CN112877226A - 一株经紫外诱变育种产生的耐酒精型陆生伊萨酵母及其在葡萄酒酿造中的应用 - Google Patents
一株经紫外诱变育种产生的耐酒精型陆生伊萨酵母及其在葡萄酒酿造中的应用 Download PDFInfo
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Abstract
本发明公开了一株经紫外诱变育种产生的耐酒精型陆生伊萨酵母及其在葡萄酒酿造中的应用。所述陆生伊萨酵母保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏名称为UV5,保藏编号:CGMCC No.20635。相较于原始陆生伊萨酵母菌株,UV5菌株的酒精耐受性可达11%,表现出优良的酒精耐受性,以其酿造出的葡萄酒花果香气浓郁、风味复杂。UV5应用于葡萄酒酿造中,可以生产出具有独特风格特征的葡萄酒,促进葡萄酒产业发展。
Description
技术领域
本发明涉及微生物技术领域。本发明具体涉及一株经紫外诱变育种产生的耐酒精型陆生伊萨酵母及其在葡萄酒酿造中的应用。
背景技术
葡萄酒的酿造是一个微生物相互作用的过程,酵母菌在酿造过程中发挥主导作用。而其中,酿酒酵母主要负责完成酒精发酵,是葡萄酒酿造过程中的优势菌种,但除酿酒酵母外,还有许多非酿酒酵母也参与其中,非酿酒酵母菌主要通过代谢和自溶的方法来提高葡萄酒的香气,参与风味物质的形成。与酿酒酵母单一发酵相比,恰当应用非酿酒酵母参与酿造的葡萄酒品质高、香气复杂性更好,拥有丰富的花香、果香和坚果香。陆生伊萨酵母(Issatchenkia terricola)应用于葡萄酒发酵中,可以使酒种游离单萜和非类异戊二烯的含量增加,带来丰富的花香、果香,以及酒种降低苹果酸的含量,减少苹果酸带来的生涩感,提升葡萄酒的口感。但是陆生伊萨酵母在葡萄酒酿造中的应用受很多因素影响,如酒精度、二氧化硫、温度以及渗透压等。其中,酒精是造成许多非酿酒酵母在发酵中后期菌群数量下降和死亡的重要因素。从自然界分离出的酵母菌,有些需要通过人工选育来获得理想的性能。酵母的改造选育技术主要包括自然选育、诱变育种以及基因重组等,诱变育种因过程简练、效果突出而被人们广泛使用。诱变育种包括物理诱变、化学诱变以及生物诱变。物理诱变中经常使用的诱变因子包括紫外线、α射线、β射线、γ射线、X射线、微波以及中子等,其中紫外诱变通常是首选方法,因为它所需要的设备简单,操作过程容易控制,而且相对安全。其原理主要是紫外线在相邻嘧啶核苷酸之间形成嘧啶二聚体,进而减轻氢键的作用,引起双链结构断裂缺失,导致碱基间的错误配对,引起突变。
发明内容
因此,本发明的目的是提供一株酒精耐受性好的陆生伊萨酵母。
本发明的另一目的是提供一株酒精耐受性好的陆生伊萨酵母在酿造葡萄酒中的应用。
针对上述目的,本发明提供的技术方案如下:
本发明提供的陆生伊萨酵母(Issatchenkia terricola),名称为UV5,属于伊萨酵母属(Issatchenkia sp.),由菌株H5经紫外诱变得到,出发菌株H5分离自山东省烟台市君顶酒庄葡萄园采摘成熟的红色酿酒葡萄品种——“赤霞珠”(Cabernet Sauvignon)自然发酵醪。该菌株已于2020年9月11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”,保藏地址:北京市朝阳区大屯路3号中国科学院微生物研究所,保藏号为CGMCCNo.20635,建议分类命名为:陆生伊萨酵母(Issatchenkia terricola)。
除非特别指明,陆生伊萨酵母(Issatchenkia terricola)UV5 CGMCC No.20635,在本文中简称陆生伊萨酵母UV5。
本发明提供的陆生伊萨酵母UV5可用于酿造葡萄酒。利用陆生伊萨酵母UV5酿造葡萄酒的优点在于:在葡萄发酵醪当中,UV5表现出更好的酒精耐受性,以其参与酿造出的葡萄酒花果香气浓郁、风味协调且复杂、口感平衡,品质高。因此,在葡萄醪中接种陆生伊萨酵母UV5,使其参与发酵,可以获得具有浓郁花果香气、结构复杂、口感柔和的优质葡萄酒,促进葡萄酒风格的多样性以及葡萄酒产业的发展。
附图说明
图1菌株UV5在WL培养基上的菌落形态。
生物保藏说明
生物材料UV5,分类命名:陆生伊萨酵母Issatchenkia terricola,于2020年09月11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,地址为: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,保藏编号为CGMCC No.20635。
具体实施方式
为了更好地理解与实施,下面结合实施例和附图详细说明本发明;所举实施例仅用于解释本发明,并非用于限制本发明的范围。
除非特别指明,以下实施例中所用的方法均为常规方法。
除非特别指明,以下实施例中所用的试剂均为分析纯级别的试剂,且可从正规渠道商购获得。
除非特别指明,以下实施例中的定量试验,均设置三次重复实验,结果取统计平均值。
实施例中涉及的培养基配方如下:
YPD液体培养基(每1L):酵母粉10g,蛋白胨20g,葡萄糖20g。
YPD固体培养基(每1L):酵母粉10g,蛋白胨20g,葡萄糖20g,琼脂20g。
葡萄酒酵母WL营养琼脂培养基(每1L):酵母粉4g,胰蛋白胨5g,葡萄糖50g,KH2PO40.55g,KCl 0.425g,CaCl2 0.125g,MgSO4 0.125g,FeCl3 2.5mg,MnSO4 2.5mg,琼脂20g,溴甲酚绿22mg。
TTC上层培养基(每1L):葡萄糖5g,琼脂15g,TTC 0.5g。
实施例1陆生伊萨酵母UV5的诱变选育
本发明一株酒精耐受性好的的陆生伊萨酵母,是由菌株H5经紫外诱变选育得到,诱变选育过程如下:
将-80℃条件下出发菌株H5的甘油管冻存液活化。活化后按2%的比列接种于100mL液体培养基中,28℃,120r/min摇床培养,每隔2小时无菌取样,测定菌悬液在600nm处的吸光值,不接种的空白YPD液体培养基作对照,每个试验设置3个平行。以时间为横坐标,600nm处的OD值为纵坐标绘制出发菌株的生长曲线,确定其生长的对数期。
取培养至对数中期的出发菌株菌悬液稀释涂布于YPD固体培养基上,打开皿盖,将平板放置于20w的紫外灯下距离50cm处照射不同时间(0、10、20、30、40、50、60、70、80、90、100、110、120s),辐照处理后,用锡纸包裹(防止光复活)培养基在28℃条件下恒温避光培养2天,进行菌落计数,计算致死率,选择致死率在80%左右的平板进行筛选。挑取菌株活化培养,将菌株的活化液稀释涂布到TTC下层培养基中,28℃培养48h后倒入TTC上层培养基,再于28℃下保温(阴暗处)3h后,选择颜色深的菌落。挑选出的菌株活化后在WL营养琼脂平板上Z字划线,28℃恒温静置培养2天,观察菌落大小形态以及是否有杂菌污染,无污染的进行下一步试验。将筛选得到的菌株分别划线于酒精体积分数为4%v/v、6%v/v、8%v/v、10%v/v、12%v/v的YPD固体平板上,然后在28℃条件下培养3天,观察平板上菌落的生长情况,以未处理的平板为对照,每个处理3个平行。再将筛选得到的菌株的活化液按2%的比例接种于不同酒精浓度的YPD液体培养基中,28℃,150r/min摇床培养,以不同处理下不接菌的YPD液体培养基为对照,每3h取样测定其在600nm处的吸光值,监测不同菌株在酒精胁迫下的生长速率,每株菌设置3个重复。挑取酒精耐受性好的诱变菌株使用40%甘油冻存管于-80℃条件下保存,待分子鉴定。
提前活化待测酵母,将其接种于YPD液体培养基,于28℃,150rpm条件下恒温振荡培养12h,离心后取酵母细胞待用。使用天根酵母基因组DNA提取试剂盒提取待测酵母基因组DNA。选用引物对ITS1(5’-TCCGTAGGTGAACCTGCGG-3’)和ITS4(5’-TCCTCCGCTTATTGATATGC-3’)对酵母rDNA基因5.8S ITS区进行PCR扩增。PCR反应条件:95℃预变性5min;95℃变性1min,52℃退火2min,72℃延伸2min,循环35次;72℃补充延伸10min。PCR反应体系:10×PCR buffer(Taq buffer with KCl)5μL,25mmol/L MgCl2 6μL,10mmol/L dNTPs 1μL,10μmol/L引物各2.5μL,Taq酶2.0U,模板DNA 1μL,加双蒸水定容至50μL。取5μL扩增产物用1%的琼脂糖凝胶电泳检测。
PCR产物测序由北京擎科新业生物技术有限公司完成。
通过美国国家生物技术信息中心(NCBI)网站(https://blast.ncbi.nlm.nih.gov/Blast.cgi)中的BLAST工具搜索待测菌株目的序列,进行相似序列搜索,根据同源序列的相似性初步确定UV5菌株的种属地位,同时校正测序图谱。比较UV5与已知酵母菌对应序列的相似程度,相似度超过99%,并通过UV5在WLN鉴别培养基上的菌落形态和细胞显微形态最终鉴定待测菌株的种属地位。
其中,陆生伊萨酵母UV5(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.20635)的5.8S ITS rDNA的测序结果如SEQ ID No.1所示。
与相关模式菌株陆生伊萨酵母PMM08-3356-AL相似性:99.51%,鉴定为陆生伊萨酵母(Issatchenkia terricola)。
实施例2陆生伊萨酵母UV5和原始菌株H5小型酿酒比较试验
提前将-20℃条件下冻存的葡萄汁取出,放在4℃冰箱缓慢解冻。葡萄汁解冻后在10000r/min,4℃低温条件下离心15分钟去除沉淀,收集上清液使用0.65μm的无菌过滤器初步过滤,再使用0.45μm的无菌过滤器过滤得到无菌葡萄汁。将甘油管冻存菌液按2%的比例接种于YPD液体培养基中,28℃,120r/min活化24小时,将活化液按10%比例接入过滤除菌的赤霞珠葡萄汁中,28℃静置培养72小时得到预培养液。量筒量取200mL无菌葡萄汁分装到500mL发酵瓶中,将酵母预培养液按1%的比例接种到分装好的葡萄汁中,每组设置三个平行。接种后使用棉花塞封口并用锡箔纸和封口膜包裹,然后置于20℃条件下静止发酵16天。分别在第0、2、4、7、10、13、16天充分摇动发酵瓶取样,并测定酵母菌落数、pH、Birx值、残糖、甘油和酒精的含量。
发酵结束后,使用顶空固相微萃取(HS-SPME)进样结合气相色谱质谱仪(GC-MS)和火焰电离处理器(FID)对样品中香气物质进行检测。检测汇总结果如表1所示。
表1小型酿酒试验后酒样挥发性香气成分分类总结
小型酿酒试验后酒样挥发性香气成分结果显示,与出发菌株H5相比,香气物质种类总数相近,但是香气类型和相对含量差异明显。UV5的酯类香气含量显著增加,尤其是酯类含量占到总香气比例的88.19%,赋予了样品更加浓郁的花香、热带果香和植物香料香气,有利于增加葡萄酒香气的复杂性,促进葡萄酒风格的多样性。
序列表
<110> 中国农业大学
<120> 一株经紫外诱变育种产生的耐酒精型陆生伊萨酵母及其在葡萄酒酿造中的应用
<130> MP21006550Z
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 409
<212> DNA
<213> 耐酒精型陆生伊萨酵母(Issatchenkia terricola)
<400> 1
acctgcggaa ggatcattac tgtgatatac tttccacact gcgtgcgcgt aacaaacccc 60
taaacatgaa taacccagtc aagaatccat aagaataaaa ctttcaacaa cggatctctt 120
ggttctcgca tcgatgaaga gcgcagcgaa atgcgatacc tagtgtgaat tgcagccatc 180
gtgaatcatc gagttcttga acgcacattg cgccccctgg tattccgggg ggcatgcctg 240
tttgagcgtc gtttctatct cacgcaagtg gagctggccc ggccttggcc ccgccgaaaa 300
gaaacgaggg cgaagcgaac tatgttgtgc gccgacccca gctatcaagc tcgacctcaa 360
atcaggtagg aatacccgct gaacttaagc atatcaataa ggcggagga 409
Claims (4)
1.一株耐酒精型陆生伊萨酵母,其特征在于,所述耐酒精型陆生伊萨酵母保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏名称为UV5,保藏编号:CGMCC No.20635。
2.如权利要求1所述的耐酒精型陆生伊萨酵母在生产葡萄酒中的应用。
3.一种生产葡萄酒的方法,是利用如权利要求1所述的耐酒精型陆生伊萨酵母发酵葡萄原料,得到葡萄酒。
4.如权利要求1所述的耐酒精型陆生伊萨酵母的紫外诱变育种、筛选和鉴定方法,其特征在于,包括以下步骤:
(1)活化冻存在-80℃条件下甘油管中的出发菌株,活化后按2%的比列接种于100mLYPD液体培养基中,28℃,120r/min摇床培养,每隔2小时无菌取样,测定菌悬液在600nm处的吸光值,不接种的空白YPD液体培养基作对照,以时间为横坐标,600nm处的OD值为纵坐标绘制出发菌株的生长曲线,确定其生长的对数期;
(2)取培养至对数中期的出发菌株菌悬液稀释涂布于YPD固体培养基上,打开皿盖,将平板放置于20w的紫外灯下距离50cm处照射不同时间(0、10、20、30、40、50、60、70、80、90、100、110、120s),辐照处理后,用锡纸包裹(防止光复活)培养基在28℃条件下恒温避光培养2天,进行菌落计数,计算致死率,选择致死率在80%左右的平板进行筛选;
(3)将筛选菌株的活化液稀释涂布到TTC下层培养基中,28℃培养48h后倒入TTC上层培养基,再于28℃下保温(阴暗处)3h后,选择颜色深的菌落;
(4)将TTC培养基筛选得到的诱变菌株酵母分别划线于酒精体积分数为4%v/v、6%v/v、8%v/v、10%v/v、12%v/v的YPD固体平板上进行酒精胁迫生长,然后在28℃条件下培养3天,观察平板上菌落的生长情况,以未处理的平板为对照;
(5)将酒精胁迫生长筛选得到的3株非酿酒酵母(H5、F26、D6)的活化液按2%的比例接种于不同酒精浓度的YPD液体培养基中,28℃,150r/min摇床培养,以不同处理下不接菌的YPD液体培养基为对照,每3h取样测定其在600nm处的吸光值,监测不同菌株在酒精胁迫下的生长速率;
(6)经紫外诱变育种、筛选后得到的菌株纯化后,使用40%甘油冻存管于-80℃条件下保存,待分子鉴定;
(7)提前活化待测酵母,将其接种于YPD液体培养基,于28℃,150rpm条件下恒温振荡培养12h,离心后取酵母细胞待用;使用天根酵母基因组DNA提取试剂盒提取待测酵母基因组DNA;选用引物对对酵母rDNA基因5.8S ITS区进行PCR扩增,取扩增产物用1%的琼脂糖凝胶电泳检测;
(8)PCR产物测序;
(9)通过美国国家生物技术信息中心网站https://blast.ncbi.nlm.nih.gov/Blast.cgi中的BLAST工具搜索待测菌株目的序列,进行相似序列搜索,根据同源序列的相似性初步确定UV5菌株的种属地位,同时校正测序图谱;比较UV5与已知酵母菌对应序列的相似程度,相似度超过99%,并通过UV5在WL鉴别培养基上的菌落形态和细胞显微形态最终鉴定待测菌株的种属地位。
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