CN117603341A - 一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法 - Google Patents
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Abstract
本发明公开了一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法，包括以下步骤：S1、牛骨粗胶原蛋白制备：牛骨→预处理→破碎→沥干→超声波处理→高压蒸煮脱脂→脱矿(磷酸溶液)→中和→烘干→超微粉碎→牛骨粗胶原蛋白；S2、酶解工艺：牛骨粗胶原蛋白→按料液比1:15加水搅拌均匀→胃蛋白酶酶解→中性蛋白酶和风味蛋白酶酶解→灭酶→检测相关指标→后续工序备用；S3、发酵工艺：用副干酪乳杆菌、动物双歧杆菌乳亚种、黑曲霉按比例和浓度加入上述牛骨胶原蛋白酶解液中，边搅拌边发酵。发酵结束后经过高温巴氏灭菌，再用膜纯化、浓缩、喷雾干燥得到小分子胶原蛋白多肽。
Description
技术领域
本发明属于动物来源胶原蛋白相关生物技术领域，特别涉及一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法。
背景技术
国内胶原蛋白肽研究起步比较晚和落后，技术不成熟，存在不少缺陷。现阶段主要生产工艺为酸水解等化学工艺，生物酶工程技术也是近几年才开始形成一定规模和技术沉淀，但是缺乏精准的工艺和分子肽结构定义和定性，功能成分分析评估等。制作生产工艺比较粗放，随着发酵技术的成熟和应用推广，酶制剂+微生物发酵才慢慢得到科研机构和厂家的关注和投入，现在处于新兴和尝试阶段。因此本发明就是从技术上解决此问题和进行创新工艺的应用和转化。
另一方面，国内的胶原蛋白肽产品分离精制工艺比较粗放和落后，一般只经活性碳精制、离心分离或膜分离制成，而国外产品在此基础上进一步增加了精制工艺，提高了产品的风味可接受性，达到无臭无味，易于在各类产品体系中使用；其次，产品功能性开发研究及专用细化产品差距。因国内胶原蛋白肽产业起步晚，产品功能性机制研发投入不足，目前产品功能性开发研究较浅泛，目标活性成分不明确，往往一个产品声称具有多种功能，专用细化产品几乎没有。由于胶原蛋白肽行业时间短，市场教育不充分，行业统一标准未出台，市场尚存在不规范情况，国内消费者对其认识、消费观念与发达国家还有一定的距离。
目前，常用的制备胶原肽的方法主要有酸法、碱法和酶法。酸法和碱法均采用化学试剂促使胶原蛋白分解产生小分子肽。这两种水解方法不仅过程难以控制，而且反应比较强烈，会造成氨基酸变性，产生有毒物质等不利结果。因此，使用可替代化学法的酶法水解、微生物发酵胶原蛋白技术日益受到重视。
发明内容
针对上述问题，本发明提供了一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原蛋白多肽的工艺方法，以解决上述背景技术中提出的问题。为实现上述目的，本发明提供如下技术方案：一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原蛋白多肽的工艺方法，包括以下步骤：
S1、牛骨粗胶原蛋白制备：牛骨→预处理(剔除杂分和边角废料)→破碎→水洗沥干→超声波处理(500w,5分钟)→高压蒸煮脱脂(125℃，10min)→脱矿(1.0mo l/L磷酸溶液浸泡15分钟，更换2次溶液)→0.5％碳酸氢钠浸泡中和→烘干→超微粉碎→牛骨粗胶原蛋白；
S2、酶解工艺：牛骨粗胶原蛋白→按料液比1:15加水搅拌均匀→胃蛋白酶在PH2.5,45℃下酶解6H，调节pH至7.0左右，并将溶液升温至55℃左右→1％中性蛋白酶N100和0.5％风味蛋白酶，料液比1:5，搅拌状态下55℃酶解2H→升温至100℃灭酶10min→检测相关指标→3000-5000D分子量胶原蛋白多肽产品；
S3、发酵工艺：用微生物发酵加强胶原蛋白多肽链的分解，和生成小分子多肽。副干酪乳杆菌L9、动物双歧杆菌乳亚种KV9、黑曲霉按照2:2:5的添加比例加入上述牛骨胶原蛋白酶解液中，接种量分别是1％、1％、2.5％(菌种浓度20cfu/mL)，边搅拌边发酵，发酵温度为38℃，发酵时间12小时。发酵结束后经过高温巴氏灭菌(95℃，15min)，再用膜纯化、浓缩、喷雾干燥得到小分子胶原蛋白多肽。
本发明的技术效果和优点：
1、酶解水解+微生物发酵胶原蛋白不仅反应条件温和，具有较高的回收率，而且水解反应速率快，对环境污染较小，所得胶原肽较酸法和碱法更安全。因此，被广泛应用于功能性食品，化妆品及其他产品开发。
2、采用传统工艺生产的胶原蛋白肽苦味和腥味比较重，适口性差，消费者比较难以接受。我们的发明新工艺则解决了以上弊端和问题。
3、我们的技术发明主要通过复合生物酶制剂对牛骨来源胶原蛋白原料进行精准肽链酶切，包括外切酶、内切酶的充分配合增效作用，提高蛋白肽键肽链的解链切位、断开精准部位肽键效率和得率，包括精准肽段的获取和靶向功能成分获取，通过复合蛋白酶制剂的精准解链和高分子肽成分，进而用特定的靶向微生物进行精准匹配发酵，精细化工艺处理，产生功能性更精准和吸收作用更强的小分子肽，分子量在1000-1500道尔顿范围，更好的应用于食品、保健和医疗行业。
Claims (4)
1.一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法，其特征在于，包括以下步骤：
S1、牛骨粗胶原蛋白制备：牛骨→预处理(剔除杂分和边角废料)→破碎→水洗沥干→超声波处理(500w,5分钟)→高压蒸煮脱脂(125℃，10min)→脱矿(1.0mol/L磷酸溶液浸泡15分钟，更换2次溶液)→0.5％碳酸氢钠浸泡中和→烘干→超微粉碎→牛骨粗胶原蛋白；
S2、酶解工艺：牛骨粗胶原蛋白→按料液比1:15加水搅拌均匀→胃蛋白酶在PH2.5,45℃下酶解6H，调节pH至7.0左右，并将溶液升温至55℃左右→1％中性蛋白酶N100和0.5％风味蛋白酶，料液比1:5，搅拌状态下55℃酶解2H→升温至100℃灭酶10min→检测相关指标→3000-5000D分子量胶原蛋白多肽产品；
S3、发酵工艺：用微生物发酵加强胶原蛋白多肽链的分解，和生成小分子多肽。副干酪乳杆菌L9、动物双歧杆菌乳亚种KV9、黑曲霉按照2:2:5的添加比例加入上述牛骨胶原蛋白酶解液中，接种量分别是1％、1％、2.5％(菌种浓度20cfu/mL)，边搅拌边发酵，发酵温度为38℃，发酵时间12小时。发酵结束后经过高温巴氏灭菌(95℃，15min)，再用膜纯化、浓缩、喷雾干燥得到小分子胶原蛋白多肽。
2.根据权利要求1所述的一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法。其特征在于，通过牛骨来源粗胶原蛋白的提取和制备，以及超声波处理，提高纯度、得率和处理效率。特地添加了超声波处理装置，比非超声波处理提高5％的粗蛋白得率，以及20％的提取效率。再经过脱脂脱矿提升牛骨胶原蛋白的纯度和达到除杂的目的。最终得到本实验需要的原料：牛骨胶原蛋白粗制品。
3.根据权利要求2所述的一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法。其特征在于，利用生化酶解工艺进行酶解切断大分子胶原蛋白长肽链：把制备好的牛骨来源粗胶原蛋白，和水按照一定比例(1:15)进行混合均匀，通过一定的条件和参数设置，用胃蛋白酶在PH2.5,45℃下酶解6H，并调节pH至7.0左右，同时将溶液升温至55℃左右，进一步用1％中性蛋白酶N100和0.5％风味蛋白酶，料液比为1:5，搅拌状态下55℃酶解2H，最后升温至100℃灭酶10min，检测相关指标，得到分子量在3000-5000D的胶原蛋白多肽产品。
4.根据权利要求3所述的一种牛骨来源胶原蛋白经酶解复合微生物发酵获得胶原多肽的工艺方法。其特征在于，利用酶解+微生物发酵双重复合处理工艺，把牛骨来源的胶原蛋白大分子分解切断为功能性更强更容易被人体吸收的小分子胶原蛋白分子肽(1000D左右)。该步骤主要用微生物发酵加强胶原蛋白多肽链的分解，生成小分子胶原蛋白多肽产品(分子量1000D左右)。工艺特点：副干酪乳杆菌L9、动物双歧杆菌乳亚种KV9、黑曲霉按照2:2:5的添加比例加入上述牛骨胶原蛋白酶解液中，接种量分别是1％、1％、2.5％(菌种浓度20cfu/mL)，边搅拌边发酵，发酵温度为38℃，发酵时间12小时。发酵结束后经过高温巴氏灭菌(95℃，15min)，最后用膜纯化、浓缩、喷雾干燥等工艺处理得到小分子(1000D)胶原蛋白多肽。
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