CN114052258A - 一种鱼籽蛋白肽和枸杞黄酮组合物及其制备方法 - Google Patents
一种鱼籽蛋白肽和枸杞黄酮组合物及其制备方法 Download PDFInfo
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Abstract
本发明提供一种鱼籽蛋白肽和枸杞黄酮组合物及其制备方法;其中包被的芯材为鱼籽蛋白肽和枸杞黄酮,壁材为海藻酸钠和氯化钙溶液。本发明选用海藻酸钠和氯化钙为壁材,鱼籽蛋白肽和枸杞黄酮为芯材,蔗糖酯为乳化剂,以油脂包埋率为指标,通过实验将乳化剂添加量、芯壁材比、反应pH、包埋温度为因素,设计响应面试验,得到制备使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊的最佳制备工艺条件,并对制备的使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊包埋率和热稳定性进行检测,制备获得具有优异稳定性的微胶囊,能够延长微胶囊产品的货架期。
Description
技术领域
本发明属于天然产物应用技术领域,具体涉及一种鱼籽蛋白肽和枸杞黄酮组合物及其制备方法。
背景技术
枸杞是茄科枸杞属的多年生植物,分布于我国西北地区。枸杞叶俗称天精草,在传统中(藏)药中主要具有补肾生精、养肝、明目安神等作用,《本草纲目》中称枸杞叶“有除烦益志补五劳七伤,壮心气,除热毒,散疮肿,除风明目”之功效。研究表明枸杞具有抗衰老、抗炎、保护心脑血管、抗菌及抗病毒等作用,同时也是一种滋补强壮剂,具有很高的药用和食用价值。
枸杞黄酮是一类天然黄酮类化合物的混合物,其含有丰富的黄酮类、黄酮醇类、黄酮醇苷类、黄烷醇类等抗心脑血管疾病以及抗癌、抗氧化物质,因此枸杞黄酮具有较高的保护心脑血管、抗癌、抗氧化等多种生物活性。
申请人在之前的研究中制备了具有改善记忆的鱼籽蛋白肽,具有预防和改善记忆衰退的潜在功能;但所制备的多肽是混合物,在使用高浓度的时候会产生炎症,而且长期保存也存在问题。
发明内容
针对现有技术所存在的上述缺点,本发明的目的在于提供一种鱼籽蛋白肽和枸杞黄酮组合物及其制备方法。
本发明首先提供一种组合物,所述的组合物包含有鱼籽蛋白肽和枸杞黄酮;
所述的鱼籽蛋白肽,是将鱼的鱼籽匀浆液加入胰蛋白酶和胶原蛋白酶进行酶解,得到酶解液,离心取上清液;制备的上清液进行膜过滤,过滤液即为鱼籽蛋白肽溶液。
所述的酶解,是50℃条件下进行酶解,酶解时间为5h;
所述的膜过滤,是上清液用3500Da和360Da膜过滤;
所述的鱼籽蛋白肽,其一种多肽的氨基酸序列为LDYEFLLYMDNYEYEAPRF。
本发明所提供的组合物可作为微胶囊的芯材来使用;
本发明再一个方面提供微胶囊,所述的微胶囊的壁材为海藻酸钠和氯化钙,芯材为上述的组合物;
作为优选,其中壁材溶液中海藻酸钠和氯化钙的质量比为2:1;
本发明所提供的微胶囊,其一种制备方法如下:
1)乳化液的制备:将芯材鱼籽蛋白肽和枸杞黄酮充分混合溶解于壁材溶液中制成乳化液,调节乳化液的pH值和温度进行乳化;
所述的乳化液中芯材和壁材的质量比为1:2.76;
进一步的,所述的乳化液中添加有蔗糖酯;优选的蔗糖酯的添加量为乳化液总质量的1.45%;
更进一步的,乳化液中还添加入乳化液总质量1.5%的壳聚糖;
所述的调节乳化液pH值和温度,是调节pH值为2-5,温度为40-50℃;
优选的pH值为4.36,温度为44.73℃;
2)真空冷冻干燥处理:将乳化液预冷后放入冷冻干燥机中真空冷冻干燥完成微胶囊的制备。
本发明选用海藻酸钠和氯化钙为壁材,鱼籽蛋白肽和枸杞黄酮为芯材,蔗糖酯为乳化剂,以油脂包埋率为指标,通过实验将乳化剂添加量、芯壁材比、反应pH、包埋温度为因素,设计响应面试验,得到制备使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊的最佳制备工艺条件,并对制备的使用鱼籽蛋白肽枸杞黄酮作为芯材的微胶囊包埋率和热稳定性进行检测,制备获得具有优异稳定性的微胶囊,能够延长微胶囊产品的货架期。
附图说明
图1为本发明制备的鱼籽蛋白肽分子量分布图;
图2为反相高效液相色谱C18纯化肽段图。
具体实施方式
本发明选用海藻酸钠和氯化钙为壁材,以具有改善记忆的鱼籽蛋白肽和枸杞黄酮为芯材,蔗糖酯为乳化剂,制备的微胶囊具有改善记忆和枸杞黄酮其自身所带来的功能,且能够作为商品来长期保存。
下面结合实施例对本发明进行详细描述。
实施例1:制备枸杞黄酮
枸杞黄酮的制备方法采用已有的方法,其中一种制备方法的具体步骤如下:
1)将枸杞经人工挑拣,去除杂质后,在室内阴干,得到干燥的枸杞;
2)将枸杞放入粉碎机中进行粉碎,粉碎后过30目筛,得到枸杞粉;
3)将枸杞粉与质量浓度为70%的乙醇按1:4的料液体积比浸提,得到浸提液;
4)浸提液经转子转速为20000r/min的胶体磨研磨后,得到胶体状的枸杞浸提溶液;
5)将胶体状的枸杞浸提溶液放入高压均质机中,在10Mpa的压力下进行均质,15min后形成均一的枸杞浸提溶液;然后,采用离心机1000r/min的离心速度对均一的枸杞浸提溶液离心分离10min,得到上清液和滤渣,收集上清液,弃去滤渣;
6)将上清液以1.5t/h的流速经孔径为0.1μm的陶瓷膜在温度为15℃、压力为0.005Mpa的条件下除去悬浮物、鞣质及粘液质,收集渗透侧的溶液,得到滤液;
7)滤液经弱极性大孔吸附树脂吸附后,先用去离子水洗脱至无糖,再用2倍床层体积、质量浓度为30%的乙醇溶液洗脱吸附在大孔树脂上的枸杞黄酮,得到含有枸杞黄酮的洗脱液;
8)将含有枸杞黄酮的洗脱液在真空度为0.03Mpa、温度为30℃的条件下经真空减压浓缩后,得到枸杞黄酮浓缩液;该枸杞黄酮浓缩液在50℃温度下干燥后,即得枸杞黄酮粉末。
其中枸杞黄酮还可以使用其它市售的产品。
实施例2:功能肽的制备
1、制备功能肽的混合多肽
称取黄鱼干鱼籽100g,加入1000ml蒸馏水,利用高速剪切机剪切匀浆30min,高速剪切机转速为10000rpm,向上述匀浆液中加入3.0g胰蛋白酶和胶原蛋白酶(酶活比1:10),50℃酶解5h,边酶解边搅拌,酶解结束后加热至90℃灭酶,酶解液8000rpm离心后,上清液用3500Da过滤,过滤液再通过360Da膜过滤,截留液经浓缩至原体积的1/10,喷雾干燥得到49.8g鱼籽蛋白肽。
根据国标(GB/T 22729-2008)方法测定制备鱼籽蛋白肽的分子量,结果显示分子量小于2000Da的鱼籽蛋白肽占87.52%(图1)。
采用东莨菪碱致记忆障碍大鼠作为实验动物,水迷宫实验结果显示,使用制备的鱼籽蛋白肽的治疗组能够提高东莨菪碱致记忆障碍大鼠的记忆能力。
2、功能肽的纯化
1)将鱼籽蛋白肽用Sephadex G-25凝胶柱进行分离纯化,洗脱液选用去离子水,洗脱速度为0.4ml/min,在220nm下收集洗脱峰。共分离到五个分离峰A-E,分别测定活性,收集活性最高的组分备用。
测定结果表明在相同蛋白浓度下峰D的活性最大,将峰D进行浓缩,进一步经过Sephadex C-25阳离子交换柱进行纯化。
2)峰D经过Sephadex C-25阳离子交换柱进行纯化,得到四个峰,其中第二个峰的水迷宫实验结果表明其具有最大的活性。
3)将第二个峰的样品进行脱盐,通过反相高效液相色谱C18半制备柱继续分离纯化,得到了活性肽P1(图2);检测其IC50值为0.0623mg/ml。
纯化后的P1多肽使用电喷雾四级杆正交加速-飞行时间串联质谱仪进行测序,Q_TOF2配备钠升喷雾源。测定在离子方式下进行,进样流速为1μl/min,质谱仪Glu_fib串联质谱碎片校正。使用氮气作为雾化气体,氩气作为碰撞气体,检测温度为80℃,锥孔电压50V,毛细管电压3000v,TOF加速电压为9.1kv,MCP检测器电压为2200V。
采用Micromass的转用软件MaxEnt3,Biolynx及MasSeq对MS/MS谱进行序列分析;确定P1肽段的序列为LDYEFLLYMDNYEYEAPRF,其分子量为1800Da。
对所分离纯化的序列为LDYEFLLYMDNYEYEAPRF的多肽进行东莨菪碱致记忆障碍大鼠(模型)逃避潜伏期和跨原平台次数的检测。水迷宫实验结果显示,在逃避潜伏期(s)上,投喂P1肽(投喂量50mg/kg大鼠)的实验组(16.3土2.4)小于模型组(20.1土2.1)。在跨越平台跨越平台数(次)上,投喂P1肽的实验组(10.4土3.1)的次数也远高于模型组(6.7土1.8)。
实施例3:以枸杞黄酮和鱼籽蛋白肽作为芯材来制备微胶囊
(1)乳化液的制备
将海藻酸钠和氯化钙分别配成一定浓度溶液,待充分溶解混合,再添加一定量的壳聚糖,搅拌至均匀,以此溶液作为反应液;将鱼籽蛋白肽和枸杞黄酮浆液缓慢滴加到反应液中,用盐酸调节pH,采用超声波处理,形成乳化液。
(2)乳化液喷雾干燥处理
将乳化液倒入塑料培养皿中,-45℃预冷后放入冷冻干燥机中冷冻干燥16h后取出。
(3)微胶囊包埋率的测定
包埋率是指微胶囊产品中被包埋的鱼籽蛋白肽和枸杞黄酮浆液含量与包埋时加入的鱼籽蛋白肽和枸杞黄酮浆液的总量的比值。
包埋率越高则芯材被包埋的量越大,效果越好。
式中:W1:微胶囊鱼籽蛋白肽和枸杞黄酮浆液总质量;W2:微胶囊表面鱼籽蛋白肽和枸杞黄酮浆液质量。
(4)微胶囊最佳工艺的确定
1)芯材与壁材比例的筛选
在反应pH值为3.0、反应温度为40℃条件下,设定芯材和壁材比例分别为1:1、1:2、1:3、1:4、1:5进行试验,测定微胶囊包埋率。以对其芯材和壁材的比例进行初步筛选。
2)反应pH值的筛选
在芯材和壁材比例为1:3、反应温度为40℃条件下,设定反应pH值分别为2.0、3.0、4.0、5.0、6.0进行试验,测定微胶囊包埋率。以对其反应pH值进行初步筛选。
3)反应温度的筛选
在芯材和壁材比例为1:3、反应pH值为3.0条件下,设定反应温度分别为10℃、20℃、30℃、40℃、50℃进行试验,测定微胶囊包埋率。以对其反应温度进行初步筛选。
结果确定芯材壁材比为1:2.76,反应pH值为4.36,反应温度44.73℃,在此条件下,微胶囊包埋率为60.13%。
实施例4:使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊制备工艺对比试验
按照实施例1的步骤进行乳状液的制备,选择果胶和卡拉胶作为包被壁材,其中芯材壁材比例为1:5、反应pH值为5.0、反应温度为60℃,添加乳化剂蔗糖酯,超声波处理,形成乳化液。将乳化液进行冷冻干燥处理,进行微胶囊包埋率的测定。经过5次重复试验,得到的微胶囊产品包埋率为53.32%、54.12%、54.28%、53.43%、53.91%,均低于实施例1中60.13%的包埋率,表明果胶和卡拉胶作为壁材的包被效果比海藻酸钠和氯化钙作为壁材的包被效果差。
实施例5:使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊热稳定性对比试验
准确称取5g实施例1制备的微胶囊产品和鱼籽蛋白肽和枸杞黄酮浆液,放置于100℃烘箱中分别加热1、2、3、4、5h后,称量各样品质量减少量,并作对比。结果显示,在整个过程中微胶囊产品和鱼籽蛋白肽和枸杞黄酮浆液减少量均在上升,但是鱼籽蛋白肽和枸杞黄酮浆液的减少量始终高于微胶囊产品,而且微胶囊产品在4h后得减少量已经相对微小,而鱼籽蛋白肽和枸杞黄酮浆液的减少量始终在上升。这说明微胶囊产品热稳定性高于鱼籽蛋白肽和枸杞黄酮浆液,即产品具有良好的热稳定性。
经过对微胶囊产品进行热稳定性测定,表明微胶囊化后鱼籽蛋白肽和枸杞黄酮的组合物稳定性优于未微胶囊化的鱼籽蛋白肽和枸杞黄酮的组合物。因此,按照本发明的方法制备的微胶囊产品具有良好地热稳定性。
本发明选用海藻酸钠和氯化钙为壁材,鱼籽蛋白肽和枸杞黄酮为芯材,蔗糖酯为乳化剂,以油脂包埋率为指标,通过实验将乳化剂添加量、芯壁材比、反应pH、包埋温度为因素,设计响应面试验,得到制备使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊的最佳制备工艺条件,并对制备的使用鱼籽蛋白肽和枸杞黄酮作为芯材的微胶囊包埋率和热稳定性进行检测,制备获得具有优异稳定性的微胶囊,能够延长微胶囊产品的货架期,具有广阔的应用前景,值得推广。
Claims (10)
1.一种组合物,其特征在于,所述的组合物包含有鱼籽蛋白肽和枸杞黄酮;所述的鱼籽蛋白肽,是将鱼的鱼籽匀浆液加入胰蛋白酶和胶原蛋白酶进行酶解,得到酶解液,离心取上清液;制备的上清液进行膜过滤,过滤液即为鱼籽蛋白肽溶液。
2.如权利要求1所述的组合物,其特征在于,所述的酶解,是50℃条件下进行酶解,酶解时间为5h。
3.如权利要求1所述的组合物,其特征在于,所述的膜过滤,是先后用3500Da和360Da的膜过滤。
4.权利要求1所述的组合物,其特征在于,所述的鱼籽蛋白肽,其一种多肽的氨基酸序列为LDYEFLLYMDNYEYEAPRF。
5.权利要求1-4任一项所述的组合物作为微胶囊的芯材的应用。
6.一种微胶囊,其特征在于,所述的微胶囊的壁材为海藻酸钠和氯化钙,芯材为权利要求1-4任一项所述的组合物。
7.如权利要求6所述的微胶囊,其特征在于,所述壁材中海藻酸钠和氯化钙的质量比为2:1。
8.如权利要求6或7所述的微胶囊,其特征在于,所述的微胶囊的制备方法如下:
1)乳化液的制备:将芯材鱼籽蛋白肽和枸杞黄酮充分混合溶解于壁材溶液中制成乳化液,调节乳化液的pH值和温度进行乳化;
2)真空冷冻干燥处理:将乳化液预冷后放入冷冻干燥机中真空冷冻干燥完成微胶囊的制备。
9.如权利要求8所述的微胶囊,其特征在于,所述的乳化液中添加有蔗糖酯和壳聚糖,其中蔗糖酯的添加量为乳化液总质量的1.45%;壳聚糖的添加量为乳化液总质量的1.5%。
10.如权利要求8所述的微胶囊,其特征在于,所述的调节乳化液pH值和温度,是调节pH值为2-5,温度为40-50℃。
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