CN116509907A - 一种对口腔疾病有改善的后生元及其应用 - Google Patents
一种对口腔疾病有改善的后生元及其应用 Download PDFInfo
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- CN116509907A CN116509907A CN202310326598.2A CN202310326598A CN116509907A CN 116509907 A CN116509907 A CN 116509907A CN 202310326598 A CN202310326598 A CN 202310326598A CN 116509907 A CN116509907 A CN 116509907A
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- lactobacillus plantarum
- probiotic composition
- lactobacillus
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Abstract
本发明提供一种益生菌组合物在制备改善口腔疾病产品中的应用,所述的益生菌组合物中包括植物乳植杆菌(Lactiplantibacillus plantarum)RH03147、植物乳植杆菌(Lactiplantibacillus plantarum)HCS03‑001和罗伊氏粘液乳杆菌(Limosilactobacillus reuteri)HCS02‑001。本发明公开的益生菌组合物,可以有效的抑制变异链球菌和牙龈卟啉单胞菌。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种益生菌组合物的制备和在制备预防或治疗口腔疾病的药品中的应用。
背景技术
后生元是对宿主健康有益的无生命的微生物和/或其成分(包括代谢物、细胞组分或无细胞混合物),包括许多不同的成分,如短链脂肪酸、微生物细胞组分、胞外多糖、细胞裂解物、磷壁酸、肽聚糖、活性脂质和有机酸等。研究证明后生元具有抗炎、抑菌、免疫调节、抗氧化、抗肥胖、抗高血压、降血脂、保肝和促进伤口愈合等生物活性。一些后生元还可以积极影响宿主肠道菌群的稳态或代谢和信号途径的表达。抗炎、抑菌是后生元最为常见的生物活性。
口腔微生态是人体重要的生态系统,主要由口腔微生物群和宿主固有口腔环境构成。生理情况下,口腔微生物与宿主处于动态平衡,维持口腔健康。当平衡被打破时,可诱发口腔有害菌滋生,引起口腔疾病,如龋齿、牙周病、口腔癌、口腔黏膜炎等。
龋齿和牙周病是一种发病率极高的口腔疾病,主要由变异链球菌和牙龈卟啉单胞菌等口腔致病菌引起的。以链球菌属为例,其生长过程中产生生物被膜,被膜中含有丰富的多糖类物质,使其具有很强的粘附性。其他种类的致龋菌吸附在被膜表面形成牙菌斑,导致龋齿形成。若龈缘处存在的细菌数目升高导致牙龈发炎,会发展为牙龈炎。调整口腔微生态平衡,抑制口腔有害菌的生长,可减少龋齿等口腔疾病的发生。传统的治疗方式旨在完全消除口腔中的致龋菌,而抗生素和抑菌剂也成为了杀菌的第一选择。但是这样的治疗方式使得抗生素的滥用,也进一步破坏口腔微生态,引发的细菌耐药性等一系列副作用,并且治疗效果一般,所以利用具有抑菌能力并可以提高机体抵抗力的益生菌治疗口腔疾病已逐渐成为研究的热点。
植物乳植杆菌(Lactiplantibacillus plantarum)是一种乳酸菌,广泛存在于自然界中,特别是在各种类型的发酵食品中。植物乳杆菌定植在人和动物肠道中,具有抑制病原菌,改善肠道环境,调节肠道菌群,提高机体免疫力等诸多优良益生功能,它广泛用于食品,医药和饲料添加剂中。专利CN
109957530A公开了植物乳杆菌(Lactobacillus plantarum)HCS03-001抑口腔致病菌能力强、具有通便和抗腹泻的功效。该发明还公开了该菌株作为抑菌特性好,尤其是抑口腔致病菌能力强的添加剂植物乳杆菌冻干粉在制备食品、保健食品或药物组合物中的应用。
罗伊氏粘液乳杆菌(Limosilactobacillus reuteri)广泛存在于人和动物肠道中,来源较为丰富。其益生作用繁多,可调节肠道菌群有效预防腹泻,同时可抑制病源微生物的繁殖,减少肠道疾病的发生。罗伊氏乳杆菌具有较多益生作用,其中一项为参与胆固醇代谢,服用后经过消化系统进入肠道,具备耐酸耐胆盐特性,对机体发挥益生作用。专利CN114990011A公开了罗伊氏乳杆菌(Lactobacillus reuteri)HCS02001具有降胆固醇和抑制阴道加德纳菌的作用。但未公开其在维持口腔健康中的应用。
发明内容
为了解决上述问题,本发明提供了一种益生菌组合物,可以有效的抑制变异链球菌和牙龈卟啉单胞菌,减少龋齿和牙周病等口腔疾病的发生。有效防止传统治疗方法引发的细菌耐药性等一系列副作用,调整口腔微生态平衡,维持口腔健康。
一方面本发明提供了一种益生菌组合物在制备益生菌制剂、饮料、奶制品、糖果制品、口腔护理品、预防或治疗口腔疾病的药品中的应用,所述的益生菌组合物由植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001组成。
具体地,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001的活化方法为:
(1)取存于低温冰箱内的植物乳植杆菌RH03147、植物乳植杆菌HCS03-001、罗伊氏粘液乳杆菌HCS02-001菌种冻存管,分别立即放入37℃水浴锅内15-30s,至冻存管内液体全部融化进行菌种复苏;
(2)按照10%接种量,分别将复苏好的菌种接种至不同的培养基A中,37℃培养箱恒温静置培养17h获得种子液;
(3)按照5%接种量,将种子液分别接种至培养基A中,37℃培养箱恒温静置培养17h得三个菌种的培养液。
具体地,培养基A的配制:酵母蛋白胨12.0g/L、葡萄糖25.0g/L、酵母浸出物8.0g/L、乙酸钠8.0g/L、一水柠檬酸3.0g/L、磷酸二氢钾3.0g/L、吐温80 0.2g/L、硫酸镁0.8g/L、余量为纯化水;按照配方比例称量、加热溶解,115℃下灭菌30min,用1mol/L食品级NaOH溶液调培养基pH值至6.60。
具体地,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001按照2-4:1-4:1-2的菌数比例混合。
在一些实施例中,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001的菌数混合比例为3.5:3.5:1;
在一些实施例中,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001的菌数混合比例为2:1:1。
具体地,所述的植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001分别接种于培养液B中,接种量2-6%,35-40℃
搅动培养17h,得发酵液。
进一步优选地,所述的发酵温度为35℃;所述的接种量为5%。
具体地,所述培养液B包括酵母蛋白胨5-15.0g/L、葡萄糖15-20.0g/L、酵母浸出物5-12.0g/L、乙酸钠3-5.0g/L、一水柠檬酸2.0-3.0g/L、磷酸二氢钾2.0-3.0g/L、吐温800.1-0.2g/L、硫酸镁0.6-0.8g/L、硫酸锰0.20-0.25g/L、余量为纯化水。
所述的护理品可以是漱口水、漱口粉、牙膏、牙粉或口喷。
所述的护理品中还可以包括其他辅料,如维生素、矿物质、甜味剂、酸味剂、凉味剂、填充剂中的一种或多种。
所述的药品的剂型可以是漱剂、喷剂、药膜、气雾剂、膏剂或糊剂。
所述的药品中还可以包括其他药学上可接受的载体或赋形剂,如淀粉、糊精、蔗糖、乳糖、甘露醇、微晶纤维素、交联聚维酮、交联羧甲纤维素钠、交联羧甲淀粉钠中的一种或多种。
另一方面本发明提供一种后生元在制备口腔护理品、预防或治疗口腔疾病的药品中的应用,所述的后生元通过上述益生菌组合物进行制备。
具体地,将前述的益生菌组合物经发酵、杀菌、干燥得到产物。
进一步具体地,将益生菌组合物接种到培养液B中,35-40℃恒温发酵、浓缩、杀菌得后生元液,加入辅料后喷雾干燥得后生元粉。
优选地,所述的发酵温度为35℃。
具体地,35℃搅动培养48h,得发酵液。将发酵液过滤得滤液,滤液无色至浅黄色透明无杂质;将上清液进行2倍加热浓缩,蒸煮杀菌30min,得后生元液。
具体地,所述的辅料包括麦芽糊精,添加量为5-15%。
优选地,所述的辅料包括麦芽糊精,添加量为8%。
具体地,向后生元液中添加麦芽糊精后,混合搅拌至完全溶解,环境湿度≤50%,置于喷雾干燥机中进行喷雾干燥,进风温度180℃,出风温度110℃,得后生元粉。
具体地,所述培养液B包括酵母蛋白胨5-15.0g/L、葡萄糖15-20.0g/L、酵母浸出物5-12.0g/L、乙酸钠3-5.0g/L、一水柠檬酸2.0-3.0g/L、磷酸二氢钾2.0-3.0g/L、吐温800.1-0.2g/L、硫酸镁0.6-0.8g/L、硫酸锰0.20-0.25g/L、余量为纯化水。
所述的护理品可以是漱口水、漱口粉、牙膏、牙粉或口喷。
所述的护理品中还可以包括其他辅料,如维生素、矿物质、甜味剂、酸味剂、凉味剂、填充剂中的一种或多种。
所述的药品的剂型可以是漱剂、喷剂、药膜、气雾剂、膏剂或糊剂。
所述的药品中还可以包括其他药学上可接受的载体或赋形剂,如淀粉、糊精、蔗糖、乳糖、甘露醇、微晶纤维素、交联聚维酮、交联羧甲纤维素钠、交联羧甲淀粉钠中的一种或多种。
另一方面本发明提供了一种益生菌组合物在抑制变异链球菌和/或牙龈卟啉单胞菌中的应用,所述的益生菌组合物由植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001组成,所述的应用为非疾病诊断或治疗的应用。
本发明还提供了一种后生元在抑制变异链球菌和/或牙龈卟啉单胞菌中的应用,所述的后生元通过上述所述的益生菌组合物进行制备,所述的应用为非疾病诊断或治疗的应用。
本发明所取得的的技术效果:本发明提供了一种益生菌组合物,抑菌试验数据显示所述的植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001之间具有协同作用,菌株的组合使用比单独使用对变异链球菌和牙龈卟啉单胞菌抑制效果更好,更有利于减少龋齿和牙周病等口腔疾病的发生。有效防止传统治疗方法引发的细菌耐药性等一系列副作用,调整口腔微生态平衡,维持口腔健康。
附图说明
图1为益生菌组合物后生元对变异链球菌抑菌作用结果图。
图2为益生菌组合物后生元对龈卟啉单胞菌抑菌作用结果图。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1益生菌组合物后生元的制备
本实施例中益生菌为:植物乳植杆菌RH03147(保藏号为CGMCCNO.25775)、植物乳植杆菌HCS03-001(保藏号为CGMCC NO.16258)、罗伊氏粘液乳杆菌HCS02-001(保藏号为CGMCC NO.19746)。
益生菌组合物后生元制备的具体步骤如下:
(1)取存于低温冰箱内的植物乳植杆菌RH03147、植物乳植杆菌HCS03-001、罗伊氏粘液乳杆菌HCS02-001菌种冻存管,分别立即放入37℃水浴锅内15-30s,至冻存管内液体全部融化进行菌种复苏;按照10%接种量,分别将复苏好的菌种接种至不同的培养基A中,37℃培养箱恒温静置培养17h获得种子液;按照5%接种量,将种子液分别接种至培养基A中,37℃培养箱恒温静置培养17h得三个菌种的培养液,使最终各菌种培养液中菌浓度接近。
(2)将植物乳植杆菌RH03147、植物乳植杆菌HCS03-001、罗伊氏粘液乳杆菌HCS02-001培养液按照3.5:3.5:1的比例接种至同一培养基B中,接种量5%,35℃搅动培养17h,得一级发酵液。
(3)步骤2结束后立即将一级发酵液以5%的接种量再次转接至培养基B中,35℃搅动培养48h,得二级发酵液。
(4)将二级发酵液过滤得滤液,滤液无色至浅黄色透明无杂质;将上清液进行2倍加热浓缩,蒸煮杀菌30min,得后生元液。
(5)向后生元液中添加8%的麦芽糊精,混合搅拌至完全溶解,环境湿度≤50%,置于喷雾干燥机中进行喷雾干燥,进风温度180℃,出风温度110℃,得后生元粉。
所述的培养基A的配制:酵母蛋白胨12.0g/L、葡萄糖25.0g/L、酵母浸出物8.0g/L、乙酸钠8.0g/L、一水柠檬酸3.0g/L、磷酸二氢钾3.0g/L、吐温80 0.2g/L、硫酸镁0.8g/L、余量为纯化水;按照配方比例称量、加热溶解,115℃下灭菌30min,用1mol/L食品级NaOH溶液调培养基pH值至6.60。
所述的培养基B的配制:酵母蛋白胨15.0g/L、葡萄糖20.0g/L、酵母浸出物12.0g/L、乙酸钠5.0g/L、一水柠檬酸2.0g/L、磷酸二氢钾2.0g/L、吐温80 0.2g/L、硫酸镁0.6g/L、硫酸锰0.25g/L、余量为纯化水;按照配方比例称量、加热溶解,115℃下灭菌30min,用1mol/L食品级NaOH溶液调培养基pH值至6.80。
实施例2后生元抑制变异链球菌试验
采用双层琼脂扩散法验证后生元对变异链球菌的抑菌能力:
(1)取后生元粉样品用无菌生理盐水进行5倍稀释(质量倍数),完全溶解后备用。
将2%的水琼脂培养基倒入无菌平板中,使水琼脂刚刚没过皿底,待琼脂凝固。
(2)将变异链球菌菌株冻存管解冻后,将1mL冻存管菌液全部加入100mL BHI液体培养基中,37℃厌氧培养18-24h;在水琼脂平皿上平均放置4个牛津杯,取100μL变异链球菌菌液(浓度约为1×108CFU/mL)与10mL 1.2%的琼脂培养基混匀倒入平皿中,待琼脂凝固,取走牛津杯,形成直径为8mm的加样孔。
(3)平板中3个加样孔加入100μL后生元样品,其余1个孔中加入MRS灭菌培养基作为空白对照,置于37℃厌氧培养24h。用游标卡尺测量抑菌圈直径,加入后生元的牛津杯抑菌圈可达23.17mm,如图1所示。
BHI(合成培养基):胰蛋白胨1.0%,无水葡萄糖0.2%,氯化钠0.5%,牛心粉0.5%,十二水磷酸氢二钠0.25%,固体加琼脂粉2%,pH 7.4±0.2。
MRS培养基:酵母蛋白胨10g/L,牛肉粉3g/L,酵母浸出物4g/L,磷酸二氢钾2g/L,一水柠檬酸2g/L,乙酸钠5g/L,无水葡萄糖20g/L,硫酸镁0.58g/L,硫酸锰0.25g/L,吐温800.6g/L,番茄汁1%(v/v),pH 6.5。
实施例3后生元抑制牙龈卟啉单胞菌试验
采用双层琼脂扩散法验证后生元对牙龈卟啉单胞菌的抑菌能力:
(1)取后生元粉样品用无菌生理盐水进行5倍稀释(质量倍数),完全溶解后备用。
(2)将2%的水琼脂培养基倒入无菌平板中,使水琼脂刚刚没过皿底,待琼脂凝固。
(3)将牙龈卟啉单胞菌菌株冻存管解冻后,将1mL冻存管菌液全部加入100mLTSB液体培养基中,37℃厌氧培养18-24h;在水琼脂平皿上平均放置4个牛津杯,取100μL牙龈卟啉单胞菌(浓度约为1×108CFU/mL)与10mL1.2%的琼脂培养基混匀倒入平皿中,待琼脂凝固,取走牛津杯,形成直径为8mm的加样孔。
(4)平板中3个加样孔加入100μL后生元样品,其余1个孔中加入MRS灭菌培养基作为空白对照,置于37℃厌氧培养24h。用游标卡尺测量抑菌圈直径,加入后生元液的牛津杯抑菌圈可达23.00mm,如图2所示。
TSB培养基:胰蛋白胨17g/L,植物蛋白胨3g/L,氯化钠5g/L,磷酸氢二钾2.5g/L,无水葡萄糖2.5g/L,固体加琼脂粉20g/L,pH 7.3±0.2,灭菌放凉后加入5%无菌脱纤维羊血。
实施例4
参照实施例1的方法制备后生元,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001的菌数混合比例为2:1:1。所述培养基B的配制:酵母蛋白胨5.0g/L、葡萄糖15.0g/L、酵母浸出物5.0g/L、乙酸钠3.0g/L、一水柠檬酸3.0g/L、磷酸二氢钾2.0g/L、吐温80 0.2g/L、硫酸镁0.8g/L、硫酸锰0.2g/L、余量为纯化水;按照配方比例称量、加热溶解,115℃下灭菌30min,用1mol/L食品级NaOH溶液调培养基pH值至6.80。
利用上述后生元参照实施例2和实施例3分别进行变异链球菌、牙龈卟啉单胞菌的抑菌试验,其试验结果如下:后生元对变异链球菌的抑菌圈直径为23.22mm;对牙龈卟啉单胞菌的抑菌圈直径为23.10mm。
对比例1-10
对比例中的后生元均参照实施例1的方法进行制备。
参照实施例2的方法设置对比例1-5并进行实验,对比例设置方式和最终抑菌圈结果如下:
参照实施例3的方法设置对比例6-10并进行实验,对比例设置方式和最终抑菌圈结果如下:
Claims (10)
1.一种益生菌组合物在制备益生菌制剂、饮料、奶制品、糖果制品、口腔护理品、预防或治疗口腔疾病的药品中的应用,其特征在于,所述的益生菌组合物由植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001组成。
2.根据权利要求1所述的应用,其特征在于,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001按照2-4:1-4:1-2的菌数比例混合。
3.根据权利要求2所述的应用,其特征在于,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001的菌数混合比例为3.5:3.5:1。
4.根据权利要求2所述的应用,其特征在于,所述植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001的菌数混合比例为2:1:1。
5.一种后生元在制备口腔护理品、预防或治疗口腔疾病的药品中的应用,其特征在于,所述的后生元通过权利要求1中所述的益生菌组合物进行制备。
6.根据权利要求5所述的应用,其特征在于,将益生菌组合物经发酵、杀菌、干燥得到产物。
7.根据权利要求6所述的应用,其特征在于,将益生菌组合物接种到培养液中,35-40℃恒温发酵、浓缩、杀菌得后生元液,加入辅料后喷雾干燥得后生元粉。
8.根据权利要求7所述的应用,其特征在于,所述培养液包括酵母蛋白胨5-15.0g/L、葡萄糖15-20.0g/L、酵母浸出物5-12.0g/L、乙酸钠3-5.0g/L、一水柠檬酸2.0-3.0g/L、磷酸二氢钾2.0-3.0g/L、吐温80 0.1-0.2g/L、硫酸镁0.6-0.8g/L、硫酸锰0.20-0.25g/L、余量为纯化水。
9.一种益生菌组合物在抑制变异链球菌和/或牙龈卟啉单胞菌中的应用,其特征在于,所述的益生菌组合物由植物乳植杆菌RH03147、植物乳植杆菌HCS03-001和罗伊氏粘液乳杆菌HCS02-001组成,所述的应用为非疾病诊断或治疗的应用。
10.一种后生元在抑制变异链球菌和/或牙龈卟啉单胞菌中的应用,其特征在于,所述的后生元通过权利要求1中所述的益生菌组合物进行制备,所述的应用为非疾病诊断或治疗的应用。
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