CN114990023A - 一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌及其筛选方法与应用 - Google Patents
一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌及其筛选方法与应用 Download PDFInfo
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- CN114990023A CN114990023A CN202210720534.6A CN202210720534A CN114990023A CN 114990023 A CN114990023 A CN 114990023A CN 202210720534 A CN202210720534 A CN 202210720534A CN 114990023 A CN114990023 A CN 114990023A
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- indole
- lactobacillus reuteri
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- bile salt
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Abstract
本发明公开了一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌及其筛选方法与应用,属于微生物领域。本发明利用MRS培养基自健康人体粪便样分离筛选出保藏编号为CGMCC No.24755的菌株(L.reuteri GH226‑12),先后经形态学、生理生化和16s rRNA测序鉴定后,对分离得到的罗伊氏乳杆菌,利用UPLC‑Q‑Orbitrap MS/MS技术对其吲哚衍生物产量进行检测筛选得到。该检测方法改善了复杂培养体系中吲哚衍生物难以检测的困难,具有线性范围广、灵敏度高、样本需要量少和操作简便等优点。最后对L.reuteri GH226‑12耐酸耐胆盐情况进行评价,证实了高产吲哚衍生物的罗伊氏乳杆菌(L.reuteriGH226‑12)能够耐受人体消化道的酸碱环境,可用于食品或药品的开发。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌及其筛选方法与应用。
背景技术
研究表明,人体膳食中仅有5%左右的色氨酸在人体小肠中被吸收利用,相当比例未被吸收的色氨酸到达结肠后,会被肠道微生物利用,产生多种吲哚衍生物,发挥调节宿主免疫、改变宿主与微生物相互作用模式等功能。色氨酸的代谢路径主要包括5-羟色胺酸代谢路径、吲哚丙酮酸代谢路径以及犬尿氨酸代谢路径。其中,人体肠道微生物产生的吲哚衍生物可作为人体细胞表面芳香烃受体(AhR)的配体,不仅具有调节宿主免疫,增强肠上皮屏障的功能,还可作为神经递质调节肠道激素分泌,刺激胃肠运动,甚至可以进入循环系统清除体内游离自由基。
众多吲哚衍生物中,最具代表性的化合物为吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸,它们都具有特殊的生理功能,例如:吲哚-3-乳酸和吲哚-3-乙酸可抑制肠道致病菌的生长,改善肠道屏障和免疫功能,而吲哚-3-丁酸还可作为植物生长激素用于果蔬催熟和农产品品质的改良。虽然吲哚衍生物具有重要的生理功能,但其应用存在以下困难:(1)细菌发酵液成分复杂,其中吲哚衍生物难以精准定性定量;(2)肠道细菌产生吲哚衍生物的能力微弱,限制了该物质的进一步利用;(3)肠道是吲哚衍生物在人体发挥功能的主要部位,但吲哚衍生物难以精准有效递送,且难以持续发挥功能。
发明内容
为了克服上述现有技术的缺点,本发明的目的在于提供一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌及其制备方法与应用,对吲哚衍生物精准定性定量,提升肠道细菌生产和精准递送吲哚衍生物的能力。
为了达到上述目的,本发明采用以下技术方案予以实现:
本发明公开了一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌,该高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的分类命名为罗伊氏乳杆菌Lactobacillus reuteri,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2022年4月22日,保藏号为CGMCC No.24755。
优选地,所述罗伊氏乳杆菌能够高产的吲哚衍生物包括吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸。
本发明还公开了上述一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,所述方法具体为:采用MRS培养基对样品进行分离培养,鉴定并筛选出罗伊氏乳杆菌,再通过UPLC-Q-Orbitrap MS/MS方法筛选出高产吲哚衍生物的罗伊氏乳杆菌,进行耐酸耐胆盐能力测定,筛选出高产吲哚衍生物且具有耐酸耐胆盐能力的罗伊氏乳杆菌。
优选地,通过UPLC-Q-Orbitrap MS/MS方法筛选出高产吲哚衍生物的罗伊氏乳杆菌的步骤为:使用Hypersil Gold C18反相色谱柱、电喷雾离子源和Orbitrap质量分析器测定筛选出的罗伊氏乳杆菌样品与吲哚衍生物标准品相同质荷比和保留时间的峰面积,根据测定的标准曲线求得样品中吲哚衍生物的含量,基于吲哚衍生物的含量筛选出高产吲哚衍生物的罗伊氏乳杆菌。
优选地,耐酸耐胆盐能力测定的步骤为:挑取高产吲哚衍生物的罗伊氏乳杆菌单菌落至MRS培养基中,37℃厌氧培养24h;以体积比1:100分别接种至pH值=3或pH值=2,以及胆盐浓度为0.3%或1%的MRS培养基中,分别在0h,2h和4h进行平板计数,计算菌种对不同pH和胆盐浓度的耐受情况;同时挑取高产吲哚衍生物的罗伊氏乳杆菌单菌落到pH值=6.5的MRS培养基做阴性对照,其他条件相同;最后,计算相对存活率,筛选出高产吲哚衍生物且具有耐酸耐胆盐能力的罗伊氏乳杆菌。
优选地,Hypersil Gold C18反相色谱柱的规格为100mm×2.1mm×1.7μm,流动相A为0-20mmol/L醋酸铵水,B为0-20mmol/L醋酸铵和乙腈;进样体积为1-10μL,流速为30-600μL/min,柱温为10-40℃,梯度洗脱程序如下:
优选地,电喷雾离子源和Orbitrap质量分析器采用正离子扫描模式,数据依赖性扫描Full MS-dd MS2,喷雾电压2.5-4.5kV;电喷雾电离源的参数为:鞘气体流量为20-60arb;辅助气体流量为-10arb;毛细管温度为200-500℃;全扫描和二级扫描分辨率分别为70 000和17 500;质量采集范围为m/z 100~1500。
优选地,吲哚衍生物标准品的配制方法为:用甲醇配制吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的1mg/mL混合标准品母液,用改良M9培养基稀释成1000ng/mL、500ng/mL、100ng/mL、20ng/mL和4ng/mL工作液,制成吲哚衍生物标准品。
本发明还公开了上述一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌在食品或药品中的应用。
优选地,所述食品包括保健食品,保健食品为发酵食品或菌剂。
与现有技术相比,本发明具有以下有益效果:
本发明提供的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌,该菌株属于厚壁菌门,芽孢杆菌纲,乳杆菌目,乳杆菌科,乳杆菌属,命名为罗伊氏乳杆菌L.reuteri GH226-12,保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC No.24755。该高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌自健康人体粪便样本中分离获得,采用MRS培养基对健康人体粪便样本进行分离培养,挑选出表面湿润光滑白色或乳白色菌落,通过对菌株进行形态学、生理生化和16S rRNA基因序列及系统发育树同源性分析,最终鉴定为罗伊氏乳杆菌。利用UPLC-Q-Orbitrap MS/MS技术对吲哚衍生物产量进行检测筛选;针对人体及动物肠道的生理条件(即人体消化食物的过程最长不会超过4h,而胃液pH值在2.0-3.0之间,胆汁液浓度在0.03%-0.3%之间),经过耐酸耐胆盐实验,证实了该罗伊氏乳杆菌具有一定的耐酸、耐胆盐特性和肠道上皮细胞黏附能力,因而能够通过人体消化道进入并定植于人体肠道,发挥益生功能;能够实现肠道的精准定位,通过对膳食色氨酸的利用可以持续且高效地产生吲哚衍生物;可用于食品或药品的开发,抑制病原微生物,改善人体或动物肠道菌群多样性,调节人体或动物肠道免疫,改善人体或动物肠道健康,具有良好的市场前景。
本发明提供的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的制备方法,不同于现有的化工合成吲哚衍生物法(纯度较低,应用于人体存在一定安全隐患)以及植物和微生物提取吲哚衍生物法(产量极低,且缺乏从复杂发酵体系中准确检测吲哚衍生物的方法,应用受到极大制约),本方法基于UPLC-Q-Orbitrap MS/MS技术,建立了针对微生物发酵液中吲哚衍生物的检测方法,并应用该技术成功筛选出了1株高产吲哚衍生物的罗伊氏乳杆菌,能够从成分复杂的微生物培养液中同时检测出吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸。该方法线性范围广,灵敏度高,简便快捷,具有样本需要量少、操作流程方便以及快捷的优点,解决了目前吲哚衍生物难以检测的窘境。
保藏说明
本发明对所述的高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌进行了下述保藏:
保藏时间:2022年4月22日;保藏地点:中国,北京。北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);保藏编号:CGMCC No.24755。
附图说明
图1为本发明的罗伊氏乳杆菌(L.reuteri GH812-16)的菌落形态图;
图2为本发明的吲哚-3-乳酸标准曲线图;
图3为本发明的吲哚-3-乙酸标准曲线图;
图4为本发明的吲哚-3-丁酸标准曲线图;
图5为本发明的UHPLC-Q-Orbitrap MS/MS分析L.reuteri GH226-12发酵上清液和吲哚-3-乳酸标准品检测结果图;其中,(A)L.reuteri GH226-12发酵上清液质荷比为206.0811下的UHPLC色谱图,(B)与A色谱峰相同保留时间下L.reuteri GH226-12发酵上清液质谱图,(C)吲哚-3-乳酸标准品UHPLC色谱图,(D)吲哚-3-乳酸标准品质谱图;
图6为本发明的UHPLC-Q-Orbitrap MS/MS分析L.reuteri GH226-12发酵上清液和吲哚-3-乙酸标准品检测结果图;其中,(A)L.reuteri GH226-12发酵上清液质荷比为176.0706下的UHPLC色谱图,(B)与A色谱峰相同保留时间下L.reuteri GH226-12发酵上清液质谱图,(C)吲哚-3-乙酸标准品UHPLC色谱图,(D)吲哚-3-乙酸标准品质谱图;
图7为本发明的UHPLC-Q-Orbitrap MS/MS分析L.reuteri GH226-12发酵上清液和吲哚-3-丁酸标准品检测结果图;其中,(A)L.reuteri GH226-12发酵上清液质荷比为206.0811下的UHPLC色谱图,(B)与A色谱峰相同保留时间下L.reuteri GH226-12发酵上清液质谱图,(C)吲哚-3-乳酸标准品UHPLC色谱图,(D)吲哚-3-乳酸标准品质谱图;
图8为本发明的罗伊氏乳杆菌(L.reuteri GH226-12)的耐酸能力评价图;
图9为本发明的罗伊氏乳杆菌(L.reuteri GH226-12)的耐胆盐能力评价图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分的实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都应当属于本发明保护的范围。
需要说明的是,本发明的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象,而不必用于描述特定的顺序或先后次序。应该理解这样使用的数据在适当情况下可以互换,以便这里描述的本发明的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外,术语“包括”和“具有”以及他们的任何变形,意图在于覆盖不排他的包含,例如,包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元,而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
下面结合附图对本发明做进一步详细描述:
本发明提供的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌分离自健康婴儿粪便样本中。高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌(L.reuteri GH226-12),于2022年4月22日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地点:中国,北京。中国科学院微生物研究所,邮编为:100101,保藏编号:CGMCC No.24755。该罗伊氏乳杆菌的16S rRNA序列如SEQ ID No.1所示,Genbank比对结果显示与罗伊氏乳杆菌同源性为99%,命名为罗伊氏乳杆菌GH226-12。该菌株具有高产吲哚衍生物(吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸)的功能,且具有耐受酸性和胆盐环境的能力。本发明提供的罗伊氏乳杆菌(Lactobacillus reuteri GH226-12)在改良M9培养基12h后,其培养液中吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸浓度分别≥241.12±2.19ng/mL、109.66±1.14ng/mL和14.91±1.32ng/mL,这些浓度显著高于其他被试菌(P<0.05);在pH值2.0的人工胃液中耐受4h,存活率98%以上,在pH值3.0的人工胃液中耐受4h,存活率99%以上,在0.3%胆盐的人工肠液中耐受4h,存活率68%以上;在1%胆盐的人工肠液中耐受4h,存活率仍保持50%以上,可用于食品、药品的开发,具有良好的市场前景;
其中,所述食品包括但不限于发酵食品或菌剂,发酵食品包括但不限于发酵果蔬制品、发酵豆制品或发酵乳制品,发酵果蔬制品包括但不限于苹果、芒果、葡萄、梨、橙、胡萝卜或枸杞,发酵豆制品包括但不限于豆浆、豆奶、豆豉或豆酱,发酵乳制品包括但不限于酸奶或奶酪。
本发明提供的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,采用MRS培养基对样品进行分离培养,鉴定并筛选出罗伊氏乳杆菌,再通过UPLC-Q-Orbitrap MS/MS方法筛选出高产吲哚衍生物的罗伊氏乳杆菌,进行耐酸耐胆盐能力测定,筛选出高产吲哚衍生物且具有耐酸耐胆盐能力的罗伊氏乳杆菌。
具体步骤如下:
步骤1、对各类样利用MRS培养基对乳酸菌进行分离培养;
步骤2、对步骤1筛选出的菌株做16S rRNA基因测序,以对菌种进行鉴定,并筛选出罗伊氏乳杆菌;
步骤3、在步骤2中经过鉴定的罗伊氏乳杆菌中,筛选出具有高产吲哚衍生物(吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸)能力最强的菌株;
具体方法为:
(1)样品准备:用接种环从MRS琼脂平板上挑取步骤2筛选出的罗伊氏乳杆菌单菌落接种到5mL新鲜MRS肉汤培养基中,37℃过夜厌氧培养12h。随后按体积比1%的接种量接种到5mL MRS肉汤培养基中,37℃,厌氧培养12h后,12,000rpm离心5min收集菌体,用PBS洗涤菌体沉淀,离心弃上清。将菌体沉淀添加到5mL新鲜改良M9培养基(+0.6μmol/L色氨酸)中培养48h,离心取上清液5mL,加100-1000μL的甲醇,摇匀,用PBS缓冲液调节pH值至5-8。加入1-10mL乙酸乙酯萃取,剧烈震荡30s,静置3min,重复3次。收集乙酸乙酯层液体于15mL离心管中,加入少量无水硫酸钠干燥5min,氮气常温吹干。
(2)将上述氮气吹干的样品用100-1000μL甲醇复溶后,通过UPLC-Q-Orbitrap MS/MS技术,测定样品与标准品相同质荷比和保留时间的峰面积,根据测定的标准曲线求得样品中3种吲哚衍生物的含量,基于3种吲哚衍生物的含量筛选出产量最高的罗伊氏乳杆菌;
其中,标准品的制备方法为:用甲醇配制吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的1mg/mL混合标准品母液,置于-20℃冰箱中保存。取一定量的混合标准品母液,用改良M9培养基(即M9培养基+0.6μmol/L色氨酸)稀释成1000ng/mL、500ng/mL、100ng/mL、20ng/mL和4ng/mL的工作液,制得标准品,并绘制标准曲线。UPLC-Q-Orbitrap MS/MS技术中,使用Hypersil Gold C18反相色谱柱(100mm×2.1mm×1.7μm),流动相A为0-20mmol/L醋酸铵水,B为0-20mmol/L醋酸铵和乙腈;进样体积为1-10μL,流速为30-600μL/min,柱温为10-40℃,梯度洗脱,梯度洗脱程序如表1:
表1梯度洗脱程序
使用电喷雾离子源和Orbitrap质量分析器,采用正离子扫描模式,数据依赖性扫描Full MS-dd MS2,喷雾电压2.5-4.5kV。电喷雾电离源(ESI)参数为:鞘气体流量为20-60arb;辅助气体流量为-10arb;毛细管温度为200-500℃;全扫描和二级扫描分辨率分别为70 000和17 500;质量采集范围为m/z 100~1500。
步骤4、对步骤3中筛选出的罗伊氏乳杆菌进行耐酸耐胆盐能力测定,筛选出高产吲哚衍生物且具有耐酸耐胆盐能力的罗伊氏乳杆菌;
具体方法为:
首先,挑取步骤3中筛选出的罗伊氏乳杆菌单菌落至15mL新鲜MRS培养基中,37℃厌氧培养24h。随后1:100分别接种至pH值=3或pH值=2,以及胆盐浓度为0.3%或1%的新鲜MRS培养基中,分别在0h,2h和4h进行平板计数,计算菌种对不同pH值和胆盐浓度的耐受情况。同时,挑取步骤3中筛选出的罗伊氏乳杆菌单菌落到普通MRS(pH值=6.5)培养基做阴性对照,其他条件相同;最后,计算相对存活率,筛选出耐酸耐胆盐较高的菌株。
实施例1
1.罗伊氏乳杆菌的分离和鉴定
(1)菌株的分离
采取厌氧培养法,对益生菌进行筛选、分离和纯化。
具体步骤为:取健康人类婴儿粪便样本约1g至0.9%的生理盐水中,充分混匀;梯度稀释后,取10-3、10-4和10-5菌液,分别均匀涂布于MRS平板中,置于厌氧培养箱中37℃恒温培养,24h后通过菌落形态和镜检对菌株进行初步鉴定,从中挑选出符合罗伊氏乳杆菌特征的菌株进行划线纯化培养。
(2)菌株的形态学和生理生化鉴定
形态学鉴定内容包括观察菌株在培养基平板上形成的菌落的特征,革兰氏染色涂片于显微镜下观察菌体形态。菌落形态见图1,菌落呈圆形、乳白色、不透明,直径约1~2mm,边沿平滑,表面润湿突出有光泽。镜检菌体呈杆状,单个或成双呈现,革兰氏阳性菌。
生理生化鉴定主要使用糖发酵管,判断依据GB4789.35-2016,如下表2所示;符合国家标准要求,则初步确定为罗伊氏乳杆菌。
表2罗伊氏乳杆菌GH812-16糖发酵鉴定结果
糖类 | 纤维二糖 | 麦芽糖 | 甘露醇 | 水杨苷 | 山梨醇 | 蔗糖 | 棉子糖 |
结果 | - | + | - | - | - | + | + |
(3)菌株的16S rRNA鉴定
对挑选的目的菌株进行DNA提取与16S rDNA鉴定。采用通用引物27F:与1492R扩增其16S rRNA片段。采用琼脂糖凝胶电泳法检测DNA提取情况以及PCR扩增产物大小。将获得的目的片段进行测序分析,见表3,序列如SEQ ID No.1所示,测序结果在NCBI数据库中进行比对分析,筛选出罗伊氏乳杆菌(即L.reuteri DSM 17938、L.reuteri GH211-4、L.reuteriGH319-13、L.reuteri GH812-16、L.reuteri GH812-17、L.reuteri GH329-22和L.reuteriGH226-12,共7株罗伊氏乳杆菌)。
表3测序结果序列表
2.高产吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的罗伊氏乳杆菌的筛选
(1)用接种环从MRS琼脂平板上挑取罗伊氏乳杆菌单菌落接种到5mL新鲜MRS肉汤培养基中,37℃过夜厌氧培养12h;
(2)随后按1%的接种量接种到5mL MRS肉汤培养基37℃,厌氧培养12h后,12,000rpm离心5min收集菌体,用PBS洗涤菌体沉淀,离心弃上清;
(3)将菌体沉淀添加到5mL新鲜改良M9培养基(+0.6μmol/L色氨酸)中培养48h,离心取上清液5mL,加200μL的甲醇,摇匀,用PBS缓冲液调节pH值至6-7;
(4)加入2.5mL乙酸乙酯萃取,剧烈震荡30s,静置3min,重复3次。收集乙酸乙酯层液体于15mL离心管中,加入少量无水硫酸钠干燥5min,氮气常温吹干用150μL甲醇复溶;
(5)用甲醇配制吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的1mg/mL混合标准品母液,置于-20℃冰箱中保存。取一定量的混合标准品母液,用改良M9培养基(即M9培养基+0.6μmol/L色氨酸)稀释成1000ng/mL、500ng/mL、100ng/mL、20ng/mL和4ng/mL工作液,制得标准品,并绘制标准曲线;
(6)使用Hypersil Gold C18反相色谱柱(100mm×2.1mm×1.7μm),流动相A为10mmol/L醋酸铵水,B为10mmol/L醋酸铵和乙腈;进样体积为5μL,流速为300μL/min,柱温为30℃,梯度洗脱;
(7)使用电喷雾离子源和Orbitrap质量分析器,采用正离子扫描模式,数据依赖性扫描Full MS-dd MS2,喷雾电压3.5kV。电喷雾电离源(ESI)参数为:鞘气体流量为40arb;辅助气体流量为-10arb;毛细管温度为200-500℃;全扫描和二级扫描分辨率分别为70 000和17 500;质量采集范围为m/z 100~1500;
(8)测定样品与标准品相同质荷比和保留时间的峰面积,根据测定的标准曲线求得样品中3种吲哚衍生物的含量。基于3种吲哚衍生物的含量筛选出产量最高的罗伊氏乳杆菌。
实验结果:
(1)检测方法的标准曲线和线性范围:待测混合标准品中3个吲哚衍生物都可以有效分离,绘制标准曲线(图2、图3和图4),得到吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的拟合线性方程,分别为:y=5312.4x+569983,y=7115.6x+647542和y=4707.1x+748430。3种吲哚衍生物的标准曲线线性关系良好,相关系数R2均>0.99;
(2)方法准确度及精密度:该方法测量回收率在90~110%之间,且相对标准偏差RSD<5%,数据表明测量方法精确度良好,见表4;
表4改良M9培养基加标回收率及相对标准偏差
样本检测结果:将L.reuteri DSM 17938、L.reuteri GH211-4、L.reuteri GH319-13、L.reuteri GH812-16、L.reuteri GH812-17、L.reuteri GH329-22、L.reuteri GH226-12共7株罗伊氏乳杆菌,按如上方法对培养液中吲哚衍生物进行检测,色谱图如图5、图6和图7所示:其中,吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的特征性碎片离子分别为m/z206.0811[M+H]+、m/z 176.0706[M+H]+和m/z204.1019[M+H]+。由特征性碎片离子峰面积定量得出的吲哚衍生物分析结果如表5。可以看出L.reuteri对色氨酸的代谢能力存在菌株差异。其中罗伊氏乳杆菌GH226-12在上述实验条件下,其培养液中吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸浓度分别为241.12±2.19ng/mL、109.66±1.14ng/mL和14.91±1.32ng/mL,显著高于其他被试菌(P<0.05)。
表5罗伊氏乳杆菌培养液中吲哚-3-乳酸、吲哚-3-乙酸、吲哚-3-丁酸检测结果
3.测定罗伊氏乳杆菌GH226-12的pH耐受性
(1)向MRS培养基中添加37%浓盐酸调节酸度,分别配pH值为2.0和3.0的两种不同酸性培养基,121℃下灭菌备用;
(2)将已活化好的处于稳定期的菌种(即罗伊氏乳杆菌GH226-12)分别接种到已经准备好的酸性培养基中,以空白培养基作参照,37℃的恒温条件下培养0h、2h和4h,观察菌株的生长情况。根据GB4789.2-2010计算活菌数,并计算菌株存活率。
实验结果如图8所示,罗伊氏乳杆菌GH226-12:pH值为2.0时对罗伊氏乳杆菌抑制作用较小,4h后存活率仍保持98%以上;在pH值为3.0时,该菌4h后存活率仍保持99%以上,说明该罗伊氏乳杆菌GH226-12具有耐受高pH的能力。
4.测定罗伊氏乳杆菌GH226-12的胆盐耐受性
(1)向MRS培养基中添加牛磺脱氧胆酸钠,分别配制胆盐浓度0.3%和1.0%的两种不同培养基,用0.22μm滤膜过滤灭菌。
(2)将已活化好的处于稳定期的菌种(即罗伊氏乳杆菌GH226-12)分别接种到已经准备好的胆盐培养基中,以不含胆盐培养基作参照,37℃的恒温条件下培养0h、2h和4h,观察菌株的生长情况。根据GB4789.2-2010计算活菌数,并计算菌株存活率;
实施结果如图9所示,罗伊氏乳杆菌GH226-12:胆盐浓度为0.3%时对罗伊氏乳杆菌抑制作用较小,4h后存活率仍保持68%以上;在胆盐浓度为1.0%时,该菌4h后存活率仍保持50%以上,说明该罗伊氏乳杆菌具有耐受高浓度胆盐的能力。
以上内容仅为说明本发明的技术思想,不能以此限定本发明的保护范围,凡是按照本发明提出的技术思想,在技术方案基础上所做的任何改动,均落入本发明权利要求书的保护范围之内。
序列表
<110> 陕西科技大学
<120> 一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌及其筛选方法与应用
<141> 2022-06-20
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 662
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
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ccgagttgag agactgatcg gccacaatgg aactgagaca cggtccatac tcctacggga 120
ggcagcagta gggaatcttc cacaatgggc gcaagcctga tggagcaaca ccgcgtgagt 180
gaagaagggt ttcggctcgt aaagctctgt tgttggagaa gaacgtgcgt gagagtaact 240
gttcacgcag tgacggtatc caaccagaaa gtcacggcta actacgtgcc agcagccgcg 300
gtaatacgta ggtggcaagc gttatccgga tttattgggc gtaaagcgag cgcaggcggt 360
tgcttaggtc tgatgtgaaa gccttcggct taaccgaaga agtgcatcgg aaaccgggcg 420
acttgagtgc agaagaggac agtggaactc catgtgtagc ggtggaatgc gtagatatat 480
ggaagaacac cagtggcgaa ggcggctgtc tggtctgcaa ctgacgctga ggctcgaaag 540
catgggtagc gaacaggatt agataccctg gtagtccatg ccgtaaacga tgagtgctag 600
gtgttggagg gtttccgccc ttcagtgccg gagctaacgc attaaagcac tccgcctggg 660
ga 662
Claims (10)
1.一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌,其特征在于,该高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的分类命名为罗伊氏乳杆菌Lactobacillus reuteri,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2022年4月22日,保藏号为CGMCC No.24755。
2.如权利要求1所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌,其特征在于,所述罗伊氏乳杆菌能够高产的吲哚衍生物包括吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸。
3.权利要求1所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,其特征在于,所述方法具体为:采用MRS培养基对样品进行分离培养,鉴定并筛选出罗伊氏乳杆菌,再通过UPLC-Q-Orbitrap MS/MS方法筛选出高产吲哚衍生物的罗伊氏乳杆菌,进行耐酸耐胆盐能力测定,筛选出高产吲哚衍生物且具有耐酸耐胆盐能力的罗伊氏乳杆菌。
4.如权利要求3所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,其特征在于,通过UPLC-Q-Orbitrap MS/MS方法筛选出高产吲哚衍生物的罗伊氏乳杆菌的步骤为:使用Hypersil Gold C18反相色谱柱、电喷雾离子源和Orbitrap质量分析器测定筛选出的罗伊氏乳杆菌样品与吲哚衍生物标准品相同质荷比和保留时间的峰面积,根据测定的标准曲线求得样品中吲哚衍生物的含量,基于吲哚衍生物的含量筛选出高产吲哚衍生物的罗伊氏乳杆菌。
5.如权利要求3所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,其特征在于,耐酸耐胆盐能力测定的步骤为:挑取高产吲哚衍生物的罗伊氏乳杆菌单菌落至MRS培养基中,37℃厌氧培养24h;以体积比1:100分别接种至pH值=3或pH值=2,以及胆盐浓度为0.3%或1%的MRS培养基中,分别在0h,2h和4h进行平板计数,计算菌种对不同pH和胆盐浓度的耐受情况;同时挑取高产吲哚衍生物的罗伊氏乳杆菌单菌落到pH值=6.5的MRS培养基做阴性对照,其他条件相同;最后,计算相对存活率,筛选出高产吲哚衍生物且具有耐酸耐胆盐能力的罗伊氏乳杆菌。
7.如权利要求4所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,其特征在于,电喷雾离子源和Orbitrap质量分析器采用正离子扫描模式,数据依赖性扫描FullMS-dd MS2,喷雾电压2.5-4.5kV;电喷雾电离源的参数为:鞘气体流量为20-60arb;辅助气体流量为-10arb;毛细管温度为200-500℃;全扫描和二级扫描分辨率分别为70 000和17 500;质量采集范围为m/z 100~1 500。
8.如权利要求4所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌的筛选方法,其特征在于,吲哚衍生物标准品的配制方法为:用甲醇配制吲哚-3-乳酸、吲哚-3-乙酸和吲哚-3-丁酸的1mg/mL混合标准品母液,用改良M9培养基稀释成1000ng/mL、500ng/mL、100ng/mL、20ng/mL和4ng/mL工作液,制成吲哚衍生物标准品。
9.权利要求1所述的一株高产吲哚衍生物且具有耐酸耐胆盐特性的罗伊氏乳杆菌在食品或药品中的应用。
10.如权利要求9所述的应用,其特征在于,所述食品包括保健食品,保健食品为发酵食品或菌剂。
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