Composite probiotics and application thereof in preparation of products for preventing or treating diabetes mellitus with periodontitis

Abstract

The invention belongs to the technical field of microorganisms, and discloses compound probiotics and application thereof in preparation of products for preventing or treating diabetes mellitus with periodontitis. The compound probiotics comprise lactobacillus rhamnosus and lactobacillus reuteri, the viable bacterium ratio of the lactobacillus rhamnosus to the lactobacillus reuteri is not lower than 1: 1, and the total viable bacterium number is not lower than 1 * 10 < 9 > CFU / mL. The compound probiotics provided by the invention comprise lactobacillus rhamnosus and lactobacillus reuteri, and by controlling the proportion of the lactobacillus rhamnosus and the lactobacillus reuteri, the effect of preventing and treating diabetes mellitus with periodontitis can be achieved. The compound probiotics can be widely applied to preparation of medicines, health-care foods or nursing products for preventing or treating diabetes mellitus with periodontitis.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a composite probiotic and an application thereof in preparing a product for preventing or treating diabetes associated with periodontitis. Background Art

[0002] There are complex interaction mechanisms between diabetes and periodontitis. Diabetes can increase the inflammatory response of periodontitis, and the presence of periodontitis may also worsen the control of diabetes. This mutually reinforcing inflammatory response makes treatment more complicated and difficult. Diabetic patients with periodontitis often need stricter blood sugar control because high blood sugar levels can affect the repair and anti-infection ability of periodontal tissues. Effective blood sugar control requires not only medications, but also daily diet, exercise and lifestyle management. Secondly, the immune system function of diabetic patients may be impaired, resulting in decreased healing ability. This makes common periodontal treatment methods (such as cleaning, antibiotics, scaling and periodontal surgery) less effective than non-diabetic patients, and the recovery time is prolonged and the risk of infection is increased. Treatment requires close cooperation between endocrinologists and dentists to simultaneously manage blood sugar and periodontal health. This multidisciplinary treatment model places high demands on the communication and collaboration of the medical team, and also increases the complexity of comprehensive treatment.

[0003] Porphyromonas gingivalis (Porphyromonas gingivalis, P.gingivalis) is a common pathogen in patients with periodontitis, especially in patients with diabetes. It can aggravate the difficulty of treating diabetes with periodontitis, which is mainly reflected in the following aspects. Strong pathogenicity: Porphyromonas gingivalis is a Gram-negative bacillus with strong pathogenicity. It can invade periodontal tissues, leading to aggravated inflammatory responses and more serious damage to periodontal tissues. Drug resistance: Some strains of Porphyromonas gingivalis may be resistant, making conventional antibiotic treatment ineffective. This increases the restrictions on treatment options and requires more laboratory tests and individualized treatment plans. Inflammatory response: The presence of Porphyromonas gingivalis can lead to the persistence of a chronic inflammatory state in gingival tissue. This inflammatory response not only affects the health of periodontal tissues, but may also affect the control of systemic diabetes through inflammatory mediators. Poor response to treatment: Due to the special biological characteristics and pathogenic mechanisms of Porphyromonas gingivalis, its response to conventional periodontal treatment methods may not be as rapid and significant as other pathogens, requiring a longer treatment process and more complex treatment strategies. Therefore, periodontitis caused by Porphyromonas gingivalis increases the complexity and difficulty of treatment of type 2 diabetes by aggravating oral and systemic inflammation. In addition, Porphyromonas gingivalis can enter the bloodstream system and spread from the mouth to the liver through the blood. Once in the blood, these bacteria can penetrate into the liver tissue or colon tissue through the blood vessel wall, causing inflammatory responses and damage. Therefore, focusing on only one of the problems may not be able to fully and effectively improve the patient's overall health.

[0004] At present, the treatment of diabetes with periodontitis mainly focuses on antibiotic treatment and oral care, but there are several major disadvantages: Limitation of antibiotic use: Although antibiotics can inhibit the growth of Porphyromonas gingivalis, long-term use will cause drug resistance problems and may destroy the balance of oral microecology, leading to other adverse consequences. At present, many health products or treatments usually only focus on a single effect. For example, oral medications may only focus on improving insulin resistance, while dental treatment may only solve periodontitis. Limitations of oral care: Current oral care methods, such as brushing and gargling, can help control dental plaque and gingivitis, but they are insufficient for controlling the complex immune response and inflammation associated with periodontitis. Durability of treatment effect: Existing treatment methods often find it difficult to achieve lasting control and prevention, and patients are prone to recurrent attacks or disease progression. Therefore, the treatment of insulin-associated periodontitis requires new methods to enhance the immune regulation and inflammation control capabilities in the oral cavity without destroying the normal balance of oral microecology, so as to improve the durability of treatment effects and the quality of life of patients. Currently, research on probiotics in the treatment of diabetes and periodontitis is still in its early stages. Probiotics usually come from natural microorganisms in the human body or the food chain, have high safety and low side effects, are not prone to drug resistance like antibiotics, and have no side effects compared to hypoglycemic drugs. Some studies have begun to explore the comprehensive treatment strategy of probiotics in the combined treatment of diabetes and periodontitis. Probiotics can help improve the periodontal health and diabetes control of diabetic patients by simultaneously regulating the intestinal and oral microbiota. However, research on probiotics for the treatment of diabetes and periodontitis is limited, and the therapeutic potential of probiotics for diabetes and periodontitis needs to be developed urgently.

[0005] Traditional treatments for diabetic periodontitis often rely on separate approaches to oral care and diabetes treatment. Such separate treatments may result in poor results because they fail to simultaneously address the health issues of periodontitis and insulin resistance, thereby failing to comprehensively promote the overall health of patients with diabetic periodontitis. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a composite probiotic and its use in preparing a product for preventing or treating diabetes with periodontitis. The composite probiotic provided by the present invention can prevent and treat diabetes with periodontitis.

[0007] The invention provides a composite probiotic.

[0008] Specifically, a composite probiotic comprises Lactobacillus rhamnosus and Lactobacillus reuteri, wherein the ratio of live bacteria 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 ^ 9 CFU / mL.

[0009] In some embodiments of the present invention, the ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is (1.5-3): 1. Preferably, the ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is (2-3): 1; further preferably, the ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is (2-2.5): 1. More preferably, the ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is 7:3.

[0010] The invention also provides application of the composite probiotics.

[0011] Specifically, the use of the above-mentioned composite probiotics in the preparation of products for preventing or treating diabetes-associated periodontitis.

[0012] In some embodiments of the present invention, the product comprises a medicine, a health food or an oral care product.

[0013] Specifically, the composite probiotics are used in the preparation of products for preventing or treating colitis caused by Porphyromonas gingivalis.

[0014] In some embodiments of the present invention, the product includes a medicine, a health food or a care product.

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

[0016] In some embodiments of the present invention, the product includes a medicine, a health food or a care product.

[0017] The present invention also provides a medicine for preventing or treating diabetes mellitus associated with periodontitis.

[0018] Specifically, a drug for preventing or treating diabetes mellitus associated with periodontitis comprises the above-mentioned composite probiotics.

[0019] The invention also provides a health food for preventing or treating diabetes associated with periodontitis.

[0020] Specifically, a health food for preventing or treating diabetes with periodontitis comprises the above-mentioned composite probiotics.

[0021] The present invention also provides a care product for preventing or treating diabetes-associated periodontitis.

[0022] Specifically, a care product for preventing or treating diabetes-associated periodontitis comprises the above-mentioned composite probiotics.

[0023] Experiments have shown that the compound probiotics not only inhibit insulin resistance and increase the content of GLP-1 protein, but also inhibit alveolar bone resorption caused by periodontitis, indicating that the compound probiotics have the function of preventing and improving diabetes with periodontitis. The compound probiotics can also simultaneously regulate the balance of oral and intestinal flora to maintain a healthy oral and intestinal environment. This is particularly important for patients with diabetes and periodontitis because they are more susceptible to changes in microbial communities. The compound probiotics can also help repair and protect liver and intestinal tissues while inhibiting insulin resistance and periodontitis, helping to maintain the normal functions of these important tissues. Therefore, the compound probiotics have great potential and effects in preventing and treating diabetes with periodontitis, as well as multiple roles in maintaining oral and intestinal health.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The composite probiotics provided by the present invention include Lactobacillus rhamnosus and Lactobacillus reuteri. By controlling the ratio of the two, it can prevent and treat diabetes with periodontitis. Specifically, (1) inhibiting the gingival index, gingival sulcus bleeding index and plaque index caused by modeling in rats; (2) reducing periodontal tissue damage; (3) inhibiting alveolar bone resorption; (4) inhibiting insulin resistance; (5) inhibiting inflammatory factors (TNF-ɑ and IL-6) in serum; (6) increasing GLP-1 protein content; (7) simultaneously regulating the balance of oral and intestinal flora; (8) secreting short-chain fatty acids; (9) improving liver and intestinal tissue damage caused by Porphyromonas gingivalis. The composite probiotics can be widely used in the preparation of medicines, health foods or care products for preventing or treating diabetes with periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The periodontal tissue diagrams of each group;

[0027] Figure 2 Gingival index diagram of each group;

[0028] Figure 3 Gingival sulcus bleeding index of rats in each group;

[0029] Figure 4 is the plaque index graph of each group;

[0030] Figure 5 Figure 2 shows the inhibition of alveolar bone resorption in each group (jaw, cheek);

[0031] Figure 6 is the attachment loss distance of each group of teeth;

[0032] Figure 7 is the bone surface area / total volume (BV / TV) value of each group;

[0033] Figure 8 Figure 2 is a graph of fasting insulin levels in each group;

[0034] Fig. 9 The insulin resistance graphs of each group;

[0035] Fig.10 is the graph of GLP-1 protein content in each group;

[0036] Fig.11 This is the graph of TNF-ɑ content of inflammatory factor in each group;

[0037] Fig.12 Figure 2 shows the IL-1β protein content in each group;

[0038] Fig.13 The graph shows the content of inflammatory factor IL-6 in each group;

[0039] Fig.14 This is a graph showing the improvement of liver tissue damage in each group;

[0040] Fig.15 It is the graph of total cholesterol (TC) inhibition in each group;

[0041] Fig.16 This is a graph showing the inhibition of triglyceride (TG) in each group;

[0042] Fig.17 This is a graph showing the changes in oral flora at the phylum level in each group;

[0043] Fig.18 This is a graph showing changes in oral flora at the genus level in each group;

[0044] Fig.19This is a graph showing the changes in intestinal flora at the phylum level in each group;

[0045] Fig. 20 This is a graph showing changes in intestinal flora at the genus level in each group;

[0046] Fig.21 is the graph of acetic acid content in each group;

[0047] Fig. 22 is the graph of propionic acid content in each group;

[0048] Fig.23 is the butyrate content of each group;

[0049] Fig.24 The graph shows the improvement of colon tissue loss in each group. DETAILED DESCRIPTION

[0050] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0051] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0052] 1. Materials and Methods

[0053] 1.1 Culture medium used

[0054] MRS medium: yeast powder 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.

[0055] BHI medium: tryptone 10.0 g / L, beef heart extract powder 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-hemin chloride 1 mL / L, pH 7.2-7.4.

[0056] BHI+hemin+vitamin K1 liquid medium: weigh 19.25g BHI and add pure water to 500mL, sterilize under high pressure at 121℃ for 20min, cool to room temperature, and store at 4℃. Take 500mL BHI, add 500μL vitamin K1 and 225μL hemin and mix well.

[0057] 1.2 Bacterial culture

[0058] Porphyromonas gingivalis (P.gingivalis ATCC33277, Pg), Lactobacillus rhamnosus ATCC 53103 and Lactobacillus reuteri ATCC 23272 were purchased from Taisituo Biotechnology. Porphyromonas gingivalis was inoculated in BHI+hemin+vitamin K1 liquid culture medium and cultured at 37°C under anaerobic conditions for 48-96h. 5mL of bacterial suspension was prepared with sterile PBS (50mmol / L, pH7.4) and the concentration was adjusted to 1×10 ^9 CFU / mL.

[0059] Lactobacillus rhamnosus ATCC 53103 and Lactobacillus reuteri ATCC 23272 were inoculated in MRS solid medium for 24 h, and then single colonies were selected and inoculated in MRS liquid medium for 24 h, and the bacterial solution was obtained by centrifugation (7200 rpm for 10 min). ^9 The amount of bacterial solution was calculated by the method of using the centrifuge tubes (CFU / mL / mouse) and the remaining amount after feeding the mice each time could not be used for the next feeding, so fresh bacterial solution was used each time.

[0060] 1.3 Animal Experiment Design and Experimental Results

[0061] Thirty-two male SPF rats aged 6-7 weeks were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. After 7 days of adaptive feeding, the fasting blood glucose of all rats was tested and weighed, and then they were randomly divided into 4 groups, with 8 rats in each group. The 4 groups were blank group (NC), high-fat diet group (HFD), Porphyromonas gingivalis combined with high-fat diet group (model group (Pg+HFD)), and compound probiotic group (where probiotics included 2 groups, Pg+HFD+LAB1 (Lactobacillus rhamnosus ATCC53103: Lactobacillus reuteri ATCC23272=7:3), Pg+HFD+LAB2 (Lactobacillus rhamnosus ATCC53103: Lactobacillus reuteri ATCC23272=3:2)). Blank group (NC): normal diet, 0.2mL sterile PBS buffer. Pg+HFD group: periodontitis pathogens were inoculated by periodontal smear. Each rat was given 1×10 9 CFU (0.2 mL) of Porphyromonas gingivalis for 5 weeks. After 5 weeks, the rats were gavaged with Porphyromonas gingivalis suspension. They were prohibited from drinking water for 1 hour after each inoculation, 3 times a week until the end of the experiment (week 15). 9CFU (0.2mL) of compound probiotics for 5 weeks. After 5 weeks, the compound probiotic suspension was gavaged. Drinking water was prohibited for 1 hour after each inoculation, 3 times a week, until the end of the experiment (week 15). High-fat diet group: fed with high-fat feed. Weigh once a week, the whole experiment lasted for 15 weeks.

[0062] 1.4 Periodontal tissue pathological analysis and measurement of gingival index, gingival sulcus bleeding index, and plaque index

[0063] At week 9, rats were anesthetized by intramuscular injection of 200 mg / kg ketamine hydrochloride, and the gingival index, gingival sulcus bleeding index and plaque index were evaluated by periodontal pocket probing. The rat gingiva was scored using the examination method revised by Loe and Silness in 1967. The scoring criteria are as follows: 0 = healthy gingiva; 1 = mild gingival inflammation: the gingiva has a slight change in color and mild edema, and no bleeding on probing; 2 = moderate gingival inflammation: the gingiva is red, the edema is bright, and bleeding is on probing; 3 = severe gingival inflammation: the gingiva is obviously red and swollen or ulcerated, and there is a tendency to bleed automatically. The results are expressed as mean ± standard deviation.

[0064] Sulcus bleeding index (SBI): reflects the activity of gingivitis. 0 points: the gingival margin and gingival papilla appear healthy, and there is no bleeding after light probing of the gingival sulcus; 1 point: the gingival margin and gingival papilla are mildly inflamed, and there is no bleeding after light probing of the gingival sulcus; 2 points: the gingiva is mildly inflamed, with color changes, no swelling or edema, and punctate bleeding after probing; 3 points: the gingiva is moderately inflamed, with color changes and mild edema, bleeding after probing, and the blood overflows into the gingival sulcus; 4 points: the gingiva is severely inflamed, not only with color changes, but also with obvious swelling, bleeding after probing, and blood overflows into the gingival sulcus; 5 points: the gingiva has color changes, obvious swelling, and sometimes ulcers. Bleeding after probing or automatic bleeding. The results are expressed as mean ± standard deviation.

[0065] Plaque index (PLI): It is an evaluation based on plaque and oral hygiene. 0 points: no plaque in the gingival margin; 1 point: thin plaque on the tooth surface in the gingival margin, but it is not visible during visual examination. If the tooth surface is scraped with a probe tip, plaque can be seen; 2 points: moderate plaque can be seen on the gingival margin or adjacent surface; 3 points: a large amount of soft plaque in the gingival sulcus or in the gingival margin and adjacent surface.

[0066] Periodontal tissue pathological analysis: The right maxillary tissue of rats (maxillary bone and gums were retained) was fixed in 4% paraformaldehyde for 48 hours, then soaked in double distilled water and decalcified with Plank-Rychlo decalcification solution. After decalcification, G3430 decalcification alkaline treatment solution was used to deacidify for 40 minutes, and then rinsed under running water for 18 hours. The fixed tissue samples were then dehydrated with 75%, 85%, 95% I, 95% II, 100% I, and 100% I I ethanol solutions for 60 minutes each, placed in xylene I and II for transparentization for 45 minutes each, and then placed in paraffin I, paraffin II, and paraffin III for wax permeation for 50-60 minutes each, embedded in paraffin, and sliced. Then bake the paraffin slices, and then put the slices into xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 95% alcohol for 3 minutes, 80% alcohol for 3 minutes, and pure water for 2 minutes. Dye with hematoxylin solution for 3-5 minutes, wash with tap water for 1 minute, differentiate with 1% hydrochloric acid alcohol for a few seconds, wash with tap water, blue solution for 10-30 seconds, and rinse with running water. Finally, put the slices into eosin solution for staining for 3-5 minutes and rinse with running water. Put the slices into 80% alcohol, 95% alcohol, anhydrous ethanol, and anhydrous ethanol II for rapid dehydration in turn, and seal with ultra-clean advanced sealing glue. Observe the inflammatory infiltration of periodontal tissues (including gums, periodontal ligaments, cementum, and alveolar bone) and the integrity of periodontal tissues under a microscope.

[0067] Figure 1 The periodontal structure diagrams of each group are shown below. Figure 1 As shown in the figure, the periodontal structure in the blank group (NC) and the high-fat diet group (HFD) was intact, alveolar bone resorption was observed in the Pg+HFD group, and no inflammatory cell infiltration was observed. In the compound probiotic groups (Pg+HFD+LAB1, Pg+HFD+LAB2), the periodontal tissue structure was intact, with no obvious pathological changes.

[0068] Figure 2 Gingival index diagram of each group. Figure 3 Gingival sulcus bleeding index of rats in each group. Figure 4 The plaque index of each group is shown in Figure 2. Figure 2-Figure 4 It can be seen that the gingival index, gingival sulcus bleeding index and plaque index of the model group (Pg+HFD) are significantly higher than those of the blank group and the high-fat diet group. It shows that applying Porphyromonas gingivalis affects oral health. Lactobacillus rhamnosus ATCC53103 combined with Lactobacillus reuteri ATCC23272 in different mixing ratios (Pg+HFD+LAB1, Pg+HFD+LAB2) can significantly inhibit the three indicators of gingival sulcus bleeding index and plaque index or gingival index of rats. It shows that the composite probiotics provided by the present invention can improve oral health.

[0069] 1.5 Alveolar bone microstructure scanning

[0070] After the rats were euthanized, the maxilla of the rats was separated with a sterile scalpel, fixed in 4% paraformaldehyde for 48 hours, washed with 75% ethanol solution, stored in 75% ethanol solution at 4°C, and sent to Nanchang University for testing. Micro-CT (Micro Computed Tomography) was used to perform X-ray scanning imaging of the rat maxilla samples; the specific scanning parameters were: voltage, 78kV; current, 100μA; scanning accuracy, 9μm; number of layers, 400; camera mode, high resolution. 1) Six measurements were taken for 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 interroot region was measured to evaluate alveolar bone resorption.

[0071] Figure 5 Figure 2 shows the inhibition of alveolar bone resorption in each group (jaw, cheek). Figure 6 is the attachment loss distance of each group of teeth, Figure 7 is the bone surface area / total volume (BV / TV) value of each group. Figure 5 As shown in the figure, there were significant differences between the blank group and the experimental groups (P<0.05). The alveolar bone in the blank group (NC) and the high-fat diet group (HFD) was smooth and complete, and the teeth were closely arranged without obvious gaps. The distance from the enamel-cementum junction to the alveolar crest was 0.94 mm. In the model group (Pg+HFD), the alveolar bone was severely resorbed and was loose and porous. The roots were exposed, and the teeth tended to loosen and fall off. The average distance from the enamel-cementum junction to the alveolar crest was 2.18 mm, which was much larger 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 a good control effect on periodontitis. The average distance from the enamel-cementum junction to the alveolar crest was 1.53 mm, which could effectively reduce the absorption of alveolar bone and the loss of cementum. Secondly, the tooth attachment loss distance (AL) of rats in the Pg+HFD group was significantly higher than that in the other groups, while the bone surface area / total volume (BV / TV) was just the opposite ( Figure 6 , Figure 7 ). Compared with the model group, the compound probiotic groups (Pg+HFD+LAB1, Pg+HFD+LAB2) could significantly inhibit the tooth attachment loss distance and increase the bone surface area / total volume of rats (P<0.05), which could inhibit periodontitis.

[0072] 1.6ELISA test

[0073] Before the rats were killed, part of the whole blood was taken from the rats, and the serum was separated. The levels of TNF-α, IL-1β, IL-6, GLP-1 and fasting insulin in the serum were detected using ELISA kits. The detection method was strictly carried out according to the instructions of the kit. The insulin resistance index (HOMA-IR) was calculated according to the HomaModel method.

[0074] HOMA-IR=(fasting insulin level×fasting blood glucose level / 22.5).

[0075] in Figure 8 The fasting insulin levels of each group are shown in Figure 2. Fig. 9 The insulin resistance diagram of each group is shown in Figure 2. Fig.10 The GLP-1 protein content of each group is shown in Figure 2. Fig.11 The content of inflammatory factor TNF-ɑ in each group is shown in the figure. Fig.12 The IL-1β protein content in each group is shown in Figure 2. Fig.13 The inflammatory factor IL-6 content in each group. Figures 8 to 13 As shown, the fasting insulin levels of the blank group, high-fat diet group and blank group, model group (Pg+HFD) and compound probiotic group (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 was 10.5618, 15.58, 19.0989, 14.933 and 17.66, respectively. Compared with the high-fat diet group, the fasting insulin level of the model group was significantly reduced and the insulin resistance was significantly increased. The insulin level of the Pg+HFD+LAB1 group was significantly higher than that of the model group and the insulin resistance was significantly reduced (P<0.05), and the inflammatory factors (TNF-ɑ, IL-6) were significantly inhibited and the GLP-1 protein content was increased. The Pg+HFD+LAB2 group showed that it could not significantly inhibit insulin resistance, inflammatory factors TNF-ɑ, IL-1β, and could not significantly increase the GLP-1 protein content, but it could significantly inhibit the inflammatory factor IL-6.

[0076] From the above experiments, it can be seen that when the mixing ratio of compound probiotics is 7:3 (Pg+HFD+LAB1), it can simultaneously inhibit periodontitis and improve insulin resistance, while when the mixing ratio is 3:2 (Pg+HFD+LAB2), it can only inhibit periodontitis.

[0077] 1.7 Pathological analysis of liver and colon tissues

[0078] Before the rats were killed, part of the whole blood was taken from the rats, and the serum was separated. The total cholesterol (Total cholesterol) and triglyceride (TG) content in the serum were detected using an automatic biochemical analyzer (BK-600, Shandong Bo Ke Biological).

[0079] Pathological analysis of liver and colon tissues: The rat colon tissues were isolated and fixed in 4% paraformaldehyde, and the operation was the same as that of periodontal tissue pathological analysis (no decalcification or alkali treatment was required). The pathological damage and inflammatory cell infiltration of liver and colon tissues were observed under a microscope.

[0080] like Fig.14 As shown, the liver tissue structure of the blank group (NC) was normal, with no obvious pathological changes. Obvious fat vacuoles, a small amount of inflammatory cell infiltration, and vascular congestion were observed in the liver tissue of the high-fat diet (HFD). Fat vacuoles and a small amount of inflammatory cell infiltration were observed in the liver tissue of the Pg+HFD group. Fat vacuoles and a small amount of inflammatory cell infiltration were observed in the liver tissue of the Pg+HFD+LAB1 and Pg+HFD+LAB2 groups. The arrows indicate lymphocyte infiltration.

[0081] Fig.15 The total cholesterol (TC) inhibition diagram of each group is shown in Figure 2. Fig.16 The figure shows the inhibition of triglyceride (TG) in each group. Fig.15 and Fig.16 It can be seen that compared with the HFD group, the triglyceride (TG) in the Pg+HFD group increased significantly, the total cholesterol (TC) and triglyceride (TG) in the Pg+HFD+LAB1 group decreased significantly, and there was no significant difference in total cholesterol (TC) and triglyceride (TG) in the Pg+HFD+LAB2 group. Diabetes, obesity, etc. will increase the synthesis and storage of cholesterol, 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. Excessive triglyceride levels may lead to fatty liver and other liver problems. The results show that the compound probiotic group Pg+HFD+LAB1 can improve liver tissue damage while inhibiting insulin resistance.

[0082] Fig.24 The improvement of colon tissue loss in each group is shown in Figure 2. Fig.24 As shown, the ileum tissue structure of the blank group (NC) was normal, and no obvious pathological changes were observed. Diffuse inflammatory cells were observed to aggregate in the mucosal layer of the ileum tissue of the high-fat diet (HFD) group, forming inflammatory foci. Diffuse inflammatory cells were observed to aggregate in the ileum tissue of the Pg+HFD group to form inflammatory foci. A small amount of inflammatory cell infiltration was observed in the ileum tissue of the composite probiotic group. The arrows indicate diffuse inflammatory cell aggregation. Combined with the results in 1.6, it is shown that the composite probiotics provided by the present invention can inhibit the inflammatory factors TNF-ɑ, IL-1β or IL-6 in the serum, and the composite probiotics have the effect of inhibiting colon tissue inflammation caused by Porphyromonas gingivalis.

[0083] 1.8 16S rRNA sequencing

[0084] Oral flora: Before killing the rats, wipe the rat's mouth with normal saline, use cotton balls to keep the tooth surface dry, use tweezers to pick up a sterile moisture-absorbing paper tip (No. 20) and gently insert it into the bottom of the periodontal pocket. After 40 seconds, take it out and put it into a 2.0 mL sterile cryopreservation tube and freeze it at -80℃ for later use.

[0085] Intestinal flora: Rat feces were collected for 24 hours and stored at -80°C, and then the intestinal flora and oral flora were sequenced using 16S rRNA.

[0086] The changes in oral and intestinal flora were analyzed at the phylum and genus levels. Figure 17-Figure 20As shown. For the changes in oral flora, the phylum level is mainly composed of Proteobacteria, Firmicutes, Bacteroidota, Actinobacteriota, Fusobacteriota, Gemmatimonadota, and Cyanobacteria. The ratio of Firmicutes / Bacteroidota in the blank group, high-fat diet group, model group, Pg+HFD+LAB1, and Pg+HFD+LAB2 groups was 0.64, 0.86, 0.75, 0.62, and 0.38%, respectively. Compared with the blank group, the ratio of Firmicutes / Bacteroidota in the high-fat diet group and the model group increased, among which Proteobacteria decreased and Fusobacteriota increased in the model group. The ratio of Firmicutes / Bacteroidota decreased in the Pg+HFD+LAB2 group, while there was no significant change in the Pg+HFD+LAB1 group. At the family level, compared with the blank group, the Enterobacteriaceae and Streptococcaceae increased in the high-fat diet group, and the Porphyromonadaceae, Streptococcaceae, and Neisseriaceae increased in the model group. Compared with the model group, the Porphyromonadaceae decreased and the Thiotrichaceae increased in the Pg+HFD+LAB1 group, and the remaining families had no significant differences from the blank group. The change ratio and structure of the flora in the Pg+HFD+LAB2 group were similar to those in the model group. Some members of the Porphyromonadaceae family may be associated with human diseases such as dental caries and periodontitis, but they are also part of normal oral and intestinal microorganisms. Members of Fusobacteriota can become pathogens under certain conditions and participate in causing infections and diseases in the oral cavity, digestive tract and other parts, such as periodontitis and appendicitis. Some members of Streptococcaceae are pathogens of humans and animals and can cause a variety of infectious diseases, such as pharyngitis, pneumonia, sepsis, etc. Different members of the genus Streptococcus have different pathogenicity to host tissues. Some Neisseriaceae bacteria are pathogens of humans and animals and can cause a variety of infectious diseases, such as otitis media, eye infections, urinary tract infections, etc. For example, some species in the genus Neisseria can cause gonorrhea and meningitis.

[0087] Regarding the changes in intestinal flora, the ratios of Firmicutes / Bacteroidota were 1.42, 4.6, 13.64, 3.11, and 9.57% in the blank group, high-fat diet group, model group, Pg+HFD+LAB1, and Pg+HFD+LAB2 probiotic groups, respectively. Studies have shown that the ratio of Firmicutes / Bacteroidota in the intestinal microbiome of obese diabetic patients is significantly increased compared with that of healthy people. At the family level, compared with the blank group, the Lachnispiraceae family increased in the high-fat diet group and the model group, the Enterobacteriaceae family was abundant in the model group, and the Muribaculaceae and Prevotellaceae were reduced. Although most Enterobacteriaceae are normal members of the intestinal flora, some strains can cause infections and diseases under certain conditions, such as enteritis, urinary tract infections, etc. In the Pg+HFD+LAB1 group, Muribaculaceae and Prevotellaceae increased compared with the model group, while there was no obvious change in the Pg+HFD+LAB2 group. Muribaculaceae and Prevotellaceae play an important role in the intestinal ecosystem and have a significant impact on the host's nutritional metabolism, immune function and overall health. In summary, it is shown that the composite probiotic can simultaneously regulate the oral and intestinal flora when the ratio of Lactobacillus rhamnosus ATCC53103 and Lactobacillus reuteri ATCC23272 is 7:3, while it cannot regulate the oral and intestinal flora when the ratio is 3:2. The composite probiotics provided by the present invention can improve diabetes with periodontitis by regulating the balance of oral flora and intestinal flora.

[0088] 1.9 Short-chain fatty acid content

[0089] Before the rats were killed, the feces were collected, 30 mg was taken and placed in a 1.5 mL EP tube, 400 μL of 75% methanol water was added, vortexed for 60 s, centrifuged for 15 min (17000 g), the supernatant was taken, and filtered with a 0.22 μm organic membrane filter head. LC-MS was used to measure the types and contents of short-chain fatty acids in the feces of rats in each group.

[0090] The contents of acetic acid, propionic acid and butyric acid are as follows Figure 21-23 As shown. Figure 21-23It can be seen that compared with the blank group, the content of fatty acids in feces was significantly reduced in the high-fat diet group and the model group (P<0.05). Compared with the high-fat diet group, the content of acetic acid in the model group was significantly reduced (P<0.05), and there was no significant difference in the content of propionic acid and butyric acid. Compared with the high-fat diet group and the model group, except for propionic acid, the content of acetic acid and butyric acid in the Pg+HFD+LAB1 probiotic group increased significantly, which were 23.% and 0.04%, respectively. In the Pg+HFD+LAB2 group, there was no significant change 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 sugar levels through various pathways, such as improving glucose metabolism and promoting the effective release of insulin. Propionic acid and butyric acid can directly affect insulin secretion, promote insulin synthesis and release, and thus help maintain blood sugar stability. Short-chain fatty acids may help improve insulin sensitivity through various mechanisms, especially when the intestinal flora is well balanced and the inflammatory response is low. The above structures indicate that the Pg+HFD+LAB1 probiotic group can secrete short-chain fatty acids to improve insulin resistance.

[0091] 1.10 Data Analysis

[0092] The above experimental data were statistically analyzed using IBM SPSS Statistics 27. The t-test method was used to analyze the significance of the difference between the two groups. The Tukey test in the one-way analysis of variance (One-way Anova) was used to compare the differences between multiple groups of data. The experimental results are expressed as mean ± standard deviation. GraphPad Prism 8 was used for graphics. P < 0.05 was considered statistically significant.

[0093] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composite probiotic, characterized in that: The invention comprises Lactobacillus rhamnosus and Lactobacillus reuteri, wherein the ratio of live bacteria 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 ^9 CFU / mL.

2. The composite probiotic according to claim 1, characterized in that The ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is (1.5-3):

1.

3. The composite probiotic according to claim 2, characterized in that The ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is (2-3):

1.

4. The composite probiotic according to claim 3, characterized in that: The ratio of live bacteria of the Lactobacillus rhamnosus to the Lactobacillus reuteri is (2-2.5):

1.

5. Use of the composite probiotic according to any one of claims 1 to 4 in preparing a product for preventing or treating diabetes with periodontitis.

6. Use of the composite probiotic according to any one of claims 1 to 4 in the preparation of a product for preventing or treating colitis caused by Porphyromonas gingivalis.

7. Use of the composite probiotic according to any one of claims 1 to 4 in the preparation of a product for preventing or treating liver tissue damage caused by Porphyromonas gingivalis.

8. A drug for preventing or treating diabetes mellitus with periodontitis, characterized in that: The invention comprises the composite probiotics according to any one of claims 1 to 4.

9. A health food for preventing or treating diabetes with periodontitis, characterized in that: The invention comprises the composite probiotics according to any one of claims 1 to 4.

10. A care product for preventing or treating diabetes with periodontitis, characterized in that: The invention comprises the composite probiotics according to any one of claims 1 to 4.

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