CN106234915B - 一种高纯乳酸链球菌素溶液的生产方法 - Google Patents
一种高纯乳酸链球菌素溶液的生产方法 Download PDFInfo
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Abstract
本发明公开一种高纯乳酸链球菌素溶液的生产方法,包括如下步骤:S1、取市售的乳酸链球菌素粗粉溶于有机酸溶液中;S2、搅拌静止,置于离心萃取机中离心分层,除去杂蛋白和粗浆,取粗品溶液——乳酸链球菌素层;S3、接着对S2中所得的乳酸链球菌素层利用柱层析进行纯化除杂,干燥后得到纯度为50%~100%的乳酸链球菌素粉;S4、接着对S3中所得的乳酸链球菌素粉加入经脱色后的发酵糖醋或其它有机酸,然后会得到清澈透明的高纯乳酸链球菌素溶液。本发明方法获得的产品具有更安全健康的配料,具有更高的货架期保护,成本性价比也更高;并能够保持产品原有的色泽口感,具有非常大的应用市场,适合工业生产。
Description
技术领域
本发明属涉及一种高纯度生物防腐剂,具体涉及一种高纯乳酸链球菌素溶液的生产方法。
背景技术
乳酸链球菌素是一种高分子多肽,它的分子式为C143H228N42O37S7,由34个氨基酸组成,英文名为Nisin,Nisin在酸性条件下极为稳定,pH为2.0条件下可耐受高温处理(12l℃,15min),无活力损失,而在中性或碱性条件下即发生失活。乳酸链球菌素能有效地杀死或抑制引起食品腐败变质的革兰氏阳性菌,特别是细菌孢子,如葡萄球菌、链球菌、乳杆菌、小球菌、明串珠菌、芽抱杆菌等均对乳酸链球菌素很敏感。乳酸链球菌素作为一种天然、无毒的防腐剂,在食品工业中的应用相当广泛,如乳制品、肉制品、酿酒、果汁饮料、焙烤食品等。
日益受重视的食品安全问题,使得消费者倾向于摒弃不了解的或含化学添加剂的食品,青睐诸如天然,新鲜,绿色有机,少杂蛋白,少盐,以及无过敏原或非转基因等清洁标签的食品。现有技术中要么提纯率低,要么采用化学提纯方法提纯获得较高纯度的乳酸链球菌素,不符合消费者的需求。
发明内容
针对上述现有技术中的不足,本发明提供了一种使产品更纯、更清洁绿色的高纯乳酸链球菌素溶液的生产方法。
为实现上述目的,本发明采用了以下技术方案:
一种高纯乳酸链球菌素溶液的生产方法,包括如下步骤:
S1、取市售的乳酸链球菌素粗粉溶于有机酸溶液中;
S2、搅拌静止,置于离心萃取机中离心分层,除去杂蛋白和粗浆,取粗品溶液——乳酸链球菌素层;
S3、接着对S2中所得的乳酸链球菌素层利用柱层析进行纯化除杂,干燥后得到纯度为50%~100%的乳酸链球菌素粉;
S4、接着对S3中所得的乳酸链球菌素粉加入有机酸溶液后,得到清澈透明的高纯乳酸链球菌素溶液。
优选的,步骤S1中,所述有机酸溶液的PH控制为3~6。
进一步的,步骤S1中,所述有机酸溶液为羧酸类。
优选的,步骤S1中,所述有机酸的体积与粗粉的质量比为(5~10):1,单位为ml/mg。
优选的,步骤S3中,取50L S2所得的粗品溶液至体积为150L的层析柱中用S1中的有机酸溶液上样,采用0.2~2mol/L的盐溶液作为洗脱液梯度洗脱1小时进行柱层析,洗脱速率为1ml/min,回收干燥得到纯度为50%~100%的乳酸链球菌素纯粉。
进一步的,步骤S3中,柱层析进行65分钟之后,回收干燥得到纯度为99%及以上的乳酸链球菌素纯粉。
优选的,步骤S4中,有机酸溶液采用乳酸溶液、丙酸溶液或发酵糖醋溶液中的任一种;当采用发酵糖醋溶液时,S3中所得的乳酸链球菌素粉加入脱色后的发酵糖醋中。
进一步的,步骤S4中,发酵糖醋溶液以玉米、甘蔗或甜菜的天然材料作为培养基、以益生菌为菌种发酵而成。
进一步的,步骤S4中,所述益生菌采用凝结芽孢杆菌、丙酸杆菌和副干酪乳杆菌。
优选的,步骤S4中,采用活性炭或膨润土进行脱色。
本发明的有益效果在于:
1)、本发明提供的方法简单,将经过柱层析得到的纯度为50%~100%的乳酸链球菌素粉溶于有机酸(乳酸溶液、丙酸溶液或发酵糖醋溶液)中均可制备得到高纯乳酸链球菌素清液;当符合消费者对天然,清新,绿色有机,少杂蛋白,少盐,以及无过敏原或非转基因等清洁标签的青睐。
2)、本发明方法可获得高纯乳酸链球菌素清液,通过实验证明其具有集束的抑菌活性和强有力的扩散度,能够高效抑制革兰氏阳性菌生长,预防肿瘤,增强食品品质和保质期;同时,本发明方法获得的高纯乳酸链球菌素清液清澈透明,使用量低,不影响食品原有色泽和风味,适合高品质的绿色食品。
3)、本发明方法获得的高纯乳酸链球菌素清液在水果中可护色,稳定花青素,具有酸化作用;在透明饮料中,由于其澄清的特性和超强的孢子抑菌效果,更显示了其无与伦比的应用优势。
4)、国内普通的乳酸链球菌素具有如下的劣势:含有氯化钠及对人体存在潜在风险的杂蛋白;抑制革兰氏阳性菌的功效受脂肪及乳化剂的影响;并且加入后会溶液浑浊,溶液底部会出现明显的沉淀;而本发明方法获得的高纯乳酸链球菌素清液几乎不含杂蛋白和氯化钠杂质,是纯天然乳酸链球菌素清液;对革兰氏阳性菌的抑制效果远远超过普通乳酸链球菌素,对革兰氏阴性菌的抑制效果更强,且更少受脂肪及乳化剂的影响;并且溶液澄清,口感无味。
5)、综上所述,本发明方法获得的产品具有更安全健康的配料,具有更高的货架期保护,成本性价比也更高;并能够保持产品原有的色泽口感,具有非常大的应用市场,适合工业生产。
附图说明
图1、2为本发明应用在再制乳酪中的试验结果图。
图3为本发明应用在苹果汁中的试验结果图。
图4为本发明应用在苹果汁中的溶液状态图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明中的技术方案进行清楚、完整地描述。以下实施例仅用于更加清楚地说明本发明的技术方案,而不能以此来限制本发明的保护范围。
一种高纯乳酸链球菌素溶液的生产方法,包括如下步骤:
S1、取市售的乳酸链球菌素粗粉溶于有机酸溶液中,有机酸溶液的PH控制为3~6;
S2、搅拌静止,置于离心萃取机中离心分层,除去杂蛋白和粗浆,取粗品溶液——乳酸链球菌素层;
S3、接着对S2中所得的乳酸链球菌素层利用柱层析进行纯化除杂,干燥后得到纯度为50%~100%的乳酸链球菌素粉;
S4、接着对S3中所得的乳酸链球菌素粉加入有机酸溶液后,得到清澈透明的高纯乳酸链球菌素溶液。
步骤S1中,所述有机酸溶液为羧酸类,其中,所述有机酸的体积与粗粉的质量比为(5~10):1,单位为ml/mg。
步骤S3中,取50L S2所得的粗品溶液至体积为150L的层析柱中用S1中的有机酸溶液上样,采用0.2~2mol/L的盐溶液作为洗脱液梯度洗脱1小时进行柱层析,洗脱速率为1ml/min,回收干燥得到纯度为50%~100%的乳酸链球菌素纯粉。其中,当柱层析进行65分钟之后,回收干燥得到纯度为99%及以上的乳酸链球菌素纯粉。
步骤S4中,有机酸溶液采用乳酸溶液,丙酸溶液或发酵糖醋溶液;;当采用发酵糖醋溶液时,S3中所得的乳酸链球菌素粉加入脱色后的发酵糖醋中。其中,发酵糖醋溶液以玉米、甘蔗或甜菜的天然材料作为培养基、以益生菌为菌种发酵而成;所述益生菌采用凝结芽孢杆菌、丙酸杆菌和副干酪乳杆菌。
步骤S4中,采用传统的液体脱色技术比如活性炭或膨润土对褐色的溶液进行脱色,得到清澈透明的高纯乳酸链球菌素溶液。
其中,步骤S4中,所述发酵糖醋的加工方法,包括如下步骤:
S1、蒸煮糊化:以玉米为原料,进行蒸煮糊化,其蒸煮的温度为100~120℃,取出冷却到25~35℃;
S2、发酵培养基的制备:以g/L计,上述蒸煮糊化的原料40,酵母膏1~5,酵母蛋白胨1~5,精制玉米糖浆10~25,K2HPO4 5~10,NH3·H2O 0.3~0.6,Ca(OH)2 0.02~0.025,按0.2%~0.5%比例拌入凝固芽孢杆菌,且每隔8~10h补加一次原料;
S3、用去离子水配料,配制好培养基后经过过滤器去除大颗粒杂质后,倒入发酵罐中,进行120℃、30min的高温灭菌,降温至25~30℃;加入进行发酵培养,过程中流加淀粉水解糖15~25g/L,发酵周期为5天,得酒精发酵液;
S4、接着,调配到酒精度在8°~9°之间,泵入下一级发酵罐进入醋酸发酵阶段,加入醋酸菌和副干酪乳杆菌、丙酸杆菌协同发酵,温度控制在35℃~45℃之间,得到最终发酵液;
S5、将上述发酵液进行浓缩,过滤,分离,制得发酵玉米糖醋。
将原料玉米换成甜菜或甘蔗也可得到相应的发酵糖醋。
其中,丙酸杆菌采用经鉴定为生物安全级别(GRAS)的菌种产丙酸丙酸杆菌。所述丙酸杆菌和副干酪乳杆菌的用量比为1:(2~4)。
本发明生产所用有机原料淀粉水解糖采用葡萄糖、酵母膏来自酿酒酵母浸提物、酵母蛋白胨来自酿酒酵母细胞、精制糖浆来自淀粉加工副产物,均为天然来源,无石化来源的成分。
应用例1
再制乳酪
蜡样芽孢杆菌和酪酸梭菌导致的“起泡”是引起再制乳酪变质的主因。将上述实施例得到的高纯乳酸链球菌素清液应用在再制乳酪中。在PH5.4,105℃下作用10分钟之后,对蜡样芽孢杆菌和酪酸梭菌的含量分别进行监测,试验结果分别如图1、2所示,控制组作为参照未添加任何物质,表明,添加高纯乳酸链球菌素清液能显著的抑制这些细菌的生长,延长保质期。且在一定范围内,加入的量越多,抑制效果更明显。
应用例2
鲜榨苹果汁
导致新鲜苹果汁变味的主要腐败菌为酸土环脂芽孢杆菌。在常温下的新鲜苹果汁中分别加入普通乳酸链球菌素和本发明产品(高纯乳酸链球菌素清液),试验结果如图3、4所示,对照组作为参照未添加任何物质,图3表明仅1/4的本产品比普通乳酸链球菌素能更高效的抑制酸土环脂芽孢杆菌,使苹果汁保鲜期大幅提高,且溶液澄清。图4表明添加本发明产品的鲜榨苹果汁始终保持着透明的溶液,相反,随着时间的推移,普通乳酸链球菌素逐渐沉淀到瓶底,伴随其包含的杂蛋白很有可能导致新鲜苹果汁产生漂浮的云团,减少货架期。
以上所述仅是本发明的优选应用实施例,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变形,这些改进和变形也应视为本发明的保护范围。
Claims (7)
1.一种乳酸链球菌素溶液的生产方法,其特征在于包括如下步骤:
S1、取市售的乳酸链球菌素粗粉溶于有机酸溶液中;
S2、搅拌静止,置于离心萃取机中离心分层,除去杂蛋白和粗浆,取粗品溶液——乳酸链球菌素层;
S3、接着对S2中所得的乳酸链球菌素层利用柱层析进行纯化除杂,干燥后得到纯度为50%~100%的乳酸链球菌素粉;
S4、接着对S3中所得的乳酸链球菌素粉加入有机酸溶液后,得到清澈透明的高纯乳酸链球菌素溶液;
其中:
步骤S1中,所述有机酸溶液的PH控制为3~6;所述有机酸的体积与粗粉的质量比为( 5~10 ):1,单位为ml/mg;
步骤S3中,取50L 步骤S2所得的粗品溶液至体积为150L的层析柱中用S1中的有机酸溶液上样,采用0.2~2mol/L的盐溶液作为洗脱液梯度洗脱1小时进行柱层析,洗脱速率为1ml/min,回收干燥得到纯度为50%~100%的乳酸链球菌素纯粉。
2.根据权利要求1所述的一种乳酸链球菌溶液的生产方法,其特征在于:步骤S1中,所述有机酸溶液为羧酸类。
3.根据权利要求1所述的一种乳酸链球菌溶液的生产方法,其特征在于:步骤S3中,柱层析进行65分钟之后,回收干燥得到纯度为99%及以上的乳酸链球菌素纯粉。
4.根据权利要求1所述的一种乳酸链球菌溶液的生产方法,其特征在于:步骤S4中,有机酸溶液采用乳酸溶液、丙酸溶液或发酵糖醋溶液中的任一种;当采用发酵糖醋溶液时,S3中所得的乳酸链球菌素粉加入脱色后的发酵糖醋中。
5.根据权利要求4所述的一种乳酸链球菌溶液的生产方法,其特征在于:步骤S4中,发酵糖醋溶液以玉米、甘蔗或甜菜的天然材料作为培养基、以益生菌为菌种发酵而成。
6.根据权利要求5所述的一种乳酸链球菌溶液的生产方法,其特征在于:步骤S4中,所述益生菌采用凝结芽孢杆菌、丙酸杆菌和副干酪乳杆菌。
7.根据权利要求1所述的一种乳酸链球菌溶液的生产方法,其特征在于:步骤S4中,采用活性炭或膨润土进行脱色。
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