酱香型窖醅中产酯细菌的分离及鉴定
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摘要
为了从酱香型窖醅中筛选产酯菌株,通过筛选培养基(透明圈法)进行初筛,用指示剂法测定发酵液的总酯含量,最终选出一株产酯较高的菌株LH2,其发酵液的总酯含量达到了2.2 g/L。通过形态学观察、生理生化试验和分子生物学技术鉴定出菌株LH2为枯草芽孢杆菌(Bacillus subtilis)。采用顶空固相微萃取-气质联用(HS-SPME-GC-MS)技术对菌株LH2发酵产物的香气物质进行分析,共鉴定出21种香气成分,主要是酯类(18.70%)、酸类(2.76%)、吡嗪类(9.71%)和醇类(12.95%),并在产酯能力较强的同时也产生四甲基吡嗪等有益身体健康的物质,增加了菌株LH2在健康白酒方向进一步研究的价值。
To screen ester-producing strains from cellar fermented grains of sauce-flavor Baijiu(Chinese liquor), through preliminary screening with the selection medium(transparent circle method), and determining the total ester content by indicator method, a strain LH2 with high yield ester was selected, and the total ester content in the fermentation broth reached 2.2 g/L. The strain LH2 was identified as Bacillus subtilis by morphological observation, physiological and biochemical tests, and molecular biological technique. The aroma substances in the fermentation products of strain LH2 were analyzed by HS-SPME-GC-MS, and a total of 21 kinds of aroma substances were identified, mainly including esters(18.70%), acids(2.76%),pyrazines(9.71%) and alcohols(12.95%). The strain also produced tetramethylpyrazine and other health-beneficial substances while increasing the ester-producing ability, which increased the value of further research on strain LH2 in healthy Baijiu.
To screen ester-producing strains from cellar fermented grains of sauce-flavor Baijiu(Chinese liquor), through preliminary screening with the selection medium(transparent circle method), and determining the total ester content by indicator method, a strain LH2 with high yield ester was selected, and the total ester content in the fermentation broth reached 2.2 g/L. The strain LH2 was identified as Bacillus subtilis by morphological observation, physiological and biochemical tests, and molecular biological technique. The aroma substances in the fermentation products of strain LH2 were analyzed by HS-SPME-GC-MS, and a total of 21 kinds of aroma substances were identified, mainly including esters(18.70%), acids(2.76%),pyrazines(9.71%) and alcohols(12.95%). The strain also produced tetramethylpyrazine and other health-beneficial substances while increasing the ester-producing ability, which increased the value of further research on strain LH2 in healthy Baijiu.
引文
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[2]沈怡方.白酒生产技术全书[M].北京:中国轻工业出版社,2007:180-200.
[3]FAN W,QIAN M C.Identification of aroma compounds in Chinese'Yanghe Daqu'liquor by normal phase chromatography fractionation followed by gas chromatography olfactometry[J].Flavour Frag J,2010,21(2):333-342.
[4]于勇.酱香型酒中的乳酸及乳酸乙酯[J].酿酒,2004,31(4):21-22.
[5]谭超,吴同,李惟一,等.近红外光谱组合区间偏最小二乘法定量白酒中的乙酸乙酯[J].计算机与应用化学,2014,31(4):510-512.
[6]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB/T 26760-2011酱香型白酒[S].北京:中国标准出版社,2011.
[7]ZHOU S S,XIONG J C.Isolation and identification of ester-producing yeast in wine starter and its application in ester production[J].Mod Food Sci Technol,2010,26(1):98-108.
[8]LUO H B,WANG D D,WANG Y,et al.Screening of mold producing aroma in Chinese koji and optimization of culture conditions[J].Adv Mater Res,2014,1033:744-747.
[9]牛广杰.优良产酯酵母菌的筛选及发酵性能研究[J].酿酒,2013,40(6):56-59.
[10]杨恩超,程世春,刘光烨.己酸乙酯酯化菌分离筛选及鉴定[J].食品与发酵科技,2010,46(3):33-36.
[11]黄丹,方春玉,储玉龙,等.一株酯化酶细菌的分离、鉴定及代谢产物特征[J].四川理工学院学报(自然科学版),2010,29(3):321-323.
[12]王耀,范文来,徐岩,等.浓香型大曲中酯化酶测定方法的研究[J].酿酒,2003,30(2):18-21.
[13]李河,张宿义,敖宗华,等.浓香型白酒酯化液的研究进展[J].中国酿造,2016,35(8):5-8.
[14]NIELSEN T.Industrial application possibilities for lipase[J].Fette Seifen Anstr,1985,87(1):15-19.
[15]刘阳.中高温大曲中产酯化酶细菌的选育[D].芜湖:安徽工程大学,2013.
[16]中华人民共和国国家质量监督检验检疫总局,中国国家标准化管理委员会.GB/T 10345-2007白酒分析方法[J].北京:中国标准出版社,2007.
[17]BAEILE M F.Gram staining technique[M].New York:Methods Mycoplasmolo,1983:39-42.
[18]LI X.Studies on bacteria producing Bouquet in Lang Wine Daqu[J].%JChongqing Teacher College(Natural Science Edition),1997,14(4):20-23.
[19]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:59-63.
[20]廖永红,任文雅,孙宝国,等.米醋中蜡状芽孢杆菌DNA提取及其ERIC-PCR体系建立[J].中国食品学报,2011,11(4):7-13.
[21]XIU L,GUO K,ZHANG H.Determination of microbial diversity in Daqu,a fermentation starter culture of Maotai liquor,using nested PCR-denaturing gradient gel electrophoresis[J].World J Microb Biot,2012,28(6):2375-2381.
[22]赵兴秀,何义国,赵长青,等.产酱香功能菌的筛选及其风味物质研究[J].食品工业科技,2016,37(6):196-200.
[23]LI W,HU G,LEI L,et al.Identification and aroma impact of volatile terpenes in Moutai liquor[J].Int J Food Prop,2016,19(6):1335-1352.