CN105441365B - 一种产β-半乳糖苷酶的菌株 - Google Patents
一种产β-半乳糖苷酶的菌株 Download PDFInfo
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Abstract
本发明提供了一种产β‑半乳糖苷酶的菌株，本发明筛选所获得的产β‑半乳糖苷酶的菌株，为环状芽孢杆菌，该环状芽孢杆菌所产的β‑半乳糖苷酶，其酶活可达5U/ml及以上，作用于乳糖，能高效地生产低聚半乳糖。本发明的β‑半乳糖苷酶制备低聚半乳糖的方法具有反应时间短、提纯步骤少、酶活性源制备简单，以及产品安全可靠的优点，适于进行大规模生产。
Description
技术领域
本发明涉及一种菌株，具体涉及一种产β-半乳糖苷酶的菌株。
背景技术
低聚半乳糖(galacto-oligosaccharide，GOS)是一种具有天然属性的功能性低聚糖，天然存在于动物母乳中，尤其在人母乳中含量较多。分子结构一般是在半乳糖或葡萄糖分子上连接1～7个半乳糖基。
低聚半乳糖具有较强的耐酸性、耐热性，不被人小肠消化吸收，但可被结肠菌群发酵，对肠内的双歧杆菌和乳酸菌具有同时增殖的作用，且能抑制有害病原菌和腐败菌生长；不被突变链球菌等口腔细菌利用，可降低龋齿；促进钙、镁、钾的吸收，降低钠吸收；降低总胆固醇和甘油三酯水平，可改善脂质代谢；能有效刺激肠道蠕动，减少和防止便秘的发生，调节肠道微生态、促进肠道健康。
低聚半乳糖的安全性已经受到广泛认可，2010年日本的低聚半乳糖成为第二大功能性低聚糖产品，2008年9月我国将其列入新资源食品目录。
低聚半乳糖的制备是以乳糖为原料，经β-半乳糖苷酶催化在低聚半乳糖基上连接1-4个半乳糖分子而制得，是寡糖类混合物。目前β-半乳糖苷酶主要来源于：细菌、霉菌、酵母等微生物，其中细菌中有乳酸菌(Lactic acid bacteria)、大肠杆菌(Escherichiacoli)，霉菌中有黑曲霉(Aspergillus niger)、米曲霉(Aspergillus oryzae)、酵母中有脆壁克鲁维酵母(Kluyveromyces fragilis)、乳酸克鲁维酵母(Kluyveromyces lactis)等，放线菌中有天蓝色链霉菌(Streptomyces coelicolor)等。而利用环状芽孢杆菌生产β-半乳糖苷酶的报道却很少，仅保龄宝生物股份有限公司专利(CN 103911322 A)中有报道，以环状芽孢杆菌生产的β-半乳糖苷酶酶活可达150U/ml。
发明内容
本发明的目的在于克服现有技术存在的不足之处而提供了一种产β-半乳糖苷酶的菌株，本发明筛选所获得的产β-半乳糖苷酶的菌株，为环状芽孢杆菌，其所产的β-半乳糖苷酶酶活可达5.0U/ml，与CN 103911322 A中报道的酶活检测方法不同，且反应时间短，分离利用方便，生产低聚半乳糖转化率高，可大大节省生产成本。
为实现上述目的，所采取的技术方案：一种产β-半乳糖苷酶的菌株，所述菌株命名为环状芽孢杆菌菌株(Bacillus circulans)NTLC 3.002，保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:M 2015666。保藏地址：武汉市武昌区珞珈山武汉大学生命科学学院，邮编430072。保藏时间为2015年11月9日。
本发明提供了一种生产β-半乳糖苷酶的方法，所述方法是将上述所述的菌株接种到发酵培养基中进行发酵培养，然后分离得到β-半乳糖苷酶。
优选地，所述发酵培养基含有以下浓度的组分：乳糖5～20g/L、酵母抽提物7～20g/L、蛋白胨5～20g/L、MgSO4·7H2O 0.1～0.5g/L、Na2HPO4 0.5～2g/L和NaCl 2～10g/L，余量为水，所述发酵培养基的pH为6.5～7.5。
所述发酵培养条件为：在25～30℃，200rpm的条件下培养40h。
本发明提供了一种采用上述所述的方法得到的β-半乳糖苷酶。
本发明提供了一种制备低聚半乳糖的方法，所述方法包括以下步骤：
在浓度为300～500g/L乳糖溶液中加入如权利要求4所述的β-半乳糖苷酶，在35～40℃下反应24～36小时，然后将反应液通过活性炭脱色、离子交换、浓缩后制得低聚半乳糖；
所述反应液中β-半乳糖苷酶的浓度为0.4U/mL。
本发明提供了一种发酵液，所述发酵液是采用上述所述的菌株进行液体培养后得到的。
本发明提供了一种发酵上清液，所述发酵上清液是将上述所述的菌液中的菌体去除后得到的。去除菌体的方法如冷冻离心，过滤等。
本发明提供了上述所述的菌株在制备低聚半乳糖中的用途。
本发明提供了上述所述的菌液在制备低聚半乳糖中的用途。
本发明提供了上述所述的菌液在制备低聚半乳糖中的用途。
本发明的有益效果在于：本发明提供了一种产β-半乳糖苷酶的菌株，本发明筛选所获得的产β-半乳糖苷酶的菌株，为环状芽孢杆菌，该环状芽孢杆菌所产的β-半乳糖苷酶，其酶活可达5U/ml及以上，作用于乳糖，能高效地生产低聚半乳糖。本发明的β-半乳糖苷酶制备低聚半乳糖的方法具有反应时间短、提纯步骤少、酶活性源制备简单，以及产品安全可靠的优点，适于进行大规模生产。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例对本发明作进一步说明。
实施例1
本发明从土壤中分离筛选到一株产生β-半乳糖苷酶的菌株，是一株产β-半乳糖苷酶的低聚半乳糖生产菌，其分类命名为环状芽孢杆菌菌株(Bacillus circulans)NTLC3.002，已于2015年11月9日保存于中国武汉的中国典型培养物保藏中心，保藏编号：CCTCCNO:M 2015666。
1、β-半乳糖苷酶酶活检测方法：将ONPG(邻硝基苯β-D-半乳吡喃糖苷)用pH7.0、0.1M的磷酸钾缓冲液配成1mg/mL的溶液。取2mL该溶液在35℃水浴中保温10min，然后加入0.5mL酶液准确反应15min。加入2.5mL 0.15mol/L的碳酸钠溶液终止反应。静止2～3分钟后，在420nm处测吸光值，根据标准曲线测得酶活。
酶活力单位定义：在上述反应条件下，每分钟分解ONPG，生成1μmol邻硝基苯酚(ONP)所需要的酶量为1个酶活力单位(U)。
酶活计算：
通过ONPG溶液酶解反应后测得的吸光度根据标准曲线算出产物ONP的量，然后乘以稀释倍数10，再除以反应时间15，即为酶活。
2、发酵菌体：
使用本发明所述环状芽孢杆菌为出发菌株；
培养基，以g/L计为：乳糖5～20、酵母抽提物7～20、蛋白胨5～20、MgSO4·7H2O0.1～0.5、Na2HPO4 0.5～2、Nacl2～10，pH 6.5～7.5；在25～30℃，200rpm的条件下培养40h；冷冻离心，获得粗酶液，粗酶液中β-半乳糖苷酶活力可达5.0U/ml及以上；
3、酶转化法制备低聚半乳糖：
在乳糖溶液中加入β-半乳糖苷酶，反应体系为200L，乳糖浓度为300g/L-500g/L，β-半乳糖苷酶浓度为0.4U/ml-1.0U/ml，在35～40℃下进行24～36小时的酶反应，制得纯度60％以上低聚半乳糖，然后通过活性炭脱色、离子交换、浓缩后最终可制得低聚半乳糖产品。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (7)
1.一种产β-半乳糖苷酶的菌株，其特征在于，所述菌株命名为环状芽孢杆菌菌株(Bacillus circulans)NTLC 3.002，保藏于中国典型培养物保藏中心，保藏编号为CCTCCNO:M2015666。
2.一种生产β-半乳糖苷酶的方法，其特征在于，所述方法是将如权利要求1所述的菌株接种到发酵培养基中进行发酵培养，然后分离得到β-半乳糖苷酶。
3.根据权利要求2所述的方法，其特征在于，所述发酵培养基含有以下浓度的组分：乳糖5～20g/L、酵母抽提物7～20g/L、蛋白胨5～20g/L、MgSO4·7H2O 0.1～0.5g/L、Na2HPO40.5～2g/L和NaCl 2～10g/L，余量为水，所述发酵培养基的pH为6.5～7.5；所述发酵培养条件为：在25～30℃，200rpm的条件下培养40h。
4.一种制备低聚半乳糖的方法，其特征在于，所述方法包括以下步骤：在浓度为300～500g/L的乳糖溶液中加入如权利要求2中所述的β-半乳糖苷酶，在35～40℃下反应24～36小时，然后将反应液通过活性炭脱色、离子交换、浓缩后制得低聚半乳糖；所述反应液中β-半乳糖苷酶的浓度为0.4U/mL。
5.一种发酵液，其特征在于，所述发酵液是采用如权利要求1所述的菌株进行液体培养后得到的。
6.如权利要求1所述的菌株在制备低聚半乳糖中的用途。
7.如权利要求5所述的发酵液在制备低聚半乳糖中的用途。
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