CN108887673B - 富含超氧化物歧化酶sod的野木瓜酵素的制备方法 - Google Patents
富含超氧化物歧化酶sod的野木瓜酵素的制备方法 Download PDFInfo
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- A—HUMAN NECESSITIES
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Preparation Of Fruits And Vegetables (AREA)
Abstract
本发明公开了一种富含超氧化物歧化酶SOD的野木瓜酵素及其制备工艺,通过该制备工艺发酵的野木瓜酵素超氧化物歧化酶SOD的含量为300‑800U/mLprot。其制备方法是将前处理的野木瓜进行低温速冻后破碎打浆,然后经过复合酶制剂低温酶解,最后添加复配发酵剂,在超声波辅助下发酵得到产品,整个制备工艺发酵周期为8‑10天。本发明以野木瓜为原料,制备富含高浓度超氧化物歧化酶SOD的野木瓜酵素,不仅有效的缩短了酵素发酵周期,同时最大程度的保留了野木瓜生物活性成分,得到的野木瓜酵素口感好,营养成分高,生物活性成分浓度高,可利用其作为中间品生产野木瓜酵素口服液、饮料、酒、茶、片剂、胶囊、粉剂和食品添加剂。
Description
技术领域
本发明属于食品加工领域,具体的涉及富含超氧化物歧化酶SOD的野木瓜酵素的制备方法。
背景技术
野木瓜( Stauntonia hexaphylla Decne) ,又名五爪金龙、假荔枝、绕绕藤、乌藤等,为木瓜属(Chaeno-meles Lindl)蔷薇科(Rosaceae)贴梗海棠灌木丛植物。野木瓜皮薄、肉厚、气香、品质优良,营养极为丰富。其鲜果肉石细胞丰富,有机酸含量极高。含有丰富的皂苷,包括去甲五环三萜皂苷类化合物,是由齐墩果酸、熊果酸等以皂苷元的形式与糖结合而成,具有独特的生理活性。果用途较多,可切片晒干食用,亦可入药;鲜果放于泡菜坛内,使泡菜味正、不变质;亦可削皮切片用盐或糖腌后食用。申请人对其活性成分分析检测,结果表明:野木瓜含有多种氨基酸,齐墩果酸,三萜,有机酸,黄酮类活性化合物,特别含有极高的超氧化物歧化梅(SOD),具有良好的抗氧化性能。
超氧化物歧化酶(Superoxide Dismutase简称SOD)是一种酶制剂。它在生物界的分布极广,几乎从动物到植物,甚至从人到单细胞生物,都有它的存在。SOD被视为生命科技中最具神奇魔力的酶、人体内的垃圾清道夫。SOD是氧自由基的自然天敌,是机体内氧自由基的头号杀手,是生命健康之本。外源SOD进入人体内血液后,就能有效地保护机体细胞里的脂质、蛋白质、DNA、细胞膜等生命物质,使它们免受人体内多余的超氧阴离子自由基氧离子和羟自由基的破坏,增强机体细胞和各种重要器官的免疫功能,对治疗自身免疫性疾病,病毒性疾病,心血管疾病,如各种炎症、高血压、 血栓病、糖尿病、风湿关节炎、红斑狼疮和癌症等,有特殊功效,对辐射有防护作用。
超氧化物歧化酶(SOD)目前世界范围内的开发,大都从动物血里提取,从动物血液里提取,一则原料来源少,代价昂贵,二则动物性SOD酶存在排他性、不易常温保存、存在交叉感染的潜在危险。由于资源有限而且容易造成污染。鉴于动物血液来源困难,许多研究者开始利用微生物发酵制备SOD,因此,选择富含SOD的植物作为发酵底物,利用微生物发酵提取浓缩原理富集SOD,无血液病毒及交叉感染危险;是现在提取SOD所倡导的一种方法。
酵素通常是由微生物发酵而成,是指以一种或多种新鲜蔬菜、水果、菌菇、中草药等为原料,经多种有益菌发酵而产生的,含有丰富的维生素、酶、矿物质和次生代谢产物等营养成分的功能性微生物发酵产品。经过酵素发酵后的产物,不仅改善了食品原有的不良风味,而且还增加了新的生物活性物。酵素通常以果蔬和食用菌为原材料,自然发酵,少则几个月,多则数年,过程工艺复杂,时间长,容易杂菌污染,产品质量难以控制。现有技术中公开的专利“一种提高酵素中活性SOD酶的制备方法”专利号CN201310539885.8采用虫草菌液(富含SOD酶,同时能能产生高含量的SOD酶活性物)和云芝菌液两步发酵,在经过机械强力破碎细胞,使SOD酶从细胞内向外流出,来提高发酵液中活性SOD酶含量。
目前对于野木瓜产品的开发利用还以果干,果酒,果醋等低附加值产品为主,还没有针对其果肉中高SOD酶含量特点而开发附加值高的产品,同时野木瓜因其果肉中单宁,纤维素含量高,所以直接食用口感酸涩,无法合理的利用其营养生物活性成分,因此,针对野木瓜产品的开发利用,如何在最大程度的保留其活性成分的基础上,开发出口感好的产品市场前景好。
发明内容
本发明的目的是提供富含超氧化物歧化酶SOD的野木瓜酵素的制备方法,通过低温速冻破碎法将野木瓜果肉细胞内超氧化物歧化酶SOD释放,利用酶制剂酶解果胶,纤维素等成分,在超声波辅助作用的条件下,采用微生物发酵提取富集超氧化物歧化酶SOD,得到富含超氧化物歧化酶SOD的野木瓜酵素。一方面解决了野木瓜口感酸涩难以食用的问题,同时为SOD酶来源提供了一种安全的新途径,避免了从动物体内提取SOD酶,存在的排他性、不易保存、交叉感染等潜在的危险。
本发明的目的及解决其主要技术问题是采用以下技术方案来实现的:
A.低温速冻破碎打浆:将成熟的野木瓜清洗切开后去籽,然后置于零下18℃的低温条件下速冻3-5小时后,取出后采用破碎打浆机打浆;
B.低温酶解:将步骤A 得到的野木瓜果浆置于常温条件下,待其温度回升至15-20℃,按照野木瓜果浆总重量的0.3-0.5%添加复合酶制剂酶解5-8小时,添加时将复合酶制剂置于30℃的温水中活化10-15分钟后加入到物料中,酶解过程中在无菌环境下操作,并间隔半小时搅拌一次;
C.超声波辅助发酵:将经过酶解后的野木瓜果浆,按总重量份数的15-20%添加白糖,按总重量份数的0.3-0.5%添加固体酵素发酵剂,添加时将固体酵素发酵剂置于30℃的温糖水中活化10-15分钟后有气泡产生时加入到物料中,加入后搅拌均匀置于配置有超声波辅助发酵的不锈钢发酵罐中,控制超声波功率为100W~600W,发酵温度为20-25℃,发酵至发酵液的固形物含量为8-10%时,终止发酵;
D.过滤除菌:通过纱布粗过滤除去野木瓜果渣,将滤液在0-10℃的温度下低温沉降12小时后,采用0.2-0.45um的除菌膜过滤得到成品。
其中步骤B中复合酶制剂的组成及含量为:果胶酶1-3份,纤维素酶0.5-1份,中性蛋白酶1-2份;步骤C中固体酵素发酵剂的组成及含量比例为:酵母菌粉3-5份,乳酸菌粉1-3份,醋酸杆菌粉0.5-1份。
与现有技术相比优点和有益效果有以下几点:
(1)本发明制备的野木瓜酵素的SOD酶活性在300-800U/mLprot,现有技术中很难达到该含量,解决了现有技术中SOD酶来源难,存在交叉感染的问题,为SOD酶的提取制备提供了新途径;
(2)本发明制备野木瓜酵素的工艺中,低温速冻破碎将野木瓜果肉细胞内超氧化物歧化酶SOD释放,利用酶制剂酶解果胶,纤维素等成分,在超声波辅助作用的条件下,本发明工艺采用三重作用将细胞壁破碎,首先是采用低温速冻,利用低温速冻,使细胞壁失水,部分细胞组织破坏,其次,利用超声波在水介质中的空化作用,使细胞壁结构发生破碎,最后,在利用纤维素酶和果胶酶的作用将细胞壁酶解,将细胞膜内超氧化物歧化酶SOD释放出来,同时微生物利用细胞壁破碎后的物质,主要为低聚糖,蛋白质,有机酸,短链脂肪酸等活性化合物,作为微生物发酵所需的益生元,为微生物提供营养物质,微生物发酵过程中也是富集超氧化物歧化酶SOD的过程;同时此制备方法时间短,活性物质损失少,产品中的三萜类,黄酮类化合物含量高,也能够起到超氧化物歧化酶SOD的氧化活性,进一步增加了产品的抗氧化活性;
(3)本发明制备野木瓜酵素口感协调,酸甜可口,同时富含超氧化物歧化酶SOD,三萜,有机酸,氨基酸,黄酮类化合物,以下对制备的野木瓜酵素中的活性成分进行了检测分析,具体数据表明产品中活性成分含量较高,另一方面,改制备方法制备的野木瓜酵素口感酸甜可口,解决了野木瓜直接食用口感差,难以下咽的技术问题。
具体实施方式
以下结合实施例,对依据本发明提出的富含超氧化物歧化酶SOD的野木瓜酵素的制备方法具体实施方式、特征及其功效,详细说明如后。
实施例1
获得这种富含超氧化物歧化酶SOD的野木瓜酵素的制备方法,包括以下步骤:
A.低温速冻破碎打浆:将成熟的野木瓜清洗切开后去籽,然后置于零下18℃的低温条件下速冻3小时后,取出后采用破碎打浆机打浆;
B.低温酶解:将步骤A 得到的野木瓜果浆置于常温条件下,待其温度回升至20℃,按照野木瓜果浆总重量的0.3%添加复合酶制剂酶解8小时,添加时将复合酶制剂置于30℃的温水中活化10分钟后加入到物料中,酶解过程中在无菌环境下操作,并间隔半小时搅拌一次;
C.超声波辅助发酵:将经过酶解后的野木瓜果浆,按总重量份数的20%添加白糖,按总重量份数的0.3%添加固体酵素发酵剂,添加时将固体酵素发酵剂置于30℃的温糖水中活化15分钟后有气泡产生时加入到物料中,加入后搅拌均匀置于配置有超声波辅助发酵的不锈钢发酵罐中,控制超声波功率为100W,发酵温度为25℃,发酵至发酵液的固形物含量为8%时,终止发酵;
D.过滤除菌:通过纱布粗过滤除去野木瓜果渣,将滤液在10℃的温度下低温沉降12小时后,采用0.45um的除菌膜过滤得到成品。
其中步骤B中复合酶制剂的组成及含量为:果胶酶1份,纤维素酶1份,中性蛋白酶1份;步骤C中固体酵素发酵剂的组成及含量比例为:酵母菌粉5份,乳酸菌粉1份,醋酸杆菌粉1份。
实施例2
获得这种富含超氧化物歧化酶SOD的野木瓜酵素的制备方法,包括以下步骤:
A.低温速冻破碎打浆:将成熟的野木瓜清洗切开后去籽,然后置于零下18℃的低温条件下速冻4小时后,取出后采用破碎打浆机打浆;
B.低温酶解:将步骤A 得到的野木瓜果浆置于常温条件下,待其温度回升至18℃,按照野木瓜果浆总重量的0.4%添加复合酶制剂酶解7小时,添加时将复合酶制剂置于30℃的温水中活化12分钟后加入到物料中,酶解过程中在无菌环境下操作,并间隔半小时搅拌一次;
C.超声波辅助发酵:将经过酶解后的野木瓜果浆,按总重量份数的18%添加白糖,按总重量份数的0.4%添加固体酵素发酵剂,添加时将固体酵素发酵剂置于30℃的温糖水中活化12分钟后有气泡产生时加入到物料中,加入后搅拌均匀置于配置有超声波辅助发酵的不锈钢发酵罐中,控制超声波功率为300W,发酵温度为22℃,发酵至发酵液的固形物含量为9%时,终止发酵;
D.过滤除菌:通过纱布粗过滤除去野木瓜果渣,将滤液在5℃的温度下低温沉降12小时后,采用0.45um的除菌膜过滤得到成品。
其中步骤B中复合酶制剂的组成及含量为:果胶酶2份,纤维素酶0.8份,中性蛋白酶1.5份;步骤C中固体酵素发酵剂的组成及含量比例为:酵母菌粉4份,乳酸菌粉2份,醋酸杆菌粉0.7份。
实施例3
获得这种富含超氧化物歧化酶SOD的野木瓜酵素的制备方法,包括以下步骤:
A.低温速冻破碎打浆:将成熟的野木瓜清洗切开后去籽,然后置于零下18℃的低温条件下速冻5小时后,取出后采用破碎打浆机打浆;
B.低温酶解:将步骤A 得到的野木瓜果浆置于常温条件下,待其温度回升至15℃,按照野木瓜果浆总重量的0.5%添加复合酶制剂酶解5小时,添加时将复合酶制剂置于30℃的温水中活化15分钟后加入到物料中,酶解过程中在无菌环境下操作,并间隔半小时搅拌一次;
C.超声波辅助发酵:将经过酶解后的野木瓜果浆,按总重量份数的15%添加白糖,按总重量份数的0.5%添加固体酵素发酵剂,添加时将固体酵素发酵剂置于30℃的温糖水中活化10分钟后有气泡产生时加入到物料中,加入后搅拌均匀置于配置有超声波辅助发酵的不锈钢发酵罐中,控制超声波功率为600W,发酵温度为20℃,发酵至发酵液的固形物含量为10%时,终止发酵;
D.过滤除菌:通过纱布粗过滤除去野木瓜果渣,将滤液在0℃的温度下低温沉降12小时后,采用0.45um的除菌膜过滤得到成品。
其中步骤B中复合酶制剂的组成及含量为:果胶酶3份,纤维素酶0.5份,中性蛋白酶2份;步骤C中固体酵素发酵剂的组成及含量比例为:酵母菌粉3份,乳酸菌粉3份,醋酸杆菌粉0.5份。
以下是对采用本发明工艺方法制备的野木瓜酵素生物活性成分进行分析检测,具体实验方法和数据结果如下:
(1)SOD酶活
采用试剂盒分析,利用黄嘌呤氧化酶反应系统可以产生超氧阴离子自由基,氧化羟胺形成亚硝酸盐,呈现紫红色,用紫外测定吸光度。当样品中亚硝酸盐减少时,测定的吸光度低于对照管吸光度,说明含有SOD,从而测定SOD活力。
实验方法如下表:
计算公式:总的SOD活力=[(对照OD值-测定OD值)/对照OD值]÷50%×(反应液总体积/取样量÷匀浆液浓度)
注:每克组织在1 mL反应液中SOD抑制率达50%的所对应的SOD量为一个SOD活力单位(U/mLprot),将野木瓜酵素发酵液均配置成浓度100 μg/mL,按照上表中方法进行测定。
(2)总三萜含量测定
样品中按照顺序依次加入0.40 mL5%香草醛-冰乙酸溶液和1.00 mL高氯酸,在60℃水浴加热15 min后,放置于冰水浴中,然后加入5.00 mL冰乙酸,摇匀,做全波长扫描测定在545 nm处吸收值最大;对照品制备:精密称取5 mg齐墩果酸到5 mL的容量瓶中,加入甲醇溶解制成每1 mL含有1 mg齐墩果酸的对照品溶液,精密吸取1 mL对照品溶液置于10 mL容量瓶中,加入甲醇稀释定容,得每1 mL含有0.1 mg齐墩果酸的对照品溶液。
(3)总黄酮含量测定
样品中加0.2 mL 5%亚硝酸钠(m/v)摇匀,放置6 min,10%硝酸铝0.2 mL(m/v),摇匀,放置6 min,加入4% NaOH(m/v)2 mL,用水定容至刻度,摇匀,放置15 min。全波长扫描测定在510 nm处有最大吸收值;对照品制备:精密称取5 mg芦丁到5 mL的容量瓶中,加入甲醇溶解制成每1mL含有1 mg芦丁的对照品溶液,精密量取2.4 mL对照品溶液置于10 mL容量瓶中,加甲醇稀释定容,得每1 mL含有0.24 mg芦丁的对照品溶液。
(4)乙醇,pH值的分析参照《食用植物酵素标准》检测分析
参照以上分析方法,检测结果见下表
以上所述,仅是本发明的较佳实施例而已,并非对发明型作任何形式上的限制,任何未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,以该工艺生产的野木瓜酵素等。均仍属于本发明技术方案的范围内。
Claims (2)
1.富含超氧化物歧化酶SOD的野木瓜酵素的制备方法,其特征在于,包括以下步骤:
A.低温速冻破碎打浆:将成熟的野木瓜清洗切开后去籽,然后置于零下18℃的低温条件下速冻5小时后,取出后采用破碎打浆机打浆;
B.低温酶解:将步骤A得到的野木瓜果浆置于常温条件下,待其温度回升至15℃,按照野木瓜果浆总重量的0.5%添加复合酶制剂酶解5小时,添加时将复合酶制剂置于30℃的温水中活化15分钟后加入到物料中,酶解过程中在无菌环境下操作,并间隔半小时搅拌一次;
C.超声波辅助发酵:将经过酶解后的野木瓜果浆,按总重量份数的15%添加白糖,按总重量份数的0.5%添加固体酵素发酵剂,添加时将固体酵素发酵剂置于30℃的温糖水中活化10分钟后有气泡产生时加入到物料中,加入后搅拌均匀置于配置有超声波辅助发酵的不锈钢发酵罐中,控制超声波功率为600W,发酵温度为20℃,发酵至发酵液的固形物含量为10%时,终止发酵;
D.过滤除菌:通过纱布粗过滤除去野木瓜果渣,将滤液在0℃的温度下低温沉降12小时后,采用0.45um的除菌膜过滤得到成品;
其中所述的步骤B中复合酶制剂的组成及含量为:果胶酶3份,纤维素酶0.5份,中性蛋白酶2份;所述的步骤C中固体酵素发酵剂的组成及含量比例为:酵母菌粉3份,乳酸菌粉3份,醋酸杆菌粉0.5份。
2.根据权利要求1所述的富含超氧化物歧化酶SOD的野木瓜酵素的制备方法,获得的富含超氧化物歧化酶SOD野木瓜酵素,在生产野木瓜酵素口服液、野木瓜饮料、野木瓜酒、野木瓜固体茶、野木瓜泡腾片剂、野木瓜酵素胶囊、野木瓜酵素粉剂中的应用。
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