WO2023236452A1 - 一种防治口腔溃疡的益生菌贴剂及其制备方法与应用 - Google Patents
一种防治口腔溃疡的益生菌贴剂及其制备方法与应用 Download PDFInfo
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- WO2023236452A1 WO2023236452A1 PCT/CN2022/133142 CN2022133142W WO2023236452A1 WO 2023236452 A1 WO2023236452 A1 WO 2023236452A1 CN 2022133142 W CN2022133142 W CN 2022133142W WO 2023236452 A1 WO2023236452 A1 WO 2023236452A1
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- lactobacillus paracasei
- group
- probiotic
- cells
- oral ulcers
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
- A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
Definitions
- the present invention relates to a probiotic patch and its preparation method and application. Specifically, it relates to the application of Lactobacillus paracasei ET-22 in the preparation of medicines for preventing and treating oral ulcers, a probiotic patch containing Lactobacillus paracasei ET-22.
- the probiotic composition of ET-22, the probiotic patch made from the composition, its preparation method and its application.
- Oral ulcers are a common ulcerative damage symptom that occurs in the oral mucosa.
- the occurrence of oral ulcers is the result of a combination of factors, including local trauma, mental stress, food, drugs, malnutrition, changes in hormone levels, and lack of vitamins or trace elements.
- CN113425831A discloses a probiotic composition for treating oral ulcers and its preparation method and application.
- the probiotic composition includes the following parts by weight of raw materials: 40 to 60 parts of Baole fruit powder, 30 to 60 parts of Trichophylla phylloxera, and probiotics. 10 to 20 parts of wintersweet, 1 to 5 parts of wintersweet, 1 to 5 parts of mannose oligosaccharides, 1 to 5 parts of radix radix leaves, and 1 to 5 parts of colostrum basic protein; the composition uses antioxidants.
- One object of the present invention is to provide a probiotic for preventing and treating oral ulcers.
- Another object of the present invention is to provide the use of Lactobacillus paracasei ET-22 in the preparation of medicaments for preventing and treating oral ulcers.
- Another object of the present invention is to provide a probiotic composition containing Lactobacillus paracasei ET-22.
- Another object of the present invention is to provide a probiotic patch made of the composition.
- Another object of the present invention is to provide a method for preparing a probiotic patch.
- Another object of the present invention is to provide applications of probiotic compositions or patches.
- 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. Furthermore, Lactobacillus paracasei ET-22 and/or its extracellular metabolites are prepared into a probiotic patch, which can effectively sterilize the oral ulcer site and effectively prevent secondary infection of the wound.
- the prevention and treatment includes prevention and/or treatment.
- the present invention provides the use of Lactobacillus paracasei in the preparation of medicaments for treating oral ulcers, wherein the Lactobacillus paracasei includes Lactobacillus paracasei ET-22 cells and/or its extracellular Metabolites, the Lactobacillus paracasei ET-22 is a strain with deposit number CGMCC No. 15077.
- Lactobacillus paracasei ET-22 strain with deposit number CGMCC No. 15077 is a biological material that has been published in CN110964653A and is available to the public.
- the Lactobacillus paracasei ET-22 cells are viable cells and/or inactivated cells.
- the Lactobacillus paracasei ET-22 extracellular metabolite is prepared according to the following method:
- the bacterial cells are removed from the incubation solution of Lactobacillus paracasei ET-22 to obtain extracellular metabolites of Lactobacillus paracasei ET-22; preferably, the incubation solution is obtained by incubating Lactobacillus paracasei ET-22 in water.
- Lactobacillus paracasei ET-22 cells are dissolved into water at a concentration of 1 to 5 ⁇ 10 9 CFU/mL, and incubated with stirring at 37°C and 200 to 500 rpm for 2 to 4 hours, and the incubation is completed. After centrifugation, the bacterial cells are removed, and the extracellular metabolites of Lactobacillus paracasei ET-22 are obtained.
- the oral ulcer includes oral ulcer caused by pathogenic bacteria, oral ulcer caused by oxidative damage or oral ulcer caused by low immunity.
- the present invention also provides a probiotic composition, which includes: Lactobacillus paracasei ET-22 cells and/or extracellular metabolites thereof; the Lactobacillus paracasei ET-22 is deposited under the deposit number CGMCC Strain No.15077.
- the Lactobacillus paracasei ET-22 extracellular metabolite is prepared according to the following method:
- Lactobacillus paracasei ET-22 cells Dissolve Lactobacillus paracasei ET-22 cells into water at a concentration of 1 to 5 ⁇ 10 9 CFU/mL, stir and incubate at 37°C and 200 to 500 rpm for 2 to 4 hours, and remove by centrifugation after the incubation.
- the bacteria were cultured to obtain the extracellular metabolites of Lactobacillus paracasei ET-22.
- the present invention also provides the use of the probiotic composition in preparing a composition for preventing and treating oral ulcers.
- the oral ulcers include oral ulcers caused by pathogenic bacteria, oral ulcers caused by oxidative damage, or oral ulcers caused by low immunity.
- Lactobacillus paracasei ET-22 live bacteria can significantly prevent and treat oral ulcers caused by pathogenic bacteria or oxidative damage.
- the application of inactivated Lactobacillus paracasei ET-22 bacteria can significantly prevent and treat oral ulcers caused by low immunity.
- the extracellular metabolite of Lactobacillus paracasei ET-22 has a significant effect on preventing and treating oral ulcers.
- the composition for preventing and treating oral ulcers of the present invention is a pharmaceutical composition.
- the pharmaceutical composition is a patch.
- the composition for preventing and treating oral ulcers of the present invention can be a general cosmetic.
- the present invention also provides a method for preparing a probiotic patch, which method includes:
- step (2) can also be performed first. ), then proceed to step (1), or perform both steps at the same time.
- the present invention also provides a probiotic patch, which is prepared with the composition of the present invention, or prepared by the above-mentioned preparation method of the probiotic patch.
- the present invention also provides a method for treating oral ulcers, which method includes administering an effective amount of the composition and/or the probiotic patch to a subject; the subject includes mammal or human.
- 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 animal models that live bacteria of Lactobacillus paracasei ET-22 can significantly prevent and treat oral ulcers caused by pathogenic bacteria or oxidative damage; smearing of inactivated bacteria of Lactobacillus paracasei ET-22 can significantly prevent and treat low immunity. Oral ulcers caused by Lactobacillus paracasei ET-22 extracellular metabolites have good effects when used to prevent and/or treat oral diseases.
- Figure 1 is a graph showing the tongue damage score results of ICR mice 48 hours after modeling in the prevention group of Example 2.
- Figure 2 is a graph showing the tongue damage score results of ICR mice after 72 hours of modeling in the treatment group of Example 2.
- Figure 3 shows the results of HE staining of tongue dorsal lesion sections of mice in the blank group, model group and probiotic group 48 hours after the prevention group of Example 2 was established.
- A is the papillary protrusion of tongue tissue epithelial cells
- B is tissue hyperplasia.
- Figure 4 is a HE stained photo of a typical hamster oral mucosal tissue section 48 hours after the modeling in the prevention group of Example 2; in the figure, the white arrow points to inflammatory cell infiltration, the black arrow points to damage and shedding of the mucosal epithelium, and the gray arrow points to capillaries. Filling and expansion, gland hyperplasia (scale bar: 200 ⁇ m).
- Figure 5 is a typical immunohistochemical photo of hamster oral ulcer mucosal tissue 48 hours after the prevention group of Example 2 (scale bar: 100 ⁇ m).
- Figure 6 is a graph showing the results of LXA4 and PGE2 levels in the oral ulcer mucosal tissue of hamsters and the levels of pro-inflammatory cytokines IL-6 and IL-1 ⁇ in serum in Example 2.
- Figure 8 shows the composition of hamster oral bacteria at the phylum level in Example 2.
- Figure 9 shows the significantly different bacterial genera in the oral cavity of hamsters in Example 2.
- Figure 10 is a graph showing the changes in biofilm biomass on the simulated tooth surface under the intervention of different probiotics in Example 3;
- Figure 11 is a scanning electron microscope 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.
- Figure 12 is a diagram showing laser confocal microscopy results of simulated changes in biofilm biomass structure on the tooth surface under the intervention of different probiotics in Example 3.
- Figure 13 is a graph showing the results of changes in biofilm thickness on the simulated tooth surface under the intervention of different probiotics in Example 3.
- the prevention group divides them into the following 10 groups:
- the treatment group was divided into the following 7 groups:
- immunohistochemistry score uses the IRS (Immunoreactive Score) scoring method. After scoring the staining degree (0-3 points) and positivity rate (0-4 points) respectively, they are multiplied to obtain a comprehensive score (0-12 points); score
- IRS Immunoreactive Score
- the degree of staining is scored based on the staining characteristics of the target protein: no staining is 0 points, light yellow is 1 point, brown is 2 points, and tan is 3 points; the positive rate is based on the positive ratio of cells in the section. Scoring: 0-5% is 0 points, 6%-25% is 1 point, 26%-50% is 2 points, 51%-75% is 3 points, and >75% is 4 points. A comprehensive score of 0 is negative (-); 1-3 is weak positive (+); 4-5 is positive (++); 6-7 is strong positive (+++).
- Figure 2 is a graph showing the results of tongue damage scores of ICR mice after 72 hours of modeling in the treatment group of Example 2.
- the treatment group compared with the tongue damage scores of mice in the model group, only the ET-22 inactivated bacteria group appeared There was no significant difference (p ⁇ 0.05), but the probiotic group showed a certain cure rate.
- the metabolite of Lactobacillus paracasei ET-22 had the lowest cure rate of 11%, and the damage score decreased by at least 23%.
- the other various cure rates were in 30%-40%, and the damage score dropped by 50%-60%.
- the average damage score after treatment with 1.0 ⁇ 10 9 CFU/mL Streptococcus salivarius K12 was 2.1, 1.0 ⁇ 10 9 CFU/mL Lactobacillus paracasei ET-22 live bacteria, inactivated bacteria and extracellular metabolism
- the mean injury scores for the object groups were 1.7, 2.6, and 2.1, respectively.
- Lactobacillus paracasei ET-22 live bacteria is better than Streptococcus salivarius K12 live bacteria in preventing candidiasis albicans.
- the extracellular metabolite group of Lactobacillus paracasei and Streptococcus salivarius K12 live bacteria have the same effect in preventing candidiasis albicans. Efficacy. Compared with live bacteria, the storage method of extracellular metabolites is simpler and the application scenarios are wider.
- the tongue tissue epithelial cell papillae of the L9 live bacteria group had a certain loss, slight peeling, and tissue hyperplasia were similar to those of the model group.
- the tongue tissue epithelial cell papillary projections were obviously lost, there was no peeling, and there was a certain degree of tissue hyperplasia.
- the tongue tissue epithelial cell papillary projections were obviously lost, there was slight peeling, and severe tissue hyperplasia.
- ET-22 live bacteria group ET-22 extracellular metabolite group, AP32 live bacteria group, and K12 live bacteria group have a certain effect on the prevention of oral candidiasis and reduce inflammation and tongue tissue lesions.
- the ET-22 live bacteria group has the best therapeutic effect.
- Typical immunohistochemistry photos (scale bar: 100 ⁇ m) of hamster oral ulcer mucosal tissue 48 hours after the prevention group was established are shown in Figure 5.
- NF- ⁇ B, MMP-9, Caspase 3, and PARP were all negatively expressed in the oral mucosal tissue of hamsters in the normal control group, while the expressions of the above four proteins were significantly increased in the model group. (P ⁇ 0.05).
- the expression of NF- ⁇ B and MMP-9 in the model group was significantly increased, indicating that an inflammatory reaction occurred in the oral mucosa and the connection between the basement membrane and cells was damaged.
- FIG. 6 shows that compared with the normal control group, the LXA4 content in the model group did not change significantly. It is speculated that because the model group did not receive intervention, its LXA4 activation degree was low and inflammation subsided slowly, which also led to more severe and persistent ulcers. Compared with the model group, the LXA4 content of the ET-22 live bacteria group, ET-22 extracellular metabolite group and K12 live bacteria group was significantly increased (P ⁇ 0.05), indicating that they may exert anti-inflammatory effects by increasing LXA4 levels. Thereby reducing the symptoms of oral ulcers; there was no significant difference between the ET-22 inactivated bacteria, HN019 live bacteria, DSM17938 live bacteria, AP32 live bacteria and VC groups and the model group.
- PGE2 can aggravate the inflammatory response, promote local blood vessel dilation and increase capillary permeability.
- the PGE2 content in the model group was significantly increased, indicating severe inflammation of the oral mucosa in the model group, consistent with its severe ulcer phenotype.
- the PGE2 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 those of the model group and the control group; while the HN019 live bacteria, DSM17938 live bacteria and AP32 live bacteria groups had no significant difference from the model group.
- the levels of proinflammatory cytokines IL-6 and IL-1 ⁇ in hamster serum are shown in Figure 6 .
- the levels of pro-inflammatory cytokines IL-6 and IL-1 ⁇ in the serum of hamsters in the model group were significantly increased (P ⁇ 0.05, Figure 6), indicating that the oral ulcer modeling was successful.
- the results of the VC group are expected and consistent with the antioxidant properties of VC.
- the results of the ET-22 live bacteria and ET-22 inactivated bacteria groups show that they have similar effects to VC and can reduce oxidative stress to a certain extent. damage and has good antioxidant potential.
- This example compares the effects of Lactobacillus paracasei ET-22, ET-22 extracellular metabolites, and Streptococcus salivarius K12 on Streptococcus mutans biofilm production and biofilm structure.
- Construct a saliva-coated hydroxyapatite model Prepare a mixture of dye solution and bacterial solution for later use.
- the bacterial solution is Lactobacillus paracasei ET-22 live bacteria solution, ET-22 extracellular metabolite solution, and Streptococcus salivarius K12 solution.
- the concentration of the bacterial solution is 10 9 CFU/ml, and the concentration of the extracellular metabolite solution is the equivalent bacterial concentration.
- HA hydroxyapatite
- Figure 10 shows the changes in biofilm biomass on the simulated tooth surface under the intervention of each probiotic group in a simulated saliva environment.
- the amount of biofilm production in the ET-22 inactivated bacteria and extracellular metabolite group was significantly reduced, and the difference was extremely significant (P ⁇ 0.00001).
- the biofilm production in the ET-22 extracellular metabolite group was The amount of biofilm produced was the lowest; the amount of biofilm produced in the K12 live bacteria group was higher than that of the control group.
- FIG. 11 is a scanning electron microscope result of the changes in biofilm biomass structure on the simulated tooth surface under the intervention of different probiotics in Example 3.
- the artificial saliva culture system control group formed a cauliflower-like multi-layer accumulation.
- 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. , the inhibitory effect is not obvious. Among them, the inhibitory effect of the ET-22 extracellular metabolite group was the most obvious.
- the biofilm showed a single layer and scattered distribution, which was significantly better than the other treatment groups.
- This example uses laser confocal microscopy 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.
- the structural changes are shown in Figure 12.
- the bacterial content in the biofilm of the control group was dominated by viable bacteria (green).
- the content of dead bacteria (red) in the biofilm of the ET-22 inactivated bacteria group increased significantly.
- ET-22 extracellular metabolites significantly reduce the number of viable bacteria in the biofilm and reduce the distribution area of the biofilm.
- the changes in biofilm thickness are shown in Figure 13.
- the thickness of the biofilm in the control group was as high as 120 ⁇ m.
- Both 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 metabolite, , the average thickness of the biofilm was only 62 ⁇ m, a decrease of 48% compared with the control group.
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Abstract
提供了副干酪乳杆菌在制备用于防治口腔溃疡的药物中的应用,该副干酪乳杆菌包括副干酪乳杆菌ET-22 菌体和/或其胞外代谢物,该副干酪乳杆菌ET-22为保藏编号 CGMCC No. 15077的菌株。还提供了一种益生菌组合物及其制备方法和应用,该组合物包括:副干酪乳杆菌ET-22 菌体和/或其胞外代谢物,还包括羧甲基纤维素钠、蜂胶、冰片、润滑剂、甘油和吐温-20。还提供了一种防治口腔溃疡的益生菌贴剂及其制备方法与应用。
Description
本发明是关于一种益生菌贴剂及其制备方法与应用,具体而言,是关于副干酪乳杆菌ET-22在制备用于防治口腔溃疡的药物中的应用、一种包含副干酪乳杆菌ET-22的益生菌组合物、由所述组合物制成的益生菌贴剂、其制备方法及其应用。
口腔溃疡俗称“口疮”,是一种常见的发生于口腔黏膜的溃疡性损伤症状。口腔溃疡的发生是多种因素综合作用的结果,包括局部创伤、精神紧张、食物、药物、营养不良、激素水平改变及维生素或微量元素缺乏等。
现有技术已知多种益生菌有助于治疗口腔溃疡。CN109985179A公开了一种治疗口腔溃疡的组合物,该组合物包括以下重量份的原料:新鲜芦笋1000-15000份、唾液乳杆菌0.1-10份、乳双歧杆菌0.1-20份、副干酪乳杆菌0.1-20份、青黛0.1-50份、淀粉10-1000份;该组合物可补充人体所需微量元素,对口腔溃疡患者有内在免疫调节与口腔环境改善的作用,增强机体免疫力,对促进疮疡愈合,具体应用时是做成丸剂、散剂、胶囊剂、片剂或冲剂等口服药物。CN113425831A公开了一种治疗口腔溃疡的益生菌组合物及其制备方法和应用,所述益生菌组合物包括如下重量份原料:宝乐果粉40~60份、木姜叶柯30~60份、益生菌10~20份、柳叶蜡梅1~5份、低聚甘露糖1~5份、乌药叶1~5份、初乳碱性蛋白1~5份;该组合物采用具有抗氧化作用的宝乐果粉和木姜叶柯,达到治疗口腔溃疡的作用,可以减少益生菌的用量,低聚甘露糖还能够促进益生菌的生长,使各益生菌之间发挥协调作用,同时通过含有的柳叶蜡梅、米糠脂肪烷醇、乌药叶和初乳碱性蛋白均具有治疗口腔溃疡的作用,进而预防和改善口腔溃疡,具体应用时是做成喷雾剂。
上述现有技术的治疗口腔溃疡的含有益生菌的药物中,益生菌本身对于治疗口腔溃疡的贡献并不突出,并且,口服药物存在作用部位广、需药量大、药物利用率低等缺点,喷雾剂在喷涂后需要在一定时间内禁止饮水、进食,使用时存在诸多不便。
发明内容
本发明的一个目的在于提供一种防治口腔溃疡的益生菌。
本发明的另一目的在于提供副干酪乳杆菌ET-22在制备用于防治口腔溃疡的药物中的应用。
本发明的另一目的在于提供一种包含副干酪乳杆菌ET-22的益生菌组合物。
本发明的另一目的在于提供一种由所述组合物制成的益生菌贴剂。
本发明的另一目的在于提供一种益生菌贴剂的制备方法。
本发明的另一目的在于提供益生菌组合物或贴剂的应用。
本案发明人在研究中发现,副干酪乳杆菌(Lactobacillus paracasei)ET-22是一种能够防治口腔溃疡的益生菌,其菌体和/或其胞外代谢物能够显著防治口腔溃疡。进一步,将副干酪乳杆菌ET-22和/或其胞外代谢物制备成益生菌贴剂,能够有效对口腔溃疡部位杀菌,可有效防止伤口的二次感染。
本发明中,所述防治包括预防和/或治疗。
从而,一方面,本发明提供了副干酪乳杆菌在制备用于治疗口腔溃疡的药物中的应用,其中,所述副干酪乳杆菌包括副干酪乳杆菌ET-22菌体和/或其胞外代谢物,所述副干酪乳杆菌ET-22为保藏编号CGMCC No.15077的菌株。
保藏编号CGMCC No.15077的副干酪乳杆菌(Lactobacillus paracasei)ET-22菌株是已在CN110964653A中公布的生物材料,是公众可以得到的。
根据本发明的具体实施方案,本发明中,所述副干酪乳杆菌ET-22菌体为活菌体和/或灭活的菌体。
根据本发明的具体实施方案,本发明中,所述副干酪乳杆菌ET-22胞外代谢物是按照以下方法制备得到的:
从副干酪乳杆菌ET-22的孵育液中去除菌体,得到副干酪乳杆菌ET-22胞外代谢物;优选地,所述孵育液为副干酪乳杆菌ET-22在水中孵育获得。
优选地,将副干酪乳杆菌ET-22菌体以1~5×10
9CFU/mL的浓度溶解至水中,37℃、200~500转/分钟的条件下搅拌孵育2~4小时,孵育结束后离心去除菌体,得到副干酪乳杆菌ET-22胞外代谢物。
根据本发明的具体实施方案,本发明中,所述口腔溃疡包括由于病原菌引起的口腔溃疡、由于氧化损伤引起的口腔溃疡或免疫低下引起的口腔溃疡。
另一方面,本发明还提供了一种益生菌组合物,其包括:副干酪乳杆菌ET-22菌体和/或其胞外代谢物;所述副干酪乳杆菌ET-22为保藏编号CGMCC No.15077的菌株。
根据本发明的具体实施方案,本发明中,所述益生菌组合物还包括:羧甲基纤维素钠、蜂胶、冰片、润滑剂、甘油和吐温-20;优选地,各组分的重量份为:
根据本发明的具体实施方案,本发明的益生菌组合物中,所述各组分的重量份为:
根据本发明的具体实施方案,本发明的益生菌组合物中,所述副干酪乳杆菌ET-22菌体为活菌体和/或灭活的菌体。
根据本发明的具体实施方案,本发明的益生菌组合物中,所述副干酪乳杆菌ET-22胞外代谢物是按照以下方法制备得到的:
将副干酪乳杆菌ET-22菌体以1~5×10
9CFU/mL的浓度溶解至水中,37℃、200~500转/分钟的条件下搅拌孵育2~4小时,孵育结束后离心去除菌体,得到副干酪乳杆菌ET-22胞外代谢物。
根据本发明的具体实施方案,本发明的益生菌组合物中,所述润滑剂可为液体石蜡或口腔贴剂常用的其他润滑剂。
另一方面,本发明还提供了所述的益生菌组合物在制备用于防治口腔溃疡的组合物中的应用。
根据本发明的具体实施方案,所述口腔溃疡包括由于病原菌引起的口腔溃疡、由于氧化损伤引起的口腔溃疡或免疫低下引起的口腔溃疡。在本发明的一些具体实施方案中,副干酪乳杆菌ET-22活菌能够显著预防、治疗由于病原菌或氧化损伤引起的口腔溃疡。在本发明的一些具体实施方案中,副干酪乳杆菌ET-22灭活菌的涂抹可以显著预防、治疗免疫低下引起的口腔溃疡。在本发明的一些具体实施方案中,副干酪乳杆菌ET-22胞外代谢物对于防治口 腔溃疡具有显著的效果。
根据本发明的一些具体实施方案,本发明的用于防治口腔溃疡的组合物为药物组合物。优选地,所述药物组合物为贴剂。
根据本发明的一些具体实施方案,本发明的用于防治口腔溃疡的组合物可以为一般化妆品。
另一方面,本发明还提供了一种益生菌贴剂的制备方法,该方法包括:
(1)将副干酪乳杆菌ET-22菌体和/或其胞外代谢物、蜂胶、羧甲基纤维素钠和润滑剂混合均匀,得到混合体系A的步骤;
(2)将冰片、甘油和吐温-20隔水加热混匀,得到混合体系B的步骤;
(3)将混合体系A和混合体系B混合均匀的步骤;
(4)干燥处理步骤。
需要说明的是,本发明的制备方法的各步骤,(1)、(2)的标号并不表示这两个步骤必须按照所描述的先后顺序进行,实际操作中,也可以先进行步骤(2)、再进行步骤(1),或是两个步骤同时进行。
另一方面,本发明还提供了一种益生菌贴剂,其是以本发明所述的组合物制备得到的,或经上述益生菌贴剂的制备方法制备得到。
另一方面,本发明还提供了一种治疗口腔溃疡的方法,所述方法包括给予受试者有效量的所述的组合物和/或所述的益生菌贴剂;所述受试者包括哺乳动物或人。
综上所述,本发明提供了副干酪乳杆菌ET-22和/或其胞外代谢物在防治口腔溃疡方面的用途,并提供了所制备得到的益生菌贴剂。本发明通过动物模型证明,副干酪乳杆菌ET-22活菌能够显著预防、治疗由于病原菌或氧化损伤引起的口腔溃疡;副干酪乳杆菌ET-22灭活菌的涂抹可以显著预防、治疗免疫低下引起的口腔溃疡;副干酪乳杆菌ET-22胞外代谢物用于预防和/或治疗口腔疾病时有良好的效果。本发明所述的益生菌贴剂,与口腔黏膜的粘附性好;添加冰片还具有清热解毒、消肿止痛的作用;能够有效对口腔溃疡部位杀菌,可有效防止伤口的二次感染。
图1为实施例2预防组造模48h后ICR小鼠舌头损伤评分值结果图。
图2为实施例2治疗组造模72h后ICR小鼠舌头损伤评分值结果图。
图3为实施例2预防组造模48h后空白组、模型组和益生菌组小鼠舌背病变切片HE染色结果图;图中,A为舌组织上皮细胞乳头状突起,B为组织增生。
图4为实施例2预防组造模48h后典型的地鼠口腔粘膜组织切片的HE染色照片;图中,白色箭头指向炎性细胞浸润,黑色箭头指向粘膜上皮破损、脱落,灰色箭头指向毛细血管充盈扩张、腺体增生(标尺:200μm)。
图5为实施例2预防组造模48h后典型的地鼠口腔溃疡粘膜组织免疫组化照片(标尺:100μm)。
图6为实施例2地鼠口腔溃疡粘膜组织中的LXA4和PGE2水平及血清中促炎细胞因子IL-6和IL-1β水平结果图。
图7为实施例2地鼠血清中的SOD活性、GSH和MDA水平结果图。
图8为实施例2地鼠口腔细菌在门水平上的组成。
图9为实施例2地鼠口腔中具有显著差异的菌属。
图10为实施例3不同益生菌处干预下模拟牙齿表面生物膜生物量的变化结果图;
图11为实施例3不同益生菌处干预下模拟牙齿表面生物膜生物量结构的变化的扫描电镜结果图。
图12为实施例3不同益生菌处干预下模拟牙齿表面生物膜生物量结构的变化的激光共聚焦显微镜结果图。
图13为实施例3不同益生菌处干预下模拟牙齿表面生物膜厚度变化结果图。
为了对本发明的技术特征、目的和有益效果有更加清楚的理解,现结合具体实施例及对本发明的技术方案进行以下详细说明,应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中,各原始试剂材料均可商购获得,未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件,或按照仪器制造商所建议的条件。
除非另外专门定义,本文使用的所有技术和科学术语都与相关领域普通技术人员的通常理解具有相同的含义。
本发明的各实施例中,所述副干酪乳杆菌ET-22胞外代谢物是按照以下方法制备得到的:将副干酪乳杆菌ET-22接种至培养基中,培养后得到菌液,将ET-22菌液离心,从而获得副干酪乳杆菌ET-22菌体(菌泥);
将所获得的副干酪乳杆菌ET-22菌体以1~5×10
9CFU/mL的浓度溶解至水中,37℃、200~500转/分钟的条件下搅拌孵育3小时,孵育结束后离心去除菌体,从而得到副干酪乳杆菌ET-22胞外代谢物。
实施例1
本实施例提供了一种治疗口腔溃疡的益生菌贴剂,制备所述益生菌贴剂的原料包括副干酪乳杆菌ET-22菌体和/或其胞外代谢物、羧甲基纤维素钠、蜂胶、冰片、润滑剂、甘油和吐温-20,各原料的重量份为:
制备益生菌贴剂的方法包括以下步骤:
(1)按上述的重量份数比,将副干酪乳杆菌ET-22菌体和/或胞外代谢物、蜂胶、羧甲基纤维素钠和润滑剂,得到混合体系A;
(2)按上述的重量份数比,将冰片、甘油和吐温-20置于容器中隔水加热,搅拌混合均匀,得到混合体系B;
(3)将混合体系A和混合体系B混合均匀后,经干燥处理,制得所述治疗口腔溃疡的益生菌贴剂。
实施例2、副干酪乳杆菌ET-22防治口腔溃疡作用
1.实验动物模型
1.1免疫低下造成的口腔溃疡模型
预防组动物模型:为了在小鼠中诱发口腔念珠菌病,本发明使用了先前描述的方案,并做了一些小的修改。小鼠适应期结束后,实验组进行为期18天的益生菌干预(饮水中添加益生菌10
9CFU/ml),空白组和模型组正常饮水。第15天通过饮用水给小鼠服用15mg/ml四环素盐酸盐,并同时进行诱导免疫抑制状态,泼尼松龙(100mg/kg)皮下注射。第16天对模型组和实验组进行白色念珠菌CGMCC 2.4122感染,感染时使用4%的三氯乙醛水合物(10ml/kg)进行麻醉);然后在口腔的各个部位感染了1.0×10
9CFU/ml白色念珠菌,将白色念珠菌浸渍的棉签放置在舌背上15分钟。小鼠于术后48小时安乐死。
治疗组动物模型:为了在小鼠中诱发口腔念珠菌病,本发明使用了先前描述的方案,并做了一些小的修改。小鼠适应期结束后,通过饮用水给小鼠服用15mg/ml四环素盐酸盐,并同时进行诱导免疫抑制状态,泼尼松龙(100mg/kg)皮下注射。第2天对模型组和实验 组进行白色念珠菌CGMCC 2.4122感染,感染时使用4%的三氯乙醛水合物(10ml/kg)进行麻醉);然后在口腔的各个部位感染了1.0×10
9CFU/ml白色念珠菌,将白色念珠菌浸渍的棉签放置在舌背上15分钟。感染24小时后(第三天)益生菌干预组每12小时进行一次益生菌涂抹干预,攻击涂抹4次,涂抹方法同感染涂抹方法,小鼠于术后72小时(第5天)安乐死。
1.2氧化损伤口腔溃疡金黄地鼠模型
六周龄的雄性LVG叙利亚金黄地鼠(LVG Golden Syrian Hamster),体重(110±20)g/只,购自北京维通利华实验动物技术有限公司,饲养条件为12h光照交替,温度保持在20±2℃,湿度维持在45%-50%。地鼠在饲养室驯化一周后,随机分成10组,分别为正常对照组、模型组、ET-22活菌组、ET-22灭活菌组、ET-22胞外代谢物组以及K12、HN019、DSM17938、AP32的活菌组和维生素C阳性对照组(VC组),正常对照组有7只,其余组别各10只。正常对照组和模型组地鼠每天灌胃1ml生理盐水,ET-22活菌组每天灌胃1ml浓度为10
9CFU/ml的ET-22活菌溶液,ET-22灭活菌组和ET-22胞外代谢物组分别灌胃1ml相应浓度的ET-22灭活菌和菌胞外代谢物,VC组每天灌胃1ml浓度为20mg/ml的维生素C溶液。灌胃15天后进行口腔溃疡造模,先腹腔注射10%水合氯醛(注射量为0.3-0.5%体重)麻醉地鼠,在正常对照组地鼠颊囊内注射0.25ml PBS缓冲液(pH=7.4),其余5组地鼠均颊囊注射0.25ml甲基紫精(10mmol/L,溶于PBS);注射当天直至地鼠被处死,各组仍保持每天灌胃干预。于造模后第4天将地鼠麻醉、取血,随后用口腔拭子刮拭地鼠口腔内部(除溃疡部位),剪取拭子头放入装有核酸保护液的离心管;剪取口腔溃疡病灶组织或正常粘膜,分为两份,一份固定于4%多聚甲醛中,另一份液氮冻存。将全血分离出血清后,分装冻存于-80℃。
2.实验动物分组
预防组 将其分为以下10组:
治疗组 将其分为以下7组:
口腔病症感染严重程度评分-小鼠
各组小鼠于术后48(预防组)/72(治疗组)小时小鼠被安乐处死后,进行舌病变严重程度的评估观察。根据舌面白色凝乳样斑块的范围和严重程度,通过0到4的病变评分表达感染的宏观评估,如下所示:0分,正常;1分,白色斑块在20%以下;2分,白色斑块在90%以下但在21%以上;3分,白色斑块在91%以上;厚白色斑块状假膜占91%以上。
取小鼠全舌于舌尖中间纵切一分为二,取其中一份于常温固定在4%多聚甲醛中性缓冲液中,然后用石蜡包埋。将样品沿舌尖切割成5μm厚的连续纵截面,然后使用苏木精法进行染色。参考文献使用徕卡荧光显微镜显微镜进行观察染色。
口腔病症感染严重程度评分-金黄地鼠
将石蜡切片依次脱蜡和梯度脱水后,采用柠檬酸抗原修复缓冲液(pH=6.0)进行抗原修复,随后将切片放入3%双氧水溶液中避光孵育25min以阻断内源性过氧化物酶,再经过封闭、一抗孵育、二抗孵育等操作步骤,最后进行DAB显色和细胞核复染,脱水封片,于显微镜下观察和拍照。免疫组化评分采用IRS(Immunoreactive Score)评分法,对其染色程度(0-3分)和阳性率(0-4分)分别进行评分后,相乘得到综合评分(0-12分);评分标准如下,染色程度以目的蛋白所呈现出的染色特性计分:无着色为0分,淡黄色为1分,棕黄色为2分,棕褐色为3分;阳性率按照切片中细胞阳性比率进行计分:0-5%为0分,6%-25%为1分,26%-50%为2分,51%-75%为3分,>75%为4分。综合评分0分为阴性(-);1-3分为弱阳性(+);4-5分为阳性(++);6-7分为强阳性(+++)。
组织病变观察结果:
图1为实施例2预防组造模48h后ICR小鼠舌头损伤评分值结果图,由图1可知,预防组在手术48小时后,与模型组小鼠舌头损伤评分相比只有ET-22活菌组出现了显著差异 (p<0.05),且平均评分降低了46.23%;DSM 17938活菌组与模型组的平均评分一致;其余实验组损伤评分虽然没有出现显著差异但也多有不同程度的降低。ET-22分泌物组(胞外代谢物组)、K12活菌组、HN019活菌组、AP32活菌组分别降低了31.9%、32.26%、35.48%以及41.94%;ET-22灭活菌组和L9活菌组分别降低了16.13%和16.13%。
图2为实施例2治疗组造模72h后ICR小鼠舌头损伤评分值结果图,由图2可知,治疗组中,与模型组小鼠舌头损伤评分相比只有ET-22灭活菌组出现了显著差异(p<0.05),但益生菌组均出现了一定治愈率,副干酪乳杆菌ET-22的代谢物的治愈率最低11%,损伤评分降幅最少23%,其余各种治愈率在30%-40%,损伤评分降幅在50%-60%。
预防组损伤评分分析:
Sanae.A.Ishijima等人探究唾液链球菌K12在小鼠模型中模型组的舌头平均损伤评分为3.4,经1.5×10
9CFU/mL唾液链球菌K12治疗后平均损伤评分为2.0。
本发明此次预防实验经1.0×10
9CFU/mL唾液链球菌K12治疗后平均损伤评分为2.1,1.0×10
9CFU/mL副干酪乳杆菌ET-22活菌、灭活菌和胞外代谢物组的平均损伤评分分别为1.7、2.6和2.1。副干酪乳杆菌ET-22活菌在预防白色念株菌病方面优于唾液链球菌K12活菌,副干酪乳杆菌胞外代谢物组与唾液链球菌K12活菌在预防白色念珠病具有相同的疗效。胞外代谢物组与活菌相比其保存方法更加简单,应用场景更加广泛。
舌背病变切片观察:
预防组造模48h后,空白组、模型组和益生菌组小鼠舌背病变切片HE染色结果如图3所示;图中,A为舌组织上皮细胞乳头状突起,B为组织增生。由图3可知,与空白组相对比,模型组出现了严重的炎症浸润,ET-22活菌组、ET-22分泌物组(胞外代谢物组组)、K12活菌组、HN019活菌组、AP32活菌组炎症浸润有明显的改善,ET-22灭活菌组和L9活菌组炎症浸润也较为严重,DSM 17938活菌组的炎症浸润严重于模型组。
由图3可知,与空白组相对比,模型组舌组织上皮细胞乳头状突起严重损失,脱皮和组织增生明显。ET-22活菌组、ET-22分泌物组和AP32活菌组舌组织上皮细胞乳头状突起保存完好,无脱皮,但与空白组相比有轻微的组织增生。ET-22灭活菌组舌组织上皮细胞乳头状突起有较为明显的损失,无脱皮,出现较为明显的组织增生。L9活菌组舌组织上皮细胞乳头状突起有一定的损失,轻微脱皮,组织增生与模型组类似。K12活菌组舌组织上皮细胞乳头状突起损失明显,无脱皮,有一定的组织增生。DSM 17938活菌组舌组织上皮细胞乳头状突起损失明显,有轻微脱皮,组织增生严重。
以上可以看出,ET-22活菌组、ET-22胞外代谢物组、AP32活菌组、K12活菌组对口腔念珠病预防有一定的疗效,减轻了炎症和舌组织病变情况。ET-22活菌组的疗效效果最优。
粘膜组织切片及HE染色:
实施例2预防组造模48h后,典型的地鼠口腔粘膜组织切片的HE染色照片如图4所示;图中,白色箭头指向炎性细胞浸润,黑色箭头指向粘膜上皮破损、脱落,灰色箭头指向毛细血管充盈扩张、腺体增生(标尺:200μm)。如图4所示,正常对照组地鼠口腔粘膜上皮连续性完好,细胞和腺体形态正常,无明显炎性细胞浸润。与正常对照组相比,模型组地鼠口腔粘膜上皮破损、脱落,连续性不完整,毛细血管充盈扩张,出现大量炎性细胞浸润,表明口腔溃疡模型造模成功。与模型组相比,ET-22活菌组的干预效果最好,该组地鼠的口腔粘膜上皮连续性相对完整,炎性细胞浸润明显减少;而ET-22灭活菌组、ET-22胞外代谢物组的地鼠口腔粘膜上皮仍有部分破损,并且存在部分炎性细胞浸润。K12活菌和HN019活菌组的地鼠口腔粘膜上皮完整程度较高,但还存在少量炎性细胞浸润;DSM17938活菌和AP32活菌组的地鼠口腔粘膜上皮连续性不完整,有明显的炎性细胞浸润现象。维生素C常被认为有助于防治口腔溃疡,从图中可看出,虽然VC组的地鼠口腔粘膜上皮缺失相较模型组有所改善,但破损仍相对严重,并且有较多炎性细胞浸润。这些结果说明食用ET-22,尤其是ET-22活菌,可降低口腔溃疡的发病风险、减轻溃疡症状,并且比预先补充VC具有更好的效果。
粘膜组织免疫组化结果:
NF-κB是一种重要的转录因子,其信号通路的激活可启动炎症反应;此外,NF-κB信号通路的激活可促进MMP-9表达上调,MMP-9可降解层黏连蛋白和Ⅳ型胶原,加速炎性细胞的浸润与转移。Caspase 3的高表达可能促使细胞凋亡上调,还能水解PARP,导致PARP无法正常参与DNA的损伤修复,从而加重细胞凋亡。
预防组造模48h后典型的地鼠口腔溃疡粘膜组织免疫组化照片(标尺:100μm)如图5所示。如图5和表1所示,正常对照组地鼠口腔粘膜组织的NF-κB、MMP-9、Caspase 3、PARP均呈阴性表达,而在模型组中上述4种蛋白的表达均显著升高(P<0.05)。模型组的NF-κB和MMP-9表达显著升高,提示口腔粘膜发生炎症反应,且基膜和细胞之间的连接受到破坏。模型组的Caspase 3和PARP表达显著升高,提示粘膜细胞凋亡上调,引发溃疡粘膜的上皮损伤、脱落。这些蛋白在模型组的阳性表达与HE染色反映的结果相一致。
与模型组相比,ET-22活菌、ET-22灭活菌、ET-22胞外代谢物和K12活菌组中这4种蛋白的表达均显著下降(P<0.05);HN019活菌组中除了NF-κB之外,其余表达均显著下降;DSM17938活菌组中只有Caspase 3和PARP表达显著下降,NF-κB和MMP-9表达与模 型组无显著性差异;AP32活菌组中NF-κB、Caspase 3、PARP表达有显著下降,MMP-9表达与模型组无显著性差异;VC组中只有NF-κB和Caspase 3的表达显著下降,MMP-9和PARP的表达下调但与模型组无显著性差异(表1)。该结果表明,ET-22对口腔溃疡的预防具有积极作用。
表1口腔粘膜组织免疫组化平均评分
口腔溃疡粘膜组织的脂氧素A4(LXA4)和前列腺素E2(PGE2)水平:
LXA4是一种具有抗炎作用的内源性脂质,可抑制IL-6等促炎细胞因子的分泌。
地鼠口腔溃疡粘膜组织中的LXA4和PGE2水平结果如图6所示。图6显示,与正常对照组相比,模型组的LXA4含量无显著变化,推测由于模型组没有进行干预,其LXA4激活程度较低,炎症消退缓慢,因此也导致溃疡较严重和持久。与模型组相比,ET-22活菌组、ET-22胞外代谢物组和K12活菌组的LXA4含量显著提高(P<0.05),表明通过它们可能通过提高LXA4水平发挥抗炎作用,进而减轻口腔溃疡症状;ET-22灭活菌、HN019活菌、DSM17938活菌、AP32活菌和VC组则与模型组无显著性差异。
PGE2可以加剧炎症反应,促进局部血管扩张、毛细血管通透性增加。与正常对照组相比,模型组的PGE2含量显著升高,表明模型组口腔粘膜出现剧烈的炎症,与其严重的溃疡表型一致。与模型组相比,ET-22活菌、ET-22灭活菌、ET-22胞外代谢物、K12活菌和VC组的PGE2均显著降低(P<0.05),其水平介于模型组和对照组之间;而HN019活菌、DSM17938活菌和AP32活菌组则与模型组无显著性差异。
血清的促炎细胞因子水平:
地鼠血清中促炎细胞因子IL-6和IL-1β水平如图6所示。与正常对照组相比,模型组地鼠血清中促炎细胞因子IL-6和IL-1β水平显著升高(P<0.05,图6),提示口腔溃疡造模成功。ET-22活菌、ET-22灭活菌、ET-22胞外代谢物组和K12活菌组地鼠血清中的IL-6浓度均比模型组显著下降(P<0.05),表明ET-22与K12可以降低炎症反应,进而起到减缓口腔 溃疡的作用;此外,所有干预组地鼠血清中IL-1β的浓度均比模型组显著下降(P<0.05)。该结果提示ET-22和K12比其他组具有更好的免疫调节作用,可通过调节机体免疫抵御口腔溃疡的形成或恶化。
血清的SOD活性、MDA和GSH浓度
SOD和GSH是重要的抗氧化酶和抗氧化剂,它们的升高提示机体因产生氧化应激而刺激了抗氧化物质的升高;MDA是脂质过氧化产物,反映了氧化应激的损伤程度。
预防组造模48h后,地鼠血清中的SOD活性、GSH和MDA水平如图7所示。与正常对照组相比,模型组地鼠血清中GSH和MDA水平显著升高(P<0.05),SOD活性也有升高但不显著。与模型组相比,ET-22活菌、ET-22灭活菌和VC组的SOD活性、GSH和MDA水平均显著降低(P<0.05),ET-22胞外代谢物组仅有MDA水平出现显著下降。在其余各对照菌株组中,MDA水平均显著降低,但在SOD活性和GSH水平上并没有都出现明显的调节效果。VC组的结果在意料之中,与VC的抗氧化特性相吻合,ET-22活菌和ET-22灭活菌组的结果则表明都它们与VC效果类似,能一定程度地降低氧化应激损伤,具有良好抗氧化潜质。
口腔菌群的多样性和组成
预防组造模48h后,地鼠口腔细菌在门水平上的组成如图8所示。从门水平的组成分析可见(图8),地鼠口腔核心菌群有厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)、变形菌门(Proteobacteria)、梭杆菌门(Fusobacteriota)等,与前人的报道一致。
预防组造模48h后,地鼠口腔中具有显著差异的菌属如图9所示。从属水平分析发现(图9),模型组地鼠口腔的伯格菌属(Bergeyella)和芬戈尔德菌属(Finegoldia)的相对丰度显著高于正常对照组(P<0.05)。伯格菌属被发现与牙周病等口腔疾病密切相关;芬戈尔德菌属可以通过激活中性粒细胞诱发炎症。与模型组比较,ET-22活菌显著下调伯格菌属和芬戈尔德菌属的占比,ET-22胞外代谢物可以显著下调伯格菌属比例(P<0.05),ET-22灭活菌组则未出现显著改变。因此,ET-22活菌可能通过抑制口腔有害菌降低口腔溃疡的发病风险。
实施例3
本实施例对比了副干酪乳杆菌ET-22、ET-22胞外代谢物、唾液链球菌K12对变异链球菌生物膜生成量、生物膜结构的影响。
构建唾液包被羟磷灰石模型:配置染液与菌液的混合液备用,菌液选用副干酪乳杆菌ET-22活菌溶液、ET-22胞外代谢物溶液、唾液链球菌K12溶液,菌液浓度为10
9CFU/ml,胞外代谢物溶液浓度为等量菌浓度。准确取用羟基磷灰石(HA)珠子将其高压灭菌后,置 于24孔培养板中,每孔加入1.5ml含有0.2%蔗糖的人造唾液中浸泡37℃包衣培养,之后用1.5ml无菌PBS洗涤两次并吸干缓冲液,加入细菌悬浮液各750ml,37℃培养2h(期间选取不同时间段进行测量,每次测量前用1.5ml无菌PBS漂洗,每次漂洗10s,冲洗三次),即制成唾液包被的羟磷灰石模型,模拟牙齿表面的唾液获得性膜结构。利用结晶紫染色法,用酶标仪在595纳米处测量结果表征生物膜的生成量。使用荧光生物成像系统进行检测,为了量化HA模型上细菌生物膜的染色情况以及细菌的定植情况,手动设置每种荧光颜色的荧光强度阈值,使用Spectral instruments imaging软件程序进行荧光成像的荧光强度分析。
图10表示在模拟唾液环境中,各益生菌组干预下,模拟牙齿表面生物膜生物量的变化。由图10可知,ET-22灭活菌体和胞外代谢物组生物膜生成量显著降低,且差异极显著(P<0.00001),相比而言,ET-22胞外代谢物组生物膜生成量最低;K12活菌组生物膜生成量均高于对照组。
本实施例利用SEM分析了在模拟唾液环境中,各益生菌组干预下模拟牙齿表面生物膜生物量结构的变化。图11为实施例3不同益生菌处干预下模拟牙齿表面生物膜生物量结构的变化的扫描电镜结果图,如图11所示,在人工唾液培养体系对照组形成的是菜花状的多层堆积状生物膜立体结构,ET-22灭活菌组、胞外代谢物组均显著抑制了生物膜的含量,并破坏了菜花状的生物膜结构,K12活菌组呈现单层堆积的生物膜状态,抑制效果不明显。其中,ET-22胞外代谢物组的抑制作用效果最为明显,生物膜呈现单层、零散分布的状态,明显优于其他各处理组。
本实施例利用激光共聚焦显微镜分析了在模拟唾液环境中,各益生菌组干预下模拟牙齿表面生物膜生物量结构与厚度的变化。结构变化如图12所示,对照组的生物膜中菌含量以活菌(绿色)为主。ET-22灭活菌组生物膜中死菌(红色)含量显著升高。ET-22胞外代谢物非常显著的降低了生物膜中的活菌的数量,同时降低了生物膜的分布面积。生物膜厚度变化如图13所示,对照组生物膜厚度高达120μm,ET-22灭活菌组、胞外代谢物组均显著抑制了生物膜的厚度,其ET-22胞外代谢物处理下,生物膜的平均厚度仅为62μm,与对照组相比,下降48%。
最后应说明的是,以上仅用以说明本发明的技术方案而非限制,尽管参照较佳布置方案对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围。
Claims (11)
- 副干酪乳杆菌在制备用于防治口腔溃疡的药物中的应用,其中,所述副干酪乳杆菌包括副干酪乳杆菌ET-22菌体和/或其胞外代谢物,所述副干酪乳杆菌ET-22为保藏编号CGMCC No.15077的菌株。
- 根据权利要求1所述的应用,其中,所述副干酪乳杆菌ET-22菌体为活菌体和/或灭活的菌体;所述副干酪乳杆菌ET-22胞外代谢物是按照以下方法制备得到的:从副干酪乳杆菌ET-22的孵育液中去除菌体,得到副干酪乳杆菌ET-22胞外代谢物;优选地,所述孵育液为副干酪乳杆菌ET-22在水中孵育获得。
- 根据权利要求1或2所述的应用,其中,所述口腔溃疡包括由于病原菌引起的口腔溃疡、由于氧化损伤引起的口腔溃疡或免疫低下引起的口腔溃疡。
- 一种益生菌组合物,其包括:副干酪乳杆菌ET-22菌体和/或其胞外代谢物;所述副干酪乳杆菌ET-22为保藏编号CGMCC No.15077的菌株。
- 根据权利要求4或5所述的益生菌组合物,其中,所述副干酪乳杆菌ET-22菌体为活菌体和/或灭活的菌体;所述副干酪乳杆菌ET-22胞外代谢物是按照以下方法制备得到的:从副干酪乳杆菌ET-22的孵育液中去除菌体,得到副干酪乳杆菌ET-22胞外代谢物;优选地,所述孵育液为副干酪乳杆菌ET-22在水中孵育获得。
- 权利要求4-6任一项所述的组合物在制备用于防治口腔溃疡的组合物中的应用;优选地,所述口腔溃疡包括由于病原菌引起的口腔溃疡、由于氧化损伤引起的口腔 溃疡或免疫低下引起的口腔溃疡。
- 根据权利要求7所述的应用,其中,所述用于防治口腔溃疡的组合物为药物组合物;优选地,所述药物组合物为贴剂。
- 一种益生菌贴剂的制备方法,该方法包括:(1)将副干酪乳杆菌ET-22菌体和/或其胞外代谢物、蜂胶、羧甲基纤维素钠和润滑剂混合均匀,得到混合体系A的步骤;(2)将冰片、甘油和吐温-20隔水加热混匀,得到混合体系B的步骤;(3)将混合体系A和混合体系B混合均匀的步骤;(4)干燥处理步骤。
- 一种益生菌贴剂,其是以权利要求4-6任一项所述的组合物制备得到的,或经权利要求9所述的方法制备得到的。
- 一种治疗口腔溃疡的方法,所述方法包括给予受试者有效量的权利要求4-6任一项所述的组合物和/或权利要求10所述的益生菌贴剂;所述受试者包括哺乳动物或人。
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