CN109022330B - 一株具有高蛋白水解能力并产酪香味的乳酸乳球菌bl19及其应用 - Google Patents
一株具有高蛋白水解能力并产酪香味的乳酸乳球菌bl19及其应用 Download PDFInfo
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Abstract
本发明提供了一株具有高蛋白水解能力并产酪香味的乳酸乳球菌BL19，属于微生物技术领域，所述乳酸乳球菌BL19保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心，保藏号为CGMCC No.16242。本发明还提供了所述乳酸乳球菌BL19在制备发酵乳制品中的应用，所述乳酸乳球菌BL19能够快速发酵牛乳；所述乳酸乳球菌BL19具有高蛋白水解能力，同时可以产酪香味，赋予发酵乳制品独特的风味和口感。
Description
技术领域
本发明属于微生物技术领域，尤其涉及一株具有高蛋白水解能力并产酪香味的乳酸乳球菌BL19及其应用。
背景技术
乳酸菌(Lactic acid bacteria，LAB)因其可以在发酵过程中产生乳酸及其他代谢产物，乳酸菌常被用于食品发酵中并赋予发酵食品独特的质地、风味和营养。乳酸菌在乳制品中的主要作用是产酸并产生独特的风味物质。乳酸乳球菌、嗜热链球菌和保加利亚乳杆菌作为发酵乳工业生产中的重要的工业生产菌株，拥有广阔的市场价值。乳酸乳球菌因其独特的发酵特性，常被应用到奶酪和酸奶的生产中用以赋予发酵乳制品独特的风味和口感。
乳酸菌发酵过程中产酸快慢与发酵乳制品生产最为相关，而乳酸乳球菌产黏特性良好可以赋予发酵乳制品浓厚粘稠的口感。乳酸乳球菌特有的酪香味的产生可以赋予发酵乳制品独特的风味。近年来，随着我国发酵乳制品行业的不断发展壮大，良好发酵特性菌株不足的问题日益明显，发酵菌种产黏产酪酸性能较差且不稳定，无法满发酵乳制品行业日益扩大的需求以及消费者对发酵乳制品不断提升的品质要求。
发明内容
有鉴于此，本发明的目的在于提供一株具有高蛋白水解能力并产酪香味乳酸乳球菌BL19及其应用。
为了实现上述目的，本发明提供了以下技术方案：一株具有蛋白水解能力并产酪香味的乳酸乳球菌BL19，保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心，保藏号为CGMCC No.16242。
本申请的另一目的是提供所述的蛋白水解能力并产酪香味的乳酸乳球菌BL19在制备发酵食品中的应用。
优选的，所述发酵食品为发酵乳制品或保健食品。
优选的，当所述食品为保健食品时，所述食品还包括食品学上的载体或添加剂支撑。
优选的，所述食品的剂型为散剂、颗粒剂、片剂、胶囊剂、悬浮剂、乳剂、糖浆剂、液体剂、浸膏、茶、果冻或饮料。
优选的，当食品为发酵乳制品时，所述食品还包括明胶、果胶、羟丙基二淀粉磷酸酯和琼脂中的一种或者多种。
优选的，所述食品还包括发酵剂，所述发酵剂为保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌、干酪乳杆菌和鼠李糖乳杆菌中的一种或几种的组合。
本申请的另一目的是提供一种利用所述乳酸乳球菌BL19制备发酵乳制品的制备方法，所述方法包括：将所述的乳酸乳球菌BL19接种到灭菌乳品中，隔氧保温发酵。
可选地，所述的乳酸乳球菌BL19的接种量为0.5×109CFU/mL～3×109CFU/mL，优选为1×109CFU/mL～2×109CFU/mL；保温温度为36～38℃；保温时间为22～26h，优选为24h。
在一种可实现的方式中，利用所述乳酸乳球菌BL19发酵乳品的方法可以为：将乳酸乳球菌BL19加入灭菌牛乳中，接种量为1×109CFU/mL；摇匀震荡(30～50)s，使得所述乳酸乳球菌在乳品中分散均匀，从而有利于菌体生长和发酵；将摇匀后的乳品混合体系置于37℃恒温培养箱中，37℃培养有利于菌体的快速生长繁殖，可节约时间成本。所述乳酸乳球菌BL19在上述条件下处于最佳生长状态。
本发明的有益效果：本发明提供的具有高蛋白水解能力并产酪香味的乳酸乳球菌(Lactococcus lactis)BL19能够快速发酵牛乳；具有高蛋白水解能力，同时可以产酪香味，赋予发酵乳制品独特的风味。
生物保藏说明
本发明提供的乳酸乳球菌(Lactococcus lactis)BL19保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心，保藏编号：CGMCC No.16242，保藏日期2018年08月09日，保藏地址北京市朝阳区北辰路西路1号院3号。
附图说明
图1为乳酸乳球菌(Lactococcus lactis)BL19的16S rDNA序列与乳酸乳球菌模式菌株16S rDNA基因组序列系统发育树；
图2为乳酸乳球菌(Lactococcus lactis)BL19菌体电镜图片；
图3为乳酸乳球菌(Lactococcus lactis)BL19菌落形态图片；
图4为乳酸乳球菌(Lactococcus lactis)BL19发酵酸乳中游离氨基氮含量测定；
图5为乳酸乳球菌(Lactococcus lactis)BL19发酵酸乳电子舌测定图；
图6为乳酸乳球菌(Lactococcus lactis)BL19发酵酸乳感官评定。
具体实施方式
本发明提供了一株具有高蛋白水解能力并产酪香味的乳酸乳球菌BL19，保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心，保藏号为CGMCC No.16242。本发明中，所述乳酸乳球菌BL19具有蛋白水解能力良好。本发明中所述乳酸乳球菌BL19的形态学特征如下：细胞呈椭球状，长4.9-5.2μm，宽4.4-4.7μm，呈成对或短链状出现；在M17培养基上生长生长形成乳白色菌落，不透明，圆形，表面光滑，中央凸起，直径约为1.1～1.4mm。本发明中所述的乳酸乳球菌BL19原始来源于俄罗斯布里亚特共和国乌尔达阿伽村自然发酵奶酪样品。
本发明还提供了所述的乳酸乳球菌BL19在制备发酵食品中的应用。在本发明中，所述食品为发酵乳制品或保健食品；所述发酵乳制品优选的包括酸奶、开菲尔、发酵酪乳、酸奶酒、乳酒或嚼口奶等。本发明所述所述发酵乳制品中优选的还包括辅料；所述辅料优选的包括明胶、果胶、羟丙基二淀粉磷酸酯和琼脂中的一种或几种。本发明中所述应用优选为以乳酸乳球菌BL19为发酵菌种发酵获得发酵乳制品。
当本发明所述的食品为发酵乳制品时，所述食品优选的还包括发酵剂，所述发酵剂还包括保加利亚乳杆菌、嗜热链球菌、嗜酸乳杆菌、双歧杆菌、干酪乳杆菌和鼠李糖乳杆菌中的一种或几种。本发明对所述发酵剂中各个组分的比例关系没有特殊限定，只要能够实现发酵乳制品即可。
本发明中提供了一种利用所述乳酸乳球菌BL19制备发酵乳制品的方法，所述方法包括以下步骤：将所述乳酸乳球菌BL19接种到灭菌乳品中隔氧保温发酵。在本发明中所述活化的乳酸乳球菌优选为乳酸乳球菌的M17培养液，其中乳酸乳球菌的浓度优选为(1～10)×109cfu/ml；所述乳酸乳球菌为脱脂乳的0.03wt‰；所述隔氧保温发酵的温度优选为36～38℃，更优选为37℃，所述隔氧保温发酵的时间优选为22～26h，更优选为24h。
本发明提供包括所述的乳酸乳球菌BL19的保健食品。所述保健食品优选的还包括食品学上允许的载体或添加剂。在本发明中所述保健食品的剂型优选为散剂、颗粒剂、片剂、胶囊剂、悬浮剂、乳剂、糖浆剂和浸膏。在本发明中所述保健食品为优选为果冻或饮料；所述果冻优选为酸乳果冻；所述饮料优选的包括酸乳饮料。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
本申请以下实验例中试验菌株来源分别为：
乳酸乳球菌BL19为实施例1制取；
所述发酵剂A购自意大利乳品研发中心CSL；
所述发酵剂B购自科汉森(中国)有限责任公司。
实施例1乳酸乳球菌(Lactococcus lactis)BL19的分离纯化及鉴定
乳酸乳球菌(Lactococcus lactis)BL19分离自俄罗斯布里亚特共和国乌尔达阿伽村自然发酵奶酪样品中分离得到，具体地，是通过TPY选择性培养基分离，通过挑取单个菌株扩培得到。
将乳酸乳球菌(Lactococcus lactis)接入到M17液体培养基，37℃培养24h，继代培养3代，恢复菌株活力。对菌液进行10倍比稀释，得到10-1～10-7稀释梯度。分别吸取200μL10-4、10-5、10-6、10-7梯度稀释液，均匀涂布于M17固体培养基平皿内，37℃厌氧培养24h。挑取形态、大小、颜色不同的单克隆接种于液体培养基中，于37℃恒温厌氧培养箱中培养24h。待菌株生长良好后，进行革兰氏染色、镜检。保存分离株，并提取菌株基因组DNA用于后续测定分析。
其中M17培养基的组成是：大豆胨5g、蛋白胨2.5g、酪蛋白胨2.5g、牛肉浸粉5g、酵母浸粉2.5g、乳糖5g、抗坏血酸钠0.5g、β-甘油磷酸钠19g、硫酸镁0.25g，调节pH至7.2，琼脂12.75g，121℃、15min灭菌。
b.分子生物学鉴定
将冷冻保存的供试菌株接种于TPY增菌液体培养基中，30℃恒温培养24h，经TPY传代培养2～3代后，取2mL对数生长末期的菌体培养物置于无菌EP管内离心，经8000×g离心3min(4℃)后收集菌体，弃上清，采用乳酸菌专用CTAB冻融法提取菌株的基因组DNA。
其中，TPY增菌培养液的组成是：乳糖10g、牛肉膏5g、酵母粉5g、酪蛋白胨10g、大豆蛋白胨5g、磷酸氢二钾2.5g、磷酸二氢钾2.5g、硫酸镁0.1g、吐温800.25g、L-半胱氨酸盐酸盐0.5g、15g琼脂、蒸馏水1L，121℃、15min灭菌。
利用Sanger测序方法对乳酸乳球菌(Lactococcus lactis)BL19的16S rDNA序列进行测序，与乳酸乳球菌4株模式菌株和鱼乳球菌模式菌株的16S rDNA序列构建系统发育树。乳酸乳球菌(Lactococcus lactis)BL19与乳酸乳球菌乳酸亚种模式菌株亲缘关系较近，其系统发育树如图1所示。
c.形态学特性
本申请的乳酸乳球菌(Lactococcus lactis)BL19具有如下形态学特征：细胞呈椭球状，长4.9-5.2μm，宽4.4-4.7μm，呈成对，或短链状出现(见图2)。
d.菌落形态特性
本申请的乳酸乳球菌(Lactococcus lactis)BL19在M17培养基上生长形成乳白色菌落，不透明，圆形，表面光滑，中央凸起，直径约为1.1-1.4mm(见图3)。
实施例2乳酸乳球菌(Lactococcus lactis)BL19发酵性能研究
将筛选后得到的乳酸乳球菌(Lactococcus lactis)BL19、A与B分别进行发酵实验制备发酵乳，用活化第三代的乳酸乳球菌M17培养液(1×109cfu/ml)接种到100g的脱脂乳中，37℃静置发酵24h，即得到BL19的发酵乳。
对3株乳酸乳球菌的pH进行测定。结果显示三株菌在发酵8h后的pH值存在差异，差异显著。其中乳酸乳球菌BL19发酵牛乳产酸速率较快使发酵牛乳率先达到发酵终点。结果显示，乳酸乳球菌BL19发酵牛乳达到发酵终点所用时间最短。
表1 乳酸乳球菌(Lactococcus lactis)发酵的pH和滴定酸度的测定
乳酸乳球菌(Lactococcus lactis)BL19发酵酸乳的游离氨基氮含量测定
将筛选后得到的乳酸乳球菌(Lactococcus lactis)BL19、A与B，对3株乳酸乳球菌发酵酸乳游离氨基氮进行测定，从而评估其蛋白水解能力，结果如图4所示。结果显示，乳酸乳球菌(Lactococcus lactis)BL19发酵牛乳游离氨基氮含量较高为7.0mmol/L，均显著高于另外两株乳酸乳球菌(A菌株为1.8mmol/L,B菌株为1.9mmol/L。上述结果表明，BL19具有良好的蛋白水解能力。
乳酸乳球菌(Lactococcus lactis)BL19发酵牛乳的电子舌测定
3株乳酸乳球菌在发酵牛乳24h后，使用电子舌测定风味物质。结果如图5所示，结果显示乳酸乳球菌(Lactococcus lactis)BL19与其余两株相比无误明显的苦涩或辛辣味。
乳酸乳球菌(Lactococcus lactis)BL19发酵牛乳的感官评定
3株乳酸乳球菌(Lactococcus lactis)发酵酸乳后，20名感官评定人员对发酵豆乳进行感官评定，以评定的标准如下表2所示(总分100分)。结果如图6所示，结果显示乳酸乳球菌(Lactococcus lactis)BL19与发酵酸乳的感官评价显著高于其余两株。BL19得分最高为83分，其余两株菌得分为75和76。
表2 感官评定指标
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.一株具有蛋白水解能力并产酪香味的乳酸乳球菌BL19，保藏于中国微生物菌种保藏管理委员会普通微生物菌种保藏中心，保藏号为CGMCC16242。
2.权利要求1所述的具有蛋白水解能力并产酪香味的乳酸乳球菌BL19在制备发酵食品中的应用。
3.利用乳酸乳球菌BL19制备食品的方法，包括以下步骤：将所述乳酸乳球菌BL19接种到灭菌乳中，隔氧保温发酵22~26h；所述保温的温度为36~38℃；所述乳酸乳球菌BL19保藏号为CGMCC16242。
4.根据权利要求3所述的制备方法，其特征在于，所述乳酸乳球菌BL19接种量为0.5×109 CFU/mL~3×109 CFU/mL。
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