CN116751713A - 一株能产丁位十二内酯的戊糖片球菌及其应用 - Google Patents
一株能产丁位十二内酯的戊糖片球菌及其应用 Download PDFInfo
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- CN116751713A CN116751713A CN202310684068.5A CN202310684068A CN116751713A CN 116751713 A CN116751713 A CN 116751713A CN 202310684068 A CN202310684068 A CN 202310684068A CN 116751713 A CN116751713 A CN 116751713A
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- Prior art keywords
- pediococcus pentosaceus
- dodecalactone
- delta dodecalactone
- delta
- producing
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Abstract
本发明属于生物领域，具体涉及一种能产丁位十二内酯的戊糖片球菌。所述戊糖片球菌的保藏号：CGMCC No.25144，其筛选于泸州老窖白酒酿造中温大曲中，在实验中表明该菌株发酵液中丁位十二内酯含量为0.752mg/L，可应用于提高发酵食品中丁位十二内酯的含量，促进风味提升。
Description
技术领域
本发明属于微生物领域，具体涉及一株能产丁位十二内酯的戊糖片球菌(Pediococcus pentosaceus)及其应用。
背景技术
丁位十二内酯别名δ-十二内酯，具有奶油香味和果香香气，是GB 2760-96规定为允许使用的食用香料，主要用于配制人造奶油、桃子、椰子和梨等型香精；可用作日用香精或其它功能添加剂，有良好定香作用。其天然存在于桃子、椰子、奶酪和奶制品中，研究发现，丁位十二内酯对酸牛奶的风味影响较大(刘南南,郑福平,张玉玉,等.SAFE-GC-MS分析酸牛奶挥发性成分[J].食品科学,2014(22):4.)。
现有研究中发现，微生物可代谢产生丁位十二内酯，可用于开发通过生物转化获得天然香料的工艺路线，也可用于提升发酵食品尤其是乳制品的风味。如张丽等(张丽,杨丽丽,余群力,等.甘肃牧区优良乳酸菌株风味研究[J].食品工业,2012(4):5.)研究从甘肃牧区筛选的3株乳酸菌-乳酸乳球菌乳脂亚种、嗜酸乳杆菌、干酪乳杆菌的风味特征，结果显示乳酸、2,3-丁二酮、3-羟基-2-丁酮及δ-十二内酯、丁酸乙酯、己酸乙酯是这3株菌的主体特征风味。孟茜(孟茜.荸荠发酵乳的研制[D].江苏:扬州大学,2016.)利用嗜热链球菌和德氏乳杆菌保加利亚亚种组合制备荸荠发酵乳，发酵乳中δ-十二内酯含量有显著提高。CN111635865B公开了利用经过多轮诱变的巴氏酵母Saccharomyces pastorianus OMK-70发酵还原马索亚油中的5-羟基-2-癸烯酸δ-内酯与5-羟基-2-十二烯酸δ-内酯生成对应的丁位癸内酯及丁位十二内酯。CN108148766B公开了由霉菌OMK-25转化正烷酸或其酯生成5-羟基烷酸或其酯，再酸化或升温加热生成丁位内酯的方法。
戊糖片球菌(Pediococcus pentosaceus)属链球菌科片球菌属，为革兰氏阳性菌，因能发酵葡萄糖产生乳酸而属于乳酸菌的一种。具有抑制食源性致病菌、调节肠道免疫功能、降低胆固醇等益生功能，也可应用于发酵肉制品、发酵乳制品和发酵蔬菜等食品加工过程，在提升产品品质、保证食用安全性、提高生产效率方面有积极作用。如Gerasi等(GERASIE，LITOPOULOUTZANETAKI E，TZANETAKIS N.Microbiological study of Manura，a hardcheese made from raw ovine milk in the Greek island Sifnos[J].Internationaljournal of dairy technology，2003，56(2)：117-122.)从瑞士Manura奶酪中分离得到16株戊糖片球菌，并指出作为唯一存在于奶酪中的片球菌，是促进Manura奶酪中风味形成和加快发酵的优势菌群。
具有产丁位十二内酯能力的戊糖片球菌未见有报道，对于现已发现在食品中应用的且具有产丁位十二内酯能力的菌株如乳酸乳球菌乳脂亚种、嗜酸乳杆菌、干酪乳杆菌等，未被检测所产丁位十二内酯的含量。
发明内容
针对现有研究空白，本发明提供了一株产丁位十二内酯的戊糖片球菌(Pediococcus pentosaceus)，该菌株筛选自泸州老窖白酒酿造中温大曲中。
因此，本发明提供一株产丁位十二内酯的戊糖片球菌，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.CGMCCNO.25144，分类名称为戊糖片球菌Pediococcus pentosaceus。
本发明还提供所述戊糖片球菌在产脂肪醇，优选为产丁位十二内酯中的应用。
本发明因而也提供一种利用所述的戊糖片球菌生产脂肪醇的方法，其特征在于，将所述戊糖片球菌在发酵培养基中培养，以产生产脂肪醇。
具体地，所述脂肪醇是丁位十二内酯。
优选地，培养的条件是37℃静置培养36h，pH值为6.0。
更具体地，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。
本发有进一步提供所述的戊糖片球菌在食品加工中的应用。
优选地，所述应用是用于提高发酵食品中丁位十二内酯的含量，促进风味提升。
更具体地，所述发酵食品是奶制品。
本发明提供的戊糖片球菌是一株可应用于食品加工领域的乳酸菌，同时具有发酵产丁位十二内酯的能力，发酵液中丁位十二内酯含量为0.752mg/L，可应用于提高发酵食品中丁位十二内酯的含量，促进风味提升。
附图说明
图1为戊糖片球菌zqw50所产的丁位十二内酯GC-MS分子碎片质谱图。
图2为丁位十二内酯标准品GC-MS分子碎片质谱图。
菌株保藏信息：
本发明的戊糖片球菌已于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.25144，其分类命名为戊糖片球菌Pediococcus pentosaceus。
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泸州老窖中温大曲样品，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℃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℃下静置培养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：产丁位十二内酯能力检测
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24h，活化三代后按2％(体积比)接种量接入50mLMRS液体培养基中进行培养24h，得到待测菌液。
(2)HS-SPME/GC-MS检测丁位十二内酯方法：
运用顶空固相微萃取/气相色谱-质谱联用仪技术：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。
通过上述检测方法，从分离的菌株中筛选出一株可产丁位十二内酯的菌株，选择该菌株做进一步研究。
实施例3：细菌的分子鉴定
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。采用乳酸菌通用引物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(50l)
10×buffer(Mg2+)15μL
27F(12.5μM)2.5μL
1541R(12.5μM)2.5μL
DNA聚合酶Mix 15μL
Template DNA 0.5μL
ddH2O 14.5μL
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，鉴定其为戊糖片球菌(Pediococcus pentosaceus)，并命名为戊糖片球菌zqw50(Pediococcuspentosaceus)。
实施例4：戊糖片球菌zqw50形态特征和理化特性
1.经实验表明，戊糖片球菌zqw50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℃和42℃条件下长势均良好，45℃和48℃条件下生长非常差甚至无法生长。
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-半乳糖，D-葡萄糖，D-果糖，D-甘露糖，D-纤维二糖，D-麦芽糖，D-密二糖，D-蔗糖，D-海藻糖，D-塔格糖；N-乙酰葡萄糖胺，熊果苷，水杨苷，菊粉，D-龙胆二糖；D-木糖，苦杏仁苷，D-乳糖，葡萄糖酸钾等21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-半乳糖，D-葡萄糖，D-果糖，D-甘露糖，D-纤维二糖，D-麦芽糖，D-密二糖，D-蔗糖，D-海藻糖，D-塔格糖；N-乙酰葡萄糖胺，熊果苷，水杨苷，菊粉，D-龙胆二糖；D-木糖，苦杏仁苷，D-乳糖，葡萄糖酸钾。
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18.9g/L。
实施例5：戊糖片球菌zqw50发酵实验
将戊糖片球菌zqw50甘油保存管溶解后接种于MRS液体培养基中，并于37℃静置培养24h，活化三代后按2％(体积比)接种量接入50mL发酵培养基中进行培养36h。发酵结束后得到菌液，10000rpm离心5min后取上清。检测方法同实施例2中的GC-MS检测方法，以0.822mg/mL的2-辛醇为内标物，以原始培养基为空白对照，计算各挥发性物质的含量。
检测结果显示，戊糖片球菌zqw50发酵培养36h后可产丁位十二内酯，含量为0.752mg/L。
实施例6：戊糖片球菌zqw50发酵产其他可挥发性代谢产物的实验
发酵与检测方法同实施例5。检测结果显示，戊糖片球菌zqw50发酵培养36h后除可产0.752mg/L的丁位十二内酯外，还可产其他27种可挥发性的代谢产物，分别为：异戊醇0.884mg/L，正庚醇1.125mg/L，正辛醇0.085mg/L，1-壬醇0.315mg/L，苯乙醇1.282mg/L，香叶醇0.528mg/L；乙酸18.431mg/L，丁酸0.359mg/L，异戊酸0.413mg/L，己酸2.386mg/L，庚酸0.435mg/L，辛酸3.239mg/L，异辛酸0.422mg/L，壬酸2.776mg/L，(E)-2-癸烯酸1.898mg/L；丙酮0.343mg/L，香叶基丙酮0.265mg/L，仲辛酮0.094mg/L，苯乙酮0.037mg/L；苯乙醛1.012mg/L，2,3-二氢-2,2,6-三甲基苯甲醛0.818mg/L；3-羟基-2，2，4-三甲基戊基异丁酸酯0.014mg/L，2,2,4-三甲基-1,3-戊二醇二异丁酸酯0.058mg/L，L-抗坏血酸-2,6-二棕榈酸酯0.452mg/L；苯酚0.015mg/L，2,6-二甲基吡嗪0.313mg/L，苯并噻唑0.006mg/L。
Claims (10)
1.一种能产丁位十二内酯的戊糖片球菌（Pediococcus pentosaceus），其保藏号为CGMCC No. 25144。
2.如权利要求1所述的戊糖片球菌在产脂肪醇中的应用。
3.如权利要求2所述的应用，其特征在于，其是在产丁位十二内酯中的应用。
4.一种利用如权利要求1所述的戊糖片球菌生产脂肪醇的方法，其特征在于，将所述戊糖片球菌在发酵培养基中培养，以产生产脂肪醇。
5.如权利要求4所述的方法，其特征在于，所述脂肪醇是丁位十二内酯。
6.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36h，pH值为6.0。
7.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。
8.如权利要求1所述的戊糖片球菌在食品加工中的应用。
9.如权利要求8所述的应用，其特征在于，所述应用是用于提高发酵食品中丁位十二内酯的含量，促进风味提升。
10.如权利要求8所述的应用，其特征在于，所述发酵食品是奶制品。
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