CN116836869A - 一株产正癸醇的食窦魏斯氏菌及其应用 - Google Patents
一株产正癸醇的食窦魏斯氏菌及其应用 Download PDFInfo
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- CN116836869A CN116836869A CN202310812620.4A CN202310812620A CN116836869A CN 116836869 A CN116836869 A CN 116836869A CN 202310812620 A CN202310812620 A CN 202310812620A CN 116836869 A CN116836869 A CN 116836869A
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- weissella
- decanol
- antral
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Abstract
本发明属于生物领域，具体涉及一株产正癸醇的食窦魏斯氏菌及其应用。本发明提供的食窦魏斯氏菌zqw90，其保藏号为CGMCC No.25149，具有发酵产正癸醇的能力，在一个具体实施方式中该菌株所产正癸醇含量为0.447mg/L。该菌株来源于浓香型白酒的大曲，适应能力强，可为白酒酿造工艺优化提供菌种资源，具有较为广阔的市场应用前景。
Description
技术领域
本发明属于微生物领域，具体涉及一株可产正癸醇的食窦魏斯氏菌及其应用。。
背景技术
正癸醇分子式C10H22O，分子量158.28，有甜花香气。用于制人造玫瑰油、橙花型和金合欢型香精等。也用于制润滑油添加剂、增塑剂、胶粘剂等，天然存在：存在于甜橘油、橙花油、杏花油、黄葵子油中。现阶段是以椰子油为原料，在混合氧化物催化下，经高温高压氢化，再经减压分馏而得。
正癸醇对发酵酒风味存在影响，欧阳德文等(欧阳德文,宁亚丽,吴跃.糙米酒低温后发酵期的菌种演替及挥发性风味物质变化[J].食品研究与开发,2021,42(15):172-180.)采用高通量测序技术和气质联用技术分析糙米酒后发酵期的菌种和风味物质变化，挥发性风味物质,主要包括11种酯、3种醇、2种酮及1种酚。其中相对含量最高、种类最多的化合物为酯类其次为醇类，代表物质为β-苯乙醇、正癸醇、2-甲基-1-丁醇。
已有研究中发现具有产正癸醇能力的为梭菌纲菌株，徐鹏翔(徐鹏翔.泸型酒窖泥中梭菌群落发酵演替及代谢特性分析.江南大学,2019.)采用微生物纯培养技术从窖泥中分离梭菌纲微生物菌株，检测了其中29株代表性菌株发酵液的主要挥发性物质组成，检出发酵液中醇类主要为乙醇，苯乙醇，丙醇，正/异丁醇，2-戊醇，己醇，正辛醇，正癸醇，香叶醇，十一醇等。
但产正癸醇的乳酸菌鲜有报道。
发明内容
本发明从白酒酿造过程中的大曲中筛选获得一株具有产正癸醇的乳酸菌，可应用于酒类酿造，增加酒体风味中正癸醇的含量。
本发明提供了一株产正癸醇的食窦魏斯氏菌(Weissella cibaria)，其已于2022年6月21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(CGMCC)，保藏号：CGMCCNo.CGMCC NO.25149，分类名称为食窦魏斯氏菌Weissella cibaria。
本发明因此提供所述的食窦魏斯氏菌在产正癸醇中的应用。
进而提供一种利用所述的食窦魏斯氏菌生产正癸醇的方法，其是将所述食窦魏斯氏菌在发酵培养基中培养，以产生正癸醇。
具体地，培养的条件是35-39℃静置培养24-64h，pH值为6.0-8.0。
优选地，培养的条件是37℃静置培养48h，pH值为7.0。
一个具体实施方式中，所述发酵培养基的组成为每升培养基中含有16g葡萄糖，10g胰蛋白胨，2.5g酵母粉，5g牛肉膏，3.85g牛脑，4.9g牛心，0.5g吐温80，1g柠檬酸铵，2.5g氯化钠，2.5g无水乙酸钠，0.05g硫酸镁，0.025g硫酸锰，1.25g磷酸氢二钠，1g磷酸氢二钾。
本发明进而提供所述的食窦魏斯氏菌在白酒酿造中的应用。
具体地，将所述食窦魏斯氏菌作为增香剂应用于白酒酿造中。
更优选地，将所述食窦魏斯氏菌应用于白酒酿造曲药或用于窖池发酵中添加剂，以提高正癸醇的含量。
在具体实施方式中，所述添加剂的形式选自是液体、冻干制剂或粉末。
本发明提供的食窦魏斯氏菌zqw90具有发酵产正癸醇的能力(在一个具体实施方式中该菌株所产正癸醇含量为0.447mg/L)，来源于浓香型白酒的大曲，适应能力强，可为白酒酿造工艺优化提供菌种资源，具有较为广阔的市场应用前景。
附图说明
图1为实施例2中菌株zqw90所产的正癸醇GC-MS分子碎片质谱图。
图2为实施例2中正癸醇标准品GC-MS分子碎片质谱图。
菌株保藏信息：
本发明的凝结芽孢杆菌已于2022年6月21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，保藏单位地址为中国北京市朝阳区北辰西路1号院3号。该菌株的保藏编号为CGMCC No.25149，其分类命名为食窦魏斯氏菌(Weissella cibaria)。
具体实施方式
下面通过具体实施例对本发明做进一步的阐述，以期更好的理解本发明，但不构成对本发明的限制。
实施例中涉及的培养基配方：
MRS液体培养基：葡萄糖20g/L，蛋白胨10g/L，酵母粉4g/L，牛肉浸粉5g/L，吐温801g/L，柠檬酸三铵2g/L，醋酸钠5g/L，硫酸镁0.2g/L，硫酸锰0.05g/L，磷酸氢二钾1g/L，pH6.0，115℃下高压蒸汽灭菌20分钟。
MRS固体培养基：在MRS液体培养基基础上添加15g/L的琼脂，115℃下高压蒸汽灭菌20分钟。
发酵培养基：葡萄糖16g/L，胰蛋白胨10g/L，酵母粉2.5g/L，牛肉膏5g/L，牛脑3.85g/L，牛心4.9g/L，吐温80 0.5g/L，柠檬酸铵1g/L，氯化钠2.5g/L，无水乙酸钠2.5g/L，硫酸镁0.05g/L，硫酸锰0.025g/L，磷酸氢二钠1.25g/L，磷酸氢二钾1g/L，pH 6.0，115℃下高压蒸汽灭菌20分钟。
实施例1：菌株的分离纯化
1)分离方法：取20g泸州老窖浓香型白酒大曲样品，放入装有180mL无菌蒸馏水的锥形瓶中，恒温摇床振荡10min将样品充分打散、混匀。取1mL样品悬液，采用倍比稀释法，稀释至10-2～10-7，从每个梯度的稀释液中吸取100μL，均匀涂布于MRS固体培养基平板上，平行制备两块平板，倒置，放于37℃厌氧下培养36-48h，并及时观察。
2)划线纯化：将长出菌落的平板取出，挑取不同菌落形态的单菌落，进行二次划线，直至纯化出所有单菌落。
3)菌种保藏：将纯化完成后的每种菌株的单菌落挑入5mLMRS液体培养基中，置于37℃厌氧下静置培养20-24h，吸取1mL菌液至保菌管中，加入0.5mL60％的无菌甘油溶液，重悬，置于-80℃保存。
实施例2：产正癸醇能力检测
(1)准备待测菌液：
筛选得到的菌株甘油保存管溶解后，分别将其接种于MRS液体培养基中，并于37℃下静置培养20h，得到待测菌液。
(2)GC-MS检测正癸醇方法：
采用顶空固相微萃取法：取上清液，加入顶空瓶中，加入饱和NaCl溶液，将制备好的样品于60℃保温平衡5min后，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。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。
GC-MS检测色谱条件：
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。
以上述方法检测进行复筛，以获得一株具有产正癸醇能力的菌株zqw90，选择该菌株做进一步研究。
实施例3：菌株zqw90的分子鉴定
对目标菌株进行扩培后取对数生长期新鲜菌液，离心收集菌体，采用细菌基因组抽提试剂盒提取基因组DNA。采用芽孢杆菌通用引物27F/1541R扩增其16S rDNA全长序列，具体如下：
27F(5′-AGAGTTTGATCCTGGCTCAG-3′)
1541R(5′-AAGGAGGTGATCCAGCC-3′)
①反应体系(50μl)
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。
PCR产物的纯化按上海生工生物技术公司的小量胶回收PCR产物纯化试剂盒说明进行，测序由上海生工生物技术公司完成。
测序获得16S rDNA片段的基因序列如SEQ ID NO:1所示：
TGCGGGTGCTATAATGCAGTCGAACGCTTTGTGGTTCAACTGATTTGAAGAGCTTGCTCA
GATATGACGATGGACATTGCAAAGAGTGGCGAACGGGTGAGTAACACGTGGGAAACCT
ACCTCTTAGCAGGGGATAACATTTGGAAACAGATGCTAATACCGTATAACAATAGCAACC
GCATGGTTGCTACTTAAAAGATGGTTCTGCTATCACTAAGAGATGGTCCCGCGGTGCATT
AGTTAGTTGGTGAGGTAATGGCTCACCAAGACGATGATGCATAGCCGAGTTGAGAGACT
GATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGG
AATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGGTTT
CGGCTCGTAAAACACTGTTGTAAGAGAAGAATGACATTGAGAGTAACTGTTCAATGTGT
GACGGTATCTTACCAGAAAGGAACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTA
TGTTCCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTATTTAAGTC
TGAAGTGAAAGCCCTCAGCTCAACTGAGGAATTGCTTTGGAAACTGGATGACTTGAGT
GCAGTAGAGGAAAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAA
CACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGTTGAGGCTCGAAAGTGTGG
GTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAGTGCTAGGTGT
TTGAGGGTTTCCGCCCTTAAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGT
ACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGC
ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTTGACAA
CTCCAGAGATGGAGCGTTCCCTTCGGGGACAAGGTGACAGGTGGTGCATGGTTGTCGTC
AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTT
GCCAGCATTCAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGG
GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGCGT
ATACAACGAGTTGCCAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCG
GATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCA
CGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGAGAGTTT
GTAACACCCAAAGCCGGTGGGGTAACCTTCGGGAGCCAGCCGTCTAAGGTGGGACAGATGATTAGGGGAAGTCGAACAAGAGC。
通过NCBI的BLAST比对，确定菌株种属信息，鉴定其为食窦魏斯氏菌(Weissellacibaria)，并命名为食窦魏斯氏菌zqw90(Weissella cibaria)。
实施例4：菌株zqw90的发酵实验
将获得的食窦魏斯氏菌zqw90甘油保存管溶解后，按5％接种量接种于10mL装液量的MRS液体培养基中，37℃静置培养24h后按2％接种量接种到发酵培养基中，连续培养2天，检测方法同实施例2中的测定方法，以0.822mg/ml的2-辛醇为内标物，以未添加菌发酵的培养液为空白对照组，计算各组分的含量。食窦魏斯氏菌zqw90在持续发酵2天后，所产正癸醇产量可达0.447mg/L。
实施例5：菌株zqw90的形态特征和理化特性
1.经实验表明，食窦魏斯氏菌zqw90的培养特性：最适生长温度为37℃，pH值为7，兼性厌氧，有氧、无氧或微氧条件下均可生长。在温度耐受性方面：该菌在37℃条件下长势良好，42℃条件下仍然能够生长，45-48℃条件下无法生长。
2.该菌在37℃厌氧条件下，培养48小时，经API50CH试剂盒鉴定，可分别利用L-阿拉伯糖，D-木糖，D-葡萄糖，D-纤维二糖，D-麦芽糖；D-核糖，D-果糖，D-甘露糖，N-乙酰葡萄糖胺，水杨苷，D-龙胆二糖，葡萄糖酸钾；熊果苷，2-酮基葡萄糖酸钾等14种单一碳源产酸。其利用能力由强到弱：L-阿拉伯糖，D-木糖，D-葡萄糖，D-纤维二糖，D-麦芽糖；D-核糖，D-果糖，D-甘露糖，N-乙酰葡萄糖胺，水杨苷，D-龙胆二糖，葡萄糖酸钾；熊果苷，2-酮基葡萄糖酸钾。可利用的单一碳源种类偏少，且利用能力整体偏弱。
3.该菌在37℃微好氧条件下，利用MRS液体培养基，静置培养48小时，通过HPLC高效液相色谱检测其非挥发性代谢产物，发现该菌可代谢生产乳酸8.8g/L，乙酸0.10g/L，乙醇3.24g/L；无残糖。
实施例6：菌株zqw90发酵产其他可挥发性代谢产物的实验
食窦魏斯氏菌zqw90按实施例4的方法发酵除可产0.447mg/L的正癸醇，还可以产其他23种挥发性代谢产物，分别为：异戊醇0.693mg/L，正庚醇0.737mg/L，正辛醇0.084mg/L，1-壬醇0.407mg/L，苯乙醇1.420mg/L，香叶醇0.819mg/L，顺11，13-十四碳二烯醇0.443mg/L，乙酸12.233mg/L，丁酸0.286mg/L，己酸1.291mg/L，异辛酸0.424mg/L，辛酸1.370mg/L，壬酸1.150mg/L，仲辛酮0.313mg/L，香叶基丙酮0.414mg/L，2,3-二氢-2,2,6-三甲基苯甲醛1.706mg/L，苯乙醛0.832mg/L，乙酸2-辛酯0.037mg/L，3-羟基-2，2，4-三甲基戊基异丁酸酯0.204mg/L，2,2,4-三甲基-1,3-戊二醇二异丁酸酯0.065mg/L，9-氧代壬酸乙酯0.436mg/L，2,6-二甲基吡嗪0.330mg/L，苯并噻唑0.012mg/L。
Claims (10)
1.一株产正癸醇的食窦魏斯氏菌（Weissella cibaria），其保藏号为CGMCC No.25149。
2.如权利要求1所述的食窦魏斯氏菌在产正癸醇中的应用。
3.一种利用如权利要求1所述的食窦魏斯氏菌生产正癸醇的方法，其特征在于，将所述食窦魏斯氏菌在发酵培养基中培养，以产生正癸醇。
4.如权利要求3所述的方法，其特征在于，培养的条件是35-39°C静置培养24-64h，pH值为6.0-8.0。
5.如权利要求4所述的方法，其特征在于，培养的条件是37°C静置培养48h，pH值为7.0。
6.如权利要求4所述的方法，其特征在于，所述发酵培养基的组成为每升培养基中含有16g葡萄糖，10g胰蛋白胨，2.5g酵母粉，5g牛肉膏，3.85g牛脑，4.9g牛心，0.5g吐温80，1g柠檬酸铵，2.5g氯化钠，2.5g无水乙酸钠，0.05g硫酸镁，0.025g硫酸锰，1.25g磷酸氢二钠，1g磷酸氢二钾。
7.如权利要求1所述的食窦魏斯氏菌在白酒酿造中的应用。
8.如权利要求7所述的应用，其特征在于，将所述食窦魏斯氏菌作为增香剂应用于白酒酿造中。
9.如权利要求8所述的应用，其特征在于，将所述食窦魏斯氏菌应用于白酒酿造曲药或用于窖池发酵中添加剂，以提高正癸醇的含量。
10.如权利要求3至4任一项所述的应用，其特征在于，所述添加剂的形式选自是液体、冻干制剂或粉末。
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