CN113388554A

CN113388554A - Lactobacillus plantarum SHY130 and application thereof in relieving diabetes

Info

Publication number: CN113388554A
Application number: CN202110896915.5A
Authority: CN
Inventors: 索化夷
Original assignee: Chongqing Yuyan Biotechnology Co ltd
Current assignee: Chongqing Yichen Hemei Biotechnology Co.,Ltd.
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2021-09-14
Anticipated expiration: 2041-08-05
Also published as: CN113388554B

Abstract

The invention discloses a strain of Lactobacillus plantarum (SHY130) which is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2021372; an active ingredient of the microbial inoculum comprises the lactobacillus plantarum SHY 130. Also discloses application of the lactobacillus plantarum SHY130 or microbial inoculum in preparing products for preventing and/or relieving and/or treating diabetes. The invention also provides application of the Lactobacillus plantarum SHY130 as a functional probiotic in preparation of food, food additives, health-care products or medicines. The invention researches the action mechanism of the probiotics on regulating the intestinal islet axis, enlarges the application range of the lactobacillus plantarum SHY130, improves the utilization value of the lactobacillus plantarum SHY130 and brings a new hope for treating type II diabetes.

Description

Lactobacillus plantarum SHY130 and application thereof in relieving diabetes

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus plantarum SHY130 and application thereof in relieving diabetes.

Background

Diabetes has become one of the most serious and critical public health problems facing the world in the 21 st century. The international diabetes association statistical data in 2019 shows that about 4.63 hundred million adults suffer from diabetes globally, and the number of people suffering from diabetes is estimated to reach 7 hundred million by 2045 years. Type ii diabetes accounts for over 90% of diabetics and is mainly manifested by relative insulin deficiency due to insulin resistance and impaired islet beta cell function. Patients with type ii diabetes mellitus are at significantly increased risk of cardiovascular disease (CVD), retinopathy, neuropathy, renal failure, and other complications, and serious patients may even be life threatening. It has been found that patients with pancreatic disease are more susceptible to diabetes and that a reduction in pancreatic beta cell area of about 65% increases the risk of diabetes. Therefore, protection of beta cell function is crucial to the prevention and treatment of diabetes.

The pathogenesis of diabetes is complex. Several in vivo and in vitro studies have shown that the intestinal islet can serve as a target for enhancing pancreatic beta cell function and improving glucose homeostasis. In the gut, Short Chain Fatty Acid (SCFA) producing bacteria are able to ferment non-digestible dietary fibre, producing short chain fatty acids. Short chain fatty acids have been shown to have a number of beneficial effects on energy metabolism in mammals. SCFAs activate the G protein-coupled receptors GPR41 and GPR43, which are expressed on enteroendocrine L cells. GPR41 and GPR43 promote secretion of glucagon-like peptide-1 (GLP-1). GLP-1 is a key incretin hormone in the shaft of the islet of intestine, can activate insulin secretion, improve insulin resistance and promote pancreatic beta cell proliferation. Therefore, the insular axis of the intestine may be an effective treatment strategy for type ii diabetes.

Probiotics are defined as living microorganisms that are administered in sufficient doses to benefit the health of the host. The history of safe use of probiotics in food products and observations in clinical trials has shown that human supplementation with probiotics is generally considered safe and generally without serious side effects. In numerous animal studies and clinical trials, it has been demonstrated that probiotics such as Lactobacillus (L.) casei, L.paracasei, L.plantarum, L.acetylophilus and Bifidobacterium infarnata can regulate blood glucose levels, protect pancreatic islets, increase glucose tolerance, improve insulin resistance and remodel intestinal flora. Probiotics have a strain-or even strain-specificity and act through a variety of mechanisms. However, there has been little research on the mechanism of action of probiotics on the insular axis of the intestine in diabetes.

Therefore, the method has wide prospect in searching the regulation action mechanism of the probiotics in the islet axis and developing the probiotics with the function of reducing the hyperglycemia into a functional preparation, and is worthy of further research.

Disclosure of Invention

The invention aims to solve the problems and provides lactobacillus plantarum SHY130 and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a strain of Lactobacillus plantarum SHY130 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021372.

An agent, the active ingredient of which comprises the Lactobacillus plantarum SHY 130.

The microbial inoculum is freeze-dried powder.

The Lactobacillus plantarum SHY130 or the microbial inoculum can be applied to the preparation of products for preventing and/or relieving and/or treating diabetes.

In the above application, the diabetes is type II diabetes.

In the above application, the product is a food, a health product or a medicine.

The invention also provides application of the Lactobacillus plantarum SHY130 as a functional probiotic in preparation of food, food additives, health-care products or medicines.

The invention also provides a product, the active ingredient of which is the Lactobacillus plantarum SHY130 or any one of the microbial inoculum, and the product is used as any one of the following:

(1) preventing and/or ameliorating and/or treating diabetes;

(2) can be used as functional probiotic bacteria to be applied to food, food additive, health product or medicine.

The product is food, food additive, health product or medicine, and the diabetes is type II diabetes.

The invention has the beneficial effects that:

the invention discovers that: the results of mice in a type II diabetes model induced by high-fat diet combined with Streptozotocin (STZ) show that: the lactobacillus plantarum SHY130 can obviously relieve the weight loss of the mice; can significantly reduce Fasting Blood Glucose (FBG); can remarkably improve the oral glucose tolerance; can obviously improve insulin resistance and the content of glucagon in serum, obviously increase the positive area of islet beta cells, obviously reduce the area of islet alpha cells and improve pancreatic injury; the expression of GPR43 and GPR41 in colon can be obviously improved, the GLP-1 content in serum is obviously increased, and the damage of the colon is improved; and the structure of intestinal flora can be adjusted, the ratio of Firmicutes/bacteria is obviously reduced, and the relative abundance of SCFA producing bacteria such as Faecalibaccum, Odoribacter, Alisipes and the like is increased, so that the content of SCFA in the excrement of the mice can be obviously increased, and the pH value of the excrement is reduced. In conclusion, lactobacillus plantarum SHY130 can relieve hyperglycemia caused by type ii diabetes by regulating the insulin axis, and improve the occurrence and development of type ii diabetes, and fig. 13 is an analysis chart of the action mechanism of probiotics on the insulin axis in the present invention. Therefore, the lactobacillus plantarum SHY130 can be used for preparing health-care food and medicines for relieving type II diabetes.

The invention provides application of Lactobacillus plantarum SHY130(Lactobacillus plantarum SHY130) with the preservation number of CCTCC NO: M2021372 in preparing health-care food and medicines for relieving type II diabetes, which not only explores the action mechanism of probiotics on the axis of a regulation intestinal island, but also expands the application range of Lactobacillus plantarum SHY130, improves the utilization value of the Lactobacillus plantarum SHY130 and brings new hope for treating type II diabetes.

Drawings

FIG. 1 shows the colony morphology (a) and the gram stain result (b) of the isolated strain;

FIG. 2 shows the construction result of a phylogenetic tree of Lactobacillus plantarum SHY 130;

FIG. 3 shows the result of API 50CH reaction of Lactobacillus plantarum SHY 130;

FIG. 4 shows the results of the tolerance of Lactobacillus plantarum SHY130 to artificial gastric acid and bile salt;

FIG. 5 shows the body weight change trend (a), the final body weight (b) and the average daily food intake (c) per mouse during the treatment period;

FIG. 6 is a graph showing the effect of Lactobacillus plantarum SHY130 on FBG levels (a), OGTT (b) and its lower curve area (c) in type II diabetic mice;

FIG. 7 is a graph of the effect of Lactobacillus plantarum SHY130 on serum insulin, HOMA-IR, and glucagon levels in type II diabetic mice;

FIG. 8 is a graph showing the histopathological effect of Lactobacillus plantarum SHY130 on pancreas in type II diabetic mice;

FIG. 9 is a graph of the effect of Lactobacillus plantarum SHY130 on colon histopathology (a) and GPR43 and GPR41 expression (b, c, d) and serum GLP-1 levels (e) in type II diabetic mice;

FIG. 10 is a graph showing the effect of Lactobacillus plantarum SHY130 on the content of short-chain fatty acids and pH in feces of type II diabetic mice;

FIG. 11 is a plot of species VENN (a), Chao1 index (b) and shannon (c) index and PCOA (d) of mouse intestinal flora;

FIG. 12 is a graph showing the effect of Lactobacillus plantarum SHY130 on the composition of intestinal flora in type II diabetic mice;

FIG. 13 is a graph showing the mechanism of action of probiotic bacteria on the island axis of the intestine according to the present invention;

in the above figures, indicates that there is a significant difference p <0.05 compared to the normal group; # indicates a significant difference p <0.05 compared to the model group.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to be limiting.

The experimental methods in the following examples are all conventional methods unless otherwise specified; the chemical and biological reagents used are all conventional reagents in the field and can be obtained commercially, if not specifically indicated.

Deposit information of Lactobacillus plantarum SHY130

The strain SHY130 is delivered to China type culture Collection (CCTCC for short) for preservation in 2021 month, wherein the preservation date is as follows: 14 days 4 months 2021; the preservation number is: CCTCC NO: M2021372; and (3) classification and naming: lactobacillus plantarum SHY130(Lactobacillus plantarum SHY 130).

Example 1 isolation and identification of Lactobacillus plantarum SHY130

1 materials of the experiment

Traditional fermented yak yoghourt in Sichuan Hongyuan.

2 method of experiment

2.1 isolation and purification of lactic acid bacteria

Diluting the retrieved yak yoghourt by 10 times in gradient, and sequentially diluting to 10 times^-6.4 appropriate dilutions (10 dilutions) were selected^-3、10^-4、10^-5、10^-6) 100 mu L of the culture medium is respectively coated on MRS solid plate culture medium, after culturing for 48h at 37 ℃, single colonies with different forms are selected and strains are separated by a plate marking method. The above procedure was repeated until a purified strain was obtained, and morphological observation was performed by gram staining.

2.2PCR amplification of 16S rDNA sequences

And extracting the DNA of the purified strain by using a bacterial genome DNA extraction kit. PCR amplification was performed using a 25. mu.L reaction: mu.L of template DNA, 1. mu.L of upstream primer (10. mu.M), 1. mu.L of downstream primer (10. mu.M), and 12.5. mu.L of 2 XTAQQ PCR Master Mix were made up to 25. mu.L with sterile ultrapure water. PCR amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 1.5min, annealing at 55 ℃ for 1min, and extension at 72 ℃ for 1.5min for 30 cycles; terminal extension at 72 ℃ for 10 min. Finally, the Huada Gene science and technology company Limited is entrusted to carry out bidirectional sequencing on the PCR amplification products qualified by detection, and the sequencing result is subjected to homology comparison analysis through a BLAST program in NCBI.

2.3 construction of phylogenetic Tree

Lactic acid bacteria having a homology of more than 98% with the sequence of the obtained 16S rDNA gene of lactic acid bacteria and a standard strain in Genome were selected from GeneBank as reference sequences, and a phylogenetic tree of the obtained strains was constructed using the Neighbor-Joining method in the MEGA5.05 program.

2.4API 50CH kit identification

The separated strain is cultured for 18h at 37 ℃, and the thalli is centrifugally collected under the conditions of 3000r/min and 15min, washed by sterile physiological saline and then resuspended into a bacterial suspension. The procedure was performed with reference to merriella (france) API 50CH kit instructions.

2.5 determination of ability to mimic gastrointestinal fluid tolerance

After the strain is cultured for 18h at 37 ℃, 10mL of the strain is centrifuged at 3000r/min for 15min to collect thalli precipitate, the thalli precipitate is washed for 2 times by using equal volume of sterile normal saline, and the thalli precipitate is resuspended in equal volume of sterile normal saline to obtain bacterial suspension. Mixing the prepared bacterial suspension with artificial gastric juice (0.2% NaCl, 0.35% pepsin 1:10000) at a ratio of 1:9, uniformly mixing, placing in a water bath constant temperature oscillator at 37 ℃ for 3h at 100r/min, respectively measuring the viable count of 0h and 3h by using a plate coating method, repeating for 3 times, and taking the result as an average value.

After the strain is cultured for 18h at 37 ℃, the strain is respectively inoculated into MRS-THIO culture media containing 0.0 percent and 0.3 percent of bovine bile salt (Cas No 8008-63-7) according to the inoculum size of 2 percent, and the mixture is evenly mixed and then placed into a water bath oscillator to be cultured for 24h at 37 ℃ and 100 r/min. Using non-inoculated MRS-THIO culture medium (high-cholesterol culture medium) without ox bile salt as blank control, determining OD of culture solution containing 0.0, 0.3% ox bile salt₆₀₀nm values, repeated 3 times, the results are expressed as mean values.

3 results and analysis

3.1 colony morphology and cell morphology of the isolated strains

After the strain is purified, a single colony is formed in an MRS culture medium, the colony morphology is almost consistent, most of the colony is round and white, and the surface is smooth and moist. The purple cell morphology was observed under a microscope after gram staining, and the cells were judged as gram-positive bacteria (G)⁺). The colony morphology and gram staining of the strains are shown in FIG. 1.

3.2 sequence analysis of 16S rDNA of Strain

The results of the 16S rDNA homology analysis showed that the homology with Lactobacillus plantarum (Lactobacillus plantarum) known in the Gene Bank database was 100%.

The separated lactobacillus plantarum is named as SHY130, and the bidirectional sequence of the 16S rDNA gene amplification product of the SHY130 is shown as SEQ ID No. 1.

3.3 construction results of phylogenetic Tree of Strain

After aligning the DNA sequence of SHY130 with the BLAST program, a phylogenetic tree was constructed by the Neighbor-Joining method, as shown in FIG. 2, the SHY130 has 100% homology with L.plant strain SN13T and L.plant strain Heal19, indicating that SHY130 is Lactobacillus plantarum.

3.4 Biochemical characterization of the Strain

Phenotypic identification at the lactobacillus species level is based primarily on carbohydrate fermentation assays. The API 50CH kit was identified by the utilization of 49 different carbohydrates by the strain.

FIG. 3 shows the API 50CH reaction results (positive yellow, negative blue) for the experimental strains. Table 1 shows the results of the fermentation test of this strain on 49 carbohydrates. As can be seen from FIG. 3 and Table 1, among the 49 carbon sources tested, 25 carbohydrates were available to this strain. Through the final identification of an API lab plus system, the experimental strain is Lactobacillus plantarum (Lactobacillus plantarum), the ID value of the experimental strain is 99.90%, the T value of the experimental strain is 0.71, and the identification requirement (the ID value is more than or equal to 99.0%, and the T value is more than or equal to 0.5) is met.

TABLE 1 results of Lactobacillus plantarum SHY130 fermentation test on 49 carbohydrates

Note: "+" indicates positive reaction; "-" indicates negative reaction.

3.5 tolerance of the strains to artificial gastric acid and bile salts

Gastric juice and bile salts in the gastrointestinal tract can inhibit and stop the growth of microorganisms. In this experiment, lactobacillus plantarum SHY130 has good tolerance to gastric juice with pH 3 and 0.3% bile salts, with potentially beneficial functions to the human body (fig. 4).

Example 2 alleviation of type II diabetes in mice by Lactobacillus plantarum SHY130 through the gut island axis

1 materials of the experiment

The experimental strain is Lactobacillus plantarum SHY130(Lactobacillus plantarum SHY130), and the preservation number is CCTCC NO: M2021372.

The experimental animals were 4-week-old male C57BL/6J mice purchased from the center of Experimental animals at Chongqing university of medicine. The animals were kept in a standardized laboratory at room temperature of 22 + -2 deg.C and relative humidity of 50 + -5% for 12h light/12 h dark, and the experiment was started after one week of acclimatization.

2 method of experiment

2.1 Experimental animal grouping and handling

The mice were randomly divided into three groups of 8 per group, normal group (NC), diabetic group (D), lactobacillus plantarum SHY130(SHY 130). The experimental period was 16 weeks, during which time each group of animals had free access to water. The NC group ingested standard diet (12.79% fat, 66.67% carbohydrate, 20.54% protein), and the remaining mice were fed high fat diet (60.65% fat, 21.22% carbohydrate, 18.14% protein) for 5 weeks. Fresh Streptozotocin (STZ) of 40mg/kg BW is dissolved in sodium citrate buffer solution, and is injected intraperitoneally for 5 days continuously to induce type II diabetes of mice, and the mice of NC group are injected with the sodium citrate buffer solution. The FBG levels were measured after 1 week and mice with FBG levels above 11.1mmol/L were considered diabetic. Diabetic mice were then randomized into group D and group SHY130, the SHY130 group was gavaged 10 daily¹⁰CFU/kg BW Lactobacillus plantarum SHY130 bacterial liquid (fermented bacterial liquid prepared by using SHY130 strain separated and purified in example 1 according to a conventional method) was subjected to intragastric administration of 10mL/kg BW physiological saline daily for 10 weeks in NC group and D group.

Body weights were measured every 7 days during the experiment and food and water intake was recorded every two days during the treatment period. Mouse feces were collected at the end of week 16. After fasting overnight on the last day of the experiment, mouse FBG was determined. The animal experimental protocol has been approved by the animal care and use committee of southwest university, with approval numbers: 20210118-01.

At the end of the experiment, the mice were sacrificed after 12h fasting. Blood samples were collected, centrifuged at 3000rpm for 10min at 4 ℃ to separate serum, and stored at-80 ℃ for further analysis. Tissue samples (pancreas and colon) were rapidly removed, washed, a portion of the tissue was fixed in 4% paraformaldehyde for 48h, and the remaining tissue was frozen in liquid nitrogen and then stored at-80 ℃.

2.2 Oral Glucose Tolerance Test (OGTT)

OGTT was performed on mice at 16 weeks and body weight was measured prior to the experiment to determine glucose gavage. Mice were fasted for 12h, allowed free access to water and blood glucose was determined immediately. The mice were then gavaged with 2 g/kg. BW glucose, blood glucose levels were measured at 30, 60, 90, 120min, respectively, and blood glucose curves were plotted and area under glucose (AUC) calculated using GRAPH PAD.

2.3 measurement of serum indices

The enzyme-linked immunosorbent assay kit is adopted to detect the levels of serum insulin, glucagon and GLP-1. The steady state model assessment of insulin resistance (HOMA-IR) is calculated as follows: HOMA-IR ═ fasting blood glucose (mmol/L) × fasting serum insulin (m IU/L)/22.5.

2.4 histological analysis

Mouse liver and pancreatic tissues were dehydrated in a series of ethanol solutions, paraffin embedded, and cut into 4 μm thick sections. After deparaffinization, the sections were examined morphologically by staining with hematoxylin and eosin. The stained tissue was observed with an optical microscope and imaged for histopathological analysis.

Paraffin-embedded pancreatic and colonic sections were dewaxed and then antigen recovery, heating and sealing were performed. The primary antibody was incubated overnight at 4 ℃ and then incubated with the secondary antibody for 50 minutes at constant temperature, followed by final staining with DAPI for 10 minutes and 3 washes with PBS. Images were observed under a fluorescent microscope and positive cell areas were quantified using Image-J software.

2.5 determination of SCFAs and pH in feces

Short chain fatty acids (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid) in mouse feces were determined by gas chromatography. The fecal samples were quickly placed into centrifuge tubes in an ice-water bath, ionized water was added, and vortex mixed for 2 minutes. Then, the mixture was left standing in an ice water bath for 20 minutes and centrifuged at 4800g at 4 ℃ for 20 minutes. Transfer 2mL of supernatant into another centrifuge tube and add 0.2mL of 50% H₂SO₄And 2mL of diethyl ether, mixed well, left standing at 4 ℃ for 30min, and the supernatant was filtered through a 0.22 μm membrane and then subjected to gas chromatography.

Gas chromatography conditions: agilent DB-WAX column (30m x 0.32mm i.d.,0.25 μm), inlet temperature 220 ℃ and detector temperature 275 ℃. The carrier gas adopts high-purity nitrogen, the flow rate is 2mL/min, the split ratio is 40: 1. the flow rates of hydrogen and air were 30 and 300mL/min, respectively. The oven temperature (80 ℃) was held for 1min, then ramped up to 170 ℃ at a rate of 15 ℃/min, then ramped up to 220 ℃ at a rate of 30 ℃/min. The amount of sample was 1. mu.L.

Another part of the fecal sample was diluted with distilled water at a ratio of 1:9 and the pH was measured with a pH meter.

2.6 high throughput sequencing of intestinal flora in feces

Bacterial total DNA in the sample is extracted from the sample by using a fecal DNA extraction kit, and the quality of the DNA is evaluated by detecting the ratio of OD values A260/A280 and A260/A230 of nucleic acid. The bacterial 16S rRNA gene V3-V4 region was amplified using specific primers 341F (SEQ ID NO.2:5'-CCTACGGGNGGCWGCAG-3') and 806R (SEQ ID NO.3:5'-GGACTACHVGGGTATCTAAT-3') with barcode. The total volume of PCR amplification is 50 μ L, the first round of amplification procedure is pre-denaturation at 94 ℃ for 2min, then denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 30s, extension at 68 ℃ for 30s for 30 cycles, and finally extension at 68 ℃ for 5min, then PCR product purification is carried out by using Agencour AMPure XP magnetic beads (purchased from Beckmann Coulter trade company (China) Co., Ltd., product number: 311303), and quantification is carried out by using qubit3.0 after purification. The second round of amplification procedure was a 12 cycle of pre-denaturation at 94 ℃ for 2min, followed by denaturation at 98 ℃ for 10s, annealing at 65 ℃ for 30s, and extension at 68 ℃ for 30s, and finally extension at 68 ℃ for 5 min. The second round of amplification products were purified using Agencourt AMPure XP beads, quantified using the ABI StepOnePlus RealTime PCR System, and sequenced on machine according to the PE250 mode pooling of Novaseq 6000.

3 results and analysis of the experiments

3.1 Effect of Lactobacillus plantarum SHY130 on weight and food intake of type II diabetic mice

As shown in FIG. 5(a), the body weight of the mice in the three groups continued to increase for the first 5 weeks, and there was no significant difference between the groups. The STZ was injected at week 6, and the body weight of all HFD (high fat diet) fed groups was significantly reduced from week 7 to week 8. After lactobacillus plantarum SHY130 dryness, the weight loss of the SHY130 group mice was alleviated. The final body weight values at the end of 16 weeks are more intuitive showing the body weight of the mice in each group, as shown in fig. 5(b), the body weight of mice in group D (24.45 ± 0.85g) is significantly lower than that of NC (26.38 ± 1.28g), while the body weight of mice in group SHY130 (25.97 ± 1.28g) is significantly increased compared to that of mice in group D (p < 0.05). While the average daily food intake of three groups of mice did not differ significantly throughout the treatment, as in fig. 5 (c). The results show that lactobacillus plantarum SHY130 alleviates the diabetic characteristics of type ii diabetic mice.

3.2 Effect of Lactobacillus plantarum SHY130 on blood glucose and glucose tolerance in type II diabetic mice

As shown in fig. 6(a), FBG levels at week 6 were significantly higher in both other groups of mice than in NC group (p <0.05), indicating successful establishment of the diabetes model. After 10 weeks of treatment, FBG (17.69 + -2.46 mmol/L) in mice in group D was still significantly higher than that in group NC (5.63 + -0.51 mmol/L), while blood glucose levels (11.98 + -2.57 mmol/L) were significantly reduced (p <0.05) after 10 weeks of gavage with Lactobacillus plantarum SHY130 compared to group D.

To measure glucose homeostasis, we measured the OGTT of mice. At the end of week 16, the study found impaired glucose tolerance in type ii diabetic mice, with the AUC of the OGTT in group D mice being more than 3-fold that in group NC, as shown in fig. 6(b, c). The glucose tolerance level was significantly improved after lactobacillus plantarum SHY130 treatment (p < 0.05). The results show that the lactobacillus plantarum SHY130 has the potential of improving the hyperglycemia and the glucose tolerance of the type II diabetic mice.

3.3 Effect of Lactobacillus plantarum SHY130 on serum insulin, HOMA-IR and glucagon levels in type II diabetic mice

Insulin resistance is one of the main pathogenesis of type II diabetes, and can be evaluated through HOMA-IR, and the improvement of insulin resistance is a good way for treating diabetes. As shown in fig. 7, serum insulin, glucagon, HOMA-IR levels were significantly higher in group D mice than in group NC (p < 0.05). SHY130 group mice significantly reduced insulin, glucagon, and HOMA-IR levels compared to group D mice (p < 0.05).

3.4 Effect of Lactobacillus plantarum SHY130 on pancreatic histopathology in type II diabetic mice

H & E staining of pancreatic sections was performed to evaluate the protective effect of Lactobacillus plantarum SHY130 on islet beta cell injury in diabetic mice, as shown in FIG. 8 (a). The islets of the mice in the NC group are in circular or oval cell clusters and are dispersed in healthy acinar cells with clear boundaries. Compared with the NC group, the number of the islets in the D group is obviously reduced, and the islets are irregular and atrophied in shape. In addition, islet cells from group D mice show degenerative changes such as nuclear atrophy or nucleolysis, cellular necrosis, vacuolization, etc., indicating damage to pancreatic structures by high fat diet in combination with STZ. The lactobacillus plantarum SHY130 improves pancreatic tissue abnormality, obviously increases the number of pancreatic islets, improves the shape and reduces cell damage.

To further evaluate the effect of lactobacillus plantarum SHY130 on islets of type ii diabetic mice, we performed double immunofluorescent staining of pancreatic tissue sections with insulin and glucagon as shown in fig. 8 (b). Compared to the NC group, the area of β cells (insulin positive) was significantly reduced (p <0.05) in group D (fig. 8(c)), and the area of α cells (glucagon positive) was significantly increased (p <0.05) (fig. 8 (D)). We also observed irregular islet shape in group D mice, consistent with the H & E staining of the pancreas in fig. 8 (a). Compared with the D group, the beta cell area of the mice in the SHY130 group is remarkably increased (p <0.05), and the alpha cell area is remarkably reduced (p <0.05), which indicates that the lactobacillus plantarum SHY130 has a protective effect on pancreatic injury.

3.5 Effect of Lactobacillus plantarum SHY130 on Colon pathology and GPR43, GPR41 expression in type II diabetic mice

According to the H & E staining result of the colon tissue shown in FIG. 9(a), the colon mucosal epithelial cells of the NC group mice are tightly connected, the mucosal structure is complete, the muscle layer is thick, and the tissue is dense. In comparison to the NC group, colon tissue of mice in group D exhibited connective lesions with inflammatory cell infiltration. After the treatment of lactobacillus plantarum SHY130, the colon injury state is obviously improved.

In addition, the mechanism of lactobacillus plantarum SHY130 in improving T2DM mouse hyperglycemia was further investigated by measuring the expression levels of GPR43 and GPR41 in colon tissues. As shown in figure 9(b, c, D) immunofluorescence results, the expression of GPR43, GPR41 was significantly reduced in group D mice compared to NC group (p < 0.05). Lactobacillus plantarum SHY130 significantly enhanced GPR43, GPR41 expression compared to group D (p < 0.05).

3.6 Effect of Lactobacillus plantarum SHY130 on serum GLP-1 levels in type II diabetic mice

GLP-1 is an enteroendocrine hormone released from intestinal tract, and can stimulate glucose-dependent endogenous insulin secretion and inhibit glucagon secretion. As shown in fig. 9(e), serum GLP-1 levels were significantly reduced in mice in group D compared to group NC (p < 0.05). And the Lactobacillus plantarum SHY130 significantly improved GLP-1 levels (p < 0.05).

3.7 Effect of Lactobacillus plantarum SHY130 on the content and pH of fecal SCFAs in type II diabetic mice

The intestinal microbiota plays a key role in regulating host health, one of the mechanisms is the production of SCFAs, which have a significant impact on host metabolism and intestinal immunity, and the reduction of SCFAs is closely associated with type ii diabetes. As shown in fig. 10, total SCFAs levels in the feces of mice in group D were significantly lower than those of mice in group NC (p < 0.001). Lactobacillus plantarum SHY130 treatment significantly increased SCFAs levels compared to group D (p < 0.05). Wherein lactobacillus plantarum SHY130 treatment significantly increased fecal acetic, butyric and isovaleric acid levels (p <0.05), whereas fecal propionic, isobutyric and valeric acid levels were not significantly increased (p > 0.05).

The fecal pH value of mice in group D was significantly higher than that in group NC (p <0.001), while the pH value of mice in group lactobacillus plantarum SHY130 was significantly lower (p <0.01) compared to group D. A decrease in stool pH also means an increase in SCFAs production.

3.8 Effect of Lactobacillus plantarum SHY130 on the Overall Structure of intestinal flora in type II diabetic mice

After mass filtering, we identified 737 OTUs at a 97% similarity level. As shown in fig. 11(a), there are 307 OTUs shared among the three groups. Notably, the NC group has 159 unique OTUs, the SHY130 group has 68 unique OTUs, and the D group only finds 37.

The Chao1 index and Shannon diversity index are used as the measurement index of alpha diversity to analyze the overall structure of intestinal flora. As shown in fig. 11(b and c), there was no significant change in Shannon diversity index after lactobacillus plantarum SHY130 treatment (p >0.05), and no significant difference in Chao1 index among the three groups (p >0.05) compared to group D.

As shown in fig. 11(D), the change in the overall structure of the intestinal flora was analyzed using the PCoA map of unweighted UniFrac, and the results showed a clear separation between the structures of NC and D groups. The intestinal flora structures of the group D and the group SHY130 are also obviously changed, which shows that the lactobacillus plantarum SHY130 changes the overall structure of the intestinal flora of the type II diabetic mice.

3.9 Effect of Lactobacillus plantarum SHY130 on the intestinal flora composition in type II diabetic mice

Fig. 12(a) shows the composition of intestinal microorganisms at the phylum level in three groups of mice. Our data show that the microbiota composition at the phylum level of group D mice changed dramatically compared to the NC group, with a 89.63% reduction in the relative abundance of Bacteroidetes, an 40.28% increase in the relative abundance level of Firmicutes, and a significant increase in the ratio Firmicutes/Bacteroidetes (p < 0.01). As shown in FIG. 12(b), the ratio of Firmicutes/bacteria was adjusted by Lactobacillus plantarum SHY130 (p <0.05), resulting in an increase in the abundance of bacteria of 95.30% and a decrease in the abundance of Firmicutes of the phylum Firmicutes of 19.05%.

In addition, fig. 12(c) demonstrates the change in gut flora at the genus level in three groups of mice. As shown in fig. 12(D), the abundance of faecalibaccum, Odoribacter, Alistipes, bacteroides was significantly increased in lactobacillus plantarum SHY130 treated mice compared to group D mice. Previous studies have shown that most of the bacteria of the above 4 species are SCFA producing bacteria, which have a beneficial effect on intestinal barrier and metabolic function. Interestingly, the Dubosiella genus in the intestinal flora of mice in group D increased 4.7-fold (p <0.05) over mice in group NC, while lactobacillus plantarum SHY130 completely normalized it.

Sequence listing

<110> university of southwest

<120> lactobacillus plantarum SHY130 and application thereof in relieving diabetes

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1425

<212> DNA

<213> Artificial sequence ()

<400> 1

gctggttcct aaaaggttac cccaccgact ttgggtgtta caaactctca tggtgtgacg 60

ggcggtgtgt acaaggcccg ggaacgtatt caccgcggca tgctgatccg cgattactag 120

cgattccgac ttcatgtagg cgagttgcag cctacaatcc gaactgagaa tggctttaag 180

agattagctt actctcgcga gttcgcaact cgttgtacca tccattgtag cacgtgtgta 240

gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttgtcac 300

cggcagtctc accagagtgc ccaacttaat gctggcaact gataataagg gttgcgctcg 360

ttgcgggact taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgta 420

tccatgtccc cgaagggaac gtctaatctc ttagatttgc atagtatgtc aagacctggt 480

aaggttcttc gcgtagcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540

caattccttt gagtttcagc cttgcggccg tactccccag gcggaatgct taatgcgtta 600

gctgcagcac tgaagggcgg aaaccctcca acacttagca ttcatcgttt acggtatgga 660

ctaccagggt atctaatcct gtttgctacc catactttcg agcctcagcg tcagttacag 720

accagacagc cgccttcgcc actggtgttc ttccatatat ctacgcattt caccgctaca 780

catggagttc cactgtcctc ttctgcactc aagtttccca gtttccgatg cacttcttcg 840

gttgagccga aggctttcac atcagactta aaaaaccgcc tgcgctcgct ttacgcccaa 900

taaatccgga caacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg 960

tggctttctg gttaaatacc gtcaatacct gaacagttac tctcagatat gttcttcttt 1020

aacaacagag ttttacgagc cgaaaccctt cttcactcac gcggcgttgc tccatcagac 1080

tttcgtccat tgtggaagat tccctactgc tgcctcccgt aggagtttgg gccgtgtctc 1140

agtcccaatg tggccgatta ccctctcagg tcggctacgt atcattgcca tggtgagccg 1200

ttaccccacc atctagctaa tacgccgcgg gaccatccaa aagtgatagc cgaagccatc 1260

tttcaaactc ggaccatgcg gtccaagttg ttatgcggta ttagcatctg tttccaggtg 1320

ttatcccccg cttctgggca ggtttcccac gtgttactca ccagttcgcc actcactcaa 1380

atgtaaatca tgatgcaagc accaatcaat accagagttc gttcg 1425

<210> 2

<211> 17

<212> DNA

<213> Artificial sequence ()

<220>

<221> misc_feature

<222> (9)..(9)

<223> n is a or c or g or t or u

<220>

<221> misc_feature

<222> (13)..(13)

<223> w is a or t

<400> 2

cctacgggng gcwgcag 17

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence ()

<220>

<221> misc_feature

<222> (8)..(8)

<223> h is a or t or c

<220>

<221> misc_feature

<222> (9)..(9)

<223> v is a or g or c

<400> 3

ggactachvg ggtatctaat 20

Claims

1. A strain of Lactobacillus plantarum SHY130 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021372.

2. An agent for bacteria, which comprises as an active ingredient Lactobacillus plantarum (SHY130) according to claim 1.

3. The microbial inoculum of claim 2, wherein: the microbial inoculum is freeze-dried powder.

4. Use of Lactobacillus plantarum (SHY130) according to claim 1, or a bacterial agent according to claim 2 or 3, for the preparation of a product for the prevention and/or alleviation and/or treatment of diabetes.

5. The use of claim 4, wherein: the diabetes is type II diabetes.

6. The use of claim 4, wherein: the product is food, health product or medicine.

7. Use of Lactobacillus plantarum (SHY130) according to claim 1 as a functional probiotic for the preparation of a food product, food additive, nutraceutical or pharmaceutical product.

8. A product characterized by: the active ingredient of the Lactobacillus plantarum (Lactobacillus plantarum) SHY130 of claim 1 or the microbial inoculum of claim 2 or 3, and the use of the product is any one of the following:

(1) preventing and/or ameliorating and/or treating diabetes;

9. The product of claim 8, wherein: the product is food, food additive, health product or medicine, and the diabetes is type II diabetes.