为了解立醅期清香型白酒发酵过程中乳酸、乙酸含量的变化规律以及酒醅中细菌菌群结构随发酵时间的变化情况,从酒醅中分离出产酸能力强的优势产酸菌株,采用高效液相色谱技术分析立醅期清香型白酒酒醅大楂和二楂阶段乳酸、乙酸含量的变化情况,通过HiSeq高通量测序技术分析酒醅中细菌菌群结构随发酵时间的变化规律,从MRS培养基中分离筛选出产酸能力强的乳酸菌株,并对该菌株的环境耐受性进行进一步研究。在立醅期2个月的发酵过程中,大楂和二楂的乳酸、乙酸含量呈波浪形上升趋势,且在二楂发酵阶段,乳酸、乙酸的初始含量与酒醅大楂时期出缸时的主体酸含量基本一致;酒醅中的乳酸含量远远高于乙酸含量且二楂酒醅中的主体酸含量高于大楂酒醅;另外,二楂酒醅乳酸与乙酸的比值也始终高于大楂酒醅。乳杆菌属在发酵过程中的菌群结构变化情况与乳酸含量变化相一致,尤其在发酵后期该菌属相对丰度显著升高。从发酵后期的酒醅样品中筛选得的5个产乳酸微生物中,棒状腐败乳杆菌Lactobacillus coryniformis 和嗜酸片球菌Pediococcus acidilactici 具有较强的产酸能力和耐酸、耐醇能力。该研究对清香型白酒酒醅在立醅期的主体酸含量变化和菌群结构动态变化进行了研究,为之后的微生物代谢改造提供了基本的信息,筛选出的菌株对改善清香型白酒的风味口感具有潜在应用价值。
In order to analyze the change of lactic acid and acetic acid contents and the bacterial flora structure in fermented grains during the fermentation of Fen-flavor Baijiu in LiPeiQi, and to isolate the stronger acid-producing strains from fermented grains. This paper analyzed the change of lactic acid and acetic acid contents in the Fen-flavor fermented grains during LiPeiQi at the stage of DaCha and ErCha by HPLC, and analyzed the changes of bacterial flora structure in fermented grains during the fermentation of Fen-flavor Baijiu by HiSeq high-throughput sequencing, then the strains with strong ability that produced lactic acid was isolated and screened by MRS medium. Further, the environmental tolerance of the strain had been studied. The contents of lactic acid and acetic acid at both DaCha and ErCha showed a wavy upward trend during the two-month fermentation period, and the initial contents of lactic acid and acetic acid in ErCha were basically the same as the main acids contents of the fermented grains at the time of leaving the vat during DaCha. In addition, the ratio of lactic acid to acetic acid in ErCha was always higher than that in DaCha. The change of flora structure of Lactobacillus during fermentation were consistent with the change of the lactic acid content. In the late fermentation period, the relative abundance of Lactobacillus increased significantly. Among the five strains of lactic acid-producing microorganisms screened from the late fermentation grains, Lactobacillus coryniformis and Pediococcs acidilactici could produce acid strongly. Also, that strains could tolerate acid and alcohol strongly. The change in lactic acid and acetic acid contents and in bacterial flora structure of fermented grains during the fermentation of Fen-flavor Baijiu in LiPeiQi provided basic information for microbial metabolic transformation later, and the screened strains have potential value for improving the flavor and taste of Fen-flavor Baijiu.
[1] 杨帆, 周兴鹏, 张敬铖, 等.混蒸混烧生产清香型白酒工艺[J].食品研究与开发, 2018, 39(9):76-81.
YANG F, ZHOU X P, ZHANG J C, et al.Technology of mild aromatic Chinese spirits by fermentation of mixed-steaming and mixed-heating[J].Food Research and Development, 2018, 39(9):76-81.
[2] 谢方安.谈白酒香气成分和作用[J].酿酒,2006,33(5):52-55.
XIE F A.Aroma components and effects of Baijiu[J].Liquor Making, 2006, 33(5):52-55.
[3] 刘殿英.酸在清香型白酒质量中的关系及其工艺控制[J].技术与市场,2008,15(8):66-67.
LIU D Y.Relationship of acid in quality of Fen-flavor Liquor and its technological control[J].Technology and Market, 2008,15(8):66-67.
[4] 赖高淮.新型白酒勾调技术与生产工艺[M].北京:中国轻工业出版社,2003.
LAI G H.New blending technology and production process of Baijiu[M].Beijing:China Light Industry Press, 2003.
[5] 王元太. 清香型白酒的主要微量成分及其量比关系对感官质量的影响[J].酿酒科技, 2004(3):27-29.
WANG Y T.Effects of the main trace components and its quantity relative ratio relationship of Fen-flavor liquor on the sensory quality[J].Liquor-Making Science & Technology, 2004(3):27-29.
[6] 黄树林.试论微量成份含量与清香型白酒质量的关系[J].酿酒,1997,24(1):17-18.
HUANG S L.Relationship between the content of trace components and the quality of Fen-flavor liquor[J].Liquor Making, 1997,24(1):17-18.
[7] 沈正祥,韩建书.清香型白酒的特点优势及发展趋势[J].酿酒,1997,24(3):9-11.
SHEN Z X, HAN J S.Characteristics, advantages and development trend of Fen-flavor liquor[J].Liquor Making, 1997,24(3):9-11.
[8] 康明官.清香型白酒生产技术[M].北京:化学工业出版社,2005:230-249.
KANG M G.Production technology of Fen-flavor Liquor[M].Beijing:Chemical Industrial Press, 2005:230-249.
[9] 王丽丽.乳酸菌的分离及酸奶的发酵[J].食品安全导刊,2016(33):135.
WANG L L.Isolation of lactic acid bacteria and fermentation of yoghurt[J].China Food Safety Magazine, 2016(33):135.
[10] 沈怡方,李大和.低度白酒生产技术[M].北京:中国轻工业出版社,1996:204-206.
SHEN Y F, LI D H.Low-Alcohol Liquor Production Technology[M].Beijing:China Light Industry Press, 1996:204-206.
[11] 魏志阳, 李秋志, 邢爽, 等.HPLC法同时测定清香类酒醅中主要酸和酯类物质[J].中国酿造, 2018, 37(8):167-171.
WEI Z Y, LI Q Z, XING S, et al.Simultaneous determination of main acids and esters in fermented grains of light-flavor Baijiu by HPLC[J].China Brewing, 2018, 37(8):167-171.
[12] WILSON K.Preparation of genomic DNA from bacteria[J].Current Protocols in Molecular Biology, 2001, 56(1):1-5.
[13] YAMAMOTO S, HARAYAMA S.PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains[J].Applied and Environmental Microbiology, 1995, 61(3):1 104-1 109.
[14] 林静, 何佳茜, 尚翠玲, 等.5种常见致病菌菌液OD值与其活菌数相关性研究[J].天津农学院学报, 2017, 24(4):42-45.
LIN J, HE J X, SHANG C L, et al.Study on correlation between OD values and viable count of five common pathogenic bacteria[J].Journal of Tianjin Agricultural University, 2017, 24(4):42-45.
[15] CAO Y G, MA L, DU X W, et al.Variation of organic acids in fermented grains during fermentation of Fen liquor[J].Food Science, 2011(7):229-232.
[16] 张鑫.大曲清香白酒地上控温发酵工艺探索[J].酿酒,2015,42(4):48-53.
ZHANG X.Exploration of aboveground temperature-controlled fermentation process of Daqu of Fen flavor liquor[J].Liquor Making, 2015, 42(4):48-53.
[17] 姬侨励,贾丽艳,王军燕,等.不同生产时期清香型白酒风味物质变化规律的研究[J].酿酒科技,2019(5):22-28.
JI Q L, JIA L Y, WANG J Y, et al.Change rules of flavoring substances in Qingxiang Baijiu in different production periods[J].Liquor-Making Science & Technology, 2019(5):22-28.
[18] 刘雪婷,王子媛,刘继明,等.清香型白酒酒醅发酵菌株分离鉴定及细菌群落结构分析[J].中国酿造, 2021, 40(1):39-44.
LIU X T, WANG Z Y, LIU J M, et al.Isolation and identification of fermentation strains and analysis of bacterial community structure in fermented grains of light-flavor Baijiu[J].China Brewing, 2021, 40(1):39-43.
[19] 杨浣漪,刘同杰,张国华,等.传统酸面团中旧金山乳杆菌基因型多样性及代谢性能研究[C].中国食品科学技术学会第十四届年会暨第九届中美食品业高层论坛,2017.
YANG W Y, LIU T J, ZHANG G H, et al.Genotypic diversity and metabolic properties of Lactobacillus sanfranciscensis in traditional sour dough[C].The 14th Annual conference of Chinese Society of Food Science and Technology and the 9th Sino-US Food Industry Forum,2017.
[20] MAGNUSSON J, JONSSON H, SCHNüRER J, et al.Weissella soli sp.nov., a lactic acid bacterium isolated from soil[J].International Journal of Systematic and Evolutionary Microbiology, 2002, 52(Pt 3):831-834.
[21] 翟齐啸, 陈卫, 谭惠子,等.一种用于Prevotella copri特异性筛选的培养基及其应用:中国,CN108486015A[P].2018-09-04.
ZHAI Q X, CHEN W, TAN H Z, et al.A medium for Prevotella copri specific screening and its application:China, CN108486015A[P].2018-09-04.
