CN116891815A - 一株产棕榈酸甲酯的类肠膜魏斯氏菌及其应用 - Google Patents
一株产棕榈酸甲酯的类肠膜魏斯氏菌及其应用 Download PDFInfo
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- CN116891815A CN116891815A CN202310812634.6A CN202310812634A CN116891815A CN 116891815 A CN116891815 A CN 116891815A CN 202310812634 A CN202310812634 A CN 202310812634A CN 116891815 A CN116891815 A CN 116891815A
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- weissella
- methyl palmitate
- enteroid
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- white spirit
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
本发明属于生物领域，具体涉及一株产棕榈酸甲酯的类肠膜魏斯氏菌及其应用。所述类肠膜魏斯氏菌的保藏号：CGMCC No.25137，在实验中表明该菌株发酵液中棕榈酸甲酯含量为0.3 mg/L，其筛选于浓香型白酒的窖泥，适应能力强，不破坏微生物环境，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，具体涉及一株可产棕榈酸甲酯的类肠膜魏斯氏菌及其应用。
背景技术
棕榈酸甲酯，又名十六烷酸甲酯，易溶于醇、丙酮、氯仿和苯，能溶于醚，是一些食用的树木和蔬菜、药用的植物、观赏的植物各类野生植物等的主要成分，也是一些动物脂肪类物质的主要成分之一工业上可用作乳化剂、湿润剂、稳定剂及增塑剂的中间体，同时也是生物柴油的成分、化妆品(乳化剂，医药)的原料。。制备方法主要有两种，一是在碱性催化条件下，棕榈油与甲醇通过酯交换反应制备脂肪酸甲酯混合物，用于生物柴油；二是以浓硫酸为催化剂，棕榈酸与甲醇直接脱水酯化制备棕榈酸甲酯。
现发现棕榈酸甲酯存在于酒体中，为其香气成分。如胡江瑛(胡江瑛.干红山楂酒香气成分的GC-MS分析[J].酿酒科技，2016(9):3.)利用溶剂萃取法对发酵干红山楂酒中的香气成分进行提取，采用GC-MS进行分析，其中主要酯类成分为丁二酸单酯，苯乙醇，丁二酸二乙酯，己二酸正辛正癸酯，4-羟基丁酸内酯，羟基丁二酸二乙酯，2，3-丁二醇，9，10-二羟基甲基酯硬脂酸，棕榈酸甲酯，乳酸乙酯。以及夏禹(夏禹.板栗糯米甜酒和板栗果酒的制作及挥发性香气研究.北京林业大学,2014.)对板栗果酒中的香气成分经GC-MS分析其中棕榈酸甲酯相对含量在15.06％，为主要香气成分。
已有研究中棕榈酸甲酯可源自某些微生物如红贝俄式孔菌、纵条纹炭角菌、竹黄菌中，而具有产棕榈酸甲酯能力的细菌较少例如多粘类芽孢杆菌LRS-1(张亮,袁红,段良霞,等.多粘类芽孢杆菌LRS-1对辣椒疫霉菌胁迫下根系分泌物的影响[J].中国蔬菜,2022(9):7.)以及Brevibacillus brevis LPF-1(蓝江林刘波陈峥史怀栗丰.菌株LPF-1(Brevibacillus brevis LPF-1)降解猪粪过程中降解物质的GS-MS分析[J].福建农业学报,2011,26(6):1056-1064.)。但关于可产棕榈酸甲酯的乳酸菌鲜有报道。
发明内容
本发明从从白酒酿造过程中的窖泥中筛选获得一株具有产棕榈酸甲酯的乳酸菌zqw26，可应用于酒类酿造，增加酒体风味中棕榈酸甲酯的含量。
本发明提供了一株产棕榈酸甲酯的类肠膜魏斯氏菌，其保藏号为CGMCCNo.25137，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：CGMCC No.CGMCC NO.25137，分类名称为类肠膜魏斯氏菌Weissellaparamesenteroides。
本发明因此提供所述的类肠膜魏斯氏菌在产棕榈酸甲酯中的应用。
进而提供一种利用所述的类肠膜魏斯氏菌生产棕榈酸甲酯的方法，其是将所述类肠膜魏斯氏菌在发酵培养基中培养，以产生棕榈酸甲酯。
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类肠膜魏斯氏菌在白酒酿造中的应用。
具体地，将所述类肠膜魏斯氏菌作为白酒风味发酵剂应用于白酒酿造中。
更优选地，将所述类肠膜魏斯氏菌应用于白酒酿造曲药或用于窖池发酵中添加剂，以提高棕榈酸甲酯的含量。
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。
本发明提供的类肠膜魏斯氏菌zqw26具有发酵产棕榈酸甲酯的能力(在一个具体实施方式中该菌株所产棕榈酸甲酯含量为0.3mg/L)，在实验中表明该菌株发酵液中棕榈酸甲酯含量为0.3mg/L，其筛选于浓香型白酒的窖泥，适应能力强，不破坏微生物环境，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为菌株所产的棕榈酸甲酯GC-MS分子碎片质谱图。
图2为棕榈酸甲酯标准品GC-MS分子碎片质谱图。
菌株保藏信息：
本发明的类肠膜魏斯氏菌已于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.25137，其分类命名为类肠膜魏斯氏菌(Weissellaparamesenteroides)。
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窖泥样品，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：产棕榈酸甲酯能力检测
(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检测棕榈酸甲酯方法：
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，以0.822mg/ml的2-辛醇为内标物，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。以未添加菌发酵的培养液为空白对照组，计算各挥发性物质的含量。
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，检测结果如图1、图2所示，在分离得到的菌株中有一株菌株zqw26具有高产棕榈酸甲酯能力。选择该菌株做进一步研究。
实施例3：菌株zqw26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片段的基因序列通过NCBI的BLAST比对，确定菌株种属信息，鉴定其为类肠膜魏斯氏菌(Weissella paramesenteroides)，并命名为类肠膜魏斯氏菌zqw26。
测序获得16S rDNA片段的基因序列如SEQ ID NO:1所示：
ATTTGGTTCACCTTTAGACGGCTGGCTCCTAAAAGGTTACCCCACCGGCTTTGGGTGTTA
CAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGC
GTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATC
CGAACTGAGACATACTTTAAGAGATTAGCGCACCCTCGCGGGTTGGCGACTCGTTGTATA
TGCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCC
CACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTGAATGCTGGCAA
CTAATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTG
ACGACAACCATGCACCACCTGTCACCTTGTCCCCGAAGGGAACGTCCTATTTCTAGGATT
AGCAAGGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGC
TCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCC
CCAGGCGGAGTGCTTAATGCGTTAGCTGCGACACTCAAGGGCGGAAACCCTCGAACAT
CTAGCACTCATCGTTTACGGTGTGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACA
CTTTCGAGCCTCAACGTCAGTTACAGTCCAGAAAGCCGCCTTCGCCACTGGTGTTCTTC
CATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTTTCCTCTACTGCACTCAAGT
CATCCAGTTTCCAAAGCCATTCCTCAGTTGAGCTGAGGGCTTTCACTTCAGACTTAAATA
ACCGTCTGCGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGGAACATACGTATTAC
CGCGGCTGCTGGCACGTATTTAGCCGTTCCTTTCTGGTAAGATACCGTCACACACTGAAC
AGTTACTCTCAGTGCCGTTCTTCTCTTATAACAGTGTTTTACGAGCCGAAACCCTTCATC
ACACACGCGGCGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGC
CTCCCGTAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCG
GCTATGCATCATTGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCACCGCGGGTC
CATCTCTTAGTGATAGCAGAACCATCTTTCAACCAACAACCATGCGGTTGTTGGTATTATA
CGGTATTAGCACTTGTTTCCAAATGTTATCCCCTGCTAAGAGGTAGGTTACCCACGTGTT
ACTCACCCGTTCGCCACTCTTTGTCAGATAAAATCAAATCAGAGCAAGCTCGTCAGATCAATTAAAGACAAAGCGTTCGACTTGCATGTATAGCACGCCGCACCGCCCTT。
实施例4：菌株zqw26的发酵实验
将获得的类肠膜魏斯氏菌zqw26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。类肠膜魏斯氏菌zqw26在持续发酵2天后，所产棕榈酸甲酯产量可达0.300mg/L。
实施例5：菌株zqw26的形态特征和理化特性
1.经实验表明，类肠膜魏斯氏菌zqw26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℃条件下虽然能够生长但长势非常不好，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D-葡萄糖，D-果糖，D-甘露糖，D-纤维二糖，D-麦芽糖，D-乳糖，D-密二糖，D-蔗糖，D-海藻糖，D-松三糖，D-棉子糖，D-土伦糖，D-塔格糖；L-阿拉伯糖，D-核糖，D-半乳糖，甘露醇，山梨醇，甲基-αD-吡喃甘露糖苷，N-乙酰葡萄糖胺，苦杏仁苷，熊果苷，水杨苷，D-龙胆二糖；七叶灵柠檬酸铁，葡萄糖酸钾；丙三醇，2酮基葡萄糖酸钾等28种单一碳源产酸。其利用能力由强到弱：D-葡萄糖，D-果糖，D-甘露糖，D-纤维二糖，D-麦芽糖，D-乳糖，D-密二糖，D-蔗糖，D-海藻糖，D-松三糖，D-棉子糖，D-土伦糖，D-塔格糖；L-阿拉伯糖，D-核糖，D-半乳糖，甘露醇，山梨醇，甲基-αD-吡喃甘露糖苷，N-乙酰葡萄糖胺，苦杏仁苷，熊果苷，水杨苷，D-龙胆二糖；七叶灵柠檬酸铁，葡萄糖酸钾；丙三醇，2酮基葡萄糖酸钾。可利用的单一碳源种类较多，且利用能力整体较强。
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11.6g/L，乙醇2.69g/L；无残糖。
实施例6：菌株zqw26发酵产其他可挥发性代谢产物的实验
类肠膜魏斯氏菌zqw26按实施例4的方法发酵除可产0.300mg/L的棕榈酸甲酯，还可以产其他27种挥发性代谢产物，分别为：l-丙氨酸乙基酰胺0.331，乙醇0.720，异戊醇0.812mg/L，正庚醇0.805mg/L，正辛醇0.175mg/L，1-壬醇0.343mg/L，2-壬基醇0.255，苯乙醇1.080mg/L，香叶醇0.367mg/L，T-杜松醇0.013，乙酸12.012mg/L，丁酸0.305mg/L，异戊酸0.358，己酸1.849mg/L，异辛酸0.387mg/L，辛酸2.877mg/L，壬酸1.263mg/L，正癸酸0.247，仲辛酮0.134mg/L，香叶基丙酮0.357mg/L，3,4-二甲基苯甲醛0.346，2,3-二氢-2,2,6-三甲基苯甲醛0.995mg/L，苯乙醛0.470mg/L，3-羟基-2，2，4-三甲基戊基异丁酸酯0.244mg/L，2,2,4-三甲基-1,3-戊二醇二异丁酸酯0.610mg/L，2,6-二甲基吡嗪0.283mg/L，4-氯-3,5-二甲基苯酚0.732mg/L。
Claims (10)
1.一株产棕榈酸甲酯的类肠膜魏斯氏菌（Weissella paramesenteroides），其保藏号为CGMCC No. 25137。
2.如权利要求1所述的类肠膜魏斯氏菌在产棕榈酸甲酯中的应用。
3.一种利用如权利要求1所述的类肠膜魏斯氏菌生产棕榈酸甲酯的方法，其特征在于，将所述类肠膜魏斯氏菌在发酵培养基中培养，以产生棕榈酸甲酯。
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所述的类肠膜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，将所述类肠膜魏斯氏菌作为白酒风味发酵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，将所述类肠膜魏斯氏菌应用于白酒酿造曲药或用于窖池发酵中的发酵剂，以提高棕榈酸甲酯的含量。
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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