Inactivated lactobacillus cell and its application

Abstract

The application discloses a lactobacillus fermentum inactivated cell capable of relieving periodontitis and application thereof, and belongs to the technical field of microorganisms. The application obtains a lactobacillus fermentum CCFM1139 inactivated cell, the lactobacillus fermentum inactivated cell has the effect of relieving periodontitis, and the effect is specifically reflected in the following aspects: the periodontal probing depth can be reduced; the expression of the cytokine IL-1beta in the gingival tissue and serum is down-regulated; the inflammatory cell recruitment is reduced; the alveolar bone loss of the periodontitis rat is reduced, the rat alveolar bone absorption is effectively relieved; and the clinical attachment loss level and the probing depth are reduced. The effect of the inactivated cell in relieving periodontitis can be close to the effect of the active probiotic product, the inactivated cell has higher safety and good stability, is not controlled by the active cell number, can be synergistically used with antibiotics, is not affected by environmental factors such as production, transportation and storage, and has a very broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a strain of Lactobacillus fermentum inactivated bacteria capable of relieving periodontitis and its application, belonging to the field of microbial technology. BACKGROUND

[0002] Periodontitis is a chronic infectious disease caused by subgingival pathogenic plaque, leading to persistent inflammation of periodontal tissue, and further causing collagen fiber dissolution and destruction of periodontal membrane and alveolar bone absorption, resulting in destruction of periodontal support tissue. Periodontitis is highly prevalent in people over 35 years old, and the pathological process is repeated and difficult to cure. With the increase of the severity of periodontitis, it causes acute swelling and bleeding of the gums, gum recession, difficulty in chewing, and even loss of masticatory function and tooth loss in severe cases, affecting the daily life and aesthetics of the person, and even increasing the risk of systemic diseases.

[0003] The interaction between the host and dental plaque microorganisms largely determines the development and severity of periodontitis. Once the host's diet and other factors change and cause ecological imbalance of the dental plaque biofilm, the pathogenic microorganisms in the dental plaque biofilm will become the dominant flora. Among them, Porphyromonas gingivalis is the main suspected pathogen of periodontitis, which colonizes in the subgingival microenvironment through adhesion and coaggregation, and further invades the deep periodontal connective tissue; it can resist the host's innate and acquired immune defense response, interfere with the normal physiological function of host cells; through the secretion of virulence factors (such as endotoxin, gingivalin, fimbriae, etc.) or promoting the secretion of pro-inflammatory mediators and chemotactic factors by host cells, causing hydrolytic destruction of periodontal tissue; at the same time, the sustained inflammation activates the immune pathway activity (such as NF-κB, PI3K, etc.), leading to osteoclast differentiation, and further causing alveolar bone absorption and destruction of periodontal support tissue.

[0004] Currently, the treatment of periodontitis is mainly mechanical therapy (supragingival scaling, subgingival scaling and root planing), aiming to effectively remove periodontal pathogens in the lesion area, but this physical treatment process may be accompanied by pain and discomfort and unsatisfactory treatment prognosis, which brings heavy burden to patients and doctors. In addition, drug treatment may be supplemented to control the formation of dental plaque, but long-term use may produce drug-resistant strains, oral microecological imbalance and other adverse consequences.

[0005] Therefore, it is a research hotspot to develop new and safe and effective treatment methods for periodontitis. Lactobacillus has the effects of inhibiting the growth of periodontal pathogenic bacteria, producing antibacterial substances, regulating local immune response, and regulating bone resorption, and is gradually applied to the intervention and adjuvant therapy of oral diseases. However, live lactobacillus is greatly affected by the environment, needs cold chain transportation and special protection, and needs to ensure a certain amount of viable bacteria in the intestine to play a beneficial effect, so the commercial development is limited. Inactivated bacteria have higher safety, good stability, are not controlled by viable bacteria, can be synergized with antibiotics, are not affected by environmental factors such as production, transportation and storage, and have a very broad application prospect, so the characteristics of inactivated bacteria are also a research hotspot at present.

[0006] Inactivated bacteria also have immune regulation effects. Studies have shown that the main component of the cell wall of gram-positive bacteria is combined with the pattern recognition receptor on the surface of leukocytes, and the effective stimulating factor is produced by the activation of the two signal pathways of NF-κB and IFN-α induced STAT. Inactivated bacteria can induce the production of anti-inflammatory cytokines and promote Th2-dependent immune response. The cell wall component lipoteichoic acid LTA has been proved to have immunological activity and can stimulate mouse leukocytes to produce IFNγ in vitro.

[0007] In summary, it is urgent to find an inactivated bacteria that can fundamentally prevent and / or relieve periodontitis. SUMMARY

[0008] The application provides application of inactivated bacteria of Lactobacillus fermentum in preparation of products for relieving periodontitis, and the Lactobacillus fermentum is Lactobacillus fermentum GDMCC No: 61116.

[0009] The Lactobacillus fermentum GDMCC No: 61116 is recorded in the Chinese invention patent document with the publication number CN112608864B and is named as Lactobacillus fermentum CCFM1139.

[0010] In an embodiment of the application, the inactivation method of the inactivated bacteria comprises heat inactivation, repeated freeze-thaw inactivation, high-pressure inactivation, ultrasonic inactivation and ultraviolet inactivation.

[0011] In an embodiment of the application, the inactivation method of the inactivated bacteria is repeated freeze-thaw inactivation.

[0012] In one embodiment of the present application, the inactivation method of the inactivated bacteria is as follows: the seed liquid of Lactobacillus fermentum GDMCC No: 61116 is inoculated at a rate of 4% (v / v) and continuously cultured for three generations, then centrifuged at 3000g for 10 min at 4℃ to obtain a bacterial slurry, the bacterial slurry is treated at 75℃ for 30 min, and then alternately treated at -80℃ overnight to obtain inactivated bacteria, and the inactivation of the bacteria is verified by plating the inactivated bacteria on MRS medium and observing the growth of the inactivated bacteria.

[0013] In one embodiment of the present application, the product is a pharmaceutical product or a daily chemical product.

[0014] In one embodiment of the present application, the pharmaceutical product comprises inactivated bacteria of Lactobacillus fermentum CCFM1139 and a pharmaceutical carrier.

[0015] In one embodiment of the present application, the pharmaceutical carrier is one or more of a pharmaceutically acceptable filler, wetting agent, disintegrant, binder, lubricant or flavoring agent.

[0016] In one embodiment of the present application, the dosage form of the pharmaceutical product includes, but is not limited to, granules, capsules, tablets, pills, oral solutions.

[0017] In one embodiment of the present application, the daily chemical product is a mouthwash or toothpaste containing inactivated bacteria of Lactobacillus fermentum CCFM1139.

[0018] In one embodiment of the present application, the pharmaceutical product or daily chemical product has one or more of the following purposes (a) to (d):

[0019] (a) reducing the level of inflammatory factors in the gingival tissue;

[0020] (b) reducing the amount of alveolar bone resorption;

[0021] (c) reducing clinical attachment loss;

[0022] (d) reducing probing depth.

[0023] In one embodiment of the present application, in the product, the count of live bacteria corresponding to the equivalent mass of inactivated bacteria of Lactobacillus fermentum CCFM1139 is at least 2×10 10 CFU / mL.

[0024] In one embodiment of the present application, in the product, the count of live bacteria corresponding to the equivalent mass of inactivated bacteria of Lactobacillus fermentum CCFM1139 is at least 2×10 10 CFU / g.

[0025] The present application provides a Lactobacillus fermentum inactivated bacterial preparation, which comprises Lactobacillus fermentum CCFM1139 inactivated bacteria.

[0026] In an embodiment of the present application, the Lactobacillus fermentum inactivated bacterial preparation is a pharmaceutical product or a pharmaceutically acceptable carrier comprising Lactobacillus fermentum CCFM1139 inactivated bacteria.

[0027] In an embodiment of the present application, the Lactobacillus fermentum inactivated bacterial preparation is a daily chemical product such as mouthwash, toothpaste, oral spray, etc. comprising Lactobacillus fermentum CCFM1139 inactivated bacteria.

[0028] The present application also provides a microbial preparation containing Lactobacillus fermentum GDMCC No:61116 inactivated bacteria, which has any one or more of the following purposes (a)-(d):

[0029] (a) reducing the level of inflammatory factors in gingival tissue;

[0030] (b) reducing alveolar bone resorption;

[0031] (c) reducing clinical attachment loss;

[0032] (d) reducing probing depth.

[0033] Beneficial effects

[0034] 1. The present application obtains a Lactobacillus fermentum CCFM1139 inactivated bacteria, which has the effect of relieving periodontitis, specifically in:

[0035] (1) The Lactobacillus fermentum CCFM1139 inactivated bacteria can directly reduce the relevant clinical indicators of periodontitis. The effect is reflected in that the probing depth of the upper left and right molars of rats is reduced from 0.911 and 0.893 to 0.659 and 0.711, respectively; and the clinical attachment loss and probing depth of patients with chronic periodontitis are significantly reduced.

[0036] (2) The Lactobacillus fermentum CCFM1139 inactivated bacteria can also slow down the progression of periodontitis from the perspective of regulating immune response, inhibit the secretion of inflammatory factors, and thus reduce the alveolar bone loss caused by persistent inflammation. The effect is reflected in that the amount of IL-1β in the gingival tissue of periodontitis rats is reduced from 39.54 pg / mg to 17.95 pg / mg, and the content of IL-1β in the serum is reduced from 28.80 pg / mL to 16.75 pg / mL; and the alveolar bone resorption of periodontitis rats is reduced from 0.82 mm to 0.69 mm.

[0037] Therefore, the Lactobacillus fermentum CCFM1139 inactivated bacteria has a very broad application prospect in preparation of daily chemicals (such as mouthwash, toothpaste and the like) and medicines for relieving periodontitis.

[0038] 2. The Lactobacillus fermentum is one of probiotics, has been included in the "List of Bacteria for Food" issued by the Ministry of Health, and the inactivated bacteria no longer has the growth and reproduction ability but still retains the original structure and characteristics of the beneficial bacteria, so that the Lactobacillus fermentum inactivated bacteria obtained in the application does not bring any side effects to the human body, and when used in products (such as daily chemicals or medicines) for relieving periodontitis, the safety is higher. Meanwhile, the inactivated bacteria is not affected by environmental changes, does not need cold chain and special protection, and has a broad commercial prospect. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 It is a schematic diagram of periodontal ligation of rats.

[0040] Figure 2 It is a flow chart of the experiment.

[0041] Figure 3 It is the change of IL-1β in gingival tissue and serum.

[0042] Figure 4 It is a Micro-CT diagram of the maxilla of rats.

[0043] Figure 5 It is the microstructure change of the maxillary alveolar bone of rats.

[0044] Figure 6 It is a periodontal pathological tissue section.

[0045] Figure 7 It is the change of osteoclasts in the maxilla of rats. DETAILED DESCRIPTION

[0046] The application will be further described below in combination with specific embodiments and drawings.

[0047] The Porphyromonas gingivalis involved in the following examples is purchased from Guangdong Microbial Culture Collection Center, and the product number is GDMCC 1.851; the Fusobacterium nucleatum involved in the following examples is purchased from Guangdong Microbial Culture Collection Center, and the product number is GDMCC 1.1290; the SPF Wistar rats involved in the following examples are purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK (Jing) 2012-0001).

[0048] The preparation method of the Lactobacillus fermentum CCFM1139 live bacteria involved in the following examples is as follows:

[0049] Lactobacillus fermentum CCFM1139 was expanded to harvest the bacteria after 3 serial passages with 4% inoculation. The bacteria were harvested by centrifugation at 3000g for 10 min at 4°C, washed 3 times with pre-cooled phosphate buffered saline (PBS) at 4°C, and finally harvested by centrifugation under the same conditions. The harvested bacteria were mixed with 13% skim milk powder at 1:1 (g / v), poured into sterile Petri dishes, and stored in a -80°C freezer overnight. The freeze-drying was performed at -40°C and 4 Pa for 48 h in a freeze-drier. The freeze-dried product was ground at low intensity and sieved to obtain the bacterial powder. The bacterial powder was resuspended in PBS to a concentration of 4 x 10 9 CFU / mL before use.

[0050] The preparation method of Lactobacillus fermentum CCFM1139 inactivated bacteria involved in the following examples is as follows:

[0051] The live bacteria were prepared according to the above method, and the prepared live bacteria were inactivated by pasteurization. Then, the plate coating method was used to detect whether there were live bacteria.

[0052] The Lactobacillus fermentum CCFM1139 bacteria solution after complete sterilization was centrifuged at 3000g for 10 min at 4°C, washed 3 times with pre-cooled phosphate buffered saline (PBS) at 4°C, and finally harvested by centrifugation under the same conditions. The harvested bacteria were mixed with 13% skim milk powder at 1:1 (g / v), poured into sterile Petri dishes, and stored in a -80°C freezer overnight. The freeze-drying was performed at -40°C and 4 Pa for 48 h in a freeze-drier. The freeze-dried product was ground at low intensity and sieved to obtain the bacterial powder. The bacterial powder was resuspended in PBS to a concentration of 4 x 10 10 CFU / mL before use.

[0053] The preparation method of Porphyromonas gingivalis bacteria suspension involved in the following examples is as follows:

[0054] Porphyromonas gingivalis was inoculated into BHI liquid medium at an inoculation amount of 4% (v / v), and the bacteria were collected after anaerobic culture at 37°C for 48 h. The bacteria were washed 2 times with PBS, and the concentration was adjusted to 2 x 10 9 CFU / mL with PBS.

[0055] The preparation method of Fusobacterium nucleatum bacteria suspension involved in the following examples is as follows:

[0056] Fusobacterium nucleatum was inoculated into BHI liquid medium at an inoculation amount of 4% (v / v), and the bacteria were collected after anaerobic culture at 37°C for 24 h. The bacteria were washed 2 times with PBS buffer solution, and the concentration was adjusted to 2 x 10 9CFU / mL.

[0057] The culture media involved in the following examples are as follows:

[0058] MRS medium (per L): yeast extract 5.0 g, beef extract 10.0 g, peptone 10.0 g, glucose 20.0 g, sodium acetate anhydrous 2.0 g, di-ammonium citrate 2.0 g, potassium phosphate dibasic trihydrate 2.6 g, manganese sulfate monohydrate 0.25 g, magnesium sulfate heptahydrate 0.5 g, and Tween-80 1 mL / L, pH 6.2-6.4.

[0059] MRS solid medium (per L): yeast extract 5.0 g, beef extract 10.0 g, peptone 10.0 g, glucose 20.0 g, sodium acetate anhydrous 2.0 g, di-ammonium citrate 2.0 g, potassium phosphate dibasic trihydrate 2.6 g, manganese sulfate monohydrate 0.25 g, magnesium sulfate heptahydrate 0.5 g, and Tween-80 1 mL / L, agar powder 2 g, pH 6.2-6.4.

[0060] BHI medium (per L): tryptone 10.0 g, beef heart infusion powder 17.5 g, sodium chloride 5.0 g, yeast extract 5.0 g, glucose 2.0 g, disodium phosphate dibasic dodecahydrate 2.5 g, 0.5% vitamin K1-hemin 1 mL / L, pH 7.2-7.4.

[0061] The detection methods involved in the following examples are as follows:

[0062] Detection of probing depth (PD):

[0063] Before the rats were sacrificed, the probing depth (PD) of the maxillary model of the rats was determined using a periodontal probe.

[0064] The probing depth (PD) of the maxillary model of the rats was determined using a periodontal probe, and the specific operation method is as follows: using a periodontal probe to detect the distance from the gum margin to the bottom of the pocket or the bottom of the gingival groove at six sites of the distal, middle, and mesial of the palatal side of the left and right second molars of the maxilla;

[0065] Detection method of inflammatory factors:

[0066] The gingival tissue was mixed with 9 volumes of RIPA lysis buffer containing 1% (v / v) protease inhibitor, and homogenized in a tissue grinder. The homogenate was centrifuged at 12,000g for 15 min at 4°C, and the supernatant was collected. The total protein content in the gingival tissue was determined by BCA protein concentration kit. The bovine serum albumin standard was set to 100% purity. The content of cytokine IL-1β in the gingival tissue was determined using an enzyme-linked immunoassay kit.

[0067] Detection method of alveolar bone absorption (ABL) and bone volume percentage

[0068] Rat maxillae were soaked in 4% paraformaldehyde for 48 h, washed with PBS buffer and air-dried. Clean rat maxillae were scanned using a Micro computed tomography (Micro CT) system. The samples were scanned using the following settings: 90 kV, 88 μA, pixel size of 18 μm, and a rotation of 180°. Alveolar bone loss (ABL) was the distance between the cement-enamel junction (CEJ) and the alveolar bone crest (ABC). ABL was measured at 6 sites, i.e., distal, mesial and middle of the buccal and palatal sides of the second molar.

[0069] Periodontal tissue histopathology

[0070] For periodontal tissue histopathology, rat maxillae were decalcified in ethylenediaminetetraacetic acid (EDTA) solution (pH 7.8) and embedded in paraffin. Paraffin was cut into 4 μm sections and stained with hematoxylin and eosin (H&E). Sections were observed using a pathological section scanner at 20x magnification. Inflammatory cell infiltration was observed at the second molar furcation and the first and second molar interdental spaces at a field of view of 100 μm.

[0071] Example 1: Probing depth change

[0072] 1. Experimental method

[0073] The rats with periodontitis involved in the examples ate and drank as follows:

[0074] Twenty-four Wistar male rats were randomly divided into 4 groups (6 rats / group), namely: a control group (Control group), a model group (Model group), a CCFM1139 live bacteria group, and a CCFM1139 inactivated bacteria group. The experimental grouping and the eating and drinking of the rats are shown in Table 1. Before the experiment, the rats in each group were placed in a 22-24°C constant temperature condition with a 12-hour light and 12-hour dark alternating cycle for one week of adaptation. The experiment lasted for 5 weeks (from the 0th week to the 4th week), a total of 35 days. Figure 2 ).

[0075] Table 1: Rat grouping and eating and drinking

[0076]

[0077] High-sugar feed Keyes 2000 feed (w / w): milk powder 28%, sucrose 56%, wheat flour 6%, yeast 4%, alfalfa powder 3%, liver powder 1%, and salt 2%.

[0078] Common feed: grains (corn, secondary powder, wheat, alfalfa, soybean meal) 80%, animal protein (Peru fish meal, American chicken meal) 10%, additives (animal premix, gluten, calcium bicarbonate, stone powder, salad oil, feed-grade sodium chloride, feed-grade magnesium oxide) 10%.

[0079] The experiment lasted for 5 weeks (from the beginning of week 0 to the end of week 4), and the model group and the test group (CCFM1139 live bacteria group, CCFM1139 inactivated bacteria group).

[0080] The rats were subjected to periodontal ligation surgery at the beginning of week 0. The specific operation method is as follows: the rats were anesthetized by intraperitoneal injection of 100 / 10 mg / kg ketamine / thiamine, two 0.22 mm sterile orthodontic silk threads were passed through the interdental space between the first and second molars and the second and third molars of the rats respectively, and the silk threads were knotted on both sides, and the orthodontic silk threads were placed in the gingival sulcus as much as possible. After periodontal ligation, a high-sugar diet was used. The effect after periodontal ligation surgery is as follows Figure 1 .

[0081] The rats in each group after ligation were infected with periodontitis pathogenic bacteria, and the infection method was as follows:

[0082] The rats with periodontal ligation were irrigated with Porphyromonas gingivalis and Fusobacterium nucleatum. The specific irrigation method is as follows: 0.5 mL of periodontal pathogenic bacteria mixed solution (including 2×10 9 CFU / mL of Porphyromonas gingivalis and 2×10 9 CFU / mL of Fusobacterium nucleatum) was sucked into a sterile syringe, and the maxillary ligation site of the rats was slowly irrigated. After irrigation, the rats were fasted and watered for half an hour. Once every two days, for three times, the infection time was one week.

[0083] The CCFM1139 live bacteria group and the CCFM1139 inactivated bacteria group were prevented and treated respectively after one week of infection, and the prevention and treatment method was as follows:

[0084] After the end of pathogenic bacteria irrigation, the rats in the CCFM1139 live bacteria group were slowly irrigated with 0.5 mL of Lactobacillus fermentum CCFM1139 live bacteria with a concentration of 4×10 9 CFU / mL at the maxillary ligation site of the rats;

[0085] After the end of pathogenic bacteria irrigation, the rats in the inactivated bacteria group were slowly irrigated with 0.5 mL of Lactobacillus fermentum CCFM1139 inactivated bacteria with a concentration of 4×10 10 CFU / mL at the maxillary ligation site of the rats.

[0086] The prevention and treatment frequency was 3 times / week at week 1, and the prevention and treatment frequency was adjusted to 6 times / week from week 2 to week 4. The specific experimental process is as follows Figure 2 .

[0087] For the model group one week after infection: the ligation site of the rat's maxilla was slowly rinsed with 0.5 mL PBS, and the treatment frequency was 6 times / week.

[0088] After the experiment, the mice in each group were euthanized.

[0089] 2. Experimental Results

[0090] Before sacrificing the rats, the probing depth (PD) at the maxillary model site was measured using a periodontal probe, and the results are shown in Table 2.

[0091] Table 2. Probing depth of the left and right sides of the maxilla in each group of rats

[0092]

[0093] The results showed that the probing depth at the left and right model sites in the model group rats was 0.911 mm and 0.893 mm, respectively, and the periodontal pocket depth was significantly increased compared with the control group.

[0094] After 4 weeks of intervention with live and inactivated Lactobacillus fermentum CCFM1139, the probing depth of periodontal tissues was reduced to some extent, and the probing depth of the left maxilla of rats with periodontitis treated with inactivated bacteria was significantly reduced (P<0.05).

[0095] In summary, both live and inactivated Lactobacillus fermentum CCFM1139 can reduce the formation of periodontal pockets in rats and alleviate gingival redness and swelling to some extent.

[0096] Example 2: Effects of inactivated Lactobacillus fermentum CCFM1139 cells on the expression of inflammatory factors in periodontal tissues and serum.

[0097] 1. Experimental Methods

[0098] The specific experimental method is the same as step 1 of Example 1; after the experiment, gingival tissue was taken from each group of rats to detect the total protein content and the IL-1β content in the gingival tissue; at the same time, abdominal aortic blood was taken from each group of rats, allowed to stand at room temperature for 30 min, centrifuged at 3000×g and 4℃ for 10 min, and the pale yellow supernatant was transferred into sterile EP tubes, aliquoted and labeled, and stored in a -80℃ refrigerator. The serum IL-1β content was determined according to the instructions of the enzyme-linked immunosorbent assay kit, and the results are as follows. Figure 3 As shown.

[0099] 2. Experimental Results

[0100] according to Figure 3The results of inflammatory factors in gingival tissue were obtained. The IL-1β level in the control group was 3.38 pg / mg, while that in the model group was 39.54 pg / mg. The IL-1β level in the gingival tissue of the model group was significantly higher than that in the control group (P<0.05). Therefore, there was significant inflammation in the periodontal tissue of the rats.

[0101] Oral administration of live and inactivated Lactobacillus fermentum CCFM1139 significantly reduced the level of IL-1β in gingival tissue (P<0.05), decreasing it to 21.44 pg / mg and 17.95 pg / mg, respectively.

[0102] The serum IL-1β level also showed the same trend. Oral administration of live and inactivated Lactobacillus fermentum CCFM1139 significantly reduced the serum IL-1β level (P<0.05), decreasing from 28.80 pg / mL in the model group to 17.94 pg / mL and 16.75 pg / mL, respectively. This indicates that oral administration of live and inactivated Lactobacillus fermentum CCFM1139 can also reduce the level of inflammatory factors in serum.

[0103] In summary, both live and inactivated Lactobacillus fermentum CCFM1139 can effectively reduce local and systemic inflammation, with inactivated bacteria showing better results than live bacteria.

[0104] Example 3: Effect of inactivated Lactobacillus fermentum CCFM1139 on alveolar bone resorption (ABL)

[0105] 1. Experimental Methods

[0106] The specific experimental method is the same as step 1 of Example 1; after the experiment, the rat maxilla was taken, and the alveolar bone resorption (ABL) was measured. The results are as follows: Figures 4-5 As shown.

[0107] 2. Experimental Results

[0108] The results show:

[0109] according to Figure 4 Micro-CT of the maxilla and Figure 5 It was found that the ABL of the model group rats was 0.82 mm compared with 0.39 mm in the control group (P<0.01).

[0110] In addition, the model group rats showed significant horizontal and vertical alveolar bone resorption, and the second molar root bifurcation was exposed, indicating that the model was successfully established.

[0111] After 4 weeks of intervention with live and inactivated Lactobacillus fermentum CCFM1139, the alveolar bone resorption (ABL) of rats with periodontitis was significantly reduced, decreasing to 0.61 mm and 0.69 mm, respectively (P < 0.05). The effect of inactivated Lactobacillus fermentum CCFM1139 in reducing maxillary alveolar bone resorption in rats was similar to that of live bacteria.

[0112] Example 4: Effects of inactivated Lactobacillus fermentum CCFM1139 on periodontal tissues

[0113] 1. Experimental Methods

[0114] The specific experimental method is the same as step 1 of Example 1; after the experiment, periodontal tissues of rats in each group were taken for pathological sections, and the results are as follows. Figure 6 As shown.

[0115] 2. Experimental Results

[0116] The results show that, according to Figure 6 The periodontal tissue pathology sections yielded the following:

[0117] In the blank control group sections, normal periodontal tissue and alveolar bone structures were observed, with the junctional epithelium tightly attached to the tooth surface and showing no attachment loss; in addition, the periodontal ligament fibers were intact and neatly arranged, and there was almost no inflammatory infiltration in the gingival tissue.

[0118] The model group showed significant downward growth of the epithelium at the top, accompanied by inflammatory cell aggregation around the maxillary second molar and periodontal fiber resorption. Furthermore, there was marked bone loss at the bifurcation of the second molar root.

[0119] After 4 weeks of intervention with live and inactivated Lactobacillus fermentum CCFM1139, the alveolar bone pathological sections of rats showed that the severity of periodontitis was reduced compared with the model group, the number of inflammatory cells in the periodontal tissue was reduced, and the histopathological state of the rats in the inactivated bacterial group was close to that of the live bacterial group.

[0120] Example 5: Effects of inactivated Lactobacillus fermentum CCFM1139 cells on osteoclasts

[0121] 1. Experimental Methods

[0122] The specific experimental method was the same as step 1 of Example 1; after the experiment, periodontal tissues from each group of rats were taken for pathological sections, and each tissue section was stained with tartrate-resistant acid phosphatase (TRAP). The tissue sections were counterstained with hematoxylin. Multinucleated TRAP-positive cells formed on the alveolar bone surface around the molars were counted as active osteoclasts. The results are as follows: Figure 7 As shown.

[0123] 2. Experimental Results

[0124] The results show that, according toFigure 7 Staining osteoclasts from the maxilla of rats revealed that, compared with the control group, the osteoclasts in the model group showed more TRAP positivity after periodontitis induction.

[0125] Compared with the model group rats, after 4 weeks of intervention with live and inactivated Lactobacillus fermentum CCFM1139, the positive rate of maxillary osteoclasts in both groups was reduced, and the ability of inactivated bacteria to inhibit osteoclast formation was close to that of live bacteria.

[0126] Example 6: Application of inactivated Lactobacillus fermentum GDMCC No: 61116

[0127] 1. Preparation of mouthwash containing inactivated Lactobacillus fermentum CCFM1139 cells.

[0128] (1) Preparation of Lactobacillus fermentum CCFM1139 seed culture

[0129] Lactobacillus fermentum CCFM1139 was streaked in three zones on an MRS solid plate and grown at 37°C for 48 h. Single colonies were picked and inoculated into MRS liquid tubes and grown at 37°C for 18 h to obtain Lactobacillus fermentum CCFM1139 seed culture.

[0130] (2) The CCFM1139 seed culture obtained in step (1) was inoculated into MRS medium at an inoculation rate of 4% (v / v). After three generations of continuous culture at 37℃, CCFM1139 bacterial culture was obtained. The CCFM1139 bacterial culture was centrifuged at 3000g at 4℃ for 10 min to obtain bacterial sludge. The obtained bacterial sludge was mixed with 13% skim milk powder at a ratio of 1:1 (g / v) and treated at 75℃ for 30 min, followed by overnight treatment at -80℃. This alternating treatment was repeated three times to obtain inactivated Lactobacillus fermentum CCFM1139 cells. Subsequently, the cells were freeze-dried at -40℃ and 4Pa for 48 h. The freeze-dried product was then subjected to low-intensity pulverization and sieving to obtain probiotic freeze-dried powder.

[0131] Prepare the mouthwash ingredients according to the following proportions: 89.75% water, 8% glycerin, 2% propylene glycol, and 0.25% preservative. Heat the water and glycerin to 85°C to emulsify, stir well, and then cool to 45°C. Add the propylene glycol and preservative, along with 10% [unclear - possibly a specific amount of water or preservative]. 10 CFU (Chemical Fumed Lactobacillus fermentum) CCFM1139 inactivated bacterial powder is stirred until completely dissolved, aged, and left to stand to make probiotic mouthwash.

[0132] Meanwhile, as a control, the specific steps are the same as above, except that no lactobacillus fermentation is added, and a placebo mouthwash is prepared.

[0133] 2. The mouthwash prepared above is applied to patients with periodontitis, and the experimental method is as follows: 40 patients with chronic periodontitis are selected and randomly divided into 2 groups using SPSS, the placebo group is given a mouthwash without the addition of Lactobacillus fermentum CCFM1139 inactivated bacteria, and the experimental group is given a mouthwash with the addition of Lactobacillus fermentum CCFM1139 inactivated bacteria.

[0134] Before the experiment, the patients are given basic periodontal treatment by a periodontal doctor with rich clinical experience, including supragingival scaling, subgingival scaling and oral hygiene guidance, and then the volunteers use placebo mouthwash or probiotic mouthwash for 1 month, once before breakfast and once before dinner.

[0135] After all the volunteers take the probiotic mouthwash and placebo mouthwash for 1 month at the baseline period (the day before the basic periodontal treatment), the clinical attachment loss (CAL) and probing depth (PD) are measured.

[0136] The CAL measurement method is as follows: the distance from the cement-enamel junction to the bottom of the periodontal pocket is measured by an electronic pressure probe (Florida probe); the PD measurement method is as follows: the distance from the free gingival margin to the bottom of the periodontal pocket is measured by the Florida probe. The experimental results are shown in Table 3.

[0137] Table 3 Changes in clinical indicators of patients with chronic periodontitis

[0138]

[0139]

[0140] Note: * and ** indicate the changes in CAL and PD after taking the probiotic mouthwash for 1 month compared with the baseline period.

[0141] From the above experimental results, it can be seen that after the basic periodontal treatment, the volunteers taking the CCFM1139 inactivated bacteria mouthwash or placebo mouthwash, the CAL and PD of the volunteers are significantly decreased, and the degree of CAL gain and PD reduction of the volunteers using the mouthwash with the addition of Lactobacillus fermentum CCFM1139 inactivated bacteria is better than that of the volunteers using only ordinary mouthwash.

[0142] In summary, oral administration of Lactobacillus fermentum CCFM1139 inactivated bacteria can down-regulate the expression of local periodontal tissue and systemic inflammatory factors, and Lactobacillus fermentum CCFM1139 inactivated bacteria shows better effect in reducing the expression level of inflammatory factors; Lactobacillus fermentum CCFM1139 inactivated bacteria can effectively reduce the amount of alveolar bone absorption and alleviate the progression of periodontitis; and can effectively reduce the clinical attachment loss level and probing depth of volunteers with chronic periodontitis. It is proved that Lactobacillus fermentum CCFM1139 inactivated bacteria has good effect on relieving periodontitis whether in animal experiment or in population experiment.

[0143] Although the present application has been disclosed in the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. Lactobacillus fermentum ( Lactobacillus fermentum The application of inactivated bacterial cells in the preparation of products for relieving periodontitis is characterized by, The Lactobacillus fermentum is Lactobacillus fermentum GDMCC No: 61116; the periodontitis is caused by infection with Porphyromonas gingivalis and Fusobacterium nucleatum.

2. The application according to claim 1, characterized in that, The inactivation methods for the inactivated bacteria include: heat inactivation, repeated freeze-thaw inactivation, high-pressure inactivation, ultrasonic inactivation, and ultraviolet inactivation.

3. The application according to claim 2, characterized in that, The product in question is a pharmaceutical or daily chemical product.

4. The application according to claim 3, characterized in that, The drug comprises inactivated Lactobacillus fermentum GDMCC No: 61116 cells and a drug carrier.

5. The application according to claim 4, characterized in that, The drug carrier is one or more of the pharmaceutically acceptable fillers, wetting agents, disintegrants, binders, lubricants, or flavoring agents.

6. The application according to claim 5, characterized in that, The dosage forms of the medicine include granules, capsules, tablets, pills, and oral liquids.

7. The application according to claim 3, characterized in that, The aforementioned daily chemical product is a mouthwash or toothpaste containing inactivated Lactobacillus fermentum GDMCC No: 61116.

8. The application according to any one of claims 1 to 7, characterized in that, The product has any one or more of the following uses (a) to (d): (a) Reduces the level of inflammatory factors in gingival tissue; (b) Reduce alveolar bone resorption; (c) Reduce clinical attachment loss; (d) Reduce the depth of probing.

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