CN111449187B - 一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体及其制备方法和抗菌用途 - Google Patents
一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体及其制备方法和抗菌用途 Download PDFInfo
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Abstract
本发明公开了一种布氏乳杆菌S‑层蛋白修饰的香芹酚/β‑环糊精脂质体及其制备方法和抗菌用途,所述香芹酚/β‑环糊精脂质体主要由以下重量份比例的原料,并且利用食品上可接受的溶剂制成:布氏乳杆菌S‑层蛋白1~5份,香芹酚2~5份,β‑环糊精10~30份,尤特奇RL100,大豆卵磷脂20~50份,胆固醇4~10份。本发明通过利用β环糊精将香芹酚包埋后,再将香芹酚/β‑环糊精的包合物包封于脂质体中,进一步提高香芹酚的可溶性,从而提高脂质体中香芹酚的包封率,提高其利用率,再由布氏乳杆菌S‑层蛋白修饰至脂质体表面,可以提高其稳定性和储存性能,布氏乳杆菌S‑层蛋白与香芹酚协同抑菌,将显著提高脂质体的抑菌效果,并可对预防致病菌的产生起到可持续释放的效果。
Description
技术领域
本发明属于香芹酚脂质体技术领域,具体涉及一种S-层蛋白修饰的香芹酚/β-环糊精脂质体及其制备方法和抗菌用途。
背景技术
香芹酚(car)是一种具有麝香草酚味的无色至淡黄色液体,普遍存在于牛至、百里香、亚加菊等植物的挥发油中。目前已报道,香芹酚具有抗氧化、抗菌、驱虫、减轻大鼠脑和脊髓缺血再灌注损伤等作用。由于其无毒副作用,无残留,对环境无污染,常作为香料添加于化妆品等日用品中;常作为天然的调味香料,可去除海鲜、肉类、鱼类等食品中的腥味,增加菜肴的风味;常作为抑菌剂、防腐剂用于食物保鲜中。但是由于香芹酚不溶于水,且在空气中易挥发、易氧化,稳定性差等缺点,极大地影响了其生物利用度和适用范围。
现有技术中CN201310436851将香芹酚与β-环糊精制备成包合物来提高其稳定性。CN1618294公开了一种从天然芳香植物百里香中提取的精油作为活性成分的驱螨组合物,24h驱避率均达到100%。CN102178251A公开了一种百里香羊肉除膻调味剂,除膻效果良好,色泽优美,百里香香气适宜,口感和谐自然,具有很高的应用价值和广阔的市场前景。CN104379156A公开了一种铺地百里香提取物,用于治疗肠炎性疾病。CN104478607A公开了一种百里香酚杀菌剂及其该杀菌剂几种剂型的制备方法。该杀菌剂低毒、环保、安全、无害,是一种非常具有市场应用前景的杀菌剂。
虽然香芹酚在医学,食品和化妆品行业被广泛应用,具有较好的杀菌、增香等特点,但是香芹酚易挥发,暴露在空气中不稳定,所以要寻找有效的方法降低香芹酚在使用过程中的挥发程度,延长其贮存期。β环糊精由于生产工艺简单,成本较低,是目前工业上唯一能大量生产的环糊精类产品。β环糊精能将香芹酚包裹在内,增强香芹酚的溶解性,且不会对香芹酚的主要活性成分造成破坏。如上所述的现有技术CN201310436851,将香芹酚与β-环糊精制备成包合物来提高其稳定性,现有技术还有将香芹酚直接制成脂质体的相关研究,但是都还存在包封率低,产品抗菌性能和缓释性能差的缺陷。
乳酸菌表面覆盖一层S-层蛋白,这是一种可以经自组装而形成的对称次晶格阵列结构的蛋白或糖蛋白,由单一蛋白或糖蛋白亚基组成。S-层蛋白占细胞总蛋白的10%-15%,相对分子质量大概在40-200kDa之间。S-层蛋白可以在适当的环境中通过静电相互作用在脂质体表面自组装形成S-层蛋白包被的脂质体来将脂质用于功能化以用于各种应用。与普通脂质体相比,S-层蛋白涂层脂质体具有出色的理化和生物学稳定性。例如L.brevis和L.kefir的S-层蛋白可以用来包埋脂质体,维持其在高温、低pH等恶劣环境下的稳定性。并且发现了L.kefir糖基化的S-层蛋白对脂质体的亲和力高于非糖基化的L.brevis的S-层蛋白。但是没有做S-层蛋白修饰脂质体关于抑菌方面的研究。
发明内容
发明目的:针对上述技术问题,本发明提供了一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体及其制备方法和抗菌用途。本发明通过将香芹酚包裹在环糊精脂质体中,以减少其在使用过程中的挥发性,增强香芹酚的溶解度,提高其利用率,布氏乳杆菌S-层蛋白自组装在脂质体表面增强其稳定性以及黏附特性,达到长效缓释抗菌的效果。
技术方案:为了达到上述发明目的,本发明所采用的技术方案如下:
一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体,其主要由以下重量份比例的原料,并且利用食品上可接受的溶剂制成:
布氏乳杆菌S-层蛋白1~5份,香芹酚2~5份,β-环糊精10~30份,大豆卵磷脂20~50份,尤特奇RL100 20~50份,胆固醇4~10份。
优选,所述食品上可接受的溶剂选自无水乙醇和超纯水。
优选,先将香芹酚和β-环糊精制成香芹酚/β-环糊精包合物,然后再利用大豆卵磷脂,尤特奇RL100和胆固醇,将所述香芹酚/β-环糊精包合物装载在脂质体中,最后将S-层蛋白修饰在香芹酚/β-环糊精脂质体中,即得所述S-层蛋白修饰的香芹酚/β-环糊精脂质体。
所述的布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体的制备方法,包括以下步骤:
a、取β-环糊精和香芹酚,超纯水作为溶剂,制备香芹酚的β-环糊精包合物,即香芹酚/β-环糊精包合物;
b、将步骤a所得香芹酚/β-环糊精包合物复溶于超纯水中,制得香芹酚/β-环糊精包合物水溶液,然后制备大豆卵磷脂,尤特奇RL100和胆固醇的无水乙醇混合溶液,注入到香芹酚/β-环糊精包合物水溶液中,搅拌;
c、将b所制备的溶液减压蒸发除掉乙醇,通过离心和微孔滤膜过滤,得到香芹酚/β-环糊精脂质体溶液。
d、取布氏乳杆菌S-层蛋白冻干粉溶解在香芹酚/β-环糊精脂质体溶液中,搅拌,得到S-层蛋白修饰的香芹酚/β-环糊精脂质体。
优选,步骤a中,先将β-环糊精加入超纯水中溶胀,制成质量体积比为0.01%-99%的β-环糊精溶液,然后将香芹酚加入到所述β-环糊精溶液中,混匀后冷冻干燥,即得所述香芹酚/β-环糊精包合物。
优选,步骤b中,大豆卵磷脂,尤特奇RL100和胆固醇加入到50~100份无水乙醇中,制成混合溶液,然后以0.1~1mL/min的速度注入到香芹酚/β-环糊精包合物水溶液中,无水乙醇混合溶液与香芹酚/β-环糊精包合物水溶液的体积比为1∶(0.1-2),搅拌。
优选,步骤c中,减压蒸发温度为35~70℃,减压蒸发时间为20~40min。
优选,步骤d中,布氏乳杆菌S-层蛋白是由布氏乳杆菌所提取的可溶性蛋白,经冷冻干燥成粉末状,进一步优选,所述S-层蛋白分子量为40–200kDa。所述搅拌为20-25℃磁力搅拌1~4h。
进一步,所述布氏乳杆菌S-层蛋白的提取方法如下:
1)布氏乳杆菌培养:先后利用MRS固体培养基和MRS液体培养基,培养布氏乳杆菌,至布氏乳杆菌生长的对数末期,连续传接三代提高菌种的活力,第三代乳酸菌培养液备用。
2)S-层蛋白提取:取培养好的布氏乳杆菌,先后利用LiCl溶液和CuHCl溶液进行处理,之后收集上清液进行透析,离心,收集溶液,冷冻干燥,即为布氏乳杆菌S-层蛋白。
所得S-层蛋白通过SDS-PAGE进行分析,通过BCA蛋白浓度测定试剂盒测定溶液蛋白浓度,分别测定三次以获得精密度。
优选,步骤1)中,布氏乳杆菌菌种在MRS液体培养基中培养时间为18-23h。
优选,步骤2)中,LiCl溶液浓度为5~10mol/mL,CuHCl溶液浓度为1-6mol/mL。
其中,上述a步骤中的溶胀时间优选为1min~48h。
其中,上述a步骤中可以采用常规方法使香芹酚混匀(即完全溶解)于溶剂中,如采用静置、加热、搅拌、超声或研磨等方法使香芹酚完全溶解于溶剂中。
其中,上述c步骤的减压蒸发温度过高会导致磷脂氧化,温度过低则磷脂达不到相变温度不能形成脂质体。
其中,空白脂质体是通过大豆卵磷脂、胆固醇和尤特奇RL100加入到50~100份无水乙醇中,制成混合溶液,然后以0.1~1mL/min的速度注入到超纯水中,搅拌并减压蒸发至乙醇完全挥发。
本发明最后还提供了所述S-层蛋白修饰香芹酚/β-环糊精脂质体作为抗菌剂的应用。
本发明首先将香芹酚和β-环糊精制成香芹酚/β-环糊精包合物,然后再通过纳米脂质体对β-环糊精/香芹酚包合物进行装载以及S-层蛋白自组装在脂质体表面,最后制成粒径为纳米级的S-层蛋白修饰香芹酚/β-环糊精脂质体。该纳米脂质体可以降低香芹酚的挥发性,延长保存期,提高脂质体的。因此,选用β-环糊精/香芹酚包合物制成稳定性更高的脂质体,能够进一步提高香芹酚的包封率和缓释性能。此外,纳米脂质体由于它们的亚细胞尺寸,能加强脂质体与细菌接触的能力机制,从而香芹酚精油的抗菌效果。
有益效果:相对于现有技术,本发明具有以下优势:
1.针对香芹酚不易溶于水,易挥发,利用率低的问题,采用环糊精脂质体双层包埋,不仅大大增加了香芹酚的溶解问题,而且提高了香芹酚的利用率,减少浪费,降低成本方面起到有效的作用。
2.香芹酚在食品工业中,不仅可以用作香料的用途,还对常见食源性致病菌具有广谱抗菌特性。通过环糊精脂质体递送系统制备新型的香芹酚/β-环糊精脂质体抗菌剂,提高了香芹酚的生物活性,具有缓释特性,延长保存期。
3.本发明充分利用环糊精脂质体的特性,不仅提高了香芹酚的食用价值,而且为提高香芹酚的抗菌特性的附加价值,提供了新的出路。
4.本发明利用布氏乳杆菌的S-层蛋白修饰香芹酚/β-环糊精脂质体,能够提高稳定性以及对细菌的黏附特性,使所制备的脂质体具有更强的稳定性。
附图说明
图1:布氏乳杆菌S-层蛋白的提取。
图2:S-层蛋白修饰香芹酚/β环糊精脂质体的包封率。
图3:香芹酚脂质体和S-层蛋白修饰香芹酚/β环糊精脂质体的稳定性。
图4:不同脂质体中香芹酚释放量曲线。
具体实施方式
下面通过具体实施例对本发明所述的技术方案给予进一步详细的说明。
以下实施例中布氏乳杆菌的培养方法如下:
取布氏乳杆菌悬液稀释至一定梯度后涂布于MRS固体培养基中,于37℃恒温培养箱48h培养,挑取单菌落接种于MRS液体培养基,于37℃恒温培养箱静置培养一定时间至布氏乳杆菌生长的对数末期,连续传接三代提高菌种的活力,第三代乳酸菌培养液可放置4℃冰箱备用
实施例1
1)布氏乳杆菌S-层蛋白的提取
1.布氏乳杆菌接种于MRS液体培养基后,在37℃恒温培养箱培养20h,将菌液离心(8000r/min,4℃,15min),对菌体称重,每克菌体加10mol/mL的LiCl溶液,在37℃恒温培养箱作用0.5h,离心(8000r/min,4℃,15min)后弃上清;每克再加入6mol/mL的CuHCl溶液,37℃作用1h后,离心(8000r/min,4℃,15min)收集上清;将上清移入14kDa截留分子量的透析袋,于去离子水中进行4℃透析,每隔4h换一次水;透析48h后,离心(12000r/min,4℃,15min)收集溶液,即为S-层蛋白,将所得蛋白进行冷冻干燥后存放于-20℃冰箱备用,并通过SDS-PAGE进行分析,结果如图1所示。通过BCA蛋白浓度测定试剂盒测定溶液蛋白浓度,分别测定三次以获得精密度。
2)香芹酚/β环糊精包合物的制备
1.β-环糊精溶液制备:称取12份的β-环糊精于120份超纯水中,搅拌溶解后制得β-环糊精溶液备用;
2.香芹酚/β-环糊精溶液制备:取β-环糊精溶液添加香芹酚4份进行磁力搅拌2h,10000rpm/min离心10min,通过0.22μm的滤膜过滤,得到滤液,即为香芹酚/β-环糊精包合物水溶液;
3)S-层蛋白修饰香芹酚/β环糊精脂质体的制备
称取大豆卵磷脂20份,尤特奇RL100 20份和胆固醇20份。加入100份无水乙醇溶液中溶解,混匀后,制成混合溶液,然后以0.5mL/min的速度注入到香芹酚/β环糊精包合物水溶液或者超纯水中,无水乙醇混合溶液与香芹酚/β环糊精包合物水溶液的体积比为1∶2,磁力搅拌15min后,旋转蒸发去除无水乙醇至溶液为100份形成香芹酚/β环糊精脂质体或者空白脂质体。减压蒸发温度为50℃,减压蒸发时间为30min。将所得香芹酚/β环糊精脂质体液体离心,用0.22μm无菌滤膜进行过滤,得滤液。取S-层蛋白冻干粉溶解在香芹酚/β-环糊精脂质体中,22℃磁力搅拌2h,得到S-层蛋白修饰的香芹酚/β-环糊精脂质体,放置于温度为4℃条件下保存备用。
实施例2
1)布氏乳杆菌S-层蛋白的提取
1.布氏乳杆菌接种于MRS液体培养基后,在37℃恒温培养箱培养21h,将菌液离心(8000r/min,4℃,15min),对菌体称重,每克菌体加8mol/mL的LiCl溶液,在37℃恒温培养箱作用0.5h,离心(8000r/min,4℃,15min)后弃上清;每克再加入5mol/mL的CuHCl溶液,37℃作用1h后,离心(8000r/min,4℃,15min)收集上清;将上清移入14kDa截留分子量的透析袋,于去离子水中进行4℃透析,每隔4h换一次水;透析48h后,离心(12000r/min,4℃,15min)收集溶液,即为S-层蛋白,将所得蛋白进行冷冻干燥后存放于-20℃冰箱备用,并通过SDS-PAGE进行分析。通过BCA蛋白浓度测定试剂盒测定溶液蛋白浓度,分别测定三次以获得精密度。
2)香芹酚/β环糊精包合物的制备
1.β-环糊精溶液制备:称取10份的β-环糊精于100份超纯水中,搅拌溶解后制得β-环糊精溶液备用;
2.香芹酚/β-环糊精溶液制备:取β-环糊精溶液添加香芹酚3份进行磁力搅拌2h,10000rpm/min离心10min,通过0.22μm的滤膜过滤,得到滤液,即为香芹酚/β-环糊精包合物水溶液;
3)S-层蛋白修饰香芹酚/β环糊精脂质体的制备
称取大豆卵磷脂15份,尤特奇RL100 15份和胆固醇8份。加入80份无水乙醇溶液中溶解,混匀后,制成混合溶液,然后以0.08mL/min的速度注入到香芹酚/β环糊精包合物水溶液或者超纯水中,无水乙醇混合溶液与香芹酚/β环糊精包合物水溶液的体积比为1∶0.1,磁力搅拌15-60min后,旋转蒸发去除无水乙醇至溶液总体积为20ml形成香芹酚/β环糊精脂质体或者空白脂质体。减压蒸发温度为40℃,减压蒸发时间为40min。将所得香芹酚/β环糊精脂质体液体离心,用0.22μm无菌滤膜进行过滤,得滤液。取S-层蛋白冻干粉溶解在香芹酚/β-环糊精脂质体中,22℃磁力搅拌3h,得到S-层蛋白修饰的香芹酚/β-环糊精脂质体,放置于温度为4℃条件下保存备用。
实施例3
1)布氏乳杆菌S-层蛋白的提取
1.布氏乳杆菌接种于MRS液体培养基后,在37℃恒温培养箱培养22h,将菌液离心(8000r/min,4℃,15min),对菌体称重,每克菌体加6mol/mL的LiCl溶液,在37℃恒温培养箱作用0.5h,离心(8000r/min,4℃,15min)后弃上清;每克再加入3mol/mL的CuHCl溶液,37℃作用1h后,离心(8000r/min,4℃,15min)收集上清;将上清移入14kDa截留分子量的透析袋,于去离子水中进行4℃透析,每隔4h换一次水;透析48h后,离心(12000r/min,4℃,15min)收集溶液,即为S-层蛋白,将所得蛋白进行冷冻干燥后存放于-20℃冰箱备用,并通过SDS-PAGE进行分析。通过BCA蛋白浓度测定试剂盒测定溶液蛋白浓度,分别测定三次以获得精密度。
2)香芹酚/β环糊精包合物的制备
1.β-环糊精溶液制备:称取10份的β-环糊精于100份超纯水中,搅拌溶解后制得β-环糊精溶液备用;
2.香芹酚/β-环糊精溶液制备:取β-环糊精溶液添加香芹酚3份进行磁力搅拌2h,10000rpm/min离心10min,通过0.22μm的滤膜过滤,得到滤液,即为香芹酚/β-环糊精包合物水溶液;
2)S-层蛋白修饰香芹酚/β环糊精脂质体的制备
称取大豆卵磷脂10份,尤特奇RL100 10份和胆固醇5份。加入50份无水乙醇溶液中溶解,混匀后,制成混合溶液,然后以0.05mL/min的速度注入到香芹酚/β环糊精包合物水溶液或者超纯水中,无水乙醇混合溶液与香芹酚/β环糊精包合物水溶液的体积比为1∶1,磁力搅拌15-60min后,旋转蒸发去除无水乙醇至溶液总体积为20ml形成香芹酚/β环糊精脂质体或者空白脂质体。减压蒸发温度为60℃,减压蒸发时间为20min。将所得香芹酚/β环糊精脂质体液体离心,用0.22μm无菌滤膜进行过滤,得滤液。取S-层蛋白冻干粉溶解在香芹酚/β-环糊精脂质体中,22℃磁力搅拌1.5h,得到S-层蛋白修饰的香芹酚/β-环糊精脂质体,放置于温度为4℃条件下保存备用。
对照例香芹酚脂质体的制备
称取大豆卵磷脂10份,尤特奇RL100 10份和胆固醇5份,香芹酚1份,加入50份无水乙醇溶液中溶解,混匀后,注入超纯水中制成混合溶液无水乙醇与超纯水溶液的体积比为1∶2,磁力搅拌15min后,旋转蒸发去除无水乙醇至溶液总体积为20ml形成香芹酚脂质体。将所得香芹酚脂质体液体用0.22μm无菌滤膜进行过滤,得滤液。放置于温度为4℃条件下保存备用。
实验例
取实施例1和对照例所得样品进行如下检测:
一、包封率的测定
1、用无水乙醇稀释香芹酚,准确称取一定比例的香芹酚标准品置于250mL容量瓶中,加入适量无水乙醇溶液使其溶解,并定容至刻度线,摇匀。然后,分别精密吸取一定体积的上述溶液配制0.005-0.1mg/mL香芹酚的乙醇标准溶液,于275nm处测吸光值(每个浓度重复测定3次,结果取平均值),得到标准曲线线性回归方程为y=11.219x+0.0152(R2=0.9997),其中x为香芹酚浓度(mg/mL),y为吸光值,线性浓度范围为0.061mg/mL~1.128mg/mL。取1mL S-层蛋白修饰香芹酚/β环糊精脂质体样品,10000rpm离心4分钟,除去上清液并通过超声处理将收集的沉淀物重新溶解等体积的乙醇溶液1小时。最后,将混合物在6000rpm,4℃下离心10分钟,并通过紫外分光光度计方法分析收集上清液。通过以下等式计算的脂质体中香芹酚的包封效率,则:
EE(%)=C1/C0×100%,
其中,EE为包封率(%),
C1为S-层蛋白修饰香芹酚/β环糊精脂质体中香芹酚的浓度(mg/mL),
C0为香芹酚的最初浓度(mg/mL)。
2、S-层蛋白修饰香芹酚/β环糊精脂质体的包封率
包封率是评价脂质体制剂质量好坏的最重要的指标,也是脂质体能否发挥较普通制剂高效、低毒等特点的关键。由图2可看出,香芹酚脂质体的包封率为14.3%,S-层蛋白修饰香芹酚/β环糊精脂质体的包封率为73%。因此制备S-层蛋白修饰香芹酚/β环糊精脂质体可以明显提高脂质体的包封率。
二、S-层蛋白修饰香芹酚/β环糊精蛋白脂质体的蛋白包被率
S-层蛋白的包被率即自组装于脂质体膜上的S-层蛋白的含量,是采用BCA法微量蛋白检测试剂盒进行间接定量,将S-层蛋白修饰的香芹酚/β-环糊精脂质体于15000rpm/min下离心30min后,测定上清液中游离蛋白的含量。蛋白包被率的计算公式如下:
P=(1-C1/C0)×100%
式中,P为蛋白包被率(%),
C0为S-层蛋白的初始浓度(mg/mL),
C1为游离S-层蛋白的浓度(mg/mL)。
蛋白包被率是评价蛋白脂质体制剂质量好坏的重要指标,也是脂质体能否发挥较普通制剂高效、低毒等特点的关键。结果测得S-层蛋白的吸附率为75.61%。
三、S-层蛋白修饰香芹酚/β环糊精脂质体的抗菌性能
1、实验材料
①香芹酚
②香芹酚脂质体
③S-层蛋白修饰香芹酚/β环糊精脂质体(β-CD/Car/SLP-LP)
2、实验方法
采用微量肉汤稀释法测定香芹酚、香芹酚脂质体和S-层蛋白修饰香芹酚/β环糊精脂质体的最小抑菌浓度(MIC),即先向96孔板的各孔加入100μL LB液体培养基,再向每一排第一孔加入100μL抑菌剂,用枪头充分吹打混匀后吸取100μL至第2孔,依次二倍系列稀释至同一排的第11孔,第11孔混匀后吸取100μL弃去。然后向每排的1~10孔和第12孔加入100μL经生理盐水稀释的金黄色葡萄球菌菌液(107CFU/mL),第11孔加入100μL生理盐水。将板置于37℃恒温培养箱中静置培养24h。以阴性对照孔(12孔)全部浑浊,阳性对照孔(11孔)全部清亮视为结果有效。使用酶标仪测定各孔OD600nm值,具有最低抑菌浓度的阴性孔即为香芹酚、香芹酚脂质体和S-层蛋白修饰香芹酚/β环糊精脂质体的MIC。从而对香芹酚脂质体和S-层蛋白修饰的香芹酚/β环糊精脂质体的抗菌活性进行评价。实验重复三次,结果取平均值。
3、S-层蛋白修饰香芹酚/β环糊精脂质体的抗菌性能
不同处理后的香芹酚样品的抗菌活性的变化也可以直接反映通过环糊精脂质体包埋能够提高其抑菌能力。因此测定了香芹酚、香芹酚脂质体和S-层蛋白修饰香芹酚/β环糊精脂质体的最小抑菌浓度,结果如表1所示,1-11分别是倍比稀释的抑菌溶液,12为空白对照,香芹酚溶液只有1号孔没长菌,该孔内香芹酚MIC值是320μg/mL。香芹酚脂质体在2号孔澄清,该孔内香芹酚MIC值是160μg/mL。经S-层蛋白修饰后的β-CD-香芹酚脂质体在4号孔澄清,证明其MIC值为40μg/mL。S-层蛋白修饰香芹酚/β环糊精脂质体显示增强了金黄色葡萄球菌抗菌效果,降低使用量并达到同等的抑菌效果。
表1通过最小抑制浓度(MIC)测量的S.aureus对S-层蛋白包被的β-CD-香芹酚脂质体的抗菌敏感性
四、S-层蛋白修饰香芹酚/β环糊精脂质体的稳定性能
1、实验材料
①香芹酚/β环糊精脂质体
②S-层蛋白修饰香芹酚/β环糊精脂质体
2、实验方法
通过测量S-层蛋白修饰香芹酚/β环糊精脂质体对去污剂的抗性能力的膜稳定性。简言之,将处理前后的两种脂质体分别加入到离心管中,处理组的脂质体是通过将100μL1%Triton X-100逐步移液到脂质体溶液中,经过混合震荡后,加入10μL 1%Triton100涡旋后8000rpm离心5分钟观察。
结果如图3所示,(A)、(B)、(C)、(D)均为不同浓度S-层蛋白包被的β-CD-香芹酚脂质体(30、40、50、100μg/mL表层蛋白);(E)为β-CD-香芹酚脂质体。加入10μL 1%Triton100涡旋后8000rpm离心5分钟得到上图,从图中可以看出,未经表层蛋白修饰的脂质体沉淀最多,说明脂质体膜不稳定,破坏的最严重。经S-层蛋白修饰后,A(30μg/mL)和B(40μg/mL)的脂质体几乎没有沉淀析出,代表脂质体膜稳定性得到很显著的提升。而C、D沉淀增多可能是因为表层蛋白量加大,所以沉淀增加。S-层蛋白修饰降低了膜的流动性,从而阻碍了高密度脂蛋白的脂质提取,降低了脂质体分解的风险,故而提升了其稳定性。
五、S-层蛋白修饰香芹酚/β环糊精脂质体的缓释性能
1、实验材料
①香芹酚脂质体
②S-层蛋白修饰香芹酚/β环糊精脂质体
2、实验方法
采用动态膜透析法进行微球体外释放试验。精密称量香芹酚/β环糊精脂质体粉末6份,每份约3mg,加释放介质2mL混悬,置预处理过的透析袋中,扎紧袋口,放入28mL释放介质中,将试样置恒温磁力搅拌器,控制温度37℃,转速为100rpm,定时吸取透析液并及时补充等量,过滤,稀释后测定香芹酚浓度,按一式计算累积释放百分率。每批样品重复操作次。以平均值为纵坐标,释放时间为横坐标绘制体外释放曲线。
通过测定香芹酚脂质体,S-层蛋白修饰香芹酚/β环糊精脂质体的缓释能力,可以表征环糊精脂质体的稳定性。如图4所示,通过S-层蛋白修饰的香芹酚/环糊精脂质体明显比脂质体直接包埋的香芹酚缓释性能更好。
Claims (5)
1.一种布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体的制备方法,其特征在于,其主要由以下重量份比例的原料,并且利用食品上可接受的溶剂制成:
布氏乳杆菌S-层蛋白1~5 份,香芹酚 2~5 份,β-环糊精 10 ~ 30 份,大豆卵磷脂 20~50 份,尤特奇RL100 20 ~ 50 份,胆固醇 4~10 份,包括以下步骤:
a、取β-环糊精和香芹酚,超纯水作为溶剂,制备香芹酚的β-环糊精包合物,即香芹酚/β-环糊精包合物;
b、将步骤a所得香芹酚/β-环糊精包合物复溶于超纯水中,制得香芹酚/β-环糊精包合物水溶液,然后制备大豆卵磷脂,尤特奇RL100和胆固醇的无水乙醇混合溶液,即大豆卵磷脂,尤特奇RL100和胆固醇加入到50~100份无水乙醇中,制成混合溶液,然后以0.1~1 mL/min的速度注入到香芹酚/β-环糊精包合物水溶液中,无水乙醇混合溶液与香芹酚/β-环糊精包合物水溶液的体积比为 1∶(0.1-2),搅拌;
c、将b所制备的溶液减压蒸发除掉乙醇,通过离心和微孔滤膜过滤,得到香芹酚/β-环糊精脂质体溶液,减压蒸发温度为35~70℃,减压蒸发时间为20~40min;
d、取布氏乳杆菌S-层蛋白冻干粉溶解在香芹酚/β-环糊精脂质体溶液中,搅拌,得到S-层蛋白修饰的香芹酚/β-环糊精脂质体。
2.根据权利要求1所述的布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体的制备方法,其特征在于,步骤a中,先将β-环糊精加入超纯水中溶胀,制成质量体积比为 0.01%-99%的β-环糊精溶液,然后将香芹酚加入到所述β-环糊精溶液中,混匀后冷冻干燥,即得所述香芹酚/β-环糊精包合物。
3.根据权利要求1所述的布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体的制备方法,其特征在于,步骤d中,布氏乳杆菌S-层蛋白是由布氏乳杆菌所提取的可溶性蛋白,经冷冻干燥成粉末状;所述搅拌为20-25℃磁力搅拌1~4h。
4.权利要求1-3任一项所述的制备方法得到的布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体。
5.权利要求4所述的布氏乳杆菌S-层蛋白修饰的香芹酚/β-环糊精脂质体在制备抗菌剂中的应用。
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