CN116970522A - 一株产棕榈酸乙酯的食丙二醇慢生乳杆菌及其应用 - Google Patents
一株产棕榈酸乙酯的食丙二醇慢生乳杆菌及其应用 Download PDFInfo
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- CN116970522A CN116970522A CN202310845300.9A CN202310845300A CN116970522A CN 116970522 A CN116970522 A CN 116970522A CN 202310845300 A CN202310845300 A CN 202310845300A CN 116970522 A CN116970522 A CN 116970522A
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- Prior art keywords
- propylene glycol
- lactobacillus
- ethyl palmitate
- white spirit
- chromenensis
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Abstract
本发明属于生物领域，具体涉及一株产棕榈酸乙酯的食丙二醇慢生乳杆菌及其应用。所述食丙二醇慢生乳杆菌的保藏号：CGMCC No.25140，实验表明该菌株发酵液中棕榈酸乙酯含量为1.545mg/L，其筛选于浓香型白酒酿造过程的酒醅中，可用于白酒和果酒的酿造，增加酒体风味，或用于生产食用香料，具有较好的应用价值。
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乙酯的食丙二醇慢生乳杆菌及其应用。
背景技术
棕榈酸乙酯又名十六酸乙酯、十六烷酸乙酯或软脂酸乙酯，为无色针状结晶。主要用途有：(1)具有微弱的蜡香和奶油香气，被GB2760-96规定为允许使用的食用香料，同时广泛用于香料、香精的生产中。(2)乳化剂、润滑剂、稳定剂及增塑剂合成的中间体。(3)合成蔗糖酯及蔗糖多酯的重要原料。
除此，棕榈酸乙酯广泛存在于白酒和果酒中，它可以增加酒体中的醇厚感，降低酒中的干涩味，同时增强白酒的后味。而其含量过高则会产生油腻，微涩的感官体现。因此，控制棕榈酸乙酯的含量能够有效提高白酒的口感与品质。陈华明等(陈华明,屈午,付立业,等.基于OAVs的柔和酱香型白酒和酱香型白酒风味组分的主成分分析[J].酿酒科技,2018(7):6.)采用气相色谱法对柔和酱香型和酱香型白酒中63种风味组分进行检测，根据主成分分析，发现柔和酱香型白酒区别于酱香型白酒的特征成分主要集中在第2主成分中，包括糠醛、3-羟基-2-丁酮、丁酸乙酯、己酸乙酯、棕榈酸乙酯、2-丁醇、正丙醇、丁酸以及己酸。
现有研究中，具有产棕榈酸乙酯能力的多为酵母属，郑树炀(郑树炀.山河陈醋酒精发酵菌群研究及工艺优化[D].山西农业大学,2019.)研究发现，酵母菌QY18的高粱发酵液中检出具有食醋特征性风味，分别为苯乙醇、乙酸乙酯、醋酸苯乙酯、醋酸异戊酯、棕榈酸乙酯、2-甲基丁酸、苯乙醛。齐晓茹(齐晓茹.红枣白兰地发酵过程中香气成分及真菌多样性研究[D].河北农业大学.)探索了红枣白兰地发酵过程中真菌群落结构与香气成分的相关性，结果显示乙酸异戊酯、异戊醇、棕榈酸乙酯和乙酸与酵母属有较强的相关性。关于可产棕榈酸乙酯的细菌鲜有报道。
发明内容
本发明从浓香型白酒酿造过程的酒醅中分离筛选出获得一株具有产棕榈酸乙酯的乳酸菌zqw41，可应用于白酒或果酒的酿造，增加酒体风味中棕榈酸乙酯的含量，或用于生产食用香料。
本发明提供了一株产棕榈酸乙酯的食丙二醇慢生乳杆菌(Weissella cibaria)，其保藏号为CGMCC No.25140，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.25140，分类名称为食丙二醇慢生乳杆菌Lentilactobacillus diolivorans。
本发明因此提供所述的食丙二醇慢生乳杆菌在产棕榈酸乙酯中的应用。
进而提供一种利用所述的食丙二醇慢生乳杆菌生产棕榈酸乙酯的方法，其是将所述食丙二醇慢生乳杆菌在发酵培养基中培养，以产生棕榈酸乙酯。
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食丙二醇慢生乳杆菌在白酒或果酒酿造中的应用，或在制备食用香料或香精中的应用。
具体地，将所述食丙二醇慢生乳杆菌作为白酒风味发酵剂应用于白酒或果酒酿造中。
更优选地，将所述食丙二醇慢生乳杆菌应用于白酒或果酒酿造曲药或用于窖池发酵中添加剂，以提高棕榈酸乙酯的含量。
在具体实施方式中，所述白酒风味发酵剂的形式选自是液体、冻干制剂或粉末。
本发明提供的食丙二醇慢生乳杆菌zqw41具有发酵产棕榈酸乙酯的能力，在一个实验中表明该菌株发酵液中棕榈酸乙酯含量为1.545mg/L，其筛选于浓香型白酒酿造过程的酒醅中，适应能力强，可为白酒或果酒酿造工艺优化提供菌种资源，增加酒体的风味，具有较为广阔的市场应用前景。
附图说明
图1为菌株产棕榈酸乙酯GC-MS图。
图2为菌株所产的棕榈酸乙酯GC-MS分子碎片质谱图。
图3为棕榈酸乙酯标准品GC-MS分子碎片质谱图。
菌株保藏信息：
本发明的食丙二醇慢生乳杆菌已于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.25140，其分类命名为食丙二醇慢生乳杆菌(Lentilactobacillus diolivorans)。
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泸州老窖酒醅样品，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：产棕榈酸乙酯能力检测
(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检测棕榈酸乙酯方法：
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，在分离得到的菌株中有一株具有高产棕榈酸乙酯能力，GC-MS质谱图如图1所示。选择该菌株做进一步研究。
实施例2：菌株zqw41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，鉴定其为食丙二醇慢生乳杆菌(Lentilactobacillus diolivorans)，并命名为食丙二醇慢生乳杆菌zqw41(Lentilactobacillus diolivorans)。
测序获得16S rDNA片段的基因序列如SEQ ID NO:1所示：
AACAACTCTGTCACCTTAGACGGCTGGCCCCCGAAGGTTACCTCACCGGCTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATGCTGATCCACGATTACTAGCGATTCCAACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGCTTTAAGAGATTAGCTTGACCTCGCGGTTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTGCTAGAGTGCCCAACTAAATGCTGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTCTGTCCCCGAAGGGAACGCCTAATCTCTTAGGTTGGCAGAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTGCAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTCTCCCGGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACCTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGATGTCAACAGTTACTCTGACATCCGTTCTTCTCCAACAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCTAAAGCGATAGCCGAAGCCATCTTTCAAACGAAAACCAGGCGGTTTTCGTTGTTATGCAGTATTAGCACCTGTTTCCAAGTGTTATCCTCTACTTCAAGGGCAGGTTACCCACGTGTTACTCACCAGTTCGCCACTCGTCTTAATGTCAAATCAGTCAAGTGCAAGCACCTAAAATCATTGACCAAGACGCGTTCGACTTGCATTATAGCACGCCGCCCGTCCC。
实施例4：菌株zqw41的发酵实验
将获得的食丙二醇慢生乳杆菌zqw41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。食丙二醇慢生乳杆菌zqw41在持续发酵2天后，所产棕榈酸乙酯产量可达1.545mg/L。
实施例5：菌株zqw41的形态特征和理化特性
1.经实验表明，食丙二醇慢生乳杆菌zqw41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L-阿拉伯糖，D-核糖，D-木糖，D-半乳糖，D-葡萄糖，D-果糖，D-甘露糖，甘露醇，D-纤维二糖，D-麦芽糖，D-乳糖，D-密二糖，D-海藻糖，D-土伦糖；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-棉子糖，D-龙胆二糖；甲基-βD-吡喃木糖苷，甲基-αD-吡喃葡萄糖苷，七叶灵柠檬酸铁，葡萄糖酸钾；丙三醇，D-蔗糖，淀粉等28种单一碳源产酸。其利用能力由强到弱：L-阿拉伯糖，D-核糖，D-木糖，D-半乳糖，D-葡萄糖，D-果糖，D-甘露糖，甘露醇，D-纤维二糖，D-麦芽糖，D-乳糖，D-密二糖，D-海藻糖，D-土伦糖；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-棉子糖，D-龙胆二糖；甲基-βD-吡喃木糖苷，甲基-αD-吡喃葡萄糖苷，七叶灵柠檬酸铁，葡萄糖酸钾；丙三醇，D-蔗糖，淀粉。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7.1g/L，乙酸1.25g/L，乙醇3.51g/L；无残糖。
实施例6：菌株zqw41发酵产其他产物的实验
食丙二醇慢生乳杆菌zqw41按实施例4的方法发酵除可产1.545mg/L的棕榈酸乙酯，还可以产其他32种挥发性代谢产物，分别为：1-甲氧基十一烷0.367mg/L，异戊醇0.886mg/L，正庚醇0.972mg/L，正辛醇0.129mg/L，1-壬醇0.474mg/L，2-壬基醇0.303mg/L，苯乙醇1.521mg/L，顺11，13-十四碳二烯醇0.442mg/L，乙酸5.816mg/L，丁酸0.347mg/L，己酸1.435mg/L，异辛酸0.393mg/L，辛酸4.563mg/L，壬酸4.372mg/L，正癸酸0.592mg/L，顺-5-十二碳烯酸0.389mg/L，香叶基丙酮0.352mg/L，2,3-二氢-2,2,6-三甲基苯甲醛1.740mg/L，苯乙醛0.445mg/L，乙酸2-辛酯0.128mg/L，3-羟基-2，2，4-三甲基戊基异丁酸酯0.192mg/L，2,2,4-三甲基-1,3-戊二醇二异丁酸酯0.137mg/L，磷酸三丁酯0.214mg/L，磷酸三异丁酯2.312mg/L，亚油酸乙酯0.186mg/L，十四酸乙酯0.427mg/L，反油酸乙酯1.758mg/L，苯酚0.026mg/L，4-氯-3,5-二甲基苯酚0.388mg/L，2,4-二叔丁基苯酚0.839mg/L，2,6-二甲基吡嗪0.363mg/L，苯并噻唑0.056mg/L。代谢产物种类繁多。
Claims (10)
1.一株产棕榈酸乙酯的食丙二醇慢生乳杆菌（Lentilactobacillus diolivorans），其保藏号为CGMCC No. 25140。
2.如权利要求1所述的食丙二醇慢生乳杆菌在产棕榈酸乙酯中的应用。
3.一种利用如权利要求1所述的食丙二醇慢生乳杆菌生产棕榈酸乙酯的方法，其特征在于，将所述食丙二醇慢生乳杆菌在发酵培养基中培养，以产生棕榈酸乙酯。
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所述的食丙二醇慢生乳杆菌在白酒或果酒酿造中的应用，或在制备食用香料或香精中的应用。
8.如权利要求7所述的应用，其特征在于，将所述食丙二醇慢生乳杆菌作为白酒风味发酵剂应用于白酒或果酒酿造中。
9.如权利要求8所述的应用，其特征在于，将所述食丙二醇慢生乳杆菌应用于白酒或果酒酿造曲药或用于窖池发酵中添加剂，以提高酒体中棕榈酸乙酯的含量。
10.如权利要求3至4任一项所述的应用，其特征在于，所述白酒风味发酵剂的形式选自是液体、冻干制剂或粉末。
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