CN110663807A - 一种提高鲍鱼生物制品补肾壮阳功效的方法 - Google Patents
一种提高鲍鱼生物制品补肾壮阳功效的方法 Download PDFInfo
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Abstract
本发明公开了一种提高鲍鱼生物制品补肾壮阳功效的方法,属于海洋生物技术领域。以鲍鱼蛋白为原料,集成现代生物分离技术、生物酶解技术、混菌微生物发酵技术,研制开发出高补肾壮阳功效的鲍鱼多肽生物制品,其中混菌好氧发酵菌种主要包括富硒酿酒酵母和枯草芽孢杆菌。所得鲍鱼多肽具有显著的补肾壮阳保健功效,提取液可根据应用途径做浓缩、吸附干燥、醇析或树脂纯化等处理,满足在保健、食品、医药等领域的高效利用。该工艺技术简单高效、所得产品具有显著的补肾壮阳保健功效,产品稳定性强,生产成本低,易于工业化规模生产。
Description
技术领域
本发明涉及一种提高鲍鱼生物制品补肾壮阳功效的方法,尤其是一种利用微生物协同发酵制备显著补肾壮阳保健功效的鲍鱼多肽生物制品,属于海洋生物技术领域。
背景技术
鲍鱼,古称鳆,又名镜面鱼、九孔螺、明目鱼、将军帽,是一种海生软体动物,属于腹足纲鲍科的单壳海生贝类,亦是中国传统的名贵食材,古称鳆鱼,山珍海味中的一种,素有“一口鲍鱼一口金”的说法。《食疗本草》记载,鲍鱼入肝通瘀,入肠涤垢,不伤元气。壮阳,生百脉,主治肝热上逆,头晕目眩,骨蒸劳热,青肓内障,高血压,眼底出血等症;现代研究结果表明,鲍鱼的营养价值极为丰富,含有二十多种氨基酸,每百克鲜鲍鱼肉含丰富蛋白质23.4克,脂肪3.4克,无机盐钙32毫克,3.0毫克铁,还有相当量的碘、锌、磷和维生素A、B1等,经常食用鲍鱼能养阴、平肝、固肾,可调整肾上腺分泌,调节血压,润燥利肠,治月经不调,大便秘结等疾患。
目前鲍鱼作为海珍品深受广大消费者青睐,食用方式主要以即食和干制品为主,存在着一定的弊端,一是食用方式单一,缺乏海参功能性强的活性提取物及其应用;二是烹制过或提取程中大量营养成分流失,影响功效;三是食用不便。因此,鲍鱼营养保健品的开发既能充分发挥鲍鱼的食效、药效功能,又便于消费者食用,顺应市场需求,具有较好的市场前景,这就对鲍鱼工功能活性物质的提取提出了更高的要求。
现有技术中,关于鲍鱼多肽及其在食品中的应用研究较少,如专利200510068010.X公开了一种运用复合植物蛋白酶(木瓜蛋白酶、无花果蛋白酶、菠萝蛋白酶),降解鲍鱼蛋白质,获得鲍鱼肽的制备方法和用途,制备方法是用鲜或发泡后的干鲍鱼进行冷冻干燥碎成粉末状后,用以上蛋白酶进行3∶2∶1.5配方对鲍鱼蛋白质进行降解,获得鲍鱼多肽;专利200410054770.0公开了一种鲍鱼食品及其制备方法,采用自溶与As1.398胃蛋白酶及复合风味蛋白酶双重酶解或直接用As1.398胃蛋白酶及复合风味蛋白酶酶解的方法制得鲍鱼食品原液。比较上述文献可以发现,关于鲍鱼多肽的制备及应用研究较少,尤其是缺乏提高鲍鱼多肽营养性的研究成为制约产业发展的关键瓶颈。
发明内容
为了克服上述现有技术的不足,满足鲍鱼多肽高效提取及显著补肾壮阳保健功效应用,本申请提供一种提高鲍鱼生物制品补肾壮阳功效的方法,集成现代生物分离技术、生物酶解技术、混菌微生物发酵技术,研制开发出高补肾壮阳功效的鲍鱼多肽生物制品,满足保健、医疗、食品等领域对鲍鱼多肽的应用需求,该工艺技术发酵工艺简单高效、所得产品具有显著的补肾壮阳保健功效,生产成本低,易于工业化规模生产。
本发明通过下述技术方案实现上述技术效果:
一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于制备方法包括如下步骤:
(1)溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解;
(2)酶解:向溶解后的加入鲍鱼蛋白质量比2~3%的复合蛋白酶,控温40~60℃条件下酶解4~8h;
(3)好氧发酵:向酶解后的酶解液中加入酶解液质量比2~3%的复合微生物菌剂,在28~35℃条件下好氧发酵6~12h;
(4)厌氧发酵:好氧发酵结束后加入1~2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8~16h;
(5)过滤:进行粗过滤除去发酵菌体;
(6)灭酶:在100℃条件下灭酶10min;
(7)离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
上述步骤(1)中最佳加水比例为鲍鱼内脏蛋白:水=1:8。
优选的,在鲍鱼内脏蛋白溶解过程中加入鲍鱼蛋白质量比2~3%的几丁聚糖、1~2%乳糖。
上述步骤(2)中所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1。
上述步骤(3)中所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3。
上述步骤(3)中好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min。
上述富硒酿酒酵母种子液制备方法为:在无菌操作条件下,从酵母菌YDP斜面上用接种环挑取两环菌种,接种酿酒酵母活化培养基,培养基葡萄糖浓度5%、酵母膏浓度1%;在28~32℃条件下培养24h,在培养6h、12h、18h时分别向培养基中添加10g/L的亚硒酸钠,制备酵母菌种子液,要求种子液的有效活菌数≥109cfu/mL。
上述枯草芽孢杆菌种子液制备方法为:在无菌条件下,采用LB培养基在28~32℃条件下培养24h,制得液体枯草芽孢杆菌种子液,要求种子液的有效活菌数≥109cfu/mL。
本发明提供一种提高鲍鱼生物制品补肾壮阳功效的方法,所得鲍鱼多肽生物制品具有显著的补肾壮阳保健功效,提取液可根据应用途径做浓缩、吸附干燥、醇析或树脂纯化等处理,满足在保健、食品、医药等领域的高效利用。
本发明提供一种提高鲍鱼生物制品补肾壮阳功效的方法,与现有技术相比,具有以下显著优势:
(1)本申请以鲍鱼内脏蛋白为原料,集成现代生物分离技术、生物酶解技术、混菌微生物发酵技术,显著提升了鲍鱼内脏蛋白补肾壮阳功效;尤为重要的是,本申请创造性的发现,在鲍鱼蛋白溶解液中加入一定比例的几丁聚糖和乳糖可以显著提高酶解发酵法制备的鲍鱼多肽生物制品的补肾壮阳功效;
(2)本申请利用好氧菌(富硒酿酒酵母、枯草芽孢杆菌)共同发酵鲍鱼内脏蛋白提取鲍鱼多肽,充分利用微生物间的协同作用,提升鲍鱼多肽的提取率和体外抗氧化能力,其中:富硒酿酒酵母在发酵过程中会产生各种降解细胞壁的酶类,使得更多的多肽等功能活性物质从细胞壁中释放出来,且酵母是产生能够释放结合态酚酸的酶类,有利于提升鲍鱼多肽的体外抗氧化能力;枯草芽孢杆菌在高温发酵过程中能够杀灭发酵液中的致病菌,并具有较强的产淀粉酶、蛋白酶等能力,可以将大分子淀粉、蛋白物质降解为微生物所利用;
(3),本发明采用乳酸杆菌进行厌氧发酵,提升了鲍鱼多肽的稳定性,试验结果表明,在20~80℃范围内,温度和时间对鲍鱼蛋白水解肽抗氧化性影响不大,当温度超过100℃时抗氧化性随着温度和时间的增加开始降低,稳定性高于目前报道的技术水平;
(4)采用液体发酵,提高了菌体的生长速率,缩短了发酵时间,提升了生产效率发酵工艺条件温和,生产成本低,且易于规模化生产,对提升行业经济效益及规模化推广应用具有重要作用。
具体实施方式
在本申请中,富硒酿酒酵母和枯草芽孢杆菌的活化方法如下:
(1)富硒酿酒酵母种子液制备方法为:在无菌操作条件下,从酵母菌YDP斜面上用接种环挑取两环菌种,接种酿酒酵母活化培养基,培养基葡萄糖浓度5%、酵母膏浓度1%;在28~32℃条件下培养24h,在培养6h、12h、18h时分别向培养基中添加10g/L的亚硒酸钠,制备酵母菌种子液,要求种子液的有效活菌数≥109cfu/mL;
(2)上述枯草芽孢杆菌种子液制备方法为:在无菌条件下,采用LB培养基在28~32℃条件下培养24h,制得液体枯草芽孢杆菌种子液,要求种子液的有效活菌数≥109cfu/mL。
实施例1
一种提高鲍鱼生物制品补肾壮阳功效的方法,具体工艺步骤包括:
(1)溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解;加水比例为鲍鱼内脏蛋白:水=1:8;
(2)酶解:向溶解后的加入鲍鱼蛋白质量比3%的复合蛋白酶,控温40~60℃条件下酶解6h;所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1;
(3)好氧发酵:向酶解后的酶解液中加入酶解液质量比2%的复合微生物菌剂,在28~35℃条件下好氧发酵12h;所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3;好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min;
(4)厌氧发酵:好氧发酵结束后加入2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8h;
(5)过滤:进行粗过滤除去发酵菌体;
(6)灭酶:在100℃条件下灭酶10min;
(7)离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
实施例2
一种提高鲍鱼生物制品补肾壮阳功效的方法,具体工艺步骤包括:
(1)溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解,并加入鲍鱼蛋白质量比2%的几丁聚糖和1%的乳糖;加水比例为鲍鱼内脏蛋白:水=1:8;
(2)酶解:向溶解后的加入鲍鱼蛋白质量比3%的复合蛋白酶,控温40~60℃条件下酶解6h;所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1;
(3)好氧发酵:向酶解后的酶解液中加入酶解液质量比2%的复合微生物菌剂,在28~35℃条件下好氧发酵12h;所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3;好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min;
(4)厌氧发酵:好氧发酵结束后加入2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8h;
(5)过滤:进行粗过滤除去发酵菌体;
(6)灭酶:在100℃条件下灭酶10min;
(7)离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
实施例3
一种提高鲍鱼生物制品补肾壮阳功效的方法,具体工艺步骤包括:
(1)溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解,并加入鲍鱼蛋白质量比3%的几丁聚糖和2%乳糖;加水比例为鲍鱼内脏蛋白:水=1:8;
(2)酶解:向溶解后的加入鲍鱼蛋白质量比3%的复合蛋白酶,控温40~60℃条件下酶解6h;所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1;
(3)好氧发酵:向酶解后的酶解液中加入酶解液质量比2%的复合微生物菌剂,在28~35℃条件下好氧发酵12h;所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3;好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min;
(4)厌氧发酵:好氧发酵结束后加入2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8h;
(5)过滤:进行粗过滤除去发酵菌体;
(6)灭酶:在100℃条件下灭酶10min;
(7)离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
实施例4
一种提高鲍鱼生物制品补肾壮阳功效的方法,具体工艺步骤包括:
(1)溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解,并加入鲍鱼蛋白质量比2%的几丁聚糖;加水比例为鲍鱼内脏蛋白:水=1:8;
(2)酶解:向溶解后的加入鲍鱼蛋白质量比3%的复合蛋白酶,控温40~60℃条件下酶解6h;所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1;
(3)好氧发酵:向酶解后的酶解液中加入酶解液质量比2%的复合微生物菌剂,在28~35℃条件下好氧发酵12h;所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3;好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min;
(4)厌氧发酵:好氧发酵结束后加入2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8h;
(5)过滤:进行粗过滤除去发酵菌体;
(6)灭酶:在100℃条件下灭酶10min;
(7)离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
实施例5
一种提高鲍鱼生物制品补肾壮阳功效的方法,具体工艺步骤包括:
(1)溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解,并加入鲍鱼蛋白质量比1%的乳糖;加水比例为鲍鱼内脏蛋白:水=1:8;
(2)酶解:向溶解后的加入鲍鱼蛋白质量比3%的复合蛋白酶,控温40~60℃条件下酶解6h;所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1;
(3)好氧发酵:向酶解后的酶解液中加入酶解液质量比2%的复合微生物菌剂,在28~35℃条件下好氧发酵12h;所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3;好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min;
(4)厌氧发酵:好氧发酵结束后加入2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8h;
(5)过滤:进行粗过滤除去发酵菌体;
(6)灭酶:在100℃条件下灭酶10min;
(7)离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
实施例6
试验动物:雄性ICR小鼠,体质量(20±2)g;
分组与给药:110只ICR雄性小鼠,在实验条件下适应5天后,分为7个试验组,每组10只。其中实施例1-5试验组给予本发明鲍鱼蛋白提取液,基础対照度灌胃等浓度等体积的鲍鱼蛋白溶液,空白对照组ig小鼠灌胃等体积生理盐水,灌胃剂量均为2g.Kg-1。30天后测定各项指标;
负重游泳实验:参照《保健食品检验与评价技术规范》要求对雄性小鼠进行负重游泳实验和常压耐缺氧实验。将小鼠末次灌胃给药30min后,在小鼠尾根部1/3~2/3处负荷体重的5%铅皮,在水深30cm水温(30±2)℃的水中游泳,同时准备多个60cm×40cm×50cm水槽,并用秒表记录小鼠自游泳开始至死亡的时间,小鼠头部沉入水中7s不起为计时终止时间;
常压耐缺氧实验:参照《保健食品检验与评价技术规范》要求对雄性小鼠进行常压耐缺氧实验。将小鼠末次灌胃给药1h后,5g钠石灰用纱布包裹放入250mL的广口瓶中,将小鼠放入瓶中(每瓶1只),瓶口立即涂抹凡士林盖严,用秒表记录小鼠自放入瓶中开始至出现全身痉挛症状的时间,并迅速将小鼠取出;
血清NO和睾酮(T)值测定:将小鼠经常压耐缺氧实验后休息60min,摘眼球取血存于1.5mL离心管中,4000r/min离心5min后取上层血清,按照NO和睾酮试剂盒说明书操作处理样品并用酶标仪测定含量;
试验结果如下表所示:
试验结果表明本申请鲍鱼多肽生物制品具有显著的补肾壮阳功效,以实施例2为本发明最佳实施例。
以上实施例仅用于说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对被发明进行了详细的说明,但对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而对这些修改或者替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (10)
1.一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于制备方法包括如下步骤:
溶解:将鲍鱼内脏蛋白干粉粉碎,加水搅拌至完全溶解;
酶解:向溶解后的加入鲍鱼蛋白质量比2~3%的复合蛋白酶,控温40~60℃条件下酶解4~8h;
好氧发酵:向酶解后的酶解液中加入酶解液质量比2~3%的复合微生物菌剂,在28~35℃条件下好氧发酵6~12h;
厌氧发酵:好氧发酵结束后加入1~2%的乳酸杆菌粉,在28~35℃条件下厌氧发酵8~16h;
过滤:进行粗过滤除去发酵菌体;
灭酶:在100℃条件下灭酶10min;
离心:以3000r/min速率离心分离,上清液即为鲍鱼多肽提取液。
2.根据权利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述步骤(1)中最佳加水比例为鲍鱼内脏蛋白:水=1:8。
3.根据权利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述鲍鱼内脏蛋白溶解过程中加入鲍鱼蛋白质量比2~3%的几丁聚糖、1~2%乳糖。
4.根据权利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述步骤(2)中所述复合蛋白酶为木瓜蛋白酶和胃蛋白酶的组合,其质量比为2:1。
5.根据利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述步骤(3)中所述复合微生物菌剂为富硒酿酒酵母和枯草芽孢杆菌的混合菌剂,其质量比组成为2:3。
6.根据权利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述步骤(3)中好氧发酵过程在发酵罐中进行时通风量为0.1~0.2m3/m3.min,搅拌转速控制在300~600r/min。
7.根据权利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述富硒酿酒酵母种子液制备方法为:在无菌操作条件下,从酵母菌YDP斜面上用接种环挑取两环菌种,接种酿酒酵母活化培养基,培养基葡萄糖浓度5%、酵母膏浓度1%;在28~32℃条件下培养24h,在培养6h、12h、18h时分别向培养基中添加10g/L的亚硒酸钠,制备酵母菌种子液,要求种子液的有效活菌数≥109cfu/mL。
8.根据权利要求1所述的一种提高鲍鱼生物制品补肾壮阳功效的方法,其特征在于所述枯草芽孢杆菌种子液制备方法为:在无菌条件下,采用LB培养基在28~32℃条件下培养24h,制得液体枯草芽孢杆菌种子液,要求种子液的有效活菌数≥109cfu/mL。
9.按权利要求1-8所述工艺制备的鲍鱼多肽生物制品。
10.权利要求9所述鲍鱼多肽生物制品在保健、食品、医药中的应用。
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