CN104357361B - 一株产几丁质酶的枯草芽孢杆菌及其应用 - Google Patents
一株产几丁质酶的枯草芽孢杆菌及其应用 Download PDFInfo
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Abstract
本发明的目的是提供一株产几丁质酶的枯草芽孢杆菌其应用，所述产几丁质酶枯草芽孢杆菌经等离子体诱变获得，具有更高的几丁质酶酶活力和蛋白酶酶活力，可作为发酵剂、益生菌和防腐剂，广泛应用于酶制剂和食品工业。本发明的枯草芽孢杆菌的保藏编号为CGMCC No.9166。本发明的菌株具有较高的几丁质酶酶活力和蛋白酶酶活力，且该菌株营养需要简单，繁殖力强，可作为发酵剂、益生菌和防腐剂，应用前景广阔。
Description
技术领域
本发明属于微生物应用技术领域，具体涉及一株产几丁质酶枯草芽孢杆菌及其应用。
背景技术
几丁质酶(chitinase)能催化降解甲壳素(几丁质)生成几丁寡糖，几丁寡糖具有水溶性，是甲壳素、壳聚糖产品的升级产品，对腐败菌的抑制效果显著，是性能优良的天然食品防腐保鲜剂。同时，几丁寡糖还具有能促进有益微生物(如双歧杆菌、乳酸菌)的生长，改善肠道微生物菌群，提高机体免疫力，促进肝脾抗体形成、促进钙及矿物质的吸收、降血脂、去除氧负离子自由基延缓衰老、抑制肿瘤细胞生长等功能，可应用于医药、功能性食品、化妆品等领域。
我国有广阔的海岸线和大面积水产养殖基地，几丁质资源丰富，但开发利用率低，生产水平不高，产品档次低，造成资料浪费的同时也造成了环境污染。而生产几丁寡糖就是一条利用几丁质开发高附加值产品的一条重要途径。目前，几丁寡糖的生产主要有化学降解法和生物酶法两种方法；但化学降解法成本高，不易控制，产物聚合度较低(生物活性低)且多为单糖，应用价值不高，而且会带来的严重的酸污染，其使用受到严格限制。而生物酶法具有催化效率高，反应温，具有特异性，易控制，可生成不同聚合度的几丁寡糖，具有良好的应用前景，但一个限定因素就是需要具有高几丁质酶酶活性的微生物。
发明内容
本发明的目的是提供一株产几丁质酶的枯草芽孢杆菌其应用，所述产几丁质酶枯草芽孢杆菌经等离子体诱变获得，具有更高的几丁质酶酶活力和蛋白酶酶活力，可作为发酵剂、益生菌和防腐剂，广泛应用于酶制剂和食品工业。
本发明一方面提供了一株枯草芽孢杆菌(Bacillus subtilis)PLa119，于2014年5月14日保藏于北京市朝阳区北辰西路1号院3号的的中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.9166。
本发明的枯草芽孢杆菌可应用于酶制剂、菌制剂、防腐剂和制备几丁寡糖。
本发明的另一方面提供了含有上述枯草芽孢杆菌的虾头酱。
所述的虾头酱，制备过程为：虾头清洗打浆，接入枯草芽孢杆菌PLa119，经半固态发酵，加入香辛料，制得虾头酱成品。
上述枯草芽孢杆菌可作为益生菌生产菌制剂，制备过程为：虾壳烘干，粉碎打浆，接入枯草芽孢杆菌PLa119，经半固态发酵，加入奶粉和蔗糖，制得枯草芽孢杆菌菌制剂。
本发明还提供一种几丁质酶的制备方法，是用上述的枯草芽孢杆菌PLa119接种到培养基中发酵后制备的；其中培养基组成如下：胶体几丁质12g/L、豆粕9.5g/L、蛋白胨5g/L、酵母膏4g/L、KH2PO40.3g/L、葡萄糖0.5g/L、MgSO4·7H2O 0.6g/L g/L、FeSO4·7H2O 0.01g/L、ZnSO40.01g/L。最佳发酵条件为：温度为35℃、转速为180r/min、接种量为4％、初始pH为7.0、发酵时间为48h。
本发明经等离子体诱变筛选到一株枯草芽孢杆菌PLa119。该菌株具有较高的几丁质酶酶活力和蛋白酶酶活力，且该菌株营养需要简单，繁殖力强，可作为发酵剂、益生菌和防腐剂，应用前景广阔。
具体实施方式
下面结合实施例对本发明进行详细的描述。
实施例1：枯草芽孢杆菌PLa119的选育及其产酶条件优化
1、出发菌株的枯草芽孢杆菌筛选自虾头富集过的土壤，经青岛农业大学食品微生物实验室鉴定、保藏，具有产几丁质酶和蛋白酶能力。
2、培养基
(1)富集培养基(g/L)：牛肉膏3，蛋白胨10，NaCl 5，pH 7.0。
(2)分离培养基(g/L)：胶体几丁质2.5，MgSO4·7H2O 0.5，K2HPO4，KH2PO40.3，FeSO4·7H2O 0.01，(NH4)2SO43，琼脂20。pH 7.0，121℃，灭菌20min。
(3)摇瓶筛选培养基(产酶培养基)(g/L)：胶体几丁质10，豆粕10，蛋白胨5，酵母膏5，K2HPO40.7，KH2PO40.3，葡萄糖1，MgSO4·7H2O0.5，FeSO4·7H2O 0.01，ZnSO40.01。pH 7.0，121℃，灭菌20min。
3、等离子体诱变选育
取出发菌株枯草芽孢杆菌，加入富集培养基，37℃培养24h，梯度稀释至106cfu/mL，常温等离子体诱变处理10min，将诱变后菌液涂布于分离培养基，于37℃培养72h，挑取菌落典型且圈径比较大菌株，进行重复分离纯化，本次实验共获得12株菌株，并分别依次命名为PLa111、PLa112、PLa113至PLa122。将纯化得到的菌株接种到摇瓶筛选培养基，测试各菌株在37℃、180r/min培养72h时上述12株菌的几丁质酶和蛋白酶活力，其中菌株PLa119的几丁质酶酶活力最高，达到1.92±0.02U/mL，且兼具产蛋白酶，蛋白酶活力95.66U/mL(表1)。
表1：12株菌在72h几丁质酶及蛋白酶酶活力实验结果
| 菌株 | 几丁质酶酶活力(U/mL) | 蛋白酶酶活力(U/mL) |
| 对照组 | 0.28±0.01 | 84.32±0.01 |
| PLa111 | 0.79±0.02 | 75.34±0.02 |
| PLa112 | 1.12±0.01 | 85.61±0.02 |
| PLa113 | 0.59±0.01 | 80.42±0.01 |
| PLa114 | 0.61±0.01 | 65.78±0.02 |
| PLa115 | 0.43±0.02 | 73.21±0.01 |
| PLa116 | 0.80±0.02 | 93.46±0.01 |
| PLa117 | 0.73±0.04 | 98.10±0.02 |
| PLa118 | 0.56±0.02 | 86.12±0.01 |
| PLa119 | 1.92±0.03 | 95.66±0.02 |
| PLa120 | 0.85±0.03 | 93.15±0.02 |
| PLa121 | 0.81±0.05 | 87.69±0.01 |
| PLa122 | 1.67±0.02 | 90.01±0.03 |
考察了其中三株产酶水平较好的菌株PLa112、PLa119、PLa122的酶液热稳定性，结果菌株PLa119的酶液活力稳定性最好，其在35～55℃范围内，几丁质酶活力稳定，45℃时几丁质酶活力达最高值，超过65℃时10min后酶活力有所下降。
4、产几丁质酶条件优化
通过部分因素设计试验找出了显著影响该菌产几丁质酶的主要因素为：胶体几丁质、豆粕和MgSO4，再通过中心组合试验，优化确定出最佳培养基配方为：胶体几丁质12g/L、豆粕9.5g/L、蛋白胨5g/L、酵母膏4g/L、KH2PO40.3g/L、葡萄糖0.5g/L、MgSO4·7H2O 0.6g/L g/L、FeSO4·7H2O 0.01g/L、ZnSO40.01g/L。单因素及正交实验确定的最佳发酵条件为：温度为35℃、转速为180r/min、接种量为4％、初始pH为7.0、发酵时间为48h。在此条件测得枯草芽孢杆菌PLa119的几丁质酶酶活力为2.59±0.05U/mL，蛋白酶酶活力为146±0.07U/mL。比在原始产酶培养基及发酵条件下几丁质酶活力提高了35％，蛋白酶活力提高了51％。
实施例2：枯草芽孢杆菌PLa119制备几丁寡糖实验
1、胶体几丁质的制备
将细粉几丁质用1％的醋酸溶液配制成浓度为5％的均一液。
2、几丁质酶酶液制备
枯草芽孢杆菌PLa119发酵液经离心(5000r/min、5min)去菌体，上清液经低温旋转蒸发浓缩，70％硫酸铵沉淀，Sephadex G25脱盐，经低温旋转蒸发浓缩至几丁质酶酶活力为10U/mL。
3、几丁寡糖制备
将几丁质酶液与5％的胶体几丁质按照1:10混合，37℃、pH7.0条件下保温反应10h后，加热灭酶，3000r/min离心10min，取上清液，经低温旋转蒸发浓缩，80℃烘干制得几丁寡糖粗品，经离子色谱分析验证，几丁寡糖粗品聚合度低于10。
实施例3：枯草芽孢杆菌PLa119制备无化学防腐剂虾头酱实验
取无污染虾头，室温下与水按照重量比1:1.5进行混合打浆，调pH为7.0，接入活化的枯草芽孢杆菌PLa119，35℃发酵培养48h，可获得富含氨基酸、短肽和几丁寡糖的虾头发酵液，加入黄酒10％(v/v)、糖16.8％、大蒜9.89％、生姜1.5％、味精1.25％、食盐15.98％，后酵15天，氨基酸含量为0.067±0.03g/g，几丁寡糖含量0.011±0.001g/g，制得无化学防腐剂虾头酱，
实施例4：枯草芽孢杆菌PLa119制备益生菌制剂实验
取无污染虾壳和虾头，110℃烘干，粉碎，与水按照重量比1:1.5混合打浆，接入枯草芽孢杆菌PLa119，35℃发酵培养48h，加入奶粉和蔗糖，80℃烘干制成1-10亿/g的制得枯草芽孢杆菌益生菌制剂，氨基酸含量为0.036±0.011g/g，几丁寡糖含量为0.032±0.008g/g，是良好的功能性益生菌制剂。另外，其Fe、Zn、Ca含量分别为128.1±2.9mg/kg、11.3±0.8mg/kg和387.5±1.6mg/kg，每天食用94g枯草芽孢杆菌益生菌制剂就可达到我国推荐铁(12mg/d)的摄入量，也能起到锌和钙补充剂的效果。
Claims (5)
1.一株枯草芽孢杆菌，其特征在于，所述的枯草芽孢杆菌的保藏编号为CGMCC No.9166。
2.权利要求1所述的枯草芽孢杆菌在制备酶制剂、菌制剂、防腐剂或制备几丁寡糖中的应用。
3.一种虾头酱，其特征在于，所述的虾头酱的制备过程如下：将虾头清洗打浆，接入权利要求1所述的枯草芽孢杆菌，经半固态发酵，加入香辛料，制得虾头酱成品。
4.一种菌制剂，其特征在于，所述的菌制剂的制备过程如下：将虾壳烘干，粉碎打浆，接入权利要求1所述的枯草芽孢杆菌，经半固态发酵，加入奶粉和蔗糖，制得枯草芽孢杆菌菌制剂。
5.一种几丁质酶的制备方法，其特征在于，所述的制备方法，是用权利要求1所述的枯草芽孢杆菌接种到培养基中发酵制备几丁质酶；其中培养基组成如下：胶体几丁质12g/L、豆粕9.5g/L、蛋白胨5g/L、酵母膏4g/L、KH2PO40.3g/L、葡萄糖0.5g/L、MgSO4·7H2O 0.6g/L、FeSO4·7H2O 0.01g/L、ZnSO40.01g/L。
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