洋河酒窖泥细菌群落结构与菌株产酸能力分析

【背景】窖泥微生物的种类及其代谢产物类型是影响浓香型白酒发酵过程中丁酸和己酸等白酒中主要有机酸合成的影响因素之一。【目的】揭示浓香型白酒不同窖龄窖泥细菌群落结构,研究厌氧细菌产酸性能,阐明窖泥细菌与白酒中有机酸合成的相关性。【方法】通过Illumina HiSeq高通量测序,基于16S rRNA基因序列分析不同窖龄窖泥细菌的组成。分离获得厌氧细菌,通过比较菌株产丁酸和己酸能力来分析窖泥的微生物代谢特性。【结果】洋河酒窖泥细菌主要分布于梭菌纲(Clostridia)、拟杆菌纲(Bacteroidia)、互营养菌纲(Synergistia)和芽孢杆菌纲(Bacilli)。20年窖龄的窖泥中氢孢菌属(Hydrogenispora)和瘤胃梭菌属(Ruminiclostridium)丰度显著增加。窖泥细菌间相关性分析表明,瘤胃梭菌属(Ruminiclostridium)为窖泥中影响最大的核心微生物,很多微生物与梭菌属(Clostridium)菌株之间多为相互促进关系。通过传统可培养方法共分离得到梭菌目(Clostridiales)的20株厌氧菌。其中梭菌属(Clostridium)菌株产酸能力高于其他菌属,酪丁酸梭菌(Clostridium tyrobutyricum)和丁酸梭菌(Clostridium butyricum)产丁酸和己酸的能力最强。产丁酸能力最高的菌主要分离自5年和20年窖龄窖泥,产己酸能力最高的菌分离自20年窖龄窖泥。【结论】解析了浓香型白酒不同窖龄窖泥的细菌组成,并对菌株产丁酸和己酸的能力进行了比较,为揭示窖泥微生物功能及其对白酒风味物质合成的影响奠定了相关的研究基础。     

浓香型白酒窖泥放线菌的群落结构及其多样性

以泸州老窖1、50、100和400年窖泥为研究对象,采用变性梯度凝胶电泳(DGGE)研究浓香型白酒窖泥放线菌的群落结构及其多样性。DGGE图谱显示,除1年样品外,其余窖底泥多样性指数(H)均低于同窖龄窖壁泥,但均匀度指数(EH)较高。不同窖池相同部位窖泥的群落结构变化趋势为:随窖龄的延长,窖壁泥H值逐渐上升,为1.74—2.28;窖底泥下降,为1.73—2.07。EH值均为波动下降,分别在0.986—0.991和0.971—0.994之间。窖底泥相似性系数(SC)逐渐上升,为0.46—0.82;窖壁泥为0.31—0.62。DGGE条带测序结果显示,窖泥放线菌归于Olsenella、Atopobium、Streptomyces和Corynebacterium 4个属。Olsenella和Atopobium属为共有的优势属,且在窖壁泥中的优势度(di)均随窖龄延长而降低,在窖底泥中升高。实验结果表明,浓香型白酒窖泥蕴藏着丰富的放线菌资源,群落结构和多样性存在差异,菌群演替呈现一定规律性。     

窖泥群落结构及功能微生物研究进展

窖泥是浓香型白酒的特征发酵载体,包含多种功能微生物,这些功能微生物对浓香型白酒的风格特征和品质有着重要的影响。本文对不同窖龄窖泥、不同浓香型白酒产地的窖泥及不同质量窖泥的窖泥微生物研究进展进行总结分析,并在此基础上对窖泥主体微生物的功能进行阐述。以期为窖泥微生物的研究提供思路,为浓香型白酒的质量和香味成分研究提供思考,促进窖泥微生物的研究,加深对浓香型白酒窖泥的认知。     

Characterization of eubacterial and archaeal community diversity in the pit mud of Chinese Luzhou-flavor liquor by nested PCR–DGGE

The aim of this study was to investigate and compare the microbial community structures of eubacteria and archaea in the pit mud of Chinese Luzhou-flavor liquor from the wall (C_w) and bottom (C_b) of cellar through nested PCR–denaturing gradient gel electrophoresis (DGGE). The Shannon–Wiener index (H) calculated from the DGGE profiles showed that the community diversities of eubacteria and archaea in samples from C_b were almost higher than that from C_w. In addition, cluster analysis of the DGGE profiles revealed that some differences were found in the microbial community structure in samples from different locations. The closely relative microorganisms of all eubacterial 16S rRNA gene sequences fell into four phyla (Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria), including 12 genera and 2 uncultured eubacteria. Moreover, 37.1 % eubacteria were affiliated with Clostridium. Particularly, genus Acinetobacter was absent in all samples from C_b but present in all samples from C_w. The closely relative microorganisms of all archaeal 16S rRNA gene sequences fell into four genera, which included Methanobrevibacter, Methanoculleus, Methanobacterium and Methanosaeta, while the dominant archaea in samples from C_w and C_b were similar. Results presented in this study provide further understanding of the spatial differences in microbial community structure in the pit mud, and is of great importance for the production and quality improvement of Luzhou-flavor liquor.     

浓香型白酒窖泥中可培养细菌的分离鉴定及产酸研究

为系统了解浓香型白酒窖泥中可培养细菌的多样性,采用平板稀释涂布法分离筛选窖泥中可培养菌株。扩增纯培养细菌的16SrRNA基因,测序并与EzBioCloud数据库比对,所有序列已在GenBank中注册。结果共从窖泥中筛选出42株差异性较大的菌株,其中5株与模式菌株相似性低于97％,共包括14个属,以Bacillus、Lysinibacillus、Sporosarcina、Staphylococcus四个属为主;高效液相色谱检测各个菌发酵液结果表明,有机酸包括乙酸、乳酸、酒石酸、苹果酸、柠檬酸、琥珀酸、α-酮戊二酸、草酸;其中尤以乙酸、乳酸的产量较高。     

浓香型白酒窖池微生物群落结构特征

窖池是中国白酒,尤其是浓香型大曲酒生产颇具特色的固态生物反应器,窖龄与微生物群落结构关系密切且复杂,对产品质量影响非常显著.本研究以微生物细胞膜的特征组分磷酸脂肪酸(PLFA)为指标,研究了不同窖龄(5年、100年和300 年)窖池窖泥、糟醅和黄水的微生物群落结构特征.结果表明:窖泥中总PLFA含量最高,糟醅次之,黄水最低.PLFA的组成因窖龄而异,黄水中总PLFA含量随窖池窖龄的增长而减小.窖泥中直链饱和脂肪酸含量最高,为PLFA总量的50.7%～73.9%,其中300年窖池窖泥最高.窖泥中表征革兰氏阳性(G+)厌氧细菌的PLFA含量较高,而糟醅和黄水中均以表征革兰氏阴性(G-)厌氧菌的PLFA含量较高.100年窖泥中表征G+菌、G-菌和厌氧菌的PLFA含量高于其他窖龄相应样品.5年窖窖泥、糟醅和黄水中真菌PLFA含量均高于其他窖龄相应样品.经主成分分析,5年窖和100年窖中主要变异菌群是G+菌和真菌,300年窖中主要变异菌群是细菌.描述窖池微生物群落特征的多样性指数可以选用PLFA的频次、Simpson优势度指数和Shannon多样性指数.     

浓香型白酒两个产区窖泥微生物群落结构分析

【目的】探索浓香型白酒两个典型产区窖泥微生物群落结构和多样性,分析窖泥微生物群落地域特征及对白酒风格形成的影响。【方法】分别提取四川和安徽两个产区窖泥样品总DNA,应用PCR-ARDRA和16S rRNA基因克隆测序技术对两个产区窖泥细菌和古菌进行研究。【结果】两个浓香型白酒产区窖泥细菌丰富,包括:厚壁菌门(Firmicute)、拟杆菌门(Bacteroidetes)、绿弯菌门(Chloroflexi)、互养菌门(Synergistetes)、Armatimonadetes类群和未分类细菌(Unclassified bacteria)。两个产区窖泥绝对优势种群均为厚壁菌门中梭菌纲(Clostridia)细菌,在四川产区窖泥中检出较多的互营单胞菌属(Synthrophomonas)和紫单胞菌属(Petrimonas)。古菌的群落组成较为简单,主要是甲烷囊菌属(Methanoculleus)、甲烷八叠球菌属(Methanosarcina)、甲烷鬃菌属(Methanosaeta)和甲烷杆菌属(Methanobacterium)4个产甲烷古菌类群,四川产区窖泥优势古菌为甲烷囊菌和甲烷八叠球菌,安徽产区则为甲烷八叠球菌属和甲烷鬃菌。【结论】四川和安徽两个产区窖泥微生物的16S rRNA基因克隆文库系统地反映了两者微生物群落的相似性和差异性,对揭示浓香型白酒两个产区的酒体风格差异形成有一定的参考价值。     

Microbial Diversity and Succession in the Chinese Luzhou-Flavor Liquor Fermenting Cover Lees as Evaluated by SSU rRNA Profiles

The microbial diversity and the community succession in the fermenting cover lees of Chinese Luzhou-flavor liquor were investigated by small-subunit rRNA (SSU rRNA) culture independent method. All sequences retrieved from the 1, 7 and 60 days fermented cover lees were respectively assigned into the genera of Streptococcus, Acetobacter, Arthrobacter, Bacillus, Staphylococcus, Serratia, Nocardia, Methanoculleus, Clostridium, Aneurinibacillus, Corynebacterium, Lactobacillus, Microbacterium, Trichosporon, Saccharomycopsis, Sagenomella, Talaromyces, Eurotium, Issatchenkia, Zygosaccharomyces, Saccharomyces and TM7 phylum. The fungal Issatchenkia, Saccharomycopsis and Talaromyces and the bacteria Staphylococcus and Lactobacillus were most abundant in the 1 day fermented cover lees, the fungal Issatchenkia, Saccharomyces and Talaromyces and the bacteria Bacillus and Streptococcus were dominant in the 7 days cover lees, the archaea Methanoculleus and the fungal Eurotium and Talaromyces were prevalent in the 60 days cover lees. When the microbial community profiles in three samples were compared at species level, the prokaryotic community similarity coefficient was from 0.4042 to 0.5703 and descended to 0.2222, and that of eukaryotic community was from 0.3000 to 0.6000 and followed to 0.5215. These results suggested that microbial diversity variability and community succession have happened in the cover lees associated with fermentation proceeding and such variability and succession respond for the appearance of some unique flavor of Luzhou-flavor liquor.     

浓香型白酒窖泥中兼性厌氧细菌的分离鉴定

为探讨浓香型白酒窖泥中微生物区系的构成,采用传统微生物分类鉴定法,对泸州老窖不同窖龄窖底和窖壁泥样的可培养兼性厌氧细菌进行了分类计数,并初步鉴定到属。平板涂布法计数得出,老窖底样的菌落总数最多,达3.98×103cfu/g泥,新窖壁样菌落数最少,只有0.24×103cfu/g泥;经生理生化鉴定,参照伯杰氏细菌鉴定手册等将划线单离后的菌株归为8个属,其大部分属于芽孢杆菌属(Bacillus)和芽孢乳杆菌属(Sporolac-tobacillus),也存在假单胞菌属(Pseudomonas)、梭菌属(Clostridium)等。     

Modeling of lactic acid fermentation of leguminous plant juices

Lactic acid fermentation of leguminous plant juices was modeled to provide a comparative efficiency assessment of the previously selected strains of lactic acid bacteria as potential components of starter cultures. Juices of the legumes fodder galega, red clover, and alfalfa were subjected to lactic acid fermentation in 27 variants of the experiment. Local strains (Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, and Lactobacillus sp. RS 4) and the collection strain Lactobacillus plantarum BS 933 appeared the most efficient (with reference to the rate and degree of acidogenesis, ratio of lactic and acetic acids, and dynamics of microflora) in fermenting fodder galega juice; Lactobacillus sp. RS 1, Lactobacillus sp. RS 2, Lactobacillus sp. RS 3, Lactobacillus sp. RS 4, and L. plantarum BS 933 were the most efficient for red clover juice. Correction of alfalfa juice fermentation using the tested lactic acid bacterial strains appeared inefficient, which is explainable by its increased protein content and a low level of acids produced during fermentation.     

Profiling the effects of physicochemical indexes on the microbial diversity and its aroma substances in pit mud

Microbial diversity of pit mud (PM) plays a significant role in Baijiu's flavour. Here we explored the microbial community structures and aroma substances of Wenwang Winery with high-throughput sequencing coupling with headspace solid-phase microextraction-gas chromatography-mass spectrometry. We discovered that the odorant was mainly derived from 14 aroma compounds because of their OAVs ≥ 1 (OAV, the ratio of substance concentration to aroma threshold; s, on behalf of the plural), such as ethyl hexanoate (2438), ethyl octanoate (975), caproic acid (52) and etc. Moreover we also revealed that Lactobacillaceae (97·08%) was the mainly bacterial microbial community in 2-year-old PM, companied by the primarily fungi including Aspergillaceae (55·45%), Unclassified Ascomycota (11·13%) and Dipodascaceae (5·72%). Compared with the 2-year-old PM, bacterial floras in 20-year-old PM and 30-year-old PM were more abundant (i.e. Dysgonomonadaceae, Clostridium and Synerggstaceas), while no fungi were detected. Besides, the physicochemical analysis showed that the content of Lactobacillaceae was inversely associated with moisture, pH and ammonia nitrogen. By further Spearman's correlation coefficient analysis, we verified that the content of Lactobacillaceae was positively correlated with ethyl hexanoate, while negatively correlated with ethyl octanoate and caproic acid. Meanwhile, ethyl octanoate and caproic acid were positively correlated with most flora including Ruminococcaceae, Dysgonomonadaceae and Clostridiacea, which were related to physicochemical indexes. This work demonstrates promise for adjusting the physicochemical indexes of PM to affect the micro-organisms and aroma, which may provide a reference for the production of high-quality Baijiu.     

Molecular Diversity of Lactobacillus spp. and Other Lactic Acid Bacteria in the Human Intestine as Determined by Specific Amplification of 16S Ribosomal DNA

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and Weissella. Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant’s life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.     

基于窖泥微生物相对定量与绝对定量联用的窖泥老熟特征分析

[目的] 基于传统的相对定量法,在总DNA提取过程中加入内标菌,探索不同老熟程度下窖泥微生物菌群组成特征。[方法] 通过添加外源菌丙酮丁醇梭菌（Clostridium acetobutylicum）作为内标菌,利用16S rRNA基因扩增子测序技术,基于相对丰度与绝对丰度解析不同老熟程度窖泥内的菌群结构和理化差异。[结果] 相对定量分析表明,随着窖泥老熟,窖泥微生物群落的多样性增加,己酸菌属（Caproiciproducens）、甲烷杆菌属（Methanobacterium）、甲烷八叠球菌属（Methanosarcina）和互营单胞菌属（Syntrophomonas）等菌的相对丰度显著增高。结合绝对丰度分析,老熟窖泥内的生物量分别是新窖泥与趋老熟窖泥的600倍与28倍。不同于相对定量分析,乳杆菌属（Lactobacillus）的绝对丰度在老熟窖泥内并未降低,为新窖泥的20倍。己酸菌属在老窖泥中的绝对丰度为新窖泥的25000倍;其他功能微生物包括甲烷杆菌属、甲烷八叠球菌属、甲烷短杆菌属（Methanobrevibacter）、互营单胞菌属的绝对丰度也随着窖龄的增加而显著增加。菌群结构的绝对定量动态分析表明,新窖泥转变为老熟窖泥的过程是一个菌群组成多样性不断提高、生物量也逐步增加的过程,老窖泥中己酸菌属、互营单胞菌属、多种甲烷菌等微生物绝对量的增加,推测对降低窖泥内的乳酸含量和形成窖泥稳态环境具有重要作用。[结论] 绝对定量方法的引入,可观测不同窖龄窖泥微生物生物量的差异,解析不同老熟阶段窖泥功能微生物绝对丰度的动态变化,为窖泥菌群结构和代谢功能的定量化和因果关系研究提供了新的策略。     

Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1

The fruity odor of Chinese liquor is largely derived from ester formation. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is one of the most important esters in the strong aromatic Chinese liquor (or Luzhou-flavor liquor), which is the most popular and best-selling liquor in China. In the traditional fermentation process, ethyl caproate in strong aromatic liquor is mainly produced by aroma-producing yeast, bacteria, and mold with high esterification abilities in a mud pit at later fermentation stages at the expense of both fermentation time and grains rather than by the ethanol-fermenting yeast Saccharomyces cerevisiae. To increase the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae AY15) and shorten the fermentation period, we constructed a recombinant strain EY15 by overexpressing EHT1 (encoding ethanol hexanoyl transferase), in which FAA1 (encoding acyl-CoA synthetases) was deleted. In liquid fermentation of corn hydrolysate and solid fermentation of sorghum, ethyl caproate production by EY15 was remarkably increased to 2.23 and 2.83 mg/L, respectively, which were 2.97- and 2.80-fold higher than those of the parental strain AY15. Furthermore, an increase in ethyl octanoate (52 and 43 %) and ethyl decanoate (61 and 40 %) production was observed. The differences in fermentation performance between EY15 and AY15 were negligible. This study resulted in the creation of a promising recombinant yeast strain and introduced a method that can be used for the clean production of strong aromatic Chinese liquor by ester-producing S. cerevisiae without the need for a mud pit.     

Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with <Emphasis Type="Italic">OPI1</Emphasis> deletion

During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.     

Microbial succession in the traditional Chinese Luzhou-flavor liquor fermentation process as evaluated by SSU rRNA profiles

The community succession of microbes inhabited in the fermenting lees of Luzhou-flavor liquor was investigated based on small-subunit rRNA culture independent method. All sequences recovered from fermenting lees respectively fell into the genera of Lactobacillus, Streptococcus, Bacillus, Staphylococcus, Clostridium, Pelobacter, Actobacter, Serratia, Burkholderia, Rhodoccous, Corynebacterium, Arthrobacter, Microbacterium, Curtobacterium, Leptotrichia, Methanocuuleus, Saccharomyces, Zygosaccharomyces, Saccharomycopsis, Pichia, Talaromyces, Aspergillus, Eurotium, Fomitopsis and Trichosporon. The fungal Pichia, Saccharomycopsis and Talaromyces were most abundant in the lees fermented for 1 day, the fungal Eurotium and the bacteria Burkholderia, Streptococcus and Lactobacillus were dominant in the lees fermented for 7 days, only the bacteria Lactobacillus, Burkholderia were prevalent in the lees fermented for 60 days. Most genera almost existed in the fermenting lees, while their distributions were significantly different in 1, 7 and 60 days fermented lees. The prokaryotic community similarity coefficient was from 0.5000 to 0.5455 and followed to 0.1523, and that of eukaryotic community was from 0.5466 to 0.5259 and to 0.3750 when compared at species level. These results suggested that many microbes in lees have community successions associated with fermenting and that such successions maybe contribute the fermentation process of Luzhou-flavor liquor and is main reasons that the characteristic flavor factors are produced.     

Case Study of the Distribution of Mucosa-Associated Bifidobacterium Species, Lactobacillus Species, and Other Lactic Acid Bacteria in the Human Colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

Diversity and Spatial Distribution of Prokaryotic Communities Along A Sediment Vertical Profile of A Deep-Sea Mud Volcano

We investigated the top 30-cm sediment prokaryotic community structure in 5-cm spatial resolution, at an active site of the Amsterdam mud volcano, East Mediterranean Sea, based on the 16S rRNA gene diversity. A total of 339 and 526 sequences were retrieved, corresponding to 25 and 213 unique (≥98% similarity) phylotypes of Archaea and Bacteria, respectively, in all depths. The Shannon–Wiener diversity index H was higher for Bacteria (1.92–4.03) than for Archaea (0.99–1.91) and varied differently between the two groups. Archaea were dominated by anaerobic methanotrophs ANME-1, -2 and -3 groups and were related to phylotypes involved in anaerobic oxidation of methane from similar habitats. The much more complex Bacteria community consisted of 20 phylogenetic groups at the phylum/candidate division level. Proteobacteria, in particular δ-Proteobacteria, was the dominant group. In most sediment layers, the dominant phylotypes of both the Archaea and Bacteria communities were found in neighbouring layers, suggesting some overlap in species richness. The similarity of certain prokaryotic communities was also depicted by using four different similarity indices. The direct comparison of the retrieved phylotypes with those from the Kazan mud volcano of the same field revealed that 40.0% of the Archaea and 16.9% of the Bacteria phylotypes are common between the two systems. The majority of these phylotypes are closely related to phylotypes originating from other mud volcanoes, implying a degree of endemicity in these systems.     

Evolution of the Lactic Acid Bacterial Community during Malt Whisky Fermentation: a Polyphasic Study

The development of the lactic acid bacterial community in a commercial malt whisky fermentation occurred in three broad phases. Initially, bacteria were inhibited by strong yeast growth. Fluorescence microscopy and environmental scanning electron microscopy revealed, in this early stage, both cocci and rods that were at least partly derived from the wort and yeast but also stemmed from the distillery plant. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes and sequence analysis revealed cocci related to Streptococcus thermophilus or Saccharococcus thermophilus, Lactobacillus brevis, and Lactobacillus fermentum. The middle phase began 35 to 40 h after yeast inoculation and was characterized by exponential growth of lactobacilli and residual yeast metabolism. Lactobacillus casei or Lactobacillus paracasei, L. fermentum, and Lactobacillus ferintoshensis were detected in samples of fermenting wort examined by DGGE during this stage. Bacterial growth was accompanied by the accumulation of acetic and lactic acids and the metabolism of residual maltooligosaccharides. By 70 h, two new PCR bands were detected on DGGE gels, and the associated bacteria were largely responsible for the final phase of the fermentation. The bacteria were phylogenetically related to Lactobacillus acidophilus and Lactobacillus delbrueckii, and strains similar to the former had previously been recovered from malt whisky fermentations in Japan. These were probably obligately homofermentative bacteria, required malt wort for growth, and could not be cultured on normal laboratory media, such as MRS. Their metabolism during the last 20 to 30 h of fermentation was associated with yeast death and autolysis and further accumulation of lactate but no additional acetate.