CN116218700A - 耐高酸酒酒球菌菌株及其在葡萄酒mlf中的应用 - Google Patents
耐高酸酒酒球菌菌株及其在葡萄酒mlf中的应用 Download PDFInfo
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Abstract
本发明公开了一种耐高酸酒酒球菌菌株,该菌株为SX‑a3,分类名为:酒酒球菌Oenococcus oeni,已于2022年8月10日保藏于位于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.25512;本发明还公开了耐高酸酒酒球菌菌株在葡萄酒MLF中的应用。本发明提供的耐酸酒酒球菌菌株,苹果酸‑乳酸酶活性更高,发酵效率更快,并且能够顺利启动并完成pH3.2、酒精度12.5%vol高酸葡萄酒中MLF。
Description
技术领域
本发明属于微生物菌种选育技术领域,具体涉及耐高酸酒酒球菌菌株,还涉及该菌种在葡萄酒MLF中的应用。
背景技术
在葡萄酒生产过程中,酒精发酵后的苹果酸-乳酸发酵(Malolacticfermentation,MLF)可降低葡萄酒酸度、增加香气复杂度、改善葡萄酒酸涩口感、增强葡萄酒微生物稳定性,进而提高葡萄酒的品质,是优质葡萄酒酿造的一个重要工艺过程。酒酒球菌(Oenococcus oeni)是启动和完成苹果酸-乳酸发酵的最主要菌株,通常酒酒球菌最适生长pH为4.5~5.6,但葡萄酒的低pH范围(通常小于3.5)非常不利于酒酒球菌的生长代谢。而亟需MLF改善酸涩口感的葡萄酒pH更低,在葡萄酒生产过程中这样的高酸度条件通常会造成MLF困难,一些情况下甚至无法正常启动。因此,本土优良耐酸酒酒球菌菌种的选育以及高酸度条件下的MLF工艺优化,是葡萄酒行业一直以来的研究热点,对葡萄酒产业具有极其重要的意义。
通过从我国葡萄酒产区筛选天然耐高酸的酒酒球菌菌株,对于高酸原料葡萄酒的酿造具有实际应用意义。
发明内容
本发明的第一个目的是提供耐高酸酒酒球菌菌株,提供了优良耐酸酒酒球菌,能够快速启动并完成高酸葡萄酒环境下的MLF,解决了高酸度条件通常会造成酒酒球菌MLF困难,一些情况下甚至无法正常启动的问题。
本发明的第二个目的是提供了上述耐高酸酒酒球菌菌株在葡萄酒MLF中的应用。
本发明所采用的第一种技术方案是,耐高酸酒酒球菌菌株,该菌株为SX-a3,名称为:酒酒球菌Oenococcus oeni,已于2022年8月10日保藏于位于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.25512。
本发明的第一种技术方案的特点还在于:
上述的耐高酸酒酒球菌菌株,按以下选育过程的步骤实施得到:
步骤1、菌株分离,从不同产区的野生酒酒球菌菌株挑取单菌落并活化培养;
步骤2、菌株筛选,比较分析步骤1中分离获得的酒酒球菌单菌落的耐酸能力,获得耐酸能力最高的酒酒球菌菌株,冻存保藏。
步骤1具体按以下步骤实施:
步骤1.1、利用固体ATB培养基分离不同产区葡萄酒中的野生酒酒球菌菌株,26℃培养6-7天;
步骤1.2、挑取步骤1.1中培养基中长出来的单菌落,进行种属特异性PCR鉴定;
步骤1.3、挑取步骤1.2中PCR鉴定为酒酒球菌的单菌落于液体ATB培养基中活化培养,26℃,静置培养3-5天。
步骤2具体按以下步骤实施:
步骤2.1、将步骤1中分离获得的酒酒球菌单菌落所活化的菌液,以4%v/v接种量分别接种于pH 3.0液体ATB培养基中,26℃静置培养;
步骤2.2、相同条件下培养酒酒球菌SX-1a和商业菌株31-DH,作为对照组;
步骤2.3、每隔12小时测定其在紫外分光光度计600nm下的吸光度,每个梯度每个菌株重复三次;
步骤2.4、对比获得耐酸能力最高的酒酒球菌菌株,冻存保藏。
本发明采用的第二种技术方案是,上述耐高酸酒酒球菌菌株在葡萄酒MLF中的应用。
本发明的第二种技术方案的特点还在于:
上述的耐高酸酒酒球菌菌株在葡萄酒MLF中的应用,具体按以下步骤实施:
步骤1、将获得的酒酒球菌单菌落所活化的菌液,以2%v/v接种量分别接种于pH4.8液体ATB培养基中,26℃静置培养3-5天;
步骤2、将种子液经短暂胁迫驯化后,按5%接种量(v/v)分别接种到葡萄酒酒体中,间隔1.5天测定葡萄酒中的残留的苹果酸含量。
本发明的有益效果是:
(1)本发明提供的耐酸酒酒球菌菌株,在pH 3.0ATB培养基长势良好,苹果酸-乳酸酶活性较商业菌株31-DH以及实验室前期筛选保藏优良酒酒球菌SX-1a更高,且启动并完成葡萄酒苹果酸-乳酸发酵的效率更快。
(2)本发明提供的耐酸酒酒球菌菌株,填补了本土发酵剂的开发几乎空白、没有突出风格的现状,削减严重的同质化问题,对于高酸度条件下相应的发酵调控策略提供了优良的MLF发酵剂。
附图说明
图1是本发明的酒酒球菌SX-a3与商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在pH 3.0下的生长曲线;
图2是本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在pH 3.2葡萄酒中不同发酵时间残余L-苹果酸含量检测结果图;
图3是本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在pH 3.5葡萄酒中不同发酵时间残余L-苹果酸含量检测结果图;
图4是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,葡萄酒高级酯类含量的统计结果图;
图5是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,葡萄酒高级醇类含量的统计结果图;
图6是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,葡萄酒脂肪酸类含量的统计结果图;
图7是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,葡萄酒醛酮类含量的统计结果图;
图8是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,葡萄酒萜烯类含量的统计结果图;
图9是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,葡萄酒总挥发性香气物质含量的统计结果图;
图10是在pH 3.5的葡萄酒中本发明的酒酒球菌SX-a3、商业菌株31-DH以及实验室前期保藏优良酒酒球菌SX-1a在苹果酸-乳酸发酵后,香气化合物主成分分析的因子载荷图及样品分布图。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
本发明提供了一种耐高酸酒酒球菌菌株,该菌株为SX-a3,名称为:酒酒球菌Oenococcus oeni,其核苷酸序列如SEQ ID NO:1所示。
该菌株已在国家知识产权局指定的保藏单位中国普通微生物菌种保藏中心保藏。
生物材料保藏信息:
名称:酒酒球菌Oenococcus oeni SX-a3株
保藏编号:CGMCC No.25512。
保藏单位:中国微生物菌种保藏管理委员会普通微生物中心
保藏时间:2022年8月10日
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
上述耐高酸酒酒球菌菌株的选育过程,具体按下面步骤实施:
步骤1、菌株分离,从不同产区的野生酒酒球菌菌株挑取单菌落并活化培养;
步骤1.1、利用固体ATB培养基分离不同产区葡萄酒中的野生酒酒球菌菌株,26℃培养6-7天;
步骤1.2、挑取步骤1.1中培养基中长出来的单菌落,进行种属特异性PCR鉴定;
步骤1.3、挑取步骤1.2中PCR鉴定为酒酒球菌的单菌落于液体ATB培养基中活化培养,26℃,静置培养3-5天;
步骤2、菌株筛选,比较分析步骤1中分离获得的酒酒球菌单菌落的耐酸能力,获得耐酸能力最高的酒酒球菌菌株,冻存保藏。
步骤2.1、将步骤1中分离获得的酒酒球菌单菌落所活化的菌液,以4%v/v接种量分别接种于pH 3.0液体ATB培养基中,26℃静置培养;
步骤2.2、相同条件下培养酒酒球菌SX-1a和商业菌株31-DH,作为对照组;
步骤2.3、每隔12小时测定其在紫外分光光度计600nm下的吸光度,每个梯度每个菌株重复三次;
步骤2.4、对比获得耐酸能力最高的酒酒球菌菌株,冻存保藏。
本发明还提供了上述耐高酸酒酒球菌菌株在葡萄酒MLF中的应用,具体按以下步骤实施:
步骤1、将获得的酒酒球菌单菌落所活化的菌液,以2%v/v接种量分别接种于pH4.8液体ATB培养基中,26℃静置培养3-5天;
步骤2、将种子液经短暂胁迫驯化后,按5%接种量(v/v)分别接种到葡萄酒酒体中,间隔1.5天测定葡萄酒中的残留的苹果酸含量。
ATB固体培养基的配值:10g葡萄糖,5g酵母浸粉,10g蛋白胨,0.2g MgS04·7H20,0.05g MnS04·4H20,0.5g盐酸半胱氨酸,250mL番茄汁,蒸馏水定容至1L,HCl/NaOH调节培养基pH,添加50mg/L的放线菌酮(用于抑制酵母菌生长)和50mg/L万古霉素,放线菌酮用于抑制酵母菌生长,万古霉素用于抑制乳杆菌生长。
实验验证
1、耐酸酒酒球菌菌株的分离、纯化与筛选
(1)菌株培养、分离与纯化:离心(8000×g,5min)收集来自中国不同酿酒产区(河北、山东、陕西、内蒙古和宁夏)自发MLF过程的酒样,梯度稀释后,涂布于ATB固体平皿,恒温26℃,恒温厌氧培养6天;挑取单个菌落进行平板划线,得到纯化菌株;通过种属特异性PCR鉴定固体培养基上纯化后的单菌落;挑取纯化后并鉴定为酒酒球菌的单菌落,接入到新鲜ATB培养基中扩大培养,恒温26℃,厌氧环境培养4天;将其用40%灭菌甘油按1:1的体积比混匀后,保存于-80℃超低温保存箱内。
(2)发酵选育:选取步骤(1)分离纯化的菌株,进行发酵实验,具体步如下:
菌悬液制备:将步骤(1)中分离自中国不同酿酒产区自发MLF过程中酒酒球菌的冻存液,以2%(v/v)接入到10mLpH 4.8ATB液体培养基中,26℃复苏活化,静置培养4天;将活化好的培养物以4%(v/v)接种量接种到新鲜100mL ATB液体培养基中,恒温26℃进行二次活化至OD600=1.0;
pH3.0生长曲线测定:取上述活化后的对数生长期菌液(OD600=1.0),并以酒酒球菌SX-1a和商业菌株31-DH为对照菌株,按4%的接种量接种于pH 3.0ATB液体培养基中,26℃静置培养,每隔12小时测定其在600nm下的吸光度,每个梯度每个突变菌株重复三次;选育出耐酸能力最高的酒酒球菌菌株。
结果如图1所示,菌株SX-a3在高酸下的生长活性显著高于其它菌株(数据过多其余菌株未显示)以及实验室前期保藏优良酒酒球菌SX-1a和商业菌株31-DH。
2、利用菌株SX-a3对pH 3.2葡萄酒进行苹果酸乳酸发酵
(1)种子液的制备:将保藏的菌株以2%v/v接种量接种于灭菌的50mL种子培养基(液体ATB培养基),26℃静置进行培养增殖4天,得到种子液;
(2)发酵培养:将种子液短暂胁迫驯化后按5%接种量(v/v)分别接种到pH 3.2葡萄酒酒体中;间隔1.5天测定葡萄酒中的残余苹果酸含量,利用葡萄酒全自动分析仪Y15以及L-苹果酸监测试剂盒对酒样中残留的苹果酸进行测定。
如图2所示,菌株SX-a3在pH 3.2(酒精度12.5%vol,美乐葡萄酒,酒石酸调节酒体pH至3.2)葡萄酒酒体中,相较于实验室前期保藏优良酒酒球菌SX-1a以及商业菌株31-DH,能够顺利启动并完成高酸葡萄酒的苹果酸-乳酸发酵过程,并且在4.5天基本完成高酸葡萄酒苹果酸-乳酸发酵,而实验室前期保藏优良酒酒球菌SX-1a和商业菌株31-DH无法顺利启动pH3.2下葡萄酒的苹果酸-乳酸发酵。
3、利用菌株SX-a3对pH 3.5葡萄酒进行苹果酸乳酸发酵
(1)种子液的制备:将保藏的菌株以2%v/v接种量接种于灭菌的50mL种子培养基(液体ATB培养基),26℃静置进行培养增殖4天,得到种子液;
(2)发酵培养:将种子液短暂胁迫驯化后按5%接种量(v/v)分别接种到pH 3.5葡萄酒酒体中;间隔1.5天测定葡萄酒中的残余的苹果酸含量,利用葡萄酒全自动分析仪Y15以及L-苹果酸监测试剂盒对酒样中残留的苹果酸进行测定。
如图3所示,在pH 3.5(酒精度11.3%vol,黑比诺葡萄酒)较低酸的葡萄酒酒体中,所有菌株均能够顺利启动并完成葡萄酒苹果酸-乳酸发酵,但是,菌株SX-a3完成苹果酸-乳酸发酵的时间,比实验室前期保藏优良酒酒球菌SX-1a和商业菌株31-DH提前了1.5天。
4、苹果酸-乳酸发酵后的酒体香气分析
(1)样品前处理:取5mL酒样,置于20mL的样品瓶中,分别加入1.5gNaCl和10μL内标物4-甲基-1-戊醇(浓度为100mg/L),放入搅拌转子后用瓶塞密封,启动搅拌子,40℃平衡15min后插入DVB/CAR/PDMS萃取纤维头,40℃吸附20min。
(2)GC-MS条件:不分流进样,保持载气(氦气)恒定流速1.0mL/min,柱升温程序为40℃保持3min,以4℃/min上升至120℃,再以6℃/min升至210℃,保持9min,最后以25℃/min上升至240℃,保持3min,进样器温度250℃,进样口温度230℃,离子源温度230℃,质谱扫描范围m/z 35~500。
(3)香气定性定量分析:内标法和NIST14质谱谱库查询进行香气化合物定性,使用Microsoft Office Excel 2010对所得数据进行基本统计与计算,IBM SPSS Statistics20.0分析软件进行数据的统计及差异显著性分析,并对发酵酒样的香气化合物进行主成分分析(PCA)。
苹果酸-乳酸发酵不仅能够柔和葡萄酒口感,同时也增加了葡萄酒酒体香气复杂性,苹果酸-乳酸发酵后酒体香气复杂性具有菌株特异性,由菌株SX-a3主导的葡萄酒酒体经过苹果酸-乳酸发酵后的酒体中香气更为复杂,其中,萜烯类的香气物质如图8所示、高级醇类香气如图5所示、醛酮类如图7所示、总挥发性香气物质如图9所示,含量显著高于其它处理组,而高级酯类香气如图4所示、脂肪酸类如图6所示与商业菌株31-DH相当。
此外,香气主成分分析结果如图10所示,由SX-a3处理组涵盖的香气物质种类最多,对处理组酒样中的香气成分类别和OAV>0.1的香气成分进行主成分分析(PCA),在香气类别方面提取出两个主成分PC1和PC2,贡献率分别为66.72%和22.15%,其累计贡献率为88.87%,对处理组SX-a3贡献较大的香气物质主要集中在PC1的正向端,主要以花果香的酯类物质为主,包括乙酸乙酯、异戊酸乙酯、乙酸异戊酯、己酸乙酯、辛酸乙酯、月桂酸乙酯等,其次还有醇类物质异戊醇、己醇、(E)-3-己烯-1-醇、2,3-丁二醇、苯乙醇,醛类物质辛醛、壬醛、癸醛,以及萜烯类物质香叶醇、β-大马烯酮、香叶基丙酮、β-紫罗酮、橙花叔醇、里那醇、α-萜品醇、香茅醇。
Claims (6)
1.耐高酸酒酒球菌菌株,其特征在于,该菌株为SX-a3,名称为:酒酒球菌Oenococcusoeni,已于2022年8月10日保藏于位于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.25512。
2.根据权利要求1所述的耐高酸酒酒球菌菌株,其特征在于,按以下选育过程的步骤实施得到:
步骤1、菌株分离,从不同产区的野生酒酒球菌菌株挑取单菌落并活化培养;
步骤2、菌株筛选,比较分析步骤1中分离获得的酒酒球菌单菌落的耐酸能力,获得耐酸能力最高的酒酒球菌菌株,冻存保藏。
3.根据权利要求2所述的耐高酸酒酒球菌菌株,其特征在于,所述步骤1具体按以下步骤实施:
步骤1.1、利用固体ATB培养基分离不同产区葡萄酒中的野生酒酒球菌菌株,26℃培养6-7天;
步骤1.2、挑取步骤1.1中培养基中长出来的单菌落,进行种属特异性PCR鉴定;
步骤1.3、挑取步骤1.2中PCR鉴定为酒酒球菌的单菌落于液体ATB培养基中活化培养,26℃,静置培养3-5天。
4.根据权利要求2所述的耐高酸酒酒球菌菌株,其特征在于,所述步骤2具体按以下步骤实施:
步骤2.1、将步骤1中分离获得的酒酒球菌单菌落所活化的菌液,以4%v/v接种量分别接种于pH 3.0液体ATB培养基中,26℃静置培养;
步骤2.2、相同条件下培养酒酒球菌SX-1a和商业菌株31-DH,作为对照组;
步骤2.3、每隔12小时测定其在紫外分光光度计600nm下的吸光度,每个梯度每个菌株重复三次;
步骤2.4、对比获得耐酸能力最高的酒酒球菌菌株,冻存保藏。
5.根据权利要求1所述的耐高酸酒酒球菌菌株在葡萄酒MLF中的应用。
6.根据权利要求5所述的耐高酸酒酒球菌菌株在葡萄酒MLF中的应用,其特征在于,具体按以下步骤实施:
步骤1、将获得的酒酒球菌单菌落所活化的菌液,以2%v/v接种量分别接种于pH4.8液体ATB培养基中,26℃静置培养3-5天;
步骤2、将种子液经短暂胁迫驯化后,按5%接种量(v/v)分别接种到葡萄酒酒体中,间隔1.5天测定葡萄酒中的残留的苹果酸含量。
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