浓香型白酒窖泥中梭菌的分离、鉴定及产丁酸性能的比较研究

Isolation and identification of clostridia from the pit mud of Strong-flavor Baijiu and comparative study on butyric acid production

为了研究窖泥来源的23株梭菌微生物的分类信息和挥发性代谢产物的差异,通过厌氧富集培养法从浓香型白酒窖泥内分离获得微生物,并比较不同菌株16S r DNA序列和系统发育树分析其分类地位,将不同微生物在RCM液态培养基内35℃厌氧培养5 d,其发酵液通过自动进样-气相色谱-质谱法(SPME-GC-MS)分析其可挥发代谢产物。分离得到的23株细菌分别与5类梭菌--拜氏梭菌菌群(Clostridium beijerinckii)、丁酸梭菌(C.butyricum)、还原铬梭菌(C.chromiireducens)、酪丁酸梭菌(C.tyrobutyricum)、广西梭菌(C.guangxiense)具有较高的序列同源性。其中拜氏梭菌菌群是一个包括C.beijerinckii、C.diolis、C.saccharobutylicum、C.chromiireducens、C.puniceum、C.saccharoperbutylacetonicum 6个16S r DNA序列相似度超过97%的同属近似种组成的菌群,与其近似的相关菌株基于16S rDNA序列难以鉴定到种。气质分析结果共检测到包括醇类、酯类、酸类等16种风味物质,且主要代谢产物为丁酸。研究表明,在浓香型白酒窖泥内存在比人们预估更多的梭菌类群,同时不同梭菌在产丁酸能力上存在明显差异。

The purpose of this study was to classify 23 clostridia isolated from the pit mud of Strong-flavor Baijiu and compare their metabolites.The organisms were isolated from the pit mud of Strong-flavor Baijiu using anaerobic enrichment cultivation method.The taxonomy prediction was conducted based on 16 S r DNA sequence comparison,and a phylogenetic tree was constructed.The microbes were fermented in liquid Robertson’s Cooked Meat medium(RCM)anaerobically at 35℃for 5 days,and the volatilic metabolites were analyzed using solid phase microextraction-gas chromatography-mass spectrometry(SPME-GC-MS).Twentythree Clostridium strains were isolated,they showed high similarities with 5 Clostridium species including:Clostridium beijerinckii group,C.butyricum,C.chromiireducens,C.tyrobutyricum,and C.guangxiense.The C.beijerinckii group related strains cannot be classified to species level based on their 16 S rDNA sequences because this group had 6 similar species(C.beijerinckii,C.diolis,C.puniceum,C.saccharobutylicum,and C.butyricum)with similarity of over 97%based on their 16 S rDNA sequences.A total of 16 flavor compounds including of alcohols,esters,and acids were analyzed using gas chromatography-mass spectrometry(GC-MS)analysis.The quantity of butyric acid was the highest among all.There were more Clostridium species found in pit mud of Strong-flavor Baijiu than we expected.In addition,the producing abilities of butyric acid varied significantly among these Clostridium species.