A composite probiotic and its use in the preparation of a product for preventing or treating diabetes with periodontitis

Abstract

The application belongs to the technical field of microorganisms, and discloses a compound probiotic and application of the compound probiotic in preparation of a product for preventing or treating diabetes mellitus accompanied by periodontitis. ^9 The compound probiotic provided by the application comprises Lactobacillus rhamnosus and Lactobacillus reuteri, and the live bacteria ratio of Lactobacillus rhamnosus to Lactobacillus reuteri is not less than 1:1, and the total number of live bacteria is not less than 1×10 CFU / mL. The compound probiotic provided by the application comprises Lactobacillus rhamnosus and Lactobacillus reuteri, and the live bacteria ratio of Lactobacillus rhamnosus to Lactobacillus reuteri is not less than 1:1, and the total number of live bacteria is not less than 1×10 CFU / mL. The compound probiotic provided by the application comprises Lactobacillus rhamnosus and Lactobacillus reuteri, and the live bacteria ratio of Lactobacillus rhamnosus to Lactobacillus reuteri is not less than 1:1, and the total number of live bacteria is not less than 1×10 CFU / mL. The compound probiotic provided by the application comprises Lactobacillus rhamnosus and Lactobacillus reuteri, and the live bacteria ratio of Lactobacillus rhamnosus to Lactobacillus reuteri is not less than 1:1, and the total number of live bacteria is not less than 1×10 CFU / mL.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a compound probiotic and its application in the preparation of products for the prevention or treatment of diabetes with periodontitis. Background Technology

[0002] There is a complex interaction between diabetes and periodontitis. Diabetes can increase the inflammatory response in periodontitis, while the presence of periodontitis can also worsen diabetes control. This mutually reinforcing inflammatory response makes treatment more complex and difficult. Diabetic patients with periodontitis often require stricter glycemic control because hyperglycemia affects the repair and infection-fighting ability of periodontal tissues. Effective glycemic control requires not only medication but also management of daily diet, exercise, and lifestyle. Furthermore, the immune system function of diabetic patients may be impaired, leading to reduced healing ability. This makes common periodontal treatments (such as scaling, antibiotics, root planing, and periodontal surgery) less effective than in non-diabetic patients, with prolonged recovery time and increased risk of infection. Treatment requires close collaboration between endocrinologists and dentists to manage glycemic and periodontal health simultaneously. This multidisciplinary treatment model places high demands on communication and collaboration within the medical team and increases the complexity of comprehensive treatment.

[0003] Porphyromonas gingivalis ( Porphyromonas gingivalis, P. gingivalis *Porphyromonas gingivalis* is a common pathogenic bacterium in patients with periodontitis, particularly those with diabetes. It can exacerbate the treatment difficulty of periodontitis in diabetic patients, mainly in the following aspects: High pathogenicity: *Porphyromonas gingivalis* is a Gram-negative bacillus with strong pathogenicity. It can invade periodontal tissues, leading to an exacerbated inflammatory response and more severe damage to periodontal tissues. Drug resistance: Some strains of *Porphyromonas gingivalis* may develop drug resistance, making conventional antibiotic treatment ineffective. This increases the limitation of treatment options, requiring more laboratory testing and individualized treatment plans. Inflammatory response: The presence of *Porphyromonas gingivalis* can lead to a persistent chronic inflammatory state in the gingival tissues. This inflammatory response not only affects the health of periodontal tissues but may also affect the control of systemic diabetes through inflammatory mediators. Poor treatment response: Due to the unique biological characteristics and pathogenic mechanism of *Porphyromonas gingivalis*, its response to conventional periodontal treatment methods may not be as rapid and significant as other pathogenic bacteria, requiring a longer treatment process and more complex treatment strategies. Therefore, periodontitis caused by *Porphyromonas gingivalis* increases the complexity and treatment difficulty of type 2 diabetes by exacerbating oral and systemic inflammation. Furthermore, *Porphyromonas gingivalis* can enter the bloodstream, spreading from the mouth to the liver. Once in the bloodstream, these bacteria can penetrate blood vessel walls and reach liver or colon tissues, triggering inflammatory responses and damage. Therefore, focusing on only one problem may not comprehensively and effectively improve a patient's overall health.

[0004] Current treatments for diabetes mellitus with periodontitis primarily focus on antibiotic therapy and oral care. However, these approaches have several major drawbacks: Limitations of antibiotic use: While antibiotics can inhibit the growth of *Porphyromonas gingivalis*, long-term use can lead to antibiotic resistance and may disrupt the oral microecological balance, resulting in other adverse consequences. Many current health products or treatments often focus on a single effect; for example, oral medications may only focus on improving insulin resistance, and dental treatments may only address periodontitis. Limitations of oral care: Current oral care methods, such as brushing and rinsing, while helping to control plaque and gingivitis, are insufficient for managing the complex immune responses and inflammation associated with periodontitis. Durability of treatment effects: Existing treatments often fail to achieve sustained control and prevention, leading to recurring episodes or disease progression. Therefore, treatment for diabetes mellitus with periodontitis requires new approaches to enhance oral immune regulation and inflammation control without disrupting the normal oral microecological balance, thereby improving the durability of treatment effects and the patient's quality of life. Current research on probiotics in the treatment of diabetes mellitus with periodontitis is still in its early stages. Probiotics are typically derived from the human body or natural microorganisms in the food chain, exhibiting high safety and low side effects. They are less likely to develop drug resistance like antibiotics and have no side effects compared to hypoglycemic drugs. Some studies have begun to explore comprehensive treatment strategies using probiotics in combination therapy for diabetes mellitus with periodontitis. By simultaneously regulating the gut and oral microbiota, probiotics can help improve periodontal health and diabetes control in diabetic patients. However, research on probiotic treatment for diabetes mellitus with periodontitis is limited, and the therapeutic potential of probiotics in this condition urgently needs to be explored.

[0005] Traditional treatments for diabetes with periodontitis often rely on separate approaches to oral care and diabetes management. This separate approach can be ineffective because it fails to address both periodontitis and insulin resistance simultaneously, thus failing to comprehensively promote the overall health of patients with diabetes and periodontitis. Summary of the Invention

[0006] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes a compound probiotic and its application in the preparation of products for the prevention or treatment of diabetic periodontitis. The compound probiotic provided by this invention can prevent and treat diabetic periodontitis.

[0007] This invention provides a compound probiotic.

[0008] Specifically, a complex probiotic includes Lactobacillus rhamnosus (Lactobacillus rhamnosus) Lactobacillus rhamnosus ) and Lactobacillus reuteri ( Lactobacillus reuteri The Lactobacillus rhamnosus (Lactobacillus rhamnosus ) and the aforementioned Lactobacillus reuteri ( Lactobacillus reuteri The ratio of live bacteria to total live bacteria should be no less than 1:1, and the total number of live bacteria should be no less than 1×10⁻⁶. ^ 9 CFU / mL.

[0009] In some embodiments of the present invention, the Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and the aforementioned Lactobacillus reuteri ( Lactobacillus reuteri The live bacteria ratio of Lactobacillus rhamnosus is (1.5-3):1. Preferably, the live bacteria ratio of Lactobacillus rhamnosus is (1.5-3):1. Lactobacillus rhamnosus ) and the aforementioned Lactobacillus reuteri ( Lactobacillus reuteri The live bacteria ratio of the bacteria is (2-3):1; more preferably, the *Lactobacillus rhamnosus* (Lactobacillus rhamnosus) Lactobacillus rhamnosus ) and the aforementioned Lactobacillus reuteri ( Lactobacillus reuteri The live bacteria ratio of the bacteria is (2-2.5):1. More preferably, the *Lactobacillus rhamnosus* ( Lactobacillus rhamnosus ) and the aforementioned Lactobacillus reuteri ( Lactobacillus reuteri The ratio of live bacteria in the culture medium was 7:3.

[0010] This invention also provides applications of the above-mentioned compound probiotics.

[0011] Specifically, the above-mentioned compound probiotics are used in the preparation of products for the prevention or treatment of diabetes with periodontitis.

[0012] In some embodiments of the present invention, the product includes pharmaceuticals or oral care products.

[0013] Specifically, the aforementioned compound probiotics are used in the preparation of products for the prevention or treatment of colitis caused by Porphyromonas gingivalis.

[0014] In some embodiments of the present invention, the product includes pharmaceutical or nursing products.

[0015] Specifically, the above-mentioned compound probiotics are used in the preparation of products for the prevention or treatment of liver tissue damage caused by Porphyromonas gingivalis.

[0016] In some embodiments of the present invention, the product includes pharmaceutical or nursing products.

[0017] The present invention also provides a drug for the prevention or treatment of diabetes with periodontitis.

[0018] Specifically, a medication for the prevention or treatment of diabetes with periodontitis includes the aforementioned compound probiotics.

[0019] The present invention also provides a nursing product for the prevention or treatment of diabetes with periodontitis.

[0020] Specifically, a care product for the prevention or treatment of diabetes with periodontitis includes the aforementioned compound probiotics.

[0021] Experiments have shown that this compound probiotic not only inhibits insulin resistance and increases GLP-1 protein levels, but also inhibits alveolar bone resorption caused by periodontitis, indicating that it has the function of preventing and improving periodontitis in diabetic patients. This compound probiotic can also simultaneously regulate the balance of oral and intestinal flora, maintaining a healthy oral and intestinal environment. This is particularly important for diabetic patients with periodontitis, as they are more susceptible to changes in the microbial community. Furthermore, while inhibiting insulin resistance and periodontitis, this compound probiotic can help repair and protect liver and intestinal tissues, contributing to the maintenance of the normal function of these vital tissues. Therefore, this compound probiotic has significant potential and efficacy in preventing and treating periodontitis in diabetic patients, as well as multiple roles in maintaining oral and intestinal health.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The compound probiotics provided by this invention include Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus reuteri ( Lactobacillus reuteri By controlling the ratio of the two, it can prevent and treat diabetic periodontitis. Specifically, it can (1) inhibit the gingival index, gingival bleeding index and plaque index caused by rat in vivo modeling; (2) reduce periodontal tissue damage; (3) inhibit alveolar bone resorption; (4) inhibit insulin resistance; (5) inhibit inflammatory factors (TNF-α and IL-6) in serum; (6) increase GLP-1 protein content; (7) regulate the balance of oral and intestinal flora; (8) secrete short-chain fatty acids; and (9) improve liver and intestinal tissue damage caused by Porphyromonas gingivalis. This compound probiotic can be widely used in the preparation of drugs or care products for the prevention or treatment of diabetic periodontitis. Attached Figure Description

[0024] Figure 1 Diagrams of periodontal structures in each group;

[0025] Figure 2 Gingival index graphs for each group;

[0026] Figure 3 A graph showing the gingival sulcus bleeding index of rats in each group;

[0027] Figure 4 The plaque index diagrams for each group are shown.

[0028] Figure 5 Diagrams showing the inhibition of alveolar bone resorption in each group (palatal and buccal).

[0029] Figure 6The distance of attachment loss for each group of teeth;

[0030] Figure 7 The values ​​for bone surface area / total volume (BV / TV) for each group are given.

[0031] Figure 8 A graph showing fasting insulin levels for each group;

[0032] Figure 9 Insulin resistance graphs for each group;

[0033] Figure 10 A graph showing the GLP-1 protein content of each group;

[0034] Figure 11 The graph shows the levels of the inflammatory factor TNF-α in each group;

[0035] Figure 12 A graph showing the IL-1β protein content in each group;

[0036] Figure 13 A graph showing the levels of the inflammatory factor IL-6 in each group;

[0037] Figure 14 Figures showing the improvement in liver tissue damage in each group;

[0038] Figure 15 A graph showing the inhibition of total cholesterol (TC) in each group;

[0039] Figure 16 A graph showing the inhibition of triglycerides (TG) in each group;

[0040] Figure 17 A graph showing the changes in oral microbiota at the phylum level for each group;

[0041] Figure 18 A diagram showing the changes in oral microbiota at the group / genus level;

[0042] Figure 19 A graph showing the changes in gut microbiota at the phylum level for each group;

[0043] Figure 20 A graph showing the changes in gut microbiota at the group / genus level;

[0044] Figure 21 A graph showing the acetic acid content of each group;

[0045] Figure 22 The graph shows the propionic acid content of each group;

[0046] Figure 23 The butyric acid content of each group;

[0047] Figure 24 The graph shows the improvement in colon tissue loss in each group. Detailed Implementation

[0048] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0049] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0050] 1. Experimental Materials and Methods

[0051] 1.1 Culture medium used

[0052] MRS medium: yeast extract 5.0 g / L, beef extract 10.0 g / L, peptone 10.0 g / L, glucose 20.0 g / L, anhydrous sodium acetate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, dipotassium hydrogen phosphate trihydrate 2.6 g / L, manganese sulfate monohydrate 0.25 g / L, magnesium sulfate heptahydrate 0.5 g / L, and Tween-80 1 mL / L, pH 6.2-6.4.

[0053] BHI medium: tryptone 10.0 g / L, ox heart extract 17.5 g / L, sodium chloride 5.0 g / L, yeast extract 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate dodecahydrate 2.5 g / L, L-cysteine ​​hydrochloride monohydrate 0.4 g / L, 0.5% vitamin K1-heme chloride 1 mL / L, pH 7.2-7.4.

[0054] BHI + heme chloride + vitamin K1 liquid culture medium: Weigh 19.25g BHI and add pure water to a final volume of 500mL. Autoclave at 121℃ for 20min, cool to room temperature, and store at 4℃. Take 500mL of BHI, add 500μL of vitamin K1 and 225μL of heme chloride, and mix well.

[0055] 1.2 Bacterial Culture

[0056] Porphyromonas gingivalis ( P. gingivalis *Lactobacillus rhamnosus* ATCC 33277 (Pg), *Lactobacillus rhamnosus* ATCC 53103, and *Lactobacillus reuteri* ATCC 23272 were purchased from Taisto Biotechnology. *Porphyromonas gingivalis* was inoculated into BHI + heme chloride + vitamin K1 liquid medium and cultured at 37℃ under anaerobic conditions for 48-96 h. A bacterial suspension of 5 mL was prepared with sterile PBS (50 mmol / L, pH 7.4) and adjusted to a concentration of 1×10⁻⁶. ^9 CFU / mL.

[0057] Lactobacillus rhamnosus ATCC 53103 and Lactobacillus reuteri ATCC 23272 were inoculated into MRS solid medium for 24 h, and then single colonies were selected and inoculated into MRS liquid medium for 24 h. The bacterial culture was obtained by centrifugation (7200 rpm for 10 min). The bacterial culture was then determined based on the feeding amount of rats (1×10⁻⁶). ^9 Calculate the bacterial culture (CFU / mL / mouse), aliquot it into centrifuge tubes, and ensure that any remaining amount after each feeding is not used for the next feeding, so that fresh bacterial culture is used each time.

[0058] 1.3 Animal Experiment Design and Results

[0059] Thirty-two male SPF-grade rats aged 6-7 weeks were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. After 7 days of acclimatization, fasting blood glucose levels were measured and rats were weighed. They were then randomly divided into four groups of eight rats each. The four groups were: control group (NC), high-fat diet group (HFD), *Porphyromonas gingivalis* combined with high-fat diet group (model group (Pg+HFD)), and compound probiotic group (the probiotic group consisted of two subgroups: Pg+HFD+LAB1 (*Lactobacillus rhamnosus* ATCC53103: *Lactobacillus reuteri* ATCC23272 = 7:3) and Pg+HFD+LAB2 (*Lactobacillus rhamnosus* ATCC53103: *Lactobacillus reuteri* ATCC23272 = 3:2)). The control group (NC) received a normal diet and 0.2 mL of sterile PBS buffer. The Pg+HFD group received 1 × 10⁻⁶ periodontal pathogens per rat via periodontal smear. 9 CFU (0.2 mL) of *Porphyromonas gingivalis* was administered to rats for 5 weeks, followed by gavage with *Porphyromonas gingivalis* suspension. Water intake was prohibited for 1 hour after each inoculation, three times a week, until the end of the experiment (week 15). Compound probiotic group: Rats were given 1×10⁻⁶ CFU of *Porphyromonas gingivalis* suspension via periodontal application. 9 CFU (0.2 mL) of compound probiotics were administered for 5 weeks, followed by gavage of compound probiotic suspension. Patients were prohibited from drinking water for 1 hour after each inoculation, three times a week, until the end of the experiment (week 15). High-fat diet group: fed a high-fat diet. Patients were weighed weekly for the entire 15-week experiment.

[0060] 1.4 Periodontal histopathological analysis and measurement of gingival index, gingival bleeding index, and plaque index

[0061] In week 9, rats were anesthetized by intramuscular injection of 200 mg / kg ketamine hydrochloride. Periodontal pocket probing was used to evaluate gingival index, gingival bleeding index, and plaque index. The rat gingiva was scored using the examination method revised by Loe and Silness in 1967. The scoring criteria were as follows: 0 = healthy gingiva; 1 = mild gingival inflammation: slight color change and mild edema of the gingiva, no bleeding on probing; 2 = moderate gingival inflammation: red, edematous, and shiny gingiva, bleeding on probing; 3 = severe gingival inflammation: significantly red and swollen gingiva or ulcers, with a tendency for spontaneous bleeding. Results are expressed as mean ± standard deviation.

[0062] The sulcus bleeding index (SBI) reflects the activity of gingivitis. 0 points: The gingival margin and papillae appear healthy; no bleeding occurs upon gentle probing of the sulcus. 1 point: Mild inflammation of the gingival margin and papillae; no bleeding occurs upon gentle probing of the sulcus. 2 points: Mild gingival inflammation with discoloration, no swelling or edema; pinpoint bleeding occurs upon probing. 3 points: Moderate gingival inflammation with discoloration and mild edema; bleeding occurs upon probing, with blood overflowing into the sulcus. 4 points: Severe gingival inflammation with not only discoloration but also significant swelling; bleeding occurs upon probing, with blood overflowing into the sulcus. 5 points: Discoloration of the gingiva, significant swelling, sometimes with ulceration. Bleeding occurs upon probing or spontaneous bleeding. Results are expressed as mean ± standard deviation.

[0063] Plaque Index (PLI): An evaluation of oral hygiene based on plaque. 0 points: No plaque at the gingival margin; 1 point: Thin plaque on the tooth surface at the gingival margin, not visible to the naked eye, but visible when scraped with a probe tip; 2 points: Moderate amount of plaque visible at the gingival margin or proximal surfaces; 3 points: Abundant soft plaque in the gingival sulcus or at the gingival margin and proximal surfaces.

[0064] Periodontal tissue pathological analysis: Right maxillary tissue (preserving the maxilla and gingiva) was collected from rats. The isolated rat maxillary bone tissue was fixed in 4% paraformaldehyde for 48 hours, then soaked in double-distilled water, followed by decalcification with Plank-Rychlo decalcification solution. After decalcification, the tissue was soaked in G3430 decalcification alkaline treatment solution for 40 minutes to deacidify, and then rinsed under running water for 18 hours. The fixed tissue samples were then dehydrated for 60 minutes each with 75%, 85%, 95% I, 95% II, 100% I, and 100% II ethanol solutions, cleared in xylene I and II for 45 minutes each, then permeated with paraffin I, paraffin II, and paraffin III for 50-60 minutes each, embedded in paraffin, and sectioned. The paraffin sections were then baked. Next, the sections were immersed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 3 min, anhydrous ethanol II for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, and pure water for 2 min. They were then stained with hematoxylin for 3-5 min, rinsed with tap water for 1 min, differentiated with 1% hydrochloric acid ethanol for a few seconds, rinsed with tap water, and then blued with a blueing solution for 10-30 seconds, followed by rinsing with running water. Finally, the sections were stained with eosin for 3-5 min and rinsed with running water. The sections were then rapidly dehydrated by sequentially immersing them in 80% ethanol, 95% ethanol, anhydrous ethanol, and anhydrous ethanol II, and then mounted with ultra-clean high-grade mounting adhesive. The inflammatory infiltration and integrity of the periodontal tissues (including the gingiva, periodontal ligament, cementum, and alveolar bone) were observed under a microscope.

[0065] Figure 1 For example, periodontal tissue diagrams of each group, such as Figure 1 As shown, the periodontal structures in the blank group (NC) and the high-fat diet group (HFD) were intact. Alveolar bone resorption was observed in the Pg+HFD group, but no inflammatory cell infiltration was seen. The periodontal tissues in the compound probiotic groups (Pg+HFD+LAB1 and Pg+HFD+LAB2) were intact, with no obvious pathological changes.

[0066] Figure 2 The gingival index diagrams for each group are shown. Figure 3 The graph shows the gingival sulcus bleeding index of rats in each group. Figure 4 This is a graph showing the plaque index for each group. (From...) Figures 2-4 It was found that the gingival index, gingival bleeding index, and plaque index of the model group (Pg+HFD) were significantly higher than those of the blank group and the high-fat diet group. This indicates that applying *Porphyromonas gingivalis* significantly affects oral health. Furthermore, *Lactobacillus rhamnosus* ATCC53103 combined with *Lactobacillus reuteri* ATCC23272 at different mixing ratios (Pg+HFD+LAB1, Pg+HFD+LAB2) significantly inhibited the gingival bleeding index and plaque index or gingival index in rats. This indicates that the compound probiotics provided by this invention can improve oral health.

[0067] 1.5 Alveolar bone microstructure scan

[0068] After euthanasia, the rat maxillae were separated using a sterile scalpel, fixed in 4% paraformaldehyde for 48 hours, washed with 75% ethanol solution, and stored in 75% ethanol solution at 4°C before being sent to Nanchang University for analysis. Micro-CT (Micro Computed Tomography) was used to perform X-ray imaging of the rat maxilla samples; specific scanning parameters were: voltage, 78kV; current, 100μA; scanning precision, 9μm; number of slices, 400; camera mode, high resolution. 1) Six measurements were taken from each tooth, and the average value was defined as the attachment loss distance (AL) for each rat. The bone surface area / total volume (BV / TV) of the M1 interdental region was measured to assess alveolar bone resorption.

[0069] Figure 5 Diagrams showing the inhibition of alveolar bone resorption in each group (palatal and buccal). Figure 6 The distance of attachment loss for each group of teeth. Figure 7 This represents the bone surface area / total volume (BV / TV) values ​​for each group. For example... Figure 5 As shown, there were significant differences between the control group and the experimental groups (P<0.05). In the control group (NC) and the high-fat diet group (HFD), the alveolar bone was smooth and intact, the teeth were tightly arranged, and there were no obvious gaps. The distance from the cementoenamel junction to the alveolar ridge crest was 0.94 mm. In contrast, the model group (Pg+HFD) showed severe alveolar bone resorption, exhibiting a loose and porous state. The tooth roots were exposed, and the teeth showed a tendency to loosen and fall out. The average distance from the cementoenamel junction to the alveolar ridge crest was 2.18 mm, which was much greater than that of the other experimental groups. Compared with the model group, Lactobacillus rhamnosus ATCC53103 combined with Lactobacillus reuteri ATCC23272 in different mixing ratios (Pg+HFD+LAB1, Pg+HFD+LAB2) showed good control of periodontitis, with an average distance from the cementoenamel junction to the alveolar ridge crest of 1.53 mm, which effectively reduced alveolar bone resorption and cementum loss. Secondly, the tooth attachment loss distance (AL) in the Pg+HFD group was significantly higher than that in other groups, while the bone surface area / total volume (BV / TV) was the opposite. Figure 6 , Figure 7 Compared with the model group, the compound probiotic groups (Pg+HFD+LAB1, Pg+HFD+LAB2) significantly inhibited the tooth attachment loss distance and increased the bone surface area / total volume in rats (P<0.05), thus inhibiting periodontitis.

[0070] 1.6 ELISA Detection

[0071] Before sacrifice, whole blood was collected from rats, and serum was separated. The levels of TNF-α, IL-1β, IL-6, GLP-1, and fasting insulin in the serum were detected using an ELISA kit, strictly following the kit instructions. The insulin resistance index (HOMA-IR) was calculated using the HOMA-IR method.

[0072] HOMA-IR = (fasting insulin level × fasting blood glucose level / 22.5).

[0073] in Figure 8 This is a graph showing the fasting insulin levels for each group. Figure 9 Insulin resistance graphs for each group. Figure 10 A graph showing the GLP-1 protein content of each group. Figure 11 The graph shows the levels of the inflammatory factor TNF-α in each group. Figure 12 This is a graph showing the IL-1β protein content in each group. Figure 13 This is a graph showing the levels of the inflammatory factor IL-6 in each group. (See graph for details.) Figures 8-13 As shown, the fasting insulin levels in the control group, high-fat diet group, model group (Pg+HFD), and probiotic groups (Pg+HFD+LAB1, Pg+HFD+LAB2) were 53.4, 45.2, 39.2, 50.8, and 44.83 mIU / L, respectively, and the insulin resistance levels were 10.5618, 15.58, 19.0989, 14.933, and 17.66, respectively. Compared with the high-fat diet group, the model group showed a significantly lower fasting insulin level and a significantly increased insulin resistance. The insulin level in the Pg+HFD+LAB1 group was significantly higher than that in the model group, and insulin resistance was significantly reduced (P<0.05). Furthermore, it significantly inhibited inflammatory factors (TNF-α, IL-6) and increased GLP-1 protein levels. The Pg+HFD+LAB2 group did not significantly inhibit insulin resistance, inflammatory factors TNF-α and IL-1β, and did not significantly increase GLP-1 protein levels, but it did significantly inhibit inflammatory factor IL-6.

[0074] The above experiments show that when the ratio of compound probiotics is 7:3 (Pg+HFD+LAB1), it can simultaneously inhibit periodontitis and improve insulin resistance, while when the ratio is 3:2 (Pg+HFD+LAB2), it can only inhibit periodontitis.

[0075] 1.7 Pathological analysis of liver and colon tissues

[0076] Before the rats were sacrificed, a portion of their whole blood was collected, and the serum was separated. The levels of total cholesterol and triglycerides (TG) in the serum were detected using a fully automated biochemical analyzer (BK-600, Shandong Boke Biotechnology).

[0077] Histopathological analysis of liver and colon tissues: Rat colon tissues were isolated and fixed in 4% paraformaldehyde, and the procedure was the same as for periodontal tissue histopathological analysis (no decalcification or alkali treatment was required). Histopathological damage and inflammatory cell infiltration in the liver and colon tissues were observed under a microscope.

[0078] like Figure 14 As shown, the liver tissue structure of the blank group (NC) was normal, with no obvious pathological changes. The liver tissue of the high-fat diet (HFD) group showed obvious fat vacuoles, a small amount of inflammatory cell infiltration, and vascular congestion. The liver tissue of the Pg+HFD group also showed fat vacuoles and a small amount of inflammatory cell infiltration. The liver tissues of the Pg+HFD+LAB1 and Pg+HFD+LAB2 groups also showed fat vacuoles and a small amount of inflammatory cell infiltration. Arrows indicate lymphocyte infiltration.

[0079] Figure 15 This is a graph showing the inhibition of total cholesterol (TC) in each group. Figure 16 This is a graph showing the inhibition of triglycerides (TG) in each group. (Source: [Graph showing triglyceride (TG) inhibition in each group]) Figure 15 and Figure 16 The results showed that compared with the HFD group, the Pg+HFD group had a significantly increased triglyceride (TG) level, while the Pg+HFD+LAB1 group had significantly decreased total cholesterol (TC) and triglyceride (TG) levels. In the Pg+HFD+LAB2 group, there was no significant difference in total cholesterol (TC) and triglyceride (TG) levels. Diabetes and obesity increase cholesterol synthesis and storage, thereby affecting cholesterol metabolism and increasing the burden on the liver. In the liver, the synthesis and metabolism of triglycerides require the participation of certain enzyme systems; excessively high triglyceride levels may lead to fatty liver and other liver problems. These results indicate that the Pg+HFD+LAB1 probiotic group can improve liver tissue damage while inhibiting insulin resistance.

[0080] Figure 24 The graph shows the improvement in colonic tissue loss in each group, as shown below. Figure 24 As shown, the ileum tissue structure in the blank group (NC) was normal, with no obvious pathological changes. In the high-fat diet (HFD) ileum tissue, diffuse inflammatory cell aggregation was observed in the mucosal layer, forming inflammatory foci. In the Pg+HFD group, diffuse inflammatory cell aggregation and inflammatory foci were also observed in the ileum tissue. In the compound probiotic group, a small amount of inflammatory cell infiltration was observed in the ileum tissue. The arrows indicate diffuse inflammatory cell aggregation. Combined with the results in 1.6, the compound probiotics provided by this invention can inhibit serum inflammatory factors TNF-α, IL-1β, or IL-6, and this compound probiotics has the ability to inhibit colonic tissue inflammation caused by *Porphyromonas gingivalis*.

[0081] 1.8 16S rRNA sequencing

[0082] Oral flora: Before sacrificing the rats, wipe the rats' mouths with physiological saline, isolate them with cotton balls to keep the tooth surfaces dry, and gently insert a sterile absorbent paper tip (size 20) into the bottom of the periodontal pocket with forceps. After 40 seconds, remove the paper and place it in a 2.0 mL sterile cryovial. Store at -80℃ for later use.

[0083] Gut microbiota: 24-hour rat feces were collected, stored at -80℃, and then the gut microbiota and oral microbiota were sequenced using 16S rRNA.

[0084] The changes in oral and gut microbiota were analyzed at the phylum and genus levels, and the results are as follows: Figures 17-20 As shown. Among the changes in oral flora, the phylum level is mainly influenced by Proteobacteria (…). Proteobacteria Firmicutes ( Firmicutes Bacteroidetes ( Bacteroidota ), Actinomycetes ( Actinobacteriota ), Fusobacteria ( Fusobacteriota ), Bacillus phylum ( Gemmatimonadota ), Cyanobacteria ( Cyanobacteria )composition. Firmicutes / Bacteroidota The proportions in the blank control group, high-fat diet group, model group, and Pg+HFD+LAB1 and Pg+HFD+LAB2 groups were 0.64%, 0.86%, 0.75%, 0.62%, and 0.38%, respectively. Compared to the blank control group, the proportions in the high-fat diet group and model group were significantly higher. Firmicutes / Bacteroidota The proportion of [organisms] increased, with Proteobacteria ([organisms]) being the most prevalent in the model group. Proteobacteria ) decrease, Fusobacterium phylum ( Fusobacteriota The number of Pg+HFD+LAB2 groups increased. Firmicutes / Bacteroidota The proportion decreased, while in the Pg+HFD+LAB1 group Firmicutes / Bacteroidota No significant changes were observed. At the bacterial level, compared to the control group, the high-fat diet group showed a higher proportion of Enterobacteriaceae (…). Enterobacteriaceae ) and Streptococcus ( Streptococcaceae The number of Porphyromonas family members increased in the model group. Porphyromonadaceae Streptococcal family ( Streptococcaceae Neisseriaceae ( Neisseriaceae The number of Pg+HFD+LAB1 group increased compared to the model group, with a higher proportion of Porphyromonas family ( Porphyromonadaceae ) decrease, thiophycetes ( Thiotrichaceae The number of families increased, while the remaining families showed no significant difference from the control group. The proportions and structure of the bacterial community in the Pg+HFD+LAB2 group were similar to those in the model group. Porphyromonadaceae Some members of this family may be associated with human diseases such as tooth decay and periodontitis, but they are also part of the normal oral and gut microbiota. Fusobacteriota Members of this group can become pathogens under certain conditions, participating in infections and diseases of the oral cavity, digestive tract, and other parts of the body, such as periodontitis and appendicitis. Certain... StreptococcaceaeStreptococcus genus members are pathogenic bacteria in humans and animals, causing a variety of infectious diseases such as pharyngitis, pneumonia, and sepsis. Different members of the Streptococcus genus exhibit varying degrees of pathogenicity to host tissues. Neisseriaceae Bacteria are pathogens in humans and animals, causing a variety of infectious diseases, such as otitis media, eye infections, and urinary tract infections. For example, *Neobryophyte* (… Neisseria Some species in the genus *Hymenopause* can cause gonorrhea and meningitis.

[0085] Regarding changes in gut microbiota Firmicutes / Bacteroidota The proportions of these microbiota in the control group, high-fat diet group, model group, and Pg+HFD+LAB1 and Pg+HFD+LAB2 probiotic groups were 1.42%, 4.6%, 13.64%, 3.11%, and 9.57%, respectively. This study indicates that the gut microbiome of obese diabetic patients is significantly different from that of healthy individuals. Firmicutes / Bacteroidota The ratio increased significantly. At the family level, compared with the control group and the model group, the high-fat diet group and the model group showed a significant increase in the number of Styloidea species (Synthaceae). Lachnispiraceae ) increase, Enterobacteriaceae ( Enterobacteriaceae The model group exhibits rich performance. Muribaculaceae and Prevotellaceae Reduced. Although most Enterobacteriaceae These are normal members of the gut microbiota; however, certain strains can cause infections and diseases under specific conditions, such as enteritis and urinary tract infections. In the Pg+HFD+LAB1 group... Muribaculaceae and Prevotellaceae Compared to the model group, there was an increase, while the Pg+HFD+LAB2 group showed no significant change. Muribaculaceae and Prevotellaceae It plays a vital role in the gut ecosystem, significantly impacting the host's nutritional metabolism, immune function, and overall health. In summary, this compound probiotic, with a ratio of *Lactobacillus rhamnosus* ATCC53103 to *Lactobacillus reuteri* ATCC23272 of 7:3, can simultaneously regulate both oral and gut microbiota, while a ratio of 3:2 fails to regulate either. The compound probiotic provided by this invention can improve diabetic periodontitis by regulating the balance of oral and gut microbiota.

[0086] 1.9 Short-chain fatty acid content

[0087] Before sacrificing the rats, feces were collected. 30 mg of the feces was added to 400 μL of 75% methanol-water in a 1.5 mL EP tube, vortexed for 60 s, and centrifuged for 15 minutes (17000 g). The supernatant was collected and filtered through a 0.22 μm organic membrane filter. The types and contents of short-chain fatty acids in the feces of each group of rats were measured by LC-MS.

[0088] The contents of acetic acid, propionic acid, and butyric acid are as follows: Figures 21 - 23 As shown. By Figures 21 - 23The results showed that, compared with the control group, the content of fatty acids in feces was significantly lower in both the high-fat diet group and the model group (P<0.05). Compared with the high-fat diet group, the content of acetic acid was significantly lower in the model group (P<0.05), while the contents of propionic acid and butyric acid did not differ significantly. Compared with the high-fat diet group and the model group, except for propionic acid, the contents of acetic acid and butyric acid were significantly increased in the Pg+HFD+LAB1 probiotic group, at 23% and 0.04%, respectively. In the Pg+HFD+LAB2 group, there were no significant changes in short-chain fatty acids (acetic acid, propionic acid, and butyric acid). Acetic acid is believed to affect insulin sensitivity and metabolism. It may help maintain stable blood glucose levels through multiple pathways, such as improving glucose metabolism and promoting effective insulin release. Propionic acid and butyric acid can directly affect insulin secretion, promoting insulin synthesis and release, thereby helping to maintain stable blood glucose. Short-chain fatty acids may help improve insulin sensitivity through multiple mechanisms, especially in cases of good gut microbiota balance and low inflammatory response. The above structures indicate that the Pg+HFD+LAB1 probiotic group can secrete short-chain fatty acids to improve insulin resistance.

[0089] 1.10 Data Analysis

[0090] The experimental data were statistically analyzed using IBM SPSS Statistics 27. The t-test was used to analyze the significance of differences between two groups, and the Tukey test in one-way ANOVA was used to compare differences between multiple groups. Results are expressed as mean ± standard deviation. GraphPad Prism 8 was used for plotting. P < 0.05 was considered statistically significant.

[0091] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A compound probiotic, characterized in that, The bacteria include *Lactobacillus rhamnosus* and *Lactobacillus reuteri*, wherein the ratio of live *Lactobacillus rhamnosus* to *Lactobacillus reuteri* is not less than 1:1, and the total live count is not less than 1 × 10⁻⁶. ^9 CFU / mL; the live bacteria ratio of Lactobacillus rhamnosus to Lactobacillus reuteri is 7:3; the Lactobacillus rhamnosus is ATCC 53103, and the Lactobacillus reuteri is ATCC 23272.

2. The use of the compound probiotics according to claim 1 in the preparation of a product for treating type 2 diabetes with periodontitis.

3. A drug for treating type 2 diabetes mellitus with periodontitis, characterized in that, Includes the compound probiotics as described in claim 1.

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