CN114098075A - 一种基于菠萝蜜的具有免疫活性的复合制剂及其应用 - Google Patents
一种基于菠萝蜜的具有免疫活性的复合制剂及其应用 Download PDFInfo
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- CN114098075A CN114098075A CN202111320393.0A CN202111320393A CN114098075A CN 114098075 A CN114098075 A CN 114098075A CN 202111320393 A CN202111320393 A CN 202111320393A CN 114098075 A CN114098075 A CN 114098075A
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- enzymolysis
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Abstract
本发明公开了一种基于菠萝蜜的具有免疫活性的复合制剂及其应用,属于菠萝蜜深加工技术领域。且所述复合制剂包括免疫活性提取物、助剂。本发明以菠萝蜜为来源,提取了免疫活性提取物,并将该免疫活性提取物与助剂进行复合,形成复合制剂,利用助剂对免疫活性提取物中的多肽、蛋白质、多糖进行负载,一方面避免活性物质的活性降低,另一方面,提高免疫活性提取物的加工性能,所述免疫活性提取物含有的多肽分子量为180‑2000Da,蛋白质含量为10.0%‑36.0%,多糖含量为30%‑70%,且该免疫活性提取物的淋巴细胞转化值为0.40‑0.60,NK细胞活性为40%‑65%。
Description
技术领域
本发明属于菠萝蜜深加工技术领域,具体地,涉及一种基于菠萝蜜的具有免疫活性的复合制剂及其应用。
背景技术
菠萝蜜(学名:Artocarpus heterophyllus Lam),又名木菠萝、树菠萝等,属桑科菠萝蜜属常绿乔木,为海南主要农作物之一。菠萝蜜果肉中含有丰富的糖类、蛋白质、维生素、矿物质等,味道爽口香甜。菠萝蜜种子内部含有大量的碳水化合物和蛋白,其质量约占到菠萝蜜的10%左右,可以炒食或煮食。
目前对菠萝蜜果肉的研究主要集中菠萝蜜水果片、蜜饯、果酒、菠萝蜜种子糕点等的制作工艺研究,而对于菠萝蜜活性物质的研究与开发主要集中在多糖、果胶、黄酮和多酚类物质上。有研究在菠萝蜜中提取到具有免疫活性的多糖。上述物质的提取率低,以及相关具有免疫活性的复合制剂研究较少,菠萝蜜没有得到充分有效利用。
因此,以菠萝蜜为基础,提取制备具有免疫活性的混合物质,进而评价其体内相关免疫活性,对食物来源免疫活性物质的相关研究意义重大,同时为提高菠萝蜜产品附加值,开发辅助提高免疫力产品,奠定了研究基础,并产生较好的经济影响。
发明内容
本发明的目的在于提供一种基于菠萝蜜的具有免疫活性的复合制剂及其应用,以解决上述背景提到的问题。
本发明的目的可以通过以下技术方案实现:
一种基于菠萝蜜的具有免疫活性的复合制剂,包括免疫活性提取物、助剂。
进一步地,所述免疫活性提取物含有蛋白质和多糖。
进一步地,按质量百分比计算,所述免疫活性提取物中蛋白质占干物质的含量为10.0%-36.0%,多糖占干物质的含量为30%-70%。
进一步地,所述蛋白质中分子量为180-2000Da的多肽质量分数为60-90%。
进一步地,采用淋巴细胞转化法测定上述免疫活性提取物的活性指标显示,淋巴细胞转化值为0.40-0.60。
进一步地,采用NK细胞活性测定法测定上述免疫活性提取物的活性指标显示,NK细胞活性为45-65%。
进一步地,所述免疫活性提取物通过以下步骤制成:
1)原料预处理:将菠萝蜜果肉在室温条件下破碎,按照菠萝蜜果肉与水的比例为1-5kg:10-15L进行混合,90℃老化10-30min冷却至50℃以下;再用胶体磨匀浆2-15min,得到菠萝蜜匀浆;
2)复合酶可控酶解:将菠萝蜜匀浆置于反应罐中,启动搅拌器将转速设置为20-30r/min,保持30-50℃恒温和搅拌状态,用8.0-10.0mol/L的氢氧化钠溶液调整反应罐中料液pH为3.5-5.5;投入第一次酶解复合酶,进行第一次酶解,然后升温至90-95℃,保温灭酶10-30min,冷却至50-60℃,用3-6mol/L的盐酸溶液调整反应罐中料液的pH值为7.0-9.0,投入第二次酶解复合酶,酶解结束后,将反应罐中酶解液迅速升温至90-95℃,保温灭酶10-30min,冷却,得到酶解混悬液;
3)二次加热澄清:将酶解混悬液过80目网筛,得透过液,将冷却至20℃以下的透过液再次升温至80-90℃后迅速冷却至40℃以下,过100目网筛去除絮凝物,得精滤液;
4)减压浓缩:将精滤液置于真空旋转蒸发仪中,控制转速为1000-1500rpm,温度45-65℃,浓缩至初始体积的1/2~1/6,得免疫活性提取物。
进一步地,步骤1)中菠萝蜜果肉与水的比例为1-5kg∶10L,更优选地为2-3kg∶10L。
进一步地,步骤2)中第一次酶解复合酶为果胶酶与纤维素酶按照质量比为1∶2-10混合组成,第一次酶解复合酶的加入质量为萝蜜肉重的0.2-1.0%。
进一步地,步骤2)中第一次酶解的酶解条件:时间为1.5-5h,温度为35-45℃,酶解pH为3.8-4.2;优选地酶解时间为1.5-3h。
进一步地,步骤2)中第二次酶解复合酶为胰蛋白酶和木瓜蛋白酶按照质量比为5:2混合组成,第二次酶解复合酶的加入质量为菠萝蜜果肉重的0.05-0.3%。
进一步地,步骤2)中第二次酶解复合酶的酶解条件:时间为1.5-4h,温度为50-55℃,酶解pH为7.4-7.8;优选地酶解时间为1.5-3h。
进一步地,所述助剂为麦芽糊精、壳聚糖、海藻糖、透明质酸、β-环糊精中的一种或几种的任意比混合物。
进一步地,所述复合制剂为冻干粉剂时,通过以下方法制成:
将助剂和水在25-30℃搅拌直至助剂完全溶解,然后加入免疫活性提取物,将混合液转入均质机中均质化10-20min,最后在-40℃左右下冻干,得冻干粉剂,其中,助剂、水、免疫活性提取物的用量比为9-17:70-100:100。
上述冻干粉可用做免疫调节剂,也用于医药中的抗菌,如烫伤伤口的处理,将上述冻干粉与其他药剂混合配置成喷雾剂喷于伤口,或是将上述冻干粉溶于消肿喷雾剂直接喷涂伤口。
上述冻干粉也可用于护肤品,例如将上述冻干粉与免洗面膜复配,用于皮肤修复和改善。
进一步地,所述复合制剂的剂型为粉剂时,通过以下步骤制成:
将助剂加入免疫活性提取物,加热搅拌至助剂完全溶解,经喷雾干燥塔喷雾干燥,得粉剂,其中,助剂的加入质量为免疫活性提取物10-20%。
上述粉剂可用于食品的添加。
本发明的有益效果:
(1)本发明以菠萝蜜为来源,提取了免疫活性提取物,并将该免疫活性提取物与助剂进行复合,形成复合制剂,利用助剂对免疫活性提取物中的多肽、蛋白质、多糖进行负载,一方面避免活性物质的活性降低,另一方面,提高免疫活性提取物的加工性能,可用于食品、医药和护肤品等方面;
(2)所述免疫活性提取物是将菠萝蜜匀浆经过复合酶可控酶解和二次加热澄清获得的;上述复合酶可控酶解和二次加热澄清技术,对菠萝蜜中的免疫活性物质(多肽、蛋白质、多糖)进行了有效地提取和分离,获得了免疫活性提取物,且该免疫活性提取物含有的多肽分子量为180-2000Da,蛋白质含量为10.0%-36.0%,多糖含量为30%-70%,且该免疫活性提取物的淋巴细胞转化值为0.40-0.60,NK细胞活性为45%-65%;
(3)本发明中的提取工艺简单,投入设备较少,产品利用价值高,有助于提高菠萝蜜附加值,可用于工业推广。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例1:
免疫活性提取物通过以下步骤制成:
1)原料预处理:取菠萝蜜整果去除果皮和花序轴之后得到约6.17kg菠萝蜜果肉,破碎成2cm左右的小块,加入50L水,90℃老化20min,待温度冷却至45℃,通过胶体磨均质5min;
2)复合酶可控酶解:将菠萝蜜匀浆置于反应罐中,开启搅拌器使其转速为20r/min,保持温度为40℃,利用8.0mol/L的氢氧化钠溶液调整反应罐中的料液pH为4.0;称菠萝蜜果肉质量的0.3%的第一次酶解复合酶(果胶酶:纤维素酶为2:1)投入反应罐中,保持温度为35℃,酶解pH为3.8,酶解时间为2.5h,酶解结束后,将反应罐中菠萝蜜酶解液迅速升温至90℃时,保持温度20min后,然后冷却至60℃以下,保持温度为55℃,利用6.0mol/L的盐酸溶液调整反应罐中的料液pH为8.0;称取初原料质量的0.1%的第二次酶解复合酶(胰蛋白酶:木瓜蛋白酶为5:2)投入反应罐中,保持温度为50℃,酶解pH为7.8,酶解时间为2h;酶解结束后,将反应罐中菠萝蜜酶解液迅速升温至90℃时,保持温度20min后,然后冷却至50℃,得到酶解混悬液;
3)二次加热澄清:将酶解混悬液过80目网筛,将透过液继续冷却至18℃;随后再次升温至86℃,再次冷却至51℃,溶液再次通过100目网筛过滤,得到精滤液;
4)减压浓缩:将精滤液在0.1MPa、60℃下减压浓缩至1/3体积,得免疫活性提取物。
将所得免疫活性提取物进行以下成分测定:
蛋白质含量:采用凯氏定氮法,以干基计;
蛋白质中180-2000Da的多肽含量:按照GB/T22729-2008进行测试;
多糖:采用苯酚硫酸法,且以葡萄糖计,以干基计;
上述成分含量测定结果为:蛋白质含量为18%,其中180-2000Da的多肽占74%,多糖含量为38%。
采用淋巴细胞转化法和NK细胞活性测定法测定其活性,测试结果如表1所示。
表1淋巴细胞转化值和NK细胞活性
处理方式 | 粗过滤 | 二次澄清 | 离心 |
淋巴细胞转化值 | 0.38 | 0.50 | 0.47 |
NK细胞活性% | 31 | 48 | 43 |
实施例2:
免疫活性提取物通过以下步骤制成:
1)原料预处理:取菠萝蜜整果去除果皮和花序轴之后得到约8.23kg菠萝蜜果肉,破碎成2cm左右的小块,加入48.65L水,90℃老化30min,待温度冷却至50℃,通过胶体磨均质10min;
2)复合酶可控酶解:将菠萝蜜匀浆置于反应罐中,开启搅拌器使其转速为25r/min,保持温度为50℃,利用9.0mol/L的氢氧化钠溶液调整反应罐中的料液pH为4.5;称取菠萝蜜果肉质量的1.0%的第一次酶解复合酶(果胶酶:纤维素酶为2:7)投入反应罐中,保持温度为45℃,酶解pH为4.0,酶解时间为3.0h,酶解结束后,将反应罐中菠萝蜜酶解液迅速升温至90℃时,保持温度20min后,然后冷却至60℃以下,保持温度为55℃,利用5.0mol/L的盐酸溶液调整反应罐中的料液pH为9.0;称取初原料质量的1.0%的第二次酶解复合酶(胰蛋白酶:木瓜蛋白酶为5:2)投入反应罐中,保持温度为45℃,酶解pH为7.5,酶解时间为3h;酶解结束后,将反应罐中菠萝蜜酶解液迅速升温至95℃时,保持温度20min后,然后冷却至45℃,得到酶解混悬液;
3)二次加热澄清:将酶解混悬液过80目网筛,将透过液继续冷却至18℃;随后再次升温至86℃,再次冷却至51℃,溶液再次通过100目网筛过滤,得到精滤液;
4)减压浓缩:将精滤液在0.1MPa、60℃下减压浓缩至1/6体积,得免疫活性提取物。
将所得免疫活性提取物进行以下成分测定:
蛋白质含量:采用凯氏定氮法,以干基计;
180-2000Da的多肽含量:按照GB/T22729-2008进行测试;
多糖:采用苯酚硫酸法,且以葡萄糖计,以干基计;
上述成分含量测定结果为:蛋白质含量为23%,其中180-2000Da的多肽占78%,多糖含量为56%。
采用淋巴细胞转化法和NK细胞活性测定法测定其活性,测试结果如表2所示。
表2淋巴细胞转化值和NK细胞活性
处理方式 | 粗过滤 | 二次澄清 | 离心 |
淋巴细胞转化值 | 0.33 | 0.60 | 0.40 |
NK细胞活性% | 34 | 65 | 44 |
实施例3-1:
一种基于菠萝蜜的具有免疫活性的复合制剂,通过以下步骤制成:
将海藻糖和水在25℃搅拌直至海藻糖完全溶解,然后加入实施例1制备的免疫活性提取物,将混合液转入均质机中均质化10min,最后在-40℃下冻干,得冻干粉剂,其中,海藻糖、透明质酸、水、免疫活性提取物的用量比为13:70:100。
实施例3-2:
一种基于菠萝蜜的具有免疫活性的复合制剂,通过以下步骤制成:
将壳聚糖、透明质酸和水在30℃搅拌直至壳聚糖和透明质酸完全溶解,然后加入实施例2制备的免疫活性提取物,将混合液转入均质机中均质化10-20min,最后在-40℃下冻干,得冻干粉剂,其中,壳聚糖、透明质酸、水、免疫活性提取物的用量比为5:9:80:100。
实施例4-1:
一种基于菠萝蜜的具有免疫活性的复合制剂,通过以下步骤制成:
将麦芽糊精加入实施例1制备的免疫活性提取物,加热搅拌至麦芽糊精完全溶解,经喷雾干燥塔喷雾干燥,得粉剂,其中,麦芽糊精的加入质量为免疫活性提取物10%。
实施例4-2:
一种基于菠萝蜜的具有免疫活性的复合制剂,通过以下步骤制成:
将壳聚糖、β-环糊精加入实施例2制备的免疫活性提取物,加热搅拌至壳聚糖和β-环糊精完全溶解,经喷雾干燥塔喷雾干燥,得粉剂,其中,助剂的加入质量为免疫活性提取物20%。
将实施例3-1、实施例3-2、实施例4-1和实施例4-2用于以下晒伤试验:
测试方法:选择40只小白鼠,在小白鼠背部相同部位进行刮毛(面积大小相同),进行紫外晒伤试验,其皮肤晒伤程度均在I度晒伤,将40只小白鼠分成5组,一组为对照组,间隔2h对烫伤处进行喷敷生理盐水;其余组分别使用实施例3-1、实施例3-2、实施例4-1和实施例4-2获得的复合制剂,将1g复合制剂溶于10g药用生理盐水,间隔2h对烫伤处进行喷敷;喷敷1天后2天后分别观察晒伤处发炎情况,结果如表3所示。
表3
从表3中的数据可以看出,实施例3-1、实施例3-2、实施例4-1和实施例4-2获得的复合制剂具有抗菌作用。
在说明书的描述中,参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上内容仅仅是对本发明所作的举例和说明,所属本技术领域的技术人员对所描述的具体实施例做各种各样的修改或补充或采用类似的方式替代,只要不偏离发明或者超越本权利要求书所定义的范围,均应属于本发明的保护范围。
Claims (9)
1.一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:包括免疫活性提取物、助剂;
所述免疫活性提取物通过以下步骤制成:
1)原料预处理:将菠萝蜜果肉在室温条件下破碎,与水混合后,加热老化10-30min,冷却至50℃以下,匀浆,得到菠萝蜜匀浆;
2)复合酶可控酶解:将菠萝蜜匀浆的pH调节为3.5-5.5,然后投入第一次酶解复合酶,进行第一次酶解,在90-95℃下保温灭酶,冷却至50-60℃,调节pH值为7.0-9.0,投入第二次酶解复合酶,进行第二次酶解,在90-95℃下保温灭酶,冷却,得到酶解混悬液;
3)二次加热澄清:将酶解混悬液过80目网筛,得透过液,将透过液再次升温至80-90℃后迅速冷却至40℃以下,过100目网筛,得精滤液;
4)减压浓缩:将精滤液旋转蒸发,浓缩至初始体积的1/2~1/6,得免疫活性提取物。
2.根据权利要求1所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:按质量百分比计算,所述免疫活性提取物中蛋白质含量为10.0%-36.0%,多糖含量为30%-70%。
3.根据权利要求2所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:所述蛋白质中分子量为180-2000Da的多肽质量分数为60-90%。
4.根据权利要求1所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:所述免疫活性提取物的淋巴细胞转化值为0.40-0.60。
5.根据权利要求1所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:所述免疫活性提取物的NK细胞活性为45-65%。
6.根据权利要求1所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:步骤2)中第一次酶解复合酶为果胶酶与纤维素酶按照质量比为1∶2-10混合组成,第一次酶解复合酶的加入质量为菠萝蜜果肉重的0.2-1.0%。
7.根据权利要求1所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:步骤2)中第二次酶解复合酶为胰蛋白酶和木瓜蛋白酶按照质量比为5:2混合组成,第二次酶解复合酶的加入质量为菠萝蜜果肉重的0.05-0.3%。
8.根据权利要求1所述的一种基于菠萝蜜的具有免疫活性的复合制剂,其特征在于:所述助剂为麦芽糊精、壳聚糖、海藻糖、透明质酸、β-环糊精中的一种或几种的任意比混合物。
9.根据权利要求1-8任意一项所述的复合制剂在食品、医药、护肤品方面的应用。
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CN103555800A (zh) * | 2013-10-26 | 2014-02-05 | 吉林大学 | 一种以红松籽粕为原料制备免疫活性肽的方法 |
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CN103555800A (zh) * | 2013-10-26 | 2014-02-05 | 吉林大学 | 一种以红松籽粕为原料制备免疫活性肽的方法 |
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