Application of lactobacillus rhamnosus FMBL L23004 CNN in preparation of medicine for preventing or treating diabetes

Abstract

The invention relates to the technical field of biology, and particularly provides application of lactobacillus rhamnosus FMBL L23004CNN in preparation of a medicine for preventing or treating diabetes mellitus, the lactobacillus rhamnosus FMBL L23004CNN is preserved in China Center for Type Culture Collection on June 26, 2023, the preservation number is CCTCC NO: M 20231099, and the lactobacillus rhamnosus FMBL L23004CNN has the effect of inhibiting the activity of alpha-glucosidase, alpha-amylase and dipeptidyl peptidase IV; oral glucose tolerance and insulin tolerance of T2DM mice can be remarkably reduced, abnormal fluctuation of postprandial blood glucose is reduced, fasting blood glucose, fasting insulin level and blood fat level are reduced, and insulin resistance is relieved; the composition of intestinal microbiome is obviously improved, the abundance and variety of effective microbial communities in intestinal tracts are improved, and the relative abundance of pathogenic bacteria is reduced; energy metabolism, carbohydrate metabolism and cofactor and vitamin metabolism of T2DM mice can be promoted, so that metabolic disorder caused by diabetes mellitus is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to application of Lactobacillus rhamnosus FMBL L23004 CNN in preparing a medicine for preventing or treating diabetes. Background Art

[0002] Diabetes is divided into type 1 diabetes and type 2 diabetes (T2DM), which is one of the biggest health problems in the world in the 21st century. Type 1 diabetes, also known as insulin-dependent diabetes, often occurs in children and adolescents. The onset is relatively rapid. Patients with absolute insulin deficiency are prone to ketoacidosis and must be treated with insulin. Type 2 diabetes accounts for the majority of diabetic patients. Early symptoms may not be obvious and are often discovered during physical examinations or when complications occur. The occurrence of type 2 diabetes is related to genetic factors on the one hand and to unhealthy lifestyles on the other hand; it can be treated with diet, exercise, oral hypoglycemic drugs and insulin. At present, the treatment drugs for diabetes include biguanides, sulfonylureas, non-sulfonylureas and thiazolidinediones, etc. These drugs can achieve the purpose of lowering blood sugar by promoting insulin secretion, improving glucose metabolism, improving insulin resistance and promoting glycogen synthesis. However, these drugs are also accompanied by a series of side effects, such as hypoglycemia, gastrointestinal discomfort, obesity, skin itching, weight gain, liver and kidney damage, etc. Therefore, finding "economical, natural and safe" material substitutes to alleviate or treat diabetes has become a technical problem that needs to be solved urgently by those skilled in the art.

[0003] Many important functions of human beings depend on intestinal flora. Changes in its composition, diversity and metabolites can cause a series of physiological disorders. Studies have found that the composition of intestinal flora in patients with type 2 diabetes is different from that in healthy people. For example, the number of some beneficial bacteria such as bifidobacteria and lactobacilli may decrease, while the number of some harmful bacteria such as conditional pathogens may increase. Long-term hyperglycemia, changes in dietary structure, drug treatment and other factors may cause changes in the function of intestinal flora, leading to intestinal flora imbalance, thereby reducing its fermentation ability and changing metabolites. Intestinal flora can produce short-chain fatty acids such as acetic acid, propionic acid, butyric acid by fermenting dietary fiber, etc. These short-chain fatty acids can stimulate intestinal L cells to secrete incretins such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), thereby promoting insulin secretion, inhibiting glucagon secretion, and regulating blood sugar levels. Intestinal flora imbalance can also lead to increased intestinal permeability, allowing harmful substances such as endotoxins to enter the blood circulation, causing chronic low-grade inflammation, and thus reducing insulin sensitivity. The above phenomena indicate that microbial intervention is an important potential approach to treat / alleviate T2DM.

[0004] Inspired by the traditional Chinese medicine theory of "medicine and food are of the same origin", the application of probiotics has been given new meaning. Probiotics are used as auxiliary treatment methods to make symbiotic probiotic products, such as co-fermentation with traditional Chinese medicine or co-fermentation with fruits and vegetables. The fermentation products are functional foods that play a role in alleviating T2DM. For example, invention patent CN116064285A discloses a strain of Lactobacillus rhamnosus ZJUIDS07, which can inhibit the activity of α-amylase, α-glucosidase and DPP-IV. Invention patent CN117487692A discloses a strain of Lactobacillus gasseri FLG-219, which has a hypoglycemic effect.

[0005] The research group of the inventor has long been committed to the research in the field of probiotics. In the previous research, a strain of Lactobacillus rhamnosus FMBL L23004CNN was screened from human feces and found to have an antibacterial effect. They applied for invention patent 202311683559.4, which has now been authorized. Based on the previous research, the inventor unexpectedly found that it also has the effect of lowering blood sugar and inhibiting the activity of α-glucosidase, α-amylase and dipeptidyl peptidase IV, so this application was written in the hope of obtaining protection. Summary of the invention

[0006] The primary purpose of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in the preparation of a drug for preventing or treating diabetes. The Lactobacillus rhamnosus FMBL L23004CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231099.

[0007] Preferably, the diabetes is type 2 diabetes.

[0008] The second object of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in the preparation of a blood sugar-lowering product. The Lactobacillus rhamnosus FMBL L23004CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099.

[0009] The third object of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in inhibiting α-amylase activity or preparing an α-amylase inhibitor. The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099.

[0010] The fourth object of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in inhibiting α-glucosidase activity or preparing an α-glucosidase inhibitor, wherein the Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M 20231099.

[0011] The fifth object of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in inhibiting dipeptidyl peptidase IV activity or preparing a dipeptidyl peptidase IV inhibitor. The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099.

[0012] The sixth object of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in the preparation of a product for preventing or treating insulin resistance. The Lactobacillus rhamnosus FMBL L23004CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M20231099.

[0013] The seventh object of the present invention is to provide an application of Lactobacillus rhamnosus FMBL L23004CNN in the preparation of a product for preventing or treating intestinal flora imbalance and metabolic dysfunction in diabetic patients. The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a preservation number of CCTCC NO: M 20231099.

[0014] Preferably, the Lactobacillus rhamnosus FMBLL23004 CNN can be a fermentation broth supernatant or an intracellular extract.

[0015] Preferably, the products include food, medicine, health products, dietary supplements, etc.

[0016] The beneficial effects of the present invention are as follows: (1) The present invention provides a new use of Lactobacillus rhamnosus FMBL L23004 CNN in the preparation of a drug for treating diabetes. The results show that both the fermentation supernatant and the intracellular extract have the effect of inhibiting the activities of α-glucosidase, α-amylase and dipeptidyl peptidase IV, and the inhibition rates of α-glucosidase and α-amylase are 18.62±1.74% and 67.22±5.37% respectively, and the inhibition rate of DPP-4 is 70.31±1.49%; (2) The Lactobacillus rhamnosus FMBL L23004 CNN can significantly reduce the oral glucose tolerance and insulin tolerance of T2DM mice, reduce the abnormal fluctuation of postprandial blood sugar, reduce fasting blood sugar, fasting insulin level, blood lipid level, thereby reducing insulin resistance to achieve the effect of lowering blood sugar; (3) The Lactobacillus rhamnosus FMBL L23004 CNN also had a significant impact on the composition of intestinal microorganisms, increasing the abundance and types of beneficial bacteria in the intestines of T2DM mice and reducing the relative abundance of pathogenic bacteria; (4) The Lactobacillus rhamnosus FMBL L23004 CNN can improve the metabolic disorders caused by diabetes by promoting the energy metabolism, carbohydrate metabolism, cofactor and vitamin metabolism of T2DM mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Body weight of mice in each group

[0018] Note: NC-normal group, DC-model group, FMBL-probiotic intervention group.

[0019] Figure 2 Food intake of mice in each group

[0020] Note: NC-normal group, DC-model group, FMBL-probiotic intervention group.

[0021] Figure 3 Fasting blood glucose levels of mice in each group

[0022] Note: NC-normal group, DC-model group, FMBL-probiotic intervention group.

[0023] Figure 4OGTT and area under the curve of mice in each group after 4 weeks of oral gavage intervention Note: NC-normal group, DC-model group, FMBL-probiotic intervention group; a-glucose tolerance graph, b-bar graph of area under the glucose tolerance curve; compared with the model group, **P<0.01 and *P<0.05; compared with the normal group, ##P<0.01 and #P<0.05.

[0024] Figure 5 ITT and area under the curve of mice in each group after 4 weeks of intragastric intervention Note: NC-normal group, DC-model group, FMBL-probiotic intervention group. a-insulin tolerance graph, b-area under the insulin tolerance curve bar graph. Compared with the model group, **P<0.01 and *P<0.05; compared with the normal group, ##P<0.01 and #P<0.05.

[0025] Figure 6 Four blood lipid conditions of mice in each group after 4 weeks of oral gavage intervention Note: NC-normal group, DC-model group, FMBL-probiotic intervention group; a-triglyceride content bar graph, b-total cholesterol content bar graph, c-high-density lipoprotein cholesterol content bar graph, d-low-density lipoprotein cholesterol content bar graph; compared with the model group, **P<0.01 and *P<0.05; compared with the normal group, ##P<0.01 and #P<0.05.

[0026] Figure 7 Morphological changes of liver cells in each group of mice after 4 weeks of intragastric intervention

[0027] Note: NC-normal group, DC-model group, FMBL-probiotic intervention group.

[0028] Figure 8 Analysis of intestinal microbial community composition at the genus level in each group of mice

[0029] Note: NC-normal group, DC-model group, PC-acarbose group, FMBL-probiotic intervention group

[0030] Fig. 9 The relative change ratio of relative abundance of different species in each intervention group compared with the model group (DC)

[0031] Note: NC-normal group, DC-model group, PC-acarbose group, FMBL-probiotic intervention group

[0032] Fig.10 KEGG enrichment pathway analysis of differential metabolites in feces of mice between probiotic group (FMBL) and model group (DC), positive control group (PC) and model group (DC)

[0033] Note: DC-model group, PC-acarbose group, FMBL-probiotic intervention group DETAILED DESCRIPTION

[0034] The following embodiments are provided to facilitate a better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following examples, unless otherwise specified, are all routine laboratory methods, and the experimental materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents.

[0035] The medium formula used is as follows:

[0036] MRS liquid medium (1L): peptone 10g; beef extract 10g; yeast extract 5g; glucose 20g; Tween 801mL; K2HPO42g; sodium acetate 5g; diammonium hydrogen citrate 2g; MgSO4·7H2O 0.58g; MnSO4·4H2O0.25g, deionized water 1000mL;

[0037] LAMVAB solid medium (1L): peptone 10g; beef extract 8g; yeast extract 4g; glucose 20g, Tween 801mL; K2HPO4 2g; sodium acetate 5g, diammonium hydrogen citrate 2g; MgSO4·7H2O 0.2g; MnSO4·4H2O 0.05g; agar 20g; L-cysteine ​​hydrochloride 0.5g; vancomycin hydrochloride 20mg, sterilized at 115℃ for 20min. Example 1. Hypoglycemic effect of Lactobacillus rhamnosus FMBLL23004 CNN

[0038] 1. Strains

[0039] Experimental strain: Lacticaseibacillus rhamnosus FMBL L23004 CNN was isolated from human fecal samples and deposited in the China Center for Type Culture Collection on June 26, 2023. The deposit number is CCTCC NO: M 20231099. The deposit address is: Wuhan University, Wuhan, China, Tel: (027)-68754052, E-mail: cctcc@whu.edu.cn.

[0040] The control strain: Lactobacillus rhamnosus GG was purchased from China General Microbiological Culture Collection Center.

[0041] The strain was streaked on an MRS agar plate for 2-3 times, and a single colony was picked and cultured in an MRS liquid medium at 37°C for 16 hours. The bacterial solution was inoculated into an MRS liquid medium at a 2% (v / v) inoculation amount, and cultured at 37°C for 16 hours to obtain a fermentation liquid of the strain.

[0042] (1) Extracellular supernatant (CFS): The fermentation broth was centrifuged at 6000 r / min and 4°C for 10 min, filtered through a 0.22 μm water filter membrane, and the supernatant was retained for later use.

[0043] (2) Intracellular extract (CFE): The cells were rinsed with PBS three times, the OD value was adjusted to 1.0, and the cells were disrupted twice using a cell disruptor in expert mode. The cells were centrifuged at 8000 rpm for 20 min at 4°C, filtered through a 0.22 μm filter membrane, and the supernatant was collected.

[0044] 2. Determination of α-amylase inhibitory activity

[0045] 125 μL sample solution was mixed with 1 mg / mL α-amylase solution in equal volumes, incubated in a 37°C constant temperature water bath for 15 min, then the reaction solution was added to 250 μL 1% soluble starch solution at 37°C, reacted at 37°C for 15 min, then 500 μL DNS solution was added, and after 5 min in a boiling water bath, it was quickly cooled to room temperature, diluted 20 times and allowed to stand at room temperature, and the absorbance was measured at 540 nm. PBS solution (0.1 mol / L, pH=6.8) was used as a blank control for the α-amylase solution and the sample to be tested.

[0046] α-Amylase inhibition rate = [1-(AB) / (CD)]*100%

[0047] Wherein: A is the sample group, containing sample solution and α-amylase solution; B is the normal sample group, containing sample solution but not α-amylase solution; C is the control group, containing no sample solution but α-amylase solution; D is the normal group, containing no sample solution and no α-amylase solution.

[0048] 3. Determination of α-glucosidase inhibitory activity

[0049] 50 μL of fermentation supernatant and 100 μL of 1U / mL α-glucosidase (prepared with pH 6.8, 0.1mol / L phosphate buffer) were added to a test tube, mixed and reacted at 37°C for 10 min, 50 μL of 5 mmol / L pNPG solution (prepared with pH 6.8, 0.1mol / L phosphate buffer) was added, mixed and reacted at 37°C for 30 min, and then 1 mL of 0.1 mol / L Na2CO3 solution was added, and the absorbance was measured at 400 nm. PBS was used instead of samples and α-glucosidase as controls.

[0050] α-glucosidase inhibition rate = [1-(AB) / (CD)]*100%

[0051] Wherein: A is the sample group, containing sample solution and α-glucosidase solution; B is the normal sample group, containing sample solution but not α-glucosidase solution; C is the control group, containing no sample solution but α-glucosidase solution; D is the normal group, containing no sample solution and no α-glucosidase solution.

[0052] Table 1 In vitro inhibitory activity of Lactobacillus rhamnosus against α-amylase and α-glucosidase

[0053]

[0054]

[0055] Note: abc represent significant differences, P<0.05.

[0056] The results are shown in Table 1. The inhibition rates of the fermentation supernatant of Lactobacillus rhamnosus GG on α-amylase and α-glucosidase were 43.33% and 4.23%, respectively, and the inhibition rates of the fermentation supernatant of Lactobacillus rhamnosus FMBL L23004 CNN on α-amylase and α-glucosidase were as high as 67.22% and 18.62%, respectively. The inhibition rates of α-amylase and α-glucosidase of the intracellular extract of Lactobacillus rhamnosus GG were -21.49% and -19.21%, respectively, which indicated that the intracellular extract of Lactobacillus rhamnosus GG not only failed to inhibit but promoted the activities of the two enzymes; while the inhibition rates of α-amylase and α-glucosidase of the intracellular extract of Lactobacillus rhamnosus FMBL L23004 CNN were 9.01% and 3.51%, respectively, which indicated that the fermentation broth and intracellular extract of Lactobacillus rhamnosus FMBLL23004 CNN had good ability to inhibit the activities of α-amylase and α-glucosidase.

[0057] 4. Determination of DPP-IV Inhibitory Activity

[0058] In a 96-well microplate, add 25 μl of glycine-p-nitroaniline (0.2 mM) and 25 μl of fermentation supernatant and pre-incubate at 37°C for 10 minutes. Then, add 50 μl of DPP-IV (0.01 U / mL) and incubate at 37°C for 60 minutes; then add 100 μl of sodium acetate buffer (1 M, pH 4.0) to terminate the reaction, and measure the absorbance of the sample at 405 nm. In the reaction system, a PBS solution with a pH of 6.8 and a 0.1 mol / L concentration was used as a blank control for the DPP-IV solution and the sample to be tested.

[0059] DPP-IV inhibition rate = [1-(AB) / (CD)]*100%

[0060] Wherein: A is the absorbance value of the solution containing DPP-IV and the sample to be tested; B is the absorbance value of the solution containing no DPP-IV but containing the sample to be tested; C is the absorbance value of the solution containing DPP-IV but not containing the sample; D is the absorbance value of the solution containing no DPP-IV and the sample to be tested.

[0061] Table 2 DPP-4 inhibition rate of fermentation supernatant of strains

[0062]

[0063] abc indicates significant difference, P<0.05.

[0064] The results are shown in Table 2. The inhibition rate of DPP-IV by the fermentation supernatant of Lactobacillus rhamnosus GG was 8.33%, while the DPP-IV inhibition rate of the fermentation supernatant of Lactobacillus rhamnosus FMBL L23004 CNN was as high as 70.31%. This shows that Lactobacillus rhamnosus FMBL L23004 CNN has great application potential in lowering blood sugar and improving diabetes, and can be used in the preparation of products for lowering blood sugar, improving obesity and diabetes.

[0065] Example 2: Improvement effect of Lactobacillus rhamnosus FMBL L23004 CNN on T2DM mice

[0066] 1. Preparation of bacterial culture

[0067] The strain was streaked on an MRS agar plate for 2 to 3 times, and then a single colony was picked and expanded in MRS liquid medium at 37°C for 16 hours. The bacterial solution was inoculated into MRS liquid medium at a 2% (v / v) inoculation amount, and after culturing at 37°C for 18 hours, the strain fermentation liquid was obtained. The fermentation liquid of Lactobacillus rhamnosus FMBL L23004 CNN was centrifuged (4000r / min, 3min, 4°C), the supernatant was discarded, the bacteria were washed twice with 0.9% sterile saline, and the concentration of the bacterial suspension was adjusted to 1×10 9 CFU / ml for future use.

[0068] 2. Animal Experiments

[0069] 2.1 Establishment and grouping of type 2 diabetes mouse model

[0070] Twenty-four male C57BL / 6J mice (20 g ± 1 g) aged 8 weeks were selected and purchased from Henan Sikebeis Biotechnology Co., Ltd. Ordinary maintenance feed and high-fat feed H10060 were purchased from Xinjiang Hengchao Biotechnology Co., Ltd. After one week of feed adaptation, the mice were randomly divided into a normal group (NC, 6 mice) and an experimental group (18 mice). The normal group was fed with ordinary feed, and the experimental group was fed with high-fat feed. After four weeks of diet, the mice in the experimental group were intraperitoneally injected with 45 mg / kg streptozotocin (STZ, citric acid-sodium citrate buffer, pH 4.5, intraperitoneal injection volume of 0.1 mL / 10 g in mice) in small doses multiple times. One week after injection of streptozotocin, the fasting blood glucose (FBG) of the mice exceeded 16.5 mmol / L as the standard for model success.

[0071] After the model was successfully established, the experimental group was divided into a model group (DC), a positive control group (PC) and a probiotic intervention group (FMBL), with 6 mice in each group.

[0072] The normal group and the model group were gavaged with the same dose of 0.9% sterile saline for 4 weeks, the positive control group was given 200 mg / kg / d of acarbose, and the probiotic intervention group was given 10 mg / kg / d of Lactobacillus rhamnosus FMBLL23004CNN according to the weight of the mice;

[0073] The weight of each group of mice was weighed and the food intake was recorded using an electronic balance every week. During the intervention period, the fasting blood glucose of the mice was measured once a week at a fixed time.

[0074] 2.2 Sacrifice of T2DM mice and tissue collection

[0075] One week before the end of the experiment, the feces of each mouse was collected and placed in a 2 mL sterile centrifuge tube and stored at -80 °C for subsequent 16S rRNA and non-targeted metabolomics analysis. All mice were prohibited from eating feed before being killed, but they were free to drink sterile distilled water. After the mice were euthanized, orbital blood was collected from all mice, and the blood was left at room temperature for 1 hour to allow blood coagulation. The blood was centrifuged at 4 °C and 3500 rpm for 45 minutes, and the mouse serum was collected and stored at -80 °C for subsequent indicator analysis. The livers of the dissected mice were collected, part of the liver was cut and placed in 4% paraformaldehyde solution for tissue fixation, and the rest was quickly frozen in liquid nitrogen and stored at -80 °C for later use.

[0076] 2.3 Oral glucose tolerance test

[0077] One week before the end of the experiment, the oral glucose tolerance test (OGTT) of mice was measured. The mice were fasted for 12 h and had free access to water. The body weight was measured before the experiment to determine the amount of glucose gavage. Then the mice were gavaged with 2 g / kg glucose solution. The blood glucose levels were measured at 30, 60, 90, and 120 min, and the blood glucose curve was drawn. The area under the blood glucose curve (AUC) was calculated using GraphPadPrism 8.0.2.

[0078] 2.4 Insulin tolerance test

[0079] One week before the end of the experiment, the insulin tolerance test (ITT) of mice was measured. The mice fasted for 12 hours and had free access to water. The body weight was measured before the experiment to determine the amount of insulin injection. The mice fasted for 12 hours and had free access to water. Then 1U / Kg of insulin was injected intraperitoneally. The blood glucose levels were measured at 30, 60, 90, and 120 minutes, and the blood glucose curves were drawn. The area under the blood glucose curve (AUC) was calculated using GraphPadPrism8.0.2.

[0080] 2.5 Insulin-related index determination

[0081] Mouse serum was collected and the insulin index (FINS) in fasting mouse serum was determined by enzyme-linked immunosorbent assay (ELISA) kit. The insulin sensitivity index (ISI) was expressed as the natural logarithm of the reciprocal of the product of FBG (measured before the end of the experiment) and FINS, and the insulin resistance index (HOMA-IR) was calculated as HOMA-IR = (FBG × FINS) / 22.5; the β-cell function index (HOMA-β) was calculated as HOMA-β = (FINS × 20) / (FBG-3.5).

[0082] 2.6 Serum parameter determination

[0083] Hitachi automatic biochemical analyzer was used to detect serum lipid metabolism levels, including total cholesterol (TC), total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C).

[0084] 2.7 Histopathological sections

[0085] After the mice were dissected, part of the liver tissue was fixed with 4% paraformaldehyde at 4°C for 48 h, eluted with gradient ethanol, transparentized with xylene, embedded in paraffin, sliced, dried, stained with hematoxylin and eosin, and observed and photographed under a 400x microscope.

[0086] 3. Data processing

[0087] All data are presented as mean and standard deviation (SD). One-way analysis of variance (ANOVA) test was used to determine the significant differences between groups, and statistical significance was set at P < 0.05. Statistical analysis was performed using GraphPad Prism 8.0.2 software.

[0088] 4. Results

[0089] Continuous weight loss is one of the typical symptoms of type 2 diabetes. Figure 1 As shown in the figure, the normal group was fed with ordinary feed, and the type 2 diabetes modeling mice were fed with high-fat feed. The weight of mice in the normal group slowly increased with the feeding time and finally reached 25.1g; the weight of mice fed with high-fat feed increased sharply during the modeling process, and the weight of modeling mice was as high as 25.0g in the fifth week. After three consecutive injections of STZ in the 5th week, the weight of mice in the intervention group began to decrease except for the normal group; this is consistent with the changes in clinical weight characteristics of diabetic patients, indicating that the diabetes modeling is successful in terms of weight. After the modeling was successful, the weight of mice in the model group continued to decrease, while the weight of mice in the probiotic intervention group showed a trend of first decreasing and then increasing. At the 10th week, the weight of mice in the probiotic intervention group was basically consistent with the normal group and reached 25.0g. It shows that the intervention of Lactobacillus rhamnosus FMBL L23004 CNN can effectively improve the symptoms of weight loss in type 2 diabetic mice.

[0090] like Figure 2 As shown in the figure, throughout the experimental period, the food intake of mice in the model group and the probiotic intervention group was significantly higher than that of mice in the normal group. After STZ injection at week 5, the food intake of mice in the probiotic intervention group was significantly reduced compared with the model group, and gradually approached the food intake of mice in the normal group. This indicates that the administration of Lactobacillus rhamnosus FMBL L23004 CNN alleviated the symptoms of T2DM in mice and reduced their food intake.

[0091] A high fasting blood glucose level is the most representative feature of type 2 diabetes, and the level of fasting blood glucose is also the gold standard for judging the success of the mouse diabetes model. After STZ injection in the 5th week, the model was considered to be successful when the fasting blood glucose value of the mouse was greater than 16.5 mol / L for three consecutive days. Figure 3 As shown in the figure, the blood glucose of mice in the normal group changed dynamically within the range of 5-9mmol / L throughout the experimental period. The fasting blood glucose (FBG) level of the model group was within the range of 20-25mmol / L, indicating that the model was successfully established. Compared with the model group, the blood glucose level of T2DM mice after probiotic administration intervention was significantly reduced as the intervention time increased. This result shows that Lactobacillus rhamnosus FMBL L23004 CNN intervention can effectively improve / reduce the blood glucose level of T2DM mice.

[0092] Oral glucose tolerance test results Figure 4 As shown in the figure, after successful modeling, the area under the curve (AUC) of the model group increased significantly, and the area under the curve (AUC) of the probiotic intervention group decreased significantly compared with the model group. This indicates that the model group mice need a longer recovery time to regulate blood sugar, and the intervention of Lactobacillus rhamnosus FMBL L23004 CNN can significantly reduce the oral glucose tolerance of T2DM mice and effectively improve / restore the ability of T2DM mice to regulate blood sugar.

[0093] The results are as follows Figure 5 As shown in the figure, after successful modeling, the area under the curve (AUC) of the model group increased significantly. Compared with the model group, probiotic intervention significantly reduced the insulin tolerance of T2DM mice. This shows that Lactobacillus rhamnosus FMBL L23004 CNN intervention can effectively reduce / alleviate the insulin resistance of T2DM mice.

[0094] As shown in Table 3, the FINS and HOMA-IR indexes of mice in the model group increased significantly, indicating that the mice in the model group developed insulin resistance, could not maintain normal blood sugar levels, and had problems with blood sugar regulation. Compared with the model group, the FBG, FINS index, and HOMA-IR index of mice in the probiotic intervention group decreased significantly; the ISI and HOMA-β index increased significantly. The intervention of Lactobacillus rhamnosus FMBL L23004 CNN can significantly improve the insulin sensitivity of T2DM mice, improve the insulin resistance of T2DM mice, and promote the proliferation of pancreatic β cells, thereby restoring the ability to regulate blood sugar.

[0095] Table 3 Insulin related index

[0096]

[0097] like Figure 6 As shown in the results, the levels of total triglyceride (TC), total cholesterol (TG), and low-density lipoprotein cholesterol (LDL-C) in the model group mice were significantly higher than those in the normal group, indicating that the abnormal glucose metabolism in diabetic mice did cause abnormal cholesterol metabolism. The levels of TC, TC, and LDL-C in the probiotic intervention group mice were close to those in the normal group, significantly lower than those in the model group, while the HDL-C level was significantly higher than that in the model group. The above results indicate that Lactobacillus rhamnosus FMBL L23004 CNN intervention can effectively regulate the lipid metabolism of T2DM mice, improve the levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol in diabetic mice, and has the potential to reduce the risk of cardiovascular disease and thrombosis.

[0098] The histopathological results of mouse liver were as follows Figure 7As shown. Excessive lipid accumulation and deposition in the liver caused lipid droplet vacuoles of varying sizes to appear in the liver slices of the model group mice, and the fatty degeneration was more serious. The mice showed a certain degree of liver fat metabolism disorder symptoms. Compared with the model group, the number and volume of lipid droplet vacuoles in the liver slices of the mice in the probiotic intervention group decreased, and the liver cell structure became relatively clear and complete, indicating that Lactobacillus rhamnosus FMBL L23004 CNN can significantly reduce the liver fat accumulation and fatty degeneration of T2DM mice, and show a good recovery and protection effect on the liver.

[0099] Example 3 Effects of Lactobacillus rhamnosus FMBLL23004 CNN on the intestinal flora of T2DM mice 1. Analysis of intestinal microbial diversity and community structure

[0100] Before the end of the experiment, fecal samples were obtained by stimulating the mouse anus, and the feces of each group of mice were collected with sterile tweezers, placed in 2 mL sterilized cryovials and stored in a -80°C ultra-low temperature refrigerator for analysis of the mouse intestinal flora structure.

[0101] Refer to the instructions of EZNAStoolDNAKit to extract the total DNA of bacteria in feces, select the V3-V4 hypervariable region of 16S rRNA gene for PCR amplification, and use specific primers with Barcode:

[0102] 338F5′-barcode-ACTCCTACGGGAGGCAGCAG-3′

[0103] 806R5′-GGACTACHVGGGTWTCTAAT-3′

[0104] According to the concentration of PCR products, the samples were mixed at equal concentrations. After being fully mixed, the PCR products were purified by 1×TAE 2% agarose gel electrophoresis. The sequences with the main band size between 400-450bp were selected, the target bands were recovered by gel cutting, and further purified by Axy PrepDNA Gel Extraction Kit. The DNA concentration in the extracted PCR products was quantified by Quanti Fluor TMds DNAAssay Kit. The library was constructed using the Ultra TM DNA LibraryPrep Kit for lllumina library construction kit. After the constructed library was quantified by Qubit and the library was tested, it was sequenced on the MiSeq machine. The raw data obtained by sequencing were first spliced ​​and filtered to obtain valid data. The valid data was subjected to OTUs clustering and species classification analysis. According to the OTUs clustering results, the representative sequence of each OTU was annotated with species. According to the species annotation results, the species relative abundance column cumulative graph was generated, and the LEfSe (LDAEffect Size) method was used to test the significance of differences in the species composition of the grouped samples.

[0105] 2. Results

[0106] 2.1 Effect of Lactobacillus rhamnosus FMBL L23004 CNN on the structure and composition of intestinal flora in T2DM mice

[0107] The results are as follows Figure 8 As shown in the figure, the intestinal microorganisms of the normal group mice were mainly composed of Muribaculaceae, Akkermansia, Ligilactobacillus, Lachnospiraceae_NK4A136_group, Bacteroides and other major genera. The model group was composed of Bacteroides, Faecalibaculum, Erysipelatoclostridium, Ligilactobacillus and other major genera. The positive control group was mainly composed of Romboutsia, Faecalibaculum, Muribaculaceae and other genera. The probiotic intervention group was mainly composed of Romboutsia, Faecalibaculum, Ligilactobacillus and Lactobacillus.

[0108] Muribaculaceae can produce short-chain fatty acids using endogenous (mucin polysaccharides) and exogenous polysaccharides (dietary fiber). The alleviating effect of a plant-based diet on inflammatory bowel disease, obesity, and type 2 diabetes is associated with an increase in the abundance of Muribaculaceae. Romboutsia can regulate the intestinal microbiota and lipid metabolism of obese rats by upregulating metabolic pathways such as glycerolipid metabolism, cholesterol metabolism, adipocyte lipolysis regulation, and insulin resistance. Faecalibaculum is one of the important producers of butyrate, has anti-inflammatory effects, maintains the activity of bacterial enzymes, and protects the digestive system from intestinal pathogens. Akkermansia has the effects of improving fat deposition, glucose tolerance, and low-grade inflammation. Lactobacillus has been shown to have a positive effect on the regulation of blood glucose levels, the improvement of insulin sensitivity, the reduction of inflammatory responses, and the effects on energy metabolism and fat storage. In addition, Lactobacillus may reduce the risk of inflammation and metabolic endotoxemia by maintaining the integrity of the intestinal barrier and reducing the penetration of endotoxins. These mechanisms are essential for the prevention and management of type 2 diabetes. Limosilactobacillus is a dominant lactic acid bacteria naturally present in the intestines of humans and animals, with many excellent probiotic functions such as inhibiting pathogens, improving the intestinal environment, and regulating intestinal flora. Roseburia can improve intestinal biodiversity, increase glucose tolerance, help lose weight, and revitalize colon cells.

[0109] like Figure 8 As shown in the figure, compared with the model group, the relative abundance of Romboutsia (9.9%), Faecalibaculum (9.14%), Ligilactobacillus (5.72%), Lactobacillus (5.33%), Akkermansia (1.99%), Lachnoclostridium (3.13%) and other beneficial bacteria in the intestines of mice in the probiotic intervention group increased significantly. Fig. 9As shown, compared with the model group, the relative abundance of Romboutsia, Akkermansia, Faecalibaculum, Lactobacillus and Roseburia in the intestines of mice in the positive control group increased by 3.63, 3.75, 15.72, 0.7 and 3.9 times, respectively, while the relative abundance of Limosilactobacillus decreased by 0.2 times; while the relative abundance of Romboutsia, Akkermansia, Faecalibaculum, Lactobacillus, Limosilactobacillus and Roseburia in the intestines of mice in the probiotic intervention group increased significantly by 14.3, 2.45, 8.1, 12, 2.2 and 10.6 times, respectively.

[0110] Escherichia-Shigella, Helicobacter, Fusobacterium, Streptococcus, and Erysipelatoclostridium are conditional pathogens in the intestine. Fusobacterium, Streptococcus, Escherichia-Shigella, and Helicobacter are related to inflammation of the body and gastrointestinal tract; the relative abundance of Odoribacter in the intestine is positively correlated with hypertension; the relative abundance of Helicobacter is positively correlated with diabetes, which can lead to increased blood sugar and insulin resistance. Fig. 9 As shown in the figure, compared with the normal group, the relative abundance of Erysipelatoclostridium (9.41%), Helicobacter (6.21%), Escherichia-Shigella (4.58%), Fusobacterium (0.91%), Streptococcus (1.18%) and Odoribacter (3.64%) in the intestine of the model group mice increased significantly. Compared with the model group, the relative abundance of Erysipelatoclostridium (0.018%), Helicobacter (0.525%), Escherichia-Shigella (0.01%), Fusobacterium (0%), Streptococcus (0.24%) and Odoribacter (0.07%) in the intestine of the probiotic group mice decreased significantly.

[0111] The above results indicate that Lactobacillus rhamnosus FMBL L23004 CNN intervention had a significant effect on the composition of intestinal microorganisms in diabetic mice, significantly increased the types and relative abundance of beneficial bacteria in the intestines of T2DM mice, and reduced the relative abundance of pathogenic bacteria.

[0112] Example 4: Lactobacillus rhamnosus FMBLL23004 CNN repairs metabolic disorders caused by T2DM

[0113] 1. Sample Collection and Preparation

[0114] One week before the end of the experiment, the feces of mice in each group were collected, 3 volumes of methanol solution were added, vortexed for 5 minutes, and then centrifuged at 5000r / min for 10 minutes. The supernatant was taken and filtered through a 0.22μm organic filter membrane for UPLC-Q-TOFMS detection.

[0115] 2 Experimental conditions

[0116] 2.1 Chromatographic conditions

[0117] Chromatographic column: Waters Acquity UPLC BEHC18 column (2.1 mm × 50 mm × 1.7 μm); chromatographic column temperature: 40 ° C; injection volume: 5 μL; mobile phase: A is acetonitrile, B is 0.1% formic acid aqueous solution; flow rate: 0.3 mL / min; gradient elution conditions: 0-1 min (5%-30% A), 1-3 min (30%-50% A), 3-3.6 min (50%-53% A), 3.6-4.2 min (53%-60% A), 4.2-6 min (60%-80% A), 6-8 min (80%-100% A), 8-9 min (100% A).

[0118] 2.2 Mass spectrometry conditions

[0119] Electrospray ion source (ESI); ion source temperature 120℃; mass scan range m / z 100~1000; cone gas and desolvation gas were both nitrogen, with flow rates of 50L / h and 700L / h, respectively, and the desolvation gas temperature was 350℃. The capillary voltage was 3.0kV in positive ion mode and 2.0kV in negative ion mode; the cone voltage and extraction cone voltage were 40V and 5.0V in positive and negative ion modes, respectively. Sodium formate was used to establish a mass standard curve, and real-time mass calibration was performed for leucine enkephalin (LE); argon was used as collision gas for MS / MS analysis, with a low collision energy of 5eV and a high collision energy of 10~25eV.

[0120] 2.3 Data Analysis

[0121] Import the raw data from the machine into the software Compound Discoverer TM 3.3 (version 3.3.2.31, Thermo, Waltham, USA) was used for peak detection, filtering and alignment. Peaks that were not detected in more than 50% of the QC samples were filtered. The undetected peaks were filled with missing values ​​based on the software Fill Gaps algorithm. The total peak area normalization method was used for data correction to obtain a quantitative list of metabolites. Differentials were screened by variable projection importance (VIP) value and P value. When VIP ≥ 1 and P < 0.05, the metabolite molecules were statistically significantly different. MetaboAnalyst 6.0 was used to perform functional pathway enrichment analysis on the screened differential metabolites, and the KEGG Mapper tool was used for visualization analysis.

[0122] 3. Results

[0123] Non-targeted metabolomics analysis of the feces of mice in the normal group, model group, positive control and probiotic intervention groups detected 1934, 1494, 1551 and 1500 substances, respectively. These substances mainly include organic acids and their derivatives, lipids and lipid molecules, organic heterocyclic compounds, benzene ring compounds, phenylpropane and polyketones, organic oxygen-containing compounds, organic nitrogen compounds, nucleotides and analogs, alkaloids and their derivatives. The results of differential metabolite analysis showed that a total of 898 differential metabolites were identified in the normal group VS model group, of which 304 metabolites were significantly increased and 594 metabolites were significantly decreased. A total of 131 differential metabolites were identified in the positive control group VS model group, of which 97 metabolites were significantly increased and 34 metabolites were significantly decreased. A total of 298 differential metabolites were identified in the probiotic intervention group VS model group, of which 244 metabolites were significantly increased and 54 metabolites were significantly decreased. It can be seen that probiotic intervention has a significant effect on the fecal metabolites of T2DM mice.

[0124] Compared with the normal group, the content of Apabetalone, histidine, Cinncassiol A, and valine in the model group decreased by 7.26, 9.78, 33.98, and 2.41 times, respectively, and the content of monomethyl phthalate, 7-ketocholesterol, and 5-aminovaleric acid betaine increased by 33.33, 1.65, and 3.57 times, respectively. Compared with the model group, the content of beneficial substances in the positive control group increased, and the content of harmful substances such as monomethyl phthalate decreased. Compared with the model group, the contents of beneficial substances Apabetalone, histidine, Cinncassiol A, Valylproline, vitamin B5, N-acetyltaurine, 3-hydroxyphenylacetic acid, callous acid, Equisetin, etc. in the probiotic intervention group, which are beneficial to the improvement of diabetes, increased by 5.48, 0.63, 7, 0.65, 3.21, 1.05, 3.58, 2.84, 0.9, and 2.6 times, respectively, while the contents of monomethyl phthalate, 7-ketocholesterol, and 5-aminovaleric acid betaine decreased by 63%, 20%, and 27%, respectively. It can be seen that probiotic intervention, similar to the positive control, can regulate the metabolic disorders caused by diabetes by significantly increasing the contents of beneficial substances and reducing the contents of unfavorable substances in the intestines of T2DM mice.

[0125] KEGG enrichment analysis was performed on the differential metabolites, and the results were as follows Fig.10 As shown. Compared with the model group, the fecal metabolites of mice in the probiotic intervention group were mainly enriched in metabolic pathways such as amino acid metabolism, lipid metabolism, cofactor and vitamin metabolism, carbohydrate metabolism, energy metabolism, and nucleotide metabolism, which were mainly related to the digestive system, endocrine system, and nervous system. Compared with the model group, the fecal metabolites of mice in the positive control group were mainly enriched in metabolic pathways such as amino acid metabolism, lipid metabolism, cofactor and vitamin metabolism. Compared with the positive control group, probiotic intervention further promoted the energy metabolism, carbohydrate metabolism, cofactor and vitamin metabolism of T2DM mice by participating in the regulation of the host nervous system, digestive system, and endocrine system, thereby improving the metabolic disorders caused by diabetes.

[0126] In summary, the present invention provides a new use of rhamnosus casei FMBL L23004 CNN in the preparation of a drug for treating diabetes. The results show that both the fermentation supernatant and the intracellular extract have the effect of inhibiting the activities of α-glucosidase, α-amylase and dipeptidyl peptidase IV, and the inhibition rates of α-glucosidase and α-amylase are 18.62±1.74% and 67.22±5.37%, respectively, and the inhibition rate of DPP-4 is 70.31±1.49%. The rhamnosus casei FMBL L23004CNN can significantly reduce the oral glucose tolerance and insulin tolerance of T2DM mice, reduce the abnormal fluctuation of postprandial blood sugar, reduce fasting blood sugar, fasting insulin level, blood lipid level, thereby reducing insulin resistance, so as to achieve the effect of lowering blood sugar. The rhamnosus casei FMBL L23004 CNN also had a significant impact on the composition of intestinal microorganisms, increased the abundance and types of beneficial bacteria in the intestines of T2DM mice, and reduced the relative abundance of pathogenic bacteria; the Lactobacillus rhamnosus FMBLL23004 CNN was able to improve the metabolic disorders caused by diabetes by promoting the energy metabolism, carbohydrate metabolism, cofactor and vitamin metabolism of T2DM mice.

[0127] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. Application of Lactobacillus rhamnosus FMBL L23004 CNN in the preparation of a drug for preventing or treating diabetes, characterized in that: The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099.

2. The use according to claim 1, characterized in that The diabetes is type 2 diabetes.

3. Application of Lactobacillus rhamnosus FMBL L23004 CNN in the preparation of hypoglycemic products, characterized in that: The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099.

4. Application of Lacticaseibacillus rhamnosus FMBL L23004 CNN in inhibiting α-amylase activity or preparing α-amylase inhibitors, characterized in that: The Lactobacillus rhamnosus FMBL L23004CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M20231099.

5. Use of Lacticaseibacillus rhamnosus FMBL L23004 CNN in inhibiting α-glucosidase activity or preparing α-glucosidase inhibitors, characterized in that: The Lactobacillus rhamnosus FMBL L23004CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M20231099.

6. Use of Lacticaseibacillus rhamnosus FMBL L23004 CNN in inhibiting dipeptidyl peptidase IV activity or preparing a dipeptidyl peptidase IV inhibitor, characterized in that: The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M 20231099.

7. Use of Lactobacillus rhamnosus FMBL L23004 CNN in the preparation of a product for preventing or treating insulin resistance, characterized in that: The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCC NO: M 20231099.

8. Application of Lactobacillus rhamnosus FMBL L23004 CNN in the preparation of a product for preventing or treating intestinal flora imbalance and metabolic dysfunction in diabetic patients, characterized in that: The Lactobacillus rhamnosus FMBL L23004 CNN was deposited in the China Center for Type Culture Collection on June 26, 2023, with a deposit number of CCTCCNO: M 20231099.

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