Lactobacillus plantarum for preventing prediabetes and application thereof

Abstract

The present application relates to the technical field of microorganism, and discloses a use of a composition containing lactobacillus plantarum AS21 with the preservation number of CGMCC No.24968 in the preparation of a product for preventing or improving aging-related impaired glucose tolerance, wherein the aging-related impaired glucose tolerance includes natural aging or a pre-diabetic state induced by high-fat diet combined with streptozotocin, wherein the lactobacillus plantarum AS21 reduces citrulline compensatory consumption by improving the citrulline synthesis capacity of the intestinal flora of a host and improving the liver and kidney functions of the host. The application of the lactobacillus plantarum AS21 in preventing pre-diabetes, in a natural aging mouse model and a HFD+STZ-induced aging pre-diabetes model, AS21 intervention can significantly reduce fasting blood glucose, improve oral glucose tolerance (oGTT), and enhance insulin secretion under glucose stimulation (GSIS), proving that it can effectively prevent and delay the occurrence and development of pre-diabetes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Lactobacillus plantarum* for the prevention of prediabetes and its applications. Background Technology

[0002] Against the backdrop of a rapidly aging global population, the decline in glycemic regulation associated with aging (i.e., prediabetes) has become a key risk factor for diabetes and various chronic diseases. Current interventions for glycemic regulation primarily act after the onset of the disease. For example, chemical hypoglycemic drugs (sulfonylureas, alpha-glucosidase inhibitors, etc.) mainly work by exogenously regulating blood glucose levels, but may cause adverse events such as hypoglycemia and cannot restore the body's own ability to regulate glycemic homeostasis. Exogenous citrulline supplementation has also shown hypoglycemic effects, but carries the risk of supraphysiological doses causing adverse intestinal reactions. Lifestyle interventions, on the other hand, have low adherence rates and are complex to implement. Therefore, developing safe, effective, and non-disruptive preventative strategies that fundamentally maintain glycemic homeostasis in elderly individuals is crucial for the proactive management and risk prevention of age-related glycemic abnormalities.

[0003] Probiotics have attracted widespread attention as potential metabolic regulators, and their application in gut health is relatively mature. However, there is still a significant gap in their specific prevention of age-related glucose intolerance. In particular, the functions and mechanisms of probiotic resources from special ecological environments (such as the Qinghai-Tibet Plateau) in regulating host metabolic homeostasis and preventing prediabetes have not been fully explored and applied. Summary of the Invention

[0004] To address the problems mentioned in the background art, the present invention provides the following technical solution: the use of a composition containing Lactobacillus plantarum AS21 with accession number CGMCC No. 24968 in the preparation of products for preventing or improving age-related glucose intolerance.

[0005] Preferably, the age-related glucose intolerance includes prediabetes induced by natural aging or a high-fat diet combined with streptozotocin.

[0006] Preferably, the *Lactobacillus plantarum* AS21 exerts its effect by increasing the host's intestinal flora's ability to synthesize citrulline and improving the host's liver and kidney function to reduce compensatory consumption of citrulline, thereby maintaining or increasing the level of circulating citrulline.

[0007] A microbial preparation for preventing or improving age-related glucose intolerance, comprising an effective amount of Lactobacillus plantarum AS21, the strain having the accession number CGMCC No. 24968.

[0008] Preferably, the viable bacterial count of the preparation is 1 × 10⁻⁶ per gram of preparation.8 Up to 1×10¹¹ CFU of Lactobacillus plantarum AS21.

[0009] Preferably, the microbial preparation is in the form of an oral liquid, capsule, tablet, powder, or granule.

[0010] A method for preparing a microbial preparation includes: culturing Lactobacillus plantarum AS21 with accession number CGMCC No. 24968 to obtain bacterial cells or fermentation product; and formulating the bacterial cells or fermentation product with an acceptable carrier.

[0011] Preferably, the microbial preparation is used to improve oral glucose tolerance, reduce fasting blood glucose, or enhance glucose-stimulated insulin secretion in middle-aged and elderly individuals.

[0012] Preferably, the microbial preparation is used to improve the citrulline metabolic balance in middle-aged and elderly individuals, maintain the citrulline level in the serum of middle-aged and elderly individuals, and / or increase the level of glucagon-like peptide-1.

[0013] A non-therapeutic health intervention method includes administering an effective amount of the described microbial preparation to aging individuals in need to prevent or improve their impaired glucose tolerance.

[0014] Compared with the prior art, the present invention provides the application of Lactobacillus plantarum AS21 in the prevention of prediabetes, which has the following beneficial effects:

[0015] 1. The application of *Lactobacillus plantarum* AS21 in the prevention of prediabetes: In naturally aging mouse models and HFD+STZ-induced prediabetes models, AS21 intervention significantly reduced fasting blood glucose, improved oral glucose tolerance (oGTT), and enhanced glucose-stimulated insulin secretion (GSIS) (see Appendix). Figure 8 (and related experimental data) prove that it can effectively prevent and delay the occurrence and development of prediabetes.

[0016] 2. Application of *Lactobacillus plantarum* AS21 in the prevention of prediabetes: AS21 intervention significantly increased the level of the beneficial factor adiponectin (APN) in the serum of aging individuals, and decreased the levels of leptin (LEP) and various pro-inflammatory factors (such as IL-1β, TNF-α, and IL-6) (see appendix). Figure 3 This approach works synergistically to maintain metabolic homeostasis by improving insulin sensitivity and reducing chronic inflammation.

[0017] 3. The application of *Lactobacillus plantarum* AS21 in the prevention of prediabetes elucidates for the first time that this strain maintains circulating citrulline levels through a "dual pathway": on the one hand, it improves liver and kidney function, reducing citrulline consumption caused by compensatory stress; on the other hand, it specifically activates citrulline synthesis-related genes (such as argF, glnA, argB, etc.) in the intestinal mucosal flora, increasing the supply of citrulline from intestinal flora (see Appendix). Figure 4 , 5 This mechanism is the core inventive contribution of this invention.

[0018] 4. Application of *Lactobacillus plantarum* AS21 in the prevention of prediabetes: Experiments confirmed that AS21 can significantly improve serum citrulline levels, which are significantly negatively correlated with improved glucose tolerance. Further analysis using the STC-1 cell model demonstrated that citrulline can directly stimulate enteroendocrine cells to synthesize and secrete glucagon-like peptide-1 (GLP-1), and AS21 intervention did indeed increase serum GLP-1 levels in aging mice (see appendix). Figure 6 , 7 This clarified the functional pathway from strain intervention to the ultimate improvement of blood glucose.

[0019] 5. Application of Lactobacillus plantarum AS21 in the prevention of prediabetes: Lactobacillus plantarum AS21 was isolated from traditional yak milk yogurt from the Qinghai-Tibet Plateau. It is a pure natural strain that has not been genetically modified. Animal experiments have confirmed that it has a mild effect and does not pose a risk of causing hypoglycemia or other adverse reactions, thus demonstrating good application safety. Attached Figure Description

[0020] Figure 1 The image shows the effect of AS21 on improving glucose tolerance in naturally aging male rats.

[0021] Figure 2 This figure shows the improvement in intestinal tissue structure in aging male rats after AS21 intervention.

[0022] Figure 3 This is a graph showing the regulation of serum metabolism and inflammatory factor levels by AS21 in aging male rats.

[0023] Figure 4 A graph showing serum citrulline levels and related metabolic indicators in AS21-preserving aging male rats.

[0024] Figure 5 The figure shows the effect of AS21 on the expression of citrulline synthesis genes in gut microbiota.

[0025] Figure 6 The graph shows the correlation between citrulline and glucose tolerance and its effect on GLP-1 secretion.

[0026] Figure 7A graph showing the effect of increasing serum GLP-1 levels in AS21 and its correlation with glucose tolerance.

[0027] Figure 8 This figure shows the preventive effect of AS21 in an induced prediabetes model. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Please see Figure 1-8 The present invention provides a technical solution: The *Lactobacillus plantarum* AS21 used in this invention has been deposited through patent procedures at the China General Microbiological Culture Collection Center (CGMCC). The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. The deposit date is May 26, 2022. The accession number is CGMCC No. 24968. The viability report from the collection center confirms that the strain is viable.

[0030] Example 1: The effect of Lactobacillus plantarum AS21 on improving glucose tolerance in naturally aging mice

[0031] This embodiment aims to verify the protective effect of Lactobacillus plantarum AS21 on the blood glucose regulation ability of naturally aging individuals.

[0032] Strains and Animals: Lactobacillus plantarum AS21 with accession number CGMCC No. 24968 was used. Experimental animals were 14-month-old and 18-month-old SPF-grade C57BL / 6J mice, which were randomly divided into control group and AS21 intervention group according to age and sex.

[0033] Intervention protocol: The AS21 intervention group received 0.2 mL of bacterial suspension with a viable count of 1×10^9 CFU (preparation method see Example 4) via gavage daily, while the control group received an equal volume of PBS via gavage. The intervention was administered once daily for the first two weeks, then adjusted to once every three days, and continued for 24 weeks.

[0034] Evaluation method: An oral glucose tolerance test (oGTT) was performed in week 23 of the experiment. After fasting for 14 hours, mice were administered a 20% glucose solution (2 g / kg) by gavage. Blood glucose levels were measured at 0, 15, 30, 60, 90, and 120 minutes, and the area under the curve (AUC) was calculated.

[0035] Result: As Figure 1 As shown, compared with the age-matched control group, the blood glucose levels of aged male rats (24 months old) in the AS21 intervention group were significantly lower at all time points of the oGTT, and the AUC was significantly reduced (p<0.05), indicating that AS21 can effectively improve glucose tolerance impairment caused by natural aging.

[0036] Example 2: Exploring the mechanism by which Lactobacillus plantarum AS21 exerts its effects on citrulline homeostasis and gut health

[0037] This embodiment aims to elucidate the potential mechanism by which AS21 improves glucose tolerance.

[0038] Sample Collection and Detection: At the end of the experiment in Example 1, serum, colon, and ileum tissues were collected from mice. Serum metabolites were detected by non-targeted metabolomics using LC-MS, and serum levels of citrulline, GLP-1, adiponectin, leptin, and inflammatory factors were detected by ELISA. Intestinal tissues were stained with hematoxylin and eosin (HE) and histologically scored.

[0039] Gut microbiota functional analysis: Intestinal mucosa was scraped, and DNA was extracted for metagenomic sequencing. HUMAnN2 software was used to analyze microbiota functional pathways, focusing on the expression abundance of genes related to citrulline synthesis (such as glnA, argB, argF, or argI).

[0040] result:

[0041] Improving metabolism and inflammation: AS21 intervention significantly increased serum adiponectin levels in aged male rats and decreased levels of leptin and pro-inflammatory factors IL-1β and TNF-α (see appendix). Figure 3 ).

[0042] Maintaining citrulline homeostasis: Serum citrulline levels in the AS21 intervention group were significantly higher than those in the control group (see appendix). Figure 4 Mechanistically, on the one hand, it improves liver and kidney function (reducing serum TB and UREA levels), thus decreasing the body's compensatory citrulline consumption; on the other hand, it activates the citrulline synthesis pathway in the intestinal mucosal flora, upregulating the expression of related genes and increasing fecal citrulline content (see appendix). Figure 5 ).

[0043] Improved intestinal structure and GLP-1 secretion: Mice in the AS21 intervention group had longer ileal villi, lower colonic tissue structure scores, and reduced inflammatory infiltration (see appendix). Figure 2 Correlation analysis showed a negative correlation between serum citrulline levels and glucose intolerance impairment. Further STC-1 cell experiments confirmed that citrulline treatment significantly upregulated the expression of the GLP-1 synthesis gene Gcg and promoted GLP-1 secretion (see appendix). Figure 6Consistent with this, serum GLP-1 levels in the AS21 intervention group mice were also significantly increased (see appendix). Figure 7 ).

[0044] Example 3: Preventive effect of Lactobacillus plantarum AS21 in an induced pre-aging diabetes model

[0045] This embodiment verifies the preventive effect of AS21 in a pathological induction model.

[0046] Model establishment and intervention: 15-month-old male C57BL / 6J mice were randomly divided into a normal control group (NC), a model group (Model), and an AS21 intervention group (AS21). Both the AS21 and model groups were induced using a high-fat diet (HFD) combined with low-dose streptozotocin (STZ) injections. The AS21 group received 1×10^9 CFU of AS21 live bacteria lyophilized powder via gavage daily throughout the treatment, while the control and model groups received PBS via gavage.

[0047] Evaluation indicators: Fasting blood glucose (FBG) was monitored weekly; oGTT and glucose-stimulated insulin secretion (GSIS) were measured in the later stage of modeling; serum citrulline level was measured as the experimental endpoint.

[0048] Result: As Figure 8 As shown, compared with the model group, AS21 intervention significantly delayed the rise in fasting blood glucose during modeling, improved the oGTT curve, and increased GSIS levels (p<0.05). Simultaneously, AS21 intervention maintained serum citrulline levels in the model mice. These results indicate that AS21 can effectively prevent the development of HFD-STZ-induced prediabetes in older adults.

[0049] Example 4: Microbial preparation containing Lactobacillus plantarum AS21 and its preparation

[0050] This embodiment provides a specific preparation method for the microbial preparation of the present invention.

[0051] Preparation of mycelium powder:

[0052] a. Inoculate Lactobacillus plantarum AS21 (CGMCC No. 24968) into MRS liquid medium and incubate at 37°C for 18-24 hours to obtain seed culture.

[0053] b. Transfer the seed culture to the fermentation medium at an inoculum volume of 2% (v / v) for scale-up culture, under the same conditions as above, until the bacterial OD of the culture reaches the target level. 600 The value reached 8.5-9.0.

[0054] c. Collect the cells by centrifuging the fermentation broth at 4℃ and 8000 rpm for 10 minutes.

[0055] d. Wash the bacterial cells twice with sterile physiological saline, add a freeze-drying protectant (such as 10% skim milk powder) to suspend, and perform vacuum freeze-drying to obtain AS21 bacterial powder with a viable count ≥1×10^11 CFU / g.

[0056] Oral capsule preparation: The above AS21 bacterial powder and pharmaceutical excipient microcrystalline cellulose are mixed evenly at a ratio of 1:1 (by weight), and then filled into hard gelatin capsules using a capsule filling machine. Each capsule contains no less than 5 × 10^9 CFU of live AS21 bacteria.

[0057] Preparation of bacterial solution: Reconstitute AS21 bacterial powder with sterile PBS or purified water, and dilute to the required concentration (e.g., 5×10^9 CFU / mL) for oral use.

[0058] Figure 1 Bacterial treatment improves glucose tolerance in aged male rats (24M) (AUC is the area under the curve).

[0059] Bacterial treatment improved glucose tolerance in naturally aging mice during a glucose tolerance test. Following oral glucose administration, blood glucose levels were significantly reduced at multiple time points in the bacterial treatment group.

[0060] Figure 2 Bacterial intervention resulted in longer ileal villi and lower colonic tissue abnormality scores in aged male rats.

[0061] The HE sections in the image show that the myelological structures of the ileum and colon in the bacterial treatment groups (20MP and 24MP) were more uniform and regular than those in the age-matched control groups (20MN and 24MN), with reduced inflammatory infiltration, reduced cell loss, longer intestinal villi, and lower overall colonic tissue scores. This improved intestinal tissue structure supports a reduction in systemic inflammation and the release of gut-derived GLP-1, a key hormone for glucose regulation.

[0062] Figure 3 Bacterial intervention regulates the levels of energy-regulating factors in the circulatory system of male rats and reduces inflammation.

[0063] Adiponectin (APN) promotes glucose uptake and fatty acid oxidation, and enhances insulin sensitivity; its levels decrease with age. APN levels in aged male rats were significantly lower than in 20-month-old male rats, while AS21 significantly increased adiponectin levels in 24-month-old male rats. Leptin (LEP) levels often indicate body fat status; aged individuals are prone to leptin resistance due to excessively high leptin levels, which is closely related to age-related metabolic diseases. AS21 significantly reduced serum leptin levels in aged male rats, improving metabolic homeostasis. AS21 treatment also reduced serum levels of pro-inflammatory factors such as interleukin-1β (IL-1β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in aged male rats.

[0064] Figure 4 Bacterial intervention increases circulating citrulline levels, improves liver and kidney function, and reduces compensatory citrulline consumption.

[0065] Citrulline is a non-essential amino acid that plays an important role in regulating inflammation levels, gut health, and glucose metabolism. Decreased serum citrulline levels in aged males are closely associated with impaired glycemic regulation. Non-target metabolomics showed that AS21 intervention significantly restored serum citrulline levels in aged males. Decreased serum total bilirubin (TB) and blood urea nitrogen (UREA) levels demonstrated that AS21 improved age-related liver and kidney function decline, thereby reducing compensatory expression of arginine succinate synthase 1 (Ass1) in the kidneys of aged male rats, thus reducing citrulline consumption. ELISA results of serum citrulline levels also supported the ability of AS21 intervention to maintain serum citrulline levels.

[0066] Figure 5 Microbial intervention increased citrulline synthesis in the intestinal mucosal flora.

[0067] Metagenomic analysis of the intestinal mucosal microbiota revealed that AS21 intervention significantly upregulated genes related to citrulline synthesis (glnA, argA, argB, argF (or argI), and argD) in the microbiota of aged male rats, and correspondingly, the citrulline content in feces was also significantly upregulated. AS21 increased the citrulline level in the microbiota by activating citrulline synthesis in the mucosal microbiota, further increasing the source of citrulline in the serum.

[0068] Figure 6 Serum citrulline levels are negatively correlated with impaired glucose tolerance. Citrulline supplementation can increase GLP-1 secretion in the STC-1 cell line.

[0069] Correlation analysis of citrulline with glycemic homeostasis indicators and inflammatory factors revealed a significant negative correlation between serum citrulline levels and oral glucose tolerance impairment and serum pro-inflammatory factor IL-6. In the STC-1 enteroendocrine cell line model, glucose and citrulline stimulation increased GLP-1 synthesis and secretion. qPCR analysis showed that mRNA levels increased the expression of the GLP-1 synthesis gene Gcg. Immunofluorescence staining showed that citrulline increased the amount of GLP-1 synthesized by cells. ELISA analysis of cell culture supernatant showed that Cit increased the level of glucagon-like peptide-1 (GLP-1) in the supernatant.

[0070] Figure 7 AS21 intervention increased serum GLP-1 levels.

[0071] Correlation analysis showed a negative correlation between serum GLP-1 levels and the area under the glucose tolerance curve in mice. Higher GLP-1 levels indicate stronger glycemic regulation. AS21 treatment significantly increased serum GLP-1 levels in aged mice, consistent with improved glycemic regulation.

[0072] Figure 8 Bacterial intervention also maintained the mice's ability to regulate blood glucose in the HFD-STZ-induced glucose intolerance mouse model.

[0073] AS21 intervention maintained serum citrulline levels in induced prediabetes model mice, significantly reduced fasting blood glucose during model induction, improved glucose tolerance, and enhanced insulin secretion under glucose stimulation. In summary, the induced glycemic imbalance mouse model demonstrates that AS21 intervention effectively prevented the development of prediabetes in aged mice and improved their glycemic regulation capacity.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Use of a composition comprising Lactobacillus plantarum AS21 with accession number CGMCC No. 24968 in the preparation of a product for the prevention or improvement of age-related glucose intolerance.

2. The use as described in claim 1, wherein, The age-related glucose intolerance includes prediabetes induced by natural aging or a high-fat diet combined with streptozotocin.

3. The use as described in claim 1 or 2, wherein, The *Lactobacillus plantarum* AS21 exerts its effect by enhancing the host's intestinal flora's ability to synthesize citrulline and improving the host's liver and kidney function to reduce compensatory consumption of citrulline, thereby maintaining or increasing the level of circulating citrulline.

4. A microbial preparation for preventing or improving age-related glucose intolerance, comprising 1×10 9 CFU of Lactobacillus plantarum AS21, the strain of which has the accession number CGMCC No. 24968.

5. The microbial preparation according to claim 4, wherein, The viable count of the formulation is 1 × 10¹¹ CFU of Lactobacillus plantarum AS21 per gram of formulation, and the oral intake is 1 × 10¹¹ CFU. 9 CFU.

6. The microbial preparation as described in claim 4 or 5, wherein it is in the form of an oral liquid, capsule, tablet, powder or granule.

7. A method for preparing the microbial preparation of claim 4, comprising: Lactobacillus plantarum AS21 with preservation number CGMCC No.24968 was cultured to obtain bacterial cells or fermentation products; And the preparation of the bacterial cells or fermentation product with an acceptable carrier.

8. The use as described in any one of claims 1 to 3, or the microbial preparation as described in any one of claims 4 to 6, for improving oral glucose tolerance, reducing fasting blood glucose, or enhancing glucose-stimulated insulin secretion in middle-aged and elderly individuals.

9. The use as described in any one of claims 1 to 3, or the microbial preparation as described in any one of claims 4 to 6, for improving serum citrulline homeostasis and / or increasing glucagon-like peptide-1 levels in middle-aged and elderly individuals with citrulline imbalance.

10. A non-therapeutic health intervention method comprising administering an effective amount of a microbial preparation as described in any one of claims 4 to 6 to an aging individual in need to prevent or improve their impaired glucose tolerance.

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