CN116536223A - 一种梭菌菌种及其应用 - Google Patents
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Abstract
本发明公开了一种梭菌菌种及其应用,属酿酒微生物技术领域。该梭菌(Clostridium sp.)菌种5N‑1保藏于中国普通微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.27521。本发明提供的新菌种分离自白酒黄水,能够同时产异戊醇、苯丙醇、2‑乙基‑3,5‑二甲基吡嗪和丁酸,在白酒酿造领域具备良好的应用价值。
Description
技术领域
本发明属于酿酒微生物技术领域,涉及一种梭菌菌种及其应用。
背景技术
浓香型白酒是我国传统香型白酒的典型风味代表之一,其采用多粮发酵、混蒸混烧的独特工艺,具备浓郁的窖香味和绵甜醇厚的口感,广受消费者青睐。浓香型白酒发酵过程中,糟醅水分向窖池底部渗透,形成一种棕黄色或褐色的浑浊液体,称为黄水。黄水是浓香型白酒特有的发酵副产物,其不仅含有大量醇、酸、酯、酮等风味物质,还包含来自于糟醅、窖泥的乳酸菌、己酸菌、丁酸菌、梭菌、酵母菌等对白酒发酵产量和质量具有重要影响的功能微生物。充分挖掘黄水中的微生物资源对于探明浓香型白酒香味形成机理、提升白酒品质具有重要指导意义。
梭菌(Clostridium sp.)是白酒酿造过程中的重要功能菌种,主要与浓香型白酒中醇类、酸类、酯类等呈香物质的合成密切相关,部分梭菌还能代谢产生少量醛酮类化合物、烷烃类化合物和吡嗪类化合物,进一步影响白酒品质。但由于梭菌专性厌氧的特征和尚不明确的培养条件,仅有少数菌种被分离筛选得到,白酒酿造过程中的梭菌微生物仍有待进一步开发。
发明内容
本发明的目的是提供一种能在白酒酿造过程中提升酒体风味的梭菌菌种。
为实现上述目的,本发明提供如下技术方案:
保藏说明:
菌种名称:梭菌
拉丁名:Clostridium sp.
菌株编号:5N-1
分类命名:梭菌(Clostridium sp.)
保藏机构:中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称:CGMCC
地址:北京市朝阳区北辰西路1号院3号
保藏日期:2023年06月05日
保藏中心登记入册编号:CGMCC NO.27521
所述梭菌菌种5N-1的16S rDNA核苷酸序列如SEQ ID NO:1所示。
本发明所述的梭菌菌种5N-1筛选培养方法,其包括将黄水稀释后涂布于强化梭菌琼脂(Reinforced Clostridium Agar,RCA)培养基,37℃厌氧培养48 h,四区划线纯化培养3次,得到纯培养物,对所述纯培养物进行16S rDNA扩增,并将其序列与EzBioCloud比对分析,得到所述梭菌菌种5N-1。
所述梭菌菌种5N-1的菌落形态为:菌落浅黄色,形状不规则,表面湿润,不透明。
所述梭菌菌种5N-1的菌体形态为:菌体呈杆状,0.6-0.8 μm × 2.7-13.1 μm,单个或成对排列,革兰氏阳性,芽胞近端生,胞囊膨大。
所述梭菌菌种5N-1的生长特性为:可在15~40℃,pH6.0~pH8.0条件下生长,最适生长温度为37℃,最适生长pH值为pH7.0。
所述梭菌菌种5N-1的生理生化特征为:能利用D-果糖、L-岩藻糖、L-鼠李糖、丙酮酸;碱性磷酸酶、酸性磷酸酶、N-乙酰葡萄糖胺酶阳性,氧化酶、过氧化氢酶阴性。
所述梭菌菌种5N-1同时产异戊醇、苯丙醇、2-乙基-3,5-二甲基吡嗪和丁酸。
其中,上述梭菌菌种5N-1产异戊醇的能力达到0.18 ppm。
其中,上述梭菌菌种5N-1产苯丙醇的能力达到0.20 ppm。
其中,上述梭菌菌种5N-1产2-乙基-3,5-二甲基吡嗪的能力达到0.06 ppm。
其中,上述梭菌菌种5N-1产丁酸的能力达到86.96 ppm。
本发明的有益效果为:本发明从浓香型白酒黄水中筛选得到了一株梭菌菌种5N-1,该菌同时产异戊醇、苯丙醇、2-乙基-3,5-二甲基吡嗪和丁酸,能够提升浓香型白酒酒体风味,在白酒酿造领域具有广阔应用前景。
附图说明
图1所示为本发明梭菌菌种5N-1的菌落形态
图2所示为梭菌菌种5N-1的菌体形态
图3所示为梭菌菌种5N-1的16S rDNA系统发育树
图4所示为梭菌菌种5N-1发酵液的HS-SPME峰图
图5所示为空白发酵液的HS-SPME峰图
具体实施方式
以下由特定的具体实例说明本发明的实施方式,给出的实例仅为了阐明本发明,并非为了限制本发明的范围。
若未特殊说明,下述实例中使用的化学试剂均可从商业途径得到,实例中采用的技术手段为本领域技术人员所用常规手段。
实施例中使用的强化梭菌培养基(Reinforced Clostridium Medium,RCM)组分为:蛋白胨10.0 g,牛肉粉10.0 g,酵母粉3.0 g,葡萄糖5.0 g,可溶性淀粉1.0 g,醋酸钠3.0 g,氯化钠0.5 g,L-半胱氨酸盐酸盐0.5 g,蒸馏水1000 mL,调节pH至6.6-7.0;RCA培养基组分为:在RCM培养基的基础上加入20.0 g琼脂粉。
实施例1、梭菌菌种5N-1的分离与鉴定
(一)分离
取1 mL浓香型白酒黄水样品,加入9 mL无菌生理盐水中,充分振荡混匀,制成1:10的样品匀液,重复上述操作,进行梯度稀释,取1:10 ~ 1:100000稀释液涂布于RCA培养基,于37℃厌氧培养48 h,挑取平板上的菌落进行四区划线纯化,重复3次,得到纯培养细菌,命名为5N-1。
(二)鉴定
1.菌种的形态特征
分离得到的梭菌菌种5N-1于RCA培养基37℃厌氧培养24 h,菌落浅黄色,表面湿润,不透明,形状不规则。经显微镜观察,菌体呈杆状,0.6-0.8 μm × 2.7-13.1 μm,单个或成对排列,革兰氏阳性,芽胞近端生,胞囊膨大。
2.菌种的生理生化特性
利用Biolog AN鉴定系统、API 20A、API ZYM鉴定系统分析菌种生理生化特征,分离得到的梭菌菌种5N-1能够利用D-果糖、L-岩藻糖、L-鼠李糖、丙酮酸;碱性磷酸酶、酸性磷酸酶、N-乙酰葡萄糖胺酶阳性,氧化酶、过氧化氢酶阴性。
3.16S rDNA鉴定
将菌种5N-1接种至RCA培养基,于37℃厌氧培养24 h,挑取一环菌落,用细菌基因组提取试剂盒提取基因组DNA。
利用引物27F和1492R对菌种5N-1基因组16S rDNA进行扩增,27F:AGAGTTTGATCCTGGCTCAG ,1492R:GGTTACCTTGTTACGACTT。
PCR扩增体系(共50 μL):2×PCR 预混液25 μL,引物F (10 μmol/L) 1μL,引物R(10μmol/L) 1 μL,菌种基因组DNA 2 μL (约50 ng~200 ng),加双蒸水至50 μL。扩增程序:94℃预变性5 min,94℃变性1 min,55℃退火1 min,72℃延伸1.5 min,共30个循环;72℃后延伸10 min;4℃终止反应,保存备用。
16S rDNA测序:对PCR产物进行凝胶电泳检测,并送至上海生工生物有限公司完成16S rDNA测序,得到菌种5N-1 16S rDNA序列如SEQ ID NO:1所示。
系统发育树构建:将所测定菌种的16S rDNA基因序列与EzBioCloud数据库中具有有效名称的菌株序列进行比较,利用MEGA软件中的邻接法构建系统发育树,如图3所示。
4.全基因组dDDH分析
将菌种5N-1及其相近模式菌株DSM 12992T(购自德国微生物菌种保藏中心)进行全基因组dDDH分析。结果显示其与模式菌株DSM 12992T的dDDH值20.40%,小于70%。
5.全基因组ANI分析
将菌种5N-1及其相近模式菌株DSM 12992T(购自德国微生物菌种保藏中心)进行全基因组ANI分析。结果显示其与模式菌株DSM 12992T的ANI值为80.05%。
综合菌落和菌体形态、生理生化特性、16S rDNA基因序列和全基因dDDH分析、ANI分析,将菌种5N-1鉴定为梭菌(Clostridium sp.)。
实施例2、梭菌菌种5N-1产风味物质测定
将上述菌种按照2%接种量接种于RCM培养基,于37℃厌氧培养10 d。
(一)风味成分
1.检测方法:发酵液经10000 r/min离心2 min后,利用0.22 μm滤膜过滤上清液2mL,经顶空固相微萃取-气相色谱-质谱联用(HS-SPME-GC-MS)测定其可挥发性风味成分。
(1)HS-SPME条件:取2 mL过滤后的发酵液置于20 mL顶空瓶中,加入10 μL内标(4-辛醇)。60℃平衡15 min,插入针头距离液面1 cm处,吸附40 min,进样口解析3 min。
(2)GC条件:使用TG-WAXMS (30 m×0.25 mm×0.25 μm) 色谱柱,进样口温度250℃,载气(He)流速1 mL/min,不分流模式进样;气相色谱升温程序:起始柱温40℃,保持5min,以4℃/min升至230℃,保持15 min。
(3)MS条件:质谱EI源,电子轰击能量70 eV;离子源温度200℃,四级杆温度150℃,质量数扫描范围35~350 amu。
以4-辛醇(0.5ppm)为内标,采用峰面积归一化法进行半定量分析,计算各化合物的峰面积,并与内标物质的峰面积之比计算各化合物的浓度。
2.结果:在RCM培养基中,菌株5N-1所产主要风味物质为异戊醇、苯丙醇、2-乙基-3,5-二甲基吡嗪。
(1)HS-SPME峰图
菌种 5N-1发酵液峰图见图4,空白培养基峰图见图5。
(2)风味物质信息见表1。
表1梭菌菌种5N-1风味物质信息表(ppm)
风味成分 | 5N-1 | 空白对照 |
异戊醇 | 0.18±0.05 | - |
苯丙醇 | 0.21±0.09 | 0.01±0.01 |
2-乙基-3,5-二甲基吡嗪 | 0.06±0.02 | - |
注:“-”表示没有检测出。
由表1可知,梭菌菌种5N-1产生的主要风味物质为异戊醇、苯丙醇和2-乙基-3,5-二甲基吡嗪,均为浓香型白酒中的重要风味物质。
(二)主要有机酸
1.检测方法
取上述发酵液,以0.1%的磷酸水溶液稀释10倍,振荡均匀后过0.22 μm滤膜,作为待测样品。
利用HPLC测定发酵液中主要有机酸含量。色谱程序为:色谱柱:Rezex ROA-Organic Acid H+(8%) (300×7.8 mmv);柱温:80℃;流动相:5 mmol/L H2SO4;流速:0.6mL/min;进样量:10 μL;检测器:PDA (210 nm)。
2.检测结果
梭菌菌种5N-1发酵液主要有机酸含量见表2。
表2梭菌菌种5N-1所产主要有机酸信息表(ppm)
名称 | 5N-1 | 空白对照 |
丁酸 | 198.13±6.54 | 111.17±5.55 |
乙酸 | 379.03±5.61 | 389.53±6.25 |
乳酸 | 79.77±7.2 | 76.43±0.90 |
由表2可知,梭菌菌种5N-1主要产丁酸,达86.96 ppm。
本发明筛选得到的菌种能够同时产异戊醇、苯丙醇、2-乙基-3,5-二甲基吡嗪和丁酸,有助于丰富浓香型白酒风味,具有广阔应用前景。
Claims (4)
1.梭菌(Clostridium sp.)菌种5N-1,其特征在于:该菌种在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.27521。
2.根据权利要求1所述的梭菌菌种5N-1,其特征在于:所述菌种的16S rDNA核苷酸序列如SEQ ID NO:1所示。
3.根据权利要求1所述的梭菌菌种5N-1,其特征在于:所述菌种同时产异戊醇、苯丙醇、2-乙基-3,5-二甲基吡嗪和丁酸。
4.权利要求1~3任一项所述的梭菌菌种5N-1在白酒酿造中的用途。
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