CN117286040A - 一株Starmerella bacillaris酵母及其在葡萄酒酿造中的应用 - Google Patents
一株Starmerella bacillaris酵母及其在葡萄酒酿造中的应用 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C12G1/00—Preparation of wine or sparkling wine
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- C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
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Abstract
本发明属于微生物以及酿酒技术领域,涉及一株Starmerella bacillaris酵母及其在葡萄酒酿造中的应用。该菌株已于2022年6月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏号为CGMCC No.25039;该菌株单独接种可以完成葡萄酒的酒精发酵,能够适应葡萄酒的酿造环境,且具有高产甘油、低产乙酸的功能,并且具有一定的产香功能;该菌株与酿酒酵母同时接种发酵,可以增加葡萄酒中乙基酯的含量,显著降低高级醇和己酸等的含量,增强葡萄酒的花香、果香和焦糖味,减弱化学味和脂肪味;该菌株与酿酒酵母顺序接种发酵,可以增加葡萄酒中异丁酸乙酯、萜烯和酮类化合物等的含量,降低高级醇、脂肪酸的含量,有利于葡萄酒风味的改善。
Description
技术领域
本发明涉及一株Starmerella bacillaris酵母及其在葡萄酒酿造中的应用,属于微生物以及酿酒技术领域。
背景技术
葡萄酒发酵是一个复杂的微生物过程,其中酵母菌起着基础性的作用。虽然酿酒酵母是参与酒精发酵的主要微生物,但葡萄汁中也存在许多属于非酿酒酵母属的其他酵母。非酿酒酵母(non-Saccharomyces)因常从发酵停滞或变质的酒及果实中分离到,并与乙酸、乙醛、硫化氢等异味物质的产生有关,早期被认为是有害菌株。但随着研究者们对酿酒酵母的深入研究发现,在酿酒过程中可以利用非酿酒酵母来改善葡萄酒的风味物质组成,如增加甘油、总酸、酯类的含量,降低乙酸的含量,形成稳定的吡喃花青素等,进而改善酒的整体品质。葡萄酒中常见的非酿酒酵母属主要包括有孢圆酵母属(Torulaspora)、有孢汉逊酵母属(Hanseniaspora)、假丝酵母属(Candida)、接合酵母属(Zygosaccharomyces)、毕赤酵母属(Pichia)、裂殖酵母属(Schizosaccharomyces)、梅奇酵母属(Metschnikowia)、类酵母属(Saccharomycodes)、伊萨酵母属(Issatchenkia)、隐球酵母属(Cryptococcus)、克鲁维酵母属(Kluyveromyces)和红酵母属(Rhodotorula)等。
Starmerella bacillaris常见于酿酒环境中,在土壤、果蝇和水果表面也有零星分布,是一种产酸、嗜果糖、耐冷、耐渗的酵母。Starmerella bacillaris还具有降解苹果酸、修饰花青素、释放甘露糖蛋白和胞外水解酶、高产甘油、有机酸、萜烯和内酯的能力,及低产乙醇的能力,被认为是一种对葡萄酒品质有积极影响的非酿酒酵母。
研究发现,Starmerella bacillaris通常不具备单独完成酒精发酵的能力,需要与酿酒酵母共同接种来完成酒精发酵。与酿酒酵母单独接种发酵相比,Starmerellabacillaris与酿酒酵母混合接种发酵可以增加马卡贝奥葡萄酒中乙基酯、短链脂肪酸和高级醇的含量,增加长相思葡萄酒中萜烯的含量,降低乙醛、乙酸酯和高级醇的含量,增加巴贝拉葡萄酒中甘油和酯的含量,有助于提升葡萄酒的果香。此外,Starmerella bacillaris与酿酒酵母顺序接种发酵可以显著影响白葡萄酒的滴定酸和甘油的含量,降低酯类物质的含量。
发明内容
本发明的目的在于提供一株Starmerella bacillaris WMBNX08003酵母,及其在葡萄酒发酵中的应用。
本发明的第一个目的是提供一株Starmerella bacillaris酵母,命名为WMBNX08003,其已于2022年6月9日保藏在中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No.25039。
所述的Starmerella bacillaris WMBNX08003酵母是从宁夏产区玫瑰香葡萄自然发酵过程中筛选得到的。
所述的Starmerella bacillaris WMBNX08003酵母单独接种可以完成葡萄酒的酒精发酵,能够适应葡萄酒的酿造环境,且具有高产甘油、低产乙酸的功能,并且具有一定的产香功能。
所述Starmerella bacillaris WMBNX08003酵母,可通过所有适合酵母生长的培养基及温度进行培养(其中在WL固体培养基上生长菌落如图1)。培养方式选用静置培养或振荡培养,培养时间足够微生物的生长即可。
本发明还提供所述的Starmerella bacillaris WMBNX08003酵母菌株在葡萄酒发酵中的应用。
进一步地,本发明还提供了三种利用所述的Starmerella bacillarisWMBNX08003酵母菌株在葡萄酒发酵中的接种方法,其特征在于,Starmerella bacillarisWMBNX08003酵母单独接种、Starmerella bacillaris WMBNX08003酵母和酿酒酵母同时接种、Starmerella bacillaris WMBNX08003酵母和酿酒酵母顺序接种。
本发明的有益效果:
(1)本发明所述的菌株Starmerella bacillaris WMBNX08003酵母来源于葡萄酒酿造环境中,相对于已知的Starmerella bacillaris菌株,Starmerella bacillarisWMBNX08003单独接种可以完成葡萄酒的酒精发酵,且高产甘油低产乙酸,并且具有一定的产香能力。
(2)本发明所述的菌株Starmerella bacillaris WMBNX08003酵母与酿酒酵母同时接种发酵,可以增加葡萄酒中挥发性化合物的种类和酒石酸、异丁酸乙酯、丁酸乙酯、己酸乙酯和辛酸乙酯的含量,显著降低琥珀酸、异戊醇、苯乙醇和己酸等的含量,增强了葡萄酒的花香、果香和焦糖味,减弱化学味和脂肪味。
(3)本发明所述的菌株Starmerella bacillaris WMBNX08003酵母菌株与酿酒酵母顺序接种发酵,可以增加葡萄酒中甘油、酒石酸、苹果酸、异丁醇、异丁酸乙酯、萜烯和酮类化合物等的含量,降低琥珀酸、乳酸、异戊醇、苯乙醇、脂肪酸的含量,改善了葡萄酒的口感和风味。
生物材料保藏
本发明所述的Starmerella bacillaris WMBNX08003酵母菌株,于2022年6月9日保藏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,保藏号为CGMCC No.25039。
附图说明
图1为Starmerella bacillaris WMBNX08003酵母在WL培养基上的菌落形态;
图2为Starmerella bacillaris WMBNX08003酵母在显微镜下的形态;
图3为Starmerella bacillaris WMBNX08003酵母在葡萄酒酿造过程中CO2失重;
具体实施方式
下面通过具体的实施方式或实施例对本发明作进一步的阐述,以便更好的理解本发明。实施方案应理解为说明性的,而非限制本发明的范围,本发明的实质和范围仅由权利要求书所限定。本发明所使用的酿酒酵母是可以采用任何来源的酿酒酵母菌株,本发明使用的发酵原料不限于赤霞珠葡萄。
下述实施例中对相关的操作或方法若无特别说明,都是本领域常规操作或方法。对试剂和仪器若无特别说明,则是常规可获得或商业途径可以购买得到的。
下述实施例所用培养基基本成分如下:
YPD固体培养基:酵母浸粉10g/L,蛋白胨20g/L,葡萄糖20g/L,琼脂20g/L,蒸馏水定容。
YPD液体培养基:酵母浸粉10g/L,蛋白胨20g/L,葡萄糖20g/L,蒸馏水定容。
WL培养基:酵母浸粉4.0g/L,胰蛋白陈5.0g/L,葡萄糖50.0g/L,磷酸二氢钾0.55g/L,氯化钾0.425g/L,氯化钙0.125g/L,硫酸镁0.125g/L,氯化铁0.0025g/L,硫酸锰0.0025g/L,琼脂20g/L,嗅甲酚绿22mg/L。
实施例中所述Starmerella bacillaris WMBNX08003酵母是从宁夏产区玫瑰香葡萄自然发酵过程中筛选获得的,该菌株已于2022年6月9日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC;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;邮编:100101),保藏编号为CGMCC No.25039。
实施例1:Starmerella bacillaris酵母的筛选及鉴定
玫瑰香葡萄采摘后低温冷藏寄往实验室,随机选取400g葡萄果实,手工压榨后放入已灭菌的500mL锥形瓶中于25℃的环境下自然发酵。在发酵过程隔天取1mL发酵样品,用无菌生理盐水进行梯度稀释,分别吸取0.1mL涂布于WL固体平板上,倒置于28℃恒温培养箱中,培养5天,根据平板上菌落形态特征(图1),挑取Starmerella bacillaris酵母进行发酵力酒精耐受等发酵性能评价。对综合性能好的菌株进行分子生物学鉴定,利用酵母特异分类鉴定引物NL1和NL4分别扩增酵母的26S rDNA的片段,将片段寄往苏州金唯智生物科技有限公司测序,随后进行序列比对,确定筛选所得的菌株为Starmerella bacillaris酵母。最终所得到一株酿酒性能优良的Starmerella bacillaris酵母,命名为Starmerellabacillaris WMBNX08003。将Starmerella bacillaris WMBNX08003保藏于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.25039。
1)WMBNX08003菌株在WL培养基上的菌落形态如图1所示。
2)WMBNX08003菌株在显微镜下的细胞形态如图2所示。
3)对WMBNX08003菌株的26S rDNA序列进行扩增并测序,测序结果如下(SEQ-1):
实施示例2 Starmerella bacillaris WMBNX08003酵母在葡萄酒酿造中的应用
(1)种子培养
取25mL试管,装入5mL YPD液体培养基,并分别接种上述实施例1获得的Starmerella bacillaris WMBNX08003菌株及商品酿酒酵母菌株EC1118,自然pH,30℃180rpm培养12小时获得一级种子液。将5mL一级种子液倒入盛有45mL YPD的250mL锥形瓶中,30℃ 180rpm培养18小时获得二级种子液。
(2)菌株接种
采用血球计数板法对二级种子液进行计数,吸取适量二级种子液于离心管中,8000rpm离心5分钟,获得菌泥,用无菌水洗涤2遍后接种于葡萄醪中,StarmerellabacillarisWMBNX08003菌株和酿酒酵母的接种比例为10∶1,初始接种量分别为1.0×107cells/mL和1.0×106cells/mL。
设以下对照组,对Starmerella bacillaris WMBNX08003菌株及不同时间加入酿酒酵母发酵的赤霞珠葡萄酒的常规酿酒参数和挥发性化合物进行检测。其中,EC1118:仅使用商品酿酒酵母Lalvin EC1118(Lallemand Inc.,Montreal,QC,Canada)发酵;WMBNX08003:仅使用Starmerella bacillaris WMBNX08003发酵;SI-WMBNX08003/EC1118:Starmerella bacillaris WMBNX08003与酿酒酵母EC1118同时接种发酵;SI-WMBNX08003/EC1118:先接种Starmerella bacillaris WMBNX08003,48小时后接种酿酒酵母EC1118发酵。
(3)葡萄酒发酵
赤霞珠葡萄果实(227.54g/L糖,pH 3.38,259.81mg/L酵母可同化氮)采摘后,除梗破碎后,将400g葡萄醪加入到已灭菌的500mL锥形瓶中,巴氏杀菌(68℃保持30分钟)除去葡萄果实中的野生酵母菌株,待恢复室温后添加60mg/L SO2,并接种酵母菌株,于25℃条件下发酵。酒精发酵过程中,每天早晚压帽并称重,通过测定失重来检测酒精发酵进程,当12小时内失重小于0.1g则认为酒精发酵结束,实验结果见图3。酒精发酵结束后,进行皮渣分离,离心获得澄清酒样,并添加60mg/L二氧化硫。
从图3可以看出,所有接种策略均未出现发酵停滞或延迟。Starmerellabacillaris WMBNX08003单独接种发酵需要11天完成酒精发酵,Starmerella bacillarisWMBNX08003与酿酒酵母EC1118同时接种发酵和Starmerella bacillaris WMBNX08003与酿酒酵母EC1118顺序接种发酵需要8天完成酒精发酵。
(4)酒精发酵结束后葡萄酒样品检测
采用高效液相色谱法检测乙醇、甘油、葡萄糖、果糖、柠檬酸、酒石酸、苹果酸、琥珀酸、乳酸和乙酸,使用pH计测定pH值,采用顶空固相微萃取结合气质联用方法检测葡萄酒的挥发性化合物,以相应标准品外标法定量获得各种成分的含量。实验结果如表1和表2所示。
表1 酒精发酵结束后葡萄酒的常规酿酒参数(g/L)
注:同一行不同字母表示样品存在显著性差异(P<0.05)水平差异显著。
从表1可以看出:酒精发酵结束后,所有接种策略的葡萄酒均发酵完全(残糖低于4g/L),Starmerella bacillaris WMBNX08003单独接种能够完成酒精发酵。与EC 1118单独接种发酵相比,Starmerella bacillaris WMBNX08003单独接种发酵的葡萄酒中乙醇、柠檬酸、琥珀酸、乳酸和乙酸的含量显著降低,甘油、酒石酸和苹果酸的含量显著增加;WMBNX08003/EC1118同时接种发酵的葡萄酒中柠檬酸和酒石酸的含量显著增加,苹果酸和琥珀酸的含量显著降低;WMBNX08003/EC1118顺序接种发酵的葡萄酒中甘油、酒石酸、苹果酸的含量显著增加,琥珀酸和乳酸的含量显著降低。
表2 赤霞珠葡萄酒酒精发酵结束后挥发性化合物的含量(μg/L)
注:同一行不同字母表示样品存在显著性差异(P<0.05)水平差异显著。“--“表示未检测到。
从表2可以看出:与EC1118单独接种发酵相比,Starmerella bacillarisWMBNX08003单独接种具有一定的产生挥发性化合物的能力,且低产高级醇、脂肪酸,可以有效避免的高级醇和脂肪酸浓度过高带来的溶剂味和脂肪味,同时高产萜烯类物质,可以为葡萄酒带来花香和果香;Starmerella bacillaris WMBNX08003与酿酒酵母EC1118同时接种发酵,可以增加葡萄酒中挥发性化合物的种类和异丁酸乙酯、丁酸乙酯、己酸乙酯和辛酸乙酯的含量,显著降低异戊醇、苯乙醇和己酸等的含量,增强了葡萄酒的花香、果香和焦糖味,减弱了化学味和脂肪味;Starmerella bacillaris WMBNX08003与酿酒酵母EC1118顺序接种发酵,可以增加葡萄酒中异丁酸乙酯、萜烯和酮类化合物等的含量,降低高级醇、脂肪酸的含量,有利于葡萄酒风味的改善。
Claims (7)
1.一株Starmerella bacillaris WMBNX08003酵母,于2022年6月9日保藏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,保藏号为CGMCC No.25039。
2.权利要求1所述的Starmerella bacillaris WMBNX08003酵母在葡萄酒酿造中的应用。
3.权利要求1所述的Starmerella bacillaris WMBNX08003酵母,其特征在于,所述Starmerella bacillaris WMBNX08003酵母包含Starmerella bacillaris WMBNX08003菌体的活细胞、冷冻干燥得到的Starmerella bacillaris WMBNX08003干菌体、固定化的Starmerella bacillaris WMBNX08003细胞、Starmerella bacillaris WMBNX08003的液体菌剂、Starmerella bacillaris WMBNX08003的固体菌剂,或者以其他任何形式存在的Starmerella bacillaris WMBNX08003菌株。
4.根据权利要求1所述的Starmerella bacillaris WMBNX08003酵母,其特征在于:所述Starmerella bacillaris WMBNX08003酵母筛选自宁夏产区玫瑰香葡萄酒自然发酵过程中,并应用于葡萄酒酿造中。
5.权利要求2所述的Starmerella bacillaris WMBNX08003酵母在葡萄酒酿造中的应用,其特征是,在葡萄酒酿造过程中Starmerella bacillaris WMBNX08003单独接种发酵,或者Starmerella bacillaris WMBNX08003酵母和酿酒酵母同时接种发酵,或Starmerellabacillaris WMBNX08003酵母和酿酒酵母顺序接种发酵。
6.权利要求5所述的接种方式不限于Starmerella bacillaris WMBNX08003和一株酿酒酵母混合接种发酵,包括Starmerella bacillaris WMBNX08003参与的多菌株发酵及Starmerella bacillaris WMBNX08003参与的所有形式的发酵。
7.根据权利要求2所述的应用,其特征在于,所述应用是应用于葡萄酒酿造中,但不仅限于葡萄酒,还包括白酒、黄酒、其它果酒等酿造酒或蒸馏酒。
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