A probiotic patch for preventing and treating oral ulcers and its preparation method and application

Abstract

The present invention provides a probiotic patch for preventing and treating oral ulcers and its preparation method and application. The present invention first provides the application of Lactobacillus paracasei in the preparation of a medicine for preventing and treating oral ulcers, wherein the Lactobacillus paracasei includes Lactobacillus paracasei ET-22 thalli and / or its extracellular metabolites, and the Lactobacillus paracasei ET-22 is a strain with a preservation number of CGMCC No.15077. The present invention also provides a probiotic composition, which includes: Lactobacillus paracasei ET-22 thalli and / or its extracellular metabolites, sodium carboxymethyl cellulose, propolis, borneol, lubricant, glycerol and Tween-20. The probiotic composition of the present invention can be made into a patch, which can effectively prevent and treat oral ulcers, is safe and reliable, and has a significant effect.

Description

Technical Field

[0001] The present invention relates to a probiotic patch and a preparation method and application thereof, and in particular to an application of Lactobacillus paracasei ET-22 in preparing a medicine for preventing and treating oral ulcers, a probiotic composition containing Lactobacillus paracasei ET-22, a probiotic patch prepared from the composition, a preparation method and an application thereof. Background Art

[0002] Oral ulcers, commonly known as "mouth sores", are a common symptom of ulcerative damage to the oral mucosa. The occurrence of oral ulcers is the result of the combined effects of multiple factors, including local trauma, mental stress, food, drugs, malnutrition, changes in hormone levels, and vitamin or trace element deficiencies.

[0003] It is known in the prior art that various probiotics are helpful in treating oral ulcers. CN109985179A discloses a composition for treating oral ulcers, which comprises the following raw materials in parts by weight: 1000-15000 parts of fresh asparagus, 0.1-10 parts of Lactobacillus salivarius, 0.1-20 parts of Bifidobacterium lactis, 0.1-20 parts of Lactobacillus paracasei, 0.1-50 parts of Indigo Naturalis, and 10-1000 parts of starch; the composition can supplement the trace elements required by the human body, has the effects of intrinsic immune regulation and oral environment improvement for patients with oral ulcers, enhances the body's immunity, and promotes the healing of ulcers. When used specifically, it is made into oral medicines such as pills, powders, capsules, tablets or granules. CN113425831A discloses a probiotic composition for treating oral ulcers, a preparation method and an application thereof. The probiotic composition comprises the following raw materials in parts by weight: 40-60 parts of tauropema powder, 30-60 parts of elizabeth chinensis leaves, 10-20 parts of probiotics, 1-5 parts of osmanthus fragrans, 1-5 parts of oligomannans, 1-5 parts of lindera agglomerata leaves, and 1-5 parts of colostrum alkaline protein. The composition adopts tauropema powder and elizabeth chinensis leaves with antioxidant effects to achieve the effect of treating oral ulcers and reduce the amount of probiotics. Oligomannans can also promote the growth of probiotics and coordinate the probiotics. At the same time, the osmanthus fragrans, rice bran fatty alkyl alcohols, lindera agglomerata leaves and colostrum alkaline protein contained in the composition all have the effect of treating oral ulcers, thereby preventing and improving oral ulcers. The composition is made into a spray when used specifically.

[0004] Among the above-mentioned prior art drugs containing probiotics for treating oral ulcers, the contribution of probiotics themselves to the treatment of oral ulcers is not outstanding. In addition, oral drugs have disadvantages such as a wide site of action, a large amount of drug required, and a low drug utilization rate. After spraying, drinking water and eating are prohibited for a certain period of time, which causes many inconveniences during use. Summary of the invention

[0005] An object of the present invention is to provide a probiotic 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 preparation of a medicament for preventing and treating oral ulcers.

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

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

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

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

[0011] The inventors of this case found in their research that Lactobacillus paracasei ET-22 is a probiotic that can prevent and treat oral ulcers, and its bacteria and / or its extracellular metabolites can significantly prevent and treat oral ulcers. Further, Lactobacillus paracasei ET-22 and / or its extracellular metabolites are prepared into probiotic patches, which can effectively sterilize the oral ulcer site and effectively prevent secondary infection of the wound.

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

[0013] Thus, on the one hand, the present invention provides the use of Lactobacillus paracasei in preparing a medicine for treating oral ulcers, wherein the Lactobacillus paracasei includes Lactobacillus paracasei ET-22 bacteria and / or its extracellular metabolites, and the Lactobacillus paracasei ET-22 is a strain with a preservation number of 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 available to the public.

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

[0016] According to a specific embodiment of the present invention, in the present invention, the extracellular metabolite of Lactobacillus paracasei ET-22 is prepared according to the following method:

[0017] The bacteria are removed from the incubation liquid of Lactobacillus paracasei ET-22 to obtain extracellular metabolites of Lactobacillus paracasei ET-22; preferably, the incubation liquid is obtained by incubating Lactobacillus paracasei ET-22 in water.

[0018] Preferably, the Lactobacillus paracasei ET-22 cells are concentrated at 1 to 5 × 10 9 The mixture was dissolved in water at a concentration of CFU / mL, and incubated with stirring at 37°C and 200-500 rpm for 2-4 hours. After the incubation, the bacteria were removed by centrifugation to obtain 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 oral ulcer caused by pathogenic bacteria, oral ulcer caused by oxidative damage or oral ulcer caused by immunosuppression.

[0020] On the other hand, the present invention further provides a probiotic composition, comprising: Lactobacillus paracasei ET-22 bacteria and / or its extracellular metabolites; the Lactobacillus paracasei ET-22 is a strain with a preservation number of 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, lubricant, glycerol and Tween-20; preferably, the weight parts of each component are:

[0022]

[0023] According to a specific embodiment of the present invention, in the probiotic composition of the present invention, the weight proportions of the components are:

[0024]

[0025] According to a specific embodiment of the present invention, in the probiotic composition of the present invention, the Lactobacillus paracasei ET-22 bacteria are live bacteria and / or inactivated bacteria.

[0026] 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 prepared according to the following method:

[0027] Lactobacillus paracasei ET-22 cells were added at a rate of 1-5 × 10 9 The mixture was dissolved in water at a concentration of CFU / mL, and incubated with stirring at 37°C and 200-500 rpm for 2-4 hours. After the incubation, the bacteria were removed by centrifugation to obtain extracellular metabolites of Lactobacillus paracasei ET-22.

[0028] 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.

[0029] On the other hand, the present invention also provides the use of the probiotic composition in preparing a composition for preventing and treating oral ulcers.

[0030] According to a specific embodiment of the present invention, the oral ulcer comprises the oral ulcer caused by pathogens, the oral ulcer caused by oxidative damage or the oral ulcer caused by immunosuppression. In some specific embodiments of the present invention, the viable bacteria of Lactobacillus paracasei ET-22 can significantly prevent and treat the oral ulcer caused by pathogens or oxidative damage. In some specific embodiments of the present invention, the smearing of the inactivated bacteria of Lactobacillus paracasei ET-22 can significantly prevent and treat the oral ulcer caused by immunosuppression. In some specific embodiments of the present invention, the extracellular metabolites of Lactobacillus paracasei ET-22 have a significant effect for preventing and treating oral ulcers.

[0031] 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.

[0032] According to some specific embodiments of the present invention, the composition for preventing and treating oral ulcers of the present invention can be a general cosmetic.

[0033] On the other hand, the present invention also provides a method for preparing a probiotic patch, the method comprising:

[0034] (1) uniformly mixing Lactobacillus paracasei ET-22 cells and / or extracellular metabolites thereof, propolis, sodium carboxymethyl cellulose and a lubricant to obtain a mixed system A;

[0035] (2) heating and mixing borneol, glycerol and Tween-20 in water to obtain a mixed system B;

[0036] (3) a step of uniformly mixing the mixed system A and the mixed system B;

[0037] (4) Drying treatment step.

[0038] It should be noted that the numbers (1) and (2) of the steps of the preparation method of the present invention do not mean that the two steps must be performed in the described order. In actual operation, step (2) can be performed first and then step (1), or the two steps can be performed simultaneously.

[0039] On the other hand, the present invention further provides a probiotic patch, which is prepared from the composition of the present invention, or prepared by the above-mentioned probiotic patch preparation method.

[0040] In summary, the present invention provides the use of Lactobacillus paracasei ET-22 and / or its extracellular metabolites in preventing and treating oral ulcers, and provides the prepared probiotic patch. The present invention proves through an animal model that live Lactobacillus paracasei ET-22 bacteria can significantly prevent and treat oral ulcers caused by pathogens or oxidative damage; the application of inactivated Lactobacillus paracasei ET-22 bacteria can significantly prevent and treat oral ulcers caused by immunosuppression; Lactobacillus paracasei ET-22 extracellular metabolites have good effects when used to prevent and / or treat oral diseases. The probiotic patch of the present invention has good adhesion to the oral mucosa; the addition of borneol also has the effects of clearing away heat and detoxifying, reducing swelling and relieving pain; it can effectively sterilize the oral ulcer site and effectively prevent secondary infection of the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a graph showing the tongue injury score of ICR mice in the prevention group of Example 2 48 hours after modeling.

[0042] Figure 2 This is a graph showing the tongue injury score results of ICR mice in the treatment group of Example 2 72 hours after modeling.

[0043] Figure 3 This is the HE staining result of tongue dorsum lesion sections of mice in the blank group, model group and probiotic group 48 hours after modeling in the prevention group of Example 2; in the figure, A is the papillary protrusions of epithelial cells in the tongue tissue, and B is tissue hyperplasia.

[0044] Figure 4 This is a typical HE-stained photograph of a hamster oral mucosal tissue section 48 hours after modeling in the prevention group of Example 2; in the figure, the white arrow points to inflammatory cell infiltration, the black arrow points to mucosal epithelial damage and shedding, and the gray arrow points to capillary filling and dilation and glandular hyperplasia (scale: 200 μm).

[0045] Figure 5 This is a typical immunohistochemical photograph of the mucosal tissue of oral ulcer in the prevention group of Example 2 48 hours after modeling (scale: 100 μm).

[0046] Figure 6 This is a graph showing the results of Example 2 showing the levels of LXA4 and PGE2 in the mucosal tissue of oral ulcers in hamsters and the levels of pro-inflammatory cytokines IL-6 and IL-1β in serum.

[0047] Figure 7 This is a graph showing the results of SOD activity, GSH and MDA levels in hamster serum in Example 2.

[0048] Figure 8 This is the composition of hamster oral bacteria at the phylum level in Example 2.

[0049] Fig. 9 These are the bacterial genera with significant differences in the oral cavity of hamsters in Example 2.

[0050] Fig.10 This is a graph showing the changes in biomass of biofilm on simulated tooth surfaces under the intervention of different probiotics in Example 3;

[0051] Fig.11 This is a scanning electron microscopy result of the changes in the biomass structure of the biofilm on the simulated tooth surface under the intervention of different probiotics in Example 3.

[0052] Fig.12 This is a laser confocal microscopy result diagram of the changes in the biomass structure of the biofilm on the simulated tooth surface under the intervention of different probiotics in Example 3.

[0053] Fig.13 This is a graph showing the changes in biofilm thickness on the simulated tooth surface under the intervention of different probiotics in Example 3. DETAILED DESCRIPTION

[0054] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the following detailed description is now given in conjunction with specific embodiments and the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. In the embodiments, each raw reagent material can be obtained commercially, and the experimental method without specifying specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0055] Unless specifically defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the relevant art.

[0056] In each embodiment of the present invention, the extracellular metabolite of Lactobacillus paracasei ET-22 is prepared according to the following method: inoculating Lactobacillus paracasei ET-22 into a culture medium, culturing to obtain a bacterial solution, and centrifuging the ET-22 bacterial solution to obtain Lactobacillus paracasei ET-22 bacterial bodies (bacterial mud);

[0057] The obtained Lactobacillus paracasei ET-22 cells were concentrated to 1-5×10 9 The mixture was dissolved in water at a concentration of 1.5 CFU / mL, and incubated with stirring at 37°C and 200-500 rpm for 3 hours. After the incubation, the bacteria were removed by centrifugation to obtain the extracellular metabolites of Lactobacillus paracasei ET-22.

[0058] Example 1

[0059] This embodiment provides a probiotic patch for treating oral ulcers. The raw materials for preparing the probiotic patch include Lactobacillus paracasei ET-22 bacteria and / or its extracellular metabolites, sodium carboxymethyl cellulose, propolis, borneol, lubricant, glycerol and Tween-20, and the weight parts of each raw material are:

[0060]

[0061] The method for preparing the probiotic patch comprises the following steps:

[0062] (1) according to the above-mentioned weight ratio, Lactobacillus paracasei ET-22 bacteria and / or extracellular metabolites, propolis, sodium carboxymethyl cellulose and lubricant are mixed to obtain a mixed system A;

[0063] (2) placing borneol, glycerol and Tween-20 in a container according to the above weight ratio, heating them in water bath, stirring and mixing them uniformly to obtain a mixed system B;

[0064] (3) After the mixed system A and the mixed system B are uniformly mixed, the mixed system A and the mixed system B are dried to obtain the probiotic patch for treating oral ulcers.

[0065] Example 2: Effect of Lactobacillus paracasei ET-22 on the prevention and treatment of oral ulcers

[0066] 1. Experimental Animal Model

[0067] 1.1 Oral ulcer model caused by immunosuppression

[0068] Prevention group animal model: To induce oral candidiasis in mice, the present invention used the previously described protocol with some minor modifications. After the adaptation period of mice, the experimental group received an 18-day probiotic intervention (10 9 CFU / ml), and the blank group and the model group drank water normally. On the 15th day, the mice were given 15mg / ml tetracycline hydrochloride through drinking water, and at the same time, the immunosuppressive state was induced, and prednisolone (100mg / kg) was injected subcutaneously. On the 16th day, the model group and the experimental group were infected with Candida albicans CGMCC 2.4122, and 4% chloral hydrate (10ml / kg) was used for anesthesia during infection); then 1.0×10 9 CFU / ml of Candida albicans, a cotton swab impregnated with Candida albicans was placed on the dorsum of the tongue for 15 minutes. Mice were euthanized 48 hours after surgery.

[0069] Animal model of treatment group: To induce oral candidiasis in mice, the present invention used the previously described protocol with some minor modifications. After the adaptation period of mice, mice were given 15mg / ml tetracycline hydrochloride through drinking water, and at the same time, an immunosuppressive state was induced, and prednisolone (100mg / kg) was injected subcutaneously. On the second day, the model group and the experimental group were infected with Candida albicans CGMCC 2.4122, and 4% trichloraldehyde hydrate (10ml / kg) was used for anesthesia during infection); then 1.0×10 9 CFU / ml of Candida albicans, a cotton swab soaked with Candida albicans was placed on the tongue dorsum for 15 minutes. 24 hours after infection (day 3), the probiotic intervention group was smeared with probiotics every 12 hours, and the attack smear was 4 times. The smear method was the same as the infection smear method. The mice were euthanized 72 hours after surgery (day 5).

[0070] 1.2 Oxidative damage oral ulcer model in golden hamsters

[0071] Six-week-old male LVG Golden Syrian Hamsters, weighing (110±20) g / hamster, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and kept under 12h light alternation, temperature maintained at 20±2℃, and humidity maintained at 45%-50%. After acclimation in the breeding room for one week, the hamsters were randomly divided into 10 groups, namely normal control group, model group, ET-22 live bacteria group, ET-22 inactivated bacteria group, ET-22 extracellular metabolite group, live bacteria groups of K12, HN019, DSM17938, AP32, and vitamin C positive control group (VC group). There were 7 hamsters in the normal control group and 10 hamsters in each of the other groups. The hamsters in the normal control group and the model group were gavaged with 1 ml of normal saline every day, and the ET-22 live bacteria group was gavaged with 1 ml of 10 9CFU / ml of ET-22 live bacteria solution, ET-22 inactivated bacteria group and ET-22 extracellular metabolite group were gavaged with 1ml of corresponding concentration of ET-22 inactivated bacteria and bacterial extracellular metabolites, and VC group was gavaged with 1ml of 20mg / ml vitamin C solution every day. Oral ulcer model was established 15 days after gavage, and hamsters were anesthetized by intraperitoneal injection of 10% chloral hydrate (injection volume of 0.3-0.5% body weight), and 0.25ml PBS buffer (pH=7.4) was injected into the cheek pouch of hamsters in the normal control group, and 0.25ml methyl viologen (10mmol / L, dissolved in PBS) was injected into the cheek pouch of hamsters in the other 5 groups; from the day of injection until the hamsters were killed, each group still maintained daily gavage intervention. On the 4th day after modeling, the hamsters were anesthetized and blood was collected. Then, the inside of the hamster's mouth (except the ulcer area) was scraped with an oral swab, and the swab head was cut and placed in a centrifuge tube filled with nucleic acid protection solution; oral ulcer lesion tissue or normal mucosa was cut and divided into two parts, one fixed in 4% paraformaldehyde and the other frozen in liquid nitrogen. After the whole blood was separated from the serum, it was packaged and frozen at -80℃.

[0072] 2. Experimental Animal Grouping

[0073] The prevention group was divided into the following 10 groups:

[0074]

[0075] The treatment group was divided into the following 7 groups:

[0076]

[0077] Oral infection severity scoring - mice

[0078] 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. Macroscopic evaluation of infection was expressed by lesion scores from 0 to 4, according to the extent and severity of white curd-like plaques on the tongue, as follows: 0 points, normal; 1 point, white plaques less than 20%; 2 points, white plaques less than 90% but more than 21%; 3 points, white plaques more than 91%; thick white plaque-like pseudomembranes accounting for more than 91%.

[0079] The whole tongue of the mouse was cut into two parts longitudinally in the middle of the tongue tip, and one of them was fixed in 4% paraformaldehyde neutral buffer at room temperature and then embedded in paraffin. The sample was cut into 5 μm thick continuous longitudinal sections along the tongue tip and then stained using the hematoxylin method. References Leica fluorescence microscope was used for observation and staining.

[0080] Oral infection severity score - golden hamster

[0081] After the paraffin sections were dewaxed and gradiently dehydrated in sequence, antigen retrieval was performed using citric acid antigen retrieval buffer (pH=6.0), and then the sections were placed in 3% hydrogen peroxide solution and incubated in the dark for 25 minutes to block endogenous peroxidase, and then subjected to the steps of blocking, primary antibody incubation, secondary antibody incubation, and finally DAB color development and cell nucleus counterstaining, dehydration, sealing, observation under a microscope, and photography. Immunohistochemistry was scored using the IRS (Immunoreactive Score) scoring method. The staining degree (0-3 points) and positive rate (0-4 points) were scored separately, and then multiplied to obtain a comprehensive score (0-12 points); the scoring criteria were as follows: the staining degree was scored according to the staining characteristics of the target protein: no staining was 0 points, light yellow was 1 point, brownish yellow was 2 points, and brownish brown was 3 points; the positive rate was scored according to the positive ratio of cells in the section: 0-5% was 0 points, 6%-25% was 1 point, 26%-50% was 2 points, 51%-75% was 3 points, and >75% was 4 points. The comprehensive score was 0 points for negative (-); 1-3 points for weak positive (+); 4-5 points for positive (++); 6-7 points for strong positive (+++).

[0082] Tissue lesion observation results:

[0083] Figure 1 This is the result graph of tongue injury score of ICR mice in the prevention group of Example 2 48 hours after modeling. Figure 1 It can be seen that 48 hours after the operation, only the ET-22 live bacteria group showed significant difference in tongue injury scores compared with the model group (p<0.05), and the average score decreased by 46.23%; the average score of the DSM 17938 live bacteria group was consistent with that of the model group; although there was no significant difference in the injury scores of the other experimental groups, they also decreased to varying degrees. The ET-22 secretion group (extracellular metabolite group), K12 live bacteria group, HN019 live bacteria group, and AP32 live bacteria group decreased by 31.9%, 32.26%, 35.48%, and 41.94%, respectively; the ET-22 inactivated bacteria group and L9 live bacteria group decreased by 16.13% and 16.13%, respectively.

[0084] Figure 2 This is a graph showing the tongue injury score of ICR mice in the treatment group of Example 2 72 hours after modeling. Figure 2 It can be seen that in the treatment group, only the ET-22 inactivated bacteria group showed significant difference compared with the tongue injury score of the model group mice (p<0.05), but the probiotic groups all showed a certain cure rate. The cure rate of the metabolite of Lactobacillus paracasei ET-22 was the lowest at 11%, and the damage score decreased by the least 23%. The other cure rates were between 30% and 40%, and the damage score decreased by 50% to 60%.

[0085] Analysis of injury scores in the prevention group:

[0086] Sanae.A.Ishijima et al. investigated the effect of Streptococcus salivarius K12 on the tongue of the mouse model. The average lesion score of the tongue in the model group was 3.4. After 1.5×10 9 The average lesion score after treatment with CFU / mL S. salivarius K12 was 2.0.

[0087] The present invention has been tested in this prevention experiment with 1.0×10 9 The average lesion score after treatment with S. salivarius K12 was 2.1 and 1.0 × 10 9 The average injury scores of live Lactobacillus paracasei ET-22, inactivated bacteria, and extracellular metabolites were 1.7, 2.6, and 2.1, respectively. Live Lactobacillus paracasei ET-22 was superior to live Streptococcus salivarius K12 in preventing candida albicans. The extracellular metabolites of Lactobacillus paracasei and live Streptococcus salivarius K12 had the same efficacy in preventing candida albicans. Compared with live bacteria, the storage method of the extracellular metabolites was simpler and its application scenarios were wider.

[0088] Observation of tongue dorsum lesions in sections:

[0089] 48 hours after modeling in the prevention group, HE staining results of tongue dorsum lesion sections of mice in the blank group, model group and probiotic group were as follows: Figure 3 As shown in the figure, A is the papillary protrusions of the tongue epithelial cells, and B is tissue hyperplasia. Figure 3 It can be seen that compared with the blank group, the model group had severe inflammatory infiltration, the inflammatory infiltration of the ET-22 live bacteria group, ET-22 secretion group (extracellular metabolite group), K12 live bacteria group, HN019 live bacteria group, and AP32 live bacteria group was significantly improved, the inflammatory infiltration of the ET-22 inactivated bacteria group and the L9 live bacteria group was also relatively severe, and the inflammatory infiltration of the DSM 17938 live bacteria group was more serious than that of the model group.

[0090] Depend on Figure 3 It can be seen that compared with the blank group, the model group had severe loss of epithelial papillary processes in the tongue tissue, and obvious desquamation and tissue hyperplasia. The papillary processes of the epithelial cells in the tongue tissue of the ET-22 live bacteria group, the ET-22 secretion group, and the AP32 live bacteria group were well preserved without desquamation, but there was slight tissue hyperplasia compared with the blank group. The papillary processes of the epithelial cells in the tongue tissue of the ET-22 inactivated bacteria group had a relatively obvious loss, no desquamation, and a relatively obvious tissue hyperplasia. The papillary processes of the epithelial cells in the tongue tissue of the L9 live bacteria group had a certain loss, slight desquamation, and tissue hyperplasia similar to the model group. The papillary processes of the epithelial cells in the tongue tissue of the K12 live bacteria group were significantly lost, without desquamation, and had a certain amount of tissue hyperplasia. The papillary processes of the epithelial cells in the tongue tissue of the DSM 17938 live bacteria group were significantly lost, with slight desquamation and severe tissue hyperplasia.

[0091] From the above, it can be seen that the ET-22 live bacteria group, ET-22 extracellular metabolite group, AP32 live bacteria group, and K12 live bacteria group have certain effects on the prevention of oral candidiasis, reducing inflammation and tongue tissue lesions. The ET-22 live bacteria group has the best effect.

[0092] Mucosal tissue sections and HE staining:

[0093] Example 2: 48 hours after modeling in the prevention group, HE staining photos of typical hamster oral mucosa tissue sections are shown in Figure 4 As shown in the figure, the white arrow points to the infiltration of inflammatory cells, the black arrow points to the damage and shedding of the mucosal epithelium, and the gray arrow points to the filling and dilation of capillaries and glandular hyperplasia (scale: 200μm). Figure 4 As shown in the figure, the continuity of the oral mucosal epithelium of the hamsters in the normal control group was intact, the cells and glands were normal in morphology, and there was no obvious inflammatory cell infiltration. Compared with the normal control group, the oral mucosal epithelium of the hamsters in the model group was damaged and detached, the continuity was incomplete, the capillaries were filled and dilated, and a large number of inflammatory cells infiltrated, indicating that the oral ulcer model was successfully established. Compared with the model group, the intervention effect of the ET-22 live bacteria group was the best. The continuity of the oral mucosal epithelium of the hamsters in this group was relatively intact, and the inflammatory cell infiltration was significantly reduced; while the oral mucosal epithelium of the hamsters in the ET-22 inactivated bacteria group and the ET-22 extracellular metabolite group was still partially damaged, and there was some inflammatory cell infiltration. The oral mucosal epithelium of the hamsters in the K12 live bacteria and HN019 live bacteria groups was relatively intact, but there was still a small amount of inflammatory cell infiltration; the oral mucosal epithelium of the hamsters in the DSM17938 live bacteria and AP32 live bacteria groups was incomplete, and there was obvious inflammatory cell infiltration. Vitamin C is often considered to help prevent and treat oral ulcers. As can be seen from the figure, although the loss of oral mucosal epithelium in the VC group of hamsters was improved compared with the model group, the damage was still relatively serious, and there were more inflammatory cells infiltrating. These results show that consuming ET-22, especially ET-22 live bacteria, can reduce the risk of oral ulcers, alleviate ulcer symptoms, and have a better effect than pre-supplementation with VC.

[0094] Results of immunohistochemistry of mucosal tissues:

[0095] NF-κB is an important transcription factor, and the activation of its signaling pathway can initiate inflammatory responses. In addition, the activation of the NF-κB signaling pathway can promote the upregulation of MMP-9 expression, which can degrade laminin and type IV collagen, accelerating the infiltration and metastasis of inflammatory cells. The high expression of Caspase 3 may promote the upregulation of cell apoptosis and hydrolyze PARP, resulting in PARP being unable to participate in DNA damage repair normally, thereby aggravating cell apoptosis.

[0096] Typical immunohistochemical photos of oral ulcer mucosal tissues in the prevention group 48 hours after modeling (scale: 100 μm) Figure 5 As shown. Figure 5 As shown in Table 1, the NF-κB, MMP-9, Caspase 3, and PARP in the oral mucosal tissue of the hamsters in the normal control group were negatively expressed, while the expression of the above four proteins in the model group was significantly increased (P < 0.05). The expression of NF-κB and MMP-9 in the model group was significantly increased, indicating that the oral mucosa had an inflammatory reaction and the connection between the basement membrane and cells was damaged. The expression of Caspase 3 and PARP in the model group was significantly increased, indicating that the apoptosis of mucosal cells was upregulated, causing epithelial damage and shedding of the ulcerative mucosa. The positive expression of these proteins in the model group was consistent with the results of HE staining.

[0097] Compared with the model group, the expression of these four proteins in the ET-22 live bacteria, ET-22 inactivated bacteria, ET-22 extracellular metabolites and K12 live bacteria groups were significantly decreased (P < 0.05); except for NF-κB, the expression of the other proteins in the HN019 live bacteria group was significantly decreased; in the DSM17938 live bacteria group, only Caspase 3 and PARP expression was significantly decreased, and the expression of NF-κB and MMP-9 was not significantly different from the model group; in the AP32 live bacteria group, the expression of NF-κB, Caspase 3, and PARP was significantly decreased, and the expression of MMP-9 was not significantly different from the model group; in the VC group, only the expression of NF-κB and Caspase 3 was significantly decreased, and the expression of MMP-9 and PARP was downregulated but not significantly different from the model group (Table 1). The results show that ET-22 has a positive effect on the prevention of oral ulcers.

[0098] Table 1 Average scores of oral mucosal tissue immunohistochemistry

[0099]

[0100] Lipoxin A4 (LXA4) and prostaglandin E2 (PGE2) levels in oral ulcer mucosal tissue:

[0101] LXA4 is an endogenous lipid with anti-inflammatory properties that can inhibit the secretion of pro-inflammatory cytokines such as IL-6.

[0102] The results of LXA4 and PGE2 levels in the mucosal tissue of oral ulcers in hamsters are as follows Figure 6 shown. Figure 6The results showed that compared with the normal control group, the LXA4 content in the model group did not change significantly. It is speculated that since the model group did not undergo intervention, the LXA4 activation level was low and the inflammation subsided slowly, which also led to more severe and persistent ulcers. Compared with the model group, the LXA4 content in the ET-22 live bacteria group, the ET-22 extracellular metabolite group, and the K12 live bacteria group increased significantly (P < 0.05), indicating that they may play an anti-inflammatory role by increasing the level of LXA4, thereby alleviating the symptoms of oral ulcers; the ET-22 inactivated bacteria, HN019 live bacteria, DSM17938 live bacteria, AP32 live bacteria, and VC groups had no significant differences from the model group.

[0103] PGE2 can aggravate the inflammatory response, promote local vasodilation, and increase capillary permeability. Compared with the normal control group, the PGE2 content in the model group was significantly increased, indicating that the oral mucosa of the model group had severe inflammation, which was consistent with its severe ulcer phenotype. Compared with the model group, the PGE2 levels of ET-22 live bacteria, ET-22 inactivated bacteria, ET-22 extracellular metabolites, K12 live bacteria, and VC groups were significantly reduced (P < 0.05), and their levels were between the model group and the control group; while there was no significant difference between the HN019 live bacteria, DSM17938 live bacteria, and AP32 live bacteria groups and the model group.

[0104] Serum pro-inflammatory cytokine levels:

[0105] The levels of proinflammatory cytokines IL-6 and IL-1β in hamster serum Figure 6 Compared with the normal control group, the levels of proinflammatory cytokines IL-6 and IL-1β in the serum of the model group were significantly increased (P<0.05, Figure 6 ), indicating that the oral ulcer model was successfully established. The IL-6 concentration in the serum of hamsters in the ET-22 live bacteria, ET-22 inactivated bacteria, ET-22 extracellular metabolites group, and K12 live bacteria group was significantly lower than that in the model group (P < 0.05), indicating that ET-22 and K12 can reduce the inflammatory response and thus play a role in alleviating oral ulcers; in addition, the IL-1β concentration in the serum of hamsters in all intervention groups was significantly lower than that in the model group (P < 0.05). The results suggest that ET-22 and K12 have better immunoregulatory effects than other groups, and can resist the formation or deterioration of oral ulcers by regulating the body's immunity.

[0106] Serum SOD activity, MDA and GSH concentrations

[0107] SOD and GSH are important antioxidant enzymes and antioxidants. Their increase indicates that the body has stimulated the increase of antioxidant substances due to oxidative stress; MDA is a product of lipid peroxidation and reflects the degree of damage caused by oxidative stress.

[0108] After 48 hours of modeling, the SOD activity, GSH and MDA levels in the serum of the hamsters in the prevention group were as follows: Figure 7 As shown. Compared with the normal control group, the levels of GSH and MDA in the serum of the model group were significantly increased (P < 0.05), and the SOD activity was also increased but not significantly. Compared with the model group, the SOD activity, GSH and MDA levels of the ET-22 live bacteria, ET-22 inactivated bacteria and VC groups were significantly decreased (P < 0.05), and only the MDA level in the ET-22 extracellular metabolite group showed a significant decrease. In the other control strain groups, the MDA level was significantly reduced, but there was no obvious regulatory effect on the SOD activity and GSH level. The results of the VC group were as expected, which was consistent with the antioxidant properties of VC. The results of the ET-22 live bacteria and ET-22 inactivated bacteria groups showed that they were similar to VC in effect, could reduce oxidative stress damage to a certain extent, and had good antioxidant potential.

[0109] Diversity and composition of oral flora

[0110] 48 hours after modeling in the prevention group, the composition of oral bacteria in the hamster at the phylum level was as follows Figure 8 As shown. From the composition analysis at the phylum level, it can be seen that ( Figure 8 ), the core flora of hamster oral cavity included Firmicutes, Bacteroidota, Proteobacteria, Fusobacteriota, etc., which was consistent with previous reports.

[0111] 48 hours after the model was established in the prevention group, the bacterial genera with significant differences in the oral cavity of the hamsters were as follows Fig. 9 As shown. Subordinate level analysis found that ( Fig. 9 ), the relative abundance of Bergeyella and Finegoldia in the oral cavity of hamsters in the model group was significantly higher than that in the normal control group (P < 0.05). Bergeyella has been found to be closely related to oral diseases such as periodontal disease; Finegoldia can induce inflammation by activating neutrophils. Compared with the model group, ET-22 live bacteria significantly downregulated the proportion of Bergeyella and Finegoldia, and ET-22 extracellular metabolites significantly downregulated the proportion of Bergeyella (P < 0.05), while the ET-22 inactivated bacteria group did not show significant changes. Therefore, ET-22 live bacteria may reduce the risk of oral ulcers by inhibiting harmful oral bacteria.

[0112] Example 3

[0113] This example compares the effects of Lactobacillus paracasei ET-22, ET-22 extracellular metabolites, and Streptococcus salivarius K12 on the amount of biofilm produced and the biofilm structure of Streptococcus mutans.

[0114] Constructing saliva-coated hydroxyapatite model: prepare a mixture of dye solution and bacterial solution for use, the bacterial solution selected is Lactobacillus paracasei ET-22 live bacteria solution, ET-22 extracellular metabolite solution, Streptococcus salivarius K12 solution, the bacterial solution concentration is 10 9 CFU / ml, the concentration of the extracellular metabolite solution is the equivalent bacterial concentration. Hydroxyapatite (HA) beads were accurately taken and sterilized by high pressure, and then placed in a 24-well culture plate. 1.5 ml of artificial saliva containing 0.2% sucrose was added to each well and soaked in 37°C coating culture. After that, 1.5 ml of sterile PBS was used to wash twice and the buffer was aspirated. 750 ml of bacterial suspension was added each time and cultured at 37°C for 2 hours (different time periods were selected for measurement during this period, and 1.5 ml of sterile PBS was used to rinse before each measurement, each rinsing for 10 seconds, and rinsed three times). The saliva-coated hydroxyapatite model was made to simulate the salivary acquired membrane structure on the tooth surface. The crystal violet staining method was used to measure the results at 595 nanometers with an enzyme marker to characterize the amount of biofilm generated. The fluorescence bioimaging system was used for detection. In order to quantify the staining of bacterial biofilms on the HA model and the colonization of bacteria, the fluorescence intensity threshold of each fluorescent color was manually set, and the fluorescence intensity analysis of fluorescence imaging was performed using the Spectral instruments imaging software program.

[0115] Fig.10 It indicates the changes in the biomass of biofilm on the surface of simulated teeth under the intervention of each probiotic group in a simulated saliva environment. Fig.10 It can be seen that the biofilm production in the ET-22 inactivated bacteria and extracellular metabolite groups was significantly reduced, and the difference was extremely significant (P<0.00001). In comparison, the biofilm production in the ET-22 extracellular metabolite group was the lowest; the biofilm production in the K12 live bacteria group was higher than that in the control group.

[0116] This example uses SEM to analyze the changes in the biomass structure of the biofilm on the simulated tooth surface under the intervention of each probiotic group in a simulated saliva environment. Fig.11 The scanning electron microscope results of Example 3 show the changes in the biomass structure of the biofilm on the simulated tooth surface under the intervention of different probiotics. Fig.11 As shown in the figure, in the control group of the artificial saliva culture system, a cauliflower-like multi-layer stacked biofilm structure was formed. The ET-22 inactivated bacteria group and the extracellular metabolite group significantly inhibited the content of the biofilm and destroyed the cauliflower-like biofilm structure. The K12 live bacteria group showed a single-layer stacked biofilm state, and the inhibitory effect was not obvious. Among them, the inhibitory effect of the ET-22 extracellular metabolite group was the most obvious, and the biofilm showed a single-layer, scattered distribution state, which was significantly better than the other treatment groups.

[0117] This example uses a laser confocal microscope to analyze the changes in the biomass structure and thickness of the biofilm on the simulated tooth surface under the intervention of each probiotic group in a simulated saliva environment. Fig.12 As shown in the figure, 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 bacteria group increased significantly. The extracellular metabolites of ET-22 significantly reduced the number of live bacteria in the biofilm and reduced the distribution area of ​​the biofilm. Fig.13 As shown, the thickness of the biofilm in the control group was as high as 120 μm, and the ET-22 inactivated bacteria group and the extracellular metabolite group significantly inhibited the thickness of the biofilm. Under the treatment of ET-22 extracellular metabolites, the average thickness of the biofilm was only 62 μm, a decrease of 48% compared with the control group.

[0118] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of extracellular metabolites of Lactobacillus paracasei ET-22 as active ingredients in the preparation of a medicament for preventing and treating oral ulcers, wherein: The oral ulcer is caused by Candida albicans or caused by oxidative damage, and the Lactobacillus paracasei ET-22 is a strain with a preservation number of CGMCC No.15077; The extracellular metabolites of Lactobacillus paracasei ET-22 are prepared according to the following method: removing the bacteria from the incubation liquid of Lactobacillus paracasei ET-22 to obtain the extracellular metabolites of Lactobacillus paracasei ET-22; wherein the incubation liquid is obtained by incubating Lactobacillus paracasei ET-22 in water.

2. The use according to claim 1, wherein: The extracellular metabolites of Lactobacillus paracasei ET-22 are prepared according to the following method: Lactobacillus paracasei ET-22 cells were added at a rate of 1-5 × 10 9 The mixture was dissolved in water at a concentration of CFU / mL, and incubated with stirring at 37°C and 200-500 rpm for 2-4 hours. After the incubation, the bacteria were removed by centrifugation to obtain extracellular metabolites of Lactobacillus paracasei ET-22.

3. Use of a probiotic composition in the preparation of a medicament for preventing and treating oral ulcers; The oral ulcer is an oral ulcer caused by Candida albicans or an oral ulcer caused by oxidative damage; The drug is a patch; The probiotic composition comprises: extracellular metabolites of Lactobacillus paracasei ET-22; the Lactobacillus paracasei ET-22 is a strain with a preservation number of CGMCC No.15077; the extracellular metabolites of Lactobacillus paracasei ET-22 are prepared according to the following method: removing the bacteria from an incubation solution of Lactobacillus paracasei ET-22 to obtain the extracellular metabolites of Lactobacillus paracasei ET-22; wherein the incubation solution is obtained by incubating Lactobacillus paracasei ET-22 in water; The probiotic composition further comprises: sodium carboxymethyl cellulose, propolis, borneol, lubricant, glycerin and Tween-20; The weight parts of each component are:

4. The use according to claim 3, wherein: The weight portion of the extracellular metabolites of Lactobacillus paracasei ET-22 is 18-20 parts.

5. The use according to claim 3, wherein: The weight portion of sodium carboxymethyl cellulose is 14-20 parts.

6. The use according to claim 3, wherein: The weight portion of propolis is 25-35 portions.

7. The use according to claim 3, wherein: The extracellular metabolites of Lactobacillus paracasei ET-22 are prepared according to the following method: Lactobacillus paracasei ET-22 cells were added at a rate of 1-5 × 10 9 The mixture was dissolved in water at a concentration of CFU / mL, and incubated with stirring at 37°C and 200-500 rpm for 2-4 hours. After the incubation, the bacteria were removed by centrifugation to obtain extracellular metabolites of Lactobacillus paracasei ET-22.

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