CN107828686A - 一种高产纳豆激酶的枯草芽孢杆菌亚种及其应用 - Google Patents
一种高产纳豆激酶的枯草芽孢杆菌亚种及其应用 Download PDFInfo
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Abstract
本发明公开了一种高产纳豆激酶的枯草芽孢杆菌亚种及其应用，属于生物技术领域。通过从豆豉中分离、纯化，采用奶粉培养基初筛、液体发酵培养基复筛得到一株纤溶活性强的纳豆激酶生产菌株，通过生理生化鉴定与16S rRNA鉴定为枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS‑1，保藏编号为CGMCC NO：14434。利用该菌种与液体发酵培养基生产含有纳豆激酶发酵液，酶活高达29020U/g，利用发酵液制备高纯纳豆激酶粉，酶活高达527230u/g，以纳豆冻干粉和高纯纳豆激酶粉为主要原料制备的一种可预防心脑血栓疾病的保健品具有纳豆激酶活性高，食疗效果好，无副作用的特点。
Description
技术领域
本发明属于生物技术领域，涉及一种高产纳豆激酶的枯草芽抱杆菌亚种及其应用。
背景技术
截止到2016年，我国60岁以上老年人口为2.2亿人，占总人口的16.1％，中国已进入老龄化社会。血栓性疾病是中老年人群的高发病，是现今仅次于癌症的第二大病症，具有很高的死亡威胁。目前，溶解血栓是治疗这一类疾病的重要手段，当今用于临床治疗的药物包括链激酶、尿激酶、组织型纤溶酶原激活剂、单链尿激酶型血纤溶酶原活剂、化学修饰的血纤溶酶一链激酶激活剂复合物等，但是这些药物都存在明显不足：全身性出血副作用、药物半衰期短、价格昂贵等，因此开发新型溶血栓药物已成为当前重要研究课题之一。
纳豆激酶(Nattokinase，NK)是一种枯草杆菌蛋白激酶，是在纳豆发酵过程中由纳豆枯草杆菌产生的一种丝氨酸蛋白酶，具有溶解血栓、降低血粘度，改善血液循环，软化和增加血管弹性等作用。纳豆激酶对纤溶蛋白原不敏感，但可直接水解交联纤维蛋白，将其转变为可溶性物质，达到溶栓目的。此外纳豆激酶可以间接促进组织型纤溶酶原激活剂(t-PA)的生成，并可以激活体内的纤溶酶原转变成纤溶酶而作用于纤维蛋白，所以纳豆激酶具有直接溶解纤维蛋白及通过调节和尿激酶间接溶解纤维的功能。纳豆激酶只溶解血栓的主体物质纤维蛋白，不水解血浆纤维蛋白原，所以不会引发出血的危险，同时，还能激活增强人体自身的溶栓能力，从而起到持久平稳的溶栓作用。然而目前国内的纳豆激酶产品活性普遍比较低，筛选具有高活力纳豆激酶生产能力的菌株，具有重要意义。
发明内容
本发明的目的是提供一种枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1，该菌株具备高产纳豆激酶的特征，将该菌株应用于纳豆激酶的液态发酵，提高了发酵液中纳豆激酶含量，以解决纳豆激酶大规模工业液态发酵缺乏优良菌种、纳豆激酶生产成本高的实际问题。该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。保藏编号：CGMCC No.14434，保藏时间：2017年07月17日。建议的分类命名：枯草芽孢杆菌Bacillus subtilis.
本发明还有一个目的在于提供一种枯草芽孢杆菌亚种Bacillus subtilissubsp.UJS-1在制备纳豆激酶预防心脑血栓疾病保健品与食品中的应用。
一种利用枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1制备纳豆激酶发酵液与提纯品中的方法，按照下述步骤进行：
1)含有纳豆激酶发酵液的制备：
将枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1菌株接种到LB平板培养基上37℃活化24小时，接种到种子液二次活化后按5％的接种量接种到液体发酵培养基中，37℃，200rpm条件下发酵48h，取发酵液离心，利用琼脂糖——纤维蛋白平板法测得发酵上清液酶活为29020u/ml。
2)纳豆激酶的提纯：
将发酵液在4℃，10000rpm，20min条件下离心，收集发酵上清。向上清液中分多次加入硫酸铵粉末，达到30％饱和度。4℃低速颠倒混合盐析6小时，4℃，10000rpm，20min条件下离心，收集上清液。向上清液中分多次加入硫酸铵粉末，达到75％饱和度。4℃低速颠倒混合盐析6小时，4℃，10000rpm，20min条件下离心，收集沉淀。用适量PH7.2的PBS缓冲液溶解沉淀，-80℃冷冻，送冻干机冻干，收集冻干粉即为高纯度的纳豆激酶粉，利用琼脂糖——纤维蛋白平板法测得高纯纳豆激酶粉酶活为527230u/g。
一种可预防心脑血栓疾病的纳豆激酶保健品的制备的制备方法，按照下述步骤进行：
1)以质量百分比计，将干燥后含水量在4-6％的实施例3中制备的高纯纳豆激酶粉5％，纳豆冻干粉35％，山楂粉10％，蓝莓粉10％，麦芽糊精10％，维生素C 1％，乳酸钙1％，甘露糖醇4％，木糖醇4％，山梨糖醇4％，蜂蜜干粉6％，大豆蛋白粉6％在混料机混合均匀；
2)经过公知方法的造粒，干燥后加入4％的硬脂酸镁，混合均匀后，送入压片机压片，每片500mg。
为了达到上述目的，本发明采取以下技术措施：
一种枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1，其筛选过程如下：
1)利用LB培养基分离豆豉中的枯草芽孢杆菌，经形态学鉴定，初步认为是枯草芽孢杆菌。
LB培养基：蛋白胨1％，氯化钠0.5％，酵母粉1％，琼脂1.8％，PH 7.0-7.2。
2)利用奶粉培养基对分离得到的菌株进行初筛，选择形成透明溶圈早且透明溶圈大的菌株。
奶粉培养基：脱脂奶粉2％，蛋白胨1％，氯化钠0.5％，酵母粉1％，琼脂1.8％，PH7.0-7.2。
3)利用发酵培养基进行液体发酵复筛，对发酵上清测定纤溶活力，选择纤溶活力高的菌株。
发酵培养基：芸豆粉4％，葡萄糖4％，氯化钠1.5％，磷酸氢二钾0.1％，磷酸二氢钾0.1％，PH 7.0-7.2。
对复筛得到菌株进行生理生化鉴定与16S rRNA分子生物学鉴定，确认是枯草芽孢杆菌亚种。该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。保藏编号：CGMCC No.14434，保藏时间：2017年07月17日。建议的分类命名：枯草芽孢杆菌Bacillus subtilis.
枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1的形态和特征为：Bacillussubtilis subsp.UJS-1菌株在LB平板上生长良好，菌落灰白色，呈不规则圆形，表面粗糙并形成褶皱，中央形成乳头状突起，边缘不整齐，无光泽，不透明。在奶粉平板上生长18h后可见明显水解溶圈；该菌株为革兰氏阴性菌，杆状、两端钝圆，能产生芽抱，芽抱圆形或椭圆形，中生。
Bacillus subtilis subsp.UJS-1菌株的生理生化特征：见表1
表1，Bacillus subtilis subsp.UJS-1菌株的生理生化试验
注：“+”标示生化反应或者革兰氏染色为阳性；“-”标示生化反应或革兰氏染色为阴性。
与现有技术相比，本发明具有以下有点：
1)本发明的枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1来源于豆豉，未经过基因工程与遗传工程改造，作为发酵食品和保健品菌株安全性高。
2)本发明的枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1产纳豆激酶能力强，单位活性较高，是开发纳豆激酶食品和保健品的重要条件。
附图说明
图1为一种枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1在奶粉培养基上的菌落形态示意图。
图2为MEGA5.05软件基于16S rRNA序列用邻近算法构建的系统进化树。显示筛选得到的UJS-1菌株属于Bacillus subtilis属，图下标尺标示碱基替换的百分率。
图3为尿激酶活力与实际溶圈面积呈线性关系示意图。
具体实施方式
实施例1：
一种枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1，其筛选过程如下：
取20-30粒豆豉，加入20ml无菌生理盐水，震荡后静置，用接种环蘸取上清在LB平板分区划线，分离单菌落。用接种环挑取单菌落，参照《伯杰细菌鉴定手册》第八版，观察是否能拉丝，选择能够拉丝的革兰氏阳性芽孢菌单菌落，到新的LB平板进行分区划线，分离纯化单菌落，挑取纯化的单菌落用生理盐水稀释后在奶粉平板上分区划线。从豆豉浸出液中筛选到20株拉丝革兰氏阳性菌株，根据奶粉平板上单菌落周围水解溶圈的情况选取5株，将其编号为UJS-1、UJS-2、UJS-3、UJS-4、UJS-5。利用发酵培养基对这5株菌株进行复筛，取上清液测定其纤溶活性，选取纤溶活性最高的一株。连续平板传代10次，验证遗传稳定性。
实施例2：
16S rRNA的PCR扩增，其步骤如下：
利用细菌基因组DNA提取试剂盒(天根生化科技有限公司)提取UJS-1菌株基因组DNA，用TE缓冲液稀释到200ng/μl，作为PCR扩增模板。配制以下反应体系：2×PCR mix 10μl，基因组DNA 2μl，上游引物(5'-AGAGTTTGATCCTGGCTCA-3')1μl，下游引物(5'-GGTTACCTTGTTACGACTT-3')1μl，纯水6μl。按以下程序进行扩增：94℃预变性3min，35个循环(94℃预变性20s，55℃退火20s，延伸1min)，72℃延伸5min，4℃保存。PCR扩增结束后取3μl反应液进行1.5％琼脂糖电泳检测。紫外检测表明扩增16S rRNA条带大小约为1500bp。
实施例3：
纳豆激酶纤溶活性测定，其步骤如下：
1)琼脂糖-纤维蛋白平板的制备：
用血清瓶量取25ml PBS缓冲液配制的1％琼脂糖溶液，降温到50-55℃，吸取200ul的0.5％纤维蛋白原溶液，在瓶底、中、上部，分三次快速将纤维蛋白原溶液打入，并快速剧烈搅动5-8次，将60ul的20U/ml凝血酶溶液按照纤维蛋白原溶液加样方法打入并剧烈搅动，立刻将血清瓶中溶液从培养皿壁快速倒入培养皿中，晃动几下后静置1h，用直径2mm的玻璃管在平板上打孔。
2)尿激酶标准曲线的制作：
配制不同活性的尿激酶标准品(10、20、40、60、80IU/ml)，各取10μl加入琼脂糖-纤维蛋白平板的加样孔，37℃孵育15h后测定透明溶圈的直径，计算各溶圈的实际面积(总面积-加样孔面积)，尿激酶活力与溶圈实际面积呈线性关系，y＝1.0373X+33.169，R2＝0.9914。
3)样品纳豆激酶纤溶活性的测定：
将1ml发酵液10000rpm，4℃条件下离心5min，取上清液用PBS缓冲液稀释400、800、1600倍，分别取10μl加入琼脂糖-纤维蛋白平板加样孔，37℃孵育15h后测定透明溶圈的直径，计算各溶圈的实际面积(总面积-加样孔面积)，从标准曲线查得酶活。此方法测定纳豆激酶纤溶活性方法简单，直观。
对复筛得到菌株进行生理生化鉴定与16S rRNA分子生物学鉴定，确认是枯草芽孢杆菌亚种。该菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所。保藏编号：CGMCC No.14434，保藏时间：2017年07月17日。建议的分类命名：枯草芽孢杆菌Bacillus subtilis.
枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1的形态和特征为：Bacillussubtilis subsp.UJS-1菌株在LB平板上生长良好，菌落灰白色，呈不规则圆形，表面粗糙并形成褶皱，中央形成乳头状突起，边缘不整齐，无光泽，不透明。在奶粉平板上生长18h后可见明显水解溶圈；该菌株为革兰氏阴性菌，杆状、两端钝圆，能产生芽抱，芽抱圆形或椭圆形，中生。
Bacillus subtilis subsp.UJS-1菌株的生理生化特征：见表1
表1，Bacillus subtilis subsp.UJS-1菌株的生理生化试验
注：“+”标示生化反应或者革兰氏染色为阳性；“-”标示生化反应或革兰氏染色为阴性。
实施例4：
一种利用枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1制备纳豆激酶发酵液与提纯品中的方法，按照下述步骤进行：
1)含有纳豆激酶发酵液的制备：
将枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1菌株接种到LB平板培养基上37℃活化24小时，接种到种子液二次活化后按5％的接种量接种到液体发酵培养基中，37℃，200rpm条件下发酵48h，取发酵液离心，利用琼脂糖——纤维蛋白平板法测得发酵上清液酶活为29020u/ml。
2)纳豆激酶的提纯：
将发酵液在4℃，10000rpm，20min条件下离心，收集发酵上清。向上清液中分多次加入硫酸铵粉末，达到30％饱和度。4℃低速颠倒混合盐析6小时，4℃，10000rpm，20min条件下离心，收集上清液。向上清液中分多次加入硫酸铵粉末，达到75％饱和度。4℃低速颠倒混合盐析6小时，4℃，10000rpm，20min条件下离心，收集沉淀。用适量PH7.2的PBS缓冲液溶解沉淀，-80℃冷冻，送冻干机冻干，收集冻干粉即为高纯度的纳豆激酶粉，利用琼脂糖——纤维蛋白平板法测得高纯纳豆激酶粉酶活为527230u/g。
实施例5：
一种可预防心脑血栓疾病的纳豆激酶保健品的制备的制备方法，按照下述步骤进行：
1)将干燥后含水量在4-6％的实施例3中制备的高纯纳豆激酶粉5％，纳豆冻干粉35％，山楂粉10％，蓝莓粉10％，麦芽糊精10％，维生素C 1％，乳酸钙1％，甘露糖醇4％，木糖醇4％，山梨糖醇4％，蜂蜜干粉6％，大豆蛋白粉6％在混料机混合均匀。
2)经过公知方法的造粒，干燥后加入4％的硬脂酸镁，混合均匀后，送入压片机压片，每片500mg。
Claims (3)
1.一种产纳豆激酶的枯草芽孢杆菌菌株，其特征在于：保藏编号：CGMCC No.14434，建议的分类命名：枯草芽孢杆菌亚种Bacillus subtilis。
2.一种利用权利要求1所述的产纳豆激酶的枯草芽孢杆菌菌株制备纳豆激酶发酵液与提纯品中的方法，其特征在于按照下述步骤进行：
1)含有纳豆激酶发酵液的制备：
将枯草芽孢杆菌亚种Bacillus subtilis subsp.UJS-1菌株接种到LB平板培养基上37℃活化24小时，接种到种子液二次活化后按5％的接种量接种到液体发酵培养基中，37℃，200rpm条件下发酵48h，取发酵液离心，利用琼脂糖——纤维蛋白平板法测得发酵上清液酶活为29020u/ml；
2)纳豆激酶的提纯：
将发酵液在4℃，10000rpm，20min条件下离心，收集发酵上清；向上清液中分多次加入硫酸铵粉末，达到30％饱和度；4℃低速颠倒混合盐析6小时，4℃，10000rpm，20min条件下离心，收集上清液；向上清液中分多次加入硫酸铵粉末，达到75％饱和度；4℃低速颠倒混合盐析6小时，4℃，10000rpm，20min条件下离心，收集沉淀；用适量PH7.2的PBS缓冲液溶解沉淀，-80℃冷冻，送冻干机冻干，收集冻干粉即为高纯度的纳豆激酶粉，利用琼脂糖——纤维蛋白平板法测得高纯纳豆激酶粉酶活为527230u/g。
3.一种可预防心脑血栓疾病的纳豆激酶保健品的制备方法，其特征在于按照下述步骤进行：
1)以质量百分比计，将干燥后含水量在4-6％的权利要求2过程中制备的高纯纳豆激酶粉5％，纳豆冻干粉35％，山楂粉10％，蓝莓粉10％，麦芽糊精10％，维生素C 1％，乳酸钙1％，甘露糖醇4％，木糖醇4％，山梨糖醇4％，蜂蜜干粉6％，大豆蛋白粉6％在混料机混合均匀；
2)经过公知方法的造粒，干燥后加入4％的硬脂酸镁，混合均匀后，送入压片机压片，每片500mg。
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