Application of Lactobacillus gasseri derived from breast milk in weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage and inhibition of intestinal pathogenic bacteria

Abstract

The present invention discloses the application of a Lactobacillus gasseri strain derived from breast milk in weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage and inhibition of intestinal pathogenic bacteria, belonging to the fields of microbial technology and pharmaceutical technology. The present invention provides a new use of Lactobacillus gasseri in weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage and inhibition of intestinal pathogenic bacteria, which can effectively reduce the prevention of weight gain, increase in body fat percentage, elevation of low-density lipoprotein cholesterol and triglycerides, inhibition of postprandial blood glucose and insulin resistance index elevation, inhibition of intestinal mucosal damage and growth of intestinal pathogenic bacteria caused by high-fat diet, and effectively improve the occurrence of chronic diseases in high-fat diet-fed mice. Therefore, Lactobacillus gasseri FN136 has great application prospects in the preparation of products for weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage and inhibition of intestinal pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to the application of a Lactobacillus gasseri strain derived from breast milk in weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage and inhibition of intestinal pathogenic bacteria, belonging to the fields of microbial technology and pharmaceutical technology. Background Art

[0002] Probiotics are defined as "live microorganisms that are beneficial to the human body after ingestion of a certain amount", and one of the important criteria for judging probiotics is their colonization ability in the human stomach and intestines. Breast milk is a good source of new probiotics. In addition to providing essential nutrients for newborns, breast milk also contains beneficial bacteria such as Lactobacillus and Bifidobacterium, which can be transferred to the baby's oral cavity and intestines through breastfeeding and interactively protect the baby from early infections and inflammation. They may also be one of the reasons why breastfed children are less likely to be obese than formula-fed children. Lactobacillus gasseri is one of the common strains in breast milk. Lactobacillus gasseri derived from breast milk has a long history of infant consumption and has good probiotic development value.

[0003] Studies have shown that Lactobacillus gasseri BNR17 derived from Korean breast milk can synthesize oligosaccharides from glucose (Microbiology. 2010; 156: 1264-1274), thus improving the occurrence of animal obesity, but its effect in humans has only been reported to have significant effects in the treatment of irritable bowel syndrome (Food Sci Biotechnol (2018) 27(3): 853–857), and it is still necessary to further screen suitable strains or find the best application objects. The differences in the flora of different populations are not related to diet and host genotype, but are related to the geographical location where they live, and have different effects on the host body composition (Elife, 10, e70349). Therefore, Lactobacillus gasseri isolated from the breast milk of Chinese mothers may be more suitable for the physiological needs of the Chinese population.

[0004] Type 2 diabetes is a chronic disease caused by insufficient insulin use or reduced efficiency, commonly seen in adults. This disease is caused by a combination of genetic and environmental factors such as lifestyle, overnutrition, and lack of physical activity. The initial symptoms are often mild, and many people are not discovered until complications occur or during a routine physical examination. The typical symptoms of type 2 diabetes are known as "polyuria, polydipsia, polyphagia, and weight loss"; but there may be no symptoms or mild symptoms in the early stage of the disease. As the disease progresses, the symptoms of hyperglycemia will become more obvious. Severe cases can develop ketoacidosis, hyperglycemic hyperosmolar coma, and are prone to combined infections, which can be life-threatening in severe cases. According to the current data, the incidence of diabetes in our country shows a rapid growth trend. The incidence of type 2 diabetes among adults in our country is approximately 10.6%, becoming one of the major diseases threatening the health of the Chinese people.

[0005] The increasing prevalence of obesity is a global concern, and obesity is characterized by the accumulation of visceral fat. The main hazards of obesity are the increased risks of diseases such as hypertension, dyslipidemia, cardiovascular diseases, and type 2 diabetes. Gut microbiota dysbiosis is closely related to these complications caused by obesity, especially type 2 diabetes. Compared with healthy individuals, the occurrence of type 2 diabetes is associated with an increase in the numbers of Proteobacteria and Bacteroidetes and a decrease in the number of Firmicutes in the gut microbiota, and the numbers of Proteobacteria and Bacteroidetes are positively correlated with blood glucose levels. Lipopolysaccharides on the cell walls of Proteobacteria and Bacteroidetes can induce intestinal leakage, cause metabolic endotoxemia and chronic inflammation, and lead to insulin resistance. Probiotics have important adjuvant application value in improving obesity and its complications due to their potential role in reshaping the gut microbiota.

[0006] Meanwhile, probiotics affect blood lipids by metabolizing bile acids in the small intestine. Primary bile acids are synthesized in the liver using cholesterol as a substrate and contain cholic acid and chenodeoxycholic acid in their structures. They are conjugated with glycine or taurine to produce water solubility and are secreted into bile and then released into the small intestine. 95% of the primary bile acids are reabsorbed in the ileum in conjugated form, and 5% are decomposed by small intestinal bacteria into secondary bile acids and enter the colon, being excreted out of the body with feces. The increased excretion amount helps promote the synthesis of primary bile in the liver, thereby reducing blood cholesterol.

[0007] The intestine is not only a digestive organ but also the largest immune detoxification organ in the human body. The health of the intestine is closely related to the body's immunity, skin health, mental state, etc. Therefore, the intestine is also called "the first line of defense for physical health".

[0008] As is well known, screening microorganisms in the natural environment has uncertainty, that is, it is very difficult to screen exactly the same microbial strains in the same screening object, which has non-reproducibility. Moreover, on this basis, it is even rarer to screen strains with multiple functions in actual situations.

[0009] In summary, it is necessary to further develop a multi-functional probiotic that has good application prospects in aspects such as weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage, and inhibition of intestinal pathogenic bacteria. Summary of the Invention

[0010] The problem to be solved by the present invention is to provide a Lactobacillus gasseri FN136 with probiotic characteristics and capable of weight loss, blood lipid reduction, prevention and treatment of type 2 diabetes, alleviation of intestinal leakage, and inhibition of intestinal pathogenic bacteria.

[0011] The present invention provides Lactobacillus gasseri FN136, which was deposited at the Guangdong Microbial Culture Collection Center on July 23, 2024, with the deposit number GDMCC No: 64909, and the deposit address is the 5th floor, Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, Guangdong Province. For convenience of description, Lactobacillus gasseri FN136 will hereinafter be simply referred to as "Lactobacillus gasseri FN136".

[0012] The Lactobacillus gasseri FN136 is a strain of Lactobacillus gasseri with potential probiotic properties isolated and screened from the breast milk of healthy mothers in Gannan Tibetan Autonomous Prefecture, China.

[0013] The present invention provides a probiotic preparation containing Lactobacillus gasseri FN136.

[0014] In one embodiment of the present invention, the probiotic preparation contains live cells and / or cell metabolites and / or inactivated cells and / or freeze-dried cells of the Lactobacillus gasseri FN136.

[0015] In one embodiment of the present invention, the number of Lactobacillus gasseri FN136 in the probiotic preparation is ≥ 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0016] The present invention also provides a drug containing Lactobacillus gasseri FN136.

[0017] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier and / or excipient.

[0018] In one embodiment of the present invention, the pharmaceutical dosage form includes dosage forms such as granules, capsules, tablets, pills or oral liquids, etc.

[0019] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, acid-base regulators, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants and flavoring agents, etc.

[0020] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate and / or microcrystalline cellulose, etc.

[0021] In one embodiment of the present invention, the binder is cellulose derivatives, alginates, gelatin and / or polyvinylpyrrolidone, etc.

[0022] In one embodiment of the present invention, the wetting agent is water, ethanol, starch and / or syrup, etc.

[0023] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and / or sodium bicarbonate, etc.

[0024] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, colloidal silica, and / or polyethylene glycol, etc.

[0025] In one embodiment of the present invention, the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, citric acid, tartaric acid, and / or sodium bicarbonate, etc.

[0026] The present invention also provides the use of Lactobacillus gasseri FN136 in the preparation of a drug for weight loss and / or lipid-lowering and / or prevention and treatment of type 2 diabetes and / or alleviation of intestinal leakage and / or inhibition of intestinal pathogenic bacteria in the target population.

[0027] In one embodiment of the present invention, the drug further contains a pharmaceutically acceptable carrier and / or excipient.

[0028] In one embodiment of the present invention, the pharmaceutical dosage form includes dosage forms such as granules, capsules, tablets, pills, or oral liquids, etc.

[0029] In one embodiment of the present invention, the acceptable excipients include one or more commonly used thickeners, antioxidants, acid-base regulators, emulsifiers, preservatives, fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents, etc.

[0030] In one embodiment of the present invention, the filler is starch, sucrose, lactose, calcium sulfate, and / or microcrystalline cellulose, etc.

[0031] In one embodiment of the present invention, the binder is cellulose derivatives, alginates, gelatin, and / or polyvinylpyrrolidone, etc.

[0032] In one embodiment of the present invention, the wetting agent is water, ethanol, starch, and / or syrup, etc.

[0033] In one embodiment of the present invention, the disintegrant is sodium carboxymethyl starch, carboxypropyl cellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate, and / or sodium bicarbonate, etc.

[0034] In one embodiment of the present invention, the lubricant is talc, calcium stearate, magnesium stearate, colloidal silica, and / or polyethylene glycol, etc.

[0035] In one embodiment of the present invention, the flavoring agent is simple syrup, sucrose, lecithin, orange peel syrup, cherry syrup, lemon, fennel, peppermint oil, sodium alginate, gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, citric acid, tartaric acid, and / or sodium bicarbonate, etc.

[0036] In one embodiment of the present invention, the enteropathogenic bacteria include, but are not limited to, one or more of Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, Proteus mirabilis, and Bacillus cereus.

[0037] In one embodiment of the present invention, the prevention and / or treatment of type 2 diabetes includes, but is not limited to, at least one of the following functions:

[0038] (1) Lowering fasting blood glucose;

[0039] (2) Alleviating the abnormally increased food intake caused by type II diabetes;

[0040] (3) Alleviating the abnormally increased water intake caused by type II diabetes.

[0041] The present invention also provides foods, functional foods, foods for special medical purposes, or health products containing the Lactobacillus gasseri FN136, etc.

[0042] In one embodiment of the present invention, the dosage forms of the health products include dosage forms such as granules, capsules, tablets, pills, or oral liquids, etc.

[0043] In one embodiment of the present invention, the foods include, but are not limited to, fermented foods or beverages prepared by fermenting with the Lactobacillus gasseri FN136.

[0044] The present invention also provides the application of the Lactobacillus gasseri FN136 in the preparation of functional foods or health products that help regulate body fat and / or help regulate the intestinal flora and / or help maintain a healthy blood lipid level (cholesterol / triglyceride) and / or help maintain a healthy blood glucose level.

[0045] Biological material preservation

[0046] Lactobacillus gasseri FN136, taxonomically named Lactobacillus gasseri, was deposited at the Guangdong Microbial Culture Collection Center on July 23, 2024, with the deposit number GDMCC No: 64909, and the deposit address is the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0047] Beneficial effects

[0048] (1) The isolated Lactobacillus gasseri FN136 of the present invention is a functional lactobacillus with good ability to tolerate the adverse environment of the human digestive tract. For example, it has excellent gastrointestinal fluid tolerance and bile salt tolerance, can inhibit harmful pathogenic bacteria in the intestine, can adhere well to human intestinal epithelial cells, and has good probiotic characteristics.

[0049] (2) The Lactobacillus gasseri FN136 of the present invention has good ability to reduce weight, lower blood lipid, prevent and treat type 2 diabetes, relieve intestinal leakage and inhibit intestinal pathogenic bacteria. Comparing the screened Lactobacillus gasseri FN136 with Lactobacillus gasseri BNR17, it has great advantages in preventing obesity and excessive body fat percentage, improving postprandial blood glucose response and insulin resistance, improving blood lipid level, improving chronic inflammation, improving plasma zonulin and lipopolysaccharide, etc., and can be used as a microbial drug or product with good industrial prospects. Description of the Drawings

[0050] Figure 1 : Colony morphology of Lactobacillus gasseri FN136.

[0051] Figure 2 : Microscopic examination picture of the cells of Lactobacillus gasseri FN136 after Gram staining.

[0052] Figure 3 ; Effect of Lactobacillus gasseri FN136 on preventing obesity and excessive body fat percentage in mice.

[0053] Figure 4 : Effect of Lactobacillus gasseri FN136 on improving postprandial blood glucose response and insulin resistance in mice.

[0054] Figure 5 : Effect of Lactobacillus gasseri FN136 on improving blood lipid level in mice.

[0055] Figure 6 : Effect of Lactobacillus gasseri FN136 on improving chronic inflammation in mice caused by high-fat diet.

[0056] Figure 7 : Effect of Lactobacillus gasseri FN136 on improving intestinal leakage in mice caused by high-fat diet. Detailed Embodiments

[0057] The present invention will be further described below in conjunction with the drawings in the specification and specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under the conventional conditions in the art. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as familiar to those skilled in the art.

[0058] Lactobacillus gasseri BNR17 (KCTC 10902BP) was obtained from the Korean Collection for Type Cultures (KCTC); Lactobacillus formatics FN136 was isolated from the breast milk of healthy mothers in Gannan Tibetan Autonomous Prefecture, Gansu Province (42 days after childbirth). Bacillus cereus ATCC 14579, Listeria monocytogenes ATCC 13932, Proteus mirabilis BNCC 107943, Staphylococcus aureus ATCC 25923, and Salmonella enterica ATCC 14028 were obtained from the American Type Culture Collection.

[0059] The culture media involved in the following examples are as follows:

[0060] MRS medium: 10 g of soy peptone, 5 g of beef extract, 5 g of yeast extract, 20 g of glucose, 1 mL of Tween-80, 2 g of sodium dihydrogen phosphate, 5 g of anhydrous sodium acetate, 2 g of citric acid triammonium, 0.02 g of manganese sulfate, 0.1 g of magnesium sulfate, 1 L of distilled water, adjust the pH to about 6.8, 15 g of agar, sterilize at 121 °C for 15 min.

[0061] The preparation method of the ninhydrin reagent involved in the following examples is: mix 250 μL of a citric acid buffer solution (0.5 M, pH 5.5) containing 1% (weight / volume ratio) of ninhydrin, 600 μL of glycerol, and 100 μL of citric acid buffer solution.

[0062] The detection methods involved in the following examples:

[0063] Oral glucose tolerance test method:

[0064] In the eighth week of the experiment, the mice in each group were fasted overnight for 12 h. Measure the fasting blood glucose of the mice (0 h), and then intragastrically administer a glucose aqueous solution at 2.0 g of glucose / kg body weight. Measure the blood glucose at 15 min, 30 min, 60 min, 90 min, and 120 min after intragastric administration, collect blood from the tail vein, and measure the blood glucose concentration with a blood glucose meter and blood glucose test strips. Draw a blood glucose change curve and calculate the area under the blood glucose curve.

[0065] Insulin resistance index method:

[0066] On the day after the last determination of the oral glucose tolerance test. Measure serum insulin and glucagon using a hypersensitive mouse insulin ELISA kit. Calculate the insulin resistance index: fasting blood glucose (nmol / L) × insulin (mU / L) / 22.5.

[0067] Example 1: Screening and identification of Lactobacillus gasseri FN136

[0068] 1. Screening of Lactobacillus gasseri FN136

[0069] (1) Sample source

[0070] The strain used in the present invention was isolated from the breast milk of healthy mothers in Gannan Tibetan Autonomous Prefecture, Gansu Province.

[0071] (2) Isolation and purification of the strain

[0072] Take about 10 mL of breast milk and add it to an anaerobic tube, immediately place it in dry ice and mail it to the laboratory for strain isolation. Take 1 mL of breast milk and centrifuge it at 15000×g for 5 min, discard the supernatant, add it to 10 mL of MRS broth medium, vortex and mix well, and then enrich and culture it in an anaerobic incubator at 37°C for 48 h; then suck 1 mL of the enriched solution in a laminar flow hood, perform ten-fold serial dilution with sterile saline, and select 10 -6 、10 -7 、10 -8 Three dilution gradients, take 100 μL of the bacterial solution for each gradient and spread it on MRS agar medium, and culture it anaerobically at 37°C for 48 h. After the culture is completed, select the plate with 50 - 150 single colonies growing from the agar medium, pick the colonies with different morphologies, sizes and colors, and streak and purify them on the MRS agar plate multiple times until the colony morphology on the whole plate is consistent, and pick the single colonies for enrichment culture in MRS broth medium. The obtained strain was suspended in PBS containing 20% glycerol and stored frozen at -80°C.

[0073] MRS medium: 10 g of soy peptone, 5 g of beef extract, 5 g of yeast extract, 20 g of glucose, 1 mL of Tween - 80, 2 g of sodium dihydrogen phosphate, 5 g of anhydrous sodium acetate, 2 g of citric acid triammonium, 0.02 g of manganese sulfate, 0.1 g of magnesium sulfate, 1 L of distilled water, adjust the pH to about 6.8, 15 g of agar, sterilize at 121°C for 15 min.

[0074] 2. Identification of Lactobacillus gasseri FN136

[0075] (1) Colony characteristics

[0076] After Lactobacillus gasseri FN136 was cultured on MRS agar medium for 48 h, its diameter was between 0.3 - 2 mm, the front morphology was round, the side morphology was convex, the edge was neat, milky white, opaque, the surface was moist and smooth, see Figure 1 .

[0077] (2) Bacterial cell characteristics:

[0078] Colony smear of Lactobacillus gasseri FN136: Gram-positive rod-shaped bacteria, the bacterial cells were about 0.9 - 1.2 μm wide and 3 - 8 μm long, and no spores were formed, see Figure 2 .

[0079] (3) Growth characteristics:

[0080] This strain is a facultative anaerobe, with the best growth at a temperature of 30 - 37°C, the highest and lowest initial growth pH values being 8.0 and 5.0 respectively, and the optimal initial growth pH being 7.0.

[0081] (4) Amplification of Lactobacillus - specific primers and 16S rDNA identification

[0082] The genomic DNA of the target strain was extracted using an Ezup column bacterial genomic DNA extraction kit. The extracted Lactobacillus genomic DNA was used as a template for PCR amplification, and Lactobacillus - specific primers were used for PCR experiments. Subsequently, universal bacterial primers 27F and 1492R were used for PCR experiments of 16S rDNA. After the PCR reaction amplification was completed, the PCR products were taken for agarose gel detection and photography. The amplified fragment length was about 1.2 Kbp. The primers sent the PCR products to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing, and the results were as shown in SEQ ID NO.1. Sequence alignment was performed on the NCBI website using EZBioCloud, and the results showed that the sequence had a homology of over 99% with the 16S rDNA sequence of Lactobacillus gasseri.

[0083] 16S rDNA sequence SEQ ID NO.1:

[0084]

[0085] Combining the sequence alignment results and physiological and biochemical results of strain FN136, the screened Lactobacillus FN136 was determined to be Lactobacillus gasseri FN136.

[0086] Example 2: Confirmation of the tolerance of Lactobacillus gasseri FN136 strain to digestive juices and its adhesion to mucin

[0087] The Lactobacillus gasseri FN136 strain and Lactobacillus gasseri BNR17 were subjected to experiments on the tolerance to digestive juices and the adhesion to mucin; the specific steps were as follows:

[0088] 1. Detection of tolerance to human gastric juice:

[0089] The Lactobacillus gasseri FN136 strain and Lactobacillus gasseri BNR17 were respectively activated in MRS medium. The activation conditions were: inoculated at 10% (v / v) in MRS medium and cultured at 37°C for 16 hours. After two generations of activation culture, the bacteria were washed twice. The washing method was: centrifuged at 12000×g for 10 min at 4°C, the bacteria were resuspended with PBS and centrifuged again to collect the bacterial cells, at 3×10 7CFU / mL was suspended in 2 mL of simulated gastric juice (pH = 3) and incubated at 37 °C for 3 h. At the same time, the viable cell rate was determined by the pour plate method using MRS agar medium. Each sample was repeated 3 times and the average value was calculated. The survival rate of the strain in simulated gastric juice was calculated according to the following formula.

[0090] Survival rate (%) = LogN1 / LogN0 × 100;

[0091] Wherein, N1 represents the number of viable bacteria in the strain system after treatment with simulated gastric juice; N0 represents the initial number of viable bacteria in the strain system.

[0092] 2. Detection of human intestinal juice tolerance:

[0093] The Lactobacillus gasseri FN136 strain and Lactobacillus gasseri BNR17 were respectively activated and cultured for two generations in MRS medium containing 0.3% (weight / volume ratio) bile salts. The activation conditions were: inoculated at 10% (v / v) in MRS medium and cultured at 37 °C for 16 hours; after activation and culture, the bacteria were washed twice. The washing method was: centrifuged at 12000×g for 10 min at 4 °C, resuspended the bacteria with PBS and then centrifuged again, collected the bacterial cells, and suspended them at 3×10 7 CFU / mL was suspended in 2 mL of simulated artificial intestinal juice and incubated at 37 °C for 3 h. The bacterial suspension was diluted and inoculated on MRS agar, and cultured under anaerobic conditions at 37 °C for 36 - 48 h. The viable cell rate was determined by the pour plate method using MRS agar medium. The survival rate of the strain in simulated artificial intestinal juice was calculated according to the following formula.

[0094] Survival rate (%) = LogN1 / LogN0 × 100.

[0095] Wherein, N1 represents the number of viable bacteria in the strain system after treatment with simulated artificial intestinal juice; N0 represents the initial number of viable bacteria in the strain system.

[0096] 3. Detection of mucin adhesion:

[0097] Lactobacillus gasseri BNR17 or Lactobacillus gasseri FN136 was cultured in MRS broth for 24 h and washed twice. The washing method: centrifuged at 12000×g for 10 min at 4 °C, resuspended the bacteria with PBS and then centrifuged again, collected the bacterial cells, and the bacterial amount: OD600 0.5 ± 0.05 was suspended in PBS containing 0.05% Tween 20 to obtain a bacterial suspension.

[0098] 10 mg of freeze-dried porcine gastric mucin was suspended in 10 mL of 50 mM Tris-HCl (pH 9.0), gently rotated (250 rpm) at 4 °C for 12 hours, then centrifuged at 16000×g for 10 minutes at 4 °C, and the supernatant was used as the mucin solution and stored at 4 °C;

[0099] Add 100 μL of mucin solution (1 mg / mL) to each well of a 96-well plate and store it in an incubator at 4 °C for 12 h; discard the mucus and wash it twice with 0.1% bovine serum albumin PBS; add 200 μL of 2% bovine serum albumin PBS and incubate it in a humidified chamber at room temperature for 2 h; wash it twice with 0.1% bovine serum albumin PBS;

[0100] Add 100 μL of bacterial suspension to each well of the above 96-well plate and incubate it overnight (16 h) at 4 °C; wash the wells with PBST and check the binding situation under an inverted microscope; pour out the solution, and after the wells are dry, measure the OD with an ELISA instrument 405 ; all measurements were performed in sextuplicate.

[0101] The specific results are shown in Table 1:

[0102] Table 1: Acid and bile salt tolerance and mucin adhesion of Lactobacillus gasseri FN136

[0103]

[0104] Note: *p < 0.05, **p < 0.01, indicating a significant difference in the activity between Lactobacillus gasseri BNR17 and Lactobacillus gasseri FN136 (n = 6, t-test).

[0105] a. Incubate the bacteria in artificial gastric juice (pH = 3.0) for 3 h, centrifuge, discard the supernatant, and determine the viable bacteria by plate counting.

[0106] b. Incubate the bacteria in MRS medium containing 3% (w / w) bile salt for 3 h, centrifuge, discard the supernatant, and determine the viable bacteria by plate counting.

[0107] The results showed that:

[0108] (1) The number of viable bacteria of Lactobacillus gasseri FN136 showed a downward trend after surviving for 3 h under the condition of simulated gastric juice (pH = 3.0), and the survival rate at 3 h was 81.0%, which was 1.21 times that of Lactobacillus gasseri BNR17.

[0109] (2) Lactobacillus gasseri FN136 was maintained in artificial intestinal juice for 3 h, and its survival rate was as high as 65.9%, which was 1.24 times that of Lactobacillus gasseri BNR17.

[0110] (3) The adhesion amount of Lactobacillus gasseri FN136 on the surface of porcine gastric mucus matrix was 0.081 (OD 405 ), which was 1.29 times that of Lactobacillus gasseri BNR17 on the surface of porcine gastric mucus matrix.

[0111] From the above experimental results, it can be seen that the Lactobacillus gasseri FN136 provided by the present invention has excellent gastrointestinal fluid tolerance and mucin adhesion, can enter the human intestine in a live state, and survive in the gastrointestinal organs of humans or animals to exert health effects, and the above characteristics are the basis for the strain to be used as a probiotic.

[0112] Example 3: Evaluation of the bile salt degradation activity and α-amylase inhibitory activity of Lactobacillus gasseri

[0113] The hydrolysis rates of glycochenodeoxycholic acid and taurodeoxycholic acid and the inhibitory activity experiments on α-amylase of Lactobacillus gasseri FN136 and Lactobacillus gasseri BNR17 were detected respectively; the specific steps are as follows:

[0114] 1. Determination of bile salt hydrolase activity:

[0115] First, the activated Lactobacillus gasseri BNR17 or Lactobacillus gasseri FN136 (activation conditions: inoculated at 10% (v / v) in MRS medium, cultured at 37 °C for 16 hours, and the number reached 1×10 10 CFU / mL) was resuspended in 1 mL of phosphate buffer solution (PBS, 0.1 M, pH 6.0) containing 10 mM dithiothreitol (Phygene, product number: PH0362). The cells were lysed by ultrasonic lysis technology, and the cell-free extract was separated by centrifugation (rotation speed: 12000×g, 10 min, 4 °C).

[0116] 100 μL of the cell-free extract was mixed with 180 μL of phosphate buffer (company: Phygene, product number: PH1822) (0.1 M, pH 6.0), 10 μL of paraffin oil (company: Xilong Scientific, product number: 8012-95-1), and 10 μL of glycochenodeoxycholic acid (company: Macklin, product number: 16409-34-0) (10 mM) or taurodeoxycholic acid (company: Aladdin, product number: 14605-22-2) (10 mM); the mixture was incubated at 37 °C for 30 min; then the mixture was mixed with 300 μL of 15% (volume fraction) trichloroacetic acid to terminate the reaction; the supernatant was separated again by centrifugation (rotation speed: 25000×g, 10 min, 4 °C), 50 μL of the supernatant was mixed with 950 μL of ninhydrin reagent, reacted in boiling water for 15 minutes, immediately cooled in ice water, and the absorbance value at 570 nm was measured.

[0117] The preparation method of ninhydrin reagent is as follows: Mix 250 μL of citric acid buffer solution (0.5 M, pH 5.5) containing 1% (weight / volume ratio) of ninhydrin, 600 μL of glycerol and 100 μL of citric acid buffer solution. Convert the absorbance value into the concentration of amino acids through a pre-established standard curve. Use an enhanced BCA protein detection kit (company: Wuhan Sevier Biotechnology Co., Ltd., product number: G2026-200T) to measure the protein concentration in the supernatant.

[0118] BSH (taurocholate hydrolase) activity: It is defined by the number of micromoles of amino acids released per mg of protein per minute, and the unit is U / mg.

[0119] 2. Determination of α-amylase inhibitory activity:

[0120] Collect the cell-free supernatant from the overnight (cultured at 37 °C for 16 h) Lactobacillus gasseri BNR17 or Lactobacillus gasseri FN136 culture by centrifugation (5000×g, 10 min, 4 °C), and use the supernatant to evaluate the strain's effect on α-glucosidase; the specific steps are as follows:

[0121] First, activate (condition: inoculate 10% (v / v) into MRS medium and culture at 37 °C for 16 hours) Lactobacillus gasseri BNR17 or Lactobacillus gasseri FN136 (the number reaches 1×10 10 CFU / mL), and resuspend it in 1 mL of phosphate buffer solution (PBS, 0.1 M, pH 6.0) containing 10 mM dithiothreitol (Phygene, product number: PH0362). Break the cells by ultrasonic lysis technology and separate the cell-free supernatant by centrifugation (rotation speed: 12000×g, 10 min, 4 °C).

[0122] Mix 30 μL of cell-free supernatant, 50 μL of PBS (0.1 M, pH 6.8) and 50 μL of p-nitrophenyl α-D-glucopyranoside (25 mM), and then react at 37 °C for 10 min to obtain a mixture;

[0123] Subsequently, mix 30 μL of α-glucosidase (0.4 U / mL) with the above mixture, react at 37 °C for 30 min, and add 50 μL of Na2CO3 to terminate the reaction;

[0124] Evaluate the inhibitory effect of the strain on α-glucosidase by measuring the A405 absorbance value of the mixture, and evaluate the inhibition rate through the following equation:

[0125] Inhibition rate (%) = [1 - (A 样品 - A 空白 ) / (A 对照 - A 空白)]×100。

[0126] The results are shown in Table 2 as follows:

[0127] Table 2: Hydrolysis rates of strains on glycochenodeoxycholic acid and taurodeoxycholic acid and inhibitory activities on α-amylase

[0128]

[0129] Note: *p < 0.05, **p < 0.01, indicating significant differences in activities between Lactobacillus gasseri BNR17 and Lactobacillus gasseri FN136 (n = 6, t-test).

[0130] Table 2 shows that Lactobacillus gasseri FN136 has the activity of hydrolyzing glycochenodeoxycholic acid and taurodeoxycholic acid, especially with stronger hydrolyzing activity on glycochenodeoxycholic acid. Its hydrolyzing activity on glycochenodeoxycholic acid is 2.18 times that of Lactobacillus gasseri BNR17 (p < 0.01), and its effect on hydrolyzing taurodeoxycholic acid is 1.24 times that of Lactobacillus gasseri BNR17 (p < 0.05). The culture supernatant of Lactobacillus gasseri FN136 also has α-amylase inhibitory activity, and its activity is 1.27 times that of Lactobacillus gasseri BNR17.

[0131] From the above experimental results, it can be seen that the Lactobacillus gasseri FN136 provided by the present invention has excellent activity of hydrolyzing bile salts, which is one of the bases for its cholesterol-lowering effect in vivo. The metabolites of Lactobacillus gasseri FN136 also have the activity of inhibiting α-amylase, and this characteristic is one of the bases for the blood glucose-lowering effect of this strain.

[0132] Example 4: Evaluation of the antibacterial effect of Lactobacillus gasseri FN136 strain

[0133] 1. Preparation of strain fermentation broth

[0134] Lactobacillus gasseri FN136 and Lactobacillus gasseri BNR17 stored in glycerol were respectively streaked on MRS agar medium and anaerobically cultured at 37°C for 18 h; single colonies were picked and inoculated into MRS broth medium and anaerobically cultured at 37°C for 48 h. The cultured bacterial fermentation broth was placed in a centrifuge and centrifuged at 12000×g for 10 min, the supernatant was aspirated, the pH was adjusted to 7.0, and after filtration with a 0.22 μm sterile filter, it was stored in a refrigerator at 4°C for standby.

[0135] 2. Effect of strains on inhibiting pathogenic bacteria

[0136] The Oxford cup method was used to detect the inhibitory effect of Lactobacillus gasseri FN136 against several major intestinal pathogenic bacteria (including Staphylococcus aureus ATCC25923, Salmonella enterica ATCC14028, Listeria monocytogenes ATCC13932, Proteus mirabilis BNCC107943, and Bacillus cereus ATCC14579).

[0137] Specifically, 100 μL of each indicator bacteria suspension cultured in LB medium at 37 °C for 24 h (the concentration of each indicator bacteria was adjusted to: OD600 = 0.5) was dropped onto the LB plate, and evenly spread with a spreading rod until there were no visible water droplets. Then, a sterilized Oxford cup was taken with sterile forceps and gently placed on the surface of the LB solid medium. 200 μL of the fermentation broth (bacteria concentration: OD600 = 0.5) was aspirated and injected into the placed and stable Oxford cup.

[0138] The medium added with the supernatant was placed in a 4 °C refrigerator for 8 h, and then the petri dish after complete diffusion treatment was placed in a 37 °C constant temperature incubator. After culturing for 16 h - 24 h, the diameter of the inhibition zone was recorded. Each sample was repeated 3 times and the average value was calculated.

[0139] The experimental results are shown in Table 3 as follows:

[0140] Table 3: Antibacterial activity (diameter of inhibition zone, mm)

[0141]

[0142] Note: *p < 0.05, **p < 0.01, indicating a significant difference in the activity between Lactobacillus gasseri BNR17 and Lactobacillus gasseri FN136 (n = 6, t-test).

[0143] The results showed that the antibacterial effect of the fermentation supernatant of Lactobacillus gasseri FN136 screened in the present invention was better than that of Lactobacillus gasseri BNR17. Among them, the antibacterial effect of Lactobacillus gasseri FN136 against Bacillus cereus ATCC 14579 was 1.18 times that of Lactobacillus gasseri BNR17; the antibacterial effect of Lactobacillus gasseri FN136 against Listeria monocytogenes ATCC 13932 was 1.18 times that of Lactobacillus gasseri BNR17; the antibacterial effect of Lactobacillus gasseri FN136 against Proteus mirabilis BNCC 107943 was 1.11 times that of Lactobacillus gasseri BNR17; the antibacterial effect of Lactobacillus gasseri FN136 against Staphylococcus aureus ATCC 25923 was 1.38 times that of Lactobacillus gasseri BNR17; the antibacterial effect of Lactobacillus gasseri FN136 against Salmonella enterica ATCC 14028 was 1.15 times that of Lactobacillus gasseri BNR17.

[0144] From the above experimental results, it can be seen that Lactobacillus gasseri FN136 provided by the present invention has a better effect of inhibiting pathogenic bacteria compared with Lactobacillus gasseri BNR17, and this characteristic is the basis for the strain to be used as a probiotic.

[0145] Example 5: Sensitivity of Lactobacillus gasseri FN136 to Antibiotics

[0146] The MIC (minimum inhibitory concentration) is considered to be the lowest antibiotic concentration that can inhibit the growth of the strain and is used to evaluate the antibiotic resistance of the selected strain. The MIC is the amount of antibiotic contained in 1 mL of the highest dilution tube that completely inhibits bacterial growth.

[0147] The MICs of Lactobacillus gasseri FN136 and Lactobacillus gasseri BNR17 against different antibiotics were detected respectively, specifically as follows:

[0148] Nine antibiotics (vancomycin, chloramphenicol, penicillin, streptomycin, gentamicin, kanamycin, erythromycin, trimethoprim, and ampicillin) were dissolved and filtered with an appropriate solution.

[0149] Lactobacillus gasseri FN136 strain and Lactobacillus gasseri BNR17 with an OD600 of 0.5 were respectively inoculated into MRS broth medium supplemented with different final concentrations (2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg / mL) of antibiotics and anaerobically cultured at 37 °C for 24 - 36 h. Each sample was repeated 3 times. After incubation, the absorbance value at OD610nm in the 96-well plate was measured.

[0150] The results are shown in Table 4:

[0151] Table 4: Sensitivity results of strains to different antibiotics

[0152]

[0153] The results showed that Lactobacillus gasseri FN136 screened by the present invention was sensitive to these antibiotics to varying degrees, and Lactobacillus gasseri BNR17 was resistant to gentamicin, streptomycin, and trimethoprim.

[0154] From the above experimental results, it can be seen that Lactobacillus gasseri FN136 provided by the present invention is more sensitive to the above antibiotics compared with Lactobacillus gasseri BNR17, indicating that this probiotic has the potential to be used as a live bacterial product.

[0155] Example 6: Effects of Lactobacillus gasseri FN136 on Body Weight and Body Fat Rate of Mice Fed a High-Fat Diet

[0156] 1. Preparation of Bacterial Suspension

[0157] First, activate Lactobacillus casei FN136 (activation conditions: inoculate at 10% (v / v) in MRS medium and culture at 37°C for 16 hours until the quantity reaches 1×10 10 CFU / mL), and then resuspend it in 1 mL of phosphate buffer solution (PBS, 0.1 M, pH 6.0) (Phygene, product number: PH0362) to obtain a bacterial suspension with a concentration of 1×10 9 CFU / mL;

[0158] Prepare a bacterial suspension with the same concentration (1×10 9 CFU / mL) of Lactobacillus casei BNR17 using the same method as above.

[0159] 2. Animal experiment grouping

[0160] Purchase 6-week-old C57BL / 6 mice from Beijing Speywood and raise them in a temperature-controlled SPF animal house with a 12-hour light-dark cycle and free access to food. The feed formula is shown in Table 5. Start the animal experiment after one week of adaptive feeding. In this experiment, the treatment of experimental animals and all experimental procedures are carried out in accordance with the standard regulations of the Animal Experiment Committee.

[0161] Table 5: Feed formula (example)

[0162]

[0163]

[0164] Divide the C57BL / 6J mice into four groups as shown in Table 6 (12 mice in each group):

[0165] ① Blank control group (LFD): Fed with normal feed;

[0166] ② Model control group (HFD): Fed with high-fat feed;

[0167] ③ HFD + Lactobacillus casei FN136 treatment (HFD + FN136): On the basis of the HFD control group, treat the HFD group with Lactobacillus casei FN136. Specifically: start the intervention after the end of adaptive feeding and intragastrically administer the Lactobacillus casei FN136 bacterial suspension;

[0168] ④ HFD + Lactobacillus casei BNR17 treatment (positive control): On the basis of the HFD control group, treat with Lactobacillus casei BNR17. Specifically: start the intervention after the end of adaptive feeding and intragastrically administer the Lactobacillus casei BNR17 bacterial suspension.

[0169] Table 6: Experimental grouping

[0170]

[0171] 3. Measurement Indicators

[0172] (1) Body weight measurement: Weigh the body weight of the mice once every 7 days, record the weighing results, and calculate the body weight growth rate. The calculation formula is:

[0173] Body weight growth rate = 100 × final body weight / initial body weight;

[0174] (2) Body fat rate measurement: Sacrifice the mice at the end of the eighth week of feeding with a high-fat diet, dissect and weigh the weights of the peritesticular fat and abdominal fat, and calculate using the following formula:

[0175] Body fat rate = 100 × (weight of peritesticular fat + weight of abdominal fat) / body weight;

[0176] 4. Measurement Results

[0177] The body weight of the mice is as shown in A in Figure 3 . After 8 weeks of feeding with a high-fat diet, the average body weight of the Lactobacillus gasseri FN136 treatment group is 34.6 g, which is 1.15 times the average body weight (27.1 g) of the Lactobacillus gasseri BNR17 treatment group. The body weight growth rate of the Lactobacillus gasseri FN136 treatment group is lower than that of the Lactobacillus gasseri BNR17, but it does not reach a statistically significant difference.

[0178] Example 7: Regulation of blood glucose in mice by Lactobacillus gasseri FN136

[0179] 1. Preparation of Bacterial Suspension

[0180] Same as Example 6, and the experimental grouping is shown in Table 6.

[0181] 2. Animal Experiment Grouping

[0182] Same as Example 6.

[0183] 3. At the 8th week of the experiment, an oral glucose tolerance test (OGTT) was performed on the mice in each group. That is, the mice in each group were fasted overnight for 12 h, and their fasting blood glucose (0 h) was measured. Then, a glucose aqueous solution was intragastrically administered at 2.0 g glucose / kg body weight. The blood glucose of the mice in each group was measured by collecting blood from the tail vein using a blood glucose meter and a matching test strip at 0 min before gavage, 15 min, 30 min, 60 min, 90 min, and 120 min after gavage. The results are shown in Table 7. On the day after the last measurement of the oral glucose tolerance test, the serum insulin and glucagon were measured using a hypersensitive mouse insulin ELISA kit, and the insulin resistance index (HOMA-IR index) was calculated. The results are shown in Table 7.

[0184] Table 7: Indexes for successful establishment of a high-fat-induced insulin resistance mouse model

[0185]

[0186] 4. Measurement Results

[0187] As Figure 4 shown, after intragastric administration of glucose to mice, the blood glucose reached the peak at about 15 min. The peak blood glucose in the HFD group was significantly higher than that in the healthy control group of mice. Treatment with Lactobacillus gasseri FN136 and BNR17 could significantly inhibit the increase in blood glucose (p < 0.05). The area under the blood glucose curve in the HFD group was significantly higher than that in the healthy control group (P < 0.01). The area under the blood glucose curve in the Lactobacillus gasseri FN136 group (1217) returned to the healthy state, and the effect of FN136 was better than that of BNR17 (p < 0.05), which was 0.81 times that of the area under the blood glucose curve in the BNR17 group (1507).

[0188] The insulin resistance index (20.4) of mice in the HFD group was significantly higher than that of mice in the healthy control group (P < 0.001). Treatment with FN136 (9.1) and the BNR17 treatment group (15.0) significantly inhibited the increase in the insulin resistance index (p < 0.01). The effect of FN136 was better than that of BNR17 (p < 0.05), which was 0.53 times that of BNR17.

[0189] Example 8: Regulation of blood lipids in mice by Lactobacillus gasseri FN136

[0190] 1. Preparation of bacterial suspension

[0191] It was the same as in Example 6, and the experimental grouping is shown in Table 6.

[0192] 2. Animal experiment grouping

[0193] It was the same as in Example 6.

[0194] 3. Measurement indexes

[0195] (1) Measurement of serum low-density lipoprotein cholesterol level:

[0196] After feeding with high-fat diet for eight weeks, the mice were sacrificed, and the blood samples of each group of mice were collected and centrifuged at 4 °C, 1500 × g for 10 min. The serum low-density lipoprotein cholesterol level was detected using an ELISA kit (Company: Solarbio, Catalog number: BC5330-50T / 48S).

[0197] (2) Measurement of serum triglyceride level:

[0198] After feeding with high-fat diet for eight weeks, the mice were sacrificed, and the blood samples of each group of mice were collected and centrifuged at 4 °C, 1500 × g for 10 min. The serum triglyceride level was detected using an ELISA kit (Company: Nanjing Jiancheng, Catalog number: A110-1-1).

[0199] 4. Experimental results

[0200] As Figure 5 shown, the triglyceride content in the blood of mice in the HFD group was 1.25 mmol / L, and the low-density lipoprotein content was 1.85 mmol / L, with significantly increased contents (p < 0.05);

[0201] After intervention with Lactobacillus gasseri FN136, the triglyceride content in the blood of mice decreased to 0.96 mmol / L, and the low-density lipoprotein content decreased to 1.29 mmol / L; after intervention with BNR17, the triglyceride content in the blood of mice decreased to 1.04 mmol / L, and the low-density lipoprotein content decreased to 1.45 mmol / L. It can be seen that the intervention of Lactobacillus gasseri significantly improved this situation.

[0202] Moreover, the improvement effect of Lactobacillus gasseri FN136 was better than that of the BNR17 group. The low-density lipoprotein level in the FN136 treatment group was 0.68 times that of the HFD group, while the level in the BNR17 treatment group was 0.76 times that of the HFD group.

[0203] Example 9: Relief of intestinal leakage in mice by Lactobacillus gasseri FN136

[0204] 1. Preparation of bacterial suspension

[0205] The same as in Example 6.

[0206] 2. Grouping of animal experiments

[0207] The same as in Example 6.

[0208] 3. Measurement indexes

[0209] Mucosal barrier and chronic inflammation: After eight weeks of feeding with high-fat diet, the mice were sacrificed, and blood samples of each group of mice were collected and centrifuged at 4°C, 15,000×g for 10 min. Referring to the instructions of the ELISA kit, the levels of zonulin, lipopolysaccharide, lipopolysaccharide-binding protein, and IL-6 in the plasma were detected.

[0210] 4. Experimental results

[0211] As Figure 6 and 7 shown, there was obvious intestinal mucosal barrier damage in the blood of mice in the HFD group, mainly manifested as significantly increased levels of plasma zonulin (content: 130.72), lipopolysaccharide (content: 632.46), IL-6 (content: 42.37), and high-sensitivity C-reactive protein (content: 7.71) (p < 0.05);

[0212] After the intervention of Lactobacillus gasseri FN136, these four indicators in the blood of mice were significantly reduced (p<0.05). The effects of FN136 plasma zonulin (content: 103.03), lipopolysaccharide (content: 488.14), IL-6 (content: 33.37), and high-sensitivity C-reactive protein (content: 5.46) were superior to those of BNR17 (p<0.05) plasma zonulin (content: 96.00), lipopolysaccharide (content: 520.25), IL-6 (content: 30.84), and high-sensitivity C-reactive protein (content: 6.65), indicating that FN136 inhibited mucosal barrier damage.

[0213] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Lactobacillus gasseri ( Lactobacillus gasseri ) FN136, characterized in that, The Lactobacillus gasseri was deposited in the Guangdong Microbiological Culture Collection Center on July 23, 2024, with the collection number GDMCC No: 64909.

2. A probiotic preparation, characterized in that: Contains the Lactobacillus gasseri FN136 according to claim 1.

3. The probiotic preparation according to claim 2, characterized in that The probiotic preparation contains living cells and / or cell metabolites of the Lactobacillus gasseri FN136. The cell metabolites are cell-free extracts separated after breaking cells, or supernatants obtained by centrifuging cultured bacterial fermentation broth.

4. The probiotic preparation according to claim 2 or 3, characterized in that: The number of Lactobacillus gasseri FN136 in the probiotic preparation is ≥1×10 6 CFU / mL or 1×10 6 CFU / g.

5. A medicament containing the Lactobacillus gasseri FN136 according to claim 1.

6. The drug according to claim 5, characterized in that The medicine also contains pharmaceutically acceptable carriers and / or excipients.

7. Use of the Lactobacillus gasseri FN136 according to claim 1 in the preparation of a drug for weight loss and / or lowering blood lipids and / or preventing and treating type 2 diabetes and / or alleviating intestinal leakage and / or inhibiting intestinal pathogens in a target population, wherein the intestinal pathogens are one or more of Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, Proteus mirabilis and Bacillus cereus.

8. Food, feed, feed additive, or food additive containing the Lactobacillus gasseri FN136 according to claim 1.

9. The food, feed, feed additive, or food additive according to claim 8, characterized in that: The food is a functional food.

10. The food, feed, feed additive, or food additive according to claim 8, characterized in that: The food is a special medical purpose formula food.

11. The food, feed, feed additive, or food additive according to claim 8, characterized in that: The food is a health product.

12. Use of Lactobacillus gasseri FN136 according to claim 1 in the preparation of functional foods that help regulate body fat and / or help regulate intestinal flora and / or help maintain healthy blood lipid levels and / or help maintain healthy blood sugar levels.

13. Use of the Lactobacillus gasseri FN136 according to claim 1 in the preparation of a health product that helps regulate body fat and / or helps regulate intestinal flora and / or helps maintain healthy blood lipid levels and / or helps maintain healthy blood sugar levels.

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