Use of lactobacillus crispatus in blood glucose regulation, improvement of cognitive dysfunction in diabetes and treatment of vaginitis

Abstract

This invention relates to the field of microbial technology, specifically disclosing *Lactobacillus curvature* and its application in blood glucose regulation, improvement of diabetic cognitive impairment, and treatment of vaginitis. In this invention, *Lactobacillus curvature* A21013 and A21033 are discovered for the first time to possess multiple functions: they can lower blood glucose, maintain the morphology and function of pancreatic β-cells, improve diabetic cognitive impairment, and inhibit the growth and reproduction of harmful vaginal pathogens and oral pathogens, providing a dual prevention and treatment approach for patients with diabetes, cognitive impairment, and vaginitis.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Lactobacillus curvature and its application in blood glucose regulation, improvement of diabetic cognitive impairment, and treatment of vaginitis. Background Technology

[0002] With improved living standards and an accelerating aging population, the incidence of diabetes is rising year by year, with type 2 diabetes accounting for over 90% of all diabetes cases. Type 2 diabetes is characterized by progressive insufficiency of insulin secretion from β-cells, insulin resistance, and genetically related secretion defects, often accompanied by metabolic syndrome. Current blood glucose management mainly relies on oral hypoglycemic agents (such as metformin) and dietary control, but these methods generally suffer from significant drug side effects and poor long-term patient adherence. In recent years, probiotics have been considered to have the potential to assist in regulating blood glucose; however, most of the currently disclosed strains have failed to stabilize blood glucose within a healthy range (3.9-6.1 mmol / L), and generally exhibit weak intestinal colonization and short-lasting effects.

[0003] Cognitive impairment, a significant central nervous system complication of diabetes, is receiving increasing attention. Long-term hyperglycemia and related metabolic abnormalities can lead to a decline in various cognitive abilities, including memory, attention and executive function, language skills, and information processing speed, imposing a significant burden on patients and society. Currently, there is a lack of effective interventions for this complication, particularly research on improving diabetes-related cognitive impairment through probiotics, which remains largely unexplored.

[0004] On the other hand, female reproductive health is closely related to the balance of the vaginal microbiota. Under healthy conditions, vaginal lactobacilli maintain an acidic environment through mechanisms such as lactic acid production, hydrogen peroxide production, and competitive adsorption, inhibiting the excessive growth of pathogenic microorganisms. Once the microbiota is imbalanced, it can easily lead to bacterial vaginosis, candidal vaginitis, etc., and the resistance rate of commonly used azole drugs is increasing year by year, with a high recurrence rate, making their treatment also challenging.

[0005] Lactobacillus curvaturei ( Lactobacillus crispatus As one of the dominant probiotics in the vagina, *Lactobacillus curvatureensis* has been extensively studied in maintaining reproductive tract health, but its functions are mostly focused on its antibacterial effects against common pathogens in vitro. Furthermore, no *Lactobacillus curvatureensis* strain has been found that can significantly lower blood sugar while simultaneously and effectively inhibiting vaginal pathogens. Moreover, there are no studies on its ability to inhibit respiratory and oral pathogens such as *Klebsiella pneumoniae* subsp. *p.* and *Streptococcus pharyngitis*. It is noteworthy that, to date, there is no oral bifunctional *Lactobacillus curvatureensis* preparation that simultaneously covers blood sugar regulation and vaginitis prevention; synergistic intervention strategies targeting metabolic diseases and reproductive tract infections still need to be developed. Summary of the Invention

[0006] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide the application of *Lactobacillus curvature* in blood glucose regulation, improvement of diabetic cognitive impairment, and treatment of vaginitis. In this invention, *Lactobacillus curvature* A21013 and A21033 are discovered for the first time to possess multiple functions: they can lower blood glucose, maintain the morphology and function of pancreatic β-cells, improve diabetic cognitive impairment, and inhibit the growth and reproduction of harmful vaginal pathogens and oral pathogens, providing a dual preventive and therapeutic approach for patients with diabetes, cognitive impairment, and vaginitis.

[0007] In a first aspect, the invention provides the use of *Lactobacillus curvatureii*, its cultures, or metabolites in the preparation of medicaments for the prevention and / or treatment of vaginal diseases in women.

[0008] In this invention, the term "culture" refers to a metabolically active mixed system dominated by a target microbial population, obtained through inoculation and cultivation under artificially controlled conditions (such as at least one of a specific culture medium, temperature, pH, and gaseous environment). That is to say, the culture contains live bacteria.

[0009] In this invention, the term "metabolite" refers to all chemical substances produced or transformed by microorganisms through a series of enzyme-catalyzed biochemical reactions (metabolic pathways) during their growth, reproduction, and maintenance of life activities, including primary metabolites and secondary metabolites.

[0010] In some embodiments of the present invention, the vaginal disease includes bacterial or fungal vaginal diseases.

[0011] In some embodiments of the present invention, the vaginal disease is vaginitis.

[0012] In some embodiments of the present invention, the vaginal disease includes bacterial vaginosis, fungal vaginitis, or mixed vaginitis.

[0013] In some embodiments of the present invention, the vaginal disease is a vaginal disease or vaginitis caused by at least one of Streptococcus vaginalis, Gardnerella vaginalis, Candida albicans, and Staphylococcus aureus.

[0014] A second aspect of the invention provides the use of *Lactobacillus curvatureii*, its cultures, or metabolites in the preparation of antibacterial products.

[0015] In some embodiments of the present invention, the antibacterial product includes: in vivo antibacterial drugs and in vitro bactericidal products.

[0016] In some embodiments of the present invention, the in vitro sterilization product includes a surface sterilizer.

[0017] In some embodiments of the present invention, the in vivo antibacterial drug is used to inhibit the growth of oral or genital microorganisms.

[0018] In some embodiments of the present invention, the microorganisms include at least one of bacteria and fungi.

[0019] In some embodiments of the present invention, the bacteria include at least one of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus vaginalis, Klebsiella pneumoniae subsp., Streptococcus pharyngitis, Streptococcus gossyflores, and Gardnerella vaginalis.

[0020] In some embodiments of the present invention, the fungus includes Candida albicans.

[0021] A third aspect of the invention provides the use of *Lactobacillus curvatureii*, its cultures, or metabolites in the preparation of medicaments for regulating blood glucose.

[0022] A fourth aspect of the invention provides the use of *Lactobacillus curvatureii*, its cultures, or metabolites in the preparation of medicaments for treating or improving cognitive impairment.

[0023] In some embodiments of the present invention, the cognitive impairment is diabetic cognitive impairment.

[0024] In some embodiments of the present invention, the curly-shaped Lactobacillus has all the effects described above.

[0025] In some embodiments of the present invention, the *Lactobacillus curvaturei* is selected from at least one of the *Lactobacillus curvaturei* with accession numbers GDMCC NO: 63543 and GDMCC NO: 63544.

[0026] In some embodiments of the present invention, Lactobacillus curvature A21013 and A21033 with accession numbers GDMCC NO: 63543 and GDMCC NO: 63544 have been disclosed in Chinese Patent CN 117165477 B, which is incorporated herein by reference.

[0027] In some embodiments of the present invention, the product or drug also contains pharmaceutically acceptable excipients.

[0028] In some embodiments of the present invention, the excipients are rationally selected based on factors such as the product form, intended use, and route of administration of the product or drug, including but not limited to: diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0029] In some embodiments of the present invention, a second active substance is also added to the product or drug.

[0030] In some embodiments of the present invention, the second active substance comprises a drug having at least one of the following functions (1)-(4):

[0031] (1) Prevention and / or treatment of vaginal diseases in women;

[0032] (2) Antibacterial;

[0033] (3) Regulate blood sugar;

[0034] (4) To treat or improve cognitive impairment.

[0035] In some embodiments of the present invention, the dosage form of the product or drug includes: solution, powder, tablet, granule, capsule, aerosol, ointment or suppository.

[0036] In some embodiments of the present invention, the product or drug is administered via the gastrointestinal tract.

[0037] In some embodiments of the present invention, the product or drug is administered orally.

[0038] The beneficial effects of this invention are:

[0039] 1. This invention is the first to discover that Lactobacillus curvature A21013 and A21033 can produce high levels of glucagon-like peptide-1 (GLP-1), thereby significantly improving pancreatic β-cell function, maintaining healthy blood glucose levels, improving pancreatic resistance and glucose tolerance, protecting pancreatic β-cells from damage, and maintaining their normal morphology and function.

[0040] 2. This invention is the first to discover that Lactobacillus curvature A21013 and A21033 can improve cognitive impairment in diabetes patients, and the effect is significant.

[0041] 3. This invention is the first to discover that Lactobacillus curvature A21013 and A21033 have the effect of inhibiting vaginal bacteria / fungi, thereby maintaining the balance of vaginal flora and alleviating vaginitis.

[0042] 4. This invention is the first to discover that Lactobacillus curvature A21013 and A21033 have the function of inhibiting oral pathogens and can prevent and / or improve oral health.

[0043] 5. The Lactobacillus curlis A21013 and A21033 in this invention have multiple effects, overcoming the shortcomings of existing technologies where Lactobacillus curlis has a single effect or no related effects, and providing a new direction for the treatment of related diseases. Attached Figure Description

[0044] Figure 1 The secretory capacity of Lactobacillus curvature A21013 and A21033 GLP-1.

[0045] Figure 2 Fasting blood glucose levels in mice of different experimental groups at week 7.

[0046] Figure 3 The fasting blood glucose (FBG), insulin (INS), and glycated hemoglobin (GHb) levels of mice in different experimental groups at week 9.

[0047] Figure 4 The levels of HOMA-IR and C-peptide (CP) in mice from different experimental groups.

[0048] Figure 5 H&E staining images of pancreatic tissue from mice in different experimental groups.

[0049] Figure 6 The results of open field experiments (total distance and dwell time) for mice in different experimental groups are shown.

[0050] Figure 7 The results of the Y-maze experiment for mice in different experimental groups (exploration time of novel arm, number of entries, and spontaneous alternation).

[0051] Figure 8 Results of the experiment to identify new objects.

[0052] Figure 9 These are representative images showing the inhibitory effects of different experimental groups on Candida albicans hyphae.

[0053] Figure 10 The results show the concentration of hydrogen peroxide produced by Lactobacillus curvature A21033 and A21013.

[0054] Biological Preservation Information

[0055] Lactobacillus crispatus A21013 and A21033 (both taxonomically named Lactobacillus crispatus) are deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on June 8, 2023, with accession numbers GDMCC No:63543 and GDMCC No:63544, respectively. Detailed Implementation

[0056] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0057] In the following embodiments, the *Lactobacillus curvature* used are *Lactobacillus curvature* A21013 and A21033, both of which are disclosed in Chinese Patent CN 117165477 B, with corresponding accession numbers GDMCC NO: 63543 and GDMCC NO: 63544, which are incorporated herein by reference.

[0058] In the following examples, the modified peptone yeast extract glucose (MPYG) solid culture medium used, calculated per L, consisted of the following components: 5.00 g tryptone, 3.00 g peptone, 2.00 g soybean peptone, 1.00 g polypeptone, 10.00 g yeast extract, 5.00 g beef extract, 5.00 g glucose, 0.50 ml Tween 80, 0.50 g maltose, 0.50 g cellobiose, 0.50 g soluble starch, 0.50 mL glycerol, 2.00 g K₂HPO₄, 0.50 g cysteine, 0.25 g Na₂S, 1.00 mg resazurin, 40.00 mL salt solution, 10.00 mL trace element mixture, 10.00 mL vitamin mixture, 10.00 mL heme chloride, 0.20 mL vitamin K1 mixture, and 15 g agar.

[0059] The salt solution contains the following components per 1 L: CaCl2·2H2O 0.25 g, MgSO4·7H2O 0.50 g, K2HPO4 1.00 g, KH2PO4 1.00 g, NaHCO3 10.00 g, and NaCl 2.00 g.

[0060] The trace element mixture, calculated per 1 L, contains the following components: 1.50 g of hyponitrotriacetic acid, 3.00 g of MgSO4·7H2O, 0.50 g of MnSO4·H2O, 1.00 g of NaCl, 0.10 g of FeSO4·7H2O, 0.18 g of CoSO4·7H2O, 0.10 g of CaCl2·2H2O, 0.18 g of ZnSO4·7H2O, 0.01 g of CuSO4·5H2O, 0.02 g of KAl(SO4)2·12H2O, 0.01 g of H3BO3, 0.01 g of Na2MoO4·2H2O, 0.03 g of NiCl2·6H2O, and 0.30 mg of Na2SeO3·5H2O.

[0061] The vitamin mixture contains the following components per 1 L: Biotin 2.00 mg, Folic Acid 2.00 mg, Pyridoxine Hydrochloride 10.00 mg, Thiamine Hydrochloride Dihydrate 5.00 mg, Riboflavin 5.00 mg, Niacin 5.00 mg, D-Calcium Pantothenate 5.00 mg, Vitamin B12 0.10 mg, Para-aminobenzoic Acid 5.00 mg, and Lipoic Acid 5.00 mg.

[0062] The preparation method of the vitamin K1 mixture is as follows: dissolve 0.1 g of vitamin K1 (purchased from Hopebio) in 20 mL of 95% ethanol and filter to sterilize.

[0063] The preparation method of heme chloride is as follows: 0.05 g of heme chloride is dissolved in 1 mL of 1 mol / L NaOH, diluted to 100 mL with distilled water, and then filtered to remove bacteria.

[0064] Example 1

[0065] In this embodiment, the in vitro GLP-1 secretion capacity of Lactobacillus curvature A21013 and A21033 was tested.

[0066] The specific experimental method is as follows:

[0067] Glyceryl truncatum A21013 and A21033 were removed from the -80℃ freezer and streaked onto MPYG solid medium, then cultured facultatively at 37℃ for 48 h. Single colonies were then transferred to MPYG broth (i.e., MPYG liquid medium, with agar removed from the solid medium) and cultured facultatively at 37℃ for 48 h. This was then inoculated again into fresh MPYG broth and cultured facultatively at 37℃ for another 48 h. After culture, 1 mL of the bacterial suspension was centrifuged (6000 rpm, 1 min), and the fermentation supernatant was collected and filtered through a 0.22 μm filter for sterilization.

[0068] The GLP-1 level in the sterilized fermentation supernatant was measured using a mouse glucagon-like peptide-1 (GLP-1) ELISA kit (purchased from Jianglai Biotechnology). Blank MPYG medium was used as a negative control.

[0069] The results are as follows Figure 1 As shown.

[0070] It was found that the GLP-1 content in the fermentation supernatant of Lactobacillus curvature A21013 and A21033 reached 1307 pg / mL and 1265 pg / mL, respectively.

[0071] Example 2

[0072] In this embodiment, lyophilized powders of Lactobacillus curlis A21013 and A21033 were prepared. The specific preparation method is as follows:

[0073] Single colonies of activated *Lactobacillus curvatureii* A21013 or A21033 were picked and cultured in 15 mL of MPYG liquid medium at 37 °C under anaerobic conditions for 12 h to obtain seed culture 1. Then, at an inoculum rate of 3%, this was inoculated into 150 mL of MRS liquid medium and cultured in anaerobic conditions at 37 °C for 20–22 h to obtain seed culture 2. Finally, at an inoculum rate of 3%, this was inoculated into 300 mL of MPYG liquid medium and cultured in anaerobic conditions at 37 °C for 20–24 h to obtain the scaled-up bacterial culture.

[0074] Centrifuge the bacterial culture at 5000 rpm for 5 min at 4 °C in a refrigerated centrifuge, and discard the supernatant. Wash the bacterial cells twice with physiological saline, and centrifuge again at 5000 rpm for 5 min at 4 °C. Weigh the wet weight of the bacterial cells. Then, add sterile 12% skim milk solution at a 1:1 volume ratio as a lyophilization protectant to resuspend the bacterial cells. Aliquot the cells into EP tubes, place them at -20 °C for 30 min, and then store them at -80 °C for 1 h. Place the EP tubes into lyophilization bottles, and then freeze-dry them in a freeze dryer for 30 h to obtain the final lyophilized bacterial powder.

[0075] Example 3

[0076] In this embodiment, the in vivo blood glucose regulation function of Lactobacillus curvature A21013 and A21033 was tested.

[0077] The specific testing method is as follows:

[0078] Fifty SPF-grade, 6-week-old male C57BL / 6J mice were randomly selected and acclimatized for one week. During the acclimatization period, a 12-hour light cycle was maintained, with a temperature of 20-26 °C and humidity of 40%-60%. Mice had free access to food and water. After the acclimatization period, the mice were randomly divided into 5 groups:

[0079] Control group (i.e. normal group): fed with basal maintenance diet and gavage with 0.2 mL of physiological saline;

[0080] Model group: fed a high-fat diet (60% fat content) and administered 0.2 mL of physiological saline by gavage;

[0081] A21013 intervention group: fed a high-fat diet (60% fat content) and administered 0.2 mL of lyophilized bacterial powder suspension by gavage (A21013 lyophilized bacterial powder was dissolved in physiological saline to a concentration of 1×10⁻⁶). 10 (cfu / mL)

[0082] A21033 intervention group: fed a high-fat diet (60% fat content) and administered 0.2 mL of lyophilized bacterial powder suspension by gavage (A21033 lyophilized bacterial powder was dissolved in physiological saline to a concentration of 1×10⁻⁶). 10 (cfu / mL)

[0083] Control group: fed a high-fat diet (60% fat content) and administered 0.2 mL of commercially available Lactobacillus flavus strain 1 lyophilized powder by gavage (the commercially available Lactobacillus flavus strain 1 lyophilized powder was dissolved in physiological saline to a concentration of 1×10⁻⁶). 10 (cfu / mL).

[0084] Fasting blood glucose levels in mice were measured before feeding them a high-fat diet. Mice with abnormal blood glucose levels (outside the normal range) were not used in the experiment. On days 14 and 17 of high-fat diet feeding, streptozotocin (STZ) (dissolved in citrate buffer at a dose of 80 mg / kg) was injected intraperitoneally into all groups except the control group. The control group received an equal volume of citrate buffer intraperitoneally. Mice were fasted the day before injection. On days 20 and 21, fasting blood glucose levels in the model group (i.e., high-fat diet-fed) was measured twice consecutively. A fasting blood glucose concentration greater than 7.0 mmol / L and a 2-hour post-feeding blood glucose concentration greater than 11.0 mmol / L were considered successful model establishment. The entire experimental period lasted 9 weeks. However, during blood glucose testing in week 7, the commercially available Lactobacillus flavus strain 1 failed to improve blood glucose levels, resulting in poor mouse condition. Therefore, the experiment in the control group was terminated prematurely.

[0085] Fasting blood glucose (FBG) in mice was measured at weeks 7 and 9 using a glucose assay kit (GOD-POD colorimetric method) (purchased from Regen Biotech). In week 9, the levels of insulin (INS) and glycated hemoglobin (GHb) in mice were measured using a mouse insulin (INS) ELISA kit and a mouse glycated hemoglobin (GHb) ELISA kit, respectively.

[0086] The results are as follows Figure 2 and Figure 3 As shown.

[0087] In this embodiment, a type 2 diabetic mouse model was established using a high-fat diet and STZ to evaluate the in vivo effects of *Lactobacillus curvaturei* A21013 and A21033 on pancreatic β-cell growth and hypoglycemic function. Results showed that, compared to the model group, at week 7, the blood glucose level in the commercially available *Lactobacillus curvaturei* strain 1 intervention group did not decrease, while blood glucose levels were significantly improved after gavage administration of A21013 and A21033, indicating differences in efficacy between different strains of the same *Lactobacillus curvaturei*.

[0088] After 9 weeks of gavage administration, compared with the model group, the A21013 and A21033 intervention groups reduced fasting blood glucose levels in mice by 75% and 74%, respectively, maintaining blood glucose at a healthy level. However, due to the lack of improvement in blood glucose levels and the poor condition of the mice in the commercially available Lactobacillus curvature strain 1 intervention group, the experiment could not continue, and therefore no relevant data were recorded.

[0089] In addition, after 9 weeks of gavage, compared with the model group, A21013 and A21033 reduced glycated hemoglobin by 59% and 35%, and insulin by 26% and 27%, respectively, in mice.

[0090] C-peptide (CP) level is an important indicator for assessing pancreatic β-cell function. Therefore, the mouse C-peptide (CP) level was further measured using a mouse C-peptide (CP) ELISA kit (purchased from Jianglai Biotechnology), and the insulin resistance index (HOMA-IR) of the mice was calculated based on the fasting blood glucose and fasting insulin results. The calculation formula is as follows:

[0091] HOMA-IR = .

[0092] Meanwhile, pancreatic tissue from each group of mice was sectioned and hematoxylin-eosin staining (HE staining) was performed to assess the structure and function of mouse pancreatic β cells.

[0093] The results are as follows Figure 4 and Figure 5 As shown.

[0094] It was found that after 9 weeks of gavage, compared with the model group, the insulin resistance index of the A21013 and A21033 intervention groups decreased by 77% and 66%, respectively, indicating a significant improvement in islet resistance. Simultaneously, the C-peptide (CP) levels in these two groups of mice increased by 60% and 57%, respectively. Combined with the results of pancreatic tissue pathology sections, it was found that the model group exhibited severe atrophy of islet β cells, accompanied by a reduction in islet β cell area. However, after gavage administration of A21013 and A21033, the islet structure remained relatively intact, indicating that A21013 and A21033 can protect islet β cells from damage and maintain their normal morphology and function.

[0095] Example 4

[0096] In this embodiment, the preventive or therapeutic effects of Lactobacillus curvature A21013 and A21033 on diabetic cognitive impairment were tested.

[0097] The specific testing method is as follows:

[0098] The model establishment method and group dosing regimen are the same as in Example 3 above, and the modeling is also based on a high-fat diet.

[0099] The experiment lasted for 9 weeks. One week before the end of the experiment, an open field test was conducted to assess the movement and exploration abilities of the mice in each group. The open field test chamber was 50 cm long, 50 cm wide, and 40 cm high, with a gray interior to ensure a clear viewing angle and prevent mouse behavior from being affected by color. The camera was placed in the center of the test chamber, and the shooting angle and focus were adjusted to ensure that the viewing angle covered the entire test area. Each group of mice underwent acclimatization feeding and testing to adapt to the test chamber environment and reduce stress responses. After the acclimatization test, the mice were placed in one corner of the open field, and timing and video recording began immediately. Smart 3.0 analysis software was used for real-time video recording and analysis, recording the mice's movement trajectory in the open field over 10 minutes, including the time spent in the central area of ​​the open field (s) and the distance traveled. After each mouse was tested, the test chamber was thoroughly cleaned with 75% alcohol to eliminate animal odors and prevent interference from the previous mouse's excrement and odors.

[0100] The results are as follows Figure 6 As shown.

[0101] It was observed that the model group mice exhibited signs of aging, such as slowed movement and reduced exploratory activity. In contrast, the Lactobacillus curvature A21033 intervention group showed more active movement and exploratory behavior. The distance traveled and the time spent in the central area were significantly higher in the A21033 group than in the model group, indicating that A21033 intervention can alleviate anxiety, improve cognition, and enhance spontaneous activity and motor ability in diabetic mice. The A21013 group showed no difference from the model group, suggesting that different Lactobacillus curvature strains have different effects on improving cognitive impairment in diabetic mice.

[0102] Based on the results of the open field experiment, it can be concluded that Lactobacillus curvature A21033 has the potential value in improving cognitive impairment in diabetic patients. Therefore, Y-maze and new object recognition tests were further conducted on mice in the A21033 intervention group.

[0103] The Y-maze test is used to assess the spatial reference memory ability of mice. Before the experiment, the three arms of the Y-maze are randomly assigned as the starting arm, the novelty arm, and the other arms, with the central triangular area as a blank area. The settings of each area cannot be changed during the experiment. One hour before the experiment, the mice are moved to the test room to acclimatize to the environment and reduce stress. The experiment is conducted in a quiet environment to minimize interference from external factors on the mouse's behavior. During the training phase, the novelty arm is closed with a partition, leaving only the other two arms open for the mouse to familiarize itself with (considered as familiar arms). Specifically, the mouse is placed facing the wall in the starting arm and allowed to freely explore the two familiar arms for 5 minutes. After training, the animal is returned to its cage. One hour later, the partition of the novelty arm is opened, and the mouse is again placed in from the starting arm, allowing it to freely explore the three arms for 5 minutes. Data such as the number of times the mouse enters each arm, the dwell time, and the exploration distance are recorded and analyzed. The results are then calculated and analyzed using the following formula:

[0104] Novel arm exploration time percentage (Time in novel arm) = ×100%;

[0105] Percentage of entries in the novel arm = ×100%;

[0106] Spontaneous alternative percentage = ×100%.

[0107] After each mouse is tested, the test chamber is thoroughly cleaned with 75% alcohol to eliminate animal odors and prevent the excrement and odors of the previous mouse from interfering with the experimental results.

[0108] The results showed that mice in group A21033 had a stronger ability to explore novel arms compared to mice in the model group.

[0109] The novel object recognition test is used to assess the learning, memory, and cognitive abilities of mice. Before the experiment, mice are allowed to acclimatize to their environment. Two identical objects (A) are placed diagonally in an open field, and the mice are allowed to explore freely for 5 minutes. The animals are then returned to their cages. One hour later, one of the objects (A) is replaced with a new object (B), and the mouse is allowed to explore freely for 8 minutes. The time taken to explore the new object (s) and the time taken to explore the old object (s) are recorded, and the novel object discrimination index is calculated using the formula:

[0110] New object discrimination index = ×100%.

[0111] After each mouse is tested, the test chamber is thoroughly cleaned with 75% alcohol to eliminate animal odors and prevent the excrement and odors of the previous mouse from interfering with the experimental results.

[0112] The results are as follows Figure 7 and 8 As shown.

[0113] It can be observed that, compared with the model group, the intervention of A21033 significantly improved the mice's ability to explore new objects, indicating that the mice's cognition and memory were significantly enhanced after the intervention of A21033.

[0114] Example 5

[0115] In this embodiment, the antibacterial effects of Lactobacillus curvature A21013 and A21033 were tested.

[0116] After activating *Lactobacillus curvature* A21013 and A21033 according to the above steps, the bacterial cells were collected for later use. Commercially available *Staphylococcus aureus* ATCC 6538, *Streptococcus vaginalis*, *Staphylococcus epidermidis* ATCC 12228, *Klebsiella pneumoniae* subsp. (GDMCC NO. 800954), *Streptococcus pharyngitis* (CICC 25340), and *Streptococcus griseus* (GDMCC NO. 1.1089) were used. Before use, each bacterium was biochemically or sequenced to confirm its authenticity. The cells were activated using the corresponding culture media and transferred to obtain seed culture. Then, at an inoculum volume of 2%, the seed culture was inoculated into agar-containing medium that had been sterilized at high temperature and cooled to approximately 50°C. After thorough mixing, the mixture was poured into square plates and allowed to air dry. Using a sterile punch, holes were sequentially punched in a square agar plate containing pathogenic bacteria. Approximately 100 μL of *Lactobacillus curvaturei* A21013 and A21033 bacterial suspensions were added to each hole. After overnight incubation at 37°C, the antibacterial effect was observed, and the size of the inhibition zone (mm) was recorded. A culture medium without *Lactobacillus curvaturei* A21013 and A21033 was used as a blank control. Two-component and three-component mixed groups were also set up. In the two-component mixed groups, the volume ratios of A21013 to A21033 were 1:1, 1:2, and 2:1, respectively. The three-component mixed group consisted of a mixture of A21013, A21033, and commercially available *Lactobacillus curvaturei* strain 1 in a volume ratio of 1:1:1.

[0117] The results are shown in the table below.

[0118] Table 1. Inhibitory effects of Lactobacillus curvature A21013 and A21033 on pathogenic bacteria such as vaginal bacteria, oral bacteria, and Klebsiella pneumoniae subsp.

[0119]

[0120] The "-" indicates that there is no inhibition zone.

[0121] The results showed that both *Lactobacillus curvaturei* A21013 and A21033, as well as the two-component mixture, exhibited significant inhibitory effects on vaginal pathogens such as *Streptococcus vaginalis* and *Staphylococcus epidermidis*, indicating that *Lactobacillus curvaturei* A21013 and A21033 play a role in improving vaginal health. Furthermore, *Lactobacillus curvaturei* A21013, A21033, and the two-component mixture also showed significant inhibitory effects on *Klebsiella pneumoniae* subsp. *p.* and *Streptococcus pharyngitis*. This suggests that *Lactobacillus curvaturei* A21013, A21033, and the two-component mixture have potential in the prevention / treatment of vaginitis, pneumonia, and oral health.

[0122] To further verify the potential of Lactobacillus curvature A21013 and A21033 in the prevention / treatment of vaginitis, the inventors further verified their inhibitory effect on Candida albicans (purchased from GDMCC, number GDMCC NO:2.194), a common pathogen of vaginitis.

[0123] After activating Lactobacillus curvature A21013 and A21033 according to the above steps, the bacterial cells were collected and resuspended in PBS to obtain 1×10⁻⁶ cells. 8 CFU / mL of *Lactobacillus curvatureii* suspension. Take 100 μL of 1×10⁻⁶ CFU / mL of the suspension. 6 CFU / mL of *Candida albicans* culture and 100 μL of *Lactobacillus curvatureii* suspension were added to a 96 empty plate and incubated at 37 °C for 24 hours. The absorbance (OD value) at 600 nm was measured using a spectrophotometer. The inhibition rate of *Lactobacillus curvatureii* against *Candida albicans* was calculated using a culture medium without *Lactobacillus curvatureii* suspension as a blank control. Furthermore, another commercially available *Lactobacillus curvatureii* strain (commercially available *Lactobacillus curvatureii* strain 2) was introduced for comparison.

[0124] Inhibition rate (%) = × 100%.

[0125] The results are shown in the table below.

[0126] Table 2. Antibacterial effect of Lactobacillus curvaturea A21013 and A21033 co-cultured with Candida albicans

[0127]

[0128] It can be found that Lactobacillus curvature A21013 and A21033 both have good antibacterial effects against Candida albicans (inhibition rate >95%), and are significantly better than existing commercially available Lactobacillus curvature strains (such as 1 and 2).

[0129] In response, the inventors further explored its antibacterial mechanism against Candida albicans.

[0130] The specific experimental steps are as follows:

[0131] (1) Inhibition ability against Candida albicans hyphae:

[0132] Preparation of 3-(N-morpholino)propanesulfonic acid-RPMI1640 (MOPS-RPMI1640) medium: Add 3-(N-morpholino)propanesulfonic acid (MOPS) to RPMI1640 medium to prepare RPMI1640 medium containing MOPS at a final concentration of 0.165 mol / L, and filter to sterilize for later use.

[0133] Prepare MRS medium: Weigh 10.0 g peptone, 5.0 g sodium acetate, 5.0 g beef extract, 1.0 mL Tween-80, 20.0 g glucose, 2.0 g K2HPO4, 4.0 g yeast extract, 0.05 g MnSO4·4H2O, 2.0 g triammonium citrate, 0.2 g MgSO4, and 12.0 g agar powder (omit agar powder for liquid medium). Add 800 mL distilled water, mix until the solutes are completely dissolved, adjust the pH to 6.2±0.2, bring the volume to 1 L, and autoclave at 121 ℃ for 20 min.

[0134] Prepare YPD medium: 2% (w / v) glucose, 2% (w / v) tryptone, and 1% (w / v) yeast extract. For solid medium, add an additional 1.5%-2% (w / v) agar powder; for broth, do not add agar powder. Autoclave at 115 °C for 20 min.

[0135] Candida albicans was activated by streaking glycerol tubes onto YPD fixed plates and incubated at 37°C for 24 h. Single colonies were then transferred to YPD liquid medium and incubated at 37°C for another 24 h. After centrifugation to collect Candida albicans and prepare a bacterial suspension, the density of Candida albicans in the suspension was adjusted to 2 × 10⁻⁶ using MOPS-RPMI 1640 medium. 7 CFU / mL was prepared for use. Lactobacillus curlis A21033 and A21013 were activated by streaking with glycerol onto MRS solid plates and cultured anaerobically at 37 °C for 24 h. Single colonies were then inoculated into 5 mL of MRS liquid medium and cultured for 24 h to obtain the seed culture. OD was adjusted accordingly. 600=1.0, 2% inoculum was added to 2 mL of MRS liquid medium and cultured for 48 h. Then, the fermentation broth of *Lactobacillus crenata* A21033 and A21013 was centrifuged, and the fermentation supernatant was collected by aseptic filtration through a 0.22 μm membrane for later use. Hyphae inhibition experiment: 100 μL of sterilized fermentation supernatant of A21033 and A21013 and 100 μL of *Candida albicans* suspension were placed in 96-well plates (experimental group), and a blank control group (i.e., 100 μL MOPS-RPMI1640 + 100 μL *Candida albicans* suspension) and a negative control group (i.e., 100 μL MRS liquid medium + 100 μL *Candida albicans* suspension) were set up. Each group was set with 3 replicates, and incubated at 37 ℃ for 3 h. After incubation, crystal violet staining was performed to observe the number of hyphae. Multiple fields of view were randomly selected under a microscope for observation and counting, and the hyphal growth inhibition rate was calculated.

[0136] Mycelial growth inhibition rate = × 100%.

[0137] The results are as follows Figure 9 As shown.

[0138] Calculations showed that Lactobacillus curlis A21033 and A21013 significantly inhibited the growth of Candida albicans hyphae, with average inhibition rates of 78.72% and 79.78%, respectively.

[0139] (2) Inhibition ability against Candida albicans biofilm:

[0140] Add 100 μL of *Candida albicans* suspension to each well of a 96-well plate, followed by 100 μL of sterilized fermentation supernatant from *Lactobacillus cristata* A21013 and A21033 (experimental groups). A blank control group (100 μL YPD liquid medium + 100 μL *Candida albicans* suspension) and a negative control group (100 μL MRS liquid medium + 100 μL *Candida albicans* suspension) were also set up. Each group was divided into three replicates. The plates were incubated at 37 °C for 24 h. The plates were washed three times with sterile PBS to remove planktonic cells, and then fixed with 100% methanol for 30 min. The methanol was discarded, and the plates were stained with 0.1% crystal violet for 5 min. The crystal violet was discarded, and the plates were washed three times with PBS, followed by elution with 33% glacial acetic acid to remove the bound crystal violet solution. The absorbance at 570 nm was measured using a microplate reader, and the biofilm inhibition rate was calculated according to the formula in the above examples.

[0141] Biomembrane inhibition rate = × 100%.

[0142] The results showed that Lactobacillus curvature A21033 and A21013 could significantly inhibit the biofilm formation of Candida albicans, with average inhibition rates of 55.59% and 45.29%, respectively.

[0143] (3) Determination of hydrogen peroxide production capacity of Lactobacillus curvature A21013 and A21033:

[0144] Single colonies of Lactobacillus curvature A21013 and A21033 were picked and inoculated into MRS liquid medium and cultured at 37 ℃ for 24 h. OD was adjusted. 600 =0.8-1.0, then added to 4 mL of fresh MRS liquid medium at a 2% inoculum size, incubated for 24 h, centrifuged at 8000 rpm for 2 min at 4℃, and the fermentation supernatant was collected by aseptic filtration through a 0.22 μm membrane for later use. Hydrogen peroxide was detected using a hydrogen peroxide assay kit (microplate method) (purchased from Nanjing Jiancheng).

[0145] The results are as follows Figure 10 As shown.

[0146] Lactobacillus curvature A21013 and A21033 can produce hydrogen peroxide at concentrations of 49.59 mM and 46.19 mM, respectively, thereby inhibiting the growth of pathogenic bacteria in the vagina.

[0147] Therefore, it can be seen that Lactobacillus curvaturea A21033 and A21013 can inhibit the growth of Candida albicans by inhibiting its hyphal growth and biofilm formation, as well as by producing hydrogen peroxide.

[0148] The inventors further tested the inhibitory effects of Lactobacillus curvature A21013 and A21033 on another common pathogen causing vaginitis, Gardnerella vaginalis (purchased from GDMCC, GDMCC NO: 1.962).

[0149] The specific steps are as follows: After culturing Gardnerella vaginalis in BR liquid medium containing 5% sheep serum, the bacterial concentration is adjusted to 10. 8CFU / mL. Add 100 μL of *Gardnerella vaginalis* suspension to each well of a 96-well plate, followed by 100 μL of sterilized fermentation supernatant from *Lactobacillus curvatureii* A21013 and A21033, respectively. Set up a blank control (100 μL BR liquid medium with 5% sheep serum + 100 μL *Gardnerella vaginalis* suspension) and a negative control (100 μL MRS liquid medium + 100 μL *Gardnerella vaginalis* suspension). Incubate at 37 °C for 24 h. Wash three times with sterile PBS to remove airborne cells, then fix with 100% methanol for 30 min. Discard the methanol and stain with 0.1% crystal violet for 5 min. Discard the crystal violet, wash three times with PBS, and then elute the bound crystal violet solution with 33% glacial acetic acid. Measure the absorbance at 570 nm using a microplate reader, and then calculate the biofilm inhibition rate according to the formula in the above examples.

[0150] The BR medium was prepared as follows: 12g tryptone, 5g peptone, 3g yeast extract, 3g beef extract, 1g soluble starch, 5g NaCl, pH adjusted to 7.3±0.2, and brought to a final volume of 1 L. It was then autoclaved at 121 ℃ for 20 min.

[0151] The results showed that Lactobacillus curvaturea A21033 and A21013 could significantly inhibit the formation of Gardnerella vaginalis biofilm, with average inhibition rates of 50.01% and 40.01%, respectively.

[0152] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of *Lactobacillus curvatureii* or its ferments in the preparation of products that inhibit bacteria and regulate blood sugar, characterized in that, The *Lactobacillus curvature* is selected from at least one of *Lactobacillus curvature* A21013 and *Lactobacillus curvature* A21033; Lactobacillus curvaturea A21013, taxonomically named Lactobacillus crispatus It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 8, 2023, with accession number GDMCC No:63543; Lactobacillus curvaturea A21033, taxonomically named Lactobacillus crispatus It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 8, 2023, with accession number GDMCC No:63544; The antibacterial product is an in vivo antibacterial drug or an in vitro bactericidal product. The in vivo antibacterial drug is used to inhibit the growth of oral or reproductive tract microorganisms; The microorganisms are bacteria and fungi; Among them, the bacteria that inhibit bacteria in vivo or kill bacteria in vitro are selected from at least one of the following: Staphylococcus epidermidis, Streptococcus vaginalis, Klebsiella pneumoniae subsp., Streptococcus pharyngitis, Streptococcus gossy, and Gardnerella vaginalis. The fungus in question is Candida albicans.

2. The use of *Lactobacillus curvatureii* or its ferments in the preparation of medicaments for the prevention and / or treatment of vaginal diseases in women, characterized in that, The vaginal disease mentioned is a bacterial or fungal vaginal disease; The *Lactobacillus curvature* is selected from at least one of *Lactobacillus curvature* A21013 and *Lactobacillus curvature* A21033; Lactobacillus curvaturea A21013, taxonomically named Lactobacillus crispatus It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 8, 2023, with accession number GDMCC No:63543; Lactobacillus curvaturea A21033, taxonomically named Lactobacillus crispatus It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 8, 2023, with accession number GDMCC No:63544; The bacteria are selected from at least one of the following: Staphylococcus epidermidis, Streptococcus vaginalis, Klebsiella pneumoniae subsp., Streptococcus pharyngitis, Streptococcus gossy, and Gardnerella vaginalis. The fungus in question is Candida albicans.

3. The application according to claim 2, characterized in that, The vaginal disease mentioned is vaginitis.

4. The application according to claim 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The drug also contains a second active substance. The second active substance includes a drug having at least one of the following functions (1)-(4): (1) Prevention and / or treatment of vaginal diseases in women; (2) Antibacterial; (3) Regulate blood sugar; (4) To treat or improve cognitive impairment.

6. The application according to claim 2, characterized in that, The dosage forms of the drug include: solution, powder, tablet, granule, capsule, aerosol, ointment or suppository.

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