Lactic acid bacteria capable of reducing fat, resisting inflammation and reducing blood sugar and application of lactic acid bacteria

Abstract

The invention relates to a lactic acid bacterium capable of reducing fat, resisting inflammation and reducing blood sugar, which is classified and named as pediococcus acidilactici XJ335, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.30093, the preservation date is March 22, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The strain can achieve the fat reducing effect by adjusting the abundance of intestinal flora; the levels of IL-6 and TNF-alpha in serum of diabetic mice are remarkably improved through regulation, so that the anti-inflammatory effect is achieved; various cardiovascular diseases of atherosclerosis are prevented by adjusting the levels of TC, TG, HDL-C and LDL-C; by reducing FINS and HOMA-IR levels and improving HOMA-IS, HOMA-beta and QUICKI value levels, insulin resistance of a body is reduced, insulin sensitivity and pancreas islet beta cell functions of the body are improved, gene expression related to blood sugar regulation is regulated, and an excellent blood sugar reducing effect is achieved in the body.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of lactic acid bacteria having fat-reducing, anti-inflammatory and blood sugar-lowering effects and application thereof. Background Art

[0002] Diabetes is a metabolic disorder caused by multiple factors and characterized by chronic hyperglycemia. Long-term high blood sugar in diabetes causes chronic damage and dysfunction of the eyes, kidneys, heart, blood vessels, and nerves, eventually leading to complications such as uremia, stroke, myocardial infarction, and even life-threatening. Diabetes is mainly divided into two types according to different pathogenic mechanisms: insufficient insulin secretion (type I) and insulin resistance (type II). Type II diabetes accounts for 90% of diabetic patients. It is a chronic disease caused by the combined effects of multiple genetic and environmental factors, and its main manifestation is insulin resistance.

[0003] At present, people regulate blood sugar levels by taking oral hypoglycemic drugs. Commonly used drugs are divided into insulin secretion promoters, insulin sensitizers, and α-glucosidase inhibitors according to their different mechanisms of action. However, although these drugs can treat symptoms, they also have side effects such as diarrhea and headache.

[0004] In recent years, there have been some studies and reports on the regulation of blood sugar by probiotics, but there are not many studies, and the hypoglycemic mechanism of probiotics is not clear. Different hypoglycemic probiotics have different hypoglycemic mechanisms, and in practical applications, different hypoglycemic strategies are used for different patients. For example, lactic acid bacteria in the existing technology ("Study on the Effects of Lactobacillus G15 and Q14 on Type II Diabetes in Rats and Its Intestinal Flora Improvement", Tian Peijun,) can improve the glucose tolerance of diabetic rats and reduce glycosylated hemoglobin. Others have found that the proliferation of bifidobacteria in the intestine can promote the synthesis and secretion of GLP-1, thereby improving blood sugar and insulin tolerance. It can be seen that for different patients, probiotics with different hypoglycemic mechanisms can be used to improve diabetic symptoms through different pathways. Therefore, seeking probiotics with different hypoglycemic mechanisms is of great significance for the widespread application of probiotics to lower blood sugar. Summary of the invention

[0005] The present invention aims to provide a strain of lactic acid bacteria with fat-reducing, anti-inflammatory and blood sugar-lowering effects. The strain has good acid and bile salt resistance, can improve the body's glucose tolerance, promote the repair of β cells, relieve fat deposition, achieve fat-reducing effects, and also has anti-inflammatory effects.

[0006] Another object of the present invention is to provide applications of the lactic acid bacteria.

[0007] The purpose of the present invention is achieved through the following technical solutions: A strain of lactic acid bacteria with fat-reducing, anti-inflammatory and blood sugar-lowering effects, characterized in that: the lactic acid bacteria are classified and named Pediococcus acidilactici ( Pediococcus acidilactici )XJ335, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30093, the deposit date is March 22, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The lactic acid Pediococcus strain XJ335 of the present invention is separated and screened from Xinjiang fermented yogurt, has excellent alpha-amylase inhibition effect, and the inhibition rate reaches 44.03%.

[0009] XJ335 has no obvious hemolysis, good antibiotic sensitivity and excellent safety.

[0010] When lactic acid bacteria XJ335 is administered orally to a type 2 diabetic mouse model, the fasting blood sugar of the mice can be significantly reduced, and the hyperglycemia symptoms of type 2 diabetic mice can be alleviated.

[0011] In addition, XJ335 effectively improved the glucose tolerance of type 2 diabetic mice.

[0012] XJ335 can also effectively reduce insulin resistance, improve insulin sensitivity and pancreatic β-cell function in type 2 diabetic mice.

[0013] XJ335 is adjusted Firmicutes (Firmicutes), Bacteroidota (Bacteroidetes), achieving the effect of fat reduction.

[0014] In addition, it also achieves a synergistic anti-inflammatory effect by downregulating the concentrations of pro-inflammatory factors TNF-α and IL-6 while increasing the concentration of anti-inflammatory factor IL-10.

[0015] The application of the above-mentioned Pediococcus acidilactici XJ335 in the preparation of medicines or foods for improving symptoms of hyperglycemia.

[0016] The use of the above-mentioned Pediococcus acidilactici XJ335 in the preparation of medicines or foods for preventing or treating inflammation caused by diabetes.

[0017] The application of the above-mentioned Pediococcus acidilactici XJ335 in the preparation of drugs or foods for regulating glucose and lipid metabolism.

[0018] The above-mentioned Pediococcus acidilactici XJ335 is used in the preparation of medicines or foods for reducing vascular damage and preventing and treating cardiovascular diseases.

[0019] The present invention has the following technical effects: The lactic acid bacteria XJ335 of the present invention can produce the following effects in the body of patients with type II diabetes: (1) It can achieve fat-reducing effects by regulating the abundance of intestinal flora and inhibiting the accumulation of lipids in the liver; (2) It significantly improved the levels of IL-6 and TNF-α in the serum of diabetic mice by regulating, thereby achieving an anti-inflammatory effect; (3) By regulating the levels of TC, TG, HDL-C, and LDL-C, it can reduce the atherosclerosis index and vascular damage, thereby preventing various cardiovascular diseases caused by atherosclerosis. It can also reduce liver cell damage in mice and improve liver function in diabetic mice. (4) By lowering the levels of FINS and HOMA-IR and increasing the levels of HOMA-IS, HOMA-β and QUICKI, the body's insulin resistance is reduced, while the body's insulin sensitivity and pancreatic β-cell function are improved, and the expression of genes related to blood sugar regulation is regulated, achieving an excellent blood sugar-lowering effect in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Pictures of the results of the hemolytic test of the initial screening strains.

[0021] Figure 2 : Effects of Pediococcus acidilactici on body weight in type 2 diabetic mice.

[0022] Figure 3 :Effects of Pediococcus acidilactici on fasting blood glucose in type 2 diabetic mice.

[0023] Figure 4 : Changes in glucose tolerance and area under the curve in type II diabetic mice fed with Pediococcus acidilactici.

[0024] Figure 5 : Changes in organ indexes of type II diabetic mice fed with Pediococcus acidilactici.

[0025] Figure 6 :Changes in serum lipid levels and atherosclerosis index in type II diabetic mice fed with Pediococcus acidilactici.

[0026] Figure 7 : Changes in serum inflammatory indicators in type II diabetic mice fed with Pediococcus acidilactici.

[0027] Figure 8 :Changes in fasting insulin, glucagon-like peptide levels and related indexes in type II diabetic mice fed with Pediococcus acidilactici.

[0028] Fig. 9 :Pathological observation of pancreatic tissue of type II diabetic mice fed with Pediococcus acidilactici by H&E staining.

[0029] Fig.10: Pathological observation of liver tissue of type II diabetic mice fed with Pediococcus acidilactici by Oil Red O staining.

[0030] Fig.11 : Principal component analysis of intestinal microbiota in type 2 diabetic mice fed with Pediococcus acidilactici.

[0031] Fig.12 : Type II diabetic mice fed with Pediococcus acidilactici (A) Bar graph of relative abundance of species at the phylum level; (B) Ratio of Firmicutes / Bacteroidetes; (CF) Bar graph of species abundance of specific phyla in each group.

[0032] Fig.13 : Bar graph of relative abundance of species at genus level in type II diabetic mice fed with Pediococcus acidilactici.

[0033] Fig.14 : Bar graph of the abundance of specific metabolic pathways in type 2 diabetic mice fed with Pediococcus acidilactici (a: alanine, aspartate, and glutamate metabolism; b: amino sugar and nucleotide sugar metabolism; c: fructose and mannose metabolism; d: glycolysis / gluconeogenesis; e: pyruvate metabolism; f: glycine, serine, and threonine metabolism).

[0034] Fig.15 : Relative mRNA expression levels of insulin signaling-related genes in type 2 diabetic mice fed with Pediococcus acidilactici.

[0035] Fig.16 : Relative mRNA expression levels of glucose production-related genes in type II diabetic mice fed with Pediococcus acidilactici.

[0036] In the above figures, * indicates significant difference compared with the model group (p<0.05); ** indicates extremely significant difference compared with the model group (p<0.01); *** indicates extremely significant difference compared with the model group (p<0.001); **** indicates extremely significant difference compared with the model group (p<0.0001); # indicates significant difference compared with the normal control group (p<0.05); ## indicates extremely significant difference compared with the normal control group (p<0.01); ### indicates extremely significant difference compared with the normal control group (p<0.001); #### indicates extremely significant difference compared with the normal control group (p<0.0001). DETAILED DESCRIPTION

[0037] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above-mentioned contents of the present invention.

[0038] Example 1 Strain screening and identification Traditional fermented yogurt was obtained from different regions of Xinjiang Uygur Autonomous Region as samples. 5 g of the sample was added to 45 mL of nutrient broth medium and cultured in a shaking incubator at 37 °C for 48 h. The enriched culture solution was diluted in a test tube containing 9 mL of saline. 0.1 mL (10 -3 , 10 -4 , 10 -5 The sample dilution solution was spread on MRS plate medium, cultured anaerobically at 37℃ for 24~48h, and the number of live colonies of 30~300 was counted. Colonies with different characteristics were selected and inoculated into MRS liquid medium, and then streaked and purified on solid medium. The purified colonies were divided into two parts, one for Gram staining and DNA identification, and the other part was subcultured in MRS liquid medium, added with 20% glycerol and stored at -80℃ for future use.

[0039] After multiple plate separation observations, 9 lactic acid bacteria with hypoglycemic effect were screened from traditional yogurt, numbered as XJ810, XZ306, XZ4410, XJ335, XJ243, XJ244, XJ402, XZ2010, and XZ502. The obtained strains were further identified by 16S rDNA sequencing. The 16S rDNA of lactic acid bacteria was amplified by PCR, the PCR was sequenced, and compared with the NCBI database. The sequences were compared and identified as Lactobacillus plantarum, Pediococcus acidilactici, Pediococcus acidilactici, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus mucosa reuteri, Weissella fusion, and Leuconostoc pseudomesenteroides. The developmental tree made according to the sequencing results was as follows: Figure 1 shown.

[0040] Safety Testing: (1) Hemolysis test: The activated probiotics were streaked on Columbia blood agar plates and the pathogenic strain Staphylococcus aureus SD was inoculated as a control at the same time. The plates were placed at 37°C for 48 h. Finally, hemolysis was observed around the colonies and recorded by taking photos.

[0041] The results are as follows Figure 1 As shown in the figure, Staphylococcus aureus showed obvious β-hemolysis, while strains XJ810, XJ243, XJ244 and XZ4410 had certain hemolysis, and strains XJ335, XZ502, XZ2010 and XZ306 had no obvious hemolysis. That is, among the above strains, XJ335, XZ502, XZ2010 and XZ306 had obvious hemolysis safety.

[0042] (2) The antibiotic susceptibility of the above four strains with hemolytic safety was further tested: the sensitivity of probiotics to each antibiotic was evaluated according to the standards of the Clinical and Laboratory Standards Institute (CLSL). 100 μL of cultured bacteria with a concentration of about 10 9 The four probiotics with hemolytic safety were aseptically coated on the sterilized and cooled MRS solid culture medium, and then 10 drug-sensitive paper strips of penicillin, ampicillin, ceftriaxone, tetracycline, erythromycin, ciprofloxacin, lincomycin, compound cinomeprazole, chloramphenicol and gentamicin were respectively attached to the surface of the culture medium. After being placed upright for a period of time, they were inverted and cultured at 37°C for 48 hours, and the diameter of the transparent circle around the paper strip was observed and measured. The results are shown in Table 1 (S, R, and I represent sensitive (Susceptible), resistant (Resistant) and intermediate sensitive (Intermediate) respectively).

[0043] Table 1:

[0044] The results showed that XJ335 had good antibiotic sensitivity.

[0045] Example 2 Simulated gastrointestinal fluid and bile salt tolerance test During the test, XZ306, also a species of Pediococcus acidilactici, was used as a microbial control for the same test.

[0046] (1) Gastrointestinal fluid resistance test Preparation of artificial gastric juice: Artificial gastric juice is composed of 0.2% NaCl and 0.35% pepsin. The NaCl and pepsin required for the experiment are weighed according to the corresponding mass-to-volume ratio for preparation. The pH of the artificial gastric juice is adjusted to 2.5 with 1 mol / L HCl, and then filtered with a 0.22 μm filter membrane for sterilization and set aside.

[0047] In the clean bench, 5 mL of bacterial culture medium was transferred to a 10 mL sterile centrifuge tube and centrifuged at 3000 r / min for 10 min. The upper culture medium was discarded and the bacteria were collected. An equal volume (5 mL) of sterile saline was added and mixed to prepare a bacterial suspension. Then 1 mL of the bacterial suspension was mixed with 9 mL of artificial gastric juice at pH 2.5. At this time, 1 mL of the above mixture was taken as the sample treated with artificial gastric juice for 0 h, and the remaining 9 mL of the mixture was placed in a constant temperature water bath shaker (37 oC, 150 r / min) for 3 h. The samples at 0 h and 3 h were diluted 10 times, and the number of viable bacteria was determined by plate coating method at appropriate gradients. o C for 48 h, and the survival rate (%) was calculated according to the formula:

[0048] After testing and calculation, in the 3h simulated gastrointestinal fluid, the survival rate of XJ335 was 85.30±8.25%, while the survival rate of XZ306 was 60.00±15.65%.

[0049] (2) Bile salt tolerance test The bacterial liquid of two generations of continuous activation was inoculated into MRS medium without bile salt (0.0%) and MRS medium containing 0.3% bile salt at a volume fraction of 2% (v / v). Each group of experiments was repeated three times in three parallels. The blank control was the liquid culture medium without inoculation, and the positive control was the liquid culture medium containing (0.0%) bile salt. The culture was cultured at 37℃ for 24 h, and the absorbance of the culture medium at 0 and 24 h was measured at a wavelength of 600 nm, and calculated according to the following formula:

[0050] After testing and calculation, the concentration was 0.3% after 24 hours ( w / v ) bile salt tolerance was 56.99±6.51%, while that of XZ306 was 33.11±8.58%.

[0051] It can be seen that XJ335 has excellent acid resistance and excellent bile salt resistance. After being applied to the body, it can withstand the influence of the human gastrointestinal tract, ensure high activity and thus exert corresponding effects in the body.

[0052] The lactic acid bacteria XJ335 was biologically preserved in the General Microbiology Center of the China Culture Collection Administration, with the preservation number CGMCC No.30093, the preservation date March 22, 2024, and the preservation address No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0053] Example 3 Mechanism of action and effect verification of Pediococcus acidilactici XJ335 in type 2 diabetes model 1. Establishment and application of type II diabetes model: Establishment of T2DM mouse model: 6-week-old SPF-grade male C57BL / 6J mice were provided by Hunan Slake Jingda Laboratory Animal Co., Ltd. and were adaptively fed for 1 week in a climate-controlled environment (22 ± 1 ℃ and 55 ± 5% humidity, 12-h light / dark cycle). Then, the mice were randomly divided into 2 groups; the group of 6 mice with normal diet served as the control group (control). The other mice were fed HFD to establish the T2DM model. After six weeks of controlled feeding, the HFD-fed mice were fasted for 12 h and injected intraperitoneally with STZ (streptozotocin) dissolved in citrate buffer (pH 4.5) at a dose corresponding to 50 mg / kg body weight (BW) for three consecutive days. The control group was injected with an equal amount of citrate buffer. Blood from the tail vein was taken to measure the fasting blood glucose (FBG) level, and FBG levels ≥ 11.1 mmol / L indicated that the T2 DM mouse model was successfully established. Diabetic mice were randomly divided into 4 groups (n = 6): diabetic model group (Model), positive control group (Metformin, 150 mg / kg BW), XJ335 group (XJ335) and XZ306 group (XZ306) (XJ335 and XZ306 are both selected lactic acid bacteria with hypoglycemic effect, so the corresponding mouse in vivo experiments were also carried out together). The corresponding preparations were gavaged once a day. Sterile water was given to the normal group and the diabetic group. The mice in the normal group were fed with ordinary food, and the mice in the other groups continued to be fed with high-fat diet. Body weight (BW) and FBG were recorded weekly. Four weeks later, the mice were anesthetized by injection and killed by cervical dislocation, and the tissues were weighed and collected, and blood samples were collected. The liver tissue was stored at -80 °C, and the pancreas and part of the liver tissue were fixed in 10% formaldehyde solution. The collected blood was allowed to stand at room temperature for 30 minutes and then centrifuged at 6000 rpm for 10 minutes to collect serum for subsequent tests. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Southwest University, Chongqing, China.

[0054] Preparation and use: The strain was cultured in MRS broth at 37 °C for 18 h. The cells were harvested by centrifugation at 6500 g for 5 min at 4 °C. The collected cells were washed twice with sterile PBS and adjusted to 5 × 10 9 CFU mL -1 Fresh bacterial suspension was prepared and administered by gavage every day, with 200 μL per mouse to ensure that each mouse contained 1×10 9 CFU mL -1 .

[0055] 2. Body weight, fasting blood sugar and glucose tolerance test: Body weight was measured once a week using an electronic balance. Fasting (12 h) blood glucose was determined weekly. Blood samples were obtained from the tip of the tail vein and blood glucose was measured using a glucometer.

[0056] like Figure 2 As shown in the figure, during the experiment, the weight of mice in the drug administration group and the bacteria feeding group increased to varying degrees compared with the model group, indicating that bacteria feeding and drug administration can alleviate the weight loss of mice caused by diabetes, and the alleviation effect of strain XJ335 is the most obvious.

[0057] Changes of fasting blood glucose in mice Figure 3 As shown, during the 4-week experimental period, the fasting blood glucose levels of the bacteria-fed and drug-treated groups showed a downward trend and were significantly lower than that of the model group, indicating that both bacteria-fed and metformin hydrochloride can alleviate the hyperglycemia symptoms of type 2 diabetic mice, and the effect of the XJ335 group is equivalent to that of metformin hydrochloride.

[0058] Due to the disorder of blood sugar regulation mechanism in patients with type 2 diabetes, blood sugar content will rise rapidly after a large amount of glucose is ingested at one time, and will continue to remain at a high level after 2 hours, which is manifested as a decrease in the body's tolerance to glucose. Glucose tolerance was evaluated after 4 weeks of treatment. The specific steps were to measure the glucose tolerance test (OGTT) in animals fasted for 12 hours at different time points (0, 30, 60, 90 and 120 minutes) after intraperitoneal injection of glucose (2 mg / kg body weight), and evaluate the area under the curve (AUC) of each group of glucose values. The results are shown in Figure 2. Figure 4 As shown, within 120 minutes after taking a large amount of glucose, the blood glucose level in the model group was the highest and decreased slowly, while the drug-treated group and the bacteria-fed group significantly inhibited the increase in blood glucose, and the blood glucose decreased rapidly. In the bacteria-fed group, XJ335 had the best effect, which was equivalent to the drug-treated group. The area under the curve (AUC) is often used to evaluate the body's ability to handle glucose. An increase in the AUC value indicates a decrease in the body's glucose tolerance, and vice versa. Figure 4 It can be seen that the area under the curve of the bacteria-feeding and drug-administered groups was significantly lower than that of the model group, indicating that bacteria-feeding and drug-administered groups effectively improved the glucose tolerance of diabetic mice, and the effect of XJ335 in the bacteria-feeding group was more significant. This indicates that taking XJ335 effectively improves the body's glucose tolerance.

[0059] 3. Organ index determination: Mice were killed by cervical dislocation and dissected to obtain liver, kidney, small intestine, colon content and cecum content. Mouse organ tissues were rinsed in 4°C precooled physiological saline to remove blood and adherent connective tissue, wiped dry with filter paper, weighed and the organ index was calculated according to the following formula:

[0060] result Figure 5 As shown, the organ indexes of the model group showed a significant increase, while the organ indexes of the mice in the drug-treated group and the bacteria-fed group showed a significant decrease compared with the model group. The increase of the organ index of diabetic mice was inhibited, and the related organs could be adsorbed and protected to achieve the effect of alleviating diabetic complications. XJ335 had the most significant effect on alleviating the increase of the organ index of mice, and the overall effect was better than that of the drug-treated group and the XZ306 group.

[0061] 4. Serum biochemical analysis: (1) Serum samples were obtained from mice that had been fasted overnight. Serum total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured using the corresponding detection kits according to the instructions, and the mouse atherosclerosis index was calculated (atherosclerosis index (AI) = [total cholesterol (TC) - high-density lipoprotein (HDL)] ÷ high-density lipoprotein (HDL)).

[0062] The results are as follows Figure 6As shown, compared with the Control group, the serum TC, TG, and LDL-C levels in the Model group increased by 35.64±7.53%, 34.11±10.98%, and 69.05±17.56% (p<0.01), and the HDL-C level decreased by 20±11.22%. Compared with the Model group, TC level in the Metformin group was decreased by 21.94±5.12%, TG level was decreased by 49.83±5.00%, LDL-C level was decreased by 30.48±2.39, and HDL-C level was increased by 28.59±9.21%; compared with the Model group, TC level in the XZ306 group was decreased by 24.35±4.31%, TG level was decreased by 56.75±4.94%, LDL-C level was decreased by 51.22±4.11%, and HDL-C level was increased by 36.72±6.60%; compared with the Model group, TC level in the XJ335 group was decreased by 24.06±3.60%, TG level was decreased by 61.07±8.92%, LDL-C level was decreased by 42.35±3.13%, and HDL-C level was increased by 41.41±13.34% (p<0.01). This shows that after intervention with Pediococcus acidilactici, the blood lipids of diabetic mice were significantly improved, and it can alleviate liver cell damage in mice and improve liver function of diabetic mice to a certain extent. Among them, the effect of XJ335 is extremely excellent.

[0063] The arteriosclerosis index is calculated based on TC and HDL-C, which can measure the degree of arteriosclerosis and mainly reflects the ratio of risk factors to protective factors in lipids. The results show that compared with the Model group, the arteriosclerosis index of each intervention group was significantly reduced (p<0.01), which indicates that lactic acid bacteria XJ335 may play a certain role in preventing cardiovascular diseases. It can reduce vascular damage and thus prevent diabetic complications and various cardiovascular diseases such as atherosclerosis.

[0064] (2) Inflammatory factors can induce insulin resistance through signaling pathways. Inhibition of proinflammatory cytokines and chemokines is a way for beneficial microorganisms to prevent inflammation. TNF-α, IL-1β and IL-6 are key triggers of the pancreatic inflammatory microenvironment. The production of proinflammatory factors will aggravate the induction of IR, and TNF-α will increase β-cell apoptosis and reduce glucose-stimulated pancreatic secretion, thereby affecting normal glucose metabolism. IL-6 is a multifunctional cytokine that inhibits glycogen synthesis and promotes the development of T2DM by interfering with insulin receptor signaling and impairing β-cell function.

[0065] (3) ELISA kits were used to measure the levels of fasting insulin (FINS), glucagon-like peptide-1 (GLP-1), and inflammatory factors (TNF-α, IL-6, and IL-10) in mouse serum.

[0066] The results are as follows Figure 7 As shown in the figure, compared with the Control group, the Model group significantly increased the levels of IL-6 and TNF-α in the serum of mice, and significantly reduced the level of IL-10 (p<0.01). Compared with the Model group, mice fed with Pediococcus acidilactici (XZ306 and XJ33) had lower levels of IL-6 and TNF-α, and the IL-10 level of Pediococcus acidilactici XJ335 also increased. The above results indicate that Pediococcus acidilactici XJ335 can significantly improve the levels of IL-6 and TNF-α in the serum of diabetic mice, thereby achieving an anti-inflammatory effect.

[0067] At the end of the study, the homeostasis model insulin resistance index (HOMA-IR), β-cell function index (HOMA-β) and quantitative insulin sensitivity test index (QUICKI) values ​​were calculated: HOMA-IR = fasting blood glucose level (mmol / L) × fasting pancreatic islet level (mIU / L) / 22.5 HOMA-β = 20 × fasting insulin level (mIU / L) (fasting blood glucose level (mmol / L) - 3.5) QUICKI = 1 / (Log fasting blood glucose level (mg / dL) + Log fasting insulin level (μU / mL) Insulin is secreted by pancreatic β cells and can control the synthesis and metabolism of carbohydrates in the body. GLP-1 can bind to the GLP-1 receptor on pancreatic β cells and maintain glucose homeostasis in type 2 diabetic mice by regulating the synthesis and secretion of insulin. The FINS, GLP-1, and corresponding insulin sensitivity index values ​​of diabetic mice before and after intervention are shown in Figure 2. Figure 8As shown in the figure, compared with the Control group, the FINS and HOMA-IR values ​​of the mice in the Model group were significantly increased (p<0.01), while the HOMA-IS, HOMA-β, QUICKI and GLP-1 values ​​were significantly decreased (p<0.01), indicating that the diabetic mice developed insulin resistance and insufficient insulin secretion as the weight of the disease increased. Compared with the Model group, the FINS and HOMA-IR values ​​of the Pediococcus acidilactici intervention groups (XZ306 and XJ335) were significantly decreased (p<0.0001), the HOMA-IS, HOMA-β and QUICKI values ​​of the XJ335 group were significantly increased (p<0.001), and the GLP-1 value of the XJ335 group was significantly higher than that of the drug group, while the effect of the XZ306 group was not significant. It can be seen that the XJ335 group had a better effect in improving insulin levels, with the Metformin group decreasing by 25.47±4.36% and the XJ335 group decreasing by 37.30±6.45%; and the XJ335 group had a more significant effect in improving glucagon-like peptide-1, with the GLP-1 of this group of mice increasing by 24.13±13.91% compared with the Model.

[0068] 5. Tissue Collection and Pathological Analysis The fixed pancreatic tissue was embedded in paraffin, cut into 3 μm thick sections, and stained with hematoxylin and eosin (H&E) for optical microscopy examination; fresh tissue from the right lobe of the liver of each group of mice was taken, frozen and stained with Oil Red O to observe the changes in lipid droplets in hepatocytes; images of liver and pancreatic tissue were obtained under an inverted microscope at 200 times magnification. Fig. 9 As shown, (a) is the Control group, (b) is the Model group, (c) is the Metformin group, (d) is the XZ306 group, and (e) is the XJ335 group. It can be seen that the pancreatic cells of the mice in the Control group are arranged regularly and have a complete structure, the edges of the islets are clear, the pancreatic cells are round or oval clusters, and the number of islet cells is large. Compared with the Control group, the islet cells of the mice in the Model group showed a large number of apoptosis, the islet cells were atrophied and damaged, the islet cell structure was loosely distributed, and the islet cells showed obvious necrosis, indicating that diabetes destroyed the pancreatic structure of the mice. Compared with the Model group, the pancreatic tissue structure of the mice in the intervention group was significantly improved, the islet cell structure was complete, the arrangement was regular, the cell nucleolus was clear, the boundary between the islet cells and the acini was more obvious, and the empty run was reduced. Compared with the Metformin intervention group, the number of islet cells in the mice in the XJ335 group increased significantly. The above results show that after intervention with Pediococcus acidilactici, especially XJ335, the pancreatic tissue pathological damage of diabetic mice was significantly improved, the boundaries of pancreatic β cells were clear, which promoted the repair of β cells and maintained the integrity of the cell structure.

[0069] Oil Red O staining was used to visualize the fat particles in the liver of mice. The red color in the slices represents fat droplets, and the more red the more fat droplets accumulate in the liver. Fig.10 As shown, (a) is the Control group, (b) is the Model group, (c) is the Metformin group, (d) is the XZ306 group, and (e) is the XJ335 group. It can be seen that the Model group mice have the most lipid droplets, indicating that type 2 diabetes can cause fat deposition in the liver, severe fat degeneration, and affect glucose and lipid metabolism. The mice in the Metformin group improved after oral administration of Metformin, and there were different degrees of improvement after intervention with different lactic acid Pediococci. Among them, the area of ​​lipid droplets in the XJ335 group was significantly improved, which was significantly different from the Model group, and the accumulation of liver fat was reduced. The area of ​​lipid droplet deposition in the XJ335 group mice was also different from that in the Model group, which shows that different lactic acid Pediococci can effectively alleviate the fat deposition phenomenon caused by diabetes in mice to varying degrees, among which the effect of the lactic acid Pediococcus XJ335 group was the most significant.

[0070] 6. Fecal DNA extraction and high-throughput sequencing: Total DNA extraction and high-throughput sequencing of intestinal microorganisms in mouse feces were performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The Illumina MiSeq platform performed statistical analysis of diversity and community structure based on operational taxonomic units (OTUs) and taxonomic information during sequencing.

[0071] The results are as follows Fig.11 As shown in the figure, most samples in the Control group were significantly separated from the Model group and the Metformin group, indicating that there were differences in the intestinal flora species between T2DM and normal mice. The sample areas of the Model group and the Metformin group partially overlapped, indicating that the two groups had similar flora structures, that is, the drug metformin hydrochloride could significantly alleviate the hyperglycemia symptoms of diabetic mice, but it could not repair the damaged intestinal flora structure of diabetic mice. The lactic acid Pediococcus group (XZ306 and XJ335) was significantly separated from the Model group and the Metformin group, indicating that lactic acid Pediococcus intervention could change the intestinal flora structure of diabetic mice. The healthy group mice were significantly separated from the bacteria treatment group XJ335, the drug (metformin hydrochloride) treatment group and the type 2 diabetes model group (p = 0.001), indicating that the composition of the intestinal flora of mice could be affected by gavage of lactic acid Pediococcus XJ335 to type 2 diabetic mice.

[0072] from Fig.12 It can be seen that the Model group and the Metformin group Firmicutes The relative abundance increased. Bacteroidota, Actinobacteriota, DesulfobacterotaCompared with the Model group, the relative abundance of Firmicutes and Desulfobacterota The relative abundance increased. Bacteroidota, Actinobacteriota The relative abundance of XZ306 and XJ335 groups decreased. Firmicutes The relative abundance decreased. Actinobacteriota , Desulfobacterota The relative abundance of lactic acid bacteria in the other two groups increased compared with that in the Model group, except for XZ306. Bacteroidota During the experiment, the Model group and the Metformin group Firmicutes The relative abundance increased. Bacteroidota The relative abundance of the bacteria decreased, so it is speculated that the bacterial flora of diabetic mice is disordered under the induction of a high-fat diet, which promotes the growth of Firmicutes and inhibits the growth of Bacteroidetes, causing the body to absorb a large amount of energy from food and store it, causing symptoms such as obesity, insulin resistance, and increased blood sugar. Compared with the Control group, the F / B values ​​of the Model group, Metformin group, and XZ306 group were all increased, but the ratio of Firmicutes / Bacteroidetes in the XJ335 group was not significantly different from that in the Control group (p>0.05). This shows that lactic acid Pediococcus XJ335 can accumulate fat and liver lipids, and has the effect of alleviating obesity and lowering blood lipids.

[0073] Actinobacteriota It plays a vital role in maintaining intestinal permeability, downregulating inflammatory processes, and directly participating in the mechanism of neurogenesis. Studies have found that Actinobacteriota It is a protective factor for chronic complications of diabetes. As the abundance of the bacterial flora increases, the risk of chronic complications of diabetes can be reduced. From the figure, we can see that lactic acid Pediococcus lactis XJ335 can increase the Actinobacteriota abundance, thereby reducing the risk of chronic complications of diabetes.

[0074] The above results indicate that Pediococcus acidilactici XJ3356 can significantly improve the imbalance of intestinal flora caused by T2DM, reduce the abundance of harmful bacterial genera while increasing the abundance of beneficial bacterial genera. like Fig.13 As shown in the figure, the microbial intestinal flora and dominant genus of each group were analyzed at the genus level. The relative distribution bar graph of species at the genus level of each experimental group showed that after the intervention of Pediococcus acidilactici, the intestinal microbial structure at the genus level was improved, especially XJ335. Compared with the Model group, the XZ306 group Ileibacterium , norank_f__ Muribaculaceae, Bifidobacterium The relative abundance decreased. Lactobacillus , unclassified_f__ Lachnospiraceae,and Pediococcus Relative abundance increased; XJ335 group Ileibacterium、Lactobacillus The relative abundance decreased. Faecalibaculum, norank_f__Muribaculaceae, unclassified_f__ Lachnospiraceae and Pediococcus The relative abundance increased; the study believes Ileibacterium The bacterial genus levels were positively correlated with blood lipid levels and metabolic disorders, which was consistent with our research conclusions.

[0075] In addition, we found that the three groups of mice treated with P. acidilactici had higher levels of Pediococcus spp. Pediococcus ), which proves from another perspective that the lactic acid Pediococcus we administered has a certain intestinal colonization ability and can achieve the effect of alleviating diabetes by improving the intestinal flora structure.

[0076] like Fig.14 As shown, the lactic acid Pediococcus XJ335 group significantly increased glycolysis / gluconeogenesis, pyruvate metabolism, fructose and mannose metabolism, amino sugar, nucleotide sugar metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism. This suggests that lactic acid Pediococcus XJ335 gavage intervention may be able to improve glycolysis / gluconeogenesis, pyruvate metabolism, fructose and mannose metabolism, amino sugar, nucleotide sugar metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism and other carbohydrate metabolism pathways and amino acid metabolism pathways in diabetic mice, thereby alleviating the hyperglycemia symptoms of diabetic mice.

[0077] 7. Metabolic profile analysis of mouse cecal contents: Enrichment analysis and pathway analysis were performed on the characteristic metabolites, and a total of 58 metabolic pathways were found to be disturbed by T2DM, of which 15 pathways were significantly affected by type 2 diabetes (p<0.05), including steroid hormone biosynthesis, arginine and proline metabolism, D-amino acid metabolism, β-alanine metabolism, cysteine ​​and methionine metabolism, primary bile acid biosynthesis, cytochrome P450 xenobiotic metabolism, lysine degradation, arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, drug metabolism-cytochrome P450, nucleotide sugar biosynthesis, histidine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis and lysine biosynthesis.

[0078] Metabolic pathways closely related to glucose and lipid metabolism in type 2 diabetes were selected based on Impact, FDR value and number of matching metabolites. We found that after intervention with Pediococcus acidilactici XJ335, pathways involved in arginine and proline metabolism (0.28), β-alanine metabolism (0.059), nucleotide metabolism (0.039) and glutathione metabolism (0.074) in T2DM were significantly disturbed.

[0079] The differential metabolites were analyzed by statistically analyzing the bonds in each group, and the results are shown in Table 2.

[0080] Table 2:

[0081] As shown in the table, compared with the Model group, XJ335 significantly reduced 2-(3-hexyl-4-methyl-2,5-dioxypyrrole-1-yl)-3-hydroxybutyric acid, 8-oxyguanosine, choline glutamate, N-glutamine proline, proline valine valine, and significantly increased 5-aminopentylamide, Beta-D-Glucopyranosyl-11-hydroxyjasmonic acid, and proline leucine; Metformin significantly reduced 1,11-undecanedicarboxylic acid, 11-epicocortisol, 3-hydroxy-N-desethyllidocaine, 8-oxyguanosine, α-naphthoflavone, argininosuccinic acid, choline glutamate, L-phosphoarginine, proline valine valine, raftansine, saxitoxin, and thromboxane B2, and significantly increased 5-aminopentylamide and Beta-D-Glucopyranosyl-11-hydroxyjasmonic acid.

[0082] These metabolites are mainly related to metabolic pathways such as steroid hormone biosynthesis, bile secretion, cortisol synthesis and secretion, cytochrome P450 xenobiotic metabolism, drug metabolism-cytochrome P450, lysine degradation, arginine biosynthesis, alanine, aspartate and glutamate metabolism, D-amino acid metabolism, arginine and proline metabolism, arachidonic acid metabolism, and 5-hydroxytryptamine synapse, further confirming that different intervention methods have different metabolic pathways for improving symptoms in diabetic mice. XJ335 mainly improved the amino acid metabolic pathway in diabetic mice.

[0083] 8. Real-time RT-qPCR detection of mRNA expression of genes related to blood glucose regulation in liver tissue Total RNA was extracted from liver samples according to the instructions in the TRIzol kit (Beijing Tsingke Biotech Co., Ltd.), and quantified by spectrophotometry by measuring the optical density at 260 and 280 nm. cDNA was synthesized from RNA samples using a cDNA synthesis kit (Beijing Tsingke Biotech Co., Ltd.). RT-qPCR reactions were performed using the Bio-Rad CFX system, and the reaction system preparation and reaction conditions involved are shown in the table. GAPDH was used as the internal reference gene, and the relative expression of the target gene was calculated using the 2−ΔΔCt method, with three parallel experiments set up for each sample.

[0084] Different lactic acid bacteria have different mechanisms and effects on diabetic mice. Fig.15 , 16 As shown, after the intervention of Pediococcus acidilactici XZ306 in mice, the expression of G6pase was significantly upregulated, and the upregulation results of Irs2, Akt2 and Ampk were not significant. After the intervention of Pediococcus acidilactici XJ335 in mice, the expression of Irs2 and G6pase was significantly upregulated, and Akt2 and Ampk were upregulated but the results were not significant. This indicates that Pediococcus acidilactici XJ335 may improve insulin resistance by promoting the expression of IRS genes and then activating Akt. It also shows that the mechanism of action of Pediococcus acidilactici XZ306 and XJ335 is completely different, and its final effect is not as good as that of XJ335.

Claims

1. A strain of lactic acid bacteria with fat-reducing, anti-inflammatory and blood sugar-lowering effects, characterized in that: The lactic acid bacteria are classified and named Pediococcus acidilactici ( Pediococcus acidilactici )XJ335, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30093, the deposit date is March 22, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of Pediococcus acidilactici XJ335 as claimed in claim 1 in the preparation of medicines or foods for improving symptoms of hyperglycemia.

3. Use of Pediococcus acidilactici XJ335 as claimed in claim 1 in the preparation of medicines or foods for preventing or treating inflammation caused by diabetes.

4. Use of Pediococcus acidilactici XJ335 as claimed in claim 1 in the preparation of drugs or foods for regulating fat metabolism.

5. Use of the Pediococcus acidilactici XJ335 as claimed in claim 1 in the preparation of drugs or foods for reducing vascular damage and preventing and treating cardiovascular diseases.

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