Application of Lactobacillus casei GG002 and its bacteriocin solution, lyophilized powder and postbiotics in the treatment of oral ulcers

Abstract

The present invention provides Lactobacillus casei ( Lacticaseibacillus casei ) Application of GG002 and its bacteriocin solution, lyophilized powder and postbiotics in treating oral ulcers, belonging to the field of microbial technology. The present invention finds that Lactobacillus casei GG002 can inhibit Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, and has the effect of treating oral ulcers. The present invention further provides a bacteriocin solution, lyophilized powder and postbiotics of Lactobacillus casei GG002, which can effectively treat oral ulcers.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Lactobacillus casei GG002 and bacteriocin solution, lyophilized powder and postbiotics thereof in treating oral ulcers. Background Art

[0002] Oral ulcers, also known as "mouth sores", are a common oral mucosal disease that often appears on the mucosa of the lips, palate, tongue and cheeks in the mouth. They are round or oval in shape, with a white or yellow center, slightly red and swollen surrounding areas, and accompanied by a clear sense of pain. Various factors such as genetics, diet, and decreased immunity may induce the development of oral ulcers, which are characterized by periodicity, recurrence, and self-limitation. The treatment measures for oral ulcers are mainly local anti-inflammatory, pain relief, and promotion of ulcer healing. Systemic treatments are mostly to extend the interval period. Treatment is based on the severity of the ulcer and the patient's wishes. The main methods include local drug therapy, systemic drug therapy, and physical therapy, such as the use of watermelon frost, laser irradiation, etc.

[0003] As a kind of microorganisms that live in the human intestine, probiotics are increasingly attracting widespread attention from researchers in this field due to their relative safety and diverse efficacy. Clinical studies have shown that probiotics play a pivotal role in preventing or reducing oral diseases. Lacticaseibacillus casei ) GG002 is a probiotic isolated and purified from yogurt, and is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC for short), with the deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, deposit number: CGMCC No: 29392, deposit date December 25, 2023. Lactobacillus casei GG002 has significant whitening and anti-freckle and anti-aging effects, and has high safety, but it has not yet been used in the study of oral ulcers. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the use of Lactobacillus casei GG002 (CGMCC No. 29392) and related preparations thereof in the preparation of a medicament for treating oral ulcers. The strain can inhibit Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, and effectively treat oral ulcers.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides Lactobacillus casei ( Lacticaseibacillus casei ) Use of GG002 or a preparation related to Lactobacillus casei GG002 in the preparation of a medicament for treating oral ulcers, wherein the preservation number of the Lactobacillus casei GG002 is CGMCC No.29392.

[0007] Preferably, the Lactobacillus casei GG002 inhibits Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis.

[0008] Preferably, the Lactobacillus casei GG002-related preparation includes any one or more of a Lactobacillus casei GG002 bacteriocin solution, a Lactobacillus casei GG002 freeze-dried powder, and a Lactobacillus casei GG002 postbiotic.

[0009] Preferably, the method for preparing the Lactobacillus casei GG002 bacteriocin solution comprises the following steps: fermenting and culturing Lactobacillus casei GG002, centrifuging the fermentation broth to obtain a fermentation supernatant, adding ammonium sulfate to the fermentation supernatant, stirring and centrifuging to obtain a precipitate, and drying and re-dissolving the precipitate to obtain a bacteriocin solution.

[0010] Preferably, the preparation method of the freeze-dried powder of Lactobacillus casei GG002 comprises the following steps: fermenting and culturing Lactobacillus casei GG002 with a fermentation medium, centrifuging the fermentation liquid, mixing the bacterial mud with a freeze-drying protective agent, and freeze-drying to obtain the freeze-dried powder of Lactobacillus casei GG002; the fermentation medium comprises a basal medium and a drug stock solution; the drug stock solution is obtained by decocting houttuynia cordata, purslane, dandelion and honeysuckle.

[0011] Preferably, the drug stock solution is 10-30wt% of the basal culture medium.

[0012] Preferably, the lyoprotectant comprises, by weight: 8-12 parts of skim milk, 6-10 parts of trehalose, 4-6 parts of sucrose, 1-3 parts of sorbitol, 0.05-0.15 parts of vitamin C, 0.05-0.15 parts of sodium glutamate, 0.2-0.8 parts of sodium alginate, 15-25 parts of drug stock solution, and 70-90 parts of water.

[0013] Preferably, the preparation method of the Lactobacillus casei GG002 postbiotic comprises the following steps: Lactobacillus casei GG002 is fermented and cultured using a postbiotic culture medium, and the fermentation liquid is spray-dried to obtain the Lactobacillus casei GG002 postbiotic; the postbiotic culture medium comprises skim milk, sucrose, yeast extract, a drug stock solution and water; the drug stock solution is obtained by decocting houttuynia cordata, purslane, dandelion and honeysuckle.

[0014] Preferably, the postbiotic culture medium comprises, by weight, 80-120 parts of skim milk, 50-70 parts of sucrose, 140-160 parts of yeast extract, 180-220 parts of drug stock solution and 480-500 parts of water.

[0015] Preferably, in the drug stock solution, the mass ratio of Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle is 30-40:10-20:15-25:5-15.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The Lactobacillus casei GG002 and related preparations thereof of the present invention can be used to prepare medicines for treating oral ulcers. The Lactobacillus casei GG002 bacteriocin solution, Lactobacillus casei GG002 freeze-dried powder or Lactobacillus casei GG002 postbiotics provided by the present invention can effectively inhibit Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, can significantly reduce the ulcer area, accelerate the healing of oral ulcers, significantly reduce the MDA and CAT levels in serum, and have high safety and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Detection results of SOD, MDA, GSH-Px and CAT levels in rat serum.

[0019] Biological Deposit Description

[0020] Lactobacillus casei GG002, classified as Lactobacillus casei ( Lacticaseibacillus casei ), depository: China General Microbiology Center of Microbiological Culture Collection (CGMCC for short), depository address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No: 29392, deposit date December 25, 2023. DETAILED DESCRIPTION

[0021] The present invention provides Lactobacillus casei ( Lacticaseibacillus casei ) Application of GG002 in the preparation of a drug for treating oral ulcers, wherein the deposit number of the Lactobacillus casei GG002 is CGMCC No. 29392. The Lactobacillus casei GG002 of the present invention can effectively inhibit Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, significantly reduce the ulcer area, accelerate the healing of oral ulcers, and significantly reduce the levels of MDA and CAT in serum.

[0022] The present invention also provides Lactobacillus casei ( Lacticaseibacillus casei ) Use of a GG002-related preparation in the preparation of a drug for treating oral ulcers, wherein the Lactobacillus casei GG002-related preparation comprises any one or more of a Lactobacillus casei GG002 bacteriocin solution, a Lactobacillus casei GG002 freeze-dried powder, and a Lactobacillus casei GG002 postbiotic.

[0023] The preparation method of the Lactobacillus casei GG002 bacteriocin solution of the invention comprises the following steps: fermenting and culturing the Lactobacillus casei GG002, centrifuging the fermentation liquid to obtain a fermentation supernatant, adding ammonium sulfate to the fermentation supernatant, stirring and centrifuging to obtain a precipitate, and drying and re-dissolving the precipitate to obtain a bacteriocin solution.

[0024] The fermentation culture of Lactobacillus casei GG002 of the present invention is preferably MRS medium. As an optional embodiment, the present invention first inoculates Lactobacillus casei GG002 in MRS solid medium for activation, and inoculates the activated Lactobacillus casei GG002 in MRS medium for expansion culture and fermentation culture, and the fermentation culture liquid is used for bacteriocin extraction. The fermentation culture temperature of Lactobacillus casei GG002 of the present invention is 36-38°C, preferably 37°C; the fermentation culture time is 15-20h, preferably 17-18h. The present invention centrifuges the fermentation broth to obtain a fermentation supernatant, and the centrifugation conditions are preferably 7000-9000rmp, 15-25min; the centrifugation speed is more preferably 8000rmp, and the centrifugation time is more preferably 20min.

[0025] The present invention preferably adds ammonium sulfate to the fermentation supernatant to a saturation of 65-75%, more preferably 70%. The present invention preferably adds ammonium sulfate and magnetically stirs overnight at low temperature (4°C), and centrifuges to discard the supernatant. The centrifugation conditions are preferably 7000-9000rmp, 10-20min, the centrifugation speed is more preferably 8000rmp, and the centrifugation time is more preferably 15min. The present invention preferably dries the precipitate at 38-42°C, and after drying, re-dissolves it in ultrapure water to obtain a bacteriocin solution. The present invention preferably uses a sterilizing filter to sterilize the obtained bacteriocin solution, and the pore size of the sterilizing filter is preferably 0.22μm.

[0026] The Lactobacillus casei GG002 bacteriocin solution prepared by the invention can effectively inhibit the growth of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis.

[0027] The preparation method of the freeze-dried powder of Lactobacillus casei GG002 of the present invention comprises the following steps: fermenting and culturing Lactobacillus casei GG002 with a fermentation medium, centrifuging the fermentation liquid, mixing the bacterial mud with a freeze-drying protective agent, and freeze-drying to obtain the freeze-dried powder of Lactobacillus casei GG002. The fermentation and culturing conditions of the present invention are preferably 36-38°C static culture for 16-20h centrifugation to obtain the bacterial mud. The centrifugation conditions of the present invention are preferably 7000-9000rmp, 10-20min, the centrifugation speed is more preferably 8000rmp, and the centrifugation time is more preferably 15min. The fermentation medium of the present invention comprises a basal culture medium and a drug stock solution; the basal culture medium is an MRS culture medium; the drug stock solution is obtained by decocting houttuynia cordata, purslane, dandelion and honeysuckle.

[0028] The drug stock solution of the present invention is 10-30wt% of the basal culture medium, preferably 15-25wt%, more preferably 20wt%.

[0029] The mass ratio of the bacterial mud to the freeze-drying protective agent of the present invention is 1:3-5, preferably 1:4.

[0030] The lyophilization protective agent of the present invention comprises (by mass): 8-12 parts of skim milk, 6-10 parts of trehalose, 4-6 parts of sucrose, 1-3 parts of sorbitol, 0.05-0.15 parts of vitamin C, 0.05-0.15 parts of sodium glutamate, 0.2-0.8 parts of sodium alginate, 15-25 parts of drug stock solution, and 70-90 parts of water, preferably 10 parts of skim milk, 8 parts of trehalose, 5 parts of sucrose, 2 parts of sorbitol, 0.1 parts of vitamin C, 0.1 parts of sodium glutamate, 0.5 parts of sodium alginate, 20 parts of drug stock solution, and 80 parts of water.

[0031] As an optional embodiment, the freeze-drying conditions of the present invention are preferably pre-freezing at -40°C for 4 hours and freeze-drying at a cold trap temperature of -70°C for 72 hours to obtain freeze-dried powder of Lactobacillus casei GG002.

[0032] The preparation method of Lactobacillus casei GG002 postbiotics of the present invention comprises the following steps: Lactobacillus casei GG002 is fermented and cultured in a postbiotic culture medium, and the fermentation liquid is spray-dried to obtain the Lactobacillus casei GG002 postbiotics.

[0033] By mass, the postbiotic medium of the present invention comprises 80-120 parts of skim milk, 50-70 parts of sucrose, 140-160 parts of yeast extract, 180-220 parts of drug stock solution and 480-500 parts of water. Preferably, the postbiotic medium comprises 100 parts of skim milk, 60 parts of sucrose, 150 parts of yeast extract, 200 parts of drug stock solution and 490 parts of water; the postbiotic medium of the present invention is sterilized at 75-85°C for 20-35 minutes before use, the sterilization temperature is preferably 80°C, and the sterilization time is preferably 25-30 minutes. The drug stock solution of the present invention is obtained by decocting Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle.

[0034] The drying air inlet temperature of the spray drying of the present invention is 160-180° C., preferably 170° C.; the feed rate is 10-11 mL / min, preferably 10.4 mL / min.

[0035] In the drug stock solution of the present invention, the mass ratio of Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle is 30-40:10-20:15-25:5-15, preferably 35:15:20:10. The preparation method of the drug stock solution of the present invention comprises: adding 2-4 times the volume of water to the raw material for soaking and decocting to obtain a first drug liquid and a drug residue; based on the volume of the raw material, the drug residue is soaked and decocted with 2-4 times the volume of water to obtain a second drug liquid and a drug residue; the first drug liquid and the second drug liquid are mixed and decocted to obtain a drug stock solution. The amount of water added each time in the present invention is preferably 3 times the volume of the raw material. The soaking time in the present invention is preferably 1-3h, more preferably 2h. Each decoction in the present invention is preferably boiled over high heat, and then decocted over low heat. Each decoction in the present invention is preferably decocted to the same mass as the raw material and then stopped. When preparing the drug stock solution, the present invention can use houttuynia cordata, purslane, dandelion and honeysuckle as raw materials respectively, and separately prepare houttuynia cordata liquid, purslane liquid, dandelion liquid and honeysuckle liquid, and then mix the four to obtain the drug stock solution; or use a mixture of houttuynia cordata, purslane, dandelion and honeysuckle as raw materials to directly prepare the drug stock solution.

[0036] The present invention preferably prepares 30-50 g of Lactobacillus casei GG002 freeze-dried powder per liter of Lactobacillus casei GG002 fermentation liquid, and the viable cell count of the Lactobacillus casei GG002 freeze-dried powder is 1×10 11 cfu / g~9×10 11 The Lactobacillus casei GG002 freeze-dried powder can effectively inhibit the growth of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, significantly reduce the ulcer area, accelerate the healing of oral ulcers, and significantly reduce the levels of MDA and CAT in serum.

[0037] The present invention preferably prepares 200-400g of Lactobacillus casei GG002 postbiotics per liter of Lactobacillus casei GG002 fermentation liquid. The Lactobacillus casei GG002 postbiotics of the present invention can effectively inhibit the growth of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, significantly reduce the ulcer area, accelerate the healing of oral ulcers, and significantly reduce the MDA and CAT levels in serum.

[0038] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] In the embodiment of the present invention, MRS liquid medium: peptone 10.0g / L, beef extract powder 5.0g / L, yeast extract powder 4.0g / L, glucose 20.0g / L, sodium acetate 5.0g / L, dipotassium hydrogen phosphate 2.0g / L, triammonium citrate 2.0g / L, manganese sulfate 0.05g / L, magnesium sulfate 0.2g / L, Tween 80 1.0g / L. BHI liquid medium: tryptone 10g, brain heart extract powder 17.5g, disodium hydrogen phosphate 2.5g, sodium chloride 5g, glucose 2g, purified water 1000g. BHI solid medium: tryptone 10g, brain heart extract powder 17.5g, disodium hydrogen phosphate 2.5g, sodium chloride 5g, glucose 2g, agar 15g, purified water 1000g.

[0040] In the embodiment of the present invention, the strains used are: Lactobacillus casei GG002 (CGMCC No. 29392), Lactobacillus casei BGP93 (purchased from Sacco, Italy), Lactobacillus paracasei LPC-37 (purchased from Danisco, Denmark), Lactobacillus rhamnosus HN001 (purchased from DuPont, USA), Lactobacillus acidophilus LA-5 (purchased from Chr. Hansen, Denmark), Streptococcus mutans (purchased from Beina Biological Co., Ltd., No. BNCC336931), Fusobacterium nucleatum (purchased from Beina Biological Co., Ltd., No. BNCC280188) and Porphyromonas gingivalis (purchased from Beina Biological Co., Ltd., No. BNCC353909).

[0041] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0042] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0043] Example 1

[0044] Antibacterial experiment of five probiotics against Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis

[0045] 1. Experimental purpose:

[0046] Lactobacillus casei GG002, Lactobacillus casei BGP93, Lactobacillus paracasei LPC-37, Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus LA-5 were used to carry out antibacterial experiments on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis to detect the antibacterial activities of the five probiotics.

[0047] 2. Test methods:

[0048] (1) Probiotic culture: Five probiotic strains were inoculated into sterilized MRS medium at 3% (v / v), cultured at 37°C for 18 h, and then stored at 4°C for use.

[0049] (2) Culture of mutans Streptococcus: 3% (v / v) of mutans Streptococcus was inoculated into BHI liquid culture medium and cultured at 37°C for 18 h. The culture medium was then stored at 4°C until use.

[0050] (3) Cultivation of Fusobacterium nucleatum and Porphyromonas gingivalis: Fusobacterium nucleatum and Porphyromonas gingivalis were inoculated into BHI liquid culture medium at 3% (v / v), cultured anaerobically at 37°C for 18 h, and then stored at 4°C for use.

[0051] (4) A sterilized Oxford cup was placed in a sterile plate. 1 ml of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were added to 100 ml of BHI solid culture medium at about 45 °C, and the mixture was immediately mixed and poured into the plate. After solidification, the Oxford cup was removed and 200 μL of the five probiotic fermentation broths were added to each well. The Streptococcus mutans plate was incubated at 37 °C for 72 h, and the Fusobacterium nucleatum and Porphyromonas gingivalis plates were incubated at 37 °C for 72 h. The results were then tested. A blank MRS culture medium was added to the control group. The antibacterial results are shown in Table 1.

[0052] Table 1 Inhibitory effects of five probiotic strains on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis

[0053]

[0054] Note: “*” indicates comparison with the control group. P <0.05.

[0055] It can be seen from the above table that Lactobacillus casei GG002 has an inhibitory effect on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, and the inhibitory effect is significant; Lactobacillus casei BGP93, Lactobacillus paracasei LPC-37, Lactobacillus rhamnosus HN001, and Lactobacillus acidophilus LA-5 have an inhibitory effect on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, but the effect is not significant compared with Lactobacillus casei GG002.

[0056] Example 2

[0057] Preparation method of Lactobacillus casei GG002 bacteriocin solution:

[0058] (1) Activation: Take the glycerol cryopreserved tube of Lactobacillus casei GG002 stored in a -80°C refrigerator and thaw it at room temperature. Then, use an inoculation loop to pick the bacteria under a sterile environment, streak inoculate on an MRS solid culture medium plate, and culture anaerobically at 37°C for 40 h. Observe the morphology of the colonies on the plate to confirm that they are Lactobacillus casei and are free of contamination.

[0059] (2) Seed culture: In a clean bench, pick a single colony from the activated MRS solid culture medium plate and inoculate it into MRS culture medium. Incubate it at 37°C for 15 h to obtain the first-level seed solution. Then, inoculate the first-level seed solution into fresh MRS culture medium at a 3.0% (v / v) inoculation rate and incubate it at 37°C for 14 h. When the OD 600 When the concentration reaches 6.0, the seed solution is obtained.

[0060] (3) Preparation of bacteriocin solution: Lactobacillus casei GG002 seed liquid was inoculated with 3% (v / v) in MRS liquid medium and cultured at 37°C for 18 h. Take 5 mL of Lactobacillus casei GG002 fermentation liquid, centrifuge at 8000 rpm for 20 min, and take the liquid as the fermentation supernatant. Add ammonium sulfate to the fermentation supernatant to make its saturation reach 70%, stir it with low temperature magnetic stirring overnight, centrifuge it at 4°C and 8000 rpm for 15 min, discard the supernatant, collect the precipitate, dry it at 40°C, dissolve it in 5 mL of ultrapure water, and filter it with a disposable filter with a pore size of 0.22 μm to obtain a sterile bacteriocin solution.

[0061] Example 3

[0062] Preparation method of Lactobacillus casei GG002 bacteriocin solution:

[0063] (1) Activation: Same as Example 2.

[0064] (2) Seed culture: Same as Example 2.

[0065] (3) Preparation of bacteriocin solution: Lactobacillus casei GG002 was inoculated with 2% (v / v) in MRS liquid culture medium and cultured at 37°C for 18 h. Take 5 mL of Lactobacillus casei GG002 fermentation liquid, centrifuge at 7000 rpm for 25 min, and take the liquid as the fermentation supernatant. Add ammonium sulfate to the fermentation supernatant to make its saturation reach 65%, stir it with low temperature magnetic stirring overnight, centrifuge it at 4°C and 7000 rpm for 20 min, discard the supernatant, collect the precipitate, dry it at 38°C, dissolve it in 5 mL of ultrapure water, and filter it with a disposable filter with a pore size of 0.22 μm to obtain a sterile bacteriocin solution.

[0066] Example 4

[0067] Preparation method of Lactobacillus casei GG002 bacteriocin solution:

[0068] (1) Activation: Same as Example 2.

[0069] (2) Seed culture: Same as Example 2.

[0070] (3) Preparation of bacteriocin solution: Lactobacillus casei GG002 was inoculated with 4% (v / v) in MRS liquid culture medium and cultured at 37°C for 18 h. Take 5 mL of Lactobacillus casei GG002 fermentation liquid, centrifuge at 9000 rpm for 15 min, and take the liquid as the fermentation supernatant. Add ammonium sulfate to the fermentation supernatant to make its saturation reach 75%, stir it with low temperature magnetic stirring overnight, centrifuge it at 4°C and 9000 rpm for 10 min, discard the supernatant, collect the precipitate, dry it at 40°C, dissolve it in 5 mL of ultrapure water, and filter it with a disposable filter with a pore size of 0.22 μm to obtain a sterile bacteriocin solution.

[0071] Example 5

[0072] Antibacterial Experiment of Extracellular Polysaccharide and Bacteriocin of Lactobacillus casei GG002

[0073] Preparation of extracellular polysaccharide solution: Lactobacillus casei GG002 was inoculated with 3% (v / v) in MRS liquid medium and cultured at 37°C for 18h. Take 5mL of Lactobacillus casei GG002 fermentation liquid, centrifuge at 8000rmp for 20min, take the liquid as the fermentation supernatant, add 3 times the volume of anhydrous ethanol to the fermentation supernatant and mix, precipitate at 4°C for 15h; then centrifuge at 4°C and 10000g for 20min, discard the supernatant, and dry the precipitate in an oven at 55°C for 10min to remove the residual liquid. Redissolve the precipitate with deionized water (the amount of deionized water is: 2mL of deionized water per 10mL of fermentation supernatant) to obtain extracellular polysaccharides. After the obtained extracellular polysaccharides are air-dried, they are dissolved with 5mL of ultrapure water, and then filtered and sterilized with a disposable filter with a pore size of 0.22μm to obtain a sterile extracellular polysaccharide solution.

[0074] Preparation of bacteriocin solution: same as in Example 2.

[0075] A sterilized Oxford cup was placed in a sterile plate, and 1 mL of Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis were added to 100 mL of 45°C BHI solid medium, and then immediately mixed and poured into the plate. After solidification, the Oxford cup was taken out, and 200 μL of Lactobacillus casei GG002 extracellular polysaccharide solution and bacteriocin solution were added to each well. The Streptococcus mutans plate was statically cultured at 37°C for 72 hours, and the Fusobacterium nucleatum and Porphyromonas gingivalis plates were anaerobically cultured at 37°C for 72 hours, and the results were detected. A blank MRS medium was added to the control group.

[0076] Table 2 Inhibitory effects of extracellular polysaccharides and bacteriocins of Lactobacillus casei GG002

[0077]

[0078] Note: “*” indicates comparison with the control group. P <0.05.

[0079] It can be seen from the above table that the bacteriocin produced by the fermentation of Lactobacillus casei GG002 has a significant antibacterial effect on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis; the extracellular polysaccharide produced by the fermentation of Lactobacillus casei GG002 has no significant inhibitory effect on the three oral pathogens.

[0080] Example 6

[0081] After Houttuynia cordata is soaked in 3 times the volume of purified water for 2 hours, it is boiled over high heat and simmered over low heat until the weight is the same as that of Houttuynia cordata, and the juice is poured out. An equal amount of purified water is added to the residue again, and it is boiled over high heat and simmered over low heat until the weight is the same as that of Houttuynia cordata, and the juice is poured out. The juices of the two decocted are concentrated to the same weight as the Houttuynia cordata raw material, which is decoction solution A. Portulaca oleracea, dandelion, and honeysuckle are prepared by the same steps to obtain decoction solution B, decoction solution C, and decoction solution D, respectively.

[0082] 1. Inhibitory effect of decoction solutions A~D combined with Lactobacillus casei GG002 on oral microorganisms

[0083] Control group: The activated Lactobacillus casei GG002 was inoculated into MRS liquid culture medium at a rate of 3% (v / v) and cultured at 37°C for 18 h.

[0084] Decoction solution group A: 20 wt% decoction solution A was added to MRS medium, sterilized at 121°C for 20 min, and activated Lactobacillus casei GG002 was inoculated at 3% (v / v) in the above sterilized medium, and cultured at 37°C for 18 h.

[0085] Decoction solution group B: 20 ​​wt% decoction solution B was added to MRS medium, sterilized at 121°C for 20 min, and activated Lactobacillus casei GG002 was inoculated at 3% (v / v) in the above sterilized medium, and cultured at 37°C for 18 h.

[0086] Decoction solution C group: 20 wt% decoction solution C was added to MRS medium, sterilized at 121°C for 20 min, and activated Lactobacillus casei GG002 was inoculated at 3% (v / v) in the above sterilized medium, and cultured at 37°C for 18 h.

[0087] Decoction solution D group: 20 wt% decoction solution D was added to MRS medium, sterilized at 121°C for 20 min, and activated Lactobacillus casei GG002 was inoculated at 3% (v / v) in the above sterilized medium, and cultured at 37°C for 18 h.

[0088] A sterilized Oxford cup was placed in a sterile plate, and 1 ml of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were added to 100 mL of 45°C BHI solid medium, and then immediately mixed and poured into the plate. After solidification, the Oxford cup was taken out, and 200 μL of the above fermentation liquid was added to each well. The Streptococcus mutans plate was statically cultured at 37°C for 72 hours, and the Fusobacterium nucleatum and Porphyromonas gingivalis plates were anaerobically cultured at 37°C for 72 hours, and the results were detected. The antibacterial results are shown in Table 3:

[0089] Table 3 Inhibitory effect of adding different decoction solutions on pathogens

[0090]

[0091] Note: “*” indicates comparison with the control group. P <0.05.

[0092] It can be seen from the above table that after fermentation with the addition of different decoctions of Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle, the inhibition zones of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were slightly larger than those of the control group, among which dandelion decoction stock solution C>Houttuynia cordata decoction stock solution A>Honeysuckle decoction stock solution D>Pursula decoction stock solution B>control group, the fermentation liquid obtained by culturing Lactobacillus casei GG002 in the culture medium containing the decoctions of Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle can improve the antibacterial effect on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis, and Lactobacillus casei GG002 has a synergistic effect with Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle.

[0093] 2. Inhibitory effects of different proportions of decoction solutions on oral microorganisms

[0094] The ratio concentration of decoction solution A (30g, 35g, 40g), decoction solution B (10g, 15g, 20g), decoction solution C (15g, 20g, 25g), and decoction solution D (5g, 10g, 15g) was used to conduct an orthogonal test with 4 factors and 3 levels, and 9 mixed solutions with different ratios were prepared to conduct antibacterial experiments on Streptococcus mutans. The experimental design and results are shown in Table 4.

[0095] Table 4 Inhibitory effects of different proportions of decoction solutions on Streptococcus mutans

[0096]

[0097] It can be seen from the above table that the inhibitory effect on Streptococcus mutans is decoction solution C>decoction solution A>decoction solution D>decoction solution B; through variance analysis, it is determined that 35g decoction solution A, 15g decoction solution B, 20g decoction solution C, and 10g decoction solution D are the best antibacterial ratios.

[0098] The antibacterial experiments on Fusobacterium nucleatum and Porphyromonas gingivalis were carried out using the optimal ratio of the decoction of Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Flos Lonicerae and two other random ratios selected above. The experimental design and results are shown in Table 5.

[0099] Table 5 Inhibitory effects of different proportions of decoction solutions on Fusobacterium nucleatum and Porphyromonas gingivalis

[0100]

[0101] From the above table, we can see that the four decoctions of Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle have inhibitory effects on Fusobacterium nucleatum and Porphyromonas gingivalis, and the combination screened for Streptococcus mutans has better effects on Fusobacterium nucleatum and Porphyromonas gingivalis than the other two combinations. Compared with the other two combinations, there are differences ( P <0.05).

[0102] 3. Effect of different drug stock solution addition amounts in MRS medium on the viable count of Lactobacillus casei GG002

[0103] Preparation of drug stock solution: Add 350g of Houttuynia cordata, 150g of Portulaca oleracea, 200g of Taraxacum mongolicum, and 100g of Honeysuckle to 3 times the volume of purified water and soak for 2h, then boil over high heat and simmer over low heat until the weight is the same as the four raw materials, then pour out the juice, continue to add 3 times the volume and simmer until the weight is the same as the four raw materials, then pour out the juice, and concentrate the juice from the two decocted juices to the same weight as the four raw materials, which is the drug stock solution. This stock solution is packaged and stored at 4°C for later use.

[0104] 10wt%, 20wt% and 30wt% of the drug stock solution were added to the MRS medium to form culture media with different ratios. The MRS medium was used as the control. Lactobacillus casei GG002 was inoculated with 3% of the above-mentioned sterilized culture medium and cultured at 37°C for 18h. The live bacteria were counted according to GB 4789.35-2016 National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination. The results are shown in Table 6.

[0105] Table 6 Effects of different drug stock solution addition amounts on the viable count of Lactobacillus casei GG002

[0106]

[0107] It can be seen from the above table that the drug solutions prepared from Houttuynia cordata, Portulaca oleracea, Taraxacum officinale and Honeysuckle did not inhibit the growth of Lactobacillus casei GG002, and the number of live bacteria increased with the increase of the amount of drug solution added. When 20% of the drug solution was added, the number of live bacteria reached 4.0×10 9 cfu / mL, increasing the concentration of the drug stock solution to 30% had little effect on bacterial growth.

[0108] Example 7

[0109] Preparation of lyophilized powder of Lactobacillus casei GG002 (the preparation method of drug stock solution is the same as step 3 of Example 6):

[0110] Add 20wt% drug stock solution to MRS medium, sterilize and inoculate Lactobacillus casei GG002 at 3% (v / v), culture at 37℃ for 18h, centrifuge at 8000rmp at 4℃ for 20min to harvest bacterial sludge. Mix the bacterial sludge and bacterial protective agent at a ratio of 1:4, pre-freeze at -40℃ for 4h, freeze-dry at -70℃ for 72h to obtain Lactobacillus casei GG002, and count the live bacteria according to GB 4789.35-2016 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test, and the live bacteria count of bacterial powder is 2.6×10 11 cfu / g.

[0111] The proportion of bacterial protective agent is: 10g skim milk, 8g trehalose, 5g sucrose, 2g sorbitol, 0.1g vitamin C, 0.1g sodium glutamate, 0.5g sodium alginate, 20g drug stock solution, and 80g water.

[0112] Example 8

[0113] Preparation of lyophilized powder of Lactobacillus casei GG002 (the preparation method of drug stock solution is the same as step 3 of Example 6):

[0114] 10wt% drug stock solution was added to MRS medium, and after sterilization, Lactobacillus casei GG002 was inoculated at 3% (v / v), cultured at 37°C for 18h, and centrifuged at 9000rmp for 10min to harvest the bacterial sludge. The bacterial sludge and bacterial protective agent were mixed at a ratio of 1:3, pre-frozen at -40°C for 4h, and freeze-dried at -70°C for 72h to obtain Lactobacillus casei GG002. The live bacteria were counted according to GB 4789.35-2016 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test, and the live bacteria count of the bacterial powder was 3.8×10 11 cfu / g.

[0115] The proportion of bacterial protective agent is as follows: 8g skim milk, 10g trehalose, 4g sucrose, 3g sorbitol, 0.15g vitamin C, 0.05g sodium glutamate, 0.5g sodium alginate, 15g drug stock solution, and 85g water.

[0116] Example 9

[0117] Preparation of lyophilized powder of Lactobacillus casei GG002 (the preparation method of drug stock solution is the same as step 3 of Example 6):

[0118] 30wt% drug stock solution was added to MRS medium, and after sterilization, Lactobacillus casei GG002 was inoculated at 3% (v / v), cultured at 37℃ for 18h, and centrifuged at 7000rmp for 20min to harvest the bacterial sludge. The bacterial sludge and bacterial protective agent were mixed at a ratio of 1:5, pre-frozen at -40℃ for 4h, and freeze-dried at -70℃ for 72h to obtain Lactobacillus casei GG002. The live bacteria were counted according to GB 4789.35-2016 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test, and the live bacteria count of the bacterial powder was 2.7×10 11 cfu / g.

[0119] The proportion of bacterial protective agent is: 12g skim milk, 6g trehalose, 6g sucrose, 1g sorbitol, 0.05g vitamin C, 0.15g sodium glutamate, 0.5g sodium alginate, 25g drug stock solution, and 75g water.

[0120] Example 10

[0121] Preparation of postbiotics of Lactobacillus casei GG002 (the preparation method of drug stock solution is the same as step 3 of Example 6):

[0122] Postbiotic medium: 200g drug stock solution, 100g skim milk, 60g sucrose, 150g yeast extract, 490g purified water, sterilized at 80℃ for 30min and set aside. Lactobacillus casei GG002 was inoculated into the sterilized postbiotic medium at 3% (v / v) and cultured at 37℃ until the pH no longer decreased (about 24h). The fermentation broth was spray dried with a drying inlet temperature of 170℃ and a feed rate of 10.4mL / min. After drying, Lactobacillus casei GG002 postbiotics were obtained.

[0123] Embodiment 11

[0124] Preparation of postbiotics of Lactobacillus casei GG002 (the preparation method of drug stock solution is the same as step 3 of Example 6):

[0125] Postbiotic medium: 180g drug stock solution, 120g skim milk, 50g sucrose, 160g yeast extract, 490g purified water, sterilized at 80℃ for 30min and set aside. Lactobacillus casei GG002 was inoculated into the sterilized postbiotic medium at 3% (v / v) and cultured at 37℃ until the pH no longer decreased (about 24h). The fermentation broth was spray dried with a drying inlet temperature of 160℃ and a feed rate of 10mL / min. After drying, Lactobacillus casei GG002 postbiotics were obtained.

[0126] Example 12

[0127] Preparation of postbiotics of Lactobacillus casei GG002 (the preparation method of drug stock solution is the same as step 3 of Example 6):

[0128] Postbiotic medium: 220g drug stock solution, 80g skim milk, 70g sucrose, 140g yeast extract, 490g purified water, sterilized at 80℃ for 30min and set aside. Lactobacillus casei GG002 was inoculated into the sterilized postbiotic medium at 3% (v / v) and cultured at 37℃ until the pH no longer decreased (about 24h). The fermentation broth was spray dried with a drying air inlet temperature of 180℃ and a feed rate of 11mL / min. After drying, Lactobacillus casei GG002 postbiotics were obtained.

[0129] Embodiment 13

[0130] 1. Study on the antibacterial effects of freeze-dried powder of Lactobacillus casei GG002 and postbiotics

[0131] Preparation of control freeze-dried powder of Lactobacillus casei GG002: 3% (v / v) Lactobacillus casei GG002 was inoculated into MRS medium, cultured at 37°C for 18h, and centrifuged at 8000rmp for 20min at 4°C to harvest the bacterial sludge. The bacterial sludge and bacterial protective agent were mixed in a ratio of 1:4, pre-frozen at -40°C for 4h, freeze-dried at -70°C for 72h, and counted according to GB 4789.35-2016 National Food Safety Standard Food Microbiology Test for Lactic Acid Bacteria, and the number of live bacteria in the bacterial powder was 5.9×10 11 cfu / g. Proportion of bacterial protective agent: skim milk 10g, trehalose 8g, sucrose 5g, sorbitol 2g, vitamin C 0.1g, sodium glutamate 0.1g, sodium alginate 0.5g, water 80g.

[0132] Preparation of freeze-dried powder of Lactobacillus casei GG002: As in Example 7, the live bacteria count was performed using the "GB 4789.35-2016 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test", and the live bacteria count of the bacterial powder was 5.8×10 11 cfu / g.

[0133] Preparation of Lactobacillus casei GG002 postbiotics: The same as Example 10, obtain Lactobacillus casei GG002 postbiotics.

[0134] Take 0.3 g of Lactobacillus casei GG002 control lyophilized powder, 0.3 g of Lactobacillus casei GG002 lyophilized powder, and 3 g of Lactobacillus casei GG002 postbiotics, add them into 10 g of sterile purified water, mix them thoroughly, and set aside.

[0135] A sterilized Oxford cup was placed in a sterile plate, and 1 mL of Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were added to 100 mL of 45°C BHI solid medium, and then immediately mixed and poured into the plate. After solidification, the Oxford cup was taken out, and 200 μL of the above three solutions were added to each well. The Streptococcus mutans plate was statically cultured at 37°C for 72 hours, and the Fusobacterium nucleatum and Porphyromonas gingivalis plates were anaerobically cultured at 37°C for 72 hours, and the results were tested. The antibacterial results are shown in Table 7:

[0136] Table 7 Inhibitory effects of Lactobacillus casei GG002 prepared by different methods on pathogens

[0137]

[0138] Note: "*" indicates that compared with the control freeze-dried powder group of Lactobacillus casei GG002, P <0.05.

[0139] As shown in the table above, the three bacterial agents all have antibacterial effects on Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis. Among them, the bacterial agents obtained by adding Chinese medicine stock solution fermentation (Lactobacillus casei GG002 freeze-dried powder, Lactobacillus casei GG002 postbiotics) have better antibacterial effects. In particular, Lactobacillus casei GG002 postbiotics contain bacteriocins produced by Lactobacillus casei GG002 and medicinal and edible components, which can synergistically enhance its antibacterial effect, with significant differences ( P <0.05).

[0140] 2. Effects of Lactobacillus casei GG002 freeze-dried powder and postbiotics on dental caries in rats

[0141] A total of 90 SPF male SD rats with a body weight of 180±20g were included. 80 rats were anesthetized with 3% sodium pentobarbital solution at 0.1mL / 100g by intraperitoneal injection. The labial mucosa of the lower incisor of the SD rats was dried with sterile cotton balls. A 3mm×3mm square filter paper was placed in 50% glacial acetic acid for 5s and then removed. It was immediately placed on the dried labial mucosa of the lower incisor of the rats and kept for 30s before the filter paper was removed. The treated area was swabbed with a sterile cotton ball containing physiological saline to remove residual acetic acid, and then the treated area was swabbed with a mixture of Streptococcus mutans, Fusobacterium nucleatum, and Porphyromonas gingivalis (1:1:1) to establish a rat oral ulcer model.

[0142] The rats with successful modeling were randomly divided into 8 groups, including model group, positive drug group, freeze-dried powder experimental group (low-dose group, medium-dose group and high-dose group), and postbiotic experimental group (low-dose group, medium-dose group and high-dose group). There were 10 rats in each group, and the remaining 10 healthy SD male rats served as the normal control group. All rats were gavaged with 1 mL of the drug at the same dose. The gavage drugs and cycles are shown in Table 8.

[0143] Table 8 Drug gavage dosage and cycle

[0144]

[0145] (1) Use a vernier caliper to measure the oral ulcer area of ​​each group of rats before administration and on the 3rd, 5th, 7th and 9th days after administration. Calculation: ulcer area = 3.14 × d1 × d2 × 1 / 4 (d1 and d2 are the maximum transverse diameter and maximum longitudinal diameter of the ulcer surface, respectively). Average food intake (g) = feed consumption per cage / number of animals per cage. The experimental results are shown in Table 9.

[0146] Table 9 Ulcer area of ​​oral ulcer model rats in each group

[0147]

[0148] Note: Compared with the model group, * P <0.05,** P <0.01.

[0149] From the above table, we can see that the model group heals slowly; the positive control group, high-dose freeze-dried powder group, medium-dose freeze-dried powder group, high-dose postbiotic group and medium-dose postbiotic group show differences on the fifth day compared with the 0 day ( P <0.05); on the 7th day, the positive control group, high-dose freeze-dried powder group, medium-dose freeze-dried powder group, high-dose postbiotic group and medium-dose postbiotic group showed significant differences compared with day 0 ( P <0.01). This indicates that the high-dose freeze-dried powder group, the medium-dose freeze-dried powder group, the high-dose postbiotic group, and the medium-dose postbiotic group can accelerate the healing of oral ulcers, and there is no difference with the positive control drug at this dose.

[0150] (2) Detection of SOD, MDA, GSH-Px and CAT levels in rat serum

[0151] After the administration, blood was collected from the abdominal aorta under sodium pentobarbital anesthesia. After the blood was placed at room temperature for 2 hours, it was centrifuged at 3500r / min for 15 minutes. The serum was divided into 1.5mL centrifuge tubes and marked, and stored in a -20℃ refrigerator. Rat serum was taken and the SOD, MDA, GSH-Px, and CAT levels in serum were determined in strict accordance with the instructions of the corresponding kits. The results are shown in Figure 1 shown.

[0152] Depend on Figure 1 It can be seen that compared with the model group, the positive control group, high-dose freeze-dried powder group, medium-dose freeze-dried powder group, high-dose postbiotic group and medium-dose postbiotic group showed significant differences in MDA concentration and CAT activity detection ( P <0.01), there were differences between the low-dose freeze-dried powder group and the low-dose postbiotic group ( P <0.05); in the detection results of SOD activity and GSH-Px activity, there was no difference between the model group and the experimental group.

[0153] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Lactobacillus casei ( Lacticaseibacillus casei ) Use of GG002 or a preparation related to Lactobacillus casei GG002 in the preparation of a medicament for treating oral ulcers, characterized in that: The deposit number of the Lactobacillus casei GG002 is CGMCC No.29392; The Lactobacillus casei GG002 inhibits Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis; The Lactobacillus casei GG002-related preparations include any one or more of Lactobacillus casei GG002 bacteriocin solution, Lactobacillus casei GG002 freeze-dried powder and Lactobacillus casei GG002 postbiotics.

2. The use according to claim 1, characterized in that: The method for preparing the Lactobacillus casei GG002 bacteriocin solution comprises the following steps: fermenting and culturing the Lactobacillus casei GG002, centrifuging the fermentation liquid to obtain a fermentation supernatant, adding ammonium sulfate to the fermentation supernatant, stirring and centrifuging to obtain a precipitate, and drying and re-dissolving the precipitate to obtain a bacteriocin solution.

3. The use according to claim 1, characterized in that: The preparation method of the Lactobacillus casei GG002 freeze-dried powder comprises the following steps: fermenting and culturing the Lactobacillus casei GG002 with a fermentation medium, centrifuging the fermentation liquid, mixing the bacterial mud with a freeze-drying protective agent, and freeze-drying to obtain the Lactobacillus casei GG002 freeze-dried powder; the fermentation medium comprises a basic medium and a drug stock solution; the drug stock solution is obtained by decocting houttuynia cordata, purslane, dandelion and honeysuckle.

4. The use according to claim 3, characterized in that: The drug stock solution is 10-30wt% of the basal culture medium.

5. The use according to claim 3, characterized in that: Calculated by weight, the lyophilization protective agent includes: 8 to 12 parts of skim milk, 6 to 10 parts of trehalose, 4 to 6 parts of sucrose, 1 to 3 parts of sorbitol, 0.05 to 0.15 parts of vitamin C, 0.05 to 0.15 parts of sodium glutamate, 0.2 to 0.8 parts of sodium alginate, 15 to 25 parts of drug stock solution, and 70 to 90 parts of water.

6. The use according to claim 1, characterized in that: The preparation method of Lactobacillus casei GG002 postbiotics comprises the following steps: Lactobacillus casei GG002 is fermented and cultured using a postbiotic culture medium, and the fermentation liquid is spray-dried to obtain Lactobacillus casei GG002 postbiotics; the postbiotic culture medium comprises skim milk, sucrose, yeast extract, a drug stock solution and water; the drug stock solution is obtained by decocting houttuynia cordata, purslane, dandelion and honeysuckle.

7. The use according to claim 6, characterized in that: In terms of mass, the postbiotic culture medium includes 80 to 120 parts of skim milk, 50 to 70 parts of sucrose, 140 to 160 parts of yeast extract, 180 to 220 parts of drug stock solution and 480 to 500 parts of water.

8. The use according to any one of claims 3 to 7, characterized in that: In the drug stock solution, the mass ratio of houttuynia cordata, purslane, dandelion and honeysuckle is 30 ~ 40:10 ~ 20:15 ~ 25:5 ~ 15.

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