CN114533768A

CN114533768A - Application of lactobacillus probiotic CGMCC No.1.13855 in preparation of diabetes treatment medicine

Info

Publication number: CN114533768A
Application number: CN202210067650.2A
Authority: CN
Inventors: ***; 刘丽云; 杨晶; 金东�
Original assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Current assignee: National Institute for Communicable Disease Control and Prevention of Chinese Center For Disease Control and Prevention
Priority date: 2022-01-20
Filing date: 2022-01-20
Publication date: 2022-05-27
Anticipated expiration: 2042-01-20
Also published as: CN114533768B

Abstract

The invention discloses an application of Lactobacillus probiotic strain in preparing a diabetes treatment medicament, wherein the preservation number of the strain is CGMCC No.1.13855, the preservation date is 12 months and 16 days in 2021, the preservation classification is named as Lactobacillus xujianguonis (Lactobacillus xujianguonis), and the preservation unit is the China general microbiological culture Collection center. The strain provided by the invention is harmless to animals and has the effect of regulating blood sugar.

Description

Application of lactobacillus probiotic CGMCC No.1.13855 in preparation of diabetes treatment medicine

Technical Field

The invention relates to probiotics and application thereof, belonging to the field of microorganisms.

Background

Lactobacillus is a lactic acid bacterium, so called because it ferments carbohydrates to produce lactic acid products, which are short rod-shaped in appearance. The lactobacillus is widely distributed in nature, and can be separated from plants, animals such as pigs, food such as beer, pickled Chinese cabbage and the like. Members of the genus lactobacillus are gram-positive bacteria, do not produce spores, have low tolerance to oxygen, are mostly anaerobic, unpowered, and are partially microaerophilic and motile. Under the condition of strict fermentation, lactobacillus can produce lactic acid as main end product from carbohydrate, so that the strain has high acid tolerance and partial strain acidophilic. Due to this property of lactobacilli, they are often used in the food fermentation industry, such as the production of cheese, yoghurt and the like. In addition, lactobacillus is considered to be a nonpathogenic and food-grade safe genus, and many strains play an important role in food microbiology and human nutrition and are used as probiotics.

Diabetes is a group of metabolic diseases characterized by hyperglycemia. The global population of diabetics is expected to increase to 6.93 billion by 2045, with over 90% of diabetics being type 2 diabetes. Obesity is often associated with type 2 diabetes before onset. Hyperglycemia is caused by a defect in insulin secretion or an impaired biological action, or both. The chronic hyperglycemia results in chronic damage and dysfunction of various tissues, particularly eyes, kidneys, heart, blood vessels and nerves. High blood glucose levels can be treated with a variety of antidiabetic agents including biguanides, sulfonylureas, thiazolidinediones, alpha-glucosidase inhibitors, and the like. However, these treatments can have side effects such as flatulence, abdominal discomfort or diarrhea. Substances having alpha-glucosidase inhibitory activity, vegetables, Chinese medicines and probiotics extracted from plants are also beginning to be used for the treatment of type 2 diabetes.

Probiotics isolated from humans, animals, dairy products and fermented foods are considered potential biotherapeutics for type 2 diabetes, especially probiotics isolated from healthy people and natural dairy products. Oral administration of lactobacillus paracasei TD062 in mice can improve lipid and glucose metabolism and reduce oxidative stress by producing alpha-glucosidase inhibitory active. Various lactic acid bacteria have been demonstrated to alleviate type 2 diabetes.

The invention aims to separate and screen a new lactobacillus species from the feces of the Qinghai wild woodchuck which only ingests plants, and the application of the new lactobacillus species in preparing a medicament for treating diabetes.

Disclosure of Invention

Based on the above purpose, the invention provides an application of a Lactobacillus probiotic strain in preparing a diabetes treatment drug, wherein the probiotic strain is CGMCC No.1.13855 in the preservation number, the preservation date is 2021 year, 12 months and 16 days, the preservation classification is named as Lactobacillus xujiangensis (Lactobacillus xujiianguonis), the preservation unit is the common microorganism center of the China Committee for culture Collection of microorganisms, the address is the institute of microbiology, China academy of sciences, No. 3 of North Chen West Lu 1 institute of south China, North Yangyang, Beijing, and the zip code is 100101.

In a preferred embodiment, the sequence of the 16S rDNA of said strain is as set forth in SEQ ID NO: 1 is shown.

In a more preferred embodiment, the diabetes therapeutic agent is an agent that reduces the glucose level in human serum.

In another more preferred embodiment, the diabetes treatment drug is a human serum insulin lowering drug.

In yet another more preferred embodiment, the diabetes treatment drug is an inflammatory factor drug that is associated with insulin resistance in human serum.

More preferably, the inflammatory factor is tumor necrosis factor-alpha and/or IL-6.

In a preferred embodiment, the diabetes treatment drug is also used as a liver disease treatment drug at the same time.

In a more preferred embodiment, the liver disease is a liver dysfunction disease caused by a disorder of lipid metabolism, hepatitis and/or liver fibrosis caused by an inflammatory factor, or liver damage caused by serum endotoxin.

In a preferred embodiment, the probiotic strain of lactobacillus is prepared as a capsule, a lyophilized powder or a bacterial suspension formulation.

The invention separates and purifies lactobacillus with probiotic characteristics from wild woodchuck feces, experiments prove that the separated lactobacillus is harmless to animals, and animal experiments prove that the lactobacillus has the effect of regulating blood sugar. The CGMCC No.1.13855 strain can obviously reduce the functions of blood sugar, blood insulin, serum IL-6 and serum TNF-alpha, and has similar blood sugar regulating effect to that of simvastatin. The physical signs of the rats ingested with the lactic acid bacteria are normal, and no abnormality is found in the pathology of the gastrointestinal liver and spleen after the experiment is finished by ingesting for five weeks. The heterotopic planting of the lactobacillus is not seen when the bacteria detection is carried out on the sterile tissue.

Drawings

FIG. 1 is a phylogenetic tree analysis (maximum likelihood method) of CGMCC No.1.13855 strain based on 16S rDNA gene;

FIG. 2 influence of CGMCC No.1.13855 strain on serum Total Cholesterol (TC) of rat in high lipid model;

FIG. 3 effect of CGMCC No.1.13855 strain on serum Triglyceride (TG) of high-fat model rat;

FIG. 4 effect of CGMCC No.1.13855 strain on serum Low Density Lipoprotein (LDL) in rat of high fat model;

FIG. 5 effect of CGMCC No.1.13855 strain on serum High Density Lipoprotein (HDL) of rat in high fat model;

FIG. 6 influence of CGMCC No.1.13855 strain on hepatic fat drop content (oil red O staining) in rat of high fat model;

FIG. 7 effect of CGMCC No.1.13855 strain on serum alanine Aminotransferase (ALT) in high fat model rats;

FIG. 8 is a graph showing the effect of CGMCC No.1.13855 on serum glutamic-oxaloacetic transaminase (AST) in rat model with high fat;

FIG. 9 influence of CGMCC No.1.13855 strain on hepatic Malondialdehyde (MDA) of rat in high fat model;

FIG. 10 is the effect of CGMCC No.1.13855 strain on hepatic superoxide dismutase (SOD) in rat model with high fat;

FIG. 11 influence of CGMCC No.1.13855 strain on hepatic glutathione peroxidase (GSH-Px) in rat of high lipid model;

FIG. 12 is a graph showing the effect of CGMCC No.1.13855 strain on interleukin 1-beta (IL-1 beta) in serum of rat in high lipid model;

FIG. 13 is the effect of CGMCC No.1.13855 strain on chemotactic protein-1 (MCP-1) in serum of rat in hyperlipidemia model;

FIG. 14 Effect of CGMCC No.1.13855 strain on serum Endotoxin (ET) of rat in high lipid model;

FIG. 15 shows the effect of CGMCC No.1.13855 strain on fasting plasma glucose of rat in high fat model;

FIG. 16 shows the effect of CGMCC No.1.13855 strain on fasting serum insulin of rat in high fat model;

FIG. 17 effect of CGMCC No.1.13855 strain on serum tumor necrosis factor-alpha (TNF-alpha) in rat of high lipid model;

FIG. 18 effect of CGMCC No.1.13855 strain on serum interleukin 6 (IL-6) in rat model with high fat.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 isolation, screening and identification of lactic acid bacteria

1. Isolation of Lactobacillus

1) Taking 100 mu L of sample from the bacteria-retaining tube, adding into an EP tube pre-filled with 900 mu L of sterile PBS, sequentially performing gradient dilution on the sample, and diluting the woodchuck fecal sample to 10^-6Doubling;

2) coating 100 mu L of samples with different dilutions on an MRS culture medium, and putting the MRS culture medium into an incubator;

3) 0.5% CO at 37 deg.C₂Culturing for 48h in the environment;

4) taking out the culture dish, picking colonies with different morphological characteristics by using an aseptic inoculating loop, transferring the colonies to a new MRS solid culture medium for purification, carrying out anaerobic culture at 37 ℃ for 48h, continuously transferring the colonies for 3 times, culturing the purified strain in liquid MRS with pH =3.5, and screening strains with excellent acid-resistant growth for experiment or frozen preservation.

2. Strain preservation

The laboratory uses MRS culture medium containing 25% glycerol as bacteria-protecting liquid to carry out the freezing preservation of the strains, and the method comprises the following steps:

1) sterilizing a 2 mL-capacity bacteria-preserving tube at 121 ℃ for 15min under high pressure for later use;

2) after lactobacillus is continuously transferred on MRS solid culture medium for 3 times, 1.5ml of sterile bacteria-protecting liquid is added to a culture dish;

3) scraping and coating the culture dish by using an L rod to fully integrate bacterial colonies into a bacteria preserving liquid;

4) transferring the bacterial liquid into a bacteria-protecting tube, mixing uniformly and preserving at-80 ℃.

3. Observation of colony appearance and cell morphology

The lactobacillus HT111-2 with the preservation number of CGMCC No.1.13855 belongs to anaerobic bacteria, grows well under anaerobic condition, forms a circular, irregular-edge, bulged-middle and rough-surface light yellow colony on a Columbia blood culture medium, and has the diameter of about 1-1.2 mm; do not grow under aerobic conditions. Under an optical microscope, the thalli are light purple after gram staining, and an end-to-end arrangement mode can appear. The thallus is rod-shaped under a transmission electron microscope, has no spores, flagella, pili and power and has the diameter of about 0.7 to 1 multiplied by 2.5 to 4.28 Mum.

4. Extraction of total DNA of bacteria

A single colony was inoculated on MRS medium, cultured overnight under anaerobic conditions at 37 ℃ and DNA was extracted according to the instructions of the bacterial genomic DNA extraction kit (TIANGEN).

5. Biochemical identification method of strain

In this study, the biochemical identification cards API 50CH and API ZYM of the "BioMerieux" bacterial system, produced by Meirieux, France, were used to carry out the biochemical identification of carbohydrate glycolysis and enzymatic reactions of strain HT111-2 and the reference strain.

As a result: according to biochemical identification, a strain with biochemical characteristics of Lactobacillus is obtained, the strain preservation number is CGMCC No.1.13855, the preservation date is 12 months and 16 days in 2021, the strain is classified and named as Lactobacillus xujiangensis (Lactobacillus xujianguonis), the preservation unit is the common microorganism center of the China Committee for culture Collection of microorganisms, the address is the institute of microbiology of the institute of China academy of sciences No. 3, North Cheng Lu No.1 of the Yangyang area, Beijing, and the postal code is 100101.

6. Bacterial universal primer 16S rRNA PCR amplification

Identification of bacterial 16S rRNA: extracting bacterial genome DNA, amplifying lactobacillus universal primer 16S rDNA PCR product sequencing, carrying out BLAST comparison on the sequence on NCBI, and carrying out preliminary identification.

The primers used in the PCR amplification of the 16S rRNA of the bacterium and the conditions of the PCR reaction used in the experiment were as follows, and the PCR system of 50. mu.L was used for all experiments. And performing BLAST comparison on the sequencing result of the lactobacillus universal primer 16S rRNA PCR product to be detected on NCBI to identify. The results suggest that the CGMCC 1.13855 strain may be a suspected new species of Lactobacillus.

Universal primer 16S rRNA PCR amplification condition

The primer sequences are as follows:

27F， 5'-AGAGTTTGATCMTGGCTCAG-3'；

1492R 5'-TACGGYTACCTTGTTACGACTT-3'；

reaction system (50 μ l):

;

amplification conditions:

。

7. phylogenetic analysis

The 16S rDNA gene of the strain CGMCC No. 1.1385516S rDNA GenBank 31-pattern strain is used as a reference sequence, the 16S rDNA gene sequence of Enterococcus. faecium ATCC 19434 is used as an ectogroup, MEGA 6.0 software is used, CLUSTAL _ W is adopted for multi-sequence alignment, and the phylogenetic tree is constructed based on the maximum likelihood method MaximumLikelihood (ML). The phylogenetic tree constructed based on the 16S rDNA gene sequence shows that the strain CGMCC No.1.13855 is clustered with adjacent lactobacillus together, and an independent branch is formed on the phylogenetic tree and is most similar to the evolutionary relationship of L.amylolytic bacteria DSM 11664 and L.hamsteri DSM 5661, which is shown in FIG. 1 (maximum likelihood method). The whole genome frame map sequence of the strain CGMCC No.1.13855 is obtained through second-generation sequencing, and is subjected to online genome hybridization with genomes of reference strains L.amylolytic bacteria DSM 11664 and L.hamsteri DSM 5661, the DDH values are 20.6 percent and 21.1 percent respectively, and the DDH values are subjected to online genome hybridization with genome sequences of 175 model strains in lactobacillus recorded in a GenBank database, and the results show that the DDH values range from 17.2 percent to 42.5 percent and are lower than 70 percent, and meet the judgment standard of new species.

In conclusion, the genomic and phylogenetic analysis suggests that the strain CGMCC No.1.13855 is a new species of Lactobacillus.

Example 2 lipid-lowering function of Lactobacillus CGMCC 1.13855 on rat serum

32 female SD rats of approximately 200 g body weight were randomly divided into four groups, one group was given normal maintenance diet, and three groups were given high fat diet (cholesterol 1%, lard 10%, 0.2 cholate, 10% egg yolk powder). Three groups of high fat diet were orally administered 10 every other day after one week of feeding⁹The CFU CGMCC No.1.13855 lactobacillus bacterial liquid is given with physiological saline every other day, and the group is given with 20mg/kg body weight of simvastatin every other day three weeks before the experiment is ended, the experiment is ended in five weeks, and the blood lipid level of the serum is detected to evaluate the preventive intervention effect of the lactobacillus CGMCC No.1.13855 on the lipid metabolism disorder model.

FIG. 2 shows the effect of CGMCC No.1.13855 on serum Total Cholesterol (TC) in high-fat model rats. The average content of serum TC in the CGMCC No.1.13855 dried group (namely HT111-2 dried group) and the simvastatin dried group is 1.79 +/-0.18 mmol/L and 1.77 +/-0.08 mmol/L, which are both obviously lower than that in the high-fat model group by 2.48 +/-0.23 mmol/L (p is less than 0.001); the average content of TC in serum of the high-fat model group is 2.48 +/-0.23 mmol/L, which is obviously higher than that of the normal control group by 1.40 +/-0.10 mmol/L (p is less than 0.001).

FIG. 3 shows the effect of CGMCC No.1.13855 on serum Triglyceride (TG) in a high-fat model rat. The average content of serum TG in the CGMCC No.1.13855 dried group (namely HT111-2 dried group) and the simvastatin dried group is 2.55 +/-0.17 mmol/L and 2.55 +/-0.08 mmol/L, which are both obviously lower than that in the high-fat model group by 3.07 +/-0.15 mmol/L (p is less than 0.001); the average content of serum TG in the high-fat model group is 3.07 +/-0.15 mmol/L, which is obviously higher than that in the normal control group by 1.87 +/-0.09 mmol/L (p is less than 0.001).

FIG. 4 shows the effect of CGMCC No.1.13855 on serum Low Density Lipoprotein (LDL) in high fat model rats. The average content of LDL in serum of the CGMCC No.1.13855 dried group (namely HT111-2 dried group) and the simvastatin dried group is 2.60 +/-0.15 mmol/L and 2.17 +/-0.32 mmol/L, which are both obviously lower than that of the high-fat model group by 3.15 +/-0.24 mmol/L (p <0.01 and p < 0.001); the average content of LDL in serum of the high-fat model group is 3.15 +/-0.24 mmol/L, which is obviously higher than that of the normal control group by 1.79 +/-0.26 mmol/L (p is less than 0.001).

FIG. 5 shows the effect of CGMCC No.1.13855 on serum High Density Lipoprotein (HDL) in a high fat model rat. The average content of serum HDL of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the average content of serum HDL of the simvastatin dry pre-group are 2.10 +/-0.22 mmol/L and 2.33 +/-0.17 mmol/L, which are both obviously higher than that of the high-fat model group by 1.51 +/-0.08 mmol/L (p <0.01 and p < 0.001); the average content of serum HDL of the high-fat model group is 1.51 +/-0.08 mmol/L, which is obviously lower than that of the normal control group by 3.10 +/-0.41 mmol/L (p is less than 0.001).

FIG. 6 shows the effect of CGMCC No.1.13855 strain on hepatic fat drop content (oil red O staining) in rat as a model of hyperlipidemia. Oil red O staining of the normal control group showed no lipid droplets within the cytoplasm. Oil red O staining in the high lipid model group showed a large number of lipid droplets within the cytoplasm. Oil red O staining in CGMCC No.1.13855 intervention group (i.e., HT111-2 intervention group) and simvastatin intervention group showed lipid droplets within the cytoplasm, but was significantly less than in the high lipid model group.

The results show that the CGMCC No.1.13855 lactobacillus strain has the functions of obviously reducing serum total cholesterol, triglyceride and low-density lipoprotein and increasing high-density lipoprotein, can reduce the fat content of the liver, and has the similar effect of reducing blood fat with simvastatin.

Rats ingested with the CGMCC No.1.13855 lactobacillus show normal behavior, and no abnormality is found in the pathology of the gastrointestinal liver and spleen after the experiment is finished by five weeks. The heterotopic planting of the lactobacillus is not seen when the bacteria detection is carried out on the sterile tissue.

Example 3 protective Effect of Lactobacillus CGMCC No.1.13855 on liver function of rat

32 female SD rats of about 200 g body weight were randomly divided into four groupsOne group was given normal maintenance diet, and three groups were given high fat diet (cholesterol 1%, lard 10%, 0.2 cholate, 10% egg yolk powder). Three groups of high fat diet were orally administered 10 every other day after one week of feeding⁹The method comprises the steps of adding a group of CFU CGMCC No.1.13855 lactobacillus bacterial liquid into a group of lactobacillus bacteria liquid every other day, adding 20mg/kg of body weight of simvastatin every other day three weeks before the experiment is ended, ending the experiment in five weeks, and detecting the levels of glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase and inflammatory factors of serum to evaluate the prevention and intervention effect of the lactobacillus CGMCC No.1.13855 on the liver function damage caused by the lipid metabolism disorder model.

FIG. 7 shows the effect of CGMCC No.1.13855 on serum alanine Aminotransferase (ALT) in high fat model rats. The average content of ALT in serum of the CGMCC No.1.13855 dried group (namely HT111-2 dried group) and the simvastatin dried group is 1.05 +/-0.09 ng/mL and 1.34 +/-0.04 ng/mL, and both the average content of ALT in serum is obviously lower than that of 1.66 +/-0.09 ng/mL (p is less than 0.001) in the high-fat model group; the mean ALT content in serum of the high fat model group is 1.66 +/-0.09 ng/mL, which is obviously higher than that of the normal control group by 0.94 +/-0.05 ng/mL (p is less than 0.001).

FIG. 8 shows the effect of CGMCC No.1.13855 on serum glutamic-oxaloacetic transaminase (AST) in high-fat model rats. The average content of the serum AST of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) and the simvastatin intervention group is 1.67 +/-0.26 ng/mL and 1.91 +/-0.34 ng/mL, which are both obviously lower than that of the high fat model group by 2.70 +/-0.12 ng/mL (p is less than 0.001); the mean content of serum AST in the high-fat model group is 2.70 +/-0.12 ng/mL, which is obviously higher than that in the normal control group by 1.51 +/-0.27 ng/mL (p is less than 0.001).

FIG. 9 shows the effect of CGMCC No.1.13855 on hepatic Malondialdehyde (MDA) in rats with high lipid model. The average content of liver MDA of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 3.12 +/-0.38 nmol/mL and 3.45 +/-0.49 nmol/mL, and both are obviously lower than that of the high-fat model group by 4.54 +/-0.20 nmol/mL (p is less than 0.01 and p is less than 0.001); the average content of liver MDA in the high-fat model group is 4.54 +/-0.20 nmol/mL, which is obviously higher than that in the normal control group by 2.63 +/-0.49 nmol/mL (p is less than 0.001).

FIG. 10 shows the effect of CGMCC No.1.13855 on liver superoxide dismutase (SOD) in high fat model rats. The average content of liver SOD in the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 1.28 +/-0.07 ng/mL and 1.18 +/-0.14 ng/mL, and both are obviously higher than that in the high-fat model group by 0.90 +/-0.05 ng/mL (p is less than 0.001); the average content of liver SOD in the high-fat model group is 0.90 +/-0.05 ng/mL, which is obviously lower than that in the normal control group by 1.32 +/-0.06 ng/mL (p is less than 0.001).

FIG. 11 shows the effect of CGMCC No.1.13855 on hepatic glutathione peroxidase (GSH-Px) in high lipid model rats. The average content of the liver GSH-Px of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 7.71 +/-1.08 ng/mL and 7.80 +/-0.63 ng/mL, which are both obviously higher than that of the high-fat model group by 4.31 +/-0.41 ng/mL (p is less than 0.001); the average content of the liver GSH-Px in the high-fat model group is 4.31 +/-0.41 ng/mL, which is obviously lower than that in the normal control group by 8.90 +/-0.48 ng/mL (p is less than 0.001).

The results show that the CGMCC No.1.13855 lactobacillus strain has the functions of obviously reducing serum glutamic-pyruvic transaminase, serum glutamic-oxalacetic transaminase and liver malondialdehyde, simultaneously increasing the functions of liver superoxide dismutase and liver glutathione peroxidase, and has the similar effect of protecting the liver function as simvastatin.

Example 4 Lactobacillus CGMCC No.1.13855 reduction of liver inflammation and inflammatory factor reduction function associated with liver fibrosis in rat serum

FIG. 12 shows the effect of CGMCC No.1.13855 on serum interleukin 1-beta (IL-1 beta) in rat of high lipid model. The average content of the serum IL-1 beta of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 20.29 +/-1.88 pg/mL and 17.98 +/-1.16 pg/mL, and both are obviously lower than 28.40 +/-1.96 pg/mL (p is less than 0.001) of the high-fat model group; the average content of IL-1 beta in serum of the high-fat model group is 28.40 +/-1.96 pg/mL, which is obviously higher than that of the normal control group by 20.06 +/-3.81 pg/mL (p is less than 0.001).

FIG. 13 shows the effect of CGMCC No.1.13855 on serum monocyte chemoattractant protein-1 (MCP-1) in rat model with high fat. The average content of MCP-1 in serum of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) is 536.98 +/-104.31 pg/mL, which is obviously lower than that of 733.69 +/-40.18 pg/mL (p is less than 0.001) of the high-fat model group; the average content of MCP-1 in serum of a simvastatin dried group is 628.89 +/-52.98 pg/mL, and the MCP-1 is not significantly different from 733.69 +/-40.18 pg/mL in a high-fat model group (p is more than 0.05); the mean content of MCP-1 in serum of the high-fat model group is 733.69 +/-40.18 pg/mL, which is remarkably higher than that of 527.28 +/-72.42 pg/mL (p is less than 0.001) of a normal control group.

The results show that the CGMCC No.1.13855 Lactobacillus strain has the function of obviously reducing serum IL-1 beta and MCP-1 inflammatory factors, and has stronger efficacy of reducing the serum inflammatory factors than simvastatin. Liver fibrosis is the final outcome of the continuous development of liver injury caused by various reasons, and MCP-l is an important macrophage chemotactic factor in tissue injury repair and plays an important role in liver injury and repair. IL-1 β is another important inflammatory cell that causes fibrosis, is a major inducer of the pro-inflammatory response, and enhances the extent of the inflammatory response by synergizing with and promoting the expression of other inflammatory factors, such as TNF- α. In conclusion, MCP-l and IL-1 β are important inflammatory factors for liver fibrosis. The CGMCC No.1.13855 lactobacillus can obviously reduce the content of IL-1 beta and MCP-1 inflammatory factor in serum, thereby reducing the occurrence of liver inflammation and liver fibrosis.

Example 5 endotoxin lowering function of Lactobacillus CGMCC No.1.13855 on rat serum

FIG. 14 shows the effect of CGMCC No.1.13855 on serum Endotoxin (ET) in rat in high lipid model. The mean content of serum ET of the CGMCC No.1.13855 intervention group (namely HT111-2 intervention group) is 36.14 +/-5.59 pg/mL, which is obviously lower than 53.95 +/-6.28 pg/mL (p is less than 0.001) of the high-fat model group; the average content of the serum ET of the simvastatin pre-treated group is 44.89 +/-4.90 pg/mL, and has no significant difference (p is more than 0.05) with 53.95 +/-6.28 pg/mL of the high-fat model group; the mean content of ET in serum of the high fat model group is 53.95 +/-6.28 pg/mL, which is obviously higher than that of the normal control group by 25.25 +/-6.26 pg/mL (p is less than 0.001).

The results show that the CGMCC No.1.13855 lactobacillus strain has the function of obviously reducing the serum Endotoxin (ET), thereby reducing the liver injury degree, and has stronger effect of reducing the serum Endotoxin (ET) than simvastatin.

Example 6 in vivo evaluation of hypoglycemic function of Lactobacillus CGMCC No.1.13855

32 female SD rats of approximately 200 g body weight were randomly divided into four groups, one group was given normal maintenance diet, and three groups were given high fat diet (cholesterol 1%, lard 10%, 0.2 cholate, 10% egg yolk powder). Three groups of high fat diet were orally administered 10 every other day after one week of feeding⁹The CFU CGMCC No.1.13855 lactobacillus bacterial liquid is given with physiological saline every other day, and the group is given with simvastatin of 20mg/kg body weight every other day three weeks before the experiment is ended, the experiment is ended in five weeks, and the blood sugar and inflammatory factor level of the fasting serum of the rat are detected to evaluate the preventive intervention effect of the lactobacillus CGMCC No.1.13855 on the metabolic disorder model.

1. Lactobacillus CGMCC No.1.13855 has effect in reducing fasting blood glucose and blood insulin of rat

FIG. 15 shows the effect of CGMCC No.1.13855 on fasting plasma glucose in rats with high fat. The average blood sugar content of the CGMCC No.1.13855 dried group (namely HT111-2 dried group) and the simvastatin dried group is 3.30 +/-0.78 mmol/L and 2.91 +/-0.72 mmol/L, which are both significantly lower than that of the high-fat model group by 5.09 +/-0.29 mmol/L (p is less than 0.001); the average blood sugar content of the high-fat model group is 5.09 +/-0.29 mmol/L, which is obviously higher than that of the normal control group by 2.67 +/-0.41 mmol/L (p is less than 0.001).

FIG. 16 shows the effect of CGMCC No.1.13855 on fasting serum insulin in rats in the high-fat model. The average content of serum insulin of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 31.45 +/-1.77 mU/L and 36.81 +/-2.23 mU/L, which are both obviously lower than that of the high-fat model group 44.84 +/-2.23 mU/L (p is less than 0.001); the mean content of serum insulin in the high-fat model group is 44.84 +/-2.23 mU/L, which is obviously higher than that in the normal control group by 25.33 +/-3.04 mU/L (p is less than 0.001).

The results show that the CGMCC No.1.13855 Lactobacillus has the function of obviously reducing blood sugar and serum insulin, and has the similar blood sugar reducing effect as simvastatin. Lipid metabolism disorders such as hypertriglyceridemia and high free fatty acid blood disease are closely related to insulin resistance. Insulin resistance decreases the efficiency of insulin in promoting glucose uptake and utilization, and compensatory hypersecretion of insulin by the body produces hyperinsulinemia to maintain the stability of blood glucose. Insulin resistance is predisposed to metabolic syndrome and type 2 diabetes. The strain of lactobacillus CGMCC No.1.13855 has the function of reducing blood fat, thereby relieving insulin resistance and causing the reduction of blood sugar and serum insulin.

Example 7 reduction of inflammatory factor associated with insulin resistance of Lactobacillus CGMCC No.1.13855 to rat serum

FIG. 17 shows the effect of CGMCC No.1.13855 on serum tumor necrosis factor-alpha (TNF-alpha) in rat model with high fat. The average content of serum TNF-alpha of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 112.54 +/-20.53 pg/mL and 159.75 +/-7.95 pg/mL, and both are obviously lower than that of 198.5 +/-13.94 pg/mL (p is less than 0.001) of the high-fat model group; the mean content of serum TNF-alpha in the high-fat model group is 198.5 +/-13.94 pg/mL, which is obviously higher than that in the normal control group of 105.44 +/-25.22 pg/mL (p is less than 0.001).

FIG. 18 shows the effect of CGMCC No.1.13855 on serum interleukin 6 (IL-6) in high-fat model rats. The average content of IL-6 in serum of the CGMCC No.1.13855 dry pre-group (namely HT111-2 dry pre-group) and the simvastatin dry pre-group is 115.43 +/-9.20 pg/mL and 132.24 +/-9.94 pg/mL, and both are obviously lower than those of the high-fat model group 155.93 +/-7.55 pg/mL (p is less than 0.001 and p is less than 0.01); the mean content of IL-6 in serum of the high-fat model group is 155.93 +/-7.55 pg/mL, which is obviously higher than that of the normal control group 114.42 +/-8.90 pg/mL (p is less than 0.001).

The results show that the CGMCC No.1.13855 Lactobacillus strain has the effects of remarkably reducing serum tumor necrosis factor-alpha and IL-6 inflammatory factors and has stronger effect of reducing the serum inflammatory factors than simvastatin. Because the production of insulin resistance is closely related to the level of serum inflammatory factors, the long-term over-secretion of serum inflammatory factors may be a major factor in the impairment of insulin secretion from islet beta cells and the production of insulin resistance. TNF-alpha and IL-6, the most representative cytokines, play a central regulatory role in inflammation and immune responses. Elevated serum levels of TNF- α and IL-6 can induce insulin resistance by interfering with insulin signaling. The CGMCC No.1.13855 Lactobacillus strain can obviously reduce the content of TNF-alpha and IL-6 inflammatory factors in serum, thereby reducing the insulin resistance and causing the blood sugar to be reduced.

Rats ingested with the CGMCC No.1.13855 lactobacillus show normal behavior, and no abnormality is found in the pathology of the gastrointestinal liver and spleen after the experiment is finished by five weeks. The bacteria detection of the sterile tissue does not show the ectopic colonization of the lactobacillus.

Sequence listing

<110> infectious disease prevention and control institute of China center for disease prevention and control

Application of <120> lactobacillus probiotic CGMCC No.1.13855 in preparation of diabetes treatment medicine

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1527

<212> DNA

<213> Lactobacillus xujianguonis

<400> 1

agagtttgat catggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

gagcagaact aacagattta cttcggtaat gacgtttcgg acgcgagcgg cggatgggtg 120

agtaacacgt gggtaacctg cccttaagtc tgggatacca cttggaaaca ggtgctaata 180

ccggataaca actagtgctg catggcacta gcttaaaagg cggcgtaagc tgtcgctaaa 240

ggatggaccc gcggtgcatt agctagttgg taaggtaacg gcttaccaag gcgacgatgc 300

atagccgagt tgagagactg atcggccaca ttgggactga gacacggccc aaactcctac 360

gggaggcagc agtagggaat cttccacaat gggcgaaagc ctgatggagc aacgccgcgt 420

gagtgaagaa ggttttcgga tcgtaaagct ctgttgttgg tgaagaagga tagaggtagt 480

aactggcctt tatttgacgg taatcaacca gaaagtcacg gctaactacg tgccagcagc 540

cgcggtaata cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg 600

cggagaaata agtctgatgt gaaagccctc ggcttaaccg aggaagtgca tcagaactgt 660

ttttcttgag tcagaagagg agagtgaact ccatgtgtag cggtggaatg cgtagatata 720

tggaagaaca ccagtggcga aggcggctct ctggtctgta actgacgctg aggctcgaaa 780

gcatgggtag cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagtgcta 840

agtgttggga ggtttccgcc tctcagtgct gcagctaacg cattaagcac tccgcctggg 900

gagtacgacc gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag 960

catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat ctggtgcaaa 1020

cctaagagat taggcgttcc cttcggggac accaagacag gtggtgcatg gctgtcgtca 1080

gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg ttattagttg 1140

ccagcattaa gttgggcact ctaatgagac tgccggtgac aaaccggagg aaggtgggga 1200

cggcgtcaag tcatcatgcc ccttatgacc tgggctacac acgtgctaca atgggcagta 1260

caacgaggag cgaacctgtg aaggcaagcg aatctctgaa agctgttctc agttcggact 1320

gtaggctgca actcgcctac acgaagctgg aatcgctagt aatcgcggat cagcacgccg 1380

cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggaa gtctgcaatg 1440

cccaaagccg gtggcctaac cttcgggaag gagccgtcta aggcagggca gatgactggg 1500

gtgaagtcgt aacaaggtaa ccgtaaa 1527

Claims

1. The application of the Lactobacillus probiotic strain in the preparation of the diabetes treatment medicine is characterized in that the preservation number of the probiotic strain is CGMCC No.1.13855, the preservation date is 12 months and 16 days 2021, the Lactobacillus strain is deposited and named as Lactobacillus xujianguonis (Lactobacillus xujianguonis), and the preservation unit is the China general microbiological culture Collection center.

2. The use according to claim 1, wherein the sequence of the 16S rDNA of the probiotic strain of Lactobacillus is as set forth in SEQ ID NO: 1 is shown.

3. The use according to claim 1 or 2, wherein the diabetes therapeutic agent is an agent that reduces the glucose level in human serum.

4. The use according to claim 1 or 2, wherein the diabetes treatment drug is a human serum insulin lowering drug.

5. The use according to claim 1 or 2, wherein the diabetes therapeutic agent is an agent that reduces inflammatory factors associated with insulin resistance in human serum.

6. The use according to claim 5, wherein said inflammatory factor is tumor necrosis factor- α and/or IL-6.

7. The use according to claim 1 or 2, wherein the diabetes treatment medicament is also used as a liver disease treatment medicament.

8. The use according to claim 7, wherein the liver disease is a liver dysfunction disease caused by a disorder of lipid metabolism, hepatitis and/or liver fibrosis caused by an inflammatory factor, or liver damage caused by serum endotoxin.

9. Use according to claim 1 or 2, characterized in that the probiotic strain of lactobacillus is prepared as a capsule, a lyophilized powder or a bacterial liquid preparation.