CN1818061A - 固定化乳糖酶生产方法 - Google Patents
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Abstract
本发明涉及的是一种固定化乳糖酶生产方法。它包括了(1)脆壁克鲁维酵母菌悬液的制备，(2)酶预破壁处理，(3)冻融破壁，(4)高压匀浆机终极破壁，(5)制备固定化载体溶液，(6)制备乳糖酶固定化载体溶液，(7)制备固定液和(8)固定化处理等过程。用固定化乳糖酶生产低乳糖奶，其特点是可以进行连续处理，固定化酶不会随液体奶流失，可以回收重复使用，降低乳糖奶的生产成本。
Description
(一)技术领域
本发明涉及的是一种微生物制剂的生产方法，具体地说是一种微生物细胞破壁及固定化乳糖酶的生产方法。
(二)技术背景
人体小肠中可分解乳糖的酶有乳糖酶(lactase)和β-半乳糖苷酶(β-galactosidase)，乳糖酶位于小肠粘膜刷状缘，而β-半乳糖苷酶位于溶酶体内，不能分解肠腔中的乳糖。乳糖酶缺乏是指小肠刷状缘绒毛膜细胞的膜结合酶即乳糖酶活性低下。乳糖酶缺乏有三种类型：(1)先天性乳糖酶缺乏：是指自出生时机体乳糖酶活性即低下或缺乏，是机体常染色体上隐性基因所致，这一类型很少见。(2)继发性乳糖酶缺乏：是指由于各种原因致使小肠上皮损伤而导致的暂时性乳糖酶活性低下，如：感染性腹泻、免疫球蛋白缺乏症、乳糜泻、局限性回肠炎、营养不良等疾病，可在机体疾病康复后恢复正常。(3)原发性乳糖酶缺乏(又称成人型乳糖酶缺乏)：是最常见的一种，不是由疾病的影响所致，主要是由于乳糖酶活性随年龄逐渐降低而引起，发生时间因种族和地区不同而不同，在婴儿断乳后就开始发生，有的20岁左右时开始出现。我国人群多在7～8岁时发生。一般认为乳糖酶缺乏是与世代形成的饮食习惯不同所造成的遗传基因突变有关。乳糖酶缺乏发生率随不同国家、不同种族人群的变化而变化。欧洲人群30％以上乳糖酶缺乏，亚洲人群乳糖酶缺乏占75％～100％，美国白人12％、黑人70％，非洲90％～100％；日本100％，中国3～13岁儿童乳糖酶缺乏发生率为87％。
牛乳中的乳糖进入小肠后，由于乳糖酶的缺乏，乳糖不能被分解成葡萄糖和半乳糖被吸收，称为乳糖消化不良和乳糖吸收不良。当乳糖进入结肠后，被细菌发酵生成短链有机酸如醋酸、丙酸、丁酸等和气体如甲烷、H2、CO2等，乳糖发酵过程可引起肠鸣、腹痛、直肠气体和渗透性腹泻，存在这些症状时称为乳糖不耐受症，严重的乳糖消化不良或吸收不良多在摄入一定量乳糖后30分钟至数小时内发生。乳糖不耐受对婴幼儿影响较大，半乳糖是大脑正常发育的重要物质，由于缺乏乳糖酶不能将乳糖分解为葡萄糖和半乳糖被吸收，因此，儿童的生长迟缓，对大脑发育也有一定的影响，并会同时伴有呕吐等现象，成人有时伴有恶心反应。乳糖酶缺乏者中有20％左右的人存在乳糖不耐受症状，而且乳糖不耐症状个体差异很大。不耐受症状的多少和严重程度与小肠内乳糖酶活性、摄入的乳糖量以及是否同时摄入其它类食品有关，因此生产低乳糖奶具有十分重要的意义。
但是，生产低乳糖奶所用的乳糖酶价格较贵，这就增加了液体奶和奶粉的生产成本，因此低乳糖奶的价格偏高，市场销售受到一定的影响，如果采用固定化乳糖酶就可解决这个问题。因为固定化乳糖酶不会随奶液流失，可以回收反复使用，大大降低生产成本。
(三)发明内容
本发明的目的在于提供一种可以连续进行处理，固定化酶不会随液体奶流失，可以回收重复使用，降低低乳糖奶的生产成本的固定化乳糖酶生产方法。
本发明的目的是这样实现的：
1、脆壁克鲁维酵母菌悬液的制备：
将培养成熟的脆壁克鲁维酵母细胞经10000r/min高速离心，获得新鲜活性菌体细胞，将菌体细胞用无菌水洗涤1-2次，然后用无菌水配制成细胞菌悬液，细胞浓度为5.0％-40.0％；
2、酶预破壁处理：
取上述菌悬液500mL，加入蛋白酶和β-葡聚糖酶，蛋白酶用量1.0-150.0u/g湿菌体，β-葡聚糖酶用量1.0-100u/g湿菌体，pH 6.0-7.0，30℃-65℃保温1-6小时；
3、冻融破壁
将经酶处理后的细胞悬液取出，置于-20℃—-50℃温度下快速冷冻，使其彻底冻结，再将冷冻的细胞置于40℃-75℃迅速融化，得到冻融细胞悬液；
4、高压匀浆机终极破壁
将冻融的细胞悬液置于液体剪切破碎装置高压匀浆机中进行二次破壁，进口处的温度维持在20℃-45℃，出口处的温度不要高于50℃，压力维持在30MPa-120MPa，得到无细胞结构的活性乳糖酶溶液，称为Ls溶液；
5、制备固定化载体溶液
称取3.5g-20.0g聚乙烯醇(PVA)，0.5g-10.0g海藻酸钠，加入100mL蒸馏水，搅拌10分钟，静止浸泡2-24小时，置于121℃温度下灭菌20分钟，冷却至10℃-35℃，此溶液为Ps溶液；
6、制备乳糖酶固定化载体溶液
量取上述Ls溶液和Ps溶液，Ps∶Ls按1∶1-1∶4，在无菌条件下彻底搅拌均匀，此溶液称为Ps-Ls溶液；
7、制备固定液
称取硼酸1.0-4.0g，氯化钙0.5-6.0g，加入100mL水，加热搅拌使其溶化，然后，121℃温度灭菌20分钟，冷却至10℃-35℃，此溶液为硼酸-氯化钙混合液；
8、固定化
将Ps-Ls溶液置于制粒机中，缓缓地滴入硼酸-氯化钙溶液中，ps-Ls溶液与硼酸-氯化钙溶液的比例按1∶1-1∶8，经2-8小时的硬化，捞出用无菌水洗涤两次，即制得固体小球状的固定化乳糖酶。
9、其他固定化方法
用其他固定化方法可以将固定化乳糖酶做成球状、丝状、柱状、条状、块状、薄膜状等等。
10、用固定化乳糖酶生产低乳糖奶
用固定化乳糖酶处理鲜牛乳、消毒乳，固定化乳糖酶和牛乳的比例为10.0％-100％，pH自然，温度4-50℃，反应时间为0.5-5小时，即可生产低乳糖液体奶和低乳糖奶粉。
本发明采用固定化技术，利用化学或物理手段将游离的微生物细胞或酶分子，定位于限定的空间区域不使细胞或酶分子泄露出来，底物分子可以自由进出，使细胞和酶保持活性并可反复使用的一种基本技术。固定化技术包括固定化酶技术和固定化细胞技术，固定化方法大致可以分成吸附法、共价结合法、交联法和包埋法等，其中以包埋法使用较为普遍。包埋法可分为凝胶包埋法和微胶囊法，就是将细胞包裹于凝胶的微小格子内或半透膜聚合物的超滤膜内，该方法操作简单，对酶和细胞活性影响较小，制作的固定化细胞载体强度较高。固定化酶具有以下优点：①易将固定化酶与底物、产物分开；②可以在较长时间内进行分批反应和装柱连续反应；③可以提高酶的稳定性；④酶反应过程能够加以严格控制；⑤产物溶液中没有酶的残留，简化了提取工艺；⑥较水溶性酶更适合于多酶反应；⑦可以增加产物的收率，提高产物的质量；⑧酶的使用效率提高，成本降低。但是，固定化酶也有其不足之处，只能用于水溶性底物，而且较适用于小分子底物，对大分子底物不适宜；与完整细胞相比，不宜多酶反应；胞内酶必须经过细胞破壁和酶的分离等。
用固定化乳糖酶生产低乳糖奶，其特点是可以进行连续处理，固定化酶不会随液体奶流失，可以回收重复使用，降低低乳糖奶的生产成本。本发明也涉及用固定化乳糖酶处理鲜牛乳、消毒乳，生产低乳糖液体奶和低乳糖奶粉。
(四)具体实施方式
取5000mL培养成熟的脆壁克鲁维酵母细胞发酵液，经10000r/min高速离心，获得新鲜活性菌体细胞，将菌体细胞用无菌水洗涤1-2次，然后用无菌水配制成细胞菌悬液，细胞浓度为5.0％-40.0％。
取菌悬液500mL，加入蛋白酶和β-葡聚糖酶，蛋白酶用量50.0u/g湿菌体，β-葡聚糖酶用量20u/g湿菌体，pH 6.5，42℃保温1.5小时。
将经酶处理后的细胞悬液取出，置于-25℃温度下快速冷冻，使其彻底冻结，再将冷冻的细胞置于50℃温度下迅速融化，得到冻融细胞悬液。
再将冻融的细胞悬液置于液体剪切破碎装置高压匀浆机中进行二次破壁，进口处的温度维持在20℃左右，出口处的温度不要高于50℃，压力维持在70MPa，得到无细胞结构的活性乳糖酶溶液，称为Ls溶液。
固定化乳糖酶的制备
称取13.0g聚乙烯醇(PVA)，5.0g海藻酸钠，加入100mL蒸馏水，搅拌10分钟，静止浸泡5小时，置于121℃温度下灭菌20分钟，冷却至35℃，此溶液为Ps溶液。
称取硼酸1.0-4.0g，氯化钙0.5-6.0g，加入100mL水，加热搅拌使其溶化，然后，121℃温度灭菌20分钟，冷却至10℃-35℃，此溶液为硼酸-氯化钙混合液。
量取上述Ls溶液和Ps溶液，Ps∶Ls按1∶2比例混合，在无菌条件下彻底搅拌均匀，此溶液称为Ps-Ls溶液。
将Ps-Ls溶液500mL置于制粒机中，缓缓地滴入硼酸-氯化钙溶液中，并不断搅拌，防止粘连。Ps-Ls溶液与硼酸-氯化钙溶液的比例按1∶4，经3小时的硬化，捞出用无菌水洗涤两次，即制得固体小球状的固定化乳糖酶。若连续快速将Ps∶Ls溶液注入硼酸-氯化钙溶液中可得到丝状的固定化乳糖酶。
将固定化乳糖酶装入容器中，然后再加入鲜牛乳，固定化乳糖酶和牛乳的比例为50.0％，pH自然，温度42℃，反应时间为2小时，即可制得低乳糖液体奶。
Claims (2)
1、固定化乳糖酶生产方法，其特征是：
(1)脆壁克鲁维酵母菌悬液的制备：
将培养成熟的脆壁克鲁维酵母细胞经10000r/min高速离心，获得新鲜活性菌体细胞，将菌体细胞用无菌水洗涤1-2次，然后用无菌水配制成细胞菌悬液，细胞浓度为5.0％-40.0％；
(2)酶预破壁处理：
取上述菌悬液500mL，加入蛋白酶和β-葡聚糖酶，蛋白酶用量1.0-150.0u/g湿菌体，β-葡聚糖酶用量1.0-100u/g湿菌体，pH6.0-7.0，30℃-65℃保温1-6小时；
(3)冻融破壁
将经酶处理后的细胞悬液取出，置于-20℃--50℃温度下快速冷冻，使其彻底冻结，再将冷冻的细胞置于40℃-75℃迅速融化，得到冻融细胞悬液；
(4)高压匀浆机终极破壁
将冻融的细胞悬液置于液体剪切破碎装置高压匀浆机中进行二次破壁，进口处的温度维持在20℃-45℃，出口处的温度不要高于50℃，压力维持在30MPa-120MPa，得到无细胞结构的活性乳糖酶溶液，称为Ls溶液；
(5)制备固定化载体溶液
称取3.5g-20.0g聚乙烯醇(PVA)，0.5g-10.0g海藻酸钠，加入100mL蒸馏水，搅拌10分钟，静止浸泡2-24小时，置于121℃温度下灭菌20分钟，冷却至10℃-35℃，此溶液为Ps溶液；
(6)制备乳糖酶固定化载体溶液
量取上述Ls溶液和Ps溶液，Ps∶Ls按1∶1-1∶4，在无菌条件下彻底搅拌均匀，此溶液称为Ps-Ls溶液；
(7)制备固定液
称取硼酸1.0-4.0g，氯化钙0.5-6.0g，加入100mL水，加热搅拌使其溶化，然后，121℃温度灭菌20分钟，冷却至10℃-35℃，此溶液为硼酸-氯化钙混合液；
(8)固定化
将Ps-Ls溶液置于成型机中，缓缓地滴入硼酸-氯化钙溶液中，Ps-Ls溶液与硼酸-氯化钙溶液的比例按1∶1-1∶8，经2-8小时的硬化，捞出用无菌水洗涤两次，即制得固体固定化乳糖酶。
2、根据权利要求1所述的固定化乳糖酶生产方法，其特征是：按固定化乳糖酶∶鲜牛乳＝1∶10-1∶100比例加入到鲜牛乳、消毒乳中，pH自然，温度4-50℃，反应0.5-5小时，生产低乳糖液体奶和低乳糖奶粉。
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