CN110710677A - 一种复合酵素及其制备方法 - Google Patents
一种复合酵素及其制备方法 Download PDFInfo
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- CN110710677A CN110710677A CN201911124084.9A CN201911124084A CN110710677A CN 110710677 A CN110710677 A CN 110710677A CN 201911124084 A CN201911124084 A CN 201911124084A CN 110710677 A CN110710677 A CN 110710677A
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Abstract
本发明公开了一种复合酵素及其制备方法，属于生物发酵技术领域。本发明采用核桃青皮15‑25份、柠檬8‑12份、石榴8‑12份、莲雾8‑12份、西瓜8‑12份、芹菜6‑10份、黑木耳6‑10份、马铃薯6‑10份、当归0.2‑1份、黄芪1‑3份、菊花2‑5份和甘草1‑3份为原料，接种酵母菌、乳酸菌进行联合发酵。通过发酵制备的酵素具备适宜的口感，又保留了原材料本身的药用功效，具有补脾养胃，生津益肺，降脂降压，促进消化，增强免疫力，缓解体力疲劳等效果。配合核桃青皮所具备的抗自由基、抑菌、镇痛、抗肿瘤等功效，使本发明酵素具备很好的保健效果，且制备方法简单，适合大规模推广应用。
Description
技术领域
本发明属于生物发酵技术领域，具体涉及一种复合酵素及其制备方法。
背景技术
酵素食品是含有丰富酶类、维生素、矿物质及多种生物活性成分的功能性发酵食品，不仅可以促进正常新陈代谢，增强机体免疫，还具有消炎、抗菌、抗衰老、解毒、抗癌等功效。由于生活压力增大、环境污染等因素造成人体内源性酶减少，食品精加工又使外源性酶受到破坏，不合理的饮食结构引起营养素缺乏，机体易出现酶失衡或作用减弱，营养不均衡而呈现亚健康状态。酵素食品可以有效调整机体酶系和营养平衡，使人体保持健康状态，因此，酵素作为一种养生食品越来越受到认可和推崇。目前已在日本、美国、中国台湾等地盛行，而在中国大陆发展相对缓慢，绝大多数酵素食品都源自进口，市场价格昂贵，对其制备方法进行科学研究具有巨大的价值。
核桃青皮，具有抗自由基、抑菌、镇痛、抗肿瘤等功效，在民间被广泛用于治疗癌症，配制药酒和利口酒等。在植物酵素研究如火如荼的背景下，将核桃青皮经微生物发酵制成核桃青皮酵素，是核桃产业颇具前景和新意的方向，同时，也将显现出其优良的环保和经济价值。然而，单单将核桃青皮作为制作酵素的原材料，其有效成分无法有效释放，并且所得酵素产品功能单一，口感欠佳。
发明内容
为解决现有技术的不足，本发明提供一种主要以核桃青皮为主要原材料，并添加西瓜、柠檬以及芹菜等新鲜果蔬，复配使用当归、黄芪以及菊花等中草药，充分利用核桃青皮中的有效物质，提供一种具有保健功效的复合酵素及其制备方法。为实现本发明的目的，所采用的技术方案是：
一种复合酵素，是由以下重量份的原料制备而成：核桃青皮15-25份、柠檬8-12份、石榴8-12份、莲雾8-12份、西瓜8-12份、芹菜6-10份、黑木耳6-10份、马铃薯6-10份、当归0.2-1份、黄芪1-3份、菊花2-5份和甘草1-3份。
一种复合酵素的制备方法，包括以下步骤：
（1）将核桃青皮、柠檬、石榴、莲雾、西瓜、芹菜、马铃薯和黑木耳清洗后加入2-4倍重量的水打浆，然后用淀粉酶、果胶酶和纤维素酶在45-50℃酶解3小时得混合果蔬汁；
（2）将当归、黄芪、甘草添加5倍重量的水浸提1小时，过滤得浸提汁；将菊花用5倍重量的水浸泡1小时，然后使用胶体磨打浆，浆液加纤维素酶于45℃酶解3小时；将当归、黄芪、甘草浸提液与菊花酶解后浆液混合得到中草药提取液；
（3）将步骤（1）所得混合果蔬汁与步骤（2）所得中草药提取液混合，密封后置于蒸气灭菌器中进行灭菌，待灭菌结束后，将混合液冷却至室温；
（4）将步骤（3）所得混合液接种酵母菌于室温下发酵60天，酵母菌接种量为0.2-1%，得初步发酵液；
（5）将步骤（4）初步发酵液加入EM菌剂以及乳酸菌，EM菌剂加入量为0.1-0.3%，乳酸菌接种量0.5-2%，并于20℃发酵120天，得二次发酵液；并将二次发酵液过200目滤布过滤、弃渣，滤液经0.2μ超滤柱超滤除菌后得到复合酵素液，将复合酵素液经低温干燥制得复合酵素粉。
步骤（3）中蒸气灭菌器的灭菌温度为120℃，灭菌时间为20分钟。
步骤（5）中所述乳酸菌为乳双歧杆菌、干酪乳杆菌、卷曲乳杆菌、罗伊氏乳杆菌、唾液乳杆菌、发酵乳杆菌、植物乳杆菌、鼠李糖乳杆菌、乳酸片球菌中的一种或几种。
一种复合酵素粉制品，是由所述复合酵素粉加工制成的复合酵素泡腾片、复合酵素颗粒冲剂、复合酵素咀嚼片、复合酵素含片或复合酵素胶囊。
核桃青皮，作为核桃的一种主要废弃物，含有大量的没食子酸、胡桃醌、鞣质、α－氢化胡桃醌－4－葡萄糖苷、胡桃醌生物碱和萘醌等活性成分，具有抗自由基、抑菌、镇痛、抗肿瘤等功效。莲雾中富含丰富的维生素C、维生素B2、维生素B6、钙、镁、硼、锰、铁、铜、锌、钼等微量元素；柠檬富含维生素C、糖类、钙、磷、铁、维生素B1、维生素B2、烟酸、奎宁酸、柠檬酸、苹果酸、橙皮苷、柚皮苷、香豆精、高量钾元素和低量钠元素等，对人体十分有益。芹菜中含有芹菜苷、佛手苷内酯和挥发油，具有降血压、降血脂、防治动脉粥样硬化的作用，对神经衰弱、月经失调、痛风、肌肉痉挛也有一定的辅助食疗作用，它还能促进胃液分泌，增加食欲。
本发明有以下几个有益效果：
1、采用自然发酵的方式，核桃青皮的有效成分难以完全释放，为提高原料利用率，本发明使用核桃青皮做为主要原材料，选用富含维生素C、微量元素的莲雾、柠檬以及芹菜等果蔬为辅料，使用酵母菌、乳酸菌联合发酵。发酵过程，微生物促进核桃青皮有效成分的充分释放，同时促进原材料代谢产生大量初级、次级代谢产物如氨基酸、维生素、有机酸、各种酶、芳香族化合物、糖醇、小分子肽以及大量的低聚糖等有机活性成分，同时还产生大量的超氧化物歧化酶以及黄酮类化合物等对人类具有保健作用的诸多宝贵因子。
2、本发明原材料添加当归、黄芪、甘草以及菊花四味中药，当归益气补血、润燥滑肠；黄芪增强免疫力、降压抗菌；甘草清热解毒、祛痰止咳；菊花散风清热，平肝明目。同核桃青皮、新鲜果蔬进行联合发酵，使中草药中的黄酮、多糖类物质充分释放。通过发酵制备的酵素具备适宜的口感，又保留了原材料本身的药用功效，具有补脾养胃，生津益肺，降脂降压，促进消化，增强免疫力，缓解体力疲劳等效果。配合核桃青皮所具备的抗自由基、抑菌、镇痛、抗肿瘤等功效，使本发明酵素具备很好的保健效果，且制备方法简单，适合大规模推广应用。
具体实施方式
下面结合具体实施例对本发明的技术方案做进一步说明，但不限于此。
实施例1
一种复合酵素，是由以下重量份的原料制备而成：核桃青皮15份、柠檬8份、石榴8份、莲雾8份、西瓜8份、芹菜6份、黑木耳6份、马铃薯6份、当归0.2份、黄芪1份、菊花2份和甘草1份。
一种复合酵素的制备方法，包括以下步骤：
（1）将核桃青皮、柠檬、石榴、莲雾、西瓜、芹菜、马铃薯和黑木耳清洗后加入2-4倍重量的水打浆，然后用淀粉酶、果胶酶和纤维素酶在45-50℃酶解3小时得混合果蔬汁；
（2）将当归、黄芪、甘草添加5倍重量的水浸提1小时，过滤得浸提汁；将菊花用5倍重量的水浸泡1小时，然后使用胶体磨打浆，浆液加纤维素酶于45℃酶解3小时；将当归、黄芪、甘草浸提液与菊花酶解后浆液混合得到中草药提取液；
（3）将步骤（1）所得混合果蔬汁与步骤（2）所得中草药提取液混合，密封后置于蒸气灭菌器中进行灭菌，待灭菌结束后，将混合液冷却至室温；
（4）将步骤（3）所得混合液接种0.2%的酵母菌于室温下发酵60天，得初步发酵液；
（5）将步骤（4）初步发酵液加入0.1%EM菌剂，0.5%的乳酸菌，并于20℃发酵120天，得二次发酵液；并将二次发酵液过200目滤布过滤、弃渣，滤液经0.2μ超滤柱超滤除菌后得到复合酵素液，将复合酵素液经低温干燥制得复合酵素粉。
步骤（3）中蒸气灭菌器的灭菌温度为120℃，灭菌时间为20分钟。
步骤（5）中所述乳酸菌为乳双歧杆菌。
一种复合酵素粉制品，是由所述复合酵素粉加工制成的复合酵素泡腾片、复合酵素颗粒冲剂、复合酵素咀嚼片、复合酵素含片或复合酵素胶囊。
实施例2
一种复合酵素，是由以下重量份的原料制备而成：核桃青皮20份、柠檬10份、石榴10份、莲雾10份、西瓜10份、芹菜8份、黑木耳8份、马铃薯8份、当归0.5份、黄芪2份、菊花3份和甘草2份。
一种复合酵素的制备方法，包括以下步骤：
（1）将核桃青皮、柠檬、石榴、莲雾、西瓜、芹菜、马铃薯和黑木耳清洗后加入2-4倍重量的水打浆，然后用淀粉酶、果胶酶和纤维素酶在45-50℃酶解3小时得混合果蔬汁；
（2）将当归、黄芪、甘草添加5倍重量的水浸提1小时，过滤得浸提汁；将菊花用5倍重量的水浸泡1小时，然后使用胶体磨打浆，浆液加纤维素酶于45℃酶解3小时；将当归、黄芪、甘草浸提液与菊花酶解后浆液混合得到中草药提取液；
（3）将步骤（1）所得混合果蔬汁与步骤（2）所得中草药提取液混合，密封后置于蒸气灭菌器中进行灭菌，待灭菌结束后，将混合液冷却至室温；
（4）将步骤（3）所得混合液接种06%的酵母菌于室温下发酵60天，得初步发酵液；
（5）将步骤（4）初步发酵液加入0.2%EM菌剂，接种1%的乳酸菌，并于20℃发酵120天，得二次发酵液；并将二次发酵液过200目滤布过滤、弃渣，滤液经0.2μ超滤柱超滤除菌后得到复合酵素液，将复合酵素液经低温干燥制得复合酵素粉。
步骤（3）中蒸气灭菌器的灭菌温度为120℃，灭菌时间为20分钟。
步骤（5）中所述乳酸菌为发酵乳杆菌。
一种复合酵素粉制品，是由所述复合酵素粉加工制成的复合酵素泡腾片、复合酵素颗粒冲剂、复合酵素咀嚼片、复合酵素含片或复合酵素胶囊。
实施例3
一种复合酵素，是由以下重量份的原料制备而成：核桃青皮25份、柠檬12份、石榴12份、莲雾12份、西瓜12份、芹菜10份、黑木耳10份、马铃薯10份、当归1份、黄芪3份、菊花5份和甘草3份。
一种复合酵素的制备方法，包括以下步骤：
（1）将核桃青皮、柠檬、石榴、莲雾、西瓜、芹菜、马铃薯和黑木耳清洗后加入2-4倍重量的水打浆，然后用淀粉酶、果胶酶和纤维素酶在45-50℃酶解3小时得混合果蔬汁；
（2）将当归、黄芪、甘草添加5倍重量的水浸提1小时，过滤得浸提汁；将菊花用5倍重量的水浸泡1小时，然后使用胶体磨打浆，浆液加纤维素酶于45℃酶解3小时；将当归、黄芪、甘草浸提液与菊花酶解后浆液混合得到中草药提取液；
（3）将步骤（1）所得混合果蔬汁与步骤（2）所得中草药提取液混合，密封后置于蒸气灭菌器中进行灭菌，待灭菌结束后，将混合液冷却至室温；
（4）将步骤（3）所得混合液接种1%的酵母菌于室温下发酵60天，得初步发酵液；
（5）将步骤（4）初步发酵液加入0.3%EM菌剂，2%的乳酸菌，并于20℃发酵120天，得二次发酵液；并将二次发酵液过200目滤布过滤、弃渣，滤液经0.2μ超滤柱超滤除菌后得到复合酵素液，将复合酵素液经低温干燥制得复合酵素粉。
步骤（3）中蒸气灭菌器的灭菌温度为120℃，灭菌时间为20分钟。
步骤（5）中所述乳酸菌为乳酸片球菌。
一种复合酵素粉制品，是由所述复合酵素粉加工制成的复合酵素泡腾片、复合酵素颗粒冲剂、复合酵素咀嚼片、复合酵素含片或复合酵素胶囊。
对比例1
本对比例采用同实施例1相同的制备方法,不同之处在于，原料中不添加核桃青皮的成分。
对比例2
本对比例采用同实施例1相同的制备方法,不同之处在于，原料中不添加当归、黄芪、甘草以及菊花四味中药。
测试例1
通过电化学方法对本发明酵素进行自由基清除活性的测定。主要测定本发明酵素对羟基自由基、超氧自由基和DPPH自由基的清除率。
（1）羟基自由基的配制
准确称取0.0028g 七水硫酸亚铁晶体，蒸馏水定容至200mL，浓度0.05mmol/L；
准确称取0.33mL30%的H2O2，蒸馏水定容至1000mL，浓度0.01%；
以上制备液储存于0～4℃冰箱中，备用。
超氧自由基的配制
准确称取0.0100g 核黄素，蒸馏水定容至10mL；
准确称取0.0104g 甲硫氨酸，蒸馏水定容至10mL；
以上制备液储存于0～4℃冰箱中，备用。
DPPH 自由基的配制
精确称取0.0043g DPPH，无水乙醇溶解定容至10mL，储存于0～4℃冰箱中，备用。
（2）采用本发明实施例1-3、对比例1、对比例2以及市售普通果蔬酵素作为实验材料，配制浓度为0.15g/ml的测试液
（3）试验方法
对羟基自由基的清除实验
本实验采用芬顿反应诱导产生羟基自由基，即双氧水与亚铁离子反应生成·OH 反应过程如下：
H2O2+ Fe2+→·OH +HO- + Fe3+
用电化学循环伏安法测定功能酵素对羟基自由基的清除作用，通过氧化峰电流值的前后变化计算清除率。
实验组：准确量取1.0mL酵素溶液+1.0mL0.05mmol/L 硫酸亚铁溶液+1.0mL0.01%H2O2溶液，震荡混合均匀；
对照组：准确量取1.0mL 不同浓度的酵素溶液+1.0mL0.05mmol/L 硫酸亚铁溶液+1.0mL 蒸馏水，震荡混合均匀。
将实验组和对照组置于37℃恒温下避光反应2h，各自移取100μL 溶液加入到电解池中进行电化学测试。
对超氧自由基的清除实验
本实验采用电化学循环伏安法测定功能酵素对超氧自由基的清除作用，通过对比实验组、对照组氧化峰电流值的变化计算其清除率。其中的超氧自由基-产生原理为：参照Beauchamp 的光化学方法，在还原剂甲硫氨酸存在下，核黄素分子吸收一个光子被还原，在O2条件下，自氧化产生超氧自由基。
实验组：准确量取1.0mL酵素溶液+1.0mL 核黄素溶液+1.0mL 甲硫氨酸，震荡混合均匀；
对照组：准确量取1.0mL酵素溶液+2.0mL 蒸馏水，震荡混合均匀。
将实验组和对照组置于37℃恒温紫外光照下（254nm 和365nm）反应2h，各自移取100μL 溶液加入到电解池中进行电化学测试。
过氧自由基的清除实验
采用电化学方法测定功能酵素对脂质过氧自由基的清除作用，根据实验组和对照组循环伏安曲线氧化峰电流值来计算自由基清除率。
实验组：准确量取1.0mL 不同浓度的酵素溶液+1.0mLDPPH 溶液，震荡混合均匀；
对照组：准确量取1.0mL 不同浓度的酵素溶液+1.0mL 乙醇，震荡混合均匀。
实验组和对照组置于37℃恒温下避光反应2h，各自移取100μL 溶液加入到电解池中进行电化学测试。
（4）计算方法
向25mL 的电解池中加入10mLpH 值为6.5 的NaH2PO4- Na2HPO4 缓冲溶液，分别移入100μL 的实验组与对照组，在-2～2V 之间以100mV/s 的扫速采用电化学循环伏安法对不同浓度的功能酵素进行测定，测定其实验组、对照组的循环伏安曲线氧化峰的电流值，每组实验重复测定四次，根据氧化峰的电流值计算出不同浓度下的自由基清除率。
自由基清除率的计算公式如下：
清除率（%）= 
×100%
注：I1为对照组氧化峰电流值，I2为试验组氧化峰电流值
（5）试验结果
通过电化学方法测试得到了各酵素溶液对羟基自由基、超氧自由基以及DPPH自由基的清除率，试验结果如下：
从试验数据我们可以看出，本发明所得酵素对羟基自由基、超氧自由基以及DPPH的最大清除率可达35.3%、46.9%和43.6%；相比较对比例1、对比例2以及市售普通果蔬酵素对自由基清除率有明显的提升。同时也可以说明，本发明原材料中，核桃青皮和四味中药相互作用，对本发明酵素功效起着关键的作用，不可省略。
需要说明的是，上述实施例仅仅是实现本发明的优选方式的部分实施例，而非全部实施例。显然，基于本发明的上述实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的其他所有实施例，都应当属于本发明保护的范围。
Claims (5)
1.一种复合酵素，其特征在于，是由以下重量份的原料制备而成，核桃青皮15-25份、柠檬8-12份、石榴8-12份、莲雾8-12份、西瓜8-12份、芹菜6-10份、黑木耳6-10份、马铃薯6-10份、当归0.2-1份、黄芪1-3份、菊花2-5份和甘草1-3份。
2.一种权利要求1所述复合酵素的制备方法，其特征在于，包括以下步骤：
（1）将核桃青皮、柠檬、石榴、莲雾、西瓜、芹菜、马铃薯和黑木耳清洗后加入2-4倍重量的水打浆，然后用淀粉酶、果胶酶和纤维素酶在45-50℃酶解3小时得混合果蔬汁；
（2）将当归、黄芪、甘草添加5倍重量的水浸提1小时，过滤得浸提汁；将菊花用5倍重量的水浸泡1小时，然后使用胶体磨打浆，浆液加纤维素酶于45℃酶解3小时；将当归、黄芪、甘草浸提液与菊花酶解后浆液混合得到中草药提取液；
（3）将步骤（1）所得混合果蔬汁与步骤（2）所得中草药提取液混合，密封后置于蒸气灭菌器中进行灭菌，待灭菌结束后，将混合液冷却至室温；
（4）将步骤（3）所得混合液接种酵母菌于室温下发酵60天，得初步发酵液；
（5）将步骤（4）初步发酵液加入EM菌剂以及乳酸菌，并于20℃发酵120天，得二次发酵液；并将二次发酵液过200目滤布过滤、弃渣，滤液经0.2μ超滤柱超滤除菌后得到复合酵素液，将复合酵素液经低温干燥制得复合酵素粉。
3.根据权利要求2所述复合酵素的制备方法，其特征在于，步骤（3）中蒸气灭菌器的灭菌温度为120℃，灭菌时间为20分钟。
4.根据权利要求2所述复合酵素的制备方法，其特征在于，步骤（5）中所述乳酸菌为乳双歧杆菌、干酪乳杆菌、卷曲乳杆菌、罗伊氏乳杆菌、唾液乳杆菌、发酵乳杆菌、植物乳杆菌、鼠李糖乳杆菌、乳酸片球菌中的一种或几种。
5.一种复合酵素粉制品，其特征在于，所述复合酵素粉制品，是由权利要求2所述复合酵素粉加工制成的复合酵素泡腾片、复合酵素颗粒冲剂、复合酵素咀嚼片、复合酵素含片或复合酵素胶囊。
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| CN111358831A (zh) * | 2020-03-12 | 2020-07-03 | 北京安洁优科技有限公司 | 一种具有缓解视疲劳作用的中药发酵液及其制备方法与应用 |
| CN111972660A (zh) * | 2020-07-30 | 2020-11-24 | 沈阳农业大学 | 一种山楂甘薯复合发酵酵素的制备方法 |
| CN114668144A (zh) * | 2022-02-22 | 2022-06-28 | 辽宁中医药大学 | 一种降糖复合植物酵素、制备方法及应用 |
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| CN111358831A (zh) * | 2020-03-12 | 2020-07-03 | 北京安洁优科技有限公司 | 一种具有缓解视疲劳作用的中药发酵液及其制备方法与应用 |
| CN111972660A (zh) * | 2020-07-30 | 2020-11-24 | 沈阳农业大学 | 一种山楂甘薯复合发酵酵素的制备方法 |
| CN114668144A (zh) * | 2022-02-22 | 2022-06-28 | 辽宁中医药大学 | 一种降糖复合植物酵素、制备方法及应用 |
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