CN115317522A - 一株鼠李糖乳杆菌r7970在制备病原菌抑制产品中的应用 - Google Patents
一株鼠李糖乳杆菌r7970在制备病原菌抑制产品中的应用 Download PDFInfo
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- CN115317522A CN115317522A CN202210871501.1A CN202210871501A CN115317522A CN 115317522 A CN115317522 A CN 115317522A CN 202210871501 A CN202210871501 A CN 202210871501A CN 115317522 A CN115317522 A CN 115317522A
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- lactobacillus rhamnosus
- pathogenic bacteria
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- pathogenic
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Abstract
本发明公开了一株鼠李糖乳杆菌R7970在制备病原菌抑制产品中的应用,本发明提供的鼠李糖乳杆菌R7970具备食用安全特性;此外,鼠李糖乳杆菌R7970还具有广谱抑制病原菌活性。因此,本发明的鼠李糖乳杆菌R7970可以有效用于制备病原菌抑制产品,该病原菌抑制产品可以达到抑制口腔致病菌及一些常见致病菌的作用,例如:可抑制口腔致病菌中具核梭杆菌、牙龈卟啉单胞菌、变异链球菌和伴放线放线杆菌的生长,抑制常见致病菌蜡样芽孢杆菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌的生长。综上,本发明的鼠李糖乳杆菌R7970可以用于预防或治疗致病菌感染的疾病,例如致病菌引起的肠道感染、腹泻、阴道感染等疾病;或者为发酵乳提供保鲜功能、抑制霉菌酵母生长等。
Description
技术领域
本发明涉及鼠李糖乳杆菌R7970新用途领域,具体涉及一株鼠李糖乳杆菌R7970在制备病原菌抑制产品中的应用。
背景技术
病原菌是一种能引起人类疾病的微生物,常见的病原菌有金黄色葡萄球菌、白色念珠菌、蜡样芽孢杆菌和大肠杆菌等。金黄色葡萄球菌可产生肠毒素,引发食物中毒、肺炎、伪膜性肠炎,甚至败血症等疾病。白色念珠菌通常会引起口腔、生殖器或者皮肤的感染。部分大肠杆菌可产生肠毒素从而导致疾病,如腹部绞痛和腹泻,有时会引起出发烧、呕吐和血性大肠炎等。蜡样芽孢杆菌引起食物中毒,中毒者症状为腹痛、呕吐腹泻。口腔中也含有潜在病原菌,如具核梭杆菌、牙龈卟啉单胞菌、伴放线放线杆菌和变异链球菌等。变异链球菌是早在1924年Clarke已经报告过变异链球菌是龋齿的病原菌。牙龈卟啉单胞菌是牙周致病菌之一,还有可能引起感染性心内膜炎,甚至是冠心病、动脉粥样硬化等常见疾病。具核梭杆菌和伴放线放线杆菌存在引起牙周病的风险。特别地,具核梭杆菌可促进癌细胞增殖、肿瘤免疫逃逸、复发和化疗耐药性等。因此控制口腔菌群的潜在致病菌数量和丰度,对人体健康具有重要意义。
目前,国内外主要采用口服抗生素的化学疗法来治疗细菌感染性疾病。长期服用抗生素会破坏机体微生态系统平衡,加剧了细菌耐药性,而益生菌疗法则为多种疾病提供了新的诊治方式。益生菌是指宿主摄入足够数量后,定殖在宿主体内,通过改变宿主菌群组成,进而改善体内微生物菌群平衡,并对其健康产生积极作用的一类活性微生物的总称。益生菌在代谢过程中会产生有机酸、细菌素、过氧化氢等物质,这类物质可抑制有害微生物的生长繁殖,因此对致病菌引起的肠道感染和腹泻、阴道菌群失调引起的细菌性阴道炎和真菌性阴道炎、口腔菌群失调引起的龋齿和牙周炎等有预防和保护作用。
一种鼠李糖乳杆菌R7970,现有技术中仅仅只公开了其具有以下应用;(a)在利用乳糖进行发酵中的应用;(b)在提高发酵产物产酸或/和产黏能力的应用;(c)在提高发酵产物产荚膜多糖能力中的应用。并且,现有技术中公开的其他鼠李糖乳杆菌通常仅仅只能实现对肠道菌群的调整,对于肠道以外的其他菌群的调整效果不佳,因此,现有技术中公开的鼠李糖乳杆菌的菌株均不具有广谱抑制病原菌活性。
发明内容
本发明的目的是提供一种鼠李糖乳杆菌R7970的新用途,具体为利用鼠李糖乳杆菌R7970的广谱抑制病原菌活性和食用安全性,提供一株鼠李糖乳杆菌R7970在制备病原菌抑制产品中的应用。
一株鼠李糖乳杆菌R7970,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.22244,分类命名为鼠李糖乳杆菌Lactobacillus rhamnosus,保藏时间为2021年4月27日,保藏地址为北京市朝阳区北辰路西路1号院3号,经保藏中心于2021年4月27日检测为存活菌株。
所述病原菌抑制产品用于预防或治疗致病菌感染的疾病,致病菌为细菌和/或真菌。
所述致病菌至少包括口腔致病菌;
优选的,所述致病菌包括具核梭杆菌、牙龈卟啉单胞菌、变异链球菌、伴放线放线杆菌、蜡样芽孢杆菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌。
本发明中的病原菌抑制产品可以是益生菌固体饮料、益生菌压片糖果、益生菌配方奶粉、功能性健康食品、膳食补充剂、保健食品等,其中还包括食品学上允许的载体或添加剂。该病原菌抑制产品为保健食品时,所述保健食品的剂型为散剂、颗粒剂、片剂、胶囊剂、悬浮剂、乳剂、糖浆剂或浸膏。
本发明中的病原菌抑制产品优选为固体饮料。
所述病原菌抑制产品中鼠李糖乳杆菌R7970菌体的添加量为(1~9)×106CFU/mL;优选的,鼠李糖乳杆菌R7970菌体的添加量为(1~3)×106CFU/mL。
所述病原菌抑制产品包括鼠李糖乳杆菌R7970菌体及其代谢产物。该鼠李糖乳杆菌R7970菌体及其代谢产物可以作为益生菌后生元使用。
所述病原菌抑制产品为粉制剂或液制剂。
所述液制剂和粉制剂均通过鼠李糖乳杆菌R7970菌体对基础培养基进行发酵而获得的发酵液制备得到,液制剂为发酵液的上清液,粉制剂为发酵液的干燥粉。
所述基础培养基包括:大豆粉1.5-3重量份、脱脂乳粉7.5-15重量份、葡萄糖1-2重量份、蒸馏水80-90重量份;
所述基础培养基中鼠李糖乳杆菌R7970菌体的接种量为1×106CFU/mL以上,发酵液的发酵终点为pH=4.50。
所述鼠李糖乳杆菌R7970与万古霉素抗生素联合在制备用于预防或治疗致病菌感染性疾病的病原菌抑制产品中的应用。
所述病原菌抑制产品在为发酵乳提供保鲜功能、抑制霉菌酵母生长中的应用。
本发明技术方案,具有如下优点:
1.本发明提供的鼠李糖乳杆菌R7970具备食用安全特性;此外,鼠李糖乳杆菌R7970还具有广谱抑制病原菌活性。因此,本发明的鼠李糖乳杆菌R7970可以有效用于制备病原菌抑制产品,该病原菌抑制产品可以达到抑制口腔致病菌及一些常见致病菌的作用,例如:可抑制口腔致病菌中具核梭杆菌、牙龈卟啉单胞菌、变异链球菌和伴放线放线杆菌的生长,抑制常见致病菌蜡样芽孢杆菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌的生长。综上,本发明的鼠李糖乳杆菌R7970可以用于预防或治疗致病菌感染的疾病,例如致病菌引起的肠道感染、腹泻、阴道感染等疾病;或者为发酵乳提供保鲜功能、抑制霉菌酵母生长等。
2.本发明提供的鼠李糖乳杆菌R7970具有耐受万古霉素抗生素的效果,因此,所述鼠李糖乳杆菌R7970可以与万古霉素抗生素联合应用在预防或治疗致病菌感染性疾病的病原菌抑制产品中。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为实施例1中液制剂对口腔致病菌的抑制率结果图;
图2为实施例1中液制剂对其他常见致病菌的抑制率结果图;
图3为实施例2中粉制剂对口腔致病菌的抑制率结果图;
图4为实施例2中粉制剂对其他常见致病菌的抑制率结果图;
图5为实施例3中鼠李糖乳杆菌R7970溶血性试验的结果图。
具体实施方式
实施例1
含鼠李糖乳杆菌R7970的液制剂,其具体组成和制备过程如下:
按照下表1中的配方,将大豆粉、脱脂乳粉和葡萄糖按比例混合于60℃的蒸馏水中,搅拌均匀后均质(160-180Bar),经过95℃、300s杀菌后降温至37±1℃,鼠李糖乳杆菌R7970接种1×106CFU/mL,发酵至pH=4.50停止发酵,降温冷却后离心取上清即为液制剂,10℃放置备用。
表1
将致病菌(包括具核梭杆菌、牙龈卟啉单胞菌、变异链球菌、伴放线放线杆菌、蜡样芽孢杆菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌)以2%的接种量接种到下表2所对应的培养基中,加入鼠李糖乳杆菌R7970液制剂,液制剂的添加量为5wt%,并设置空白组(不添加液制剂),进行共培养,分别在不同时间点采用平板倾注法统计活菌数。其中,口腔潜在致病菌(变异链球菌、牙龈卟啉单胞菌、伴放线放线杆菌和具核梭杆菌)分别共培养0.25h、1h、3h、5h、8h和24h,并进行活菌计数。其他常见致病菌(蜡样芽孢杆菌、白色念珠菌、金黄色葡萄球菌和大肠杆菌)于0h、2h、4h、6h、8h和24h进行活菌计数。
表2
根据统计的活菌数,采用下述计算方法获取抑制率(%),不同致病菌在不同时间的抑制率(%)的结果如图1-图2所示。
抑制率(%)=(空白组活菌数-实验组活菌数)/空白组活菌数*100%。
通过图1中,0.25h、1h、3h、5h、8h、24h的抑制率结果显示,含有鼠李糖乳杆菌R7970液制剂对4株口腔致病菌(变异链球菌、伴放线放线杆菌、具核梭杆菌和牙龈卟啉单胞菌)均有显著抑制作用(P<0.01)。在应用水平上,特别有意义的是,在1h后产生了较为明显的抑制效果。
通过图2中抑制率结果可知,自0h起,分别于2h、4h、6h、8h、24h的活菌计数结果显示,鼠李糖乳杆菌R7970液制剂均对白色念珠菌、大肠杆菌、蜡样芽孢杆菌和金黄色葡萄球菌有显著的抑制作用(p<0.01)。在应用水平上,特别有意义的是,在1h后产生了较为明显的抑制效果。
实施例2
含鼠李糖乳杆菌R7970的粉制剂,其具体组成和制备过程如下:
按照下表3中的配方,将大豆粉、脱脂乳粉和葡萄糖按比例混合于60℃的蒸馏水中,搅拌均匀后均质(160-180Bar),经过95℃、300s杀菌后降温至37±1℃,鼠李糖乳杆菌R7970接种1×106CFU/mL,发酵至pH=4.50停止发酵,降温冷却后喷雾干燥得到鼠李糖乳杆菌R7970粉制剂,常温(20-25℃)放置备用即可。
表3
将致病菌(包括具核梭杆菌、牙龈卟啉单胞菌、变异链球菌、伴放线放线杆菌、蜡样芽孢杆菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌)以2%的接种量接种到实施例1中表2所对应的培养基中,加入鼠李糖乳杆菌R7970粉制剂,粉制剂的添加量为1wt%,并设置空白组(不添加粉制剂),进行共培养,分别在不同时间点采用平板倾注法统计活菌数。并采用实施例1中相同计算方法获取抑制率(%),不同致病菌在不同时间的抑制率(%)的结果如图3-图4所示。
通过图3中,0.25h、1h、3h、5h、8h、24h的抑制率结果显示,含有鼠李糖乳杆菌R7970粉制剂对4株口腔致病菌(变异链球菌、伴放线放线杆菌、具核梭杆菌和牙龈卟啉单胞菌)均有显著抑制作用(P<0.01)。在应用水平上,特别有意义的是,在1h后产生了较为明显的抑制效果。
通过图4中抑制率结果可知,自0h起,分别于2h、4h、6h、8h、24h的活菌计数结果显示,鼠李糖乳杆菌R7970粉制剂均对白色念珠菌、大肠杆菌、蜡样芽孢杆菌和金黄色葡萄球菌有显著的抑制作用(p<0.01)。在应用水平上,特别有意义的是,在1h后产生了较为明显的抑制效果。
实施例3
鼠李糖乳杆菌R7970的抗生素耐受性。
依据CLSI M45-2016《苛养菌少见菌药敏判断标准》对青霉素(PEN)、亚胺培南(IP)、氨苄西林(AM)、红霉素(EM)、万古霉素(VA)、克林霉素(CM)和利奈唑胺(LZ)7种抗菌药物的最小抑制浓度进行测定,以MRS培养液为稀释液,采用试管两倍稀释法对药物进行不同浓度的稀释,于37℃厌氧培养24h,观察菌株的生长情况,其耐药性判定标准参考《苛养菌少见菌药敏判断标准》。检测结果如下表4所示。
表4
由表4可知,鼠李糖乳杆菌R7970对青霉素、亚胺培南、氨苄西林、红霉素、克林霉素和利奈唑胺6种抗生素敏感,对万古霉素具有耐受性。因此,所述鼠李糖乳杆菌R7970可以与万古霉素抗生素联合达到更好的预防或治疗致病菌感染性疾病的作用。
实施例4
鼠李糖乳杆菌R7970的食用安全性验证。
1、鼠李糖乳杆菌R7 970溶血性试验
参照GB 4789.11-2014《食品安全国家标准食品微生物学检验β型溶血性链球菌检验》检测方法。将活化后的鼠李糖乳杆菌R7970接种于MRS液体培养基中,37℃恒温培养24h后,将菌悬液点种于血平板上(3个重复),于37℃培养72h,将金黄色葡萄球菌和英诺克李斯特菌分别作为阳性和阴性对照菌株,观察是否有溶血现象。若不出现溶血圈,则说明该菌株能够作为潜在益生菌。
溶血性试验结果如图5所示。该图5中,1#为阴性对照CICC10417英诺克李斯特菌,2#为阳性对照菌CICC10473金黄色葡萄球菌,3#为鼠李糖乳杆菌R7970。
通过图5可知,经穿刺接种血平板后,鼠李糖乳杆菌R7970在穿刺处的周围未出现透明圈,鼠李糖乳杆菌R7970与阴性对照菌CICC10417英诺克李斯特菌溶血效果相似,与阳性对照菌CICC10473金黄色葡萄球菌溶血效果具有显著差异。试验结果表明,鼠李糖乳杆菌R7970没有引起溶血现象且结果呈阴性。
2、生物胺的测定
菌种产生的生物胺是由细菌底物特异性酶使氨基酸脱羧形成的。取鼠李糖乳杆菌R7970发酵液5mL离心(6 000×g、5min),取湿菌体加入体积分数为10%的三氯乙酸沸水浴提取1h,取上清液衍生,生物胺的定量分析采用高效液相色谱仪(HPLC)测定,具体参照GB5009.208-2016《食品安全国家标准食品中生物胺的测定》(第一法),其样品检出限为:腐胺、组胺、酪胺1.25mg/kg,尸胺0.75mg/kg。
在食品中,低含量的生物胺被认为是安全的,摄入过量时可能会引起特定的药理、生理和毒性效应。经HPLC分析,未检测到腐胺、组胺、酪胺以及尸胺。表明鼠李糖乳杆菌R7970是安全性菌株,可应用于相关产品行业。
3、致病性评价
3.1、腹腔注射途径干扰实验
依据《食品用菌种致病性评价程序(征求意见稿)》对鼠李糖乳杆菌R7970进行致病性评价。具体如下:受试小鼠80只雌雄各半,分别随机分成4组(每组10只),包括培养基原液对照、培养物原液、培养基5倍浓缩液对照以及培养物5倍浓缩液,每只小鼠体内注射20.0mL/kg BW剂量的不同组别的液体,观察不同液体分别对雌雄小鼠体质量以及毒副作用的影响,具体评价参照保健食品用菌种致病性评价程序附录A。
上述鼠李糖乳杆菌R7970培养物通过腹腔注射途径干扰实验小鼠,结果如表5和6所示。
表5鼠李糖乳杆菌R7970培养物对雄性小鼠体质量的影响
表6鼠李糖乳杆菌R7970培养物对雌性小鼠体质量的影响结果
由表5和表6的数据可知,实验期间,小鼠初始体质量及终体质量在培养物组和其它组间组比较均无显著差异(P>0.05),未观察到实验小鼠明显的活动或行为变化、疾病或死亡,实验证实了鼠李糖乳杆菌R7970培养物对小鼠的体质量无影响,该菌株对动物的健康状况、生长发育没有明显的口服毒性影响,与小鼠无临床症状一致。
3.2、小鼠急性毒性作用实验
受试小鼠40只,随机分成4组(每组10只),以20.0mL/kg BW的剂量给小鼠连续灌胃鼠李糖乳杆菌R7970培养物原液和5倍浓缩液3d,观察7d,具体结果如表7所示。
表7鼠李糖乳杆菌R7970培养物对小鼠急性毒性作用
小鼠在灌胃培养物原液、5倍浓缩液后,受试小鼠体征、行为未有发生疾病、中毒或死亡急等现象,证明该菌株是安全的。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.一株鼠李糖乳杆菌R7970在制备病原菌抑制产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述病原菌抑制产品用于预防或治疗致病菌感染的疾病,致病菌为细菌和/或真菌。
3.根据权利要求2所述的应用,其特征在于,所述致病菌至少包括口腔致病菌;
优选的,所述致病菌包括具核梭杆菌、牙龈卟啉单胞菌、变异链球菌、伴放线放线杆菌、蜡样芽孢杆菌、大肠杆菌、金黄色葡萄球菌和白色念珠菌。
4.根据权利要求1-3任一项所述的应用,其特征在于,所述病原菌抑制产品中鼠李糖乳杆菌R7970菌体的添加量为(1~9)×106CFU/mL;优选的,鼠李糖乳杆菌R7970菌体的添加量为(1~3)×106CFU/mL。
5.根据权利要求1-4任一项所述的应用,其特征在于,所述病原菌抑制产品包括鼠李糖乳杆菌R7970菌体及其代谢产物。
6.根据权利要求5所述的应用,其特征在于,所述病原菌抑制产品为粉制剂或液制剂。
7.根据权利要求6所述的应用,其特征在于,所述液制剂和粉制剂均通过鼠李糖乳杆菌R7970菌体对基础培养基进行发酵而获得的发酵液制备得到,液制剂为发酵液的上清液,粉制剂为发酵液的干燥粉。
8.根据权利要求7所述的应用,其特征在于,所述基础培养基包括:大豆粉1.5-3重量份、脱脂乳粉7.5-15重量份、葡萄糖1-2重量份、蒸馏水80-90重量份;
所述基础培养基中鼠李糖乳杆菌R7970菌体的接种量为1×106CFU/mL以上,发酵液的发酵终点为pH=4.50。
9.根据权利要求1-8任一项所述的应用,其特征在于,所述鼠李糖乳杆菌R7970与万古霉素抗生素联合在制备用于预防或治疗致病菌感染性疾病的病原菌抑制产品中的应用。
10.根据权利要求1所述的应用,其特征在于,所述病原菌抑制产品在为发酵乳提供保鲜功能、抑制霉菌酵母生长中的应用。
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