CN108741080B - 一种微藻dha-花青素双相纳米脂质体及其制备方法 - Google Patents
一种微藻dha-花青素双相纳米脂质体及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种微藻DHA‑花青素双相纳米脂质体及其制备方法。所述双相纳米脂质体主要由大豆磷脂、微藻DHA、花青素和十八胺组成，以柠檬酸‑柠檬酸钠体系为缓冲液；以占脂质体总质量比计，所述各组分含量：大豆磷脂4‑10mg/mL，微藻DHA2‑5mg/mL，花青素1.33‑3.33mg/mL，十八胺0.66‑1.67mg/mL，柠檬酸‑柠檬酸钠缓冲液200‑300mmol/L。其制备方法主要包括以下步骤：(1)原料溶解；(2)制膜；(3)制备花青素水相溶液；(4)洗膜；(5)高速剪切；(6)高压微射流均质；(7)将得到的微藻DHA‑花青素双相纳米脂质体进行储存、检验。本发明双相纳米脂质体可提高DHA和花青素的稳定性，改善口感、增加生物利用度；同时具有粒径均匀、包封率较高、zeta电位绝对值较大等优点。
Description
技术领域
本发明属于食品技术领域，特别是涉及一种微藻DHA-花青素双相纳米脂质体及其制备方法。
背景技术
花青素，属酚类化合物中的类黄酮，为水溶性色素，广泛存在于植物花瓣、果实的组织中及茎叶的表面细胞与下表皮层，具有强抗氧化，抗癌症和防辐射的作用，亦扮演益生元角色调节大肠生物菌群比例，是一种广为人知的保健品和护肤品成分。DHA，二十二碳六烯酸，可促进幼儿大脑发育、预防肠道菌群失调、降低肥胖和慢性炎症几率，是幼儿饮食中极需强化的脂溶性营养素，人体脑细胞中大量存在DHA，在神经原表层高度富集，约占脑细胞脂肪酸的10％，亦有临床证据表明血浆中含较多DHA的孕妇生出的胎儿中枢神经系统成熟较快。
然而，花青素易受光、氧、酶和高温的破坏，直接作为营养强化剂难度大，目前仅有少数企业直接销售花青素产品；DHA腥味重、溶解性差、极易氧化，致使营养价值与产品货架期大幅下降，应用范围严重受限。脂质体是一种具有双分子层结构的封闭囊泡，是一种人工膜，在自组装过程中可同时包埋亲脂性 (双层膜之间)和亲水性(中心)功能营养物质。自1965年被英国剑桥大学 Bangham教授团队发现以来，脂质体在医药、化妆品和基因工程等领域有了广泛应用，目前已有超过18种包埋了不同抗癌药物的脂质体产品经过了美国FDA批准上市。
动态高压微射流(dynamic high-pressure microfluidization,DHPM)是一种高压均质技术，它利用液压泵使流体产生高压，实现高速撞击、高频剪切、气蚀、高频振动、瞬时压降等综合作用，在100MPa下，时间小于5s即可达到使物料细化、乳化、均质和改性等目的。本发明将传统的薄膜分散法与DHPM技术相结合，用于制备微藻DHA-花青素纳米脂质体，既可克服微藻DHA、花青素各自的弱点，避免使用大量有毒试剂，提高脂质体安全性，又可在一定程度上减小脂质体粒径且使颗粒分散均匀，提高营养素包封率，适用于大规模连续化生产。
中国专利申请201510787539.0公开了一种靶向脂质体包裹水相纳米金复合物的制备方法，将氢化大豆磷脂，胆固醇，P-DSPE混合，加入氯仿溶解于圆底烧瓶中，然后旋转蒸发，至瓶壁上形成一层膜，放入真空干燥箱过夜，将纳米金溶液超滤离心浓缩，逐步加入硫酸铵溶液((NH4)2SO4)，然后将该含有纳米金的硫酸铵溶液加入到上述圆底烧瓶中，置于恒温水浴摇床上进行脂质体对纳米金的包裹，超声半小时后，过层析柱葡聚糖凝胶，收集脂质体层析液得到脂质体包裹纳米金的微球。
中国专利申请201711175784.1公开了一种柔性脂质体的制备方法，定量称取蛋黄卵磷脂、胆固醇、胆酸钠和维生素E，加入到圆底烧瓶中，添加氯仿- 甲醇复合溶剂使其溶解，负压旋转蒸发清除有机溶剂，直至烧瓶壁出现暗黄色均匀薄膜；加入聚乙二醇的磷酸盐缓冲液，旋转洗膜；在冰水浴中适度超声，使用0.22微米微孔滤膜滤除大颗粒；取少量维生素E溶取脂质体原液，使用超速离心将脂质体从混悬液体系中分离，使用微孔滤膜过滤，去除清液后即得柔性脂质体。
中国专利申请CN201710272152.0公开了一种含有植物提取物复合多肽脂质体组合物及其制备方法和在祛皱产品的应用，将脂质和溶剂混合均匀，形成脂质溶液；将植物提取物、复合小分子活性多肽、乳化剂加入缓冲液中，溶解完全，加热搅拌下加入脂质溶液，并持续搅拌，冰水浴下超声处理，过微孔滤膜，得到含有植物提取复合多肽脂质体组合物。
中国专利申请CN201710272152.0公开了脂质体制剂及制造，将含有治疗性试剂的溶液和含有脂质组分的溶液混合以形成囊泡；在单阶段通过单尺寸设计过滤器挤出所述囊泡，超滤。
上述的脂质体制备方法都存在这样的缺陷：包埋的内容物单一，也就决定了相关脂质体的功能单一，一些复合脂质体也只是在脂质体中心的水相中加入多种水溶性物质；另外，未经过均质处理的脂质体，往往颗粒大小不均匀，长期放置易发生聚集沉降，被包埋物释放加剧，严重影响脂质体寿命。
发明内容
本发明的目的在于提供一种微藻DHA-花青素双相纳米脂质体及其制备方法。本发明同时包埋油-水双相功能分子的脂质体，并且经过高压微射流处理得到了稳定的油-水双相脂质体，以解决现有脂质体普遍存在功能单一、颗粒大小不均匀、稳定性低等问题。
为了达到上述的目的，本发明采取以下技术方案：
一种微藻DHA-花青素双相纳米脂质体，所述双相脂质体主要由大豆磷脂、微藻DHA、花青素和十八胺组成，以柠檬酸-柠檬酸钠体系为缓冲液；以占脂质体总质量比计，所述各组分含量：大豆磷脂4-10mg/mL，微藻DHA 2-5 mg/mL，花青素1.33-3.33mg/mL，十八胺0.66-1.67mg/mL，柠檬酸-柠檬酸钠缓冲液200-300mmol/L。
优选的，以占脂质体总质量比计，所述各组分含量：大豆磷脂6-8mg/mL，微藻DHA3-4mg/mL，花青素2-2.67mg/mL，十八胺1-1.33mg/mL，柠檬酸- 柠檬酸钠缓冲液200-300mmol/L。
优选的，所述柠檬酸-柠檬酸钠缓冲液pH为3.0-4.0；更优选的，柠檬酸- 柠檬酸钠缓冲液pH为3.3-3.8。
一般脂质体采用PBS作为缓冲溶液，缓冲pH较高，而花青素在较高pH 下不稳定，易被氧化，本发明采用柠檬酸-柠檬酸钠缓冲液可以达到较低缓冲 pH，能够有效减少花青素被氧化。
本发明还提供了上述微藻DHA-花青素双相纳米脂质体的制备方法，包括如下步骤：
(1)原料溶解：将大豆磷脂、微藻DHA、十八胺混合，加入溶剂无水乙醇，搅拌至充分溶解；
(2)制膜：将步骤(1)处理后的溶液加入旋转蒸发仪中，抽真空去溶剂，容器内壁附着一层脂溶性混合物；
(3)花青素水相溶液：将花青素溶解于柠檬酸-柠檬酸钠缓冲溶液，然后用0.22μm孔径的微孔滤膜过滤以除去不溶性杂质；
(4)洗膜：将步骤(3)制得的花青素溶液加入步骤(2)得到的脂溶性混合物中进行洗膜，直至容器内壁附着物被完全洗下，获得粗脂质体；
(5)高速剪切：将步骤(4)制得的粗脂质体用高速剪切机剪切5-10min；
(6)高压微射流均质：将步骤(5)处理后的粗脂质体加入高压微射流均质机进行二次均质，得到微藻DHA-花青素双相纳米脂质体；
(7)将步骤(6)得到的微藻DHA-花青素双相纳米脂质体进行储存、检验。
优选的，所述步骤(2)旋蒸速度为30-60rpm，更优选为35-50rpm；旋蒸温度为45-60℃，更优选为40-55℃。
优选的，所述步骤(2)旋转蒸发仪抽真空处理时间为10-20min。
优选的，所述步骤(4)洗膜时间为60-120min。
优选的，所述步骤(6)高压微射流均质压力为110-130MPa，更优选为 115-125MPa。
本发明的原理是：本发明采用薄膜分散-动态高压微射流法制备了纳米级脂质体，同时包埋了微藻DHA-花青素油-水双相营养素。将DHA溶解于磷脂等油相溶液并在抽真空条件下获得脂质薄膜，花青素溶解在柠檬酸-柠檬酸钠水相溶液中，加入脂质薄膜进行洗膜，此过程磷脂分子自发组装形成双分子层闭合囊泡结构，DHA嵌入磷脂双分子层、花青素则被包埋于水相中心成为芯材。制得的粗脂质体经过高速剪切机预处理，再经过动态高压微射流均质，获得粒度分布均匀的纳米级脂质体，大大提高脂质体稳定性和包埋效率。
本发明具有以下技术特点：
1)本发明选择合适的原材料相配合，通过改良的生产工艺，制备得到的脂质体能够同时包埋油-水双相物质，使得脂质体能够同时搭载不同极性营养物质。
2)本发明油-水双相脂质体同时包埋两种营养素，在提高DHA和花青素稳定性的同时，亦可改善口感、增加生物利用度，具有实际应用价值。
3)本发明微藻DHA-花青素双相纳米脂质体具有粒径均匀、包封率高、稳定性高等优点。本发明制备的纳米脂质体平均粒度为120-140nm，分散系数小于0.30，zeta电位为15-25mV，花青素包封率为45-55％，DHA包封率为35-45％。
附图说明
图1是本发明的工艺流程图；
图2是本发明薄膜分散-动态高压微射流技术制备微藻DHA-花青素双相纳米脂质体一较佳实施例(实施例1)的透射电镜图；
图3是具体实施方式中实施例1-3制备得到的微藻DHA-花青素双相纳米脂质体的粒径(a)以及zeta电位(b)数据；
图4是薄膜分散-动态高压微射流技术制备微藻DHA-花青素双相纳米脂质体及其对照脂质体的粒径(a)以及zeta电位(b)数据；
具体实施方式
以下具体实施例是对本发明提供的方法与技术方案的进一步说明，但不应理解成对本发明的限制。
本发明公开了薄膜分散-动态高压微射流技术制备微藻DHA-花青素双相纳米脂质体，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述微藻DHA-花青素双相纳米脂质体已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
一种微藻DHA-花青素双相纳米脂质体，所述双相脂质体主要由大豆磷脂、微藻DHA、花青素和十八胺组成，以柠檬酸-柠檬酸钠体系为缓冲液；以占脂质体总质量比计，所述各组分含量：大豆磷脂4-10mg/mL，微藻DHA 2-5 mg/mL，花青素1.33-3.33mg/mL，十八胺0.66-1.67mg/mL，柠檬酸-柠檬酸钠缓冲液200-300mmol/L。所述柠檬酸-柠檬酸钠缓冲液pH为3.0-4.0。
上述微藻DHA-花青素双相纳米脂质体的制备方法，如图1所示，包括如下步骤：
(1)原料溶解：将大豆磷脂、微藻DHA、十八胺混合，加入溶剂无水乙醇，搅拌至充分溶解；
(2)制膜：将步骤(1)处理后的溶液加入旋转蒸发仪中，抽真空去溶剂，容器内壁附着一层脂溶性混合物；
(3)花青素水相溶液：将花青素溶解于柠檬酸-柠檬酸钠缓冲溶液，然后用0.22μm孔径的微孔滤膜过滤以除去不溶性杂质；
(4)洗膜：将步骤(3)制得的花青素溶液加入步骤(2)得到的脂溶性混合物中进行洗膜，直至容器内壁附着物被完全洗下，获得粗脂质体；
(5)高速剪切：将步骤(4)制得的粗脂质体用高速剪切机剪切5-10min；
(6)高压微射流均质：将步骤(5)处理后的粗脂质体加入高压微射流均质机进行二次均质，得到微藻DHA-花青素双相纳米脂质体；
(7)将步骤(6)得到的微藻DHA-花青素双相纳米脂质体进行储存、检验。
下面结合实施例，进一步阐述本发明。
实施例1：
原料溶解：将大豆磷脂、微藻DHA、十八胺按照6:3:1质量比混合，大豆磷脂浓度为8mg/mL，共制备12mL，加入12mL无水乙醇，搅拌至充分溶解；
制膜：将上述步骤处理后的溶液加入旋转蒸发仪，于55℃，40rpm条件下抽真空处理13min至圆底烧瓶内污水乙醇完全蒸发，此时瓶内壁上均匀附着有一层磷脂及微藻DHA等脂溶性的混合物；
花青素水相溶液：将花青素溶解于250mmol/L的柠檬酸-柠檬酸钠缓冲液，用0.22μm孔径的微孔滤膜过滤花青素溶液以除去不溶性杂质；
洗膜：将制得的花青素溶液加入旋转蒸发仪的圆底烧瓶内进行洗膜，条件为50℃，40rpm,时间为60min，直至圆底烧瓶内壁附着物被完全洗下，获得粗脂质体；
高速剪切：将制得的粗脂质体溶液用高速剪切机剪切5min；
高压微射流均质：将处理后的脂质体加入高压微射流均质机以120MPa压力进行二次均质；
将得到的纳米双相脂质体进行储存、检验。得到的纳米双相脂质体的TEM 图如图2所示。通过粒径和zeta电位检测，如图3所示，所得微藻DHA-花青素双相纳米脂质体粒径为126.8nm，zeta电位为22.4mV，花青素包封率为 53.2％，DHA包封率为41.2％。
实施例2：
原料溶解：将大豆磷脂、微藻DHA、十八胺按照6:3:1质量比混合，大豆磷脂浓度为8mg/mL，共制备30mL，加入30mL无水乙醇，搅拌至充分溶解；
制膜：将处理后的溶液加入旋转蒸发仪，于55℃，40rpm条件下抽真空处理13min至圆底烧瓶内乙醇完全蒸发，此时瓶内壁上均匀附着有一层磷脂及微藻DHA等脂溶性的混合物；
花青素水相溶液：将花青素溶解于200mmol/L的柠檬酸-柠檬酸钠缓冲液，用0.22μm孔径的微孔滤膜过滤花青素溶液以除去不溶性杂质；
洗膜：将制得的花青素溶液加入旋转蒸发仪的圆底烧瓶内进行洗膜，条件为45℃，40rpm,时间为60min，直至圆底烧瓶内壁附着物被完全洗下，获得粗脂质体；
高速剪切：将制得的粗脂质体溶液用高速剪切机剪切5min；
高压微射流均质：在处理后的脂质体加入高压均质机以120MPa压力进行二次均质；
将得到的纳米双相脂质体进行储存、检验。通过粒径和zeta电位检测，如图3所示，所得微藻DHA-花青素双相纳米脂质体粒径为127.3nm，zeta电位为19.4mV，花青素包封率为48.7％，DHA包封率为39.8％。
实施例3：
原料溶解：将大豆磷脂、微藻DHA、十八胺按照6:3:1质量比混合，大豆磷脂浓度为8mg/mL，共制备20mL，加入20mL无水乙醇，搅拌至充分溶解；
制膜：将处理后的溶液加入旋转蒸发仪，于50℃，40rpm条件下抽真空处理15min至圆底烧瓶内乙醇完全蒸发，此时瓶内壁上均匀附着有一层磷脂及微藻DHA等脂溶性的混合物；
花青素水相溶液：将花青素溶解于300mmol/L的柠檬酸-柠檬酸钠缓冲液，用0.22μm孔径的微孔滤膜过滤花青素溶液以除去不溶性杂质；
洗膜：将制得的花青素溶液加入旋转蒸发仪的圆底烧瓶内进行洗膜，条件为55℃，40rpm,时间为60min，直至圆底烧瓶内壁附着物被完全洗下；
高速剪切：将制得的粗脂质体溶液用高速剪切机剪切5min；
高压微射流均质：将处理后的脂质体加入高压均质机以120MPa压力进行二次均质；
将得到的纳米双相脂质体进行储存、检验。通过粒径和zeta电位检测，如图3所示，所得微藻DHA-花青素双相纳米脂质体粒径为125.6nm，zeta电位为24.9mV，花青素包封率为49.8％，DHA包封率为43.5％。
实施例4：
按照实施例1中方法，保持其他条件不变，分别不添加微藻DHA、不添加花青素、不添加DHA和花青素，由此制得对照的花青素脂质体、DHA脂质体和空白脂质体。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.一种微藻DHA-花青素双相纳米脂质体的制备方法，包括如下步骤：
(1)原料溶解：将大豆磷脂、微藻DHA、十八胺混合，加入溶剂无水乙醇，搅拌至充分溶解；
(2)制膜：将步骤(1)处理后的溶液加入旋转蒸发仪中，抽真空去溶剂，容器内壁附着一层脂溶性混合物；
(3)花青素水相溶液：将花青素溶解于柠檬酸-柠檬酸钠缓冲溶液，然后用0.22μm孔径的微孔滤膜过滤以除去不溶性杂质；
(4)洗膜：将步骤(3)制得的花青素溶液加入步骤(2)得到的脂溶性混合物中进行洗膜，直至容器内壁附着物被完全洗下，获得粗脂质体；
(5)高速剪切：将步骤(4)制得的粗脂质体用高速剪切机剪切5-10min；
(6)高压微射流均质：将步骤(5)处理后的粗脂质体加入高压微射流均质机进行二次均质，得到微藻DHA-花青素双相纳米脂质体；
(7)将步骤(6)得到的微藻DHA-花青素双相纳米脂质体进行储存、检验；
其中，所述双相脂质体主要由大豆磷脂、微藻DHA、花青素和十八胺制成，以柠檬酸-柠檬酸钠溶液为缓冲液；以占脂质体总质量比计，所述各组分含量：大豆磷脂4-10mg/mL，微藻DHA 2-5mg/mL，花青素1.33-3.33mg/mL，十八胺0.66-1.67mg/mL，余量为柠檬酸-柠檬酸钠缓冲液；柠檬酸-柠檬酸钠缓冲液的浓度为200-300mmol/L。
2.根据权利要求1所述的制备方法，其特征在于，所述步骤(2)旋蒸速度为30-60rpm，旋蒸温度为45-60℃。
3.根据权利要求1所述的制备方法，其特征在于，所述步骤(2)旋转蒸发仪抽真空处理时间为10-20min。
4.根据权利要求1所述的制备方法，其特征在于，所述步骤(4)洗膜时间为60-120min。
5.根据权利要求1所述的制备方法，其特征在于，所述步骤(6)高压微射流均质压力为110-130MPa。
6.根据权利要求1所述的制备方法，其特征在于，以占脂质体总质量比计，所述各组分含量：大豆磷脂6-8mg/mL，微藻DHA3-4mg/mL，花青素2-2.67mg/mL，十八胺1-1.33mg/mL，余量为柠檬酸-柠檬酸钠缓冲液；柠檬酸-柠檬酸钠缓冲液的浓度为200-300mmol/L。
7.根据权利要求1所述的制备方法，其特征在于，所述柠檬酸-柠檬酸钠缓冲液pH为3.0-4.0。
8.根据权利要求3所述的制备方法，其特征在于，所述柠檬酸-柠檬酸钠缓冲液pH为3.3-3.8。
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