CN116656590A - 一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物及其应用 - Google Patents
一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种提高罗伊氏乳杆菌黏附Caco‑2细胞能力的组合物,所述组合物为滇黄精总多糖和/或滇黄精水提物;提高罗伊氏乳杆菌黏附Caco‑2细胞能力的方法是将益生菌种子液接种在添加了滇黄精总多糖和/或滇黄精水提物的MRS液体培养基中培养,即可获得黏附能力提高的罗伊氏乳杆菌;其中,滇黄精总多糖的添加浓度为0.0004~0.0006 g/mL,滇黄精水提物的添加浓度为0.005~0.007 g/mL。所述组合物的应用为在制备罗伊氏乳杆菌制剂中的应用。本发明将滇黄精总多糖和水提物物与罗伊氏乳杆菌共孵育,显著增强了罗伊氏乳杆菌益生特性,提高了其对肠道Caco‑2细胞的黏附力和增殖定值能力,为肠道罗伊氏乳杆菌制剂的制备提供了新途径。
Description
技术领域
本发明属于生物技术领域,具体涉及一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物及其应用。
背景技术
益生菌是一类通过合理、足量的摄入后,对宿主产生有益作用的活性微生物。益生菌进入人和动物消化道能否黏附定植于宿主肠道黏膜上皮细胞表面,形成稳定的菌群,是其发挥生态效应的关键,不能黏附于肠上皮细胞表面的细菌,只是过路菌而不能在肠道内定植,也就不能充分发挥其生理功能。益生菌在宿主肠道中定植后,将在肠黏膜层形成生物膜或占据黏附位点,阻止病原菌对宿主肠黏膜受体的结合,甚至清除病原菌。在生产实践中,除了选择黏附定植能力强的菌株外,可以在益生菌使用过程中添加益生菌保护剂,提高益生菌的黏附与定植能力,有利于增加其竞争优势,在消化道中保持一定的数量,更好地发挥其益生作用。然而,现有保护剂存在一定局限性,一些保护剂会影响部分益生菌的生长,无法很好达到益生效果。因此,如何提高益生菌在肠道的黏附能力,为其提供有效的保护以抵御宿主机体内的不利环境是十分必要的。
本发明旨在提供一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物。
发明内容
本发明的第一目的在于提供一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物,本发明的第二目的是提供所述组合物的应用。
本发明的第一目的是这样实现的,一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物,所述组合物为滇黄精总多糖和/或滇黄精水提物;
提高罗伊氏乳杆菌黏附Caco-2细胞能力的方法是将罗伊氏乳杆菌种子液按照体积比为1∶100的接种量接种在添加了滇黄精总多糖和/或滇黄精水提物的MRS液体培养基中,于37 ℃温度下,摇床速度为180 r/min的条件下厌氧培养至对数中期即可获得黏附能力提高的罗伊氏乳杆菌;
本发明的第二目的是这样实现的,所述组合物的应用为在制备罗伊氏乳杆菌制剂中的应用。
本发明将滇黄精总多糖和水提物物与罗伊氏乳杆菌共孵育,显著增强了罗伊氏乳杆菌益生特性,提高了其对肠道Caco-2细胞的黏附力和增殖定值能力,为肠道罗伊氏乳杆菌制剂的制备提供了新途径。
附图说明
图1为实施例1制备的滇黄精总多糖和水提物对罗伊氏乳杆菌表面疏水性的影响图;
图2为实施例1制备的滇黄精总多糖和水提物对罗伊氏乳杆菌3h自凝聚率的影响图;
图3为实施例1制备的滇黄精总多糖和水提物对罗伊氏乳杆菌6h自凝聚率的影响图;
图4为实施例1制备的滇黄精总多糖和水提物对罗伊氏乳杆菌黏附Caco-2细胞的影响图;
图5为显微镜下罗伊氏乳杆菌黏附Caco-2细胞的图片。
具体实施方式
下面结合实施例对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。
本发明一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物,所述组合物为滇黄精总多糖和/或滇黄精水提物;
提高罗伊氏乳杆菌黏附Caco-2细胞能力的方法是将罗伊氏乳杆菌种子液按照体积比为1∶100的接种量接种在添加了滇黄精总多糖和/或滇黄精水提物的MRS液体培养基中,于37 ℃温度下,摇床速度为180 r/min的条件下厌氧培养至对数中期即可获得黏附能力提高的罗伊氏乳杆菌;具体方法包括如下步骤:
1、灼烧后的接种环蘸取罗伊氏乳杆菌的甘油菌于平板上划线。37 ℃厌氧培养12h后挑单菌落,接入MRS液体培养基中,37 ℃、180 r/min厌氧培养至OD600 nm值为0.6~0.8作为种子液;
2、将种子液按1∶100(体积比)接种量分别接种到MRS液体培养基(对照)、MRS滇黄精总多糖培养基(0.0004~0.0006 g/mL)、MRS滇黄精水提物培养基(0.005~0.007 g/mL)中,37 ℃、180 r/min厌氧培养至对数中期(OD600 nm=0.6~0.8);
3、取2 mL菌液12000 r/min离心10 min取沉淀,菌体用0.0067 mol/L 的PBS (磷酸盐缓冲液 PhosphateBuffered Saline),洗涤3次后用MRS培养基调整菌体OD值为0.8-1.0左右获得菌体。
滇黄精总多糖的制备方法如下:将新鲜黄精清洗干净,切制成小块,控制料液比1:20 -25g/mL,在高速榨汁机粉碎榨汁,超声50-60 min,进行离心,取上清液,过滤,操作3-4次,旋转浓缩上清液,再加入90 %的乙醇,沉淀24 h后,离心弃去上清液,60 ℃挥干乙醇后冻干,得到滇黄精总多糖。
滇黄精水提物的制备方法如下:将炮制好的滇黄精打粉,取粉末按料液比 1∶10 -15g/mL加入蒸馏水加热回流2 -3h,过滤取上清液。药渣按上述料液比再次加热回流2-3 h,将两次上清液合并加热浓缩后冷冻干燥,得到滇黄精水提物。
本发明还提供了所述组合物在制备罗伊氏乳杆菌制剂中的应用。
实施例1 滇黄精总多糖和水提物的制备
1、滇黄精总多糖的制备:将新鲜黄精清洗干净,切制成小块,控制料液比1:20 g/mL,在高速榨汁机粉碎榨汁,超声50 min,进行离心,取上清液,过滤,操作3次,旋转浓缩上清液,再加入90 %的乙醇,沉淀24 h后,离心弃去上清液,60 ℃挥干乙醇后冻干,得到滇黄精总多糖。
2、滇黄精水提物的制备:将炮制好的滇黄精打粉(新鲜滇黄精切片烘干,用黄酒浸润4 h,用高压灭菌锅蒸1 h,烘干,粉碎),取饮片粉末按料液比 1∶10g/mL加入蒸馏水加热回流2 h,过滤取上清液。药渣按上述料液比再次加热回流2 h,将两次上清液合并加热浓缩后冷冻干燥,得到滇黄精水提物。
实施例2 滇黄精总多糖和水提物的制备
1、滇黄精总多糖的制备:将新鲜黄精清洗干净,切制成小块,控制料液比1:25 g/mL,在高速榨汁机粉碎榨汁,超声60 min,进行离心,取上清液,过滤,操作3次,旋转浓缩上清液,再加入90 %的乙醇,沉淀24 h后,离心弃去上清液,60 ℃挥干乙醇后冻干,得到滇黄精总多糖。
2、滇黄精水提物的制备:将炮制好的滇黄精打粉(新鲜滇黄精切片烘干,用黄酒浸润4 h,用高压灭菌锅蒸1 h,烘干,粉碎),取饮片粉末按料液比 1∶15g/mL加入蒸馏水加热回流3 h,过滤取上清液。药渣按上述料液比再次加热回流2 h,将两次上清液合并加热浓缩后冷冻干燥,得到滇黄精水提物。
实施例3 罗伊氏乳杆菌扩大培养
挑取罗伊氏乳杆菌的单菌落接种到MRS液体培养基,37 ℃、180 r/min厌氧培养至OD600 nm值为0.6~0.8作为种子液;种子液按1∶100(体积比)接种量分别接种到MRS液体培养基(空白对照组)、MRS滇黄精总多糖培养基(0.0005 g/mL,滇黄精总多糖组-ps)和MRS滇黄精水提物培养基(0.00634 g/mL,滇黄精水提物组-pw)中,37℃、180 r/min厌氧培养至对数中期(OD600 nm=0.6~0.8)得到。
实验例1 滇黄精总多糖和水提物对伊氏乳杆菌非特异性黏附能力的影响
一、滇黄精总多糖和水提物对伊氏乳杆菌表面疏水性的影响
实验方法:
1、将实施例3伊氏乳杆菌扩大培养后的菌液在5000 r/min下离心10 min,收集菌体,PBS洗涤两次,每次20 mL。离心10 min以PBS为空白对照组,用PBS调整受试菌株液浓度600 nm波长下吸光度A值约为1.0。
2、取2 mL调好菌液浓度的菌悬液,与2 mL二甲苯混合,涡旋2 min,室温静止30min。
3、取1 mL水相,以PBS为空白对照组,在600 nm下测定吸光度A值,并记录,每株菌株平行做3管重复。
菌株表面疏水率(CSH %)计算: H %=[(A0-A)/A0]*100%
其中A0和A分别是与二甲苯混匀前和混匀后水相在600 nm下测定的A值。
实验结果:如图1所示,使用滇黄精总多糖及水提物能够提高伊氏乳杆菌的疏水率。
二、滇黄精总多糖和水提物对伊氏乳杆菌自凝聚率的影响
实验方法:
1、将实施例3 伊氏乳杆菌扩大培养后的菌液在6000 r/min下离心10 min,收集菌体,PBS洗簇两次,每次10 mL,6000 r/min离心10min。PBS为空白对照,用PBS调整菌株菌液浓度,使其在600 nm波长处A值约为1.0。
2、取4 mL调整好菌浓度的菌悬液于型号一致的试管中,室温静置,分别在静畳3h、6 h后吸取1 mL上层溶液测600 nm吸光值,每个菌株每个时间点平行做3管重复。
细菌自凝聚百分比计算: 菌株自凝聚力(%)=[1-At/A0]*100%
其中A0和A分别是自凝聚前后上清溶液在600 nm下测定的A值。
实验结果:如图2和图3所示,自凝聚实验结果显示罗伊氏乳杆菌的3 h和6 h自凝聚率得到提高。
菌株疏水性强弱与黏附能力成正比,自凝聚能力被认为与多种黏附现象有关,自凝聚率越大越有利于益生菌聚集。综上,滇黄精总多糖和水提物对罗伊氏乳杆菌的表面疏水性和自凝聚率能力均有提高,能使菌株更好地发挥益生特性。
实验例2 滇黄精总多糖和水提物对伊氏乳杆菌黏附Caco-2细胞的影响
实验方法:
1、从液氮罐中取出Caco-2细胞冻存管,快速置于37 ℃水浴锅反复摇动使其在1min中内融化;立马转移至超净台后,将Caco-2冻存液迅速转移至15 mL无菌离心管中,另加3 mL完全高糖 DMEM培养基,离心(1000 r/min,3 min)后,吸掉上清液;向离心管中加入5mL完全高糖DMEM培养基,充分混匀后转移至50 mL细胞培养瓶中;置于5 % CO2培养箱中37℃培养;隔天更换新鲜完全高糖DMEM培养基;待细胞生长状态良好(贴壁生长为单层且培养瓶底部80 %左右被细胞覆盖),PBS洗涤2-3次后(PBS每次2 mL,目的:洗去死细胞和代谢产物),用2 mL 0.25 %胰蛋白酶一EDTA进行消化(37 ℃预热),弃去消化液,加入2 mL完全高糖DMEM培养基终止消化,转移至15 mL无菌离心管,1000 r/min离心3min;弃掉上清液,重新加入4 mL新鲜完全高糖DMEM培养基,充分吹打混合均匀后分瓶(1传4),再向每瓶中加入4mL新鲜培养基。置5 %CO2培养箱37 ℃传代培养。将Caco-2细胞以3×105细胞/孔的浓度从培养瓶传代接种至6孔板中,每隔一天更换一次培养液,待细胞贴壁完全后,继续培养至极化状态时方可进行实验。在进行试验的前一天,向孔板中加入不含双抗的高糖DMEM培养基。即可进行黏附试验。
2、取2 mL实施例3制备的菌液在12000 r/min离心10 min取菌体沉淀,菌体用0.0067 mol/L PBS洗涤3次后用不含双抗的细胞培养基调整菌体OD值为0.8-1.0左右。
3、向6孔板中添加罗伊氏乳杆菌菌体之前,用无菌PBS缓冲液洗涤6孔板中单层Caco-2细胞两次,再向每孔加入2 mL浓度为108CFU/mL(Vo)的罗伊氏乳杆菌菌体;将6孔板转移至5%CO2培养箱中,37 ℃培养2 h后;用PBS溶液洗涤12孔板中每孔的单层细胞层3次以上,以洗脱没有黏附的罗伊氏乳杆菌和代谢分泌物;然后向每孔中加入500 uL 0.25 %胰蛋白酶-EDTA孵育3 min;再加入500 uL培养液终止消化;收集每个孔内溶液,进行10倍梯度稀释后,通过平板菌落计数法检测培养后的活菌数(V1)。
黏附率(%)计算方法如下:黏附率(%)=(V1/V0)*100%
实验结果:
如图4所示, 滇黄精多糖组及水提物组的Caco-2细胞黏附率显著多于空白对照组。如图5所示,显微镜下可见滇黄精多糖组及水提物组的Caco-2细胞黏附效果多于空白对照组。综合分析可知,比起单独使用罗伊氏乳杆菌,向MRS培养基中添加滇黄精总多糖或水提物能够显著提高Caco-2细胞黏附率。
Claims (4)
1.一种提高罗伊氏乳杆菌黏附Caco-2细胞能力的组合物,其特征在于,所述组合物为滇黄精总多糖和/或滇黄精水提物;
提高罗伊氏乳杆菌黏附Caco-2细胞能力的方法是将罗伊氏乳杆菌种子液按照体积比为1∶100的接种量接种在添加了滇黄精总多糖和/或滇黄精水提物的MRS液体培养基中,于37 ℃温度下,摇床速度为180 r/min的条件下厌氧培养至对数中期即可获得黏附能力提高的罗伊氏乳杆菌;
其中,滇黄精总多糖的添加浓度为0.0004~0.0006 g/mL,滇黄精水提物的添加浓度为0.005~0.007 g/mL。
2.根据权利要求1所述组合物,其特征在于,滇黄精总多糖的制备方法如下:将新鲜滇黄精清洗干净,切制成小块,控制料液比1:20 -25g/mL,在高速榨汁机粉碎榨汁,超声50-60min,进行离心,取上清液,过滤,操作3-4次,旋转浓缩上清液,再加入90 %的乙醇,沉淀24 h后,离心弃去上清液,60 ℃挥干乙醇后冻干,得到滇黄精总多糖。
3.根据权利要求1所述组合物,其特征在于,滇黄精水提物的制备方法如下:将炮制好的滇黄精打粉,取粉末按料液比 1∶10 -15g/mL加入蒸馏水加热回流2 -3h,过滤取上清液;
药渣按上述料液比再次加热回流2-3 h,将两次上清液合并加热浓缩后冷冻干燥,得到滇黄精水提物。
4.权利要求1所述组合物在制备罗伊氏乳杆菌制剂中的应用。
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