Lactobacillus plantarum TY-M21 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum TY-M21 and application of the lactobacillus plantarum TY-M21, and the preservation number of the lactobacillus plantarum TY-M21 is CGMCC (China General Microbiological Culture Collection Center) No. 29230. The lactobacillus plantarum TY-M21 has relatively high inhibitory activity on alpha-glucosidase activity, and animal experiments prove that the lactobacillus plantarum TY-M21 can be used for remarkably relieving diabetes characteristics of mice with type II diabetes, remarkably reducing fasting blood-glucose level, remarkably improving insulin resistance and serum insulin level, remarkably reducing serum Glu level and serum TG level, and remarkably improving insulin resistance and serum insulin level. The pathological injury of pancreatic tissues is obviously improved; the lactobacillus plantarum TY-M21 has the advantages that the lactobacillus plantarum TY-M21 can be used for preparing the lactobacillus plantarum TY-M21, the serum ALT level and the serum AST level can be obviously reduced, the pathological injury of liver tissues can be obviously improved, a certain protection effect on the liver is achieved, and the lactobacillus plantarum TY-M21 has

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus plantarum TY-M21 and an application thereof. Background Art

[0002] Diabetes is a general term for metabolic disorders caused by insulin deficiency or reduced insulin action due to different causes and pathogenesis, which is mainly manifested as abnormal glucose metabolism in clinical practice. Diabetes is mainly divided into two types according to different pathogenic mechanisms: insufficient insulin secretion (type I) and insulin resistance (type II). Type II diabetes accounts for about 90% of diabetic patients. It is a chronic disease caused by genetic inheritance and environmental factors, and is mainly manifested as insulin resistance, progressive increase in blood sugar and decreased glucose tolerance. Due to the complex pathogenesis and many influencing factors of the disease, there are currently no good and effective prevention and treatment measures.

[0003] At present, drugs such as acarbose can achieve the effect of lowering blood sugar by inhibiting the activity of α-glucosidase. Specifically, α-glucosidase is a key enzyme in the process of sugar metabolism. The sugars in food are catalyzed and hydrolyzed into monosaccharides that can be absorbed by the small intestine under the action of α-glucosidase. Therefore, regulating the function of α-glucosidase can effectively block the conversion of sugars in food into monosaccharides, thereby lowering blood sugar levels. Therefore, inhibiting the activity of α-glucosidase is an important means to control postprandial blood sugar and reduce diabetes. However, this type of drug often brings great side effects to the gastrointestinal tract, including nausea, flatulence, diarrhea, etc. Therefore, there is an urgent need to develop new, efficient and safe hypoglycemic substances that can prevent, alleviate or treat type 2 diabetes.

[0004] Domestic and foreign studies have shown that lactic acid bacteria have a certain effect on the prevention and treatment of diabetes. They can achieve the purpose of lowering blood sugar and protecting pancreatic islets by regulating the proportion of intestinal flora and immune system of the host, and probiotics are generally recognized as safe for consumption. Therefore, it is of great significance to screen out probiotics with high inhibitory activity on α-glucosidase, high safety, and the functions of lowering blood sugar, preventing or improving type 2 diabetes. Summary of the invention

[0005] The present invention isolates a strain of Lactobacillus plantarum from fecal samples of centenarians in Jiangjin District, Chongqing. The strain has the effects of lowering blood sugar, lowering blood lipids, lowering insulin and insulin resistance, and can be used in the prevention, improvement or treatment of diabetes.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] The present invention provides a strain of Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension. The deposit number of Lactobacillus plantarum TY-M21 is CGMCC No.29230.

[0008] Another aspect of the present invention provides a composition, which includes the Lactobacillus plantarum TY-M21 of the present invention or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension.

[0009] Preferably, the above composition further comprises: one or more strains and / or one or more synthetic small molecules with chemical activity and / or one or more natural small molecules with chemical activity and / or one or more polypeptides.

[0010] In another aspect, the present invention provides a health food, functional food or medicine, which includes the Lactobacillus plantarum TY-M21 of the present invention or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension or the composition of the present invention.

[0011] Preferably, the above-mentioned medicine, health food or functional food is in the form of tablets, powders, pills, granules, capsules, emulsions, gels, patches or liquids.

[0012] In another aspect, the present invention provides a use of Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or the composition of the present invention in the preparation of a medicament for preventing, improving or treating diabetes.

[0013] Preferably, in the above application, diabetes is type 2 diabetes.

[0014] Preferably, in the above application, the drug has one or more of the following effects: (a) having the effect of alleviating weight loss and / or increased water intake caused by diabetes; (b) having the effect of lowering blood sugar levels; (c) having the effect of lowering insulin levels; (d) having the effect of lowering insulin resistance; (e) having the effect of lowering glycosylated hemoglobin levels; (f) having the effect of improving pancreatic and / or liver damage caused by diabetes; (g) having the effect of inhibiting α-glucosidase activity.

[0015] In another aspect, the present invention provides a use of the plant lactobacillus TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or the composition of the present invention in the preparation of health food; the health food has one or more of the following functions: (a) helps maintain healthy blood sugar levels; (b) helps maintain healthy blood lipid levels; (c) helps to assist in protecting the pancreas and / or liver damage caused by diabetes; (d) helps maintain healthy insulin levels.

[0016] On the other hand, the present invention provides a use of the plant lactobacillus TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or the composition of the present invention in the preparation of functional foods; the functional foods have one or more of the following functions: (a) helping to maintain healthy blood sugar levels; (b) helping to maintain healthy blood lipid levels; (c) helping to assist in protecting the pancreas and / or liver damage caused by diabetes; (d) helping to maintain healthy insulin levels.

[0017] The preservation information of Lactobacillus plantarum TY-M21 in the present invention is as follows: Lactobacillus plantarum TY-M21 was deposited at the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC for short) on December 4, 2023; the preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation date is December 4, 2023; the preservation number is CGMCC No. 29230; the classification name is: Lactobacillus plantarum.

[0018] The beneficial effects of the present invention include: the plant lactobacillus TY-M21 of the present invention has high inhibitory activity on α-glucosidase activity, and animal experiments have confirmed that it can significantly alleviate the diabetic characteristics of type II diabetic mice (weight loss and increased water intake), significantly reduce fasting blood sugar levels, significantly improve insulin resistance and serum insulin levels, significantly reduce serum Glu levels and serum TG levels, and significantly improve pathological damage to pancreatic tissue; it can also significantly reduce serum ALT levels and serum AST levels, significantly improve pathological damage to liver tissue, and has a certain protective effect on the liver, and the plant lactobacillus TY-M21 has no side effects and is highly safe. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The colony morphology (A) and Gram infection results (B) of TY-M21 strain;

[0020] Figure 2 The figures are the weight change trend (A), final weight (B), and average daily food intake (C) and water intake (D) of each mouse during the treatment period.

[0021] Figure 3 The effect of Lactobacillus plantarum TY-M21 on fasting blood glucose level in type Ⅱ diabetic mice;

[0022] Figure 4 Effects of Lactobacillus plantarum TY-M21 on glucose tolerance (A) and the area under the curve (B) in type Ⅱ diabetic mice;

[0023] Figure 5 The effect of Lactobacillus plantarum TY-M21 on serum insulin (A) and HOMA-IR (B) in type Ⅱ diabetic mice;

[0024] Figure 6 The effect of Lactobacillus plantarum TY-M21 on serum Glu (glucose) level in type Ⅱ diabetic mice;

[0025] Figure 7 The effect of Lactobacillus plantarum TY-M21 on serum TG (triglyceride) levels in type Ⅱ diabetic mice;

[0026] Figure 8 Effects of Lactobacillus plantarum TY-M21 on serum ALT (alanine aminotransferase) (A) and AST (aspartate aminotransferase) (B) levels in type Ⅱ diabetic mice;

[0027] Fig. 9 The effect of Lactobacillus plantarum TY-M21 on pancreatic tissue pathology in type Ⅱ diabetic mice;

[0028] Fig.10 The effect of Lactobacillus plantarum TY-M21 on liver histopathology in type Ⅱ diabetic mice;

[0029] In the above figures, different lowercase letters indicate significant differences between the two groups (p<0.05). DETAILED DESCRIPTION

[0030] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0031] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0032] In the present invention, "culture" refers to a collection of microbial cell groups or growths of a certain strain within a certain time and space; "metabolites" refer to a variety of chemical substances produced by the strain in the metabolic process, which can be primary metabolites (such as sugars, amino acids, nucleotides, etc., which are necessary for the growth and reproduction of microorganisms) or secondary metabolites (such as antibiotics, vitamins, enzymes, etc.); "bacterial suspension" refers to a suspension containing microbial cells of a strain; "bacterial powder" refers to a powdery substance obtained by drying the culture of a strain (such as spray drying, freeze drying, etc.); "fermentation broth" refers to a liquid obtained by inoculating a strain into a liquid culture medium and fermenting it for a period of time.

[0033] In the present invention, it should be noted that in order to further standardize the management of the "List of Bacteria that can be used for food" and the "List of Bacteria that can be used for infant food", the review agency organized experts to update the two lists of bacteria. For the classification and naming adjustments of bacteria in the list, a 2-year transition period is set. For the updated announcement, please refer to the website: http: / / www.nhc.gov.cn / sps / s7892 / 202208 / 1d6c229d6f744b35827e98161c146afb.shtml, where the "Lactobacillus plantarum (Latin: Lactobacillus plantarum)" in the "List of Bacteria that can be used for food" is changed to "Lactobacillus plantarum (Latin: Lactiplantibacillus plantarum)", and it can be known from the above that Lactobacillus plantarum in the present invention can also be called Lactobacillus plantarum, which is a strain that can be used for food, indicating that it has no toxic side effects and is safe.

[0034] The embodiment of the present invention provides a strain of Lactobacillus plantarum TY-M21 or a culture thereof, a metabolite, a fermentation liquid, a bacterial powder or a bacterial suspension. The deposit number of Lactobacillus plantarum TY-M21 is CGMCC No.29230.

[0035] It should be noted that the Lactobacillus plantarum in the present invention was isolated from feces samples of centenarians in Jiangjin District, Chongqing. After sequencing, its 16S rDNA sequence is shown in SEQ ID No. 1, which is as follows:

[0036] GATGCGGCTGCTATAATGCAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCC.

[0037] The embodiment of the present invention further provides a composition, which includes the Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension in the present invention.

[0038] It should be noted that the Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension in the present invention can be combined with other active ingredients to form a composition to improve the efficacy, and the other active ingredients can be pharmaceutically acceptable active ingredients or edible active ingredients.

[0039] In some specific examples, the above composition also includes: one or more strains and / or one or more synthetic small molecules with chemical activity and / or one or more natural small molecules with chemical activity and / or one or more polypeptides.

[0040] It should be noted that, as mentioned above, other active ingredients can be one or more combinations of strains, chemically active synthetic small molecules, chemically active natural small molecules or polypeptides; wherein, the strains, chemically active synthetic small molecules, chemically active natural small molecules or polypeptides can be one or more types, for example, the composition can also include other 1, 2 or 3 types of probiotics; for another example, the composition can also include 1, 2 or 3 types of chemically active synthetic small molecules; for another example, the composition can also include 1, 2 or 3 types of chemically active natural small molecules; the composition can also include 1, 2 or 3 types of polypeptides. In addition, chemically active synthetic small molecules refer to chemical small molecules synthesized by chemical synthesis, and chemically active natural small molecules are plant-derived small molecules that can be isolated from plants.

[0041] The embodiment of the present invention further provides a health food, functional food or medicine, which includes the Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension or the composition of the present invention.

[0042] It should be noted that the plant lactobacillus TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension or the composition of the present invention can be added with a pharmaceutically acceptable or edible carrier to be prepared into a health food, functional food or medicine; the pharmaceutically acceptable or edible carrier can prepare the plant lactobacillus TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension or the composition of the present invention into different dosage forms for easier use, transportation or storage; in addition, the pharmaceutically acceptable or edible carriers are all well known in the art.

[0043] In some specific examples, the above-mentioned medicines, health foods or functional foods are in the form of tablets, powders, pills, granules, capsules, emulsions, gels, patches or liquid dosage forms.

[0044] It should be noted that, as mentioned above, drugs, health foods or functional foods can be in different dosage forms. For example, for health foods, it can be capsules, granules or oral liquids; for example, functional foods can be liquid dosage forms with strong fluidity, such as yogurt or lactic acid bacteria beverages, or can be gels, such as coagulated yogurt that can be eaten with a spoon, or can be probiotic powder, probiotic tablets or biscuits, etc.; for another example, for drugs, it can be tablets, or capsules, or granular dosage forms, etc.; specific dosage forms can be prepared by adding different carriers according to methods known in the art. Specifically, for example, the preparation of tablets mainly uses diluents (such as starch, dextrin, sucrose or glycosides), absorbents (calcium sulfate, calcium hydrogen phosphate or light magnesium oxide, etc.), binders (povidone, syrup or hydroxypropyl methylcellulose, etc.), wetting agents (water, etc.) or disintegrants (dry starch, sodium hydroxymethyl starch or cross-linked polyvinylpyrrolidone, etc.); for example, the preparation of liquids mainly uses solubilizers, suspending agents, emulsifiers or colorants, etc.

[0045] The embodiments of the present invention also provide a use of Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or the composition of the present invention in the preparation of a drug for preventing, improving or treating diabetes.

[0046] It should be noted that the plant lactobacillus TY-M21 of the present invention has high inhibitory activity on α-glucosidase activity, and animal experiments have confirmed that it can significantly alleviate the diabetic characteristics of type 2 diabetic mice (weight loss and increased water intake), significantly reduce fasting blood sugar levels, significantly improve insulin resistance and serum insulin levels, significantly reduce serum Glu levels and serum TG levels, and significantly improve pathological damage to pancreatic tissue; it can also significantly reduce serum ALT levels and serum AST levels, significantly improve pathological damage to liver tissue, and has a certain protective effect on the liver. The plant lactobacillus TY-M21 has no side effects and is highly safe; it can be used in the preparation of drugs for preventing, improving or treating diabetes.

[0047] In some specific examples, in the above applications, diabetes is type 2 diabetes.

[0048] In some specific examples, in the above applications, the drug has one or more of the following effects: (a) it has the effect of alleviating weight loss and / or increased water intake caused by diabetes; (b) it has the effect of lowering blood sugar levels; (c) it has the effect of lowering insulin levels; (d) it has the effect of lowering insulin resistance; (e) it has the effect of lowering glycated hemoglobin levels; (f) it has the effect of improving pancreatic and / or liver damage caused by diabetes; (g) it has the effect of inhibiting α-glucosidase activity.

[0049] It should be noted that the Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or composition in the present invention can achieve the purpose of preventing, improving or treating diabetes through the effects of (a) to (g) above.

[0050] An embodiment of the present invention also provides an application of the plant lactobacillus TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or the composition of the present invention in the preparation of health food; the health food has one or more of the following functions: (a) helps maintain healthy blood sugar levels; (b) helps maintain healthy blood lipid levels; (c) helps to assist in protecting the pancreas and / or liver damage caused by diabetes; (d) helps maintain healthy insulin levels.

[0051] It should be noted that, through the verification of the present invention, it can be known that the plant lactobacillus TY-M21 in the present invention can reduce the blood sugar level, blood lipid level, insulin level of high-fat mice and assist in protecting the pancreas and / or liver damage caused by diabetes. Therefore, it and its culture, metabolites, fermentation broth, bacterial powder or bacterial suspension or composition can be prepared into health foods that maintain healthy levels of blood sugar, blood lipids, and insulin and protect the pancreas and / or liver damage caused by diabetes, and can be used for specific groups of people to achieve the effect of health care and assist the body to maintain a healthy state.

[0052] In some specific examples, the dosage form of the above-mentioned health food can be tablets, powders, pills, granules, capsules, emulsions, gels, patches or liquid dosage forms.

[0053] An embodiment of the present invention also provides an application of the plant lactobacillus TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension or the composition of the present invention in the preparation of functional foods; the functional foods have one or more of the following functions: (a) helping to maintain healthy blood sugar levels; (b) helping to maintain healthy blood lipid levels; (c) helping to assist in protecting the pancreas and / or liver damage caused by diabetes; (d) helping to maintain healthy insulin levels.

[0054] It should be noted that, as mentioned above, the plant lactobacillus TY-M21 in the present invention can reduce the blood sugar level, blood lipid level, insulin level of high-fat mice and assist in protecting the pancreas and / or liver damage caused by diabetes, so it and its culture, metabolites, fermentation broth, bacterial powder or bacterial suspension or composition can be prepared into functional foods that maintain healthy levels of blood sugar, blood lipids, and insulin and protect the pancreas and / or liver damage caused by diabetes, and used in daily diet to help the body maintain a healthy state.

[0055] In some specific examples, the dosage form of the functional food can be tablets, powders, pills, granules, capsules, emulsions, gels, patches or liquid dosage forms.

[0056] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0057] 1. Isolation, identification and screening of Lactobacillus plantarum TY-M21

[0058] (I) Isolation and identification of Lactobacillus plantarum TY-M21

[0059] (1) Experimental Materials

[0060] Lactobacillus plantarum TY-M21: isolated from the feces of centenarians in Jiangjin District, Chongqing, and deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit number CGMCC No.29230.

[0061] (2) Isolation and purification of Lactobacillus plantarum

[0062] The stool samples of the centenarians were diluted 10-fold in a gradient manner, from 10 -4 , 10 -5 and 10 -6 ; Take the dilution of 10 -4 , 10 -5 and 10 -6 100 μL of the sample was spread on MRS solid culture medium and cultured at 37°C for 48 h. Single colonies with different morphologies were selected and the strains were isolated by the plate streak method. The above steps were repeated until the purified strains were obtained. The cell morphology of the strains was observed under a microscope after Gram staining and photographed for record.

[0063] like Figure 1 As shown, strain TY-M21 formed single colonies with consistent colony morphology on MRS solid medium, most of which were round, white, and had a smooth and moist surface ( Figure 1 A); after Gram staining, the strain cells were observed under a microscope to be purple, short rod-shaped and uniform in morphology, and were determined to be Gram-positive bacteria ( Figure 1 B).

[0064] (3) 16S rDNA gene sequence analysis

[0065] The PCR reaction used a 25 μL system: 1 μL template; 12.5 μL 2×Taq PCR Master Mix; 1 μL primers (27F, 1492R); 25 μL sterile ultrapure water; reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 40 s; 55°C annealing for 40 s; 72°C extension for 1 min, 30 cycles in total; 72°C final extension for 10 min. After the reaction, the PCR amplification product was entrusted to Sangon Biotech (Shanghai) Co., Ltd. for bidirectional sequencing, and the sequencing results were analyzed by homology comparison using the BLAST program in NCBI.

[0066] After sequencing, the obtained PCR amplification product was subjected to homology comparison analysis using the BLAST program. The results showed that the sequence of the 16S rDNA PCR amplification product of strain TY-M21 (SEQ ID NO.1) had a homology of 99.93% with the known Lactobacillus plantarum (Lactobacillus plantarum, accession number: MT544832.1) in the Gene Bank database.

[0067] (II) α-glucosidase inhibition activity test

[0068] Carbohydrates ingested by the human body must be hydrolyzed into glucose monomers by key digestive enzymes (α-amylase and α-glucosidase) in the intestines to be absorbed by the human body; α-glucosidase is a hydrolase that exists in the brush border of the small intestinal mucosa and is responsible for catalyzing the hydrolysis of carbohydrates (oligosaccharides, disaccharides, etc.). Therefore, reducing postprandial blood sugar by inhibiting α-glucosidase activity has become an effective target for clinical intervention in diabetes.

[0069] After Lactobacillus plantarum was cultured in MRS liquid medium at 37°C for 18 h, the bacterial sludge was collected by centrifugation at 8000 r / min for 15 min at 4°C, washed twice with sterile 0.1 mol / L phosphate buffer (PBS, pH 6.8), resuspended in PBS, and the lactic acid bacteria concentration was adjusted to 10 9 CFU / mL; incubate at 37℃ for 16h, centrifuge at 4℃, 8000r / min for 15min, filter the supernatant with a 0.22μm filter membrane to obtain a cell-free supernatant (CFS); take a 96-well microtiter plate, add 25μL 2.5mmol / L 4-nitrophenyl-D-pyranoglucoside (PNPG) and 25μL CFS to the wells, incubate at 37℃ for 10min, then add 50μL 0.2U / mL α-glucosidase; react at 37℃ for 15min, finally add 100μL 0.2mol / L Na2CO3 solution to terminate the reaction, and use an enzyme reader to detect the absorbance (A) of the reaction solution at a wavelength of 405nm; the inhibition rate calculation formula is as follows:

[0070]

[0071] In the formula: positive is PNPG+PBS (0.1mol / L, pH6.8)+enzyme+Na2CO3; negative is PNPG+PBS (0.1mol / L, pH6.8)+PBS (0.1mol / L, pH6.8)+Na2CO3; sample is PNPG+sample+enzyme+Na2CO3; sample blank is PNPG+sample+PBS (0.1mol / L, pH6.8)+Na2CO3.

[0072] The test results show that the inhibition rate of Lactobacillus plantarum TY-M21 on α-glucosidase activity is (19.26±8.50)%, which is an ideal strain with high efficiency in inhibiting α-glucosidase activity.

[0073] 3. Determination of drug resistance of strains

[0074] The paper diffusion method was used to determine the drug resistance of the strain (the drug resistance of the strain is an important criterion for evaluating the safety of the strain), which specifically included: culturing the strain in MRS liquid medium at 37°C for 24 hours, taking 5 mL of the bacterial solution and centrifuging it at 4000 r / min for 10 minutes, discarding the supernatant, adding 5 mL of sterile physiological saline and mixing evenly to prepare a bacterial suspension, and taking a bacterial concentration of 10 8 cfu / mL of plant lactobacillus TY-M21 bacterial suspension 100 μL was spread on MRS agar plate, antibiotic tablets (diameter = 6 mm) were placed on the surface of the agar plate, and the diameter of the inhibition zone was measured using a vernier caliper after culturing at 37°C for 48 h; the content of each antibiotic in the drug sensitivity paper used was: CTR ceftriaxone (30 μg), PIP piperacillin (100 μg), TET tetracycline (30 μg), CPZ cefoperazone (75 μg), AMP ampicillin (10 μg), CAZ ceftazidime (30 μg), E erythromycin (15 μg), PEN penicillin (10 U), CXM cefuroxime sodium (30 μg), MI minocycline (30 μg). The test results are shown in Table 1 below ("S" means sensitive, "M" means moderately sensitive).

[0075] Table 1 Drug resistance results of Lactobacillus plantarum TY-M21

[0076] antibiotic name Diameter of inhibition zone (mm) result CTR (cefotaxime) 31.43±1.69 S PIP (piperacillin) 36.04±0.53 S TET (Tetracycline) 17.29±1.52 M CPZ (Cefoperazone) 22.55±0.54 S AMP (Ampicillin) 32.57±0.59 S CAZ (Ceftazidime) 25.14±0.38 S E(erythromycin) 28.09±0.52 S PEN (Penicillin) 22.57±1.38 M CXM (Cefuroxime Sodium) 30.84±1.23 S MI (minocycline) 23.13±0.30 S

[0077] As shown in Table 1, the plant lactobacillus TY-M21 of the present invention is moderately sensitive to two antibiotics, tetracycline and penicillin, and is sensitive to eight antibiotics, including ceftriaxone, piperacillin, cefoperazone, ampicillin, ceftazidime, erythromycin, cefuroxime sodium, and minocycline, and has high safety.

[0078] 2. Animal Experiments

[0079] 1. Grouping and treatment of experimental animals

[0080] Thirty-two 5-week-old SPF-grade C57BL / 6J male mice were selected and housed in a standardized laboratory at room temperature of 25°C±2°C, relative humidity of 50%±5%, and 12h light / 12h dark. The experiment began after one week of adaptive feeding. After the adaptation period, the mice were randomly divided into a normal group, a model group, an acarbose group, and a TY-M21 group, with 8 mice in each group. The experimental period was 16 weeks, and the animals in each group had free access to water during the experiment. The normal group consumed ordinary feed, and the mice in the model group, acarbose group, and TY-M21 group were fed a high-fat diet (containing 34.9% fat, 1.3% carbohydrates, and 2.5% omega-3 fatty acids). Compound 26.3% and protein 26.2%) for 5 weeks; after fasting for 12h, all the mice were intraperitoneally injected with 100mg / kg body weight (BW) of fresh streptozotocin (STZ) dissolved in sodium citrate buffer to induce type II diabetes in mice (model group, acarbose group and TY-M21 group), and the normal group mice were injected with sodium citrate buffer; after 1 week, the fasting blood glucose levels of mice were measured, and mice with blood glucose levels exceeding 11.1mmol / L were considered to be type II diabetic mice; then the TY-M21 group was gavaged 10 times a day 9 The patients were treated with Lactobacillus plantarum TY-M21 suspension at 400 CFU / kg·BW. The acarbose group was gavaged with 40 mg / kg·BW acarbose solution every day, while the normal group and the model group were gavaged with 10 mL / kg·BW normal saline every day for 9 weeks.

[0081] Body weight was measured once a week during the experiment, and food and water intake was recorded every two days during gavage;

[0082] On the last day of the experiment, after fasting for 12 h, the mice were killed after their fasting blood glucose was measured. Blood samples were collected and centrifuged at 3000 rpm for 10 min at 4 °C. The upper serum was collected and stored at -80 °C for further analysis.

[0083] Tissue samples (pancreas and liver) were quickly removed and rinsed, and part of the tissue was fixed in 4% paraformaldehyde for 48 h. The rest of the tissue was frozen in liquid nitrogen and then stored at -80°C.

[0084] (II) Different test situations

[0085] (1) Changes in body weight, food intake, and water intake of mice

[0086] The changes in body weight of mice in each group are as follows Figure 2As shown in (A), the results showed that the body weight of the four groups of mice continued to increase in the first 5 weeks, with no significant difference among the groups; after STZ injection in the 7th week, the body weight of all high-fat diet-fed groups (model group, acarbose group and TY-M21 group) decreased significantly in weeks 8-9; after intervention with the drugs acarbose and Lactobacillus plantarum TY-M21, the weight loss of mice in the acarbose group and TY-M21 group was alleviated.

[0087] The final body weight (week 16) of each group of mice was as follows: Figure 2 As shown in (B), the results showed that the body weight of mice in the model group (20.24±2.83g) was significantly lower than that in the normal group (25.19±2.95g), while the body weight of mice in the acarbose group (23.94±1.43g) and the TY-M21 group (24.84±1.57g) was significantly increased compared with that of mice in the model group (p<0.05).

[0088] The average daily food intake of mice in each group is as follows Figure 2 As shown in (C), the results showed that during the entire treatment process, the average daily food intake of mice in the model group (10.93±1.62g) was significantly higher than that in the normal group (5.28±0.27g), while the average daily food intake of mice in the acarbose group (9.34±0.55g) and the average daily food intake of mice in the TY-M21 group (10.48±1.79g) were both reduced compared with the average daily food intake of mice in the model group (p>0.05).

[0089] The average daily water intake of mice in each group is as follows Figure 2 As shown in (D), the results showed that during the entire treatment process, the average daily water intake of mice in the model group (13.21±2.41mL) was significantly higher than that in the normal group (6.90±0.71mL), while the average daily water intake of mice in the acarbose group (8.01±0.80mL) and the average daily water intake of mice in the TY-M21 group (8.91±0.85mL) were significantly reduced compared with the average daily water intake of mice in the model group (p<0.05).

[0090] The above results showed that Lactobacillus plantarum TY-M21 alleviated the diabetic characteristics of type 2 diabetic mice, and there was no significant difference in the effect with acarbose (p>0.05).

[0091] (2) Changes in fasting blood glucose in mice

[0092] At the end of the 7th week and the 16th week, the fasting blood glucose levels of mice in each group were tested using a Roche blood glucose meter (model: 929, purchased from Roche Diagnostics (Shanghai) Co., Ltd.). The results are shown in Table 1. Figure 3As shown in the results, compared with the normal group, the fasting blood glucose levels of the other three groups of mice at the 7th week were significantly higher than those of the normal group (p<0.05), indicating that the type 2 diabetes model was successfully established. After 9 weeks of treatment, the fasting blood glucose of the model group mice (18.49±3.44mmol / L) was still significantly higher than that of the normal group (4.13±0.69mmol / L), while compared with the model group, after 9 weeks of treatment, the fasting blood glucose levels of the acarbose group (7.24±2.88mmol / L) and the TY-M21 group (7.80±3.33mmol / L) were significantly decreased (p<0.05). The results showed that Lactobacillus plantarum TY-M21 has the effect of improving hyperglycemia in type 2 diabetic mice, and there is no significant difference in the effect of acarbose (p>0.05).

[0093] (3) Oral glucose tolerance test

[0094] To detect glucose homeostasis, oral glucose tolerance test (OGTT) experiment was performed on mice in each group at week 16. The mice were fasted for 12 h and had free access to water. Then, 2 g / kg·BW glucose was gavaged into the stomach. Blood glucose levels were measured at 0 min, 30 min, 60 min, 90 min and 120 min using a Roche blood glucose meter (model: 929, purchased from Roche Diagnostics (Shanghai) Co., Ltd.). Blood glucose curves were drawn and the area under the curve (AUC) was calculated.

[0095] The results are as follows Figure 4 As shown in (A) and 4(B), the results showed that at the end of the 16th week, the study found that the glucose tolerance of type II diabetic mice was impaired, and the AUC of OGTT in the model group mice was more than 3 times that of the normal group. After treatment with Lactobacillus plantarum TY-M21, the glucose tolerance level was significantly improved (p<0.05). The results showed that Lactobacillus plantarum TY-M21 has the potential to improve the glucose tolerance of type II diabetic mice, and there is no significant difference in the effect of acarbose (p>0.05).

[0096] (4) Determination of serum indicators

[0097] The serum insulin level was detected by enzyme-linked immunosorbent assay kit (purchased from Shanghai Yuanju Biotechnology Center); the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated as follows: HOMA-IR = fasting blood glucose (mmol / L) × fasting serum insulin (mIU / L) / 22.5. The levels of Glu, ALT, AST and TG in mouse serum were determined by automatic biochemical analyzer (model: BS-460, purchased from Shenzhen Mindray Animal Medical Technology Co., Ltd.).

[0098] 1) Experiment on serum insulin and HOMA-IR levels in mice

[0099] Insulin is secreted by pancreatic β cells and can control the synthesis and metabolism of carbohydrates in the body. Insulin resistance is one of the main pathogenesis of type 2 diabetes and can be assessed by HOMA-IR. Improving insulin resistance is a good way to treat diabetes.

[0100] The serum insulin level and HOMA-IR level of each group of mice were tested. Figure 5 (A) and Figure 5 As shown in (B), the results showed that the serum insulin and HOMA-IR levels of the model group mice were significantly higher than those of the normal group (p<0.05); compared with the model group, the acarbose group and the TY-M21 group significantly reduced the insulin and HOMA-IR levels (p<0.05). The above results show that Lactobacillus plantarum TY-M21 can improve the serum insulin level of type II diabetic mice and reduce insulin resistance, and there is no significant difference in the effect of acarbose (p>0.05).

[0101] 2) Mouse serum Glu level experiment

[0102] The serum Glu levels of mice in each group were tested. Figure 6 As shown, the serum Glu level of mice in the model group was significantly higher than that in the normal group (p<0.05); compared with the model group, the Glu level in the acarbose group and the TY-M21 group was significantly reduced (p<0.05), and there was no significant difference in the drug effects of Lactobacillus plantarum TY-M21 and acarbose (p>0.05).

[0103] 3) Mouse serum TG level experiment

[0104] Triglyceride (TG) is an organic substance and a component of lipids. In normal plasma, TG maintains a dynamic equilibrium state. However, for diabetic patients, due to high blood sugar, the oxidation of sugar in mitochondria is relatively enhanced, which increases the synthesized cholesterol substances. Continuous high blood sugar will promote insulin-induced hepatic HMGCOA (3-hydroxy-3-methylglutaryl coenzyme A) reductase synthesis, thereby increasing cholesterol synthesis and causing serum TG levels to rise.

[0105] The serum TG levels of mice in each group were tested. Figure 7 As shown, the results showed that the serum TG level of mice in the model group was significantly higher than that in the normal group (p<0.05); compared with the model group, the TG level in the acarbose group and the TY-M21 group was significantly reduced (p<0.05), and there was no significant difference in the drug effects of Lactobacillus plantarum TY-M21 and acarbose (p>0.05).

[0106] 4) Mouse serum ALT and AST level experiment

[0107] Serum ALT and AST levels are important indicators for evaluating liver function. When the liver is damaged, the permeability of the liver cell membrane increases, and ALT and AST are released into the blood.

[0108] The serum ALT and AST levels of mice in each group were tested. Figure 8 As shown in (A) and 8(B), the results showed that the serum ALT and AST levels of the model group mice were significantly higher than those of the normal group (p<0.05); compared with the model group, the serum ALT and AST levels of the acarbose group and the TY-M21 group were significantly reduced (p<0.05); the above results showed that Lactobacillus plantarum TY-M21 had a protective effect on the liver of type II diabetic mice, and there was no significant difference in the drug effects of Lactobacillus plantarum TY-M21 and acarbose (p>0.05).

[0109] (5) Histopathological analysis

[0110] Mouse liver and pancreatic tissues were dehydrated in a series of ethanol solutions, embedded in paraffin, and cut into 4 μm thick sections; after dewaxing and dehydration, the sections were stained with hematoxylin and eosin (H&E) for morphological examination; the stained liver and pancreatic tissues were observed under an optical microscope, and images were taken for histopathological analysis.

[0111] 1) Pancreatic pathology analysis

[0112] Pancreatic sections were stained with H&E to evaluate the protective effect of Lactobacillus plantarum TY-M21 on pancreatic β-cell damage in diabetic mice. Fig. 9 As shown in the figure, the pancreatic tissue structure of mice in the normal group was intact, and the islet cells were normal in morphology and uniform in size. The pancreatic tissue of mice in the model group was severely damaged, the islets were significantly atrophied, the number of pancreatic β cells was significantly reduced compared with the normal group, and there were bleeding spots and inflammatory cell infiltration in the intercellular space; compared with the model group, acarbose and Lactobacillus plantarum TY-M21 intervention for 9 weeks can significantly alleviate the atrophy of the islets in type II diabetic mice, and reduce the apoptosis and cell damage of pancreatic β cells.

[0113] 2) Liver pathology analysis

[0114] H&E staining of liver sections was performed to evaluate the protective effect of Lactobacillus plantarum TY-M21 on liver cell damage in type II diabetic mice. Fig.10As shown, the results showed that the liver cell structure of the normal group mice was normal, the nuclear membrane was intact, the nucleolus was clearly visible, and there was no lipid droplet and inflammatory cell infiltration in the liver cytoplasm, while the liver cells of the model group mice showed severe damage such as necrosis, inflammatory cell infiltration and vacuolation; after the mice were gavaged with Lactobacillus plantarum TY-M21, the liver tissue hepatocytes were arranged more regularly and had normal morphology, and round vacuoles were visible in the cytoplasm of a small number of hepatocytes. In addition, the number of vacuoles in the liver tissue hepatocytes of the mice in the acarbose group was significantly reduced compared with the model group. The above results show that Lactobacillus plantarum TY-M21 can significantly improve the liver lesions of type II diabetic mice, and the improvement effect is not significantly different from that of acarbose.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A strain of Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension, wherein the deposit number of Lactobacillus plantarum TY-M21 is CGMCC No.29230.

2. A composition, characterized in that The method comprises the plant lactobacillus TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension as described in claim 1.

3. The composition according to claim 2, further comprising: One or more strains and / or one or more chemically active synthetic small molecules and / or one or more chemically active natural small molecules and / or one or more polypeptides.

4. A health food, functional food or medicine, characterized in that: The invention comprises the Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension as described in claim 1 or the composition as described in claim 2 or 3.

5. The health food, functional food or medicine according to claim 4, characterized in that: The dosage form of drugs, health foods or functional foods is tablets, powders, pills, granules, capsules, emulsions, gels, patches or liquid dosage forms.

6. Use of the Lactobacillus plantarum TY-M21 or its culture, metabolite, fermentation broth, bacterial powder or bacterial suspension according to claim 1 or the composition according to claim 2 or 3 in the preparation of a medicament for preventing, improving or treating diabetes.

7. The use according to claim 6, characterized in that: Diabetes is type 2 diabetes.

8. The use according to claim 6 or 7, characterized in that: Medicines have one or more of the following effects: (a) having the effect of alleviating weight loss and / or increased water intake caused by diabetes; (b) having the effect of lowering blood sugar levels; (c) having the effect of lowering insulin levels; (d) It has the effect of reducing insulin resistance; (e) having the effect of lowering glycated hemoglobin levels; (f) having the effect of improving pancreatic and / or liver damage caused by diabetes; (g) It has the effect of inhibiting the activity of α-glucosidase.

9. Use of the plant lactobacillus TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension according to claim 1 or the composition according to claim 2 or 3 in the preparation of health food; the health food has one or more of the following functions: (a) Helps maintain healthy blood sugar levels; (b) Helps maintain healthy blood lipid levels; (c) Helps protect the pancreas and / or liver from damage caused by diabetes; (d) Helps maintain healthy insulin levels.

10. Use of the plant lactobacillus TY-M21 or its culture, metabolite, fermentation liquid, bacterial powder or bacterial suspension according to claim 1 or the composition according to claim 2 or 3 in preparing functional foods; the functional foods have one or more of the following functions: (a) Helps maintain healthy blood sugar levels; (b) Helps maintain healthy blood lipid levels; (c) Helps protect the pancreas and / or liver from damage caused by diabetes; (d) Helps maintain healthy insulin levels.

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