CN116769617A - 产乙酯类化合物的异常威克汉姆酵母及应用 - Google Patents
产乙酯类化合物的异常威克汉姆酵母及应用 Download PDFInfo
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Abstract
本发明属于微生物技术领域，特别涉及一株从酒曲中分离得到的可同时产多种乙酯类化合物的异常威克汉姆酵母(Wickerhamomyces anomalus)。所述的异常威克汉姆酵母保藏编号为：CGMCC No.26371，其性能稳定，在好氧条件下，该菌株所产的乙酸乙酯、己酸乙酯、棕榈酸乙酯含量分别为30.219mg/L、3.356mg/L、2.031mg/L，能应用于白酒、果酒、酱油、馒头等多种食品制备中。
Description
技术领域
本发明属于微生物技术领域，特别涉及一株从酒曲中分离得到的可同时产多种乙酯类化合物的异常威克汉姆酵母。
背景技术
乙酯类化合物是一类由乙醇和酸缩合酯化得到的产物，通式为RCOOEt。常见的乙酯类化合物有甲酸乙酯、乳酸乙酯、己酸乙酯、乙酸乙酯等。大多乙酯类化合物具有果实气味或是独特芳香气味，可应用于香精香料。例如：甲酸乙酯似桃香或荔校味，己酸乙酯具有曲香、菠萝香型的香气，乙酸乙酯具有苹果和香蕉的水果香气，棕榈酸乙酯呈微弱蜡香、果爵和奶油香气。
乙酯类化合物存在于多种发酵食品中，例如研究发现葛根甜面酱挥发性风味物质酯类含量最高，其中以月桂酸乙酯、9-十六碳烯酸乙酯、棕榈酸乙酯、亚麻油酸乙酯、油酸乙酯、硬脂酸乙酯为主。乙酸乙酯是清香型白酒的主要香味成分，在浓香型、酱香型等白酒中含量均很高。现有研究中多研究发酵高产单一乙酯类化合物的菌种，比如专利CN109609393A公开了一种饲用异常威克汉姆酵母及其应用，其具有高产乙酸乙酯的能力。而关于能同时高产多种乙酯类化合物的菌种还处于研究空白阶段。
发明内容
为了获得可产多种乙酯类风味物质的功能菌，使其不仅应用于发酵食品行业，还能应用于生物来源的香精香料组合物制备，促进香料行业发展。本发明从酒曲中分离筛选得到一株可产多种乙酯类化合物的酵母菌。
本发明所述的异常威克汉姆酵母(Wickerhamomyces anomalus)，保藏编号为：CGMCC No.26371。保藏时间为2022年12月30日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(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。所述酵母菌筛选于酒曲中，名称为：异常威克汉姆酵母LZLJ1-1(Wickerhamomyces anomalus)。
其中，产乙酯类化合物的异常威克汉姆酵母LZLJ1-1的ITS序列如SEQ ID NO：1所示：
GGGGCTCGGAAGGATCATTATAGTATTCTATTGCCAGCGCTTAATTGCGCGGCGATAAACCTTACACACATTGTCTAGTTTTTTTGAACTTTGCTTTGGGTGGTGAGCCTGGCTTAC TGCCCAAAGGTCTAAACACATTTTTTTAATGTTAAAACCTTTAACCAATAGTCATGAAAATTTTTAACAAAAATTAAAATCTTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTATTGTGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCACCCTCTGGTATTCCAGAGGGTATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCTTCGGGTTTGGTATTGAGTGATACTCTGTCAAGGGTTAACTTGAAATATTGACTTAGCAAGAGTGTACTAATAAGCAGTCTTTCTGAAATAATGTATTAGGTTCTTCCAACTCGTTATATCAGCTAGGCAGGTTTAGAAGTATTTTAGGCTCGGCTTAACAACAATAAACTAAAAGTTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCTAAA
其中，上述异常威克汉姆酵母LZLJ1-1生物学特征：在YPD固体培养基上30℃培养3天，菌落大而厚，呈乳白色、圆形且边缘不整齐，表面光滑湿润，有粘性易被挑起。
其中，所述YPD固体培养基为酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L，pH 7.0，115℃下高压蒸汽灭菌20分钟。
其中，上述异常威克汉姆酵母LZLJ1-1所产乙酯类化合物包括：乙酸乙酯、己酸乙酯、棕榈酸乙酯。
本发明还提供上述异常威克汉姆酵母LZLJ1-1在发酵产乙酯类化合物上的应用。
本发明还提供上述异常威克汉姆酵母LZLJ1-1在提高发酵产品或香精香料中乙酯类化合物含量中的应用。
其中，所述发酵产品包括：白酒、果酒、酱油或面制食品中至少一种。
本发明提供了上述异常威克汉姆酵母在发酵产乙酯类化合物发酵方法，包括以下步骤：将所述异常威克汉姆酵母在发酵培养基中20℃-30℃好氧培养48h-72h，pH值为4.5-7.5，所述异常威克汉姆酵母的接种量为1％-5％。
优选地，所述发酵方法包括以下步骤：将所述异常威克汉姆酵母LZLJ1-1在发酵培养基中30℃好氧培养72h，pH值为7.0，所述异常威克汉姆酵母LZLJ1-1的接种量为1％。
优选地，所述发酵培养基的组成为每升培养基中含有麦芽浸粉130g，氯霉素0.1g。
其中，采用上述发酵方法，在好氧条件下，该菌株所产的乙酸乙酯、己酸乙酯、棕榈酸乙酯含量分别为30.219mg/L、3.356mg/L、2.031mg/L，所产乙酯类化合物占总挥发性物质含量的38.12％。
有益效果：本发明所提供的同时产多种乙酯类化合物的异常威克汉姆酵母(保藏编号为：CGMCC No.26371)，分离筛选于市售酒曲，性能稳定，能应用于白酒、果酒、酱油、面制食品等多种食品制备中。本发明所提供的菌株发酵工艺简单，在好氧条件下，该菌株所产的乙酸乙酯、己酸乙酯、棕榈酸乙酯含量分别为30.219mg/L、3.356mg/L、2.031mg/L，所产乙酯类化合物占总挥发性物质含量的38.12％，能作为研制乙酯类香料组合物的新方向。
附图说明
图1为本发明异常威克汉姆酵母LZLJ1-1菌落形态图。
本发明提供的异常威克汉姆酵母(Wickerhamomyces anomalus)，保藏编号为：CGMCC No.26371。保藏时间为2022年12月30日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(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。名称为：异常威克汉姆酵母LZLJ1-1(Wickerhamomyces anomalus)。
具体实施方式
本发明所述的异常威克汉姆酵母(Wickerhamomyces anomalus)，保藏编号为：CGMCC No.26371。保藏时间为2022年12月30日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(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。所述酵母菌筛选于酒曲中，名称为：异常威克汉姆酵母LZLJ1-1(Wickerhamomyces anomalus)。
其中，产乙酯类化合物的异常威克汉姆酵母LZLJ1-1的ITS序列如SEQ ID NO：1所示：
GGGGCTCGGAAGGATCATTATAGTATTCTATTGCCAGCGCTTAATTGCGCGGCGATAAACCTTACACACATTGTCTAGTTTTTTTGAACTTTGCTTTGGGTGGTGAGCCTGGCTTACTGCCCAAAGGTCTAAACACATTTTTTTAATGTTAAAACCTTTAACCAATAGTCATGAAAATTTTTAACAAAAATTAAAATCTTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCAACGATGAAGAACGCAGCGAAATGCGATACGTATTGTGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCACCCTCTGGTATTCCAGAGGGTATGCCTGTTTGAGCGTCATTTCTCTCTCAAACCTTCGGGTTTGGTATTGAGTGATACTCTGTCAAGGGTTAACTTGAAATATTGACTTAGCAAGAGTGTACTAATAAGCAGTCTTTCTGAAATAATGTATTAGGTTCTTCCAACTCGTTATATCAGCTAGGCAGGTTTAGAAGTATTTTAGGCTCGGCTTAACAACAATAAACTAAAAGTTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCTAAA
其中，上述异常威克汉姆酵母LZLJ1-1生物学特征：在YPD固体培养基上30℃培养3天，菌落大而厚，呈乳白色、圆形且边缘不整齐，表面光滑湿润，有粘性易被挑起。
其中，所述YPD固体培养基为酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L，pH 7.0，115℃下高压蒸汽灭菌20分钟。
其中，上述异常威克汉姆酵母LZLJ1-1所产乙酯类化合物包括：乙酸乙酯、己酸乙酯、棕榈酸乙酯。
本发明还提供上述异常威克汉姆酵母LZLJ1-1在发酵产乙酯类化合物上的应用。
本发明还提供上述异常威克汉姆酵母LZLJ1-1在提高发酵产品或香精香料中乙酯类化合物含量中的应用。
其中，所述发酵产品包括：白酒、果酒、酱油或面制食品中至少一种。
本发明提供了上述异常威克汉姆酵母在发酵产乙酯类化合物发酵方法，包括以下步骤：将所述异常威克汉姆酵母在发酵培养基中20℃-30℃好氧培养48h-72h，pH值为4.5-7.5，所述异常威克汉姆酵母的接种量为1％-5％。
其中，所述发酵方法包括以下步骤：将所述异常威克汉姆酵母LZLJ1-1在发酵培养基中30℃好氧培养72h，pH值为7.0，所述异常威克汉姆酵母LZLJ1-1的接种量为1％。
优选地，所述发酵培养基的组成为每升培养基中含有麦芽浸粉130g，氯霉素0.1g。
其中，采用上述发酵方法，在好氧条件下，该菌株所产的乙酸乙酯、己酸乙酯、棕榈酸乙酯含量分别为30.219mg/L、3.356mg/L、2.031mg/L，所产乙酯类化合物占总挥发性物质含量的38.12％。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例中涉及的培养基配方：
孟加拉红固体培养基：蛋白胨5g/L，葡萄糖10g/L，磷酸二氢钾1g/L，硫酸镁0.5g/L，孟加拉红0.03g/L，氯霉素0.1g/L，琼脂15g/L，pH 7.0，115℃下高压蒸汽灭菌20分钟。
YPD液体培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，pH 7.0，115℃下高压蒸汽灭菌20分钟。
YPD固体培养基：酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂15g/L，pH7.0，115℃下高压蒸汽灭菌20分钟。
发酵培养基：麦芽浸粉130g/L，氯霉素0.1g/L，pH 7.0，115℃下高压蒸汽灭菌20分钟。
实施例1产乙酯类化合物的酵母菌筛选
取1.5g酒曲样品(广西桂林酒曲样品)装入混有玻璃珠和50mL无菌水的250mL三角瓶中，37℃摇床上以115r/min振荡过夜，自然静置沉淀法取1mL上清，用无菌水连续稀释至10-5，每稀释梯度取0.2mL涂布于孟加拉红固体培养基平板上，30℃培养2～3天；然后挑取生长旺盛的单菌落经多次划线分离纯化后接入YPD液体培养基，培养温度30℃，摇床转速120rpm，培养2-3天后进行感官评定(对发酵液风味的感官评价，有果实气味或独特芳香气味)，根据感官评分选择适合的酵母菌，于-80℃用30％的甘油保藏以备进一步分析。
实施例2目标酵母菌分子鉴定
目标酵母菌生物学特征：在YPD固体培养基上30℃培养3天，观察菌落形态，如图1所示，菌落大而厚，呈乳白色、圆形且边缘不整齐，表面光滑湿润，有粘性易被挑起。
ITS序列扩增：吸取1mL菌液10000rpm离心5min，去上清，得菌泥，加入CTAB溶液及苯酚-氯仿—异戊醇(25：24：1)萃取核酸DNA，12000rpm离心5min取上清，加入等体积的氯仿-异戊醇(24：1)，混匀后12000rpm离心5min取上清，乙醇溶液漂洗两次，12000rpm离心5min去上清，沉淀吹干，加入50μL无菌水重悬，即得菌株DNA模板。
引物为ITS1(SEQ ID NO：2：5’-TCCGTAGGTGAACCTGCGG-3’)和ITS4(SEQ ID NO：3：5’-TCCTCCGCTTATTGATATGC-3’)，进行PCR扩增。
①反应体系(20μL)
②反应程序
PCR产物用1.0％的琼脂糖凝胶电泳分离检验，电压约11V/cm，电泳时间20min。
测序由上海生工生物技术公司完成，将获得ITS片段的基因序列通过NCBI的BLAST比对，确定目标菌株种属信息，ITS序列如SEQ ID NO：1所示，鉴定其为异常威克汉姆酵母(Wickerhamomyces anomalus)，并命名为异常威克汉姆酵母LZLJ1-1。
实施例3酵母菌挥发性代谢产物检测实验
(1)检测样品制备：将异常威克汉姆酵母LZLJ1-1(Wickerhamomyces anomalus)甘油保存管溶解后接种于5mL YPD液体培养基中，并于30℃培养12-20h，活化三代后按1％(体积比)接种量接入400mL发酵培养基中，培养温度30℃，摇床转速120rpm，培养72h。发酵结束后得到发酵菌液，10000rpm离心5min后取上清。
(2)挥发性代谢产物检测
运用顶空固相微萃取/气相色谱-质谱联用仪技术(HS-SPME/GC-MS)测定挥发性产物。
HS-SPME/GC-MS具体方法如下：
(1)HS-SPME萃取条件
取上清液，加入顶空瓶中，加入饱和NaCl溶液，用50/30μmDVB/CAR/PDMS萃取头在60℃萃取50min，萃取结束后在GC进样口250℃解吸5min。
(2)GC分析条件
气相色谱条件：HP-INNOWAX色谱柱(60m×0.25mm×0.25μm)；升温程序：起始温度40℃，保持5min，以4℃/min升到100℃，再以6℃/min升至230℃，保持10min，载气为高纯氦气(1.0mL/min)；进样口温度250℃，不分流。
质谱条件：电子电离源，电子能量70eV；电子倍增器电压350V；离子源温度230℃；传输线温度250℃；质量范围40-450m/z。
以0.822mg/mL 2-辛醇为内标物，化合物检索结果与NIST标准谱库进行匹配，相似度达到80％以上确认为目的化合物。以未添加菌发酵的培养液为空白对照组，计算物质含量。
检测结果如表1所示，异常威克汉姆酵母LZLJ1-1发酵所产的乙酸乙酯、己酸乙酯、棕榈酸乙酯含量分别为30.219mg/L、3.356mg/L、2.031mg/L，所产乙酯类化合物占总挥发性物质含量的38.12％。
表1异常威克汉姆酵母LZLJ1-1乙酯类化合物含量检测结果
需要说明的是，本说明书中描述的具体特征、结构、材料或者特点可以在任一个或多个实施例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例以及不同实施例的特征进行结合和组合。
Claims (9)
1.产乙酯类化合物的异常威克汉姆酵母(Wickerhamomyces anomalus)LZLJ1-1，其特征在于：保藏编号为：CGMCC No.26371。
2.根据权利要求1所述的产乙酯类化合物的异常威克汉姆酵母，其特征在于：所产乙酯类化合物包括：乙酸乙酯、己酸乙酯、棕榈酸乙酯。
3.权利要求1所述的产乙酯类化合物的异常威克汉姆酵母在发酵产乙酯类化合物上的应用。
4.权利要求1所述的产乙酯类化合物的异常威克汉姆酵母在提高发酵产品或香精香料中乙酯类化合物含量中的应用。
5.根据权利要求4所述的应用，其特征在于：所述发酵产品包括：白酒、果酒、酱油或面制食品中至少一种。
6.权利要求3或权利要求4应用中的发酵方法，其特征在于：包括以下步骤：将所述异常威克汉姆酵母在发酵培养基中20℃-30℃好氧培养48h-72h，pH值为4.5-7.5，所述异常威克汉姆酵母的接种量为1％-5％。
7.根据权利要求6所述的发酵方法，其特征在于：包括以下步骤：将所述异常威克汉姆酵母在发酵培养基中30℃好氧培养72h，pH值为7.0，所述异常威克汉姆酵母的接种量为1％。
8.根据权利要求7所述的发酵方法，其特征在于：所述发酵培养基的组成为每升培养基中含有麦芽浸粉130g，氯霉素0.1g。
9.根据权利要求6～8任一项所述的发酵方法，其特征在于：在好氧条件下，所述菌株所产的乙酸乙酯、己酸乙酯、棕榈酸乙酯含量分别为30.219mg/L、3.356mg/L、2.031mg/L，所产乙酯类化合物占总挥发性物质含量的38.12％。
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