CN112042852B - 一种蛋清蛋白-Nisin纳米粒子抗菌剂制备方法 - Google Patents
一种蛋清蛋白-Nisin纳米粒子抗菌剂制备方法 Download PDFInfo
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Abstract
本发明公开了一种蛋清蛋白‑Nisin纳米粒子抗菌剂制备方法,以蛋清蛋白和乳酸链球菌素(Nisin)为主要原料,通过蛋白质加热变性凝胶的过程使蛋清蛋白和Nisin自组装形成纳米粒子,减少了其因环境因素造成的活性损失,不仅提高了Nisin的溶解度,而且增强了Nisin的稳定性和长效抑菌作用。所制备的纳米粒子具有乳化性强、分散性好、水溶性强、稳定性好、制备周期短、产率高、绿色安全等特点。
Description
技术领域
本发明涉及抗菌剂及其制备方法,特别涉及一种蛋清蛋白-Nisin纳米粒子抗菌剂制备方法。
背景技术
乳酸链球菌素(Nisin)是由34个氨基酸组成的小阳离子肽,是一种天然食品防腐剂,被世界卫生组织和美国FDA认可、批准在食品中应用。Nisin的抗菌活性强,对革兰氏阳性菌抗菌谱广,并且作用机理研究较为成熟,常用在肉制品、乳制品、罐藏食品、酿造工业等。但是Nisin在应用过程中受到一定的限制,如:对革兰氏阴性菌抗菌谱窄、溶解性差、易被酶降解以及在高温条件下会失去部分活性等。所以要寻找一种能够改善Nisin的溶解性、抵抗酶攻击以及热稳定性。目前有将Nisin制成纳米颗粒或利用食品级壁材用于包埋Nisin制成纳米颗粒的报道。使用的技术各有不同,有优势也存在一些不足,如脂质体递送系统存在使用有机溶剂、成本高和不可控的分散性特点;壳聚糖-角叉菜胶和壳聚糖-聚谷氨酸递送系统方法较为复杂。
蛋清蛋白(Egg White Protein,EWP)具有优异的营养价值、可消化性、自组装和两亲性质,被认为是制备纳米级递送颗粒的理想材料。它可以提高分子的水溶性和生物利用度,保护包封组分免受酶攻击,控制释放速率和靶向递送能力,现已有关于蛋清蛋白作为递送载体的报道如递送姜黄素,胡萝ト素等功能性成分。本发明均采用绿色、安全的原材料,通过低成本、简单易操作的制备方法提高Nisin的稳定性。
发明内容
发明目的:本发明目的是提供抗菌效果好、溶解性强的蛋清蛋白-Nisin纳米粒子抗菌剂制备方法。本发明通过将Nisin包裹在蛋清蛋白中,增强Nisin的溶解度和稳定性,从而提高其生物利用率,达到长效抗菌的目的。与此同时,也改善了蛋清蛋白的乳化性。
技术方案:本发明提供一种蛋清蛋白-Nisin纳米粒子抗菌剂制备方法,包括如下步骤:
(1)制备壁材:取蛋清蛋白粉溶解于去离子水中,离心、过滤,制备蛋清蛋白水溶液;
(2)制备蛋清蛋白-Nisin混合物:加入Nisin搅拌,得到蛋清蛋白-Nisin混合物;
(3)制备蛋清蛋白-Nisin凝胶:取搅拌后混合物加热使其凝胶化,冰浴、冷藏,制得蛋清蛋白-Nisin凝胶;
(4)超声均质;
(5)离心、取上清、过膜;
(6)冻干,制得蛋清蛋白-Nisin复合物粉末。
进一步地,所述步骤(1)中溶解后调节溶液pH为2-5,过滤后调节溶液pH为2-5。
进一步地,所述步骤(1)中离心条件为6000rpm-10000rpm,时间为10-20min。
进一步地,所述步骤(2)中,蛋清蛋白和Nisin的比例为4∶1-1∶2,Nisin的浓度为2.5-20mg/mL。
进一步地,所述步骤(3)中加热的条件为70-100℃,5-120min。
进一步地,所述步骤(4)中超声均质的条件为400W-900W,1-60min。
进一步地,所述步骤(5)中离心的条件为3000-5000rpm,时间是5-15min。
有益效果:本发明具有如下优势:
1.本发明通过将蛋清蛋白加热凝胶的过程使蛋清蛋白和Nisin自组装成纳米粒子,整个制备方法及制备过程简单快捷,蛋清蛋白来源广泛,纳米粒子制备周期短、分散性好、产率高。
2.针对Nisin溶解性差的问题,合理利用蛋清蛋白体系包埋,不仅解决Nisin溶解性的问题,而且对Nisin的稳定性有所改善。
3.本发明通过将蛋清蛋白和Nisin加热凝胶制备成纳米粒子,对乳酸链球菌素起到了保护作用,减少了其因环境因素造成的活性损失,提高了其生物利用率,并且纳米粒子具备控制释放的能力,能够延长Nisin的抑菌效果。
4.本发明充分利用蛋清蛋白和Nisin的结合特性,有效增强了蛋清蛋白的乳化功能,并且在制备过程中不添加化学乳化剂,绿色安全,使所制备抗菌剂应用潜能进一步扩大。
5.本发明充分利用蛋白质资源,不仅拓宽了Nisin的应用价值,而且为提高蛋清蛋白的附加值以及开发具有多功能型的绿色食品防腐剂提供了新的思路。
附图说明
图1为样品制备后的成品图,其中,A:蛋清蛋白纳米粒子(HE);B:未经过热处理的Nisin(N);C:蛋清蛋白和Nisin混合物(EN);D:蛋清蛋白-Nisin纳米粒子(HEN);
图2为蛋清蛋白-Nisin纳米粒子的透射电镜图,其中,A:蛋清蛋白纳米粒子(HE);B:蛋清蛋白-Nisin纳米粒子(HEN);
图3为蛋清蛋白纳米粒子(HE)、蛋清蛋白和Nisin混合物(EN)和蛋清蛋白-Nisin纳米粒子(HEN)的乳化性及乳化稳定性;
图4为Nisin(N)、蛋清蛋白纳米粒子(HE)、蛋清蛋白和Nisin混合物(EN)和蛋清蛋白-Nisin纳米粒子(HEN)的差示扫描量热法;
图5为Nisin(N)、蛋清蛋白和Nisin混合物(EN)和蛋清蛋白-Nisin纳米粒子(HEN)的抑菌率。
具体实施方式
实施例1为蛋清蛋白-Nisin纳米粒子的制备方法,包括以下步骤:
1.称取蛋清蛋白粉,以去离子水作为溶剂,使蛋清蛋白溶液质量浓度为1%,并调节溶液pH=5,离心(6000g,10min)去除不溶性蛋白质,过滤,再调pH=2.0制备蛋清蛋白溶液;
2.称取乳酸链球菌素(Nisin)加到蛋清蛋白溶液中使Nisin终浓度10mg/mL,并在25℃下搅拌2h;
3.取搅拌后混合物在90℃下加热30min使其凝胶化并冰浴20min,于4℃过夜,制得蛋清蛋白-Nisin凝胶;
4.取蛋清蛋白-Nisin凝胶于600W超声处理15min;
5.取超声后样品进行低速离心(3000rpm,5min)取上清,过膜,制得蛋清蛋白-Nisin纳米粒子。
实施例2为蛋清蛋白-Nisin纳米粒子的制备方法,包括以下步骤:
1.称取蛋清蛋白粉,以去离子水作为溶剂,使蛋清蛋白溶液质量浓度为1%,并调节溶液pH=5,离心(6000g,15min)去除不溶性蛋白质,过滤,再调pH=4.0制备蛋清蛋白溶液;
2.称取乳酸链球菌素(Nisin)加到蛋清蛋白溶液中使Nisin浓度为5mg/mL,并在25℃下搅拌2h;
3.取搅拌后混合物在90℃下加热5min使其凝胶化并冰浴20min,于4℃过夜,制得蛋清蛋白-Nisin凝胶;
4.取蛋清蛋白-Nisin凝胶于600W超声处理15min;
5.取超声后样品进行低速离心(3000rpm,15min)取上清,过膜,制得蛋清蛋白-Nisin纳米粒子。
实施例3为蛋清蛋白-Nisin纳米粒子的制备方法,包括以下步骤:
1.称取蛋清蛋白粉,以去离子水作为溶剂,使蛋清蛋白溶液质量浓度为1%,并调节溶液pH=5,离心(10000g,10min)去除不溶性蛋白质,过滤,再调pH=5.0制备蛋清蛋白溶液;
2.称取乳酸链球菌素(Nisin)加到蛋清蛋白溶液中使Nisin浓度为20mg/mL,并在25℃下搅拌2h;
3.取搅拌后混合物在80℃下加热30min使其凝胶化并冰浴20min,于4℃过夜,制得蛋清蛋白-Nisin凝胶;
4.取蛋清蛋白-Nisin凝胶于600W超声处理1min;
5.取超声后样品进行低速离心(5000rpm,5min)取上清,过膜,制得蛋清蛋白-Nisin纳米粒子。
实施例4为蛋清蛋白-Nisin纳米粒子的制备方法,包括以下步骤:
1.称取蛋清蛋白粉,以去离子水作为溶剂,使蛋清蛋白溶液质量浓度为1%,并调节溶液pH=5,离心(10000g,10min)去除不溶性蛋白质,过滤,再调pH=3.0制备蛋清蛋白溶液;
2.称取乳酸链球菌素(Nisin)加到蛋清蛋白溶液中使Nisin浓度为2.5mg/mL,并在25℃下搅拌2h;
3.取搅拌后混合物在90℃下加热120min使其凝胶化并冰浴20min,于4℃过夜,制得蛋清蛋白-Nisin凝胶;
4.取蛋清蛋白-Nisin凝胶于600W超声处理60min;
5.取超声后样品进行低速离心(5000rpm,5min)取上清,过膜,制得蛋清蛋白-Nisin纳米粒子。
实施例5:性能测试
对于实施例1制得的蛋清蛋白-Nisin纳米颗粒的各项指标进行检测,测得的各项技术指标如下:
一、蛋清蛋白-Nisin纳米粒子的包封率和负载率
对于蛋清蛋白-Nisin纳米粒子,将制备好的样品过0.22μm的膜,取少量样品加入到10KDa超滤管中,在4℃条件下,以10000rpm离心10min,取滤过液,用紫外分光光度计分别在波长215nm和225nm测滤过液的吸光值取之间的差代入线性回归方程y=0.0002x+0.1213,R2=0.999(x为nisin的质量浓度μg/mL,y为吸光值)以测定游离的Nisin的浓度。通过公式计算得出蛋清蛋白-Nisin纳米粒子的包封率(EE)和负载量(LC)。
(1)和(2),分别为:
包封率(%)=总nisin含量-游离的nisin含量/总nisin×100 (1)
负载率(%)=总nisin含量-游离的nisin含量/样品质量×100 (2)
包封率和负载率是评价包封物质质量好坏的重要指标,也是载体是否充分发挥包封性能的关键。结果如表1可知,实施例4制备的样品包封率和负载率达到最佳值,分别为90.3%、23.4。
表1不同实施例制备的样品包封率和负载率
二、蛋清蛋白-Nisin纳米粒子的制备及粒径、PDI、形态
粒径和PDI是反应蛋白质纳米颗粒均匀性及其在食品工业中应用效果的重要指标。透射电镜可以观察到纳米粒子的结构形态。纳米颗粒的PDI越小说明纳米颗粒分散性越好。如图1所示,样品(从左到右)分别为蛋清蛋白纳米粒子(HE)、未经过热处理的Nisin(N)、蛋清蛋白-Nisin混合物(EN)、蛋清蛋白-Nisin纳米粒子溶液(HEN)。游离Nisin的粒径为760nm,PDI为0.86;蛋清蛋白-Nisin纳米粒子为79.43nm,PDI为0.318。说明此蛋清蛋白-Nisin纳米粒子具有优良的均匀性和分散性。如图2透射电镜图所示,可以看出图A蛋清蛋白纳米粒子表面形态呈球状,图B蛋清蛋白-Nisin纳米粒子表面出现大量聚集说明颗粒有聚集趋势。
三、乳化性及乳化稳定性
分别取3mL的蛋清蛋白-Nisin混合溶液(EN)、蛋清蛋白-Nisin纳米粒子溶液(HEN),加1mL的花生油,用均质机在室温条件下,12000r/min,均质后迅速取出100μL稀释到5mL的SDS(0.1%,w/v)溶液中,在500nm处测吸光值记为A0,等待10min再取100μL稀释到5mL的SDS(0.1%,w/v)溶液中,在500nm处测吸光值记为A10,计算出EAI和ESI。结果如图3,可以看出蛋清蛋白与Nisin经过热诱导制成纳米粒子(实质是蛋白质-蛋白质相互作用)其乳化性和乳化稳定性均得到明显的提高。
四、差示扫描量热法(DSC)
采用差示扫描量热法(DSC)分析蛋清蛋白纳米粒子(HE)、未经过热处理的Nisin(N)、蛋清蛋白和Nisin混合物(EN)和蛋清蛋白-Nisin纳米粒子(HEN)的热力学性质。从25℃~120℃以90℃的扫描速率进行分析,并在120℃下保持1min,以确保样品加热均匀。结果如图4所示,蛋清蛋白纳米粒子的变性温度为79.70℃,蛋清蛋白和Nisin混合物的变性温度为76.17℃而蛋清蛋白-Nisin纳米粒子变性温度达到了81.04℃,使得热力学性质得到改善。
五、抗菌活性测试
(1)最小抑菌浓度(MIC)和最小杀菌浓度(MBC)
根据NCCLS 2000推荐的肉汤稀释法测定颗粒的最小抑菌浓度(MIC)。以金黄色葡萄球菌为试验菌,接种于LB肉汤中,培养10-12h。采用LB肉汤二倍稀释法在96孔板中制备一系列样品浓度。在37℃培养24小时后,MIC被确定为最低样品浓度的澄清孔。为了评估最小杀菌浓度(MBC),将所有澄清孔(MIC试验)中的液体涂布到LB琼脂平板上,在37℃培养24h。MBC为无细菌生长的平板相应的样品的最低浓度。对每个样品进行三次试验。
测试结果显示,Nisin的MIC和MBC分别为156.25μg/mL和312.5μg/mL,蛋清蛋白加入后及蛋清蛋白纳米凝胶包埋并未影响Nisin的抗菌活性。
(2)抑菌率
采用试管法测OD值,计算出未经过热处理的Nisin(N)、蛋清蛋白和Nisin混合物(EN)和蛋清蛋白-Nisin纳米粒子(HEN)分别在12h、24h、36h和48h的抑菌率(Nisin终浓度均为1MIC)。如图5所示,当在12h时,抑菌效果没有显著差异,抑菌率均达到50%左右;随着时间的延长,抑菌率都在下降,蛋清蛋白-Nisin纳米粒子的抑菌率呈缓慢下降状态,而蛋清蛋白和Nisin混合物呈急速下降趋势,从侧面体现出蛋清蛋白-Nisin纳米粒子具有缓释性,延长Nisin的作用时间。
六、热稳定性研究
未经过热处理的Nisin(N)、经过高温处理的Nisin(HN)、蛋清蛋白Nisin混合物(EN)和蛋清蛋白-Nisin纳米粒子(HEN)对LB培养基中金黄色葡萄球菌的最小抑菌浓度和最小杀菌浓度值见表2。由表2可知,没有经过高温处理时,蛋清蛋白的加入并不影响到Nisin的抑菌活性;经过高温处理后,Nisin抑菌活性损失了50%,而经纳米凝胶包封处理后Nisin活性未受到影响,说明本发明方法能有效保护Nisin的抑菌活性。
表2 121°处理15min后样品对S.aureus的MIC和MBC
Claims (2)
1.一种蛋清蛋白-Nisin纳米粒子抗菌剂制备方法,其特征在于:包括如下步骤:
(1)制备壁材:取蛋清蛋白粉溶解于去离子水中,离心、过滤,制备蛋清蛋白水溶液;
(2)制备蛋清蛋白-Nisin混合物:加入Nisin搅拌,得到蛋清蛋白-Nisin混合物;
(3)制备蛋清蛋白-Nisin凝胶:取搅拌后混合物加热使其凝胶化,冰浴、冷藏,制得蛋清蛋白-Nisin凝胶;
(4)超声均质;
(5)离心、取上清、过膜;
(6)冻干,制得蛋清蛋白-Nisin复合物粉末;
所述步骤(1)中溶解后调节溶液pH为2-5,过滤后调节溶液pH为2-5;
所述步骤(2)中,蛋清蛋白和Nisin的比例为4:1-1:2,Nisin的浓度为2.5-20 mg/mL,
所述步骤(3)中加热的条件为70-100℃,5-120min;
所述步骤(4)中超声均质的条件为400W-900W,1-60min;
所述步骤(5)中离心的条件为3000-5000 rpm ,时间是5-15 min。
2.根据权利要求1所述的蛋清蛋白-Nisin纳米粒子抗菌剂制备方法,其特征在于:所述步骤(1)中离心条件为6000rpm-10000rpm,时间为10-20min。
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