Pediococcus acidilactici XZ106 and application thereof

Abstract

The invention relates to pediococcus acidilactici XZ106, which is classified and named as pediococcus acidilactici, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.30094, the preservation date is March 22, 2024, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The pediococcus acidilactici XZ106 can (1) relieve insulin resistance, regulate insulin and glucagon secretion and improve liver gluconeogenesis, so that diabetes hyperglycemia is improved; (2) weight gain, fat accumulation and liver lipid accumulation of obese mice are inhibited, glycolipid metabolism disorder of the mice is improved, and the composition has excellent effects of reducing fat and relieving obesity; (3) the level of IL-10 can be remarkably improved, and the anti-inflammatory ability of mice is improved; (4) vascular injury is reduced, so that diabetic complications and various cardiovascular diseases of atherosclerosis are prevented.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Pediococcus acidilactici XZ106 strain and an application thereof. Background Art

[0002] Type 2 diabetes mellitus (T2DM) refers to a chronic disease caused by insufficient insulin secretion and / or insulin resistance (the body's decreased sensitivity to insulin and inability to effectively utilize it) due to genetic and / or environmental factors, leading to increased blood sugar levels. As the etiology and pathogenesis of diabetes have not been fully elucidated, there is still a lack of etiological treatment. The short-term goal of diabetes treatment is to eliminate diabetes symptoms and prevent acute and severe metabolic disorders by controlling hyperglycemia and related metabolic disorders; the long-term goal is to prevent and delay the occurrence and development of chronic complications of diabetes through good metabolic control, maintain good health and learning and working ability, improve the quality of life of patients, reduce mortality and prolong life.

[0003] At present, diabetes is often treated by taking hypoglycemic drugs. Common oral hypoglycemic drugs include sulfonylureas, biguanides, α-glucosidase inhibitors, DPP-4 inhibitors, and GLP-1 receptor agonists. There are many types of these hypoglycemic drugs, but they all have different side effects. Therefore, a safe T2DM drug with no side effects is urgently needed.

[0004] In recent years, laboratory and clinical studies have found that probiotics may be a promising auxiliary treatment for patients with T2DM. However, many probiotics with hypoglycemic effects have good results in in vitro tests, but after entering the body, due to the complex blood sugar mechanism and complex metabolic environment in the body, probiotics are easily affected by processing conditions, especially extreme temperatures and the human gastrointestinal tract, which reduces their vitality or even kills them, resulting in unsatisfactory probiotic effects. Therefore, it is necessary to continue to screen out probiotics with the potential to lower blood sugar, good stability in the human gastrointestinal tract, and strong safety. Summary of the invention

[0005] The purpose of the present invention is to provide a multifunctional lactic acid bacteria, which has excellent blood sugar lowering function, links the body's insulin resistance, and has excellent acid and bile salt resistance, as well as good fat reduction, anti-inflammatory, and vascular damage reduction effects.

[0006] Another object of the present invention is to provide the application of the above-mentioned Pediococcus acidilactici.

[0007] The purpose of the present invention is achieved through the following technical solutions: A strain of Pediococcus acidilactici XZ106, characterized in that: the classification of the Pediococcus acidilactici is named Pediococcus acidilactici (Pediococcus acidilactici ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30094, the deposit date is March 22, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The invention obtains traditional fermented yogurt from different regions of Xinjiang as samples, separates and screens the obtained strain XZ106, performs homology analysis and constructs a phylogenetic tree, and confirms that the separated XZ106 is Pediococcus acidilactici in combination with morphological observation.

[0009] After incubating Pediococcus acidilactici XZ106 in simulated gastric fluid for 3 h, the number of surviving live bacteria exceeded 10 7 CFU / mL, with good gastrointestinal fluid tolerance. Tested in 0.3% bile salt concentration, it has excellent bile salt resistance.

[0010] The application of the above-mentioned Pediococcus acidilactici XZ106 in the preparation of medicines or foods for improving symptoms of hyperglycemia.

[0011] The application of the above-mentioned Pediococcus acidilactici XZ106 in the preparation of drugs or foods for regulating glucose and lipid metabolism.

[0012] The application of the above-mentioned Pediococcus acidilactici XZ106 in the preparation of anti-inflammatory drugs or foods.

[0013] The application of the above-mentioned Pediococcus acidilactici XZ106 in the preparation of medicines or foods for reducing vascular damage and preventing and treating cardiovascular diseases.

[0014] The present invention has the following technical effects: The lactic acid bacteria XZ106 of the present invention can (1) relieve insulin resistance, regulate insulin and glucagon secretion, improve liver gluconeogenesis, and thus improve diabetic hyperglycemia; (2) inhibit weight gain, fat accumulation, and liver lipid accumulation in obese mice, improve glucose and lipid metabolism disorders in mice, and have excellent fat-reducing and obesity-alleviating effects; (3) can significantly improve IL-10 levels and enhance the anti-inflammatory ability of mice; and (4) reduce vascular damage, thereby preventing diabetic complications and various cardiovascular diseases such as atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Bar graph of α-amylase inhibition rate of screened strains.

[0016] Figure 2 : Bar graph of α-glucosidase results of screened strains.

[0017] Figure 3 :Results of hemolytic test of screened strains.

[0018] Figure 4 :Effects of three strains of Pediococcus acidilactici on body weight of mice.

[0019] Figure 5 : Effects of three lactic acid pellets on fasting blood glucose changes in mice.

[0020] Figure 6 : Area under curve (AUC) of mice (# 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)).

[0021] Figure 7 :Effects of different lactic acid Pediococci on changes of mouse organ indexes.

[0022] Figure 8 :Changes of fasting insulin, glucagon-like peptide levels and related indexes in mice.

[0023] Fig. 9 : Serum lipid levels and arteriosclerosis index in mice.

[0024] Fig.10 :Changes in the level of IL-10, an inflammatory factor indicator in mouse serum.

[0025] Fig.11 :Pathological observation of H&E staining of mouse pancreatic tissue.

[0026] Fig.12 :Pathological observation of mouse liver tissue by Oil Red O staining.

[0027] Fig.13 :(A) Column chart of relative abundance of species at phylum level;(B) Ratio of Firmicutes / Bacteroidetes;(CE) Column chart of species abundance of specific phyla in each group.

[0028] Fig.14 : Bar graphs showing the abundance of specific metabolic pathways (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).

[0029] Fig.15 : Relative mRNA expression levels of insulin signaling-related genes.

[0030] Fig.16 : Relative expression levels of mRNA of genes related to glucose production.

[0031] In the above figures, * indicates a significant difference compared with the model group (p<0.05); ** indicates an extremely significant difference compared with the model group (p<0.01); *** indicates an extremely significant difference compared with the model group (p<0.001); **** indicates an extremely significant difference compared with the model group (p<0.0001). DETAILED DESCRIPTION

[0032] 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.

[0033] Example 1 Screening and identification of strains Traditional fermented yogurt was obtained from different regions of Xinjiang 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.

[0034] After multiple plate separation observations, 9 lactic acid bacteria were screened from traditional yogurt, numbered XJ810, XZ306, XZ4410, XZ106, XJ243, XJ244, XJ402, XZ2010, and XZ502. 16S rDNA sequencing was used to further identify the obtained strains. PCR amplification of the 16S rDNA of lactic acid bacteria was performed, and the PCR was sequenced and compared with the NCBI database. The sequences were compared and identified as plant lactobacillus, lactobacillus acidilactici, lactobacillus acidilactici, lactobacillus rhamnosus, lactobacillus paracasei, Lactobacillus mucosa reuteri, Weissella fusion, and pseudo-mesenteric leuconostoc.

[0035] Example 2 In vitro screening of hypoglycemic strains 100 μL of the preserved bacterial solution was activated in MRS liquid medium for two generations and then used for extraction of cell-free supernatant: the test bacterial solution was inoculated into MRS medium at a ratio of 2% and cultured at 37 °C for 24 h, followed by centrifugation at 6000 r / min for 10 min, the supernatant was collected and filtered through a 0.22 μm microporous filter membrane to obtain the cell-free supernatant (CFS).

[0036] (1) Determination of α-amylase inhibition rate: 200 μL CFS was mixed with PBS (pH 7.4) and 0.5 mg / mL α-amylase solution in equal volumes, incubated in a 37 ℃ constant temperature water bath for 10 min, then the reaction solution was added to 100 μL of 1% soluble starch solution (w / v) at 37 ℃, reacted at 37 ℃ for 3 min, and then 200 μL DNS solution was added. After reacting in a boiling water bath for 5 min, it was quickly cooled to room temperature, 2 mL of sterile distilled water was added, and the absorbance was measured at 540 nm. PBS solution (0.1 mol / L, pH = 7.4) was used as a blank control for α-amylase solution and the sample to be tested, and acarbose was used as a positive control. The calculation formula of α-amylase inhibitor was as follows:

[0037] Wherein: A is the sample group, containing the sample and α-amylase solution; B is the sample blank group, containing samples but not α-amylase solution; C is the control group, containing no sample but α-amylase solution; The results are as follows Figure 1 As shown, the α-amylase inhibition rates of strains XZ106, XZ502, XJ810, XZ2010, XJ243, XJ244, XZ306, XJ402, and XZ4410 were 43.07±2.30, 62.51±0.89, 80.12±1.42, 48.56±2.67, 47.43±1.36, 62.16±3.87, 59.55±3.52, 49.52±0.53, and 46.90±1.74, respectively.

[0038] (2) Determination of α-glucosidase inhibition rate: For the strains obtained in the initial screening, 25 μL of 0.1 mol / L PBS buffer (pH 6.8) was first added to the wells of the 96-well plate, followed by 25 μL of 20 mmol / L PNPG and 25 μL of sample; the reaction was carried out at 37°C for 10 min, and then 50 μL of 0.2U / mL α-glucosidase was added; the reaction was carried out at 37°C for 15 min, and finally 100 μL of 0.1 mol / L Na2CO3 solution was added to terminate the reaction, and the absorbance (A) of the reaction solution at a wavelength of 405 nm was detected by an ELISA reader. With 1 mg / mL acarbose as the positive control, the inhibition rate is shown as follows: ×100% Wherein: A is the sample group, containing the sample and α-amylase solution; B is the sample blank group, containing samples but not α-amylase solution; C is the control group, containing no sample but α-amylase solution; The α-glucosidase inhibition rates of strains XZ106, XZ502, XJ810, XZ2010, XJ243, XJ244, XZ306, XJ402, and XZ4410 were 14.59±1.31, 9.83±2.31, 31.69±4.16, 13.56±4.45, 10.87±1.52, 10.5±1.74, 10.47±1.76, 3.82±1.08, and 7.12±2.19, respectively. Figure 2 XJ402 was excluded based on the in vitro glucose-lowering index, and the safety of the remaining 9 lactic acid bacteria was evaluated.

[0039] Example 3 Strain safety test: (1) Hemolytic activity evaluation: Activated probiotics were streaked onto 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.

[0040] The results are as follows Figure 3 As shown in the figure, Staphylococcus aureus showed obvious β-hemolysis, while strains XJ810, XJ243, XJ244 and XZ4410 had certain hemolysis, and strains XZ106, XZ502, XZ2010 and XZ306 had no obvious hemolysis. That is, among the above strains, XZ106, XZ502, XZ2010 and XZ306 had obvious hemolysis safety.

[0041] (2) Antibiotic sensitivity test: Take 100 µL of the cultured bacteria with a concentration of about 10 9 The above four probiotics with hemolytic safety at CFU / mL 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 sensitivity results of probiotics to each antibiotic were evaluated according to the standards of the Clinical and Laboratory Standards Institute (CLSL), and the results are shown in Table 1.

[0042] Table 1:

[0043] It can be seen from the table that strains XZ106 and XZ2010 have good antibiotic sensitivity.

[0044] The same Pediococcus acidilactici XZ306 was used as a control in the screening and was subsequently tested with XZ106.

[0045] Example 4 Simulated gastrointestinal fluid tolerance test (1) Gastric 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.

[0046] 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 o C, 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:

[0047] (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:

[0048] The results are shown in Table 2.

[0049] Table 2:

[0050] It can be seen that in the simulated gastrointestinal fluid, the survival rate of strain XZ106 reached 116.67%, while that of XZ306 was only 60.00%, indicating that XZ106 has excellent acid resistance and can effectively survive and grow in the gastrointestinal fluid environment. In addition, the bile salt tolerance of XZ106 is also better than that of XZ306.

[0051] Example 5 Verification of various types of effects of Pediococcus acidilactici in the body: 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), XZ106 group (XZ106) and XZ306 group (XZ306) (XZ106 and XZ306 are both selected lactic acid 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. The tissues were weighed and collected, and blood samples were collected. The liver tissue was stored at −80 ℃, 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. The serum samples were aliquoted and stored at −80 ℃. Animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Southwest University, Chongqing, China.

[0052] 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 .

[0053] (1) Body weight, fasting blood glucose and glucose tolerance test: Body weight was measured once a week using an electronic balance. Fasting (12 h) blood glucose determination was determined weekly. Blood samples were obtained from the tip of the tail vein, and blood glucose was measured using a glucometer. Glucose tolerance was assessed after 4 weeks of treatment. Glucose tolerance test (OGTT) was determined in animals fasted for 12 h at different time points (0, 30, 60, 90, and 120 min) after intraperitoneal injection of glucose (2 mg / kg body weight). The area under the curve (AUC) of each group of glucose values ​​was evaluated from 0 to 120 min.

[0054] Effects of drug administration and bacterial administration on body weight of type 2 diabetic mice induced by high-fat diet and STZ Figure 4 As shown in the figure, during the experiment, except for the model group, the weight of all mice increased to varying degrees, indicating that feeding bacteria and drug administration can alleviate the weight loss of mice caused by diabetes, among which XZ106 has the most obvious effect.

[0055] Changes of fasting blood glucose in mice Figure 5 As shown, during the four weeks of the 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 XZ106 group was equivalent to that of metformin hydrochloride and significantly better than that of the XZ306 group.

[0056] The area under the curve (AUC) refers to the overall change in blood glucose concentration within a specific period of time. It is often used to evaluate the body's glucose tolerance, that is, the body's ability to process glucose. An increase in the AUC value indicates a decrease in the body's glucose tolerance, and vice versa. Figure 6 It can be seen that the areas under the curve of the bacteria-fed and drug-administered groups were significantly lower than those of the model group, indicating that bacteria-fed and drug-administered groups effectively improved the glucose tolerance of diabetic mice, and the XZ106 group was better among the bacteria-fed groups.

[0057] (2) Determination of mouse organ index: Under normal circumstances, the organ index is relatively constant. When animals are poisoned, their organs are damaged, thus changing the organ index. The mouse organ index is calculated using the following formula: Organ index = organ mass (mg) / body weight (g) Depend on Figure 7 It can be seen that the intervention with Pediococcus acidilactici has the functional effect of inhibiting the increase of organ indexes of liver, kidney, spleen and heart in diabetic mice, and can repair and protect related organs to achieve the effect of alleviating diabetic complications. In addition, the effect of Pediococcus acidilactici XZ106 on alleviating the increase of organ indexes in diabetic mice is most significant.

[0058] (3) Serum biochemical analysis: Biochemical analysis All serum samples were obtained from mice 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 manufacturer's instructions in the corresponding detection kits. Fasting insulin levels (FINS), glucagon-like peptide (GLP-1), and inflammatory factors (TNF-α, IL-6, and IL-10) in mouse serum were measured using ELISA kits according to the manufacturer's instructions. At the end of the study, the homeostatic model insulin resistance index (HOMA-IR), β-cell function index (HOMA-β), and quantitative insulin sensitivity test index (QUICKI) values ​​were calculated: HOMA-IR = C FINS ×C FBG / 22.5 HOMA-β=20×C FINS / (C FBG -3.5) QUICKI=1 / (Log FBG+Log FINS) 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 after intervention are shown in the following table. 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 diabetic mice developed insulin resistance and insufficient insulin secretion as their weight increased. Compared with the Model group, the FINS and HOMA-IR values ​​of the Metformin group were significantly decreased (p<0.01), and the HOMA-IS was significantly increased (p<0.01), indicating that Metformin can improve the insulin resistance of T2DM. Compared with the Model group, the FINS and HOMA-IR values ​​of the Pediococcus acidilactici intervention groups (XZ306 and XZ106) were significantly decreased (p<0.01), and the GLP-1 value of the XZ106 group was significantly increased (p<0.05), while the effect of the XZ306 group was not significant. It can be seen that the XZ106 group was more effective in improving insulin levels, with the Metformin group decreasing by 25.47±4.36%, the XZ306 group decreasing by 29.38±6.01%, and the XZ106 group decreasing by 36.80±5.81%; and the XZ106 group had a more significant effect in improving glucagon-like peptide-1, with the GLP-1 of this group of mice increasing by 65.31±14.85% compared with the Model (p<0.01).

[0059] like Fig. 9As 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 XZ106 group was decreased by 25.31±2.02%, TG level was decreased by 62.28±3.53%, LDL-C level was decreased by 51.91±2.86%, and HDL-C level was increased by 29.84±5.12% (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, XZ106 has a better overall effect.

[0060] 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 suggests that Pediococcus acidilactici may play a certain role in preventing cardiovascular diseases.

[0061] IL-10 is an anti-inflammatory factor that can inhibit inflammation and prevent insulin resistance from worsening. Fig.10 As shown. Compared with the Control group, the Model group significantly reduced the level of IL-10 (p<0.01). Compared with the Model group, XZ306 and XZ106 were gavaged with lactic acid pellets. XZ306 had no significant effect on the level of IL-10 in serum, while the IL-10 level of mice in the XZ106 group was significantly increased (p<0.01). The above results indicate that lactic acid pellets XZ106 can significantly improve the IL-10 level and enhance the anti-inflammatory ability of mice.

[0062] (4) 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. 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 tissues were obtained at 200 times magnification using an inverted microscope. Fig.11 As shown ((a)Control group; (b)Model group; (c)Metformin group; (d)XZ306 group; (e)XZ106 group).

[0063] It can be seen that the pancreatic cells of the mice in the Control group were arranged regularly and had a complete structure, the edges of the islets were clear, the pancreatic cells were round or oval cluster structures, and the number of islet cells was large. Compared with the Control group, the islet cells 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 nucleoli were clear, the boundary between the islet cells and the acinus was more obvious, and the empty run was reduced. Among them, the number of islet cells in the XZ106 group mice increased significantly, and the number of islet cells in the XZ306 group mice was smaller than that in the Metformin intervention group. The above results show that after the intervention of P. acidilactici, especially XZ106, the pathological damage of the pancreatic tissue in diabetic mice was significantly improved, the boundaries of pancreatic β cells were clear, the repair of β cells was promoted, and the integrity of the cell structure was maintained.

[0064] 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.12 As shown in the figure ((a) Control group; (b) Model group; (c) Metformin group; (d) XZ306 group; (e) XZ106 group), the mice in the Model group had 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 different lactic acid Pediococcus had different degrees of changes after intervention. Among them, the lipid droplet area in the XZ106 group was significantly improved, which was significantly different from the Model group, and the accumulation of liver fat was reduced.

[0065] (5) 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.

[0066] Firmicutes (Firmicutes) are known as "obesity bacteria" and are significantly enriched in obese people. Bacteroidota (Bacteroidetes) is just the opposite. Fig.13 As shown in the experiment, the Model group and the Metformin group Firmicutes The relative abundance increased. Bacteroidota The relative abundance decreased, so it is speculated that the microbiota 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. The ratio of Firmicutes to Bacteroidetes (F / B) between the groups was calculated, and the results are shown in the figure. 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 XZ106 group was not significantly different from that in the Control group (p>0.05). This shows that lactic acid Pediococcus XZ106 has the effect of alleviating obesity. 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 bacterial flora increases, the risk of chronic complications of diabetes can be reduced. From the figure, we can see that lactic acid bacteria XZ106 can increase the Actinobacteriota abundance, thereby reducing the risk of chronic complications of diabetes.

[0067] like Fig.14 As shown, the lactic acid Pediococcus XZ106 treatment 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 indicates that lactic acid Pediococcus XZ1006 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.

[0068] (6) Metabolic profile analysis of mouse cecal contents: According to previous studies, the blood glucose level, biochemical indexes and intestinal flora-related results of T2DM mice were effectively improved after intervention with Pediococcus acidilactici XZ106 and XZ306, while the effect of XZ306 was not significant. In order to clearly observe and compare the changes in metabolite levels and metabolic differences of Pediococcus acidilactici, this experiment detected and analyzed the liver metabolites of Control, Model, Metformin, XZ306 and XZ106, with 6 mice in each group. The results are shown in Table 3.

[0069] Table 3: Metabolic pathways to which XZ106 significantly contributes

[0070] 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 XZ106, pathways involved in steroid hormone biosynthesis (0.28), arginine and proline metabolism (0.07), β-alanine metabolism (0.24), lysine degradation (0.16), lysine biosynthesis (0.11), nucleotide metabolism (0.11), cytochrome P450 xenobiotic metabolism (0.010), alanine, aspartate and glutamate metabolism (0.14), drug metabolism-cytochrome P450 (0.014), tryptophan metabolism (0.056), phenylalanine, tyrosine and tryptophan biosynthesis (0.14), tyrosine metabolism (0.03), cofactor biosynthesis (0.076), pyrimidine metabolism (0.11) and glutathione metabolism (0.083) in T2DM were significantly disturbed.

[0071] The differential metabolites among the groups in multivariate analysis are shown in Table 4 .

[0072] Table 4:

[0073] As shown in the table, compared with the Model group, XZ106 significantly decreased 1,11-undecanedicarboxylic acid, 11-epicotin, 1-nitro-5-glutathione-6-hydroxy-5,6-dihydronaphthalene, 2-(3-hexyl-4-methyl-2,5-dioxypyrrole-1-yl)-3-hydroxybutyric acid, 3-hydroxy-N-desethyl lidocaine, 3-methyl-5-propyl-2-furanonanoic acid, 8-oxyguanosine, α-naphthoflavonoid, argininosuccinic acid, asparagine threonine aspartic acid, cholestyrenic acid, N-glutamine proline, L-phosphoarginine, phenylglucuronide, proline valine valine, raftansine, saxitoxin, thromboxane B2, secoemopetasitolide B, valine proline glutamine, and significantly increased 5-aminopentylamide, Beta-D-Glucopyranosyl-11-hydroxyjasmonic acid, N-alpha-acetyllysine, proline, leucine; XZ306 significantly reduced 1-nitro-5-glutathione-6-hydroxy-5,6-dihydronaphthalene, 3-methyl-5-propyl-2-furannonanoic acid, argininosuccinic acid, L-phosphoarginine, saxitoxin, and significantly increased 5-aminopentylamide. 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 microbial strains have different metabolic pathways for improving the symptoms of diabetic mice.

[0074] Among them, XZ106 mainly improved the steroid hormone biosynthesis and amino acid (arginine and proline metabolism, lysine degradation, lysine biosynthesis, alanine, aspartate and glutamate metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, tyrosine metabolism) related metabolic pathways in diabetic mice.

[0075] (7) 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.

[0076] The results are as follows Fig.15 , 16 As shown in the figure, after the intervention of Pediococcus acidilactici XZ106 in mice, the expression of Ampk, Akt2, G6pase and PEPCK was significantly upregulated. Some evidence shows that Akt2 is related to glucose homeostasis. In fact, reducing the expression of Akt2 functional gene will lead to decreased insulin sensitivity, resulting in its inability to lower blood sugar well and increasing the risk of type 2 diabetes. In addition, Akt can regulate lipid metabolism and glucose metabolism through steroid regulatory element binding proteins (Sterol-regulatory element binding proteins, SREBP) and forkhead box protein O1 (Forkhead Box Protein O 1, FOXO1), respectively. SREBP can indirectly promote lipid metabolism and reduce fat synthesis. FOXO1 can upregulate the expression of phosphoenolpyruvate carboxykinase (Phosphoenolpyruvate Carboxykinase, PEPCK) to further regulate glycolysis / gluconeogenesis and regulate glucose and lipid metabolism, ultimately achieving the effect of improving diabetes. In addition, the activation of AMPK causes the phosphorylation of phosphofructo-2-kinase, stimulating the production of fructose 2,6-bisphosphate, thereby promoting glycolysis. AMPK promotes glucose uptake through an insulin-independent pathway. First, it induces glucose transporter 4 (GLUT4) to transport glucose into the plasma membrane, and second, it activates GLUUT4 gene expression through transcription factor phosphorylation, thereby improving diabetes. This suggests that lactic acid bacteria XZ106 can indirectly upregulate the expression of PEPCK by upregulating Akt2, and upregulating AMPK to induce GLUT4 expression, ultimately achieving the effect of improving diabetes by regulating glucose and lipid metabolism.

[0077] Different lactic acid bacteria have different mechanisms and effects on diabetic mice. Fig.15 and 16As shown, after the lactic acid Pediococcus XZ306 intervened in mice, the expression of G6pase was significantly upregulated, while the upregulation results of Irs2, Akt2 and Ampk were not significant, which indicates that the mechanism of action of lactic acid Pediococcus XZ306 is completely different from that of XZ106, and its final effect is not as good as that of XZ106.

Claims

1. A strain of Pediococcus acidilactici XZ106, characterized in that: The lactic acid Pediococcus is classified and named as lactic acid Pediococcus ( Pediococcus acidilactici ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30094, 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 XZ106 as claimed in claim 1 in the preparation of medicines or foods for improving symptoms of hyperglycemia.

3. Use of the Pediococcus acidilactici XZ106 as claimed in claim 1 in the preparation of drugs or foods for regulating glucose and lipid metabolism.

4. Use of the Pediococcus acidilactici XZ106 as claimed in claim 1 in the preparation of anti-inflammatory drugs or foods.

5. Use of the Pediococcus acidilactici XZ106 according to claim 1 in the preparation of medicines or foods for reducing vascular damage and preventing and treating cardiovascular diseases.

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