CN105999039B - 一种酪蛋白-豆蔻精油脂质体抗菌剂的制备方法和用途 - Google Patents
一种酪蛋白-豆蔻精油脂质体抗菌剂的制备方法和用途 Download PDFInfo
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Abstract
本发明属于抗菌剂及食品添加剂或者化妆品领域，具体涉及一种酪蛋白‑豆蔻精油脂质体抗菌剂的制备及应用。本发明通过将豆蔻精油包裹在酪蛋白脂质体中，以实现减少豆蔻精油在施用过程中的挥发，从而减少豆蔻精油的浪费，提高其利用率，达到长效抗菌与高效利用的目的。
Description
技术领域
本发明属于抗菌剂及食品添加剂或者化妆品领域，具体涉及一种酪蛋白-豆蔻精油脂质体抗菌剂的制备方法及应用。
背景技术
豆蔻，多年生常绿草本植物，是姜科植物，产出暖性精油。精油无色或黄色，甜而温暖，主要成分是松油醇，桉油醇，姜烯和柠檬烯。肉豆蔻含挥发油、脂肪油、苯丙素、木脂素和黄酮等多种化学成分，具有抗菌消炎、镇痛、止泻、抗肿瘤等广泛的药理作用。豆蔻精油有良好的抗炎，镇痛，抑菌作用，对各种食源性病原菌有较好的抗菌效果，且安全、无毒副作用，在肉制品加工中应用广泛，可去异味，抑细菌，增辛香。
国内有很多关于豆蔻在医学应用的专利申请。CN103893226A公开了肉豆蔻提取物在制备抗痛风的药物、食品或保健品的应用。CN102988333A公开了豆蔻明在制备防治肠炎的药物或食品中的应用。CN102036662A公开了一种利用肉豆蔻衣木脂素预防和治疗ppAR介导的疾病的方法。CN103719464A发明了一种肉豆蔻脾胃虚弱保健茶。CN1709467A公开了一种复方草豆蔻滴丸及其制备方法。CN101508937A公开了一种超临界二氧化碳提取肉豆蔻精油的方法,可以获得高品质的肉豆寇精油产品。CN103638074A公开了一种肉豆寇挥发油在制备治疗抑郁症药物中的应用。
虽然豆蔻精油在医学，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，但是豆蔻精油易挥发，暴露在空气中不稳定，所以要寻找有效的方法降低肉桂精油在使用过程中的挥发程度，延长其贮存期。
纳米脂质体能将精油包裹在内，并且纳米脂质体不会对豆蔻精油的主要活性成分造成破坏。纳米脂质体使豆蔻精油中的活性成分与外界环境隔绝开来，可以降低豆蔻精油的挥发性，延长保存期。但由于脂质体的缓释作用，包裹精油的脂质体保质期有限。因此，选用酪蛋白制成稳定性更高的酪蛋白脂质体。此外，纳米脂质体由于它们的亚细胞尺寸，能加强脂质体的被动吸收机制，减少物质运输阻力，从而增强精油的抗菌效果。
发明内容
本发明的目的是公开一种酪蛋白-豆蔻精油脂质体的制备方法及其用途，通过将豆蔻精油包裹在酪蛋白脂质体中，以实现减少豆蔻精油在施用过程中的挥发，从而减少豆蔻精油的浪费，提高其利用率，达到长效抗菌与高效利用的目的。本发明由豆蔻精油，大豆卵磷脂，胆固醇和表面活性剂，酪蛋白制成酪蛋白-豆蔻精油脂质体。
本发明的制备方法是将豆蔻精油，大豆卵磷脂，胆固醇混合于有机溶剂，减压蒸发形成光滑的薄膜，加入水相介质溶解膜状物并超声成乳液，通过离心和微孔滤膜过滤，得到粒径为纳米级的豆蔻精油脂质体，其特征在于：将纳米级的豆蔻精油脂质体与酪蛋白和表面活性剂的溶液混合，搅拌均匀，通过低温冷冻缓慢融化的方法使酪蛋白镶嵌在脂质体上，通过温和超声使脂质体闭合，最终形成包裹豆蔻精油的酪蛋白脂质体。
本发明的大豆卵磷脂和胆固醇的质量比为5:1；用于制备精油脂质体的乳液含有豆蔻精油的浓度为4.0mg/mL；表面活性剂为十二烷基聚乙二醇醚(Brij-35)，得到包裹豆蔻精油的酪蛋白脂质体后其最终浓度为1.0mg/mL。
本发明中所述的有机溶剂为氯仿。
本发明中所用的水相介质是根据中国药典2000版标准配制的磷酸盐缓冲溶液，pH值6.5～7.4，优选7.2。
所述的酪蛋白和表面活性剂的溶液是将酪蛋白分散在Brij-35的磷酸盐缓冲液中得到的，Brij-35浓度为10mg/mL，分散后溶液中酪蛋白浓度为1.0mg/mL。
本发明中脂质体上镶嵌的蛋白为酪蛋白，豆蔻精油脂质体与酪蛋白和表面活性剂的溶液以体积比10:1混合，混合后溶液中酪蛋白浓度为0.1mg/mL，Brij-35浓度为1.0mg/mL。
本发明中低温冷冻缓慢融化是指将混合液在-18℃下冷冻2h，随后在0℃下缓慢融化2h。
本发明中温和超声是指在低功率50W条件下超声2h，温度范围0-10℃。
附图说明
图1酪蛋白-豆蔻精油脂质体的包封率。
图2酪蛋白-豆蔻精油脂质体的粒径与多分散系数(PDI)。
图3酪蛋白-豆蔻精油脂质体对大肠杆菌的抗菌效果。
图4酪蛋白-豆蔻精油脂质体对金黄色葡萄球菌的抗菌效果。
表1酪蛋白-豆蔻精油脂质体的Zeta电位。
具体实施方式
通过下面实例说明本发明的具体实施方式，但本发明的保护内容，不仅局限于此
实施例1酪蛋白-豆蔻精油脂质体的包封率
1实验材料
2实验方法
1)豆蔻精油脂质体的制备
①称取1g大豆卵磷脂，0.2g胆固醇和200mg的豆蔻精油，加入50mL氯仿使其溶解。
②在旋转蒸发仪中蒸发至溶剂蒸干，蒸发温度为10～30℃，圆底烧瓶内壁会形成光滑的薄膜；然后将所得产品放入真空干燥箱中，30℃，真空状态下干燥24h。
③加入50mL的磷酸盐缓冲液到圆底烧瓶中进行水化，水化超声波功率为100W，超声时间为5h。
④将所得混合液于细胞超微粉碎仪中以功率360W，工作10s间隙5s，粉碎30min。
⑤将所得产品进行离心，4000rpm，15min，取上层液体。
⑥将所得液体用0.22μm滤膜进行过滤，得滤液。
2)酪蛋白-豆蔻精油脂质体的制备
①按照上述豆蔻精油脂质体的制备方法，制备豆蔻精油浓度为4mg/mL的豆蔻精油脂质体。
②将酪蛋白超声分散在含10mg/mL Brij-35的磷酸盐缓冲液中，最终形成酪蛋白—Brij-35溶液，其中酪蛋白浓度为1.0mg/mL。
③在50mL豆蔻精油脂质体中添加5mL 1mg/mL的酪蛋白—Brij-35溶液，混合均匀。
④将所得混合液于超声条件下扩散10min。
⑤将所得混合液在-18℃下冷冻2h。
⑥将所得混合液在0℃下缓慢融化2h。
⑦通过温和超声(功率50W，2h，KQ-300DE，昆山市超声仪器有限公司)使脂质体闭合，所得溶液即为酪蛋白-豆蔻精油脂质体。
3)包封率的测定
用无水乙醇稀释豆蔻精油，逐级稀释成浓度分别为0.1、0.2、0.4、0.6、0.8mg/mL的标准溶液。然后，分别吸取1μL标准溶液进行GC-MS分析，对其主要成分的谱峰面积进行自动积分，绘制主成分峰面积-豆蔻精油浓度标准曲线。取1mL酪蛋白-豆蔻精油脂质体样品，13500rpm，离心3h，倒掉上清液。接着加入1mL乙醇破乳剂，超声3h。最后以10000rpm的转速离心15min，取上清液，用于GC-MS分析。制备好的脂质体样品，对精油主成分的峰面进行自动积分，再根据绘制的标准曲线，计算出脂质体中豆蔻精油的含量。则：
3酪蛋白-豆蔻精油脂质体的包封率
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1可看出，豆蔻精油脂质体的包封率为41.21％，酪蛋白-豆蔻精油脂质体的包封率为78.62％。因此制备酪蛋白-豆蔻精油脂质体可以明显提高脂质体的包封率。
实施例2酪蛋白-豆蔻精油脂质体的粒径与多分散系数(PDI)
1实验材料
①空白脂质体
②豆蔻精油脂质体
③酪蛋白-豆蔻精油脂质体
2实验方法
用美国布鲁克海文仪器公司产的，型号为BI-9000的高浓度激光粒度仪测定酪蛋白-豆蔻精油脂质体的粒径和多分散系数(PDI)值，所测样品放入样品池直接测量即可。
3酪蛋白-豆蔻精油脂质体的粒径与多分散系数(PDI)
多分散系数(PDI)直接反映酪蛋白-豆蔻精油脂质体的稳定性，因此是主要参考指标，脂质体的PDI在0～0.3范围内属于最好，在0.3～0.7范围内较差，但可以接受，当PDI>0.8时，不予考虑。如图2所示，空白脂质体的粒径为86.4nm、PDI为0.353，豆蔻精油脂质体的粒径为160.7nm、PDI为0.217，酪蛋白-豆蔻精油脂质体的粒径为601.5nm、PDI为0.019。酪蛋白-豆蔻精油脂质体的多分散系数(PDI)最小，因此制备酪蛋白-豆蔻精油脂质体可以明显提高脂质体的稳定性。
实施例3酪蛋白-豆蔻精油脂质体的Zeta电位
1实验材料
①空白脂质体
②豆蔻精油脂质体
③酪蛋白-豆蔻精油脂质体
2实验方法
用英国马尔文仪器有限公司生产的型号为Zetasirer nano ZSZeta的电位仪测量，直接将待测脂质体样品放入电位仪中测量即可。
3酪蛋白-豆蔻精油脂质体的Zeta电位
表1酪蛋白-豆蔻精油脂质体的Zeta电位
Zeta电位也可以直接反映酪蛋白-豆蔻精油脂质体的稳定性，因此也是主要参考指标，脂质体的Zeta电位的绝对值越大说明脂质体越稳定性，Zeta电位的绝对值在0～30范围内属于不稳定，在大于30时脂质体较稳定性。如表1所示，三种脂质体均带负电，空白脂质体的Zeta电位为-22.8mV，其绝对值小于30，脂质体不稳定；豆蔻精油脂质体的Zeta电位为-21.1mV，其绝对值小于30脂质体，不稳定；酪蛋白-豆蔻精油脂质体的Zeta电位为-38.2mV，其绝对值大于30且最大，脂质体最稳定。
实施例4酪蛋白-豆蔻精油脂质体的抗菌性能
1实验材料
①豆蔻精油脂质体(保存7天、30天、60天、90天)
②酪蛋白-豆蔻精油脂质体(保存7天、30天、60天、90天)
2实验方法
采用平板菌落计数法，以大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)为模式菌种测定酪蛋白-豆蔻精油脂质体的残存菌数。将大肠杆菌和金黄色葡萄球菌接种到液体培养基中，分别置于空气摇床中在37℃、150rpm的条件下震荡培养24～48h，获得对数生长期的菌悬液。取适量处于对数期的大肠杆菌和金黄色葡萄球菌分别加入含有一定量无菌磷酸盐缓冲液的试管中(细菌浓度约为105～106CFU/mL)，然后再向试管中加入浓度为20％(v/v)的豆蔻精油脂质体及酪蛋白-豆蔻精油脂质体。同时另取两个分别含有以上两种菌的试管并向其中加入等量无菌水(不加脂质体)作为对照。将各试管均置于空气摇床中在37℃、150rpm的条件下震荡反应24h。分别于不同时间点的取适量培养液进行十倍梯度稀释到合适的浓度，然后移取100μL稀释液滴到无菌固体平板培养基上，涂布均匀之后放入37℃恒温恒湿培养箱中倒置培养。24～48h后进行平板菌落计数，从而对豆蔻精油脂质体和酪蛋白-豆蔻精油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酪蛋白-豆蔻精油脂质体的抗菌性能
不同保存期的酪蛋白-豆蔻精油脂质体的抗菌活性的变化也可以间接反映脂质体的稳定性。因此对保存7天、30天、60天、90天的豆蔻精油脂质体及酪蛋白-豆蔻精油脂质体进行了抗菌性能评价，结果如图3、图4所示。保存时间为7天时，豆蔻精油脂质体与酪蛋白-豆蔻精油脂质体的抗菌活性相近，对大肠杆菌和金黄色葡萄球菌均显示了良好的抗菌效果。保存时间为30天时，豆蔻精油脂质体的抗菌效果明显降低，而酪蛋白-豆蔻精油脂质体仍然显示出良好的抗菌效果。保存时间为60天和90天时，豆蔻精油脂质体几乎没有显示抗菌效果，而酪蛋白-豆蔻精油脂质体仍然保持良好的抗菌效果。
Claims (3)
1. 一种酪蛋白-豆蔻精油脂质体抗菌剂的制备方法，将豆蔻精油，大豆卵磷脂，胆固醇混合于有机溶剂，减压蒸发形成光滑的薄膜，加入水相介质溶解膜状物并超声成乳液，通过离心和微孔滤膜过滤，得到粒径为纳米级的豆蔻精油脂质体，其特征在于：将纳米级的豆蔻精油脂质体与酪蛋白和表面活性剂的溶液混合，搅拌均匀，通过低温冷冻缓慢融化的方法使酪蛋白镶嵌在脂质体上，通过温和超声使脂质体闭合，最终形成包裹豆蔻精油的酪蛋白脂质体；低温冷冻缓慢融化是指将混合液在-18 ℃下冷冻2 h，随后在0 ℃下缓慢融化2 h；温和超声是指在低功率50 W条件下超声2 h，温度范围0-10 ℃；大豆卵磷脂和胆固醇的质量比为5:1；用于制备精油脂质体的乳液含有豆蔻精油的浓度为4.0 mg/mL；表面活性剂为十二烷基聚乙二醇醚（Brij-35），得到包裹豆蔻精油的酪蛋白脂质体后其最终浓度为1.0mg/mL；所用的水相介质是根据中国药典2000版标准配制的磷酸盐缓冲溶液，pH值6.5~7.4；所述的酪蛋白和表面活性剂的溶液是将酪蛋白分散在Brij-35的磷酸盐缓冲液中得到的，Brij-35浓度为10 mg/mL，分散后溶液中酪蛋白浓度为1.0 mg/mL；脂质体上镶嵌的蛋白为酪蛋白，豆蔻精油脂质体与酪蛋白和表面活性剂的溶液以体积比10:1混合，混合后溶液中酪蛋白浓度为0.1 mg/mL，Brij-35浓度为1.0 mg/mL。
2.如权利要求1所述的一种酪蛋白-豆蔻精油脂质体抗菌剂的制备方法，其特征在于：所述的有机溶剂为氯仿。
3.如权利要求1所述的一种酪蛋白-豆蔻精油脂质体抗菌剂的制备方法，其特征在于：所用的水相介质是根据中国药典2000版标准配制的磷酸盐缓冲溶液，pH值7.2。
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