Application of breast milk-sourced lactobacillus gasseri in reducing weight, reducing blood fat, preventing and treating type 2 diabetes mellitus, relieving intestinal leakage and inhibiting intestinal pathogenic bacteria

Abstract

The invention discloses application of breast milk-derived lactobacillus gasseri in reducing weight, reducing blood fat, preventing and treating type 2 diabetes mellitus, relieving intestinal leakage and inhibiting intestinal pathogens, and belongs to the technical field of microorganisms and the technical field of medicines. The invention provides a new application of lactobacillus gasseri in reducing weight, reducing blood fat, preventing and treating type 2 diabetes, relieving intestinal leakage and inhibiting intestinal pathogens. The traditional Chinese medicine composition can effectively prevent weight gain, body fat rate increase, low-density lipoprotein cholesterol and triglyceride increase of mice caused by high-fat diet, inhibit postprandial blood sugar and insulin resistance index increase, inhibit intestinal mucosa damage and growth of intestinal pathogenic bacteria, and effectively improve chronic diseases of mice with high-fat diet. Therefore, the lactobacillus gasseri FN136 has a huge application prospect in preparation of products for reducing weight, reducing blood fat, preventing and treating type 2 diabetes mellitus, relieving intestinal leakage and inhibiting intestinal pathogens.

Description

Technical Field

[0001] The invention relates to application of a strain of Lactobacillus gasseri derived from breast milk for reducing weight, lowering blood lipids, preventing and treating type 2 diabetes, alleviating intestinal leakage and inhibiting intestinal pathogens, and belongs to the technical field of microorganisms and the technical field of medicine. Background Art

[0002] Probiotics are defined as "live microorganisms that are beneficial to the human body when taken in a certain amount." One of the important criteria for judging probiotics is the ability to colonize 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 mouth and intestines through breastfeeding and interact to protect the baby from early infection and inflammation. They may also be one of the reasons why breastfed children are less likely to become obese than formula-fed children. Lactobacillus gasseri is one of the common bacteria in breast milk. Breast milk-derived Lactobacillus gasseri has a long history of consumption by infants and has good value for the development of probiotics.

[0003] Studies have shown that Lactobacillus gasseri BNR17 from Korean breast milk can synthesize glucose into oligosaccharides (Microbiology. 2010; 156: 1264-1274), thereby improving the occurrence of animal obesity, but its effect on humans has only been reported to be significant in the treatment of irritable bowel syndrome (Food Sci Biotechnol (2018) 27 (3): 853–857), and further screening of suitable strains or finding the best application targets are needed. The differences in microbiota among 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 Chinese mothers' breast milk may be more suitable for the physiological needs of the Chinese population.

[0004] Type 2 diabetes is a chronic disease caused by insufficient or inefficient use of insulin, and is common in adults. The disease is caused by genetics, 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 routine physical examinations. The typical symptoms of type 2 diabetes are called "three mores and one less", which refer to drinking more, eating more, urinating more, and losing weight; but in the early stages of the disease, there may be no symptoms or mild symptoms. As the disease progresses, the symptoms of hyperglycemia will become more obvious. In severe cases, ketoacidosis and hyperosmolar diabetic coma may occur, and it is easy to be combined with multiple infections, which can be life-threatening in severe cases. According to current data, the incidence of diabetes in my country is showing a rapid growth trend. The incidence of type 2 diabetes among adults in our country is about 10.6%, becoming one of the main 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 harm of obesity is that it increases the risk of diseases such as hypertension, dyslipidemia, cardiovascular disease and type 2 diabetes. Intestinal flora imbalance 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 number of Proteobacteria and Bacteroidetes and a decrease in the number of Firmicutes in the intestinal flora, while the number of Proteobacteria and Bacteroidetes is positively correlated with blood glucose levels. Lipopolysaccharides on the cell walls of Proteobacteria and Bacteroidetes can induce intestinal leakage, metabolic endotoxemia and chronic inflammation, leading to insulin resistance. Probiotics have important auxiliary application value in improving obesity and its complications because of their potential role in reshaping the intestinal flora.

[0006] At the same time, probiotics have an impact on blood lipids by metabolizing bile acids in the small intestine. Primary bile acids are synthesized in the liver using cholesterol as a substrate, and their structure contains bile acid and chenodeoxycholic acid. They combine with glycine or taurine to become water-soluble, and are secreted into bile and then released into the small intestine. 95% of primary bile acids are reabsorbed in a bound form in the ileum, and 5% are broken down by small intestinal bacteria into secondary bile acids that enter the colon and are excreted from the body with feces. Increased excretion helps promote the liver to synthesize primary bile, thereby lowering blood cholesterol.

[0007] The intestine is not only a digestive organ, but also the largest immune and detoxification organ of 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] It is well known that screening microorganisms in natural environments is uncertain, that is, it is difficult to screen exactly the same microbial strains in the same screening object, which is non-reproducible. On this basis, it is even rarer to screen multifunctional strains in actual situations.

[0009] In summary, it is necessary to further develop a multifunctional probiotic that can have good application prospects in weight loss, lowering blood lipids, preventing and treating type 2 diabetes, alleviating intestinal leakage and inhibiting intestinal pathogens. Summary of the invention

[0010] The problem to be solved by the present invention is to provide a strain of Lactobacillus gasseri FN136 which has probiotic properties and can reduce weight, lower blood lipids, prevent and treat type 2 diabetes, relieve intestinal leakage and inhibit intestinal pathogens.

[0011] The present invention provides Lactobacillus gasseri FN136, which has been deposited in the Guangdong Microbiological Culture Collection Center on July 23, 2024, with a deposit number of GDMCC No: 64909, and a deposit address of 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province. For the convenience of description, Lactobacillus gasseri FN136 is referred to as "Lactobacillus gasseri FN136" below.

[0012] The Lactobacillus gasseri FN136 is a strain of Lactobacillus gasseri with potential probiotic properties, which is 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 living 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 medicine 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 granules, capsules, tablets, pills or oral liquids.

[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.

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

[0021] In one embodiment of the present invention, the binder is a cellulose derivative, alginate, gelatin and / or polyvinyl pyrrolidone, etc.

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

[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, micro powder silica gel and / or polyethylene glycol.

[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, methylcellulose, sodium carboxymethylcellulose, citric acid, tartaric acid and / or sodium bicarbonate, etc.

[0026] The present invention also provides the use of the Lactobacillus gasseri FN136 in the preparation of a drug for reducing weight 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.

[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 granules, capsules, tablets, pills or oral liquids.

[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.

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

[0031] In one embodiment of the present invention, the binder is a cellulose derivative, alginate, gelatin and / or polyvinyl pyrrolidone, etc.

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

[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, micro powder silica gel and / or polyethylene glycol.

[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 intestinal pathogenic 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) Reduce fasting blood sugar;

[0039] (2) Alleviate the abnormal increase in food intake caused by type 2 diabetes;

[0040] (3) Alleviate the abnormal increase in water intake caused by type 2 diabetes.

[0041] The present invention also provides food, functional food, special medical formula food or health care product containing the Lactobacillus gasseri FN136.

[0042] In one embodiment of the present invention, the dosage form of the health care product includes granules, capsules, tablets, pills or oral liquids.

[0043] In one embodiment of the present invention, the food includes but is not limited to fermented food or beverage prepared by fermenting with the Lactobacillus gasseri FN136.

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

[0045] Biomaterial Deposit

[0046] Lactobacillus gasseri FN136, taxonomic name: Lactobacillusgasseri, was deposited in the Guangdong Microbiological Culture Collection Center on July 23, 2024, with the deposit number GDMCC No: 64909, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0047] Beneficial Effects

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

[0049] (2) The Lactobacillus gasseri FN136 of the present invention has good abilities of reducing weight, lowering blood lipids, preventing and treating type 2 diabetes, alleviating intestinal leakage and inhibiting intestinal pathogens. The screened Lactobacillus gasseri FN136 is compared with Lactobacillus gasseri BNR17, and has great advantages in preventing obesity and excessive body fat rate, improving postprandial blood sugar response and insulin resistance, improving blood lipid levels, improving chronic inflammation, and improving plasma zonulin and lipopolysaccharide, and can be used as a microbial drug or product with good industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0052] Figure 3 ; Effect of Lactobacillus gasseri FN136 in 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 levels in mice.

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

[0056] Figure 7 :Effect of Lactobacillus gasseri FN136 on improving intestinal leakage induced by high-fat diet in mice. DETAILED DESCRIPTION

[0057] The present invention will be further described below in conjunction with the accompanying drawings and specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions in the art. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art.

[0058] Lactobacillus gasseri BNR17 (KCTC 10902BP) was obtained from the Korean Collection of Type Cultures KCTC; Lactobacillus gasseri FN136 was isolated from breast milk of healthy mothers in Gannan Tibetan Autonomous Prefecture, Gansu Province (42 days after delivery). 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 medium involved in the following examples is as follows:

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

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

[0062] The detection methods involved in the following embodiments are:

[0063] Oral glucose tolerance test method:

[0064] In the eighth week of the experiment, mice in each group fasted overnight for 12 hours. The fasting blood glucose of mice (0 hours) was measured, and then 2.0 g glucose / kg body weight glucose aqueous solution was gavaged. Blood glucose was measured 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after gavage. Blood was collected from the tail vein, and blood glucose concentration was measured using a blood glucose meter and blood glucose test strips. The blood glucose change curve was drawn, and the area under the blood glucose curve was calculated.

[0065] Insulin resistance index method:

[0066] The day after the last oral glucose tolerance test, serum insulin and glucagon were measured using an ultrasensitive mouse insulin ELISA kit. The insulin resistance index was calculated as follows: 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 is isolated from breast milk of healthy mothers in Gannan Tibetan Autonomous Prefecture, Gansu Province.

[0071] (2) Isolation and purification of strains

[0072] Take about 10mL of breast milk and add it to an anaerobic tube. Immediately put it in dry ice and mail it to the laboratory for strain isolation. Take 1mL of breast milk and centrifuge it at 15000×g for 5min. Discard the supernatant and add it to 10mL of MRS broth medium. Vortex mix and enrich it in an anaerobic incubator at 37℃ for 48h. Then, take 1mL of the enrichment solution in the clean bench and dilute it tenfold with sterile saline. Select 10 -6 , 10 -7 , 10 -8 Three dilution gradients, 100uL of bacterial solution from each gradient was applied to MRS agar medium and cultured anaerobically at 37℃ for 48h. After the culture, a plate with 50-150 single colonies was selected from the agar medium, colonies with different morphology, size and color were picked, and streaked on the MRS agar plate for purification several times until the colonies on the entire plate were consistent in morphology, and single colonies were picked to MRS broth medium for bacterial enrichment culture. The obtained strain was suspended in PBS containing 20% ​​glycerol and stored at -80℃.

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

[0074] 2. Identification of Lactobacillus gasseri FN136

[0075] (1) Colony characteristics

[0076] After 48 hours of culture on MRS agar medium, Lactobacillus gasseri FN136 has a diameter between 0.3-2 mm, a round shape on the front, a convex shape on the side, neat edges, milky white, opaque, and a moist and smooth surface. Figure 1 .

[0077] (2) Bacterial characteristics:

[0078] Colony smear of Lactobacillus gasseri FN136: Gram-positive rod-shaped bacteria, about 0.9-1.2 μm wide, 3-8 μm long, no spore formation, see Figure 2 .

[0079] (3) Growth characteristics:

[0080] This strain is a facultative anaerobic bacterium that grows best at a temperature of 30-37°C. The highest and lowest initial growth pH are 8.0 and 5.0, and the optimal initial growth pH is 7.0.

[0081] (4) Lactobacillus-specific primer amplification and 16S rDNA identification

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

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

[0084]

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

[0086] Example 2: Confirmation of digestive juice tolerance and mucin adhesion of Lactobacillus gasseri FN136 strain

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

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

[0089] The Lactobacillus gasseri FN136 strain and the Lactobacillus gasseri BNR17 strain were activated in MRS medium respectively, and the activation conditions were as follows: 10% (v / v) was inoculated in MRS medium, cultured at 37°C for 16 hours, and the bacteria were washed twice after two generations of activation culture, and the washing method was as follows: 12000×g, 4°C centrifugation for 10 min, the bacteria were suspended in PBS and re-centrifuged, the bacteria were collected, and 3×10 7CFU / mL was suspended in 2mL simulated gastric fluid (pH=3) and incubated at 37°C for 3h. At the same time, the viable bacteria rate was determined by the MRS agar medium pouring method. Each sample was repeated 3 times and the average value was calculated. The survival rate of the strains in simulated gastric fluid was calculated according to the following formula.

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

[0091] Among them, N1 represents the number of live bacteria in the strain system after treatment with simulated gastric fluid; N0 represents the initial number of live bacteria in the strain system.

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

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

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

[0095] Among them, N1 represents the number of live bacteria in the strain system after treatment with simulated artificial intestinal fluid; N0 represents the initial number of live 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 hours, washed twice, washing method: 12000×g, 4°C centrifugation for 10 minutes, the bacteria were suspended in PBS and re-centrifuged, and the bacteria were collected. The bacterial count: 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), and after gentle rotation (250 rpm) at 4°C for 12 h, the mixture was centrifuged at 16,000 × g for 10 min at 4°C, and the supernatant was used as the mucin solution and stored at 4°C;

[0099] Add 100 μL (1 mg / mL) of mucin solution to each well of a 96-well plate and store in a 4°C incubator for 12 h; discard the mucus and wash twice with 0.1% bovine serum albumin PBS; add 200 μL of 2% bovine serum albumin PBS and incubate in a wet box at room temperature for 2 h; wash 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 overnight (16 h) at 4°C; wash the wells with PBST and check the binding status with an inverted microscope; pour out the solution, wait for the wells to dry, and 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: Gastric acid, bile salt tolerance and mucin adhesion of Lactobacillus gasseri FN136

[0103]

[0104] Note: *p<0.05, **p<0.01, indicating that the activity of Lactobacillus gasseri BNR17 and Lactobacillus gasseri FN136 was significantly different (n=6, T test).

[0105] a. Incubate the bacteria in artificial gastric juice (pH=3.0) for 3 hours, centrifuge, discard the supernatant, and count the live bacteria on a plate.

[0106] b. Place the bacteria in MRS medium containing 3% (w / w) ox bile salts, incubate for 3 hours, centrifuge, discard the supernatant, and count the live bacteria on a plate.

[0107] The results show:

[0108] (1) The number of live bacteria of Lactobacillus gasseri FN136 after surviving for 3 hours in simulated gastric fluid (pH=3.0) showed a downward trend. The survival rate at 3 hours was 81.0%, which was 1.21 times that of Lactobacillus gasseri BNR17.

[0109] (2) The survival rate of Lactobacillus gasseri FN136 was as high as 65.9% after being kept in artificial intestinal fluid for 3 hours, which was 1.24 times that of Lactobacillus gasseri BNR17.

[0110] (3) The adhesion of Lactobacillus gasseri FN136 on the surface of pig gastric mucus matrix was 0.081 (OD 405 ), which is 1.29 times the adhesion amount of Lactobacillus gasseri BNR17 on the surface of pig 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 living state, and survive in the gastrointestinal organs of humans or animals to exert health benefits, and the above characteristics are the basis of the strain as a probiotic.

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

[0113] The hydrolysis rates of Lactobacillus gasseri FN136 and Lactobacillus gasseri BNR17 on glycodeoxycholic acid and taurodeoxycholic acid and the inhibitory activity on α-amylase were tested 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: 10% (v / v)) was inoculated into MRS medium and cultured at 37°C for 16 hours until the number reached 1×10 10 CFU / mL), resuspended in 1 mL of phosphate buffer solution (PBS, 0.1 M, pH 6.0) (Phygene, Cat. No. PH0362) containing 10 mM dithiothreitol. The cells were disrupted by ultrasonic lysis technology, and the cell-free extract was separated by centrifugation (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, Catalog No.: PH1822) (0.1 M, pH 6.0), 10 μL of paraffin oil (Company: Xilong Scientific, Catalog No.: 8012-95-1) and 10 μL of glycodeoxycholic acid (Company: MacLean, Catalog No.: 16409-34-0) (10 mM) or taurodeoxycholic acid (Company: Roan, Catalog No.: 14605- 22-2) (10 mM); the mixture was incubated at 37°C for 30 min; the mixture was then mixed with 300 μL of 15% (volume fraction) trichloroacetic acid to terminate the reaction; the supernatant was separated again by centrifugation (speed of 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 min, immediately cooled in ice water, and the absorbance at 570 nm was measured.

[0117] The preparation method of ninhydrin reagent is as follows: 250 μL of citric acid buffer (0.5M, pH 5.5) containing 1% (weight / volume ratio) ninhydrin, 600 μL of glycerol and 100 μL of citric acid buffer solution are mixed. The absorbance value is converted to the concentration of amino acids by a pre-established standard curve. The protein concentration in the supernatant is determined using an enhanced BCA protein assay kit (Company: Wuhan Saiweier Biotechnology Co., Ltd., Catalog No.: G2026-200T).

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

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

[0120] The bacteria-free supernatant was collected from overnight (16 h at 37°C) cultures of Lactobacillus gasseri BNR17 or Lactobacillus gasseri FN136 by centrifugation (5000×g, 10 min, 4°C), and the supernatant was used to evaluate the strains for α-glucosidase; the specific steps were as follows:

[0121] First, activate Lactobacillus gasseri BNR17 or Lactobacillus gasseri FN136 (the number reaches 1×10 10 CFU / mL), resuspended in 1 mL of phosphate buffer solution (PBS, 0.1 M, pH 6.0) (Phygene, Cat. No. PH0362) containing 10 mM dithiothreitol. The cells were disrupted by ultrasonic lysis technology, and the bacteria-free supernatant was separated by centrifugation (speed 12000×g, 10 min, 4°C).

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

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

[0124] The inhibitory effect of the strain on α-glucosidase was evaluated by measuring the A405 absorbance value of the mixture, and the inhibition rate was evaluated by 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:

[0127] Table 2: Hydrolysis rate of glycodeoxycholic acid and taurodeoxycholic acid and inhibitory activity against α-amylase by strains

[0128]

[0129] Note: *p<0.05, **p<0.01, indicating that the activity of Lactobacillus gasseri BNR17 and Lactobacillus gasseri FN136 was significantly different (n=6, T test).

[0130] Table 2 shows that Lactobacillus gasseri FN136 has the activity of hydrolyzing glycodeoxycholic acid and taurodeoxycholic acid, especially the hydrolysis activity of glycodeoxycholic acid is stronger. Its activity of hydrolyzing glycodeoxycholic acid is 2.18 times that of Lactobacillus gasseri BNR17 (p<0.01), and its effect of 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 in 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 property is one of the bases for the strain to exert its blood sugar-lowering effect.

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

[0133] 1. Preparation of strain fermentation broth

[0134] The glycerol-free Lactobacillus gasseri FN136 and Lactobacillus gasseri BNR17 were streaked on MRS agar medium and cultured anaerobically at 37°C for 18 hours; a single colony was picked and placed in MRS broth medium and cultured anaerobically at 37°C for 48 hours. The cultured bacterial fermentation liquid was placed in a centrifuge and centrifuged at 12000×g for 10 minutes, the supernatant was aspirated, the pH was adjusted to 7.0, filtered with a 0.22μm sterile filter, and stored in a 4°C refrigerator for later use.

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

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

[0137] Specifically, take 100 μL of each indicator bacteria suspension cultured in LB medium at 37°C for 24 h (the concentration of each indicator bacteria is adjusted to: OD600 = 0.5), drop it on the LB plate, and spread it evenly with a spreading rod until no water droplets are visible. Then use sterile tweezers to take the sterilized Oxford cup, gently place it on the surface of the LB solid culture medium, absorb 200 μL of the fermentation liquid (bacterial concentration is: OD600 = 0.5), and inject it into the Oxford cup placed stably.

[0138] Place the culture medium with supernatant in a 4℃ refrigerator for 8 hours, then place the plate after diffusion treatment in a 37℃ constant temperature incubator, incubate for 16-24 hours, and record the diameter of the inhibition zone. Repeat 3 times for each sample and calculate the average value.

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

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

[0141]

[0142] Note: *p<0.05, **p<0.01, indicating that the activity of Lactobacillus gasseri BNR17 and Lactobacillus gasseri FN136 was significantly different (n=6, T test).

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

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

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

[0146] MIC (minimum inhibitory concentration) is considered to be the lowest antibiotic concentration that can inhibit the growth of strains and is used to assess the antibiotic resistance of selected strains. 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 to different antibiotics were tested respectively, specifically:

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

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

[0150] The results are shown in Table 4:

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

[0152]

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

[0154] It can be seen from the above experimental results that the Lactobacillus gasseri FN136 provided by the present invention is more sensitive to the above antibiotics than Lactobacillus gasseri BNR17, indicating that the probiotic has the potential to be used as a live bacteria product.

[0155] Example 6: Effects of Lactobacillus gasseri FN136 on body weight and body fat percentage of mice fed a high-fat diet

[0156] 1. Preparation of Bacterial Suspension

[0157] First, the activated Lactobacillus gasseri FN136 (activation conditions: 10% (v / v)) was inoculated into MRS medium and cultured at 37°C for 16 hours until the number reached 1×10 10 CFU / mL) and resuspended in 1 mL of phosphate buffer solution (PBS, 0.1 M, pH 6.0) (Phygene, Catalog No. PH0362) to obtain a concentration of 1×10 9 CFU / mL bacterial suspension;

[0158] The same concentration of bacterial suspension (1×10 9 CFU / mL).

[0159] 2. Animal Experiment Grouping

[0160] Six-week-old C57BL / 6 mice were purchased from Beijing Sibeifu and housed in a temperature-controlled SPF animal room with a 12-h light-dark cycle. The mice were allowed to eat freely and the diet formula was shown in Table 5. Animal experiments were started after one week of adaptive feeding. The treatment of experimental animals and all experimental procedures in this experiment were carried out in accordance with the standard regulations of the Experimental Animal Committee.

[0161] Table 5: Feed formula table (example)

[0162]

[0163]

[0164] C57BL / 6J mice were divided 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 diet;

[0167] ③HFD+Lactobacillus gasseri FN136 treatment (HFD+FN136): On the basis of the HFD control group, the HFD group was treated with Lactobacillus gasseri FN136, specifically: intervention began after the end of adaptive feeding, and Lactobacillus gasseri FN136 suspension was gavaged;

[0168] ④HFD+Lactobacillus gasseri BNR17 treatment (positive control): On the basis of the HFD control group, the mice were treated with Lactobacillus gasseri BNR17. Specifically, the intervention started after the end of adaptive feeding, and Lactobacillus gasseri BNR17 suspension was gavaged intragastrically.

[0169] Table 6: Experimental groups

[0170]

[0171] 3. Determination indicators

[0172] (1) Body weight measurement: Weigh the mice once every 7 days, record the weighing results, and calculate the body weight growth rate using the following formula:

[0173] Weight gain rate = 100 × final weight / initial weight;

[0174] (2) Body fat percentage determination: After eight weeks of high-fat diet feeding, mice were killed and dissected to measure the peritesticular fat and abdominal fat weights. The following formula was used to calculate the body fat percentage:

[0175] Body fat percentage = 100 × (peritoneal fat weight + abdominal fat weight) / body weight;

[0176] 4. Measurement results

[0177] Mouse weight Figure 3 As shown in A, after 8 weeks of high-fat diet feeding, the average body weight of the group treated with Lactobacillus gasseri FN136 was 34.6 g, which was 1.15 times the average body weight (27.1 g) of the group treated with Lactobacillus gasseri BNR17. The body weight growth rate of the group treated with Lactobacillus gasseri FN136 was lower than that of Lactobacillus gasseri BNR17, but it did 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, experimental grouping is shown in Table 6.

[0181] 2. Animal Experiment Grouping

[0182] Same as Example 6.

[0183] 3. In the eighth week of the experiment, oral glucose tolerance test (OGTT) was performed on each group of mice, i.e., each group of mice fasted overnight for 12h, and the fasting blood glucose (0h) of the mice was measured, and then 2.0g glucose / kg body weight glucose aqueous solution was gavaged, and blood glucose was measured by blood sampling through the tail vein of mice at 0min before gavage, 15min, 30min, 60min, 90min, and 120min after gavage using a blood glucose meter and supporting test strips. The blood glucose of each group of mice was measured at 0min before gavage, 15min, 30min, 60min, 90min, and 120min after gavage, and the results are shown in Table 7. The next day of the last oral glucose tolerance test was measured. Serum insulin and glucagon were measured using an ultrasensitive mouse insulin ELISA kit, and the insulin resistance index (HOMA-IR index) was calculated. The results are shown in Table 7.

[0184] Table 7: Successful modeling indicators of high-fat-induced insulin resistance mouse model

[0185]

[0186] 4. Measurement results

[0187] like Figure 4 The results showed that after mice were gavaged with glucose, blood glucose reached a peak at about 15 minutes. The peak blood glucose level in the HFD group was significantly higher than that in the healthy control group. Treatment with Lactobacillus gasseri FN136 and BNR17 significantly inhibited 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 a healthy state. 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 in the healthy control group (P<0.001). The FN136 (9.1) and BNR17 treatment groups (15.0) significantly inhibited the increase in the insulin resistance index (p<0.01). The effect of FN136 was superior to 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] Same as Example 6, experimental grouping is shown in Table 6.

[0192] 2. Animal Experiment Grouping

[0193] Same as Example 6.

[0194] 3. Determination indicators

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

[0196] After eight weeks of high-fat diet feeding, mice were killed, and blood samples from each group were collected and centrifuged at 4°C, 1500×g for 10 min. Serum low-density lipoprotein cholesterol levels were detected using an ELISA kit (company: Solarbio, catalog number: BC5330-50T / 48S).

[0197] (2) Determination of serum triglyceride levels:

[0198] After eight weeks of high-fat diet feeding, the mice were killed, and blood samples from each group were collected and centrifuged at 4°C, 1500×g for 10 min. Serum triglyceride levels were detected using an ELISA kit (company: Nanjing Jiancheng, item number: A110-1-1).

[0199] 4. Experimental results

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

[0201] After the intervention of Lactobacillus gasseri FN136, the triglyceride content in the blood of mice decreased to 0.96mmol / L, and the low-density lipoprotein content decreased to 1.29mmol / L; after the intervention of BNR17, the triglyceride content in the blood of mice decreased to 1.04mmol / L, and the low-density lipoprotein content decreased to 1.45mmol / 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 LDL level of the FN136-treated group was 0.68 times that of the HFD group, while the level of the BNR17-treated group was 0.76 times that of the HFD group.

[0203] Example 9: Effect of Lactobacillus gasseri FN136 on the alleviation of intestinal leakage in mice

[0204] 1. Preparation of bacterial suspension

[0205] Same as Example 6.

[0206] 2. Animal Experiment Grouping

[0207] Same as Example 6.

[0208] 3. Determination indicators

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

[0210] 4. Experimental results

[0211] like Figure 6 and 7 The results showed that there was obvious intestinal mucosal barrier damage in the blood of mice in the HFD group, mainly manifested by significant increases in 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 intervention with Lactobacillus gasseri FN136, the four indicators in the blood of mice were significantly reduced (p<0.05). The effects of FN136 on plasma zonulin (content: 103.03), lipopolysaccharide (content: 488.14), IL-6 (content: 33.37) and high-sensitivity C-reactive protein (content: 5.46) were better than 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 as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactobacillus gasseri FN136, It is 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, It is characterized in that Contains the Lactobacillus gasseri FN136 according to claim 1.

3. The probiotic preparation according to claim 2, It is characterized in that The probiotic preparation contains living cells and / or cell metabolites, and / or inactivated cells, and / or freeze-dried cells of the Lactobacillus gasseri FN136.

4. The probiotic preparation according to claim 2 or 3, It is 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, It is 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 reducing weight 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.

8. The use according to claim 7, It is characterized in that The intestinal pathogenic bacteria include, but are not limited to, one or more of Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, Proteus mirabilis and Bacillus cereus.

9. Food, functional food, special medical purpose formula food, health care product, feed, feed additive, food additive containing Lactobacillus gasseri FN136 according to claim 1.

10. Use of Lactobacillus gasseri FN136 according to claim 1 in the preparation of functional foods or health products that help regulate body fat and / or help regulate intestinal flora and / or help maintain healthy blood lipid levels (cholesterol / triglycerides) and / or help maintain healthy blood sugar levels.

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