Application of Lactobacillus fermentum ZJUIDS06 in preventing and alleviating periodontitis

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to the application of Lactobacillus fermentum ZJUIDS06 in preventing and alleviating periodontitis. The present invention provides the application of Lactobacillus fermentum ZJUIDS06 of CGMCC NO.21339 in preparing a preparation having the efficacy of preventing and / or treating periodontitis; the preparation can simultaneously inhibit the growth of three periodontal pathogens, namely Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn).

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to the application of fermented lactobacillus ZJUIDS06 in preventing and alleviating periodontitis. Background Art

[0002] Periodontitis is an inflammatory infectious disease caused by pathogenic microorganisms. Periodontitis usually causes the dissolution and destruction of collagen fibers in the gums and periodontal membranes, resulting in alveolar bone resorption, accompanied by symptoms such as bad breath, swollen and bleeding gums, loose teeth, and chewing dysfunction. It is the primary cause of tooth loss in adults. More and more studies have confirmed that bacteria that cause periodontitis are closely related to a variety of systemic diseases, such as cardiovascular disease, diabetes, Alzheimer's disease, premature birth, etc. Periodontitis also increases the incidence of certain malignant tumors, such as lung cancer and colon cancer. As my country enters an aging society, periodontitis has become an oral problem that needs to be solved urgently.

[0003] Currently, known factors that induce periodontitis include dental plaque microorganisms, host immune inflammatory responses, and genetic factors. The main pathogens include Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), etc. Among them, the key pathogen (Pg) colonizes with the help of accessory pathogens (Fn, etc.), resulting in impaired innate host defense and the occurrence of periodontal inflammation, which leads to periodontal dysbiosis. Gingival crevicular fluid (GCF) is the result of inflammatory tissue destruction. Periodontal inflammation increases the flow of gingival crevicular fluid, thereby bringing degraded collagen and heme-containing compounds into the gingival crevice. These molecules are selectively used by proteases, causing the dysbiosis to worsen. In contrast, the healthy flora cannot take advantage of the new environmental conditions and are eliminated by competition. This imbalance exacerbates the dysbiosis, which further exacerbates inflammation and ultimately leads to periodontitis in susceptible populations.

[0004] At present, the main clinical treatments for periodontitis include subgingival scaling, PDT photodynamic antibacterial therapy, and semiconductor laser therapy. However, it is difficult for existing treatments for periodontitis to take into account both bone regeneration and inflammation suppression in the same step, and they generally have disadvantages such as long treatment cycles and high costs. The European Federation of Periodontology (EFP) published clinical guidelines for the treatment of S3-level periodontitis in 2023. The guidelines state that "you can consider using probiotics as an adjuvant treatment for PMPR under professional guidance." Probiotics as an auxiliary means of non-surgical periodontal treatment have become a current research hotspot. A large amount of clinical data has confirmed that there is potential and room for exploration. There are a number of literature and patents based on probiotics for the adjuvant treatment of periodontitis that have been publicly released. For example, CN113046258B discloses a strain of Lactobacillus rhamnosus CCFM1138, which can inhibit the formation of dual biofilms of Porphyromonas gingivalis and Fusobacterium nucleatum in vitro, and significantly reduce weight loss and periodontal damage caused by periodontitis modeling in rats; CN116763760A discloses an electrospun film combination loaded with Lactobacillus plantarum DP189 strain, which can improve the symptoms of periodontal tissue in rats; CN116024128A provides a probiotic composition, which combines Lactobacillus plantarum, Lactobacillus salivarius and Streptococcus thermophilus strain P0012 to synergistically improve the antibacterial activity against oral pathogens; CN115919903A discloses an oral film loaded with Lactobacillus paracasei LC88, which can make the lactobacillus have a higher survival rate, proliferation rate and stability in the oral cavity, adjust the oral flora to a greater extent, improve oral problems and relieve periodontitis. The query found that the probiotics used in the patents for probiotics-assisted treatment of periodontitis are mostly Lactobacillus plantarum, Lactobacillus paracasei, etc., while there are fewer studies on Lactobacillus fermentum.

[0005] However, the strains mentioned in the above patents only have the effect of inhibiting the growth of one or two periodontal pathogens and cannot achieve the purpose of treating periodontitis. There are almost no public patents for fermented Lactobacillus that can simultaneously inhibit the growth of multiple periodontitis pathogens and inhibit biofilm formation, and has a low level of biofilm formation itself.

[0006] The invention of CN112608864B, "A strain of fermented lactobacillus capable of preventing and / or treating periodontitis and its application", informs that fermented lactobacillus CCFM1139 has the effect of alleviating periodontitis, which is specifically reflected in: inhibiting the formation of mixed bacterial biofilm of Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum, reducing the amount of biofilm by 52.45%; reducing the content of TNFα from 131.37pg / mL to 83.31pg / mL, and the content of IL 8 from 147.70pg / mL to 121.12pg / mL in the periodontitis cell model; reducing the colonization of Porphyromonas gingivalis and Fusobacterium nucleatum in the oral cavity by 1 to 2 orders of magnitude; and reducing the amount of alveolar bone resorption from 1838.0μm to 805.7μm. It is not informed that fermented lactobacillus CCFM1139 has an inhibitory effect on Aggregatibacter actinomycetemcomitans (Aa).

[0007] The invention of CN117683698A, "A plant lactobacillus JYLP-376 and postbiotic agent, preparation method and application", discloses that: plant lactobacillus (lactobacillus plantarum) JYLP-376 improves recurrent oral ulcers, and the postbiotic agent of plant lactobacillus JYLP-376 has a good inhibitory effect on common oral bacteria such as oral streptococci, lactobacillus, oral Candida albicans, periodontal suspected pathogens and actinomycetemcomitans. It is not disclosed that plant lactobacillus JYLP-376 has an inhibitory effect on Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn), and it is not disclosed that it has tolerance to lysozyme.

[0008] The invention of 202110145017.6 discloses a strain of human-derived fermented Lactobacillus ZJUIDS06 (with a deposit number of CGMCCNO.21339), which has good lipid-lowering ability in vivo. The strain also has strong antioxidant and cholesterol-lowering effects, good acid and bile salt resistance, no antibiotic resistance in the culture medium, and inhibits harmful pathogens in the intestine, proving that the strain has good probiotic properties. The strain was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 9, 2020, with the deposit number: CGMCC NO.21339. Summary of the invention

[0009] The technical problem to be solved by the present invention is to provide a new use of fermented lactobacillus ZJUIDS06 (CGMCC NO.21339), that is, to provide the use of fermented lactobacillus ZJUIDS06 in preventing and alleviating periodontitis.

[0010] That is, in view of the current lack of research on the efficacy of fermented Lactobacillus in the prevention and / or treatment of periodontitis, the present invention provides a strain of fermented Lactobacillus ZJUIDS06 with the efficacy of preventing and / or treating periodontitis and its application, and investigates the effect of fermented Lactobacillus on alleviating periodontitis by detecting the antibacterial effect on oral pathogens and the inhibitory effect on the level of biofilm formation of pathogens.

[0011] In order to solve the above technical problems, the present invention provides the use of Lactobacillus fermentum ZJUIDS06 of CGMCC NO.21339 in preparing a preparation (live bacteria preparation) with the efficacy of preventing and / or treating periodontitis; that is, the use of Lactobacillus fermentum ZJUIDS06 in preparing a preparation with the efficacy of inhibiting oral pathogens.

[0012] As an improvement of the application of the present invention, the growth of three periodontal pathogens, namely Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn), can be inhibited simultaneously.

[0013] As a further improvement of the application of the present invention, the present invention has the effects of preventing and treating the periodontal pathogen biofilm formed by periodontal pathogens.

[0014] As a further improvement of the application of the present invention: preparing a product having the function of improving (preventing and / or treating) periodontitis.

[0015] As a further improvement of the application of the present invention: the preparation / product is any of the following:

[0016] Lactobacillus fermentum ZJUIDS06 is (1~2)×10 11 CFU / kg probiotic tablets;

[0017] Lactobacillus fermentum ZJUIDS06 is 10 9 ~10 10 CFU / mL of Lactobacillus fermentum ZJUIDS06 starter culture;

[0018] Lactobacillus fermentum ZJUIDS06 functional fermented milk prepared by using the Lactobacillus fermentum ZJUIDS06 starter;

[0019] Lactobacillus fermentum ZJUIDS06 powder;

[0020] The number of viable bacteria of fermented Lactobacillus ZJUIDS06 is ≥10 9 CFU / g probiotic solid beverage;

[0021] Chewing gum with live bacterial count of fermented Lactobacillus ZJUIDS06 ≥5.0x10 cfu / g.

[0022] That is, the present invention provides: the use of fermented lactobacillus for preparing probiotic tablets in food, the use of fermented lactobacillus for preparing live bacterial preparations with the efficacy of preventing and / or treating periodontitis, the use of fermented lactobacillus for preparing functional fermented milk products with the efficacy of preventing and / or treating periodontitis, the use of fermented lactobacillus for preparing bacterial powder with the efficacy of preventing and / or treating periodontitis, and the use of fermented lactobacillus for preparing probiotic solid beverages with the efficacy of preventing and / or treating periodontitis.

[0023] The invention finds that: Lactobacillus fermentum has the effect of inhibiting the growth of oral pathogenic bacteria and can also inhibit the formation of biofilm of pathogenic bacteria, which has obvious advantages over other lactic acid bacteria; Lactobacillus fermentum has obvious advantages over other lactic acid bacteria in terms of its own biofilm formation ability and will not aggravate the disorder of oral flora; the culture solution of Lactobacillus fermentum has no antibiotic resistance, indicating that the strain does not carry drug-resistant genes; Lactobacillus fermentum has lysozyme tolerance, indicating that it has strong colonization ability; Lactobacillus fermentum has high hydrophobicity and can adhere to the oral cavity to play a probiotic role; Lactobacillus fermentum has strong self-aggregation and copolymerization ability, indicating that it can prevent pathogenic bacteria from adhering to the oral cavity.

[0024] The fermented lactobacillus strain with the efficacy of preventing and / or treating periodontitis of the present invention has the function of inhibiting oral pathogens, can inhibit the formation of pathogenic bacteria biofilm, has lysozyme, has no antibiotic resistance in the culture fluid, has strong self-polymerization and copolymerization ability, and has strong hydrophobicity. Therefore, the fermented lactobacillus strain ZJUIDS06 of the present invention can be widely used in the development of probiotics and other related products.

[0025] The strain of the present invention has obvious advantages over other lactic acid bacteria in inhibiting the growth of periodontal pathogens, and can inhibit the formation of biofilm while inhibiting bacteria, making it easier to colonize in the periodontium and promote the discharge of planktonic pathogens from the oral cavity. It has no antibiotic resistance and high lysozyme tolerance. The fermented lactobacillus of the present invention can provide a basis for the development of probiotic products.

[0026] Compared with Lactobacillus fermentum CCFM1139 and Lactobacillus plantarum JYLP-376, the strain of the present invention has a more comprehensive antibacterial effect on relieving periodontitis. In addition, the present invention supplements the evaluation of the colonization effect of lactic acid bacteria in the oral cavity, has a higher tolerance to lysozyme in the oral cavity, better hydrophobicity, and is easier to colonize and play a probiotic role. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings;

[0028] Figure 1 The agar inhibition zone effect diagram of different forms of fermented Lactobacillus ZJUIDS06 on Aggregatibacterium spp.

[0029] Figure 1 Medium: 1- Lactobacillus supernatant, 2- Lactobacillus suspension, 3- Lactobacillus cell solution, 4- PBS, 5- MRS, 6- Metronidazole solution.

[0030] Figure 2 It is a mixed bacterial biofilm in a 96-well plate;

[0031] Figure 2 middle:

[0032] The upper and lower rows, from left to right, are MRS solution, blank, and ZJUIDS06 bacterial suspension; that is, the upper and lower rows have the same components and are only repeated to show the formation of biofilm.

[0033] Figure 3 This is an example of a plate showing antibiotic sensitivity of Lactobacillus fermentum ZJUIDS06;

[0034] Figure 3 Chinese: 5-Cefuroxime sodium, 6-Cefuroxime, 7-Tetracycline, 8-Erythromycin.

[0035] Figure 4 This is a comparison chart of the lysozyme tolerance of fermented Lactobacillus ZJUIDS06, MRS and metronidazole solution.

[0036] Figure 5 The graphs show the changes in the autoagglutination rate of Lactobacillus fermentum ZJUIDS06 within 24 hours, and the coagglutination changes of Lactobacillus fermentum ZJUIDS06 and Porphyromonas gingivalis;

[0037] Note: ZJUIDS06 is the self-agglutination rate of the strain, and ZJUIDS06&Pg is the co-agglutination rate of the strain and pathogenic bacteria. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0039] Example 1. Antibacterial effect test of fermented Lactobacillus ZJUIDS06 (CGMCC NO.21339) on periodontal pathogens 1.1 Preparation of lactic acid bacteria in different forms

[0040] A single colony of fermented Lactobacillus ZJUIDS06 was picked up and placed in 50 ml of MRS liquid culture medium, placed in a 37° C. incubator for aerobic culture for 18 h, and then inoculated into MRS liquid culture medium at a rate of 1% (v / v). After aerobic culture at 37° C. for 18 h, a bacterial suspension was obtained.

[0041] The bacterial suspension was centrifuged at 5000×g for 5 min at 4°C, and the supernatant was collected as the metabolic supernatant of fermented Lactobacillus ZJUIDS06.

[0042] The bacterial cells obtained by centrifugation were resuspended in PBS buffer (0.01 M, pH 7.2). The number of viable cells in the suspension was about 10 8 CFU / mL, and obtain bacterial solution.

[0043] 1.2 Preparation of pathogenic bacteria solution

[0044] Mix BHI broth powder, agar powder and distilled water in a ratio of 3.7g:2g:100ml. Autoclave at 121℃ for 15 minutes and then reduce to 50℃. Add sterile defibrinated sheep blood at a concentration of 1:100 and mix evenly by shaking. Pour onto a sterile plate and wait for it to cool and solidify to obtain BHI blood agar plate medium. Store in a 4℃ refrigerator away from light for later use.

[0045] Weigh 3.7 g of BHI broth powder, dissolve it in 100 ml of distilled water, sterilize it by high pressure at 121° C. for 15 minutes, and cool it to room temperature to prepare BHI broth medium for later use.

[0046] The three frozen standard pathogenic strains (Aggregatibacter actinomycetemcomitans NCTC9710, Fusobacterium nucleatum ATCC25586 and Porphyromonas gingivalis ATCCW83) were activated 2-3 times on BHI blood agar plate medium. The activated single colonies were picked out and placed in BHI broth medium and cultured anaerobically at 37°C for 18 hours. The bacterial cells were collected by centrifugation and resuspended in PBS buffer (0.01M). The number of viable cells in the suspension was about 10 7 CFU / mL.

[0047] Description: Porphyromonas gingivalis ATCCW83, i.e., Porphyromonas gingivalis ATCC BAA-308, W 83.

[0048] 1.3 Agar diffusion method for determination of antibacterial activity of lactic acid bacteria

[0049] The internationally accepted agar diffusion method was used to determine the antibacterial activity of lactic acid bacteria.

[0050] Add the pathogenic bacteria suspension at 1% (v / v) to the sterilized BHI blood agar plate medium cooled to about 55°C, mix well and pour into the culture dish (15mL / dish), and remove the sterile Oxford cup placed in advance after condensation. Add the prepared fermented Lactobacillus ZJUIDS06 metabolic supernatant, bacterial suspension and bacterial solution to the cup holes (200μL / hole), and use uninoculated MRS liquid culture medium (pH6.2) and PBS buffer (0.01M) as blank controls, and clinical treatment drug metronidazole solution (0.05g / 100mL) as a positive control. Culture anaerobically at 37°C, and measure the diameter of the inhibition zone after 24h. Figure 1 As shown, observe whether there is an obvious inhibition zone around the small hole and measure the diameter of the inhibition zone. Repeat three times for each.

[0051] The results are shown in Table 1. The various forms of Lactobacillus fermentum ZJUIDS06 have significant inhibitory effects on Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum. It can be seen that the different forms of this strain have antibacterial properties. Among them, the antibacterial effect of the supernatant of this strain is significantly better than that of other forms, and the difference in antibacterial effect with the positive control group is small.

[0052] Table 1 Antibacterial effect of fermented Lactobacillus ZJUIDS06 on periodontal pathogens

[0053]

[0054] Example 2. Experimental study on the preventive and therapeutic effects of fermented Lactobacillus ZJUIDS06 on biofilm formation of periodontal pathogens. Referring to the preparation method of lactic acid bacteria supernatant in 1.1 of Example 1, fermented Lactobacillus ZJUIDS06 supernatant was obtained.

[0055] Refer to the preparation method of pathogenic bacteria solution in 1.2 of Example 1 to obtain a pathogenic bacteria solution.

[0056] 2.1 Prevention trial design

[0057] 40 μL of Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans bacterial solution were added to each well of a 96-well plate, and then 80 μL of the filtered Lactobacillus supernatant was added, and the mixture was co-cultured at 37°C for 48 h to obtain a mixed pathogenic bacteria biofilm. Figure 2 As shown in the figure, the negative control group used the same volume of blank MRS liquid culture medium instead of lactobacillus supernatant.

[0058] 120 μL of Porphyromonas gingivalis, Fusobacterium nucleatum or Aggregatibacter actinomycetemcomitans bacterial solution was added to each well of a 96-well plate, followed by 80 μL of filtered Lactobacillus supernatant, and co-cultured anaerobically at 37°C for 48 h to obtain a single pathogenic bacteria biofilm. The negative control group was replaced with the same volume of blank MRS liquid culture medium instead of Lactobacillus supernatant.

[0059] 2.2 Treatment trial design

[0060] The difference from the "prevention test design" is that after adding the pathogenic bacteria solution to the 96-well plate, it is first cultured anaerobically at 37°C for 24 hours, and then 80 μL of the filtered lactobacillus supernatant is added and co-cultured anaerobically at 37°C for 24 hours. The other test steps are the same.

[0061] 2.3 Measurement of biofilm reduction by crystal violet staining

[0062] After the incubation, the biofilm was washed twice with PBS buffer (0.01M), and after the biofilm was left to dry at room temperature, 100 μL of 0.1% crystal violet aqueous solution was added to each well, and the biofilm was stained for 30 minutes. After staining, it was washed twice with PBS. 100 μL of 95% ethanol was added to dissolve it, and the OD 600 The absorbance value was read to obtain the amount of biofilm after mediation, and the reduction amount of single pathogenic bacteria / mixed pathogenic bacteria biofilm after mediation by Lactobacillus fermentum ZJUIDS06 was calculated.

[0063] The amount of biofilm reduction after mediation (%) = (the amount of biofilm in the blank control group - the amount of biofilm after mediation) / the amount of biofilm in the blank control group.

[0064] The calculation results are shown in Table 2. In both the prevention test and the treatment experiment, Lactobacillus fermentum ZJUIDS06 had an inhibitory effect on the biofilm formation of single strains / mixed strains of pathogenic bacteria, and the inhibitory effect was better in the treatment experiment.

[0065] Table 2 Inhibitory effect of fermented Lactobacillus ZJUIDS06 on biofilm formation (biofilm reduction after mediation)

[0066]

[0067] Example 3: Confirmation of the biofilm-forming ability of Lactobacillus fermentum ZJUIDS06

[0068] In order to ensure that probiotics do not promote the formation of oral biofilm in the oral environment, their own biofilm formation ability should be as low as possible. Therefore, the crystal violet staining method in Example 2 was used to determine the biofilm formation ability of lactic acid bacteria. The number of viable cells was about 10 8200 μL of the bacterial solution with CFU / mL was added to a 96-well plate and cultured at 37°C for 24 h. After the culture, the biofilm was washed twice with PBS, and after natural air drying, 100 μL of 0.1% crystal violet solution was added to each well for staining for 30 min. After washing with PBS to remove the floating color, 100 μL of 95% ethanol was added to dissolve it, and the OD was read by an enzyme reader. 600 Absorbance value. Fusobacterium nucleatum, which has a strong film-forming ability, was used as the control bacteria.

[0069] The results are shown in Table 3. The film-forming ability of Lactobacillus fermentum ZJUIDS06 is significantly lower than that of Fusobacterium nucleatum, and its ability to form biofilm itself is low, indicating that it may not promote the formation of biofilm when acting in the oral cavity.

[0070] Table 3 Biofilm formation ability of Lactobacillus fermentum ZJUIDS06

[0071]

[0072] Example 4: Confirmation of antibiotic sensitivity of Lactobacillus fermentum ZJUIDS06

[0073] According to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI), the paper diffusion method was used to detect the antibiotic sensitivity of the strain. After centrifugation and washing of the bacteria, the concentration of the bacterial suspension was controlled to reach 10 8 CFU / mL. Take 100μL of bacterial solution and evenly spread it on MRS solid culture medium. Then use sterile tweezers to gently press the antibiotic paper against the surface of the plate and place it in a 37℃ incubator for 24 hours. Use a vernier caliper to measure and record the diameter of the inhibition zone. The antibiotic sensitivity of the strain can be determined based on the diameter of the inhibition zone. Figure 3 According to the "Antimicrobial Susceptibility Test Implementation Standard" (17th Edition) formulated by CLSI, the resistance of lactic acid bacteria to antibiotics is determined and divided into three levels: sensitive (S), intermediate (I) and resistant (R).

[0074] The diameter of the inhibition zone of the sensitivity of Lactobacillus fermentum ZJUIDS06 to antibiotics is shown in Table 4. According to the CLSI (2017) drug sensitivity test standard, Lactobacillus fermentum ZJUIDS06 is sensitive to cephalexin, ampicillin, cefuroxime sodium, ceftazidime, tetracycline, azithromycin, lincomycin and cephradine. It is moderately sensitive to neomycin. The experimental results show that Lactobacillus fermentum ZJUIDS06 is sensitive to common antibiotics and has good safety.

[0075] Table 4 Results of antibiotic sensitivity of Lactobacillus fermentum ZJUIDS06

[0076]

[0077] Note: S, sensitive; I, intermediate; R, resistant

[0078] With the widespread use of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria is becoming more and more serious. Long-term intake of drug-resistant lactic acid bacteria will bring great difficulties to clinical treatment. The fermented lactobacillus ZJUIDS06 provided by the present invention is sensitive to common antibiotics and will not cause harm to human health.

[0079] Example 5: Confirmation of lysozyme tolerance of Lactobacillus fermentum ZJUIDS06

[0080] The lysozyme in saliva has a strong dissolving effect on the acetylamino polysaccharide components on the wall of Gram-positive cocci, thereby causing the death of bacteria. By measuring the tolerance of Lactobacillus fermentum ZJUIDS16 to lysozyme, it is possible to judge whether the strain has the ability to survive when applied to the oral cavity. Lysozyme was purchased from Sangon Biotechnology (Shanghai) Co., Ltd. with a CAS number of 12650-88-3. The tolerance of Lactobacillus fermentum ZJUIDS16 to lysozyme was investigated by spectrophotometry. Referring to the preparation method of the lactobacillus cell solution in Example 1, the lactobacillus cell solution was inoculated into a 96-well plate, and different concentrations of lysozyme MRS solution were added respectively so that the final concentration of lysozyme in each well was 0.4, 0.8, 1.2, 1.6, 2.0, and 3.0 mg / mL, respectively. The blank group was replaced by MRS, and the positive control group was replaced by metronidazole solution. After culturing at 37°C for 24 hours at OD 600 Measure the absorbance.

[0081] like Figure 4 As shown in the figure, the growth of fermented Lactobacillus ZJUIDS06 is basically unaffected when the lysozyme concentration is 0-3.0 mg / mL, and the maximum tolerance concentration range is higher than 60%. The tolerance of the blank group and the positive control group is far less than that of fermented Lactobacillus ZJUIDS06. It is inferred that the critical tolerance value of ZJUIDS06 to lysozyme is higher than 3.0 mg / mL, which is much higher than the lysozyme concentration of 1-57 μg / mL in the human oral cavity. This shows that fermented Lactobacillus ZJUIDS06 has good survival ability in the oral cavity.

[0082] Example 6: Verification of in vitro hydrophobicity and acid-base charge of Lactobacillus fermentum ZJUIDS06

[0083] The surface properties of bacteria, such as surface hydrophobicity and surface acid-base charge, are related to the nonspecific adhesion between strains and between strains and the host. The higher the surface hydrophobicity of the strain, the stronger the adhesion to oral epithelial cells, which is more conducive to the colonization of beneficial strains in the oral cavity.

[0084] The hydrophobicity of fermented Lactobacillus ZJUIDS06 has been reported in 202110145017.6. The comparison of its acid charge and base charge with the patented strain (patent number ZL201711410714.X) plantarum Lactobacillus ZY08 is as follows:

[0085] Table 5. Surface properties of fermented Lactobacillus ZJUIDS06

[0086]

[0087] This indicated that Lactobacillus fermentum ZJUIDS06 had a strong colonization ability.

[0088] Example 7: Verification of the self-polymerization and copolymerization ability of Lactobacillus fermentum ZJUIDS06

[0089] Lactobacillus self-aggregates and uses the extracellular substances it produces to form a protective film, which protects the strain against environmental changes and helps colonization. Studies have shown that lactobacillus with strong self-aggregation ability usually have a strong ability to adhere to epithelial cells. The copolymerization ability of lactobacillus with pathogenic bacteria is the key to the oral probiotic properties of lactobacillus. Studies have reported that the copolymerization ability of lactic acid bacteria with pathogenic bacteria is related to its ability to inhibit the formation of pathogenic biofilms. After entering the mouth, probiotics can copolymerize with planktonic pathogenic bacteria strains, and the copolymers formed are easily discharged from the mouth with the flow of saliva, thereby achieving the purpose of inhibiting pathogenic bacteria in the mouth.

[0090] The self-aggregation ability of the target bacteria was determined by spectrophotometry. 8 CFU / mL) was vortexed for 10 seconds and allowed to stand at 37°C for 24 hours. 1 mL of the top layer of solution was taken every 2 hours and the OD 600 The self-aggregation capacity is determined by measuring the residual absorbance and is expressed by the following formula:

[0091] Ac(%)=(1-A xh / A0)×100

[0092] Where: Ac is the self-aggregation rate; A0 is the initial OD 600 Value; A xh is the OD of the upper lactic acid bacteria solution after static incubation for xh 600 value.

[0093] The copolymerization ability of fermented Lactobacillus ZJUIDS06 and Porphyromonas gingivalis was determined by spectrophotometry. 8 CFU / mL) and Porphyromonas gingivalis bacterial solution (10 7 CFU / mL) and vortex for 10 seconds to measure the initial OD 600The mixed bacterial solution was placed at 37°C for 24 hours and the OD value was measured every 2 hours. 600 The copolymerization ability is expressed by the following formula:

[0094] OD(%)=(1-OD xh / OD0)×100

[0095] Where: OD is the coaggregation rate; OD0 is the initial OD 600 Value; OD xh is the OD of the mixed bacterial solution in the upper layer after static incubation for xh 600 value.

[0096] Figure 5 The results showed that ZJUIDS06 had a certain self-aggregation performance. After 8 hours of cultivation, the self-aggregation and copolymerization abilities of the fermented Lactobacillus ZJUIDS06 exceeded 20%, indicating that the strain had strong self-aggregation and copolymerization abilities, strong colonization ability in the oral cavity, and was not easy to form biofilms to cause oral diseases. In addition, the fermented Lactobacillus ZJUIDS06 had a strong copolymerization ability with Porphyromonas gingivalis, which may play a role in preventing pathogenic bacteria from adhering to and colonizing the oral cavity, and had a high development potential.

[0097] Example 8: Preparation of probiotic tablets using Lactobacillus fermentum ZJUIDS06

[0098] 1. Processing process of probiotic tablets:

[0099] Raw materials → weighing → screening → premixing → dry mixing → tableting → packaging → storage;

[0100] 2. Operation points

[0101] 1) Ingredients: Weigh the raw materials according to the weight components and mix them evenly; in this embodiment, the weight of each kilogram (kg) of tablets is: the ratio of each active probiotic and the total number of live bacteria are: Lactobacillus fermentum: 3.0×10 12 CFU; fructooligosaccharide 400g, xylo-oligosaccharide 40g, the balance is tablet excipients (microcrystalline cellulose and magnesium stearate), base powder water activity (Aw) 0.15-0.18;

[0102] 2) Sieve: the ingredients are sieved through a 20-mesh sieve;

[0103] 3) Premixing of probiotic powder and prebiotics (fructoligosaccharides, xylo-oligosaccharides);

[0104] 4) Add the remaining ingredients and dry mix for 25 to 30 minutes;

[0105] 5) Tableting: The tablet press pressure was adjusted to 55 kN for tableting; the obtained tablets had a diameter of 15 to 25 mm and a tablet weight of about 0.75 g;

[0106] 6) Packaging: PVDC film, aluminum foil film or aluminum foil bag to make probiotic tablets. Ensure cold chain configuration (2-8°C) during transportation and sales.

[0107] After testing, the survival rate of Lactobacillus fermentum in the tablets obtained in this example was about 1.2×10 11 CFU / kg.

[0108] Example 9: Preparation of a fermented Lactobacillus ZJUIDS06 starter culture having the efficacy of preventing and / or treating periodontitis

[0109] The original strain of fermented Lactobacillus ZJUIDS06 was inoculated into 11% skim milk (sterilized at 115℃ for 20min), cultured aerobically at 37℃ for 18-24h until curdling, and activated for two generations continuously as the mother starter. The mother starter was inoculated into 11% skim milk (sterilized at 115℃ for 20min) at a rate of 3%-5%, and cultured at 37℃ for 18-24h until curdling, at which time the number of viable bacteria can reach 10 9 -10 10 CFU / mL, and the fermented Lactobacillus ZJUIDS06 starter culture was obtained.

[0110] Example 10: Preparation of functional fermented milk using Lactobacillus fermentum ZJUIDS06 with the efficacy of preventing and / or treating periodontitis

[0111] The conventionally pretreated milk was heated to about 60°C, 6% sucrose was added, and after being fully dissolved, homogenized under a pressure of 20MPa. Then, after heat treatment at 95°C for 5 minutes, the milk was cooled to 38°C. The fermented Lactobacillus ZJUIDS06 starter prepared according to Example 9 was added at a 5% inoculum amount, and fermented at 37°C for 14-18 hours until the milk curdled. After cooling, the milk was refrigerated at 4°C to obtain the functional fermented milk of Lactobacillus ZJUIDS06.

[0112] Example 11: Preparation of bacterial powder using Lactobacillus fermentum ZJUIDS06 with efficacy in preventing and / or treating periodontitis

[0113] Use an inoculation loop to inoculate the fermented lactobacillus ZJUIDS06 into 50mL MRS liquid culture medium, and place it in a 37°C incubator for 18 hours. Then transfer it to a sterilized 250mL MRS liquid culture medium with a 5% inoculation amount. Place it in a bacterial incubator at 37°C and culture it for 24 hours. Centrifuge the resulting fermented liquid, then discard the supernatant to collect the bacterial precipitate, rinse the bacterial body twice with sterile phosphate buffer (PBS), each time for 10 minutes, and the centrifugation speed is 5000r / min. The fermented lactobacillus ZJUIDS06 bacterial precipitate can be obtained.

[0114] Weigh 4.1g of trehalose, 2.8g of sodium glutamate, and 8g of sucrose respectively and dissolve them in 10mL of 40℃ distilled water, filter and sterilize them with a 0.22um microporous filter membrane, then add them to 90mL of sterile water for later use. Dissolve 15g of skim milk powder in distilled water to obtain 100mL of 15% skim milk powder solution, sterilize at 110℃ for later use.

[0115] The fermented Lactobacillus ZJUIDS06 bacterial precipitate prepared above was fully mixed with the protective agent solution at a ratio of 1g:5ml to obtain a bacterial suspension. 5mL of the bacterial suspension was divided into 10mL sterile stoppered glass bottles, pre-frozen at -70°C, taken out after 2 hours and freeze-dried in a vacuum freeze dryer, the freeze-drying conditions were vacuum degree 5Pa, partition heating temperature 20°C, cold trap temperature -55°C, and freeze-dried for 30 hours and then crushed to obtain the fermented Lactobacillus ZJUIDS06 bacterial powder.

[0116] Example 12: Preparation of probiotic solid beverage using fermented Lactobacillus ZJUIDS06 with efficacy in preventing and / or treating periodontitis

[0117] The steps of preparing probiotic solid beverage by fermenting Lactobacillus ZJUIDS06 are as follows:

[0118] (1) Preparation of probiotic powder: Prepare probiotic powder of Lactobacillus fermentum ZJUIDS06 with reference to Example 11

[0119] (2) The formula of solid beverage is as follows: edible corn starch (balance), white sugar (10%), oligofructose

[0120] (6%), isomaltooligosaccharide (9%), citric acid, edible flavor, probiotic powder (2-5%).

[0121] (3) Add according to the formula and mix evenly to ensure that the number of viable bacteria of fermented Lactobacillus ZJUIDS06 is 10 9 CFU / g and above.

[0122] (4) Each small package is packaged according to 10 / 20g, and 10-20 small packages are packaged into a large package.

[0123] Example 13: Preparation of probiotic chewing gum using fermented Lactobacillus ZJUIDS06 bacterial powder

[0124] The purpose of this embodiment is to provide a plate-type chewing gum containing highly active coagulant and fermented lactobacillus, which is composed of the fermented lactobacillus ZJUIDS06 powder described in Example 11 and gum matrix, powdered sugar, maltose, calcium carbonate, glucose, flavors and other ingredients. Except for the gum matrix and the powder, the necessary components in the preparation process of the plate-type chewing gum are all auxiliary materials, which are the same as in the prior art. The formula includes the following components in weight percentage: 0.1% coagulant and fermented lactobacillus ZJUIDS06 powder, 29.9% gum matrix, and 70% sugar and other ingredients.

[0125] (1) Preparation of resin matrix: Vinyl acetate, ester gum, butyl phthalate and chicle gum are prepared into chewing gum matrix. When preparing, each raw material is put into a kneader, and emulsifier and plasticizer are added at the same time. The kneading temperature is strictly controlled within the range of 80-120°C to form a whole material;

[0126] (2) Mixing: Using a mixer, add the heated and dissolved chewing gum base first, then sugar, glucose, maltose, flavorings, etc., and stir evenly in a certain order;

[0127] (3) Forming and packaging: The mixed raw materials have a uniform texture and a moisture content of 2.1%. They are cut into rectangular pieces of 8 x 25 x 5 cm by a dicing machine, and then loaded into an extruder for extrusion. After extrusion, they are pressed into sheets of a certain thickness by a multi-stage roller machine, and then cut into appropriate shapes by a cutting machine. They are then kept in a conditioning room at a temperature of 21 to 24°C and a relative humidity of 45 to 50% for 6 hours before packaging.

[0128] (4) The number of live bacteria in the plate-type chewing gum containing highly active Bacillus coagulans provided in this embodiment is 5.0×10 cfu / g. The plate-type chewing gum containing highly active Bacillus coagulans provided in this embodiment has the health functions of cleaning the oral cavity, refreshing breath, antagonizing Streptococcus mutans, and preventing dental caries.

[0129] Example 14: Preparation of probiotic mouthwash using Lactobacillus fermentum ZJUIDS06 with efficacy in preventing and / or treating periodontitis

[0130] A probiotic mouthwash composition having the effect of preventing and / or treating periodontitis, comprising the following raw materials in parts by weight:

[0131] Phase A: water 87.96%, glycerol 8%, sucralose 0.03%, cetylpyridinium chloride 0.05%;

[0132] Phase B: propylene glycol 2%, preservative 0.25%, methyl diisopropyl propionamide 0.05%, fragrance 0.05%;

[0133] Phase C: 1% Japanese evening cherry blossom extract, 0.5% fermentation supernatant of Lactobacillus fermentum ZJUIDS06, 0.1% xylitol, and 0.01% glucose oxidation.

[0134] The aromatic agent is essence, and the preservative is PEG-40 hydrogenated castor oil.

[0135] A method for preparing a probiotic mouthwash composition having the efficacy of preventing and / or treating periodontitis comprises the following steps:

[0136] Put phase A into the emulsifying pot, heat to 80-85℃, stir evenly, and cool down; pre-mix phase B, stir and dissolve evenly, and set aside; cool phase A in the emulsifying pot to 45℃, put phase B into the emulsifying pot, and stir until completely dissolved; add raw materials of phase C into the emulsifying pot, and stir until completely dissolved; inspect the discharged material, let it age and stand still, fill and package after passing the inspection, and put it into storage after passing the inspection.

[0137] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of Lactobacillus fermentum ZJUIDS06 in the preparation of a preparation having the efficacy of preventing and / or treating periodontitis; The periodontitis is caused by three periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum. The preparation is fermented lactobacillus ZJUIDS06 probiotic tablets, fermented lactobacillus ZJUIDS06 starter, and fermented lactobacillus ZJUIDS06 bacterial powder prepared by using fermented lactobacillus ZJUIDS06.

2. The use according to claim 1, characterized in that: The number of viable bacteria in the fermented Lactobacillus ZJUIDS06 probiotic tablets is (1-2)×10 11 CFU / kg; The number of viable bacteria in the fermentation culture of Lactobacillus fermentum ZJUIDS06 is 10 9 ~10 10 CFU / mL.

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