CN114869917A - 植物乳杆菌n13在制备防治阴道炎或抑制病原菌药物中的用途 - Google Patents
植物乳杆菌n13在制备防治阴道炎或抑制病原菌药物中的用途 Download PDFInfo
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- CN114869917A CN114869917A CN202210791819.9A CN202210791819A CN114869917A CN 114869917 A CN114869917 A CN 114869917A CN 202210791819 A CN202210791819 A CN 202210791819A CN 114869917 A CN114869917 A CN 114869917A
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- lactobacillus plantarum
- pathogenic bacteria
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- inhibition
- vaginitis
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Abstract
本发明植物乳杆菌N13及其在制备防治阴道炎或抑制病原菌药物中的用途属于微生物技术领域。本发明一方面请求保护保藏编号为CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途,另一方面请求保护所述植物乳杆菌N13在制备妇科外用洗剂方面的用途,以及非治疗目的地抑制病原菌方面的用途。本发明通过大量实验证实N13具有优异的阴道上皮细胞粘附力,可有效抑制致病菌繁殖,对常见的3大病原菌有较强的抑菌能力,并经人体试验证实其具有显著的抗阴道炎的能力。
Description
技术领域
本发明属于微生物技术领域,具体涉及植物乳杆菌N13及其在制备防治阴道炎或抑制病原菌药物中的用途。
背景技术
阴道炎是妇科疾病中的常见病、多发病,易感染,发病率逐年上升,其具有复发率高、根治困难等特点。阴道炎常见诸如排尿困难、瘙痒和分泌物等生殖道症状。最常见的原因是外阴道感染,其有三个常见原因:念珠菌病、细菌性阴道疾病,以及滴虫病,其中前两者都是由微生物所造成。
白色念珠菌通常在正常阴道环境中存在,但数量很少,不引起疾病,当阴道正常环境被破坏时,该菌大量繁殖并进入细胞引起疾病。
大肠埃希菌属肠道正常菌群,可因入侵肠外组织及免疫力下降等因素而成为条件致病菌,引起多部位感染,是泌尿系统感染分离率最高的病原菌。
金黄色葡萄球菌是一种常见条件致病菌,是引起院内感染和社区感染的主要病原菌。金葡菌与尿路感染关系密切,也是引起女性生殖道感染较为常见的一种病原菌。
阴道的防御系统主要基础是阴道鳞状上皮细胞内的糖原经乳杆菌作用,分解成乳酸,使阴道局部形成弱酸性环境(pH≤4.5,多在3.8-4.4),可以抑制潜在病原菌的过渡生长,因而,此环境对嗜酸性的乳酸菌来说则是理想的环境。乳酸菌通过替代、竞争排斥机制阻止致病微生物粘附于阴道上皮细胞。同时,分泌过氧化氢、细菌素、类细菌素和生物表面活性剂等抑制致病微生物生长,从而维持阴道微生态的平衡。而植物乳杆菌是乳酸菌的一种,与其它乳酸菌的区别在于其活菌数比较高,能大量产酸、过氧化氢等活性物质,此外,植物乳杆菌还具有免疫调节作用,抑制肿瘤细胞的形成;因此,开发具有抑制阴道病原菌的植物乳杆菌用于预防或治疗阴道炎的复发意义重大。
发明内容
为了解决现有技术问题中常规植物乳杆菌不具有抑制念珠菌、维持阴道酸性环境并成功定植于阴道等能力的技术问题,本发明旨在提供植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途。
本发明的技术方案如下:
保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途。
所述防治阴道炎指:提升阴道清洁度、和/或,降低阴道白细胞数。
所述病原菌选自:白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌中的一种或一种以上。
所述药物包括药效活性成分;所述药效活性成分包括保藏编号为CGMCC No.20496的植物乳杆菌N13;
优选地,所述药物还包括药用辅料。
保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备妇科外用洗剂方面的用途。
所述妇科外用洗剂还包括:药用辅料。
所述药用辅料为表面活性剂;
优选地,所述表面活性剂为苯扎溴铵。
保藏编号为 CGMCC No.20496的植物乳杆菌N13在非治疗目的地抑制病原菌方面的用途。
所述病原菌选自:白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌中的一种或一种以上。
所述植物乳杆菌N13抑制白色念珠菌的抑菌圈为8.0mm或15.9mm;
所述植物乳杆菌N13抑制大肠杆菌的抑菌圈为37mm;
所述植物乳杆菌N13抑制沙门氏菌的抑菌圈为22mm;
所述植物乳杆菌N13抑制金黄色葡萄球菌的抑菌圈为22mm。
本发明还请求保护保藏编号为CGMCC No.20496的植物乳杆菌N13在防治阴道炎或抑制病原菌方面的用途。
所述植物乳杆菌N13通过生物样品分离出的菌株低pH值实验的初筛得到50余株植物乳杆菌菌株,进一步地,以拮抗白色念珠菌为主要筛选指标,进行金黄色葡萄球菌、大肠杆菌及沙门氏菌的拮抗筛选,最后通过对Hela细胞粘附能力的筛选考察,得到一株具有预防和治疗白色念珠菌的植物乳杆菌N13。
与现有技术相比,本发明提供的植物乳杆菌,对白色念珠菌具有较强的拮抗作用,其拮抗能力优于普通常用的植物乳杆菌和商业鼠李糖乳杆菌LGG。
所述的植物乳杆菌N13具有较强的Hela细胞粘附能力,平均每个细胞粘附19个植物乳杆菌N13,与市售对照菌相比,具有优异的阴道上皮细胞粘附力。
所述的植物乳杆菌N13与普通植物乳杆菌及市售对照菌LGG相比,具有优异的乳酸产酸能力及耐酸能力,在维持阴道酸性环境,抑制致病菌繁殖并成功定植于阴道方面发挥了重要作用。
所述植物乳杆菌N13具有较强的耐酸及耐胆盐的能力,对常见的3大病原菌有较强的抑菌能力,并且对常用抗生素敏感,能够顺利到达肠道,说明该菌株是具有开发应用潜力的安全益生菌,可用于食品或口服药用制品。
本发明的另一个目的是提供植物乳杆菌N13在制备预防或治疗阴道炎药物组合物及食品中的应用。
所述药用组合物可包含口服剂型或外用剂型;口服剂型例如为锭剂、胶囊、溶液剂及粉剂等。外用剂型例如乳膏、喷雾剂、洗剂、凝胶或霜剂等。
所述食品组合物可包含但不限于发酵乳、酸奶、奶酪、乳制饮品、乳茶、咖啡、固体饮料或以上的组合。
附图说明
图1为本发明的实验例4中植物乳杆菌N13在体外模拟的人工胃液及人工肠液的耐受性测试中的存活率柱形图。
具体实施方式
下面结合具体实施例和实验例对本发明作进一步详细的描述,但本发明的实施方式不限于此。
生物材料的来源
下述实验例中涉及的实验动物为普通级新西兰种成年健康雌性实验用家兔,购自鲁抗医药集团;下述实验例中涉及的白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌均购自广东微生物菌种保藏中心;下述实验例中涉及的伊红美蓝培养基,BG培养基均购自青岛海博生物技术有限公司;DMEM培养基购自gibco公司;
下述实验例中涉及的培养基如下:
MRS液体培养基:葡萄糖20g/L,酵母蛋白胨10g/L,牛肉浸粉10g/L,酵母浸粉5g/L,无水乙酸钠5g/L,吐温-80 1g/L,柠檬酸氢二铵2g/L,磷酸氢二钾2g/L,七水硫酸镁0.58g/L,一水硫酸锰0.19g/L,水1000mL,pH6.8。
13号液体培养基:葡萄糖10g/L,蛋白胨10g/L,酵母抽提物5g/L,水1000mL,pH7.0。
LB液体培养基:胰蛋白胨10g/L,酵母5g/L,NaCl5g/L,水1000mL,pH7.2。
固体培养基中加1.5%的琼脂粉。
第1组实施例、本发明的植物乳杆菌N13的制药用途
本组实施例提供保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途。
在一些实施例中,所述防治阴道炎指:提升阴道清洁度、和/或,降低阴道白细胞数。
在另一些实施例中,所述病原菌选自:白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌中的一种或一种以上。
在具体的实施例中,所述药物包括药效活性成分;所述药效活性成分包括保藏编号为CGMCC No.20496的植物乳杆菌N13;
优选地,所述药物还包括药用辅料。
在具体的实施例中,所述药用辅料选自:溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏合剂、整合剂、渗透促进剂、pH值调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂、反絮凝剂、助滤剂、释放阻滞剂。
根据本发明的内容,出于实际生产应用中的不同需求,再结合药物制备领域的常规技术手段(例如,《制剂技术百科全书》、《药物制剂技术》等),本领域技术人员可对上述药用辅料进行选择和调配,并将CGMCC NO.20496的植物乳杆菌(Lactobacillus plantarum)菌株N13制成不同的剂型,例如粉剂、片剂、注射剂、口服液等。
第2组实施例、本发明N13菌株的妇科外用洗剂制备用途
本组实施例提供保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备妇科外用洗剂方面的用途。
在一些具体的实施例中,所述妇科外用洗剂还包括:药用辅料。
在更具体的实施例中,所述药用辅料为表面活性剂;
优选地,所述表面活性剂为苯扎溴铵。
第3组实施例、本发明的N13菌株的抑菌用途
本组实施例提供保藏编号为 CGMCC No.20496的植物乳杆菌N13在非治疗目的地抑制病原菌方面的用途。
在一些实施例中,所述非治疗目的地抑制病原菌方面的用途指:用植物乳杆菌N13抑制非人源样本或非动物源样本中的病原菌。
在另一些实施例中,所述非治疗目的地抑制病原菌方面的用途指:以植物乳杆菌N13为拮抗菌,以病原菌为指示菌,进行抑菌圈实验。
在一些实施例中,所述病原菌选自:白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌中的一种或一种以上。
在更具体的实施例中,所述植物乳杆菌N13抑制白色念珠菌的抑菌圈为8.0mm或15.9mm;
所述植物乳杆菌N13抑制大肠杆菌的抑菌圈为37mm;
所述植物乳杆菌N13抑制沙门氏菌的抑菌圈为22mm;
所述植物乳杆菌N13抑制金黄色葡萄球菌的抑菌圈为22mm。
第4组实施例、本发明的食品
本组实施例提供一种食品。本组所有的实施例都具备如下共同特征:所述食品包括保藏编号为CGMCC No.20496的植物乳杆菌N13。
在具体的实施例中,所述食品还包括食用辅料。
在另一些实施例中,所述食用辅料选自:漂白剂、防腐剂、抗氧化剂、着色剂、甜味剂、酸味剂、增味剂、护色剂等。
根据本发明的内容,出于实际生产应用中的不同需求,再结合食品生产加工工艺领域的常规技术手段(例如,《食品生产概论》、《食品与食品生产百科全书》、《食品加工技术》等),本领域技术人员可对上述食用辅料进行选择和调配,并将CGMCC NO.20496的植物乳杆菌(Lactobacillus plantarum)菌株N13制成不同的剂型,例如发酵乳制品、发酵豆制品、发酵果蔬制品、发酵肉制品、发酵饮料、益生菌发酵剂、益生菌固体饮料等。所述发酵乳制品包括常温酸奶、低温酸奶、搅拌型酸奶、凝固型酸奶、饮用性酸奶、乳酪、乳酸菌饮料等。
在更具体的实施例中,所述食品为固体饮料。
实验例1、对预防或治疗阴道炎的植物乳杆菌N13的筛选
(1)低pH值植物乳杆菌菌株的初筛
130株植物乳杆菌来源于微康益生菌菌种资源库,从菌种库甘油管出发,进行一代活化培养后,以2%接种量转接2代(10ml/15ml离心管),培养16h后测量其pH值
实验结果表明:经过本轮初筛,筛选出pH值低于3.9的菌株50株。
(2)抑制白色念珠菌植物乳杆菌N13的筛选
将(1)中筛选出的50株菌株分别接种到MRS液体培养基中,37℃静置培养24h,1%接种量;白色念珠菌100ul接种于13号液体培养基中37℃摇床培养16h。
双层平板法:将13号固体培养基冷却至55℃左右,分别与指示病原菌菌悬液按一定比例混匀,使指示菌的活菌数在105CFU/mL数量级,然后迅速倾注于平板中,待培养基冷却凝固后,吸取菌泥点板,于37℃培养箱培养5h后,将MRS培养基倾注于点菌的13号固体培养基上,37℃兼性厌氧培养24h左右,观察并记录抑菌能力强的抑菌圈情况。实验结果如下表1所示:
对50株菌株通过白色念珠菌的拮抗筛选实验,优选出10株抑制白色念珠菌效果较好的植物乳杆菌菌株,且比商业菌株LGG拮抗白色念珠菌能力更强。
采用牛津杯打孔法评价了植物乳杆菌N13对白色念珠菌的拮抗作用。将MRS固体培养基冷却至55℃左右,分别与指示病原菌菌悬液按一定比例混匀,使指示菌的活菌数在105CFU/mL数量级,然后迅速倾注于预先放置牛津杯的平板中,待培养基冷却凝固后,取出牛津杯,每孔中加入150μL植物乳杆菌发酵液,恒温箱培养24h,测量抑菌圈直径(mm),结果如下表2所示。
(3)对Hela细胞粘附能力强的植物乳杆菌N13筛选
Hela细胞的复苏与传代培养:从液氮中取出冻存Hela细胞的冻存管,迅速放入盛有37℃温水的保温杯中,然后在细胞操作室中将冻存的细胞接种到盛有37℃预温的含有10%小牛血清和0.5%双抗的DMEM细胞培养液中,置于37℃含5%CO2培养箱中培养。传2-3代后生长旺盛的细胞,方可进行后续试验。
Hela细胞悬液的制备:待细胞培养瓶中的Hela细胞长到80%满时,用1mL0.25%胰蛋白酶-EDTA溶液消化2min后,吸除消化液,立即加入新鲜的含有10%胎牛血清的DMEM培养基轻轻吹打混匀,使细胞脱离培养瓶,并转移细胞液于15mL无菌离心管中,低速离心后,弃去上清液,加入含有10%胎牛血清的DMEM培养基,使用移液枪轻轻吹打混匀后取1mL悬液用血球计数板在倒置显微镜下计数。根据计数结果,加入适量细胞培养液配制成1*105个/mL细胞悬液备用。
粘附实验:取500μL浓度为105个/mL HeLa细胞接种于24孔培养板中,置于37℃、5%CO2孵育箱中过夜培养至汇合单层细胞形成,将培养的HeLa细胞用PBS清洗3次并加入500μL无双抗、无小牛血清的DMEM基础培养基。然后再加入制备好的500μL浓度为108CFU/mL活化过夜培养的植物乳杆菌N13(对照孔加入2mL无双抗无小牛血清的DMEM细胞基础培养液),轻柔晃动细胞培养板,使植物乳杆菌均匀分散开后,置于37℃、5%CO2环境中孵育4h,然后用 PBS清洗4次,0.25%的胰酶消化4~5min,再用350μL DMEM 培养基终止消化,并吹打混匀,取100μL104倍稀释菌液涂布MRS平板,37℃培养计数,取3次平行测定的平均值。
如表3所示,结果表明:Hela细胞经复苏,在细胞培养瓶中生长情况良好,第三天出现角状形态,并开始分化。粘附实验结果表明,采用与阴道上皮细胞同源性最高的宫颈癌上皮细胞Hela细胞为体外实验模型,对植物乳杆菌粘附上皮细胞的能力进行筛选。不同植物乳杆菌粘附Hela细胞的能力不同。有些分离株粘附能力很强,每个Hela细胞可以粘附十多个植物乳杆菌;有些分离株则粘附能力较弱或者没有粘附能力。由下表实验结果可知,N13菌株粘附Hela细胞能力最强,综合拮抗白色念珠菌的筛选实验结果可知,植物乳杆菌N13菌株拮抗白色念珠菌及粘附Hela细胞能力最强。
最终筛选出:抑制白色念珠菌能力和Hela细胞粘附能力双高的菌株为N13,该菌株N13即记载于中国专利申请CN113957006A中的植物乳杆菌N13。
实验例2、植物乳杆菌N13对其它病原菌的拮抗能力
考察植物乳杆菌N13抑制其它3种致病菌(金黄色葡萄球菌、大肠杆菌、沙门氏菌)的拮抗作用,进一步验证植物乳杆菌N13能够预防或治疗阴道炎。
指示菌悬液的制备:将大肠杆菌、沙门氏菌和金黄色葡萄球菌分别在平板培养基上活化,37℃培养24h,挑取菌苔至生理盐水制备菌悬液,调整菌液浓度至108CFU/mL。植物乳杆菌发酵液制备:将乳酸杆菌按1%接种至MRS液体培养基中,37℃厌氧培养24h,调整菌液浓度至108CFU/mL。
抑菌实验:将伊红美蓝琼脂培养基、BG琼脂培养基、LB琼脂培养基冷却至55℃左右,依序分别与大肠杆菌、沙门氏菌、金黄色葡萄球菌菌悬液按一定比例混匀,使指示菌的活菌数在106CFU/mL数量级,然后迅速倾注于预先放置牛津杯的平板中,待培养基冷却凝固后,取出牛津杯,于每孔注入150uL植物乳杆菌N13发酵液,37℃过夜培养后,测量抑菌圈直径。
结果如表4所示,植物乳杆菌N13对大肠杆菌、金黄色葡萄球菌和沙门氏菌均有不同程度的抑制作用,其中对大肠杆菌的抑菌能力最强。
实验例3、植物乳杆菌N13模拟消化耐受性测定
模拟人工胃液:将PBS调节pH至3后灭菌,加入0.3%胃蛋白酶,充分溶解后,用0.22μm微孔滤膜过滤除菌备用;
模拟人工肠液:将PBS调节pH至8后灭菌,加入0.1%胰蛋白酶和0.3%的胆粉,充分溶解后,用0.22μm微孔滤膜过滤除菌备用;
植物乳杆菌N13活化培养2代。活化后的植物乳杆菌N13菌液离心,弃上清,收集菌体,制备菌悬液调整活菌数为108CFU/mL。取1mL菌悬液离心后,去掉上清液,收集菌体后,分别接入1mL配制耐受消化溶液,并充分混匀,37℃条件下孵育,同时分别取0h和3h的消化液检测活菌数,计算存活率,结果见图1。其中,菌株存活率计算公式为:N1/N0×100%,式中N0表示菌株0h的活菌数(CFU/mL),N1表示菌株3h的活菌数(CFU/mL)。
实验结果如图1所示,植物乳杆菌N13在体外模拟的人工胃液及人工肠液的耐受性测试中,其存活率均较高。
实验例4、植物乳杆菌N13抗生素敏感性试验
将植物乳杆菌N13在MRS固体平板上划线活化后,挑取菌苔至生理盐水中制备菌悬液,调整菌悬液浓度为108CFU/mL,取100μL菌悬液,用无菌棉签均匀涂布于MRS固体平板上,将抗生素药敏试纸片有序置于平板表面,置于厌氧条件下,37℃培养24-36h后,用游标卡尺测量抑菌圈直径。根据美国临床和实验标准协会CLSI的评价标准,判断抗生素对植物乳杆菌的耐药性,结果见表5。
注:S-敏感;I-中度耐药。
表5结果表明植物乳杆菌N13对测试的15种常用抗生素均敏感,表明该菌株是具有开发应用潜力的安全益生菌。
实验例5、植物乳杆菌N13人体试验
(1)受试者的选择
筛选年龄在20-50岁之间的已婚女性志愿者,排除药物过敏的患者,排除患有严重心、脑、肝、肾疾病或免疫性疾病的患者,排除处于妊娠期或哺乳期的患者,排除入组前一周内采用抗菌药物进行治疗的患者,进行妇科检查和阴道分泌物五联查的检查,根据检查结果,选定受试者共38人,其中患有阴道炎症的受试者18人,阴道健康的受试者20人。
妇科检查包括:分泌物、附件、宫颈、外阴、阴道、子宫、既往史、其它现有症状。
阴道分泌物五联检包括:BV、滴虫、念珠菌、乳酸杆菌、白细胞。
阴道清洁度判定:清洁度分为4度,分别为:
I度:白细胞0-5/HP,属正常。
Ⅱ度:白细胞5-15/HP,可见少量球菌,属基本正常。
Ⅲ度:白细胞15-30/HP,球菌较多,提示有阴道炎。
Ⅳ度:白细胞>30/HP,有大量球菌,提示有较重的阴道炎。
(2)药物试验
受试样品:将植物乳杆菌N13分别经发酵培养、离心浓缩、深冷速冻或真空冷冻干燥、粉碎或低温喷雾干燥制成菌粉;外用洗液:以菌粉为原料,添加麦芽糊精,经混合、包装制成活菌数≥5.0*109CFU/g的植物乳杆菌N13产品。无菌注射用水1mL/瓶。内服胶囊:将上述植物乳杆菌N13产品交由江苏微康益生菌有限公司生产;0.5g/粒,每粒内含植物乳杆菌N13活菌应不低于1*109CFU。注:以上产品经过活菌计数,杂菌,霉菌检验合格后,进行人体试验。
用药方法:受试者在给药前清洗外阴,将植物乳杆菌N13菌粉溶于无菌注射用水中,用给药器将植物乳杆菌N13溶液推入阴道内,同时口服植物乳杆菌N13胶囊1粒/日,睡前使用。
试验周期:每晚口服及阴道给药一次,连续给药10天。
(3)试验结果
表6分别给出了18名阴道炎受试者和20名阴道健康受试者使用植物乳杆菌N13菌粉前后相关检查结果。
试验结果表明:试验过程中及试用后,受试者均未出现胃部不适症状,也未出现阴道瘙痒、灼痛、红肿等过敏不适的症状,不良反应为零,说明该植物乳杆菌N13的安全性。使用植物乳杆菌N13菌粉后,阴道炎患者和阴道健康者的阴道炎症或阴道清洁度均有不同程度的改善,说明了该植物乳杆菌N13的有效性。
实验例6、植物乳杆菌N13在生态妇科外用药物组合物洗剂中的应用
将活化好的植物乳杆菌N13接种于发酵培养基(以MRS培养基作为发酵培养基)。接种量1.0%。37℃发酵24h,再将温度降至32℃继续培养,经检测还原糖耗尽,结束发酵,将发酵液离心,收集离心液,得到所说的益生菌发酵培养物。
发酵完成后加入苯扎溴铵,使苯扎溴铵的浓度为0.115ml/100ml,搅拌均匀,同时,使得最终药物组合物洗剂活菌含量为50亿CFU/mL。即得到本药物组合物洗剂。
将本药物组合物洗剂采用双层平板法分别倾注于溶解的MRS固体培养基后,混匀,倾注于平板上,在MRS平板上再分别倾注混有金黄色葡萄球菌的LB培养基、大肠杆菌的LB培养基、白色念珠菌的13号培养基,植物乳杆菌N13的MRS培养基。37℃培养箱兼性厌氧培养24h后,观察MRS平板上植物乳杆菌N13生长情况并测量抑菌圈直径。
实验结果(表7)表明:含植物乳杆菌N13的生态妇科外用药物组合物洗剂对金黄色葡萄球菌、大肠杆菌、白色念珠菌均具有抑制作用,且该药物组合物对植物乳杆菌N13的生长无抑制作用。
含植物乳杆菌N13药物组合物洗剂多次完整皮肤刺激实验:取实验用家兔3只,体重范围在3.2kg-3.3kg,试验前日,将家兔背部脊柱两侧被毛减掉,去毛范围左右约3cm*3cm。正式实验时,采用自身对照法。取0.5ml样品涂在右侧暴露的皮肤上,在涂抹后4h,用蒸馏水去残留物。每只实验动物每天涂抹一次,连续14天,在每次涂抹后24h观察结果,左侧暴露皮肤作为对照。在观察期内,动物均未出现红斑及水肿,也未见其它异常及中毒现象。
实验例7、植物乳杆菌N13在益生菌固体饮料中的应用
含植物乳杆菌N13的益生菌固体饮料由以下原料制备而成:菌粉(30%)、低聚果糖、鼠李糖乳杆菌、赤藓糖醇、麦芽糊精、蓝莓水果粉、植物乳杆菌N13、鼠李糖乳杆菌。制备此固体饮料的具体方法为:称取麦芽糊精按1:4-1:6重量比投入70℃水中,充分搅拌至完全溶解后过80目筛,得到浆液代用;称取菊粉、低聚果糖、脱脂乳粉、赤藓糖醇、蓝莓水果粉,投入沸腾制粒机中,设置温度65℃,引风频率28Hz,混料20min至均匀,得到混合物料;更改设置沸腾制粒机温度至55℃,引风频率28 Hz,将混合浆液在压力0.1Mpa,流速80r/min条件下喷入沸腾制粒机中的混合物料中,喷浆时间50min至物料出现颗粒度均匀的状态,得到造粒体;更改设置沸腾制粒机温度65℃,引风频率25 Hz,将造粒体干燥20min后停止加热,待料温降至50℃以下后收料;将所收料与植物乳杆菌N13,鼠李糖乳杆菌均匀混合后分装。并对3个批次固体饮料的活菌量进行活菌检测,结果表明本发明的益生菌固体饮料,其益生菌活菌量大于1×1010CFU/g。
Claims (10)
1.保藏编号为CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途。
2.根据权利要求1所述的保藏编号为CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途,其特征在于,所述防治阴道炎指:提升阴道清洁度、和/或,降低阴道白细胞数。
3.根据权利要求1或2所述的保藏编号为CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途,其特征在于,所述病原菌选自:白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌中的一种或一种以上。
4.根据权利要求1或2所述的保藏编号为CGMCC No.20496的植物乳杆菌N13在制备防治阴道炎或抑制病原菌的药物中的用途,其特征在于,所述药物包括药效活性成分;所述药效活性成分包括保藏编号为CGMCC No.20496的植物乳杆菌N13;
和/或,所述药物还包括药用辅料。
5.保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备妇科外用洗剂方面的用途。
6.根据权利要求5所述的保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备妇科外用洗剂方面的用途,其特征在于,所述妇科外用洗剂还包括:药用辅料。
7.根据权利要求6所述的保藏编号为 CGMCC No.20496的植物乳杆菌N13在制备妇科外用洗剂方面的用途,其特征在于,所述药用辅料为表面活性剂;
和/或,所述表面活性剂为苯扎溴铵。
8.保藏编号为 CGMCC No.20496的植物乳杆菌N13在非治疗目的地抑制病原菌方面的用途。
9.根据权利要求8所述的保藏编号为 CGMCC No.20496的植物乳杆菌N13在非治疗目的地抑制病原菌方面的用途,其特征在于,所述病原菌选自:白色念珠菌、大肠杆菌、沙门氏菌、金黄色葡萄球菌中的一种或一种以上。
10.根据权利要求9所述的保藏编号为 CGMCC No.20496的植物乳杆菌N13在非治疗目的地抑制病原菌方面的用途,其特征在于,所述植物乳杆菌N13抑制白色念珠菌的抑菌圈为8.0mm或15.9mm;
所述植物乳杆菌N13抑制大肠杆菌的抑菌圈为37mm;
所述植物乳杆菌N13抑制沙门氏菌的抑菌圈为22mm;
所述植物乳杆菌N13抑制金黄色葡萄球菌的抑菌圈为22mm。
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