Probiotic patch for preventing and treating oral ulcers, method for producing the same, and use thereof

Abstract

Provided are a probiotic patch for preventing and treating oral ulcers, a method for producing the same, and use thereof. First, the present invention provides a use of Lactobacillus paracasei in the production of a medicament for preventing and treating oral ulcers, wherein the Lactobacillus paracasei contains Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites, and the Lactobacillus paracasei ET-22 is a strain with the deposit number CGMCC No. 15077. Further, the present invention provides a probiotic composition containing Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites, sodium carboxymethyl cellulose, propolis, borneol, a lubricant, glycerin, and Tween 20. The probiotic composition of the present invention can be made into a patch, can effectively prevent and treat oral ulcers, is safe and reliable, and has a remarkable effect.

Description

Technical Field

[0001] The present invention relates to a probiotic patch, a method for producing the same, and use thereof. Specifically, it relates to the use in the production of a medicament for preventing and treating oral ulcers with Lactobacillus paracasei ET-22, a probiotic composition containing Lactobacillus paracasei ET-22, a probiotic patch produced from the composition, a method for producing the same, and use thereof.

Background Art

[0002] Oral ulcers, commonly known as "aphthae", are common ulcerative injury symptoms that occur on the oral mucosa. The occurrence of oral ulcers is due to the combined action of various factors including local trauma, mental stress, food, drugs, malnutrition, changes in hormone levels, and deficiencies of vitamins and trace elements.

[0003] In the prior art, it is known that various probiotics are useful for the treatment of oral ulcers. CN109985179A discloses a composition for treating oral ulcers, which contains raw materials such as 1000 - 15000 parts by weight of fresh asparagus, 0.1 - 10 parts by weight of Lactobacillus salivarius, 0.1 - 20 parts by weight of Bifidobacterium lactis, 0.1 - 20 parts by weight of Lactobacillus paracasei, 0.1 - 50 parts by weight of indigo naturalis, and 10 - 1000 parts by weight of starch. The composition can supplement trace elements necessary for the human body, has the effects of internal immune regulation and oral environment improvement on oral ulcer patients, enhances the body's immunity, and promotes the healing of sores. When specifically used, it is made into oral medicaments such as pills, powders, capsules, tablets or granules. CN113425831A discloses a probiotic composition for treating oral ulcers, its manufacturing method and use, which contains raw materials such as 40 - 60 parts by weight of Borojo powder, 30 - 60 parts by weight of Lithocarpus litseifolius (Hance) Chun, 10 - 20 parts by weight of probiotics, 1 - 5 parts by weight of Chimonanthus salicifolius, 1 - 5 parts by weight of oligomannose, 1 - 5 parts by weight of the leaves of Lindera aggregata (Sims.) Kosterm., and 1 - 5 parts by weight of colostrum alkaline protein. The composition plays a role in treating oral ulcers by Borojo powder and Lithocarpus litseifolius (Hance) Chun having antioxidant effects, can reduce the usage amount of probiotics, promotes the growth of probiotics by oligomannose, can exert a synergistic effect among various probiotics, and further prevents and improves oral ulcers because the included Chimonanthus salicifolius, rice bran aliphatic alcohol, the leaves of Lindera aggregata and colostrum alkaline protein all have the effect of treating oral ulcers. When specifically used, it is made into a spray.

[0004] In the above-mentioned probiotic-containing medicine for treating oral ulcers in the prior art, the probiotics themselves do not significantly contribute to the treatment of oral ulcers. In addition, oral medicines have drawbacks such as a wide range of action sites, a large required dosage, and low drug utilization rate. Sprays require refraining from drinking water and eating for a certain period of time after spraying, and there are many inconveniences when using them.

Summary of the Invention

[0005] One object of the present invention is to provide probiotics for preventing and treating oral ulcers.

[0006] Another object of the present invention is to provide the use of Lactobacillus paracasei ET-22 in the manufacture of a medicine for preventing and treating oral ulcers.

[0007] Another object of the present invention is to provide a probiotic composition containing Lactobacillus paracasei ET-22.

[0008] Another object of the present invention is to provide a probiotic patch produced from the above composition.

[0009] Another object of the present invention is to provide a method for manufacturing a probiotic patch.

[0010] Another object of the present invention is to provide the use of the probiotic composition or the patch.

[0011] Through the research of the present inventors, it has been discovered that Lactobacillus paracasei ET-22 is a probiotic that can prevent and treat oral ulcers, and its cells and / or its extracellular metabolites can significantly prevent and treat oral ulcers. Furthermore, it has been discovered that by using Lactobacillus paracasei ET-22 and / or its extracellular metabolites as a probiotic patch, the oral ulcer site can be effectively sterilized, and secondary infection of the wound can be effectively prevented.

[0012] In the present invention, the prevention and treatment include prevention and / or treatment.

[0013] Accordingly, the present invention provides, on one hand, the use in the manufacture of a medicament for treating oral ulcers with Lactobacillus paracasei, wherein the Lactobacillus paracasei comprises Lactobacillus paracasei ET-22 bacterial cells and / or its extracellular metabolites, and the Lactobacillus paracasei ET-22 is a strain with the deposit number CGMCC No. 15077.

[0014] The Lactobacillus paracasei ET-22 strain with the deposit number CGMCC No. 15077 is a biological material disclosed in CN110964653A and is accessible to the general public.

[0015] According to a specific embodiment of the present invention, in the present invention, the Lactobacillus paracasei ET-22 bacterial cells are viable bacterial cells and / or inactivated bacterial cells.

[0016] According to a specific embodiment of the present invention, in the present invention, the Lactobacillus paracasei ET-22 extracellular metabolites are produced by the following method.

[0017] Remove the bacterial cells from the incubation broth of Lactobacillus paracasei ET-22 to obtain the extracellular metabolites of Lactobacillus paracasei ET-22. Preferably, the incubation broth is obtained by incubating Lactobacillus paracasei ET-22 in water.

[0018] Preferably, dissolve the Lactobacillus paracasei ET-22 bacterial cells in water at a concentration of 1-5×10 9 CFU / mL, incubate with stirring at 37°C and 200-500 revolutions per minute for 2-4 hours, and after the incubation is completed, centrifuge to remove the bacterial cells to obtain the extracellular metabolites of Lactobacillus paracasei ET-22.

[0019] According to a specific embodiment of the present invention, in the present invention, the oral ulcer includes an oral ulcer caused by a pathogenic bacterium, an oral ulcer caused by oxidative damage, or an oral ulcer caused by immune deficiency.

[0020] In another aspect, the present invention provides a probiotic composition comprising Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites, wherein the Lactobacillus paracasei ET-22 is a strain with the accession number CGMCC No. 15077.

[0021] According to a specific embodiment of the present invention, in the present invention, the probiotic composition further comprises sodium carboxymethyl cellulose, propolis, borneol, a lubricant, glycerin, and Tween 20. Preferably, the parts by weight of each component are as follows. Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites 15-20 parts Sodium carboxymethyl cellulose 10-20 parts Propolis 20-40 parts Borneol 2-5 parts Lubricant 2-5 parts Glycerin 8-12 parts Tween 20 1-3 parts

[0022] According to a specific embodiment of the present invention, in the probiotic composition of the present invention, the parts by weight of each component are as follows. Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites 18-20 parts Sodium carboxymethyl cellulose 14-20 parts Propolis 25-35 parts Borneol 2-5 parts Lubricant 2-5 parts Glycerin 8-12 parts Tween 20 1-3 parts

[0023] According to a specific embodiment of the present invention, in the probiotic composition of the present invention, the Lactobacillus paracasei ET-22 cells are viable cells and / or inactivated cells.

[0024] According to a specific embodiment of the present invention, in the probiotic composition of the present invention, the extracellular metabolite of Lactobacillus paracasei ET-22 is produced by the following method.

[0025] Dissolve Lactobacillus paracasei ET-22 cells in water at a concentration of 1 to 5×10 9 CFU / mL, incubate with stirring at 37°C under the condition of 200 to 500 revolutions per minute for 2 to 4 hours. After the incubation, centrifuge to remove the cells to obtain the extracellular metabolite of Lactobacillus paracasei ET-22.

[0026] According to a specific embodiment of the present invention, in the probiotic composition of the present invention, the lubricant may be liquid paraffin or other lubricants commonly used in oral patches.

[0027] In another aspect, the present invention provides the use of the probiotic composition in the manufacture of a composition for preventing and treating oral ulcers.

[0028] According to a specific embodiment of the present invention, the oral ulcers include oral ulcers caused by pathogenic bacteria, oral ulcers caused by oxidative damage, or oral ulcers caused by immune deficiency. In some specific embodiments of the present invention, viable Lactobacillus paracasei ET-22 can significantly prevent and treat oral ulcers caused by pathogenic bacteria or oxidative damage. In some specific embodiments of the present invention, the application of inactivated Lactobacillus paracasei ET-22 can significantly prevent and treat oral ulcers caused by immune deficiency. In some specific embodiments of the present invention, the extracellular metabolite of Lactobacillus paracasei ET-22 has a significant effect on the prevention and treatment of oral ulcers.

[0029] According to some specific embodiments of the present invention, the composition for preventing and treating oral ulcers of the present invention is a pharmaceutical composition. Preferably, the pharmaceutical composition is a patch.

[0030] According to some specific embodiments of the present invention, the composition for preventing and treating oral ulcers of the present invention may also be a general cosmetic.

[0031] In another aspect, the present invention (1) A step of uniformly mixing Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites, propolis, sodium carboxymethyl cellulose and a lubricant to obtain a mixed system A, (2) A step of heating borneol, glycerin and Tween 20 by decocting and uniformly mixing them to obtain a mixed system B, (3) A step of uniformly mixing the mixed system A and the mixed system B, (4) A step of drying to provide a method for manufacturing a probiotic patch.

[0032] Note that for each step of the manufacturing method of the present invention, the signs (1) and (2) do not indicate that these two steps must be performed in the order in which they are described. In actual operation, step (2) may be performed first, and then step (1), or the two steps may be performed simultaneously.

[0033] In another aspect, the present invention provides a probiotic patch manufactured from the composition described in the present invention or manufactured by the method for manufacturing the probiotic patch described above.

[0034] In another aspect, the present invention provides a method for treating oral ulcers, which includes administering an effective amount of the composition and / or the probiotic patch to a subject, and the subject includes mammals or humans.

[0035] As described above, the present invention provides the use of Lactobacillus paracasei ET-22 and / or its extracellular metabolites in the prevention and treatment of oral ulcers, and further provides a probiotic patch produced thereby. The present invention has demonstrated, by means of an animal model, that viable Lactobacillus paracasei ET-22 can significantly prevent and treat oral ulcers caused by pathogenic bacteria or oxidative damage, that application of heat-inactivated Lactobacillus paracasei ET-22 can significantly prevent and treat oral ulcers caused by immunosuppression, and that the extracellular metabolites of Lactobacillus paracasei ET-22 have a good effect for the prevention and / or treatment of oral diseases. The probiotic patch described in the present invention has good adhesion to the oral mucosa, and by adding borneol, it also has the effects of clearing heat and detoxifying, resolving swelling and relieving pain, can effectively sterilize the oral ulcer site, and can effectively prevent secondary infection of the wound.

Brief Description of the Drawings

[0036]

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Modes for Carrying Out the Invention

[0037] To more clearly understand the technical features, objectives, and beneficial effects of the present invention, the technical solution of the present invention will be described in detail below with reference to specific examples. However, it should be understood that these examples are only for explaining the present invention and do not limit the scope of the present invention. Each starting reagent material in the examples is commercially available, and the experimental methods for which specific conditions are not specified are the common methods and common conditions well known in the art, or are carried out according to the conditions recommended by the equipment manufacturer.

[0038] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art.

[0039] In each embodiment of the present invention, the Lactobacillus paracasei ET-22 extracellular metabolite is produced by inoculating Lactobacillus paracasei ET-22 into a medium for culture to obtain a bacterial solution, centrifuging the ET-22 bacterial solution to obtain Lactobacillus paracasei ET-22 bacterial cells (slime).

[0040] The obtained Lactobacillus paracasei ET-22 bacterial cells are dissolved in water at a concentration of 1 to 5×10 9 CFU / mL, incubated with stirring at 37°C and 200 to 500 revolutions per minute for 3 hours, and after the incubation, centrifuged to remove the bacterial cells to obtain the Lactobacillus paracasei ET-22 extracellular metabolite.

Example

[0041] Example 1 This example provides a probiotic patch for treating oral ulcers. The raw materials for producing the probiotic patch include Lactobacillus paracasei ET-22 bacterial cells and / or its extracellular metabolite, sodium carboxymethylcellulose, propolis, borneol, lubricant, glycerin, and Tween 20. The parts by weight of each raw material are as follows. Lactobacillus paracasei ET-22 bacterial cells and / or its extracellular metabolite 18 parts Sodium carboxymethylcellulose 15 parts Propolis 30 parts Borneol 5 parts Lubricant 5 parts Glycerin 10 parts Tween 20 2 parts

[0042] The method for producing the probiotic patch includes the following steps. (1) Mix Lactobacillus paracasei ET-22 bacterial cells and / or extracellular metabolite, propolis, sodium carboxymethylcellulose, and lubricant in the ratio of the above parts by weight to obtain a mixed system A. (2) In the ratio of the above parts by weight, borneol, glycerin and Tween 20 were placed in a container, heated by simmering, stirred and uniformly mixed to obtain a mixed system B. (3) After uniformly mixing the mixed system A and the mixed system B, through a drying process, a probiotic patch for treating the oral ulcer was manufactured.

Example

[0043] Example 2 Preventive and Therapeutic Effects of Lactobacillus paracasei ET-22 on Oral Ulcers 1. Experimental Animal Model 1.1 Oral Ulcer Model Induced by Immunosuppression Preventive group animal model: To induce oral candidiasis in mice, the present invention adopted a protocol with some modifications to the previously described one. After the mice were acclimated, in the experimental group, probiotics intervention (adding 10 9 CFU / ml of probiotics to drinking water) was carried out for 18 days, and in the blank group and the model group, they drank water normally. On the 15th day, 15 mg / ml of tetracycline hydrochloride was administered to the mice by drinking water, and at the same time, prednisolone (100 mg / kg) was subcutaneously injected to induce an immunosuppressed state. On the 16th day, the model group and the experimental group were infected with Candida albicans CGMCC 2.4122. At the time of infection, after anesthetizing with 4% chloral hydrate (10 ml / kg), 1.0×10 9 CFU / ml of Candida albicans was infected at each site of the oral cavity, and a cotton swab impregnated with Candida albicans was placed on the dorsal surface of the tongue for 15 minutes. The mice were euthanized 48 hours after the operation.

[0044] Therapeutic group animal model: To induce oral candidiasis in mice, the present invention adopted a protocol with some modifications to the previously described one. After the mice were acclimated, they were administered 15 mg / ml of tetracycline hydrochloride by drinking water, and at the same time, immunosuppression was induced by subcutaneous injection of prednisolone (100 mg / kg). On the second day, the model group and the experimental group were infected with Candida albicans CGMCC 2.4122. At the time of infection, after anesthesia with 4% chloral hydrate (10 ml / kg), 1.0×10 9 CFU / ml of Candida albicans was infected, and a cotton swab impregnated with Candida albicans was placed on the dorsal surface of the tongue for 15 minutes. 24 hours after infection (the third day), the probiotics intervention group was intervened by applying probiotics once every 12 hours, for a total of 4 applications. The application method was the same as the application method at the time of infection. The mice were euthanized 72 hours after the operation (the fifth day).

[0045] 1. Oral ulcer golden hamster model due to oxidative damage Six-week-old male LVG Golden Syrian Hamsters, with a body weight of (110±20) g per animal, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The breeding conditions were maintained with 12-hour alternating light irradiation, a temperature of 20±2°C, and a humidity of 45%-50%. After the hamsters were acclimated in the breeding room for one week, they were randomly divided into 10 groups, namely the normal control group, the model group, the ET-22 live bacteria group, the ET-22 inactivated bacteria group, the ET-22 extracellular metabolite group, K12, HN019, DSM17938, the AP32 live bacteria group, and the vitamin C positive control group (VC group). The normal control group had 7 animals, and the remaining groups each had 10 animals. The hamsters in the normal control group and the model group were given 1 ml of physiological saline, and the ET-22 live bacteria group was given 1 ml of a concentration of 10 91 ml of the corresponding concentration of inactivated ET-22 bacteria and extracellular metabolites of bacteria, and 1 ml of vitamin C solution with a concentration of 20 mg / ml in the VC group were forcibly administered orally to the live ET-22 bacteria solution at CFU / ml, the inactivated ET-22 flora, and the extracellular metabolites of ET-22, respectively, every day. After 15 days of forced oral administration, a model of oral ulcer was constructed. First, 10% chloral hydrate (injection volume: 0.3 - 0.5% body weight) was intraperitoneally injected to anesthetize the hamsters. 0.25 ml of PBS buffer (pH = 7.4) was injected into the cheek pouches of the hamsters in the normal control group, and 0.25 ml of methyl viologen (10 mmol / L, dissolved in PBS) was injected into the cheek pouches of the hamsters in the remaining 5 groups. From the day of injection until the disposal of the hamsters, each group maintained the forced oral administration intervention every day. On the 4th day after the model was constructed, the hamsters were anesthetized and blood was collected. Then, the inside of the hamsters' mouths (excluding the ulcer site) was wiped with an oral swab, the swab head was cut off and placed in a centrifuge tube containing nucleic acid preservation solution, and the lesion tissue or normal mucosa of the oral ulcer was excised and divided into two parts. One part was fixed with 4% paraformaldehyde, and the other part was cryopreserved in liquid nitrogen. After separating the serum from the whole blood, it was dispensed and cryopreserved at 80°C.

[0046] 2. Grouping of experimental animals The prevention group was divided into the following 10 groups.

Table 1

[0047] The treatment group was divided into the following 7 groups.

Table 2

[0048] Scoring of the severity of oral disease infection - mouse Mice in each group were euthanized 48 (prevention group) / 72 (treatment group) hours after surgery, and the severity of tongue lesions was evaluated and observed. Based on the range and severity of white card-like plaques on the tongue surface, the macroscopic evaluation of infection was expressed by a lesion score of 0-4, as follows: 0 points: normal, 1 point: white plaque is 20% or less, 2 points: white plaque is 21% or more and 90% or less, 3 points: white plaque is 91% or more, 4 points: thick white plaque-like pseudomembrane is 91% or more.

[0049] The entire tongue of the mouse was longitudinally cut in half at the middle of the tongue tip, and one of them was fixed with 4% paraformaldehyde neutral buffer at room temperature and then embedded in paraffin. After the sample was cut into 5-μm-thick continuous longitudinal sections along the tongue tip, it was stained with hematoxylin method. Referring to the literature, observation staining was performed with a Leica fluorescence microscope.

[0050] Oral disease infection severity scoring - Golden hamster After the paraffin sections were sequentially dewaxed and gradient dehydrated, antigen activation was performed with antigen activation citrate buffer (pH = 6.0). Then, the sections were placed in 3% hydrogen peroxide solution and incubated for 25 min in the dark to block endogenous peroxidase. Furthermore, through operation steps such as blocking, primary antibody incubation, secondary antibody incubation, etc., finally, DAB color development and nuclear counterstaining were performed, dehydrated and sealed, and observed and photographed under a microscope. Immunohistochemical scoring adopted the IRS (Immunoreactive Score) scoring method. After scoring the staining degree (0-3 points) and positive rate (0-4 points) respectively, they were multiplied to obtain the comprehensive score (0-12 points). The scoring criteria were as follows. The staining degree was scored according to the staining characteristics presented by the target protein, no staining: 0 points, light yellow: 1 point, light brown: 2 points, dark brown: 3 points. The positive rate was scored according to the ratio of positive cells in the section, 0-5%: 0 points, 6%-25%: 1 point, 26%-50%: 2 points, 51%-75%: 3 points, >75%: 4 points. The comprehensive score was defined as 0 points negative (-), 1-3 points weakly positive (+), 4-5 points positive (++), 6-7 points strongly positive (+++).

[0051] Observation results of tissue lesions: Figure 1 is a result diagram of the tongue injury scoring values of ICR mice 48 h after the construction of the prevention group model in Example 2. As can be seen from Figure 1, in the prevention group, 48 hours after the operation, compared with the mice in the model group, there was a significant difference in the tongue injury score only in the ET-22 viable bacteria group (p<0.05), and the average score decreased by 46.23%. The viable bacteria group of DSM 17938 had the same average score as the model group, and there was no significant difference in the injury scores of the remaining experimental groups, but they mostly decreased to varying degrees. The ET-22 secretion group (extracellular metabolite group), K12 viable bacteria group, HN019 viable bacteria group, and AP32 viable bacteria group decreased by 31.9%, 32.26%, 35.48%, and 41.94% respectively, and the ET-22 inactivated bacteria group and L9 viable bacteria group decreased by 16.13% and 16.13% respectively.

[0052] Figure 2 is a result diagram of the tongue injury scoring values of ICR mice 72 h after the construction of the treatment group model in Example 2. As can be seen from Figure 2, in the treatment group, compared with the mice in the model group, there was a significant difference in the tongue injury score only in the ET-22 inactivated bacteria group (p<0.05). The probiotics groups all showed a certain cure rate. The cure rate by the metabolites of Lactobacillus paracasei ET-22 was at least 11%, the reduction range of the injury score was at least 23%, the cure rates of the remaining groups were 30%-40%, and the reduction ranges of the injury scores were 50%-60%.

[0053] Analysis of the injury score of the prevention group: According to the research of Sanae.A.Ishijima et al., in the mouse model, the average tongue injury score of the model group was 3.4, and the average injury score after treatment with Streptococcus salivarius K12 at 1.5×10 9 CFU / mL was 2.0.

[0054] In the prevention experiment of the present invention this time, 1.0×10 9The average injury score after treatment with Streptococcus salivarius K12 at 1.0×10 9 CFU / mL was 2.1. The average injury scores of viable Lactobacillus paracasei ET-22, heat-inactivated Lactobacillus paracasei ET-22, and extracellular metabolites were 1.7, 2.6, and 2.1, respectively. Viable Lactobacillus paracasei ET-22 was superior to viable Streptococcus salivarius K12 in preventing Candida albicans disease. Extracellular metabolites of Lactobacillus paracasei ET-22 and viable Streptococcus salivarius K12 had the same therapeutic effect in preventing Candida albicans disease. Compared with viable bacteria, extracellular metabolites had a simpler storage method and a wider application scenario.

[0055] Observation of tongue dorsum lesion sections: Forty-eight hours after the prevention group model was constructed, the HE staining results of mouse tongue dorsum lesion sections in the blank group, model group, and probiotics group are shown in Figure 3. In the figure, A is the papilla of tongue tissue epithelial cells, and B is tissue hyperplasia. As can be seen from Figure 3, compared with the blank group, severe inflammatory infiltration appeared in the model group, and the inflammatory infiltration was significantly improved in the ET-22 viable bacteria group, ET-22 secretion group (extracellular metabolites group), K12 viable bacteria group, HN019 viable bacteria group, and AP32 viable bacteria group. The inflammatory infiltration in the ET-22 heat-inactivated bacteria group and L9 viable bacteria group was also relatively severe, and the inflammatory infiltration in the DSM 17938 viable bacteria group was more serious than that in the model group.

[0056] As can be seen from Fig. 3, compared with the blank group, in the model group, the papillae of the tongue tissue epithelial cells were significantly lost, and exfoliation and tissue hyperplasia were obvious. In the ET-22 viable bacteria group, ET-22 secretion group, and AP32 viable bacteria group, the papillae of the tongue tissue epithelial cells were completely preserved and there was no exfoliation, but there was slight tissue hyperplasia compared with the blank group. In the ET-22 inactivated bacteria group, the papillae of the tongue tissue epithelial cells were relatively clearly lost and there was no exfoliation, but relatively obvious tissue hyperplasia appeared. In the L9 viable bacteria group, the papillae of the tongue tissue epithelial cells were lost to a certain extent, there was slight exfoliation, and the tissue hyperplasia was similar to that of the model group. In the K12 viable bacteria group, the papillary projections of the cells of the tongue tissue epithelium were significantly lost and there was no exfoliation, but there was a certain degree of tissue hyperplasia. In the DSM 17938 viable bacteria group, the papillae of the tongue tissue epithelial cells were significantly lost, there was slight exfoliation, and the tissue hyperplasia was severe.

[0057] As can be seen from the above, the ET-22 viable bacteria group, ET-22 extracellular metabolite group, AP32 viable bacteria group, and K12 viable bacteria group have a certain therapeutic effect on the prevention of oral candidiasis and reduce inflammation and tongue tissue lesions. The therapeutic effect of the ET-22 viable bacteria group is the best.

[0058] Mucosal tissue section and HE staining: Forty-eight hours after the construction of the prevention group model in Example 2, the HE staining photographs of typical hamster oral mucosal tissue sections are shown in Fig. 4. In the figure, the white arrow indicates inflammatory cell infiltration, the black arrow indicates mucosal epithelial damage and shedding, and the gray arrow indicates capillary engorgement and glandular hyperplasia (scale: 200 μm). As shown in Fig. 4, in the normal control group, the continuity of the hamster oral mucosal epithelium was perfect, the morphology of cells and glandular ducts was normal, and there was no obvious inflammatory cell infiltration. Compared with the normal control group, the hamster oral mucosal epithelium in the model group was damaged and shed, the continuity was incomplete, the capillaries were engorged and dilated, and a large number of inflammatory cell infiltrations appeared, indicating that the construction of the oral ulcer model was successful. Compared with the model group, the intervention effect of the ET-22 viable bacteria group was the best. The continuity of the hamster oral mucosal epithelium in this group was relatively complete, and the inflammatory cell infiltration was significantly reduced. However, in the ET-22 inactivated bacteria group and the ET-22 extracellular metabolite group, there were some damages in the hamster oral mucosal epithelium and some inflammatory cell infiltrations. In the K12 viable bacteria group and the HN019 viable bacteria group, the integrity of the hamster oral mucosal epithelium was relatively high, but there were a small number of inflammatory cell infiltrations. In the DSM17938 viable bacteria group and the AP32 viable bacteria group, the continuity of the hamster oral mucosal epithelium was incomplete, and there was an obvious inflammatory cell infiltration phenomenon. Vitamin C is usually considered to be helpful for the prevention and treatment of oral ulcers. As can be seen from the figure, in the VC group, the deletion of the hamster oral mucosal epithelium was improved compared with the model group, but it was still damaged relatively severely and there were many inflammatory cell infiltrations. These results indicate that by ingesting ET-22, especially viable ET-22 bacteria, the risk of oral ulcer development can be reduced, ulcer symptoms can be alleviated, and it has a better effect than pre-supplementing VC.

[0059] Immunohistochemical results of mucosal tissue: NF-κB is an important transcription factor. Activation of its signaling pathway can initiate an inflammatory response. Moreover, activation of the NF-κB signaling pathway can promote the upregulated expression of MMP-9, which can degrade laminin and type IV collagen to accelerate the infiltration and migration of inflammatory cells. High expression of Caspase 3 can promote the upregulation of apoptosis and can also hydrolyze PARP, so that PARP can no longer participate normally in DNA damage repair, leading to the progression of apoptosis.

[0060] Immunohistochemical photographs (scale: 100 μm) of typical hamster oral ulcer mucosal tissues 48 h after the construction of the prevention group model are shown in Figure 5. As shown in Figure 5 and Table 1, in the normal control group, NF-κB, MMP-9, Caspase 3, and PARP in the hamster oral mucosal tissues were all negative expressions, while in the model group, the expressions of the above four proteins all increased significantly (P < 0.05). The significant increase in the expressions of NF-κB and MMP-9 in the model group suggested that an inflammatory reaction occurred in the oral mucosa and the connection between the basement membrane and cells was disrupted. The significant increase in the expressions of Caspase 3 and PARP in the model group suggested that the apoptosis of mucosal cells was upregulated, causing epithelial damage and shedding of the ulcerated mucosa. The positive expressions of these proteins in the model group were consistent with the results by HE staining.

[0061] Compared with the model group, the expression of these four proteins in the ET-22 live bacteria group, ET-22 inactivated bacteria group, ET-22 extracellular metabolite group, and K12 live bacteria group all decreased significantly (P<0.05). In the HN019 live bacteria group, the expression of all except NF-κB decreased significantly. In the DSM17938 live bacteria group, only the expression of Caspase 3 and PARP decreased significantly, but there was no significant difference in the expression of NF-κB and MMP-9 compared with the model group. In the AP32 live bacteria group, the expression of NF-κB, Caspase 3, and PARP decreased significantly, but there was no significant difference in the expression of MMP-9 compared with the model group. In the VC group, only the expression of NF-κB and Caspase 3 decreased significantly, and the expression of MMP-9 and PARP was downregulated but there was no significant difference compared with the model group (Table 1). These results indicate that ET-22 plays a positive role in the prevention of oral ulcers.

Table 3

[0062] Levels of lipoxin A4 (LXA4) and prostaglandin E2 (PGE2) in oral ulcer mucosal tissue: LXA4 is an endogenous lipid with anti-inflammatory effects and can inhibit the secretion of pro-inflammatory cytokines such as IL-6.

[0063] The results of the LXA4 and PGE2 levels in the hamster oral ulcer mucosal tissue are shown in Figure 6. As shown in Figure 6, compared with the normal control group, there was no significant change in the LXA4 content in the model group. Since no intervention was performed on the model group, it was speculated that the activation degree of LXA4 was low and the resolution of inflammation was slow, resulting in severe and persistent ulcers. Compared with the model group, the LXA4 content in the ET-22 live bacteria group, ET-22 extracellular metabolite group, and K12 live bacteria group increased significantly (P<0.05), indicating that these groups may exert anti-inflammatory effects by increasing the LXA4 level and then reducing oral ulcer symptoms. There was no significant difference between the ET-22 inactivated bacteria group, HN019 live bacteria group, DSM17938 live bacteria group, AP32 live bacteria group, and VC group and the model group.

[0064] PGE2 can exacerbate the inflammatory response, promote local vasodilation, and increase capillary permeability. Compared with the normal control group, the content of PGE2 in the model group was significantly increased, and severe inflammation appeared in the oral mucosa of the model group, indicating that it was consistent with the severity of the ulcer phenotype. Compared with the model group, PGE2 was significantly decreased in the ET-22 live bacteria group, ET-22 inactivated bacteria group, ET-22 extracellular metabolite group, K12 live bacteria group, and VC group (P<0.05), and its level was between the model group and the control group. However, there was no significant difference between the HN019 live bacteria group, DSM17938 live bacteria group, and AP32 live bacteria group and the model group.

[0065] Levels of pro-inflammatory cytokines in serum: The levels of pro-inflammatory cytokines IL-6 and IL-1β in hamster serum are shown in Figure 6. Compared with the normal control group, the levels of pro-inflammatory cytokines IL-6 and IL-1β in the hamster serum of the model group were significantly increased (P<0.05, Figure 6), indicating that the model of oral ulcer was successfully constructed. The IL-6 concentrations in the hamster serum of the ET-22 live bacteria group, ET-22 inactivated bacteria group, ET-22 extracellular metabolite group, and K12 live bacteria group were all significantly lower than those in the model group (P<0.05), indicating that ET-22 and K12 can reduce the inflammatory response and thus act to relieve oral ulcers. Furthermore, the concentrations of IL-1β in the hamster serum of all intervention groups were all significantly lower than those in the model group (P<0.05). This result suggested that ET-22 and K12 had better immunomodulatory effects than other groups and could inhibit the formation or exacerbation of oral ulcers by regulating the body's immunity.

[0066] SOD activity, MDA, and GSH concentrations in serum SOD and GSH are important antioxidant enzymes and antioxidants, and an increase in these indicates that the body has stimulated an increase in antioxidants due to the occurrence of oxidative stress. MDA is a lipid peroxidation product and reflects the degree of damage caused by oxidative stress.

[0067] The SOD activity, GSH, and MDA levels in the serum of hamsters 48 h after the construction of the prevention group model are shown in Fig. 7. Compared with the normal control group, in the model group, the GSH and MDA levels in the serum of hamsters significantly increased (P < 0.05), and the SOD activity also increased but not significantly. Compared with the model group, in the ET-22 viable bacteria group, ET-22 inactivated bacteria group, and VC group, the SOD activity, GSH, and MDA levels all significantly decreased (P < 0.05), but in the ET-22 extracellular metabolite group, only the MDA level significantly decreased. In each of the remaining control strain groups, the MDA level significantly decreased, but no obvious regulatory effect was observed on either the SOD activity or GSH level. The results of the VC group were consistent with the antioxidant properties of VC as expected, and the results of the ET-22 viable bacteria group and ET-22 inactivated bacteria group indicated that they were similar to the effects of VC, could reduce oxidative stress damage to a certain extent, and had good antioxidant potential.

[0068] Diversity and composition of the oral microbiota The composition of the hamster oral bacteria at the phylum level 48 h after the construction of the prevention group model is shown in Fig. 8. As is evident from the phylum-level composition analysis (Fig. 8), the oral core microbiota of hamsters includes the phyla Firmicutes, Bacteroidota, Proteobacteria, Fusobacteriota, etc., which is consistent with previous reports.

[0069] The bacterial genera with significant differences in the hamster oral cavity 48 h after preventive group model construction are shown in Fig. 9. As is clear from the genus-level analysis (Fig. 9), the relative abundances of Bergeyella and Finegoldia in the oral cavity of the model group hamsters were significantly higher than those of the normal control group (P<0.05). Bergeyella has been found to be closely related to oral diseases such as periodontal disease, and Finegoldia can induce inflammation by activating neutrophil cells. Compared with the model group, viable ET-22 bacteria significantly decreased the occupancy rates of Bergeyella and Finegoldia, and the extracellular metabolites of ET-22 significantly decreased the ratio of Bergeyella (P<0.05), but no significant changes were observed in the inactivated ET-22 bacterial flora. Therefore, viable ET-22 bacteria may reduce the risk of oral ulcer development by suppressing oral harmful bacteria.

Example

[0070] Example 3 This example compared the effects of Lactobacillus paracasei ET-22, extracellular metabolites of ET-22, and Streptococcus salivarius K12 on the biofilm formation amount and biofilm structure of Streptococcus mutans.

[0071] Construction of a saliva-coated hydroxyapatite model: A mixed solution of a staining solution and a bacterial solution was prepared for use. As the bacterial solutions, a viable ET-22 bacteria solution of Lactobacillus paracasei, an extracellular metabolite solution of ET-22, and a solution of Streptococcus salivarius K12 were selected, and the bacterial solution concentration was 10 9CFU / ml, and the concentration of the extracellular metabolite solution was taken as the equivalent bacterial concentration. Hydroxyapatite (HA) beads were accurately taken and autoclaved, then placed in a 24-well culture plate. 1.5 ml of artificial saliva containing 0.2% sucrose was added to each well and immersed, and cultured at 37 °C for coating. Then, it was washed twice with 1.5 ml of sterile PBS, the buffer was aspirated and dried, 750 ml of the bacterial suspension was added to each well, and cultured at 37 °C for 2 h (during which, different time points were selected for measurement, and before each measurement, it was washed with 1.5 ml of sterile PBS, washed 3 times for 10 s each time). A saliva-coated hydroxyapatite model was created to simulate the salivary acquired film structure on the tooth surface. The amount of biofilm formation was represented by the result measured at 595 nm by a microplate reader using the crystal violet staining method. To detect using a fluorescence bioimaging system and quantify the staining status of the bacterial biofilm and the colonization status of bacteria in the HA model, the fluorescence intensity threshold for each fluorescence color was manually set, and fluorescence intensity analysis of the fluorescence imaging was performed using the Spectral instruments imaging software program.

[0072] Figure 10 shows the changes in the biomass of the simulated tooth surface biofilm due to the intervention of each probiotic group in the simulated saliva environment. As can be seen from Figure 10, the amount of biofilm formation in the ET-22 inactivated cell group and the extracellular metabolite group decreased significantly, and the difference was extremely significant (P < 0.00001). Comparatively, the amount of biofilm formation in the ET-22 extracellular metabolite group was the lowest. The amount of biofilm formation in the K12 live bacteria group was higher than that in the control group in all cases.

[0073] In this example, SEM was used to analyze the change in the biomass structure of the simulated tooth surface biofilm due to the intervention of each probiotic group in a simulated saliva environment. Fig. 11 is a scanning electron microscope result diagram of the change in the biomass structure of the simulated tooth surface biofilm due to the intervention of different probiotic groups in Example 3. As shown in Fig. 11, in the artificial saliva culture system control group, a cauliflower-like multi-layer deposited biofilm three-dimensional structure was formed. However, in both the ET-22 inactivated bacterial group and the extracellular metabolite group, the content of the biofilm was significantly suppressed, the cauliflower-like biofilm structure was destroyed, and the K12 live bacterial group showed a monolayer-deposited biofilm state, and the inhibitory effect was not obvious. Here, the inhibitory effect of the ET-22 extracellular metabolite group was the most significant, the biofilm showed a monolayer and scattered state, and it was significantly superior to other treatment groups.

[0074] In this example, a laser confocal microscope was used to analyze the change in the biomass structure and thickness of the simulated tooth surface biofilm due to the intervention of each probiotic group in a simulated saliva environment. As shown in Fig. 12 for the structural change, the bacterial content in the biofilm of the control group was mainly live bacteria (green). The content of dead bacteria (red) in the biofilm of the ET-22 inactivated bacterial group increased significantly. The ET-22 extracellular metabolite extremely significantly reduced the number of live bacteria in the biofilm and decreased the distribution area of the biofilm. As shown in Fig. 13 for the change in the thickness of the biofilm, the thickness of the biofilm in the control group was as high as 120 μm. However, in both the ET-22 inactivated bacterial group and the extracellular metabolite group, the thickness of the biofilm was significantly suppressed. The average thickness of the biofilm was only 62 μm after the treatment with the ET-22 extracellular metabolite, which was 48% lower than that of the control group.

[0075] It should be noted that the above is only for explaining the technical solution of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Use in the manufacture of a medicament for preventing and treating oral ulcers with Lactobacillus paracasei, wherein the Lactobacillus paracasei contains Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites, and the Lactobacillus paracasei ET-22 is the strain with the deposit number CGMCC No. 15077.

2. The Lactobacillus paracasei ET-22 cells are viable cells and / or inactivated cells, The extracellular metabolites of Lactobacillus paracasei ET-22 are produced by removing the cells from the incubation broth of Lactobacillus paracasei ET-22 to obtain the extracellular metabolites of Lactobacillus paracasei ET-22. Preferably, the incubation broth is obtained by incubating Lactobacillus paracasei ET-22 in water. The use according to Claim 1.

3. The oral ulcers include oral ulcers caused by pathogenic bacteria, oral ulcers caused by oxidative damage, or oral ulcers caused by immune deficiency. The use according to Claim 1 or 2.

4. A probiotic composition containing Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites, and the Lactobacillus paracasei ET-22 is the strain with the deposit number CGMCC No. 15077.

5. Further comprising sodium carboxymethylcellulose, propolis, borneol, lubricant, glycerin and Tween20, Preferably, the parts by weight of each component are 15-20 parts, for example 18-20 parts of Lactobacillus paracasei ET-22 cells and / or its extracellular metabolites 10-20 parts, for example 14-20 parts of sodium carboxymethylcellulose 20-40 parts, for example 25-35 parts of propolis 2-5 parts of borneol 2-5 parts of lubricant 8-12 parts of glycerin 1-3 parts of Tween20 The probiotic composition according to Claim 4.

6. The Lactobacillus paracasei ET-22 cells are viable cells and / or inactivated cells, The extracellular metabolite of Lactobacillus paracasei ET-22 is produced by removing the bacterial cells from the incubation broth of Lactobacillus paracasei ET-22 to obtain the extracellular metabolite of Lactobacillus paracasei ET-22. Preferably, the incubation broth is obtained by incubating Lactobacillus paracasei ET-22 in water. The probiotic composition according to claim 4 or 5.

7. Preferably, the oral ulcer includes an oral ulcer caused by a pathogenic bacterium, an oral ulcer caused by oxidative damage, or an oral ulcer caused by immune deficiency. Use in the manufacture of a composition for preventing and treating the oral ulcer of the composition according to any one of claims 4 to 6.

8. The composition for preventing and treating the oral ulcer is a pharmaceutical composition. Preferably, the pharmaceutical composition is a patch. The use according to claim 7.

9. (1) A step of uniformly mixing Lactobacillus paracasei ET-22 bacterial cells and / or its extracellular metabolite, propolis, sodium carboxymethyl cellulose, and a lubricant to obtain a mixed system A. (2) A step of heating borneol, glycerin, and Tween 20 by decocting and uniformly mixing them to obtain a mixed system B. (3) A step of uniformly mixing the mixed system A and the mixed system B. (4) A step of drying treatment A method for manufacturing a probiotic patch, comprising:

10. A probiotic patch produced from the composition according to any one of claims 4 to 6, or produced by the method according to claim 9.

11. A method for treating an oral ulcer, comprising administering to a subject an effective amount of the composition according to any one of claims 4 to 6 and / or the probiotic patch according to claim 10, wherein the subject includes a mammal or a human. A method for treating an oral ulcer.

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