CN104095242B - 一种复合锌硒多肽化合物及其制备方法 - Google Patents

一种复合锌硒多肽化合物及其制备方法 Download PDF

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CN104095242B
CN104095242B CN201410336871.0A CN201410336871A CN104095242B CN 104095242 B CN104095242 B CN 104095242B CN 201410336871 A CN201410336871 A CN 201410336871A CN 104095242 B CN104095242 B CN 104095242B
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曾庆祝
许庆陵
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Abstract

本发明提供了一种复合锌硒多肽化合物及其制备方法,该化合物由鱼肉与蛋白酶在一定条件下水解得到多肽,再与无机锌、无机硒分别进行配合反应制得;该化合物不仅含有丰富的多肽成分,还含有重要微量元素锌、硒,具有同时满足机体补充多肽和补充有机硒、有机锌等多重作用。

Description

一种复合锌硒多肽化合物及其制备方法
技术领域
本发明涉及化学领域,具体涉及复合微量元素多肽化合物及其制备。
背景技术
硒是生物体必需的微量元素之一。具有抗脂质过氧化、保护生物膜的作用。缺硒会导致许多疾病如克山病、糖尿病、心血管疾病、白内障、肿瘤、易衰老等。因此,硒在人类防癌抗癌、抗衰老、抗氧化、预防和治疗心血管疾病、克山病和大骨节病等方面起到了重要的作用,微量元素硒与生物体,特别是与人体健康的关系,已经引起了人们极大的兴趣。动物饲料中硒摄入量为0.1~0.2mg·kg-1,低于0.05mg·kg-1就会出现硒缺乏症。适量补硒对提高机体免疫能力、调节机体对自由基的防御功能、抑制衰老、预防疾病等均有重要意义。硒以无机和有机两种形式存在,同其他微量元素一样,动物对于无机硒和有机硒的利用率差别很大。有机硒会主动穿过肠壁,能有效地把硒元素贮存、积累在靶组织内,供机体利用,而无机硒则被动地扩散通过肠壁,具有强的助氧化作用,对脂肪造成氧化性应激而自我抵消,产生毒副作用,人体对其吸收和利用率低。正是因为无机硒化合物的毒性大、活性低,应用受到限制,因此开发有机硒、寻找生物活性高且毒性小的有机硒化合物并成为补硒剂非常必要。
锌(Zn)是人和动物必须的微量元素,被称为生命元素。目前,有机锌主要是氨基酸鳌合物,如锌、硒或钙等微量元素的氨基酸鳌合物。食用微量元素的氨基酸螯合物不仅可以补充人体或动物所需的微量元素,还可以同时补充人体或动物所需的氨基酸。国知局于2006年2月22日授权公告了一项发明专利“一种有机硒的制备工艺”(申请号200410016499.1),该专利公开了一种蛋氨酸硒的制备方法:将蛋氨酸盐和四氯化硒在水溶液或有机溶液中反应0.5~10小时,控制反应时pH2~9、温度-10~100℃,最后经过滤、浓缩和干燥即可。国知局于1997年4月2日公开了一项发明专利申请“氨基酸锌的制备方法及其应用”(申请号94114854.8),该申请公开了一种三甘氨酸锌鳌合物的制备方法,即将氨基酸和氧化锌按2∶1的摩尔比混合,在水溶液中反应一段时间后浓缩,最后用酒精析出反应产物。国知局于2006年2月11日公开了一项发明专利申请“氨基酸鳌合物的制备方法”(申请号200380105098.7),该申请公开了一种制备金属氨基酸鳌合物的方法,即将摩尔比为1∶1~1∶4的金属碳酸盐和酸性氨基酸(如谷氨酸、天冬氨酸等)在0~100℃的水溶液中反应,最后取反应物上清经冷冻干燥即可。上述方法所用的螯合剂都是非天然的单一成分的氨基酸,所得鳌合物营养成分单一,在实际使用中,往往需要几种氨基酸微量元素螯合物的配合使用才能体现出较好的效果,合成及应用成本都较高。而且,由于氨基酸的制备是常采用化学酸水解法,难免会引入化学污染物或者化学残留物,需要加以严密的监控与检测。随着小分子多肽可以被机体整体吸收理论的证实,有关多肽的开发就成为营养学或食品科学领域新的关注焦点。由于纯品肽的化学合成难度大、成本高,而且化学合成肽往往比从动植物中制备肽的生物活性要低,所以,通过化学合成肽与微量元素反应制备金属配合物的应用也会受到一定限制。本专利申请人(曾庆祝、许庆陵)于2011年11月30日获得了一件国家发明专利“一种锌-多肽配合物的制备方法及其应用”(专利ZL200810028217.8),该专利公开了一种锌-多肽配合物的制备方法和应用,即将低值鱼肉与碱性蛋白酶在一定条件下水解得到特定多肽,再与醋酸锌在适宜条件下进行配合反应制得一种具有增强机体免疫力、增强GSH-PX活力和SOD活力、清除超氧自由基等功效的多肽锌配合物。
发明内容
本发明的目的在于提供一种复合锌硒多肽化合物及其制备方法,即通过从罗非鱼、金枪鱼等淡水或海水鱼蛋白中制备出特定天然多肽,进而合成一种富含锌硒的复合锌硒多肽化合物,可同时满足机体补充多肽和补硒、补锌的多重作用。
为实现上述目的,本发明采用了如下技术方案:
一种复合锌硒多肽化合物,该化合物由鱼肉与蛋白酶在一定条件下水解得到多肽,再与无机锌、无机硒分别进行配合反应制得;其中,所述鱼肉是指罗非鱼等淡水鱼肉或金枪鱼等海水鱼肉;所述蛋白酶优选碱性蛋白酶Alcalase或木瓜蛋白酶;所述无机锌是醋酸锌、氧化锌、碳酸锌、柠檬酸锌或葡萄糖酸锌,优选醋酸锌或氧化锌;所述无机硒是亚硒酸钠。
一种复合锌硒多肽化合物的制备方法,包括以下步骤:
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,同时按照原料:水为1:1的重量比例补充水量;
(2)酶水解:取上述磨细后的原料,按总原料:水=1:2~1:3的重量比例再补加水,搅拌均匀,用碳酸氢钠调节pH值至8.0-9.0,再按照300-1000U/g原料蛋白加入碱性蛋白酶Alcalase或木瓜蛋白酶,在45~60℃恒温酶解罐中进行酶解,低速(15-20r/min)连续转动搅拌或每隔10分钟间歇搅拌一次,酶解3-5h;
(3)硒锌多肽化合物制备:
①用柠檬酸、盐酸或醋酸调节步骤(2)所得多肽液的pH值至5.5-6.5,优选6.0;
②加入醋酸锌或氧化锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速(15-20r/min)搅拌或间歇搅拌;其中,加入醋酸锌或氧化锌与原料鱼肉质量比为1:20;
③配合反应结束后采用真空浓缩(温度为50℃~80℃),获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品;
④称取一定量锌-多肽配合物溶于水中,加入适量冰醋酸或柠檬酸或盐酸调节pH值至5.0-6.0;将亚硒酸钠溶于少量水中(亚硒酸钠与水的重量比为5:1),缓慢加入到锌-多肽配合物溶液中(加入的亚硒酸钠与锌-多肽配合物的摩尔比为1:1),在40℃~60℃温度下反应24h-48h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀;
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
本发明的有益效果是:
本发明所述的富含锌硒的复合锌硒多肽化合物不仅含有丰富的多肽成分,还含有重要微量元素锌、硒,具有同时满足机体补充多肽和补硒、补锌的多重作用。
具体实施方式
下面结合五个制备实例来对本发明作进一步详细说明。
例1
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,同时按照原料:水为1:1的重量比例补充水量。
(2)酶水解:取上述磨细后的原料,按总原料:水=1:3的重量比例再补加水,搅拌均匀,用碳酸氢钠调节pH至8.5,再按照500U/g原料蛋白加入碱性蛋白酶Alcalase2.4LFG,在55℃恒温酶解罐中进行酶解,低速(15-20r/min)连续转动搅拌或每隔10分钟间歇搅拌一次,酶解4h。
(3)硒锌多肽化合物制备:
①用醋酸调节步骤(2)所得多肽液的pH至6.5;
②加入醋酸锌或氧化锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速(15-20r/min)搅拌或间歇搅拌;其中,加入醋酸锌或氧化锌与原料鱼肉质量比为1:20。
③配合反应结束后采用真空浓缩(温度为50℃~80℃),获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品。
④称取一定量锌-多肽配合物溶于水中,加入适量冰醋酸调节pH值至5.0-6.0。将亚硒酸钠溶于少量水中(亚硒酸钠与水的重量比为5:1),缓慢加入到锌-多肽配合物溶液中(加入的亚硒酸钠与锌-多肽配合物的摩尔比为1:1),在40℃~60℃温度下反应48h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀。
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
例2
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,在磨细过程中按照原料:水为1:1的重量比例补充水量。
(2)酶水解:取上述磨细后的原料,按总原料:水=1:2.5的重量比例比例再补加水,搅拌均匀,用碳酸氢钠调节pH至8.5,再按照1000U/g原料蛋白加入碱性蛋白酶Alcalase2.4LFG,在55℃恒温酶解罐中进行酶解,低速(15-20r/min)连续转动搅拌或每隔10分钟间歇搅拌一次,酶解3.5h。
(3)硒锌多肽化合物制备:
①用醋酸调节步骤(2)所得多肽液的pH至6.5;
②加入醋酸锌或氧化锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速(15-20r/min)搅拌或间歇搅拌;其中,加入醋酸锌或氧化锌与原料鱼肉质量比为1:20。
③配合反应结束后采用真空浓缩(温度为50℃~80℃),获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品。
④称取一定量锌-多肽配合物溶于水中,加入适量冰醋酸调节pH值至5.0-6.0。将亚硒酸钠溶于少量水中(亚硒酸钠与水的重量比为5:1),缓慢加入到锌-多肽配合物溶液中(加入的亚硒酸钠与锌-多肽配合物的摩尔比为1:1),在60℃温度下反应24h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀。
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
例3
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,在磨细过程中按照原料:水为1:1的重量比例补充水量。
(2)酶水解:取上述磨细后的原料,按总原料:水=1:2.5的重量比例再补加水,搅拌均匀,用碳酸氢钠调节pH至8.5,再按照1000U/g原料蛋白加入碱性蛋白酶Alcalase2.4LFG,在55℃恒温酶解罐中进行酶解,低速(15-20r/min)连续转动搅拌或每隔10分钟间歇搅拌一次,酶解3.5h。
(3)硒锌多肽化合物制备:
①用柠檬酸调节步骤(2)所得多肽液的pH至6.5;
②加入柠檬酸锌或氧化锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速(15-20r/min)搅拌或间歇搅拌;其中,加入柠檬酸锌或氧化锌与原料鱼肉质量比为1:20。
③配合反应结束后采用真空浓缩(温度为50℃~80℃),获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品。
④称取一定量锌-多肽配合物溶于水中,加入适量柠檬酸调节pH值至5.0。将亚硒酸钠溶于少量水中(亚硒酸钠与水的重量比为5:1),缓慢加入到锌-多肽配合物溶液中(加入的亚硒酸钠与锌-多肽配合物的摩尔比为1:1),在50℃温度下反应36h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀。
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
例4
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,在磨细过程中按照原料:水为1:1的重量比例补充水量。
(2)酶水解:取上述磨细后的原料,按总原料:水=1:2.5的重量比例再补加水,搅拌均匀,用碳酸氢钠调节pH至8.5,再按照1000U/g原料蛋白加入碱性蛋白酶Alcalase2.4LFG,在55℃恒温酶解罐中进行酶解,低速(15-20r/min)连续转动搅拌或每隔10分钟间歇搅拌一次,酶解3.5h。
(3)硒锌多肽化合物制备:
①用盐酸调节步骤(2)所得多肽液的pH至6.5;
②加入氧化锌或碳酸锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速(15-20r/min)搅拌或间歇搅拌;其中,加入氧化锌或碳酸锌与原料鱼肉质量比为1:20。
③配合反应结束后采用真空浓缩(温度为50℃~80℃),获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品。
④称取一定量锌-多肽配合物溶于水中,加入适量盐酸调节pH值至5.0-6.0。将亚硒酸钠溶于少量水中(亚硒酸钠与水的重量比为5:1),缓慢加入到锌-多肽配合物溶液中(加入的亚硒酸钠与锌-多肽配合物的摩尔比为1:1),在50℃温度下反应24h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀。
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
例5
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,在磨细过程中按照原料:水为1:1的重量比例补充水量。
(2)酶水解:取上述磨细后的原料,按总原料:水=1:3的重量比例再补加水,搅拌均匀,再按照1000U/g原料蛋白加入木瓜蛋白酶,在55℃恒温酶解罐中进行酶解,低速(15-20r/min)连续转动搅拌或每隔10分钟间歇搅拌一次,酶解3-4h。
(3)硒锌多肽化合物制备:
①用醋酸调节步骤(2)所得多肽液的pH至6.5;
②加入氧化锌或醋酸锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速(15-20r/min)搅拌或间歇搅拌;其中,加入氧化锌或醋酸锌与原料鱼肉质量比为1:20。
③配合反应结束后采用真空浓缩(温度为50℃~80℃),获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品。
④称取一定量锌-多肽配合物溶于水中,加入适量冰醋酸调节pH值至5.0-6.0。将亚硒酸钠溶于少量水中(亚硒酸钠与水的重量比为5:1),缓慢加入到锌-多肽配合物溶液中(加入的亚硒酸钠与锌-多肽配合物的摩尔比为1:1),在50℃温度下反应30h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀。
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
以上所述者,仅为本发明的较佳实施例而已,不能以此限定本发明实施的范围,即大凡依本发明申请专利范围及发明说明内容所作的简单的等效变化与修饰,皆仍属本发明专利涵盖的范围内。

Claims (8)

1.一种复合锌硒多肽化合物,该化合物由鱼肉与蛋白酶在一定条件下水解得到多肽,再与无机锌、无机硒分别进行配合反应制得;其中,所述鱼肉是指淡水鱼肉或海水鱼肉;所述蛋白酶是碱性蛋白酶Alcalase或木瓜蛋白酶;所述无机锌是醋酸锌、氧化锌、碳酸锌、柠檬酸锌或葡萄糖酸锌;所述无机硒是亚硒酸钠;所述复合锌硒多肽化合物的制备方法,包括以下步骤:
(1)鱼肉预处理:冷冻鱼肉,用绞肉机或斩拌机快速切碎,再采用胶体磨磨细,同时按照原料:水为1:1的重量比例补充水量;
(2)酶水解:取上述磨细后的原料,按总原料:水=1:2~1:3的重量比例再补加水,搅拌均匀,用碳酸氢钠调节pH值至8.0-9.0,再按照300-1000U/g原料蛋白加入碱性蛋白酶Alcalase或木瓜蛋白酶,在45~60℃恒温酶解罐中进行酶解,低速连续转动搅拌或每隔10分钟间歇搅拌一次,酶解3-5h;
(3)硒锌多肽化合物制备:
①用柠檬酸、盐酸或醋酸调节步骤(2)所得多肽液的pH值至5.5-6.5;
②加入醋酸锌或氧化锌于多肽液中,升温至95℃进行配合反应20-30min,反应过程中低速搅拌或间歇搅拌;其中,加入醋酸锌或氧化锌与原料鱼肉质量比为1:20;
③配合反应结束后采用真空浓缩,获得锌-多肽配合物浓缩产品,再加入无水乙醇使其完全沉淀并清洗,分离沉淀后真空干燥获得锌-多肽配合物粉末产品;
④称取一定量锌-多肽配合物溶于水中,加入适量冰醋酸或柠檬酸或盐酸调节pH值至5.0-6.0;将亚硒酸钠溶于少量水中,缓慢加入到锌-多肽配合物溶液中,在40℃~60℃温度下反应24h-48h,减压蒸馏浓缩,再加入无水乙醇,使其完全沉淀;
⑤离心分离,沉淀用无水乙醇洗涤3次,加少量水溶解,用去离子水透析24h,未透液经浓缩、真空或冷冻干燥即得。
2.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述鱼肉进一步选用罗非鱼肉或金枪鱼肉。
3.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述无机锌进一步选用醋酸锌或氧化锌。
4.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述步骤(2)中的低速连续转动搅拌和步骤(3)中的低述搅拌是指搅拌速度为15-20r/min。
5.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述步骤(3)第③项中的采用真空浓缩的温度为50℃~80℃。
6.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述步骤(3)第①项中用柠檬酸、盐酸或醋酸调节步骤(2)所得多肽液的pH值至6.0。
7.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述步骤(3)第④项中将亚硒酸钠溶于少量水时,亚硒酸钠与水的重量比为5:1。
8.根据权利要求1所述的一种复合锌硒多肽化合物,其特征在于:所述步骤(3)第④项中加入亚硒酸钠与锌-多肽配合物的摩尔比为1:1。
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Assignee: Guangdong Xinjing Traditional Chinese Medicine Development Co.,Ltd.

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Denomination of invention: A compound zinc-selenium polypeptide compound and its preparation method

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Assignee: Shenzhen Yuanling Technology Co.,Ltd.

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