 -1 Up to 10 -5 Dilution gradient of bacterial suspension. Select dilution of 10 -3 , 10 -4 , 10 -5 200 μL of each of the three gradient bacterial suspensions were added to mMRS solid medium (Huankai Biology, China), spread evenly with a sterile spreader stick, and cultured at 37°C under anaerobic conditions for 48 h. Typical colonies on the mMRS solid medium plate were picked and streaked to purify and culture to obtain pure lactic acid bacteria colonies, which were then preserved and bacterial DNA was extracted. Bacterial DNA was amplified by PCR, and the PCR amplification primers were: forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 1492R: 5'-CTACGGCTACCTTGTTACGA-3'. PCR reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 45 s, a total of 35 cycles, and 72°C extension for another 10 min. PCR products were sequenced by 16S rDNA, and the sequences were compared with the NCBI database and homology analysis was performed. The isolated lactic acid bacteria strains were subjected to in vitro experiments, and one lactic acid bacteria strain was initially selected for subsequent experiments. The 16S rRNA gene sequence of the selected lactic acid bacteria strain was used to construct a phylogenetic tree and verify it using MALDI-TOF MS (BRUKER, Germany).

[0028] The present invention screened a probiotic lactic acid bacteria with anti-HBV function, named Bifidobacterium longum ( Bifidobacterium longum )GY2, phylogenetic tree as Figure 1 As shown, the results of MALDI-TOF MS (BRUKER, Germany) mass spectrometry verification showed that the identified bacterial species were consistent.

[0029] Bifidobacterium longum ( Bifidobacterium longum) GY2 was deposited on July 16, 2024 in the Guangdong Microbiological Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, Postal Code: 510070, and the deposit number is: GDMCC No: 64873.

[0030] Example 2 Screening of Anti-HBV Lactic Acid Bacteria

[0031] (1) Preparation of lactic acid bacteria fermentation supernatant

[0032] The activated lactic acid bacteria GY2 and GY5 were inoculated into mMRS broth medium at a volume fraction of 2%, and the cells were cultured in an anaerobic workstation at 37°C for 48 h to allow the cells to be in the logarithmic growth phase. The cells were centrifuged at 3,000 r / min and 4°C for 10 min, and the supernatant was taken to adjust the pH to 7.2-7.4. The supernatant was then filtered through 0.45 μm and 0.22 μm microporous filter membranes in turn, and stored at -80°C after aliquoting.

[0033] (2) Culture of HepG2.2.15 cells

[0034] Add HepG2.2.15 cells to a T25 cell culture flask and add complete cell culture medium to 5 mL. The culture conditions are 37°C and 5% CO2. The culture medium is changed every 2 to 3 days and the cells are passaged every 4 to 5 days.

[0035] (3) Cytotoxicity test of lactic acid bacteria fermentation broth

[0036] The toxicity of fermentation supernatant to HepG2.2.15 cells was detected by CCK-8 method to screen out the lactic acid bacteria fermentation supernatant with the maximum safe concentration for cells.

[0037] Take 1×10 4 100 μL of cell suspension per well was added to a 96-well plate, and the cells were cultured at 37°C and 5% CO2 for 24 h. The old culture medium was discarded, and the cells were rinsed 2-3 times with an appropriate amount of PBS buffer and discarded. 100 μL of lactic acid bacteria fermentation supernatant diluted with pure cell culture medium (DMEM, Huankai Biology, China) was added, and the culture plate was gently shaken to mix. Only 100 μL of pure DMEM was added to the blank group. Three wells were repeated for each group. The cells were cultured at 37°C and 5% CO2 for 24-96 h. Then 10 μL of CCK-8 reagent was added, and A450 nm was measured after incubation at 37°C for 2 h. The experiment was repeated three times.

[0038]

[0039] In the formula, S1 represents cell survival rate (%);

[0040] As represents the absorbance of the experimental wells (the absorbance of the wells containing cells, culture medium, CCK-8, and test compounds);

[0041] Ab represents the absorbance of blank wells (absorbance of wells containing culture medium and CCK-8);

[0042] Ac represents the absorbance of control wells (absorbance of wells containing cells, culture medium and CCK-8).

[0043] The results of the cytotoxicity test of lactic acid bacteria fermentation supernatant on HepG2.2.15 cells are as follows Figure 2 As shown in A in the figure, the concentration of lactic acid bacteria fermentation broth within the range of 100% ± 10% cell activity was selected to establish its maximum safe concentration for cells as the basis for subsequent experiments, so as to exclude the possibility that the toxic effects of drugs or fermentation broth on cells may affect the monitoring results of HBV DNA.

[0044] (4) Determination of anti-HBV performance

[0045] HepG2.2.15 cells were collected at 1×10 5 100 μL of lactic acid bacteria fermentation supernatant (maximum safe dilution concentration) was added at non-toxic concentrations, and 3 parallel wells were set for each concentration. At the same time, 3 wells of cell negative control group and 3 wells of lamivudine (LAM, 125 μg / mL) group were set as positive control group. Continue to culture for 24-96 hours, collect the culture fluid, and detect the expression level of HBV DNA by qPCR. The experiment was repeated three times.

[0046] The primers used in qPCR were forward primer HBx-F: 5'-GGCCATCAGCGCATGC-3', reverse primer HBx-R: 5'-CGCTGCGAGCAAAACA-3', and probe primer HBx-Probe: 5'-R-CTCTGCCGATCCATACTGCGGAACTC-Q-3'. The qPCR reaction system was: 2×Probe qPCR SuperMix 10 μL, HBx-F and HBx-R final concentrations were 0.2~10 μmol / L, 10 μmol / L HBx- Probe 0.4 μL, ROX Reference Dye (to be selected according to the machine model) 0.4 μL, and RNase-Free Water to make up the volume to 20 μL. The reaction program was: 50℃, 2 min; 94℃, 2 min; 94℃ 15s, 60℃ 20 s, 72℃ 30 s, 45 cycles.

[0047] The levels of HBeAg and HBsAg were determined by enzyme-linked immunosorbent assay.

[0048] In clinical practice, the level of HBV DNA, HBeAg and / or HBsAg is used to determine the infectiousness of HBV infection and to assess the prognosis of HBV-related diseases. Figure 2 As shown, in the in vitro experiment of lactic acid bacteria provided by the present invention, Figure 2 As shown in Figures C and D, the inhibition rate of extracellular HBV DNA in the GY2-treated group was 25.25%±1.36% compared with the control group (P<0.05). Figure 2 As shown in Figures E and F, the relative extracellular expression levels of HBeAg and HBsAg were 22.51% ± 1.57% (P<0.05) and 13.68% ± 0.31% (P<0.05), respectively. This indicates that probiotic lactic acid bacteria GY2 can inhibit the replication of HBV DNA and the expression of HBeAg and HBsAg to a certain extent, and its application in antiviral function deserves further study.

[0049] Example 3 Evaluation of the probiotic properties of lactic acid bacteria

[0050] (1) Artificial gastric juice and intestinal juice tolerance test

[0051] The activated lactic acid bacteria GY2 was inoculated into sterile mMRS liquid culture medium at a volume fraction of 2%, and cultured at 37°C under anaerobic conditions for 48 h. The culture medium was centrifuged at 3000 r / min for 10 min to collect the bacteria, resuspended and washed with sterile saline, and centrifuged twice to make a bacterial solution with a concentration of 1×10 8 CFU / mL of bacterial suspension. Then, 1 mL of bacterial suspension was inoculated into a 9 mL filter-sterilized pH 3.0 artificial gastric fluid test tube, mixed thoroughly and cultured anaerobically at 37°C. Samples were taken at 0 h after the start of the experiment and 3-5 h after incubation to determine the number of viable bacteria. Then, 1 mL of the bacterial suspension treated with gastric juice was aseptically aspirated and inoculated into 9 mL filter-sterilized pH 8.0 artificial intestinal fluid, and continued to be cultured anaerobically in a 37°C constant temperature incubator. Samples were taken after 3-5 h to determine the number of viable bacteria. Each experiment was repeated three times.

[0052] Preparation of artificial simulated gastric fluid: Use sterilized PBS buffer (pH 7.2) to prepare pepsin (1:10000) into a pepsin solution with a concentration of 3 mg / mL, adjust the pH value to 3.0 with 1 mol / L hydrochloric acid solution, and then filter through a 0.22 μm microporous filter membrane for sterilization before use.

[0053] Preparation of artificial simulated intestinal fluid: Use sterilized PBS buffer (pH 7.2) to prepare trypsin (1:250) into a solution with a concentration of 1 mg / mL, then add 0.3% ox bile salt, and adjust the pH value to 8.0 with 1 mol / L sodium hydroxide, then filter through a 0.22 μm microporous filter membrane for sterilization and set aside.

[0054] The calculation formula is as follows:

[0055]

[0056] In the formula, S2 represents the survival rate of lactic acid bacteria (%);

[0057] N t It indicates the number of viable lactic acid bacteria (CFU) after 3 h or 4 h of treatment;

[0058] N0 represents the number of viable lactic acid bacteria (CFU) at 0 h of treatment.

[0059] The selected strains were subjected to artificial gastric juice and intestinal juice simulation experiments. The results were as follows: Figure 3 As shown in A, after being treated with gastric juice for 3 h and intestinal juice for 4 h, the survival rates of GY2 were 70.67% and 84.67%, respectively, which indicates that the lactic acid bacteria GY2 provided by the present invention has a certain gastrointestinal juice tolerance ability and can survive and function in the gastrointestinal tract.

[0060] (2) Determination of hydrophobicity of lactic acid bacteria

[0061] The activated lactic acid bacteria GY2 was inoculated into sterile mMRS liquid medium at a volume fraction of 2%, and cultured at 37°C under anaerobic conditions for 48 h. 1 mL of the lactic acid bacteria suspension was taken and centrifuged at 3,000 r / min for 10 min. The supernatant was removed and washed twice with sterile PBS (pH 7.2). 595nm Adjust to 0.8~1.0. Add 1 mL of ethyl acetate, dichloromethane, chloroform and n-hexane to 3 mL of lactic acid bacteria suspension, react at 37°C for 10 min, balance the temperature, shake and mix, incubate at 37°C for 3 h, and measure the OD of the aqueous solution. 595nm The experiment was repeated three times. The hydrophobicity of lactic acid bacteria was calculated as follows:

[0062]

[0063] In the formula, H represents the hydrophobicity of lactic acid bacteria (%);

[0064] G0 and G t Respectively represent the OD of the bacterial solution before and after the lactic acid bacteria are mixed with organic solvents (ethyl acetate, dichloromethane, chloroform and n-hexane)595 nm value;

[0065] The hydrophobicity of lactic acid bacteria is one of the most important factors that determine its non-specific adhesion to various biological and non-biological surfaces and interfaces. It is also one of the main factors affecting the absorption and degradation of hydrophobic organic substances by bacteria. The high hydrophobicity of lactic acid bacteria is conducive to the survival of strains in the intestine and the maintenance of intestinal flora homeostasis. The hydrophobicity of the selected strains was measured, and the results are as follows: Figure 3 As shown in B, GY2 exhibits high hydrophobicity, and its hydrophobicity rates to ethyl acetate, dichloromethane, chloroform and n-hexane are 64.28%, 90.88%, 91.61% and 14.32%, respectively.

[0066] (3) Determination of aggregation properties of lactic acid bacteria

[0067] Self-aggregation: The activated lactic acid bacteria GY2 was inoculated into sterile mMRS liquid culture medium at a volume fraction of 2%, and cultured at 37°C under anaerobic conditions for 48 h. 1 mL of the lactic acid bacteria suspension was taken, centrifuged at 3,000 r / min for 10 min, the supernatant was aspirated, and the cells were washed twice with sterile PBS. 3 mL of sterile PBS was added to the cells, vortexed for 10 s, and 200 μL of the bacterial solution was taken to measure its absorbance at 600 nm. After that, the bacterial suspension was placed at room temperature for 3 h and 9 h, and the experiment was repeated to measure A. t The experiment was repeated three times. The percentage of lactic acid bacteria self-aggregation was calculated as follows:

[0068]

[0069] In the formula, S3 represents the self-polymerization ability of lactic acid bacteria (%);

[0070] A0 represents the absorbance value of lactic acid bacteria at 0 h;

[0071] At represents the absorbance value of lactic acid bacteria at different time t (3 h and 9 h).

[0072] Coaggregation: Enterohemorrhagic Escherichia coli ( Enterohemorrhagic Escherichia coli )O157:H7 was used to evaluate the coaggregation ability of lactic acid bacteria. Equal volumes of lactic acid bacteria and pathogenic bacteria suspension were mixed, vortexed for 10 s, incubated at 37°C for 3 h and 9 h, and 200 μL of the upper suspension was gently taken to measure the absorbance at 600 nm. The experiment was repeated three times. The percentage of lactic acid bacteria coaggregation was calculated as follows:

[0073]

[0074] In the formula, S4 represents the coaggregation ability of lactic acid bacteria (%);

[0075] AP Indicates the initial absorbance value of lactic acid bacteria;

[0076] A E It indicates the initial absorbance value of pathogenic bacteria O157:H7;

[0077] A mix It represents the absorbance value of the mixed bacterial suspension of lactic acid bacteria and pathogenic bacteria O157:H7 after culturing for 3 and 9 hours.

[0078] The hydrophobicity and self-aggregation ability of lactic acid bacteria are positively correlated with the biofilm formation ability of microorganisms, which can reflect the adhesion and colonization ability of strains. The present invention measures the aggregation rate of strains at 3 h and 9 h respectively, and the results are as follows Figure 3 As shown in C, with the passage of time, the self-aggregation rate and co-aggregation rate of lactic acid bacteria showed an upward trend. The self-aggregation rate of GY2 reached 23.11% and 63.61 at 3 h and 9 h, respectively. At the same time, the co-aggregation rate with pathogenic bacteria O157:H7 reached 71.51% at 9 h, indicating a certain degree of antagonism against enterohemorrhagic Escherichia coli. This shows that the lactic acid bacteria GY2 provided by the present invention can effectively antagonize pathogenic bacteria in the intestine.

[0079] (4) Antibacterial activity of lactic acid bacteria fermentation supernatant

[0080] The activated lactic acid bacteria GY2 was inoculated into sterile mMRS liquid culture medium at a volume fraction of 2%, and cultured under anaerobic conditions at 37°C for 48 h. The supernatant was centrifuged at 3,000 r / min for 10 min at 4°C and filtered through a 0.22 μm microporous filter for sterilization before use. The viable count of pathogenic indicator bacteria was adjusted to about 10 8 CFU / mL, draw 200 μL and spread evenly on the nutrient agar plate, carefully place the sterile Oxford cup on the plate, draw 200 μL of lactic acid bacteria fermentation supernatant into the Oxford cup, diffuse in a 4℃ refrigerator for 10 h. Carefully place in a 37℃ constant temperature incubator and measure the antibacterial zone diameter. Each experiment was repeated three times.

[0081] Table 1 Antibacterial test of lactic acid bacteria GY2 fermentation supernatant

[0082]

[0083] Note: “-” indicates no obvious antibacterial effect; “+” indicates the antibacterial circle diameter d<10 mm; “++” indicates 10 mm≤d≤15 mm; “+++” indicates 15 mm≤d≤20 mm; “++++” indicates d>20 mm.

[0084] The ability of probiotics to produce antimicrobial compounds may constitute a key feature for effectively excluding pathogens from surviving in the intestine and expressing probiotic effects on the host. As can be seen from Table 1, lactic acid bacteria GY2 has strong antibacterial activity against a variety of foodborne pathogens, and the diameter of the antibacterial zone is greater than 20 mm, but E. sakazakii , P. cocovenenans and S. haemolyticus No obvious antibacterial activity.

[0085] Example 4 Safety Evaluation of Lactic Acid Bacteria

[0086] (1) Antibiotic sensitivity test of lactic acid bacteria

[0087] Whether lactic acid bacteria are sensitive to antibiotics is an important indicator for evaluating bacterial safety. S. aureus )ATCC ® 25923 was used as the quality control bacteria, and 11 commonly used antibiotics were selected: Penicillin G (PEN, 10 μg), Erythromycin (ERY, 15 μg), Spectinomycin (SPE, 100 μg), Oxacillin (OXA, 1 μg), Clarithromycin (CLA, 15 μg), Vancomycin (VAN, 30 μg), Clindamycin (CLT, 2 μg), Tetracycline (TET, 30 μg), Minocycline (MIN, 30 μg), Ciprofloxacin (CIP, 5 μg), and Nitrofurantoin (NIT, 300 μg) according to the Clinical and Laboratory Standards Association (CLI). The antibiotic sensitivity test paper standard published by the CLSI was used to determine the sensitivity of probiotic lactic acid bacteria GY2 to antibiotics.

[0088] Table 2 Antibiotic sensitivity test

[0089]

[0090] Note: S stands for sensitive; R stands for insensitive.

[0091] It can be seen from Table 2 that GY2 is sensitive to multiple antibiotics, which indicates that the antiviral lactic acid bacteria provided by the present invention are relatively safe.

[0092] (2) Hemolysis test

[0093] During the metabolism process, lactic acid bacteria may produce hemolytic toxins to dissolve red blood cells, leading to anemia and other reactions. Under sterile conditions, lactic acid bacteria GY2 was inoculated into a sheep blood plate, cultured at 37°C for 48 hours, and hemolysis was observed. Hemolysis will form three characteristics on the blood plate: α hemolysis, a grass-green hemolysis ring will appear around the colonies, and this type of bacteria is generally opportunistic; β hemolysis, a wider transparent hemolysis ring will appear around the colonies, generally speaking, it is highly pathogenic; γ hemolysis, no hemolysis ring will appear around the colonies, and generally speaking, the strain is non-pathogenic. The results are as follows: Figure 4 As shown, S. aureus As a positive control, no hemolytic ring appeared around the GY2 colonies, so the results showed that the lactic acid bacteria GY2 provided by the present invention were all γ-hemolytic, had no hemolytic activity, and were non-pathogenic.

[0094] Example 5 Evaluation of anti-HBV in vivo

[0095] (1) Experimental animal grouping, tumor volume, and organ index determination

[0096] Four-week-old male BALB / c-nude mice (from Guangdong Medical Experimental Animal Center) were selected, all of which were SPF grade. The nude mice were placed in a controlled environment (temperature 23±3℃, relative humidity 60%±10%, light / dark cycle 12 / 12h, free access to water and food during the experiment) for feeding. After one week of adaptive feeding, the nude mice were randomly divided into 4 groups: the healthy control group (Control) was fed with 0.1 mL / mouse saline per day; the model control group (Model) was fed with 0.1 mL / mouse saline per day; the drug prevention group (tenofovir alafenamide, TAF) was fed with the same volume of TAF at the requirement of 100 mg / (kg / mouse) per day; the probiotic lactic acid bacteria GY2 prevention group (GY2) was fed with 10 9 CFU / (0.1 mL / mouse) of GY2. Except for the healthy control group, each nude mouse in the other three groups was subcutaneously implanted with 2×10 HepG2.2.15 cells. 6 The nude mice were gavaged daily starting from the second day after implantation. The nude mice were weighed and the tumor diameter was measured every 4 days during the feeding period. The experimental design was approved by the Experimental Animal Management and Ethics Committee of Guangdong Institute of Microbiology, and the nude mice were maintained in accordance with standard guidelines.

[0097] The time for nude mice to form tumors was about 37 days (tumor diameter > 5 mm was considered successful). After the experiment, blood was collected from the nude mice through the orbital vein and they were killed. The liver, spleen, kidney, small intestine and colon were immediately removed, rinsed and weighed with pre-cooled sterile PBS, frozen in dry ice, and stored at -80°C until analysis. HBV DNA and HBsAg levels in serum and liver tissue were measured. The organ index (%) was calculated according to the following formula:

[0098]

[0099] Wherein, I represents the organ index (%), W represents the weight of the nude mouse organ (g), and W0 represents the weight of the nude mouse (g).

[0100] Table 3 Organ indexes of nude mice in each group (n=7)

[0101]

[0102] Note: Compared with the healthy control group (Control), * express P< 0.05, ** express P< 0.01, *** express P< 0.001; Difference compared with the model control group (Model), # express P< 0.05.

[0103] The spleen is the largest immune organ in tumor-bearing nude mice and plays an immune role through humoral immunity. The spleen coefficient can reflect the changes in the immune function of tumor-bearing nude mice to a certain extent. Studies have shown that under the condition of no other interference, the stronger the spleen coefficient, the stronger the immune function of tumor-bearing nude mice. Figure 5 It can be seen that the weight of subcutaneous tumor-bearing nude mice was significantly reduced. Compared with the healthy control group (Control), the liver index of other groups was significantly reduced, and only the spleen index of the lactic acid bacteria treatment group (GY2) was significantly reduced; at the same time, the lactic acid bacteria treatment group (GY2) was able to delay the occurrence of tumors, which was statistically significant. Therefore, it can be preliminarily explained that the lactic acid bacteria GY2 provided by the present invention can alleviate the weight loss of tumor-bearing nude mice and improve their immune function, and at the same time, it can also delay the occurrence of hepatocellular carcinoma.

[0104] (2) Determination of HBV DNA, HBeAg, and HBsAg in serum

[0105] 50 μL of serum was taken from each nude mouse in each group and HBV DNA was extracted after pretreatment. The HBV DNA content in the serum was determined by qPCR. The specific conditions and operations were the same as those in Example 2. The HBsAg and HBeAg content in the serum were determined by enzyme-linked immunosorbent assay. The experimental results are shown in Figure 6As shown, the HBV DNA expression levels in the serum of tumor-bearing nude mice in the model control group (Model), drug group (TAF) and lactic acid bacteria group (GY2) were 6.04 ± 0.34 (lg IU / mL), 4.38 ± 0.21 (lg IU / mL) and 5.16 ± 0.71 (lg IU / mL), respectively; the HBeAg content was 41.39 ± 4.36 IU / L, 8.78 ± 1.77 IU / L and 27.4 ± 23.24 IU / L, respectively; the HBsAg content was 34.41 ± 1.59 mg / L, 9.41 ± 7.81 mg / L and 20.23 ± 5.79 mg / L, respectively; and the HBV DNA, HBsAg and HBeAg expression levels in the serum of the treatment groups (TAF and GY2) were significantly lower than those in the Model group. This indicates that the lactic acid bacteria GY2 provided by the present invention can also exert an antiviral effect on HBV in vivo.

[0106] (3) Determination of cytokines in serum

[0107] The main biological function of cytokines is to mediate and regulate immune response and inflammatory response. 50 μL of serum was taken from each nude mouse in each group, and the cytokine content was determined by enzyme-linked immunosorbent assay. Figure 7 As shown, compared with the Control group, the pro-inflammatory factor (IL-6) in the modeling group was significantly upregulated, while the anti-inflammatory factor (IL-10) was significantly downregulated, but there was no significant difference between the pro-inflammatory factor and anti-inflammatory factor of the nude mice treated with lactic acid bacteria GY2 and the Control group. This shows that the lactic acid bacteria GY2 provided by the present invention participates in the immune regulation of the host body in vivo, and in this process it can inhibit the expression of pro-inflammatory factors and increase the expression level of anti-inflammatory factors.

[0108] Example 6 Verification of lactic acid bacteria specific molecular targets

[0109] (1) Discovery of specific new molecular targets

[0110] Firstly, a bacterial genome extraction kit (Huankai Biology, China) was used to extract and purify bacterial genomic DNA, and the whole genome of lactic acid bacteria GY2 was sequenced. Then, bioinformatics analysis was performed using the GenBank database to screen out specific gene fragments of GY2. The nucleotide sequence of the gene fragment of GY2 is shown in SEQ ID NO.1.

[0111] SEQ ID NO.1:

[0112]

[0113] (2) Primer validity test

[0114] According to the sequence SEQ ID NO.1 in (1), a specific PCR amplification primer set (including a forward primer and a reverse primer) was designed. The primer set sequence is shown in Table 4 below.

[0115] Table 4 Specific sequence PCR detection primer set

[0116]

[0117] (1) Preparation of DNA template: GY2 was cultured in sterile mMRS liquid medium, and its bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit as the template to be tested.

[0118] (2) PCR amplification: The PCR amplification system for GY2 was 10 μL of 2×Taq PCR MasterMix, 0.5 μL of Primer F (10 μmol / L), 0.5 μL of Primer R (10 μmol / L), 1 ng of template DNA, and RNase-Free Water to make up the volume to 20 μL. The PCR amplification program for GY2 was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 10 min.

[0119] (3) Perform gel electrophoresis on the PCR amplification products and observe the amplified bands of each group of products. If a single amplified band appears in GY2 and there is no amplified band in other groups, it means that the corresponding target is a strain-specific molecular target. In the present invention, 133 strains of bifidobacteria (including 84 strains of long bifidobacterium), 23 strains of other non-target lactic acid bacteria and 24 strains of non-lactic acid bacteria, a total of 34 species and 180 different bacteria were selected for GY2 to verify the specific molecular target of the target bacteria GY2.

[0120] In step (1), the DNA template is prepared by extracting the genomic DNA of each bacterium; in step (2), the primers used in the PCR amplification are the primers in the primer set. A blank control is set, and the template of the blank control group is RNase-Free Water without genome.

[0121] The verification results of the specific molecular target of the target strain GY2 are shown in Table 5 below. The results show that only the target strain has two single amplification bands at the same time, and other non-target strains do not contain the target bands. This result indicates that only the target strain contains the specific molecular target in this method.

[0122] Table 5 Results of the specific molecular target evaluation test for target bacteria GY2

[0123] ;

[0124] ;

[0125] .

[0126] Note: N indicates blank control, “-” indicates negative, and “+” indicates the appearance of the target band.

[0127] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention. The protection scope of the present invention shall be subject to the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A strain of Bifidobacterium longum ( Bifidobacterium longum )GY2, the deposit number is: GDMCC No: 64873.

2. Use of the Bifidobacterium longum GY2 according to claim 1 in the preparation of anti-hepatitis B virus medicines.

3. The use of Bifidobacterium longum GY2 according to claim 1 in the preparation of a medicine for preventing and controlling pathogenic bacteria, wherein the pathogenic bacteria is Yersinia enterocolitica ( Yersinia enterocolitica ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Escherichia coli ( Escherichia coli ), Salmonella typhimurium ( Salmonella typhimurium )、Bacillus subtilis( Bacillus subtilis ), Bacillus cereus ( Bacillus cereus ), Staphylococcus aureus ( Staphylococcus aureus ) and Listeria monocytogenes ( Listeria monocytogenes ).

4. The use according to claim 3, characterized in that: The Escherichia coli includes enterohemorrhagic Escherichia coli ( Enterohemorrhagic Escherichia coli )O157:H7.

5. A primer set for detecting the specific molecular target of Bifidobacterium longum GY2 according to claim 1, characterized in that: The primer set includes a primer pair whose nucleotide sequences are shown as SEQ ID NO.2-3 and a primer pair shown as SEQ ID NO.4-5.

6. The primer set according to claim 5, characterized in that: The specific molecular target of Bifidobacterium longum GY2 has a nucleotide sequence as shown in SEQ ID NO.

1.

7. Use of the primer set according to claim 5 or 6 in detecting Bifidobacterium longum GY2.

8. A method for detecting Bifidobacterium longum GY2 according to claim 1, characterized in that: The following steps are involved: Extract DNA from the microorganism of the sample to be tested, perform PCR amplification on it using the primer set described in claim 5 or 6, and perform gel electrophoresis on the amplified product. If two bands of 246 bp and 274 bp are obtained at the same time, it is determined that Bifidobacterium longum GY2 is detected in the sample; if two bands of 246 bp and 274 bp are not obtained at the same time, it is determined that Bifidobacterium longum GY2 is not detected in the sample.

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