Microbial diversity analysis of vineyards in the Xinjiang region using high‐throughput sequencing

The microbial diversity of soil, grape leaves and grape fruits from three wine‐growing areas in Xinjiang, China were analysed using high‐throughput sequencing. Results show that five fungal phyla and 237 fungal genera, as well as eight bacterial phyla and 314 bacterial genera were detected in 27 samples. For both fungi and bacteria, the microbial diversity was most complex in the soil, followed by grape leaves and fruit. In the soil samples, Ascomycota, Sordariales, Tetracladium and Geomyces were the dominant fungal genus, while Arthrobacter, Kaistobacter and Skermanella were the dominant bacterial genus. With regard grape leaves and fruit, Aureobasidium, Cryptococcus, Aspergillus and Pleosporaceae were the dominant fungal genus, while Pseudomonas, Sphingomonas and Adhaeribacter were the dominant bacterial genus. Different geographical locations were associated with differences in microbial diversity. The complex microflora of vineyards provides a basis for further microbial screening and its potential utilization in different regions. © 2018 The Institute of Brewing & Distilling     

High throughput sequencing of the bacterial composition and dynamic succession in Daqu for Chinese sesame flavour liquor

Sesame flavour liquor is a traditional Chinese distilled spirit with fruity, sweaty, roasted sesame and floral aromas. High temperature Daqu production is one of the key processes in the formation of sesame flavour liquor. The composition and changes in the microbial community during the making of Daqu have a significant impact on the quality of Daqu liquor. In this study, microbial diversity based on high throughput sequencing technology was used to reveal the bacterial community structure and succession law in the four critical stages of high temperature Daqu production. The results show that Firmicutes had a significant advantage (76.7%-98.2%) in the four stages as Proteobacteria and Actinobacteria reached peak values in the first and second periods, and decreased in the later periods. At the genus level, Kroppenstedtia, Lactobacillus, Weissella, Lentibacillus, Bacillus and Saccharopolyspora were detected as the main bacterial groups in the high temperature Daqu of Chinese sesame flavour liquor. The abundance of Lactobacillus and Weissella was greater than that of other microbes in the Daqu entry period. During the first Daqu flipping, the number of bacterial genera reached a peak in the production process. Then, the bacterial diversity continued to decrease until the last period, while Kroppenstedtia, Saccharopolyspora and Lentibacillus adapted to the high temperature environment and accumulated during the second Daqu flipping and the Daqu exit. This research used high throughput sequencing technology to reveal, for the first time, the bacterial composition and dynamic succession in high temperature Daqu production of Chinese sesame flavour liquor. This work will contribute to a deeper understanding of the correlation between the formation of flavour substances in sesame flavour liquor and the microorganisms used in its production. © 2019 The Institute of Brewing & Distilling     

襄阳地区高温大曲和中高温大曲真菌多样性解析

采用Illumina MiSeq高通量测序技术对采集自湖北省襄阳市某酒厂的高温大曲和中高温大曲的真菌群落结构进行解析。结果表明,高温大曲真菌类群以嗜热真菌属（Thermomyces）（57.43%）和嗜热子囊菌属（Thermoascus）（30.73%）为主,而中高温大曲真菌类群以嗜热真菌属（Thermomyces）（48.05%）、嗜热子囊菌属（Thermoascus）（17.72%）和曲霉菌属（Aspergillus）（14.56%）为主。虽然两类大曲共有核心操作分类单元（OUT）包含的序列数占总序列数的64.21%,但经主坐标分析（PCoA）和聚类分析（CA）发现两类大曲在空间排布上呈现明显分离趋势。经线性判别分析效应量（LEfSe）分析发现,导致两类大曲真菌群落结构存在差异的菌属为嗜热真菌属（Thermomyces）。一些真菌类群在高温大曲和中高温大曲中均存在,但两类大曲亦具有各自较为独特的真菌类群。     

Bacterial communities during the process of high‐temperature Daqu production of roasted sesame‐like flavour liquor

Bacterial communities in fermentative high‐temperature Daqu play an important role in the Chinese roasted sesame‐like flavour liquor brewing process. A culture‐independent method of 16S rDNA clone library was applied to investigate the changes in bacterial diversity during the process of a Daqu fermentation. The results indicated that the types of bacteria species gradually increased with prolonged fermentation time. The first dominant bacterium, Pantoea sp. (73.68%), in the raw material was only detected at the beginning of the fermentation stage (8 days) in small quantities. The second dominant bacterium, Weissella sp. (16.99%) and the third, Lactobacillus sp. (4.57%), were both detected in the beginning fermentation sample (3.22, 11.29%), in the ripening fermentation sample (24 days; 6.89, 25.11%) and in the drying stage (49 days; 3.74, 7.47%). In the beginning fermentation, Thermoactinomyces sp. (47.57%) was detected as the first predominant bacterium, and it was also detected in the ripening fermentation stage (4.93%) and in the drying stage (15.68%). The bacterial communities in the Daqu fermentation process affected the physicochemical index of Daqu. A molecular analysis of the bacterial community from the Daqu fermentation process will benefit studies on the aroma production mechanisms from these bacteria. Copyright © 2015 The Institute of Brewing & Distilling     

Bacterial and fungal diversity in the traditional Chinese strong flavour liquor Daqu

In this study, the diversity of bacterial and fungal communities in the strong flavour Daqu production process was investigated using polymerase chain reaction denatured gradient gel electrophoresis (PCR‐DGGE). The band number, dominance, diversity and similarity of the 16S rDNA differed in the DGGE patterns, reflecting the wide diversity of the microbial communities present in the different phases. The band abundance and dominance of the eukaryotic microbial community varied in the different phases. To provide insight into factors contributing to this diversity, the physicochemical properties of Daqu were explored. The core temperature of Daqu gradually increased to a maximum during the Dinghuo phase followed by a decline. The moisture and starch content decreased throughout fermentation. The total acid, reducing sugar, amino nitrogen, α‐amylase activity, glucoamylase activity, protease activity, bacterial and mould number increased gradually to a maximum value at the end of the Peijun phase before decreasing in the Dinghuo phase increasing slightly in the Huanluo phase and then remaining almost unchanged. The esterase activity exhibited a similar change to that of other enzymes and then slightly increased after the Peijun stage. For yeasts, the highest number was obtained at the Peijun stage and then decreased. Correlation analysis revealed that the diversity of both bacteria and fungi was negatively correlated with moisture and starch and positively correlated with other indices. The reducing sugar, amino nitrogen, α‐amylase and protease activities showed significant positive correlation with bacteria, and glucoamylase and esterase showed significant correlation with fungi. © 2019 The Institute of Brewing & Distilling     

基于高通量测序技术分析牛栏山大曲微生物多样性

为了探究牛栏山大曲的微生物菌群结构,为制曲工艺及成品曲质量评估提供理论依据,该研究采用高通量测序方法对牛栏山低温大曲原核16S rDNA V3/V4区和真核ITS1区进行微生物多样性分析。研究结果表明,从牛栏山大曲中共获得358种原核操作分类单元（OTU）,乳酸菌类群是大曲中主体原核生物,维斯氏菌（Weissella confuse）、耐酸乳杆菌（Lactobacillus acetotolerans）和戊糖片球菌（Pediococcus pentosaceus）分别占大曲总OTU的22.68%、16.54%和9.84%,同时大曲中高温放线菌（Kroppenstedtia eburnea）可占14.78%;而真核多样性较低,仅获得69种OTU,扣囊覆膜酵母（Saccharomycopsis fibuligera）和库氏毕赤酵母（Pichia kudriavzevii）是主体真核微生物,其余真核微生物含量较低;综上得出,牛栏山大曲中原核微生物多样性要远高于真核微生物,且大曲主体微生物较为明显。     

应用Illumina MiSeq高通量测序技术解析梅干菜中细菌多样性

采用Illumina MiSeq高通量测序技术对恩施地区梅干菜中细菌多样性进行解析,同时使用传统纯培养技术对乳酸菌进行分离鉴定。结果表明,恩施地区梅干菜中含量最高的优势细菌门为硬壁菌门（Firmicutes）,其平均相对含量高达60.70%。优势细菌属为乳酸杆菌属（Lactobacillus）、假单胞菌属（Pseudomonas）、葡萄球菌属（Staphylococcus）、魏斯氏菌属（Weissella）、鞘脂单胞菌属（Sphingomonas）、嗜冷杆菌属（Psychrobacter）、四联球菌属（Tetragenococcus）和棒状杆菌属（Corynebacterium）,其平均相对含量分别为60.50%、8.69%、2.86%、1.73%、1.28%、1.07%、1.03%、1.00%。采用传统培养方法从梅干菜分离出5种共12株乳酸菌,经分子生物学鉴定分别为植物乳杆菌（Lactobacillus plantarum）、短乳杆菌（Lactobacillus brevis）,副干酪乳杆菌副干酪亚种（Lactobacillus paracasei subsp. paracasei）、食品乳杆菌（Lactobacillus alimentarius）和融合魏斯氏菌（Weissella confusa）。恩施地区梅干菜中的细菌主要以乳酸菌为主,且乳酸杆菌多样性较高。     

Changes in microbial community during fermentation of high‐temperature Daqu used in the production of Chinese ‘Baiyunbian’ liquor

Daqu is a fermentation starter used in the production of Chinese liquor. The present study investigated changes in the microbial community during the fermentation of Baiyunbian high‐temperature Daqu during a cycle time of 140 days. Two methods were used, a culture‐dependent method (viable cell counting) and a culture‐independent method (high‐throughput sequencing of bacterial 16S rRNA gene and fungal internal transcribed spacer region). It was found that thermotolerant microorganisms such as Bacillus , Thermomyces and Actinobacteria were dominant in all stages of Daqu fermentation, especially during the high‐temperature period of days 7–38. In the initial 110 days of fermentation, operational taxonomic units and number of bacteria were higher than those of fungi, but the opposite was observed towards the end of fermentation (day 140). In mature Daqu , the predominant bacterial species were Bacillus and unclassified Thermoactinomycetaceae, followed by Thermoactinomyces , Kroppenstedtia and Saccharopolyspora ; the predominant fungi were Aspergillus , unclassified Trichocomaceae, Thermomyces , Rhizopus , Monascus and Candida , among others. It is possible that the addition of mother Daqu , temperature and storage time played critical roles in the microbial composition of Daqu . Collectively, the above findings provide important information that can be used to optimize conditions for large‐scale production of Daqu . Copyright © 2017 The Institute of Brewing & Distilling     

基于高通量测序分析紫贻贝冻藏过程中菌群组成变化

以蒸汽开壳后获得的新鲜紫贻贝肉(FS)、-18℃冻藏1周紫贻贝肉(FS-1)、冻藏3周紫贻贝肉(FS-3)和冻藏9周紫贻贝肉(FS-9)为研究对象,采用DNA抽提试剂盒提取贝肉表面微生物基因进行高通量测序分析。各样本的有效序列区间为31 896~42 812条、操作分类单元(Operational taxonomic units,OTU)范围为289~681,其中OTU最少的是冻藏9周样品,最多的是新鲜紫贻贝肉。分析多样性指数可知,菌群丰富度最低的是冻藏3周紫贻贝肉。在属水平上,各样本优势菌群中均包含弧菌属(Vibrio)。随冻藏时间的延长,葡萄球菌属(Staphylococcus)的占比逐渐降低。冻藏9周时新增希瓦菌属(Shewanella)且相对丰度很大,另外仅新鲜紫贻贝和冻藏9周样本的优势菌群中含有不动杆菌属(Acinetobacter)。     

襄阳地区中高温大曲曲皮和曲心真菌多样性解析

该研究采用MiSeq高通量测序技术对襄阳地区中高温大曲曲皮和曲心的真菌多样性进行解析,同时结合多元统计学手段对其曲皮和曲心中真菌类群的差异进行甄别。结果表明,曲心主要以子囊菌门（Ascomycota）为主,其平均相对含量为94.32%,而曲皮主要以子囊菌门和毛霉菌门（Mucoromycota）为主,其平均相对含量分别为54.01%和38.98%。经Wilcoxon检验发现,根霉属（Rhizopus）、双足囊菌属（Dipodascus）和毕赤酵母属（Pichia）在曲皮中的相对含量显著偏高（P＜0.05）,平均相对含量分别为38.83%、8.09%和5.35%,而嗜热真菌属（Thermomyces）、热子囊菌属（Thermoascus）、踝节菌属（Rasamsonia）和根毛霉属（Rhizomucor）在曲心中相对含量显著偏高（P＜0.05）,平均相对含量分别为56.67%、16.86%、5.55%和2.01%。主坐标分析结果显示,曲皮和曲心中的真菌类群结构存在明显的差异,且线性判别分析效应大小（LEfSe）分析结果表明,这种差异主要是由Rhizomucor和Thermoascus及若干低丰度菌属造成的。     

Analysis of the mould microbiome and exogenous enzyme production in Moutai‐flavor Daqu

The objective of this study was to isolate and screen the mould microbiome of five Moutai‐flavor Daqu samples and to characterize and quantify the associated exogenous enzymes. In all, 11 and 18 mould genera were identified in the samples by, respectively, a culture‐dependent method and a culture‐independent high throughput sequencing method. Species of Aspergillus and Rhizopus were the dominant mould genera in the Moutai‐flavor Daqu samples. Analysis of enzyme production showed that Aspergillus versicolor in Daqu ZX01 exhibited the highest neutral proteinase activity (2671.0 ± 73.9 U/g) and Rhizomucor pusillus in Daqu DYT01 exhibited the highest saccharifying amylase production ability (1724.6 ± 11.0 U/g). Copyright © 2017 The Institute of Brewing & Distilling     

酱香型白酒大曲和糟醅的细菌多样性分析

采用Illumina HiSeq 2500测序系统全面剖析酱香型白酒中的高温制曲和发酵酿酒阶段的细菌群落结构。结果显示：酱香型白酒高温制曲阶段和发酵酿酒阶段大曲和糟醅的细菌菌群在数量及结构分布上均具有多样性特点,菌群种类基本一致。两个阶段的优势菌门均为厚壁菌门Firmicutes、变形菌门Proteobacteria、放线菌门Actinobacteria、拟杆菌门Bacteroidetes 4?类。高温制曲和发酵酿酒生态环境之间存在差异,导致微生物优势菌不同。另外,随着发酵时间的推移,酱香型白酒酿造过程中微生物群落呈现一定的变化规律,大曲样本的细菌菌类变化与贮存时间有关,优势菌属有Clostridium sensu stricto 1、Escherichia-Shigella、Lactobacillus、Streptococcus、Actinobacillus、Bifidobacterium、Peptoclostridium、Mycoplasma、Citrobacter等,诸如Escherichia-Shigella、Actinobacillus、Bifidobacterium、Citrobacter等细菌随贮存时间的延长而增加,Clostridium sensu stricto 1、Mycoplasma等表现为先上升后下降,而Lactobacillus、Peptoclostridium等无明显变化,Streptococcus等细菌呈现下降趋势。而糟醅微生物随窖池发酵（起窖）及高温堆积（下窖）的工艺而不断变化,发酵酿酒阶段的优势微生物为Escherichia-Shigella、Lactobacillus、Clostridium sensu stricto 1、Streptococcus、Actinobacillus、Bifidobacterium、Alternaria。相对丰度较高的细菌菌属中Escherichia-Shigella、Lactobacillus在发酵前期菌数先升高,中期从第2轮次开始下降,第3轮次下窖时达到最低,后期逐渐上升。Clostridium sensu stricto 1、Streptococcus菌群丰度变化规律复杂,呈现先下降后上升的趋势。结果表明：高温制曲与发酵酿酒阶段的优势菌菌属基本一致,说明大曲是后续糟醅发酵的微生物来源。另外,细菌的生长耐受性范围较广,随着时间的推移,温度和水分达到相对平衡,细菌群落也逐渐得到了调整,形成单一菌类为主导的环境,更有利于酱香酒的酿造。     

基于高通量测序技术分析浓香型白酒大曲真菌菌群多样性

采用高通量测序技术对15份浓香型白酒大曲真菌多样性进行分析。结果表明,不同样品真菌群落丰度和多样性存在显著差异。主坐标分析（PCoA）结果表明,样品NX-1、NX-3、NX-4、NX-10、NX-11真菌组成相似、样品NX-2、NX-8真菌组成相似、其余样品真菌组成存在差异。15份大曲样品中共检测到393个操作分类单元（OTUs）,隶属于6个门19个纲和117个属,其中子囊菌门（Ascomycota）、酵母纲（Saccharomycetes）和散囊菌纲（Eurotiomycetes）在15份大曲样品中的相对丰度均较高。在属分类水平上,15份大曲样品出现明显差异,样品NX-1、NX-4、NX-11中的热子囊菌属（Thermoascus）、嗜热真菌属（Thermomyces）,样品NX-3、NX-10、NX-13、NX-6和NX-14中的覆膜孢酵母属（Saccharomycopsis）、毕赤酵母属（Pichia）,样品NX-5、NX-12、NX-15、NX-7中的曲霉属（Aspergillus）,样品NX-9中的曲霉属和根毛霉属（Rhizomucor）、样品NX-2中的威克汉姆酵母属（Wickerhamomyces）、酵母属（Saccharomyces）,及样品NX-8中的耐碱酵母属（Galactomyces）和双足酵母属（Dipodascus）相对丰度较高。     

基于高通量测序分析不同品种水牛生乳中细菌多样性

目的 利用16S rRNA高通量测序研究广西水牛研究所种畜厂内摩拉、尼里-拉菲及三品杂3品种水牛生鲜乳中的细菌多样。方法 提取当天采集的生水牛乳样本细菌总DNA,PCR扩增细菌的16S rDNA,纯化PCR扩增目的片断,将PCR产物与pMD18-T载体连接,构建其菌群的16S rDNA文库。用Miseq对其16S rRNA基因的V3-V4可变区进行测序以及BLAST比对。结果 3品种27个样本共获得1223个OUTs, M、N、S独有的OTU分别为295、41和42,共有OTU为511。物种组成与丰度差异显示：3品种优势菌门为：变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)。优势菌属为：金黄杆菌属(Chryseobacterium)、不动杆菌属(Acinetobacter)、乳球菌属(Lactococcus)、假单胞菌属(Pseusomonas)。样本层级聚类显示：3品种样本间菌落组成相似度较高。PCA与PLS-DA分析表明：3品种样本组内菌群多样性差异不显著,但组间菌群丰度差异明显。结论 摩拉、尼里-拉菲及三品杂水牛生鲜乳中细菌菌群均呈现较丰富的多样性,其共有的优势菌群为金黄杆菌(Chryseobacterium)、不动杆菌(Acinetobacter)、乳球菌(Lactococcus)、假单胞菌(Pseusomonas)。各品种间菌群组成种类整体相似,但其主要菌落丰富度差异明显。     

基于高通量测序技术分析汉中地区泡菜水细菌多样性

该研究采用Illumina MiSeq高通量测序技术对陕西省汉中地区泡菜水中的细菌构成进行解析,并采用生物信息学方法对其多样性进行分析,同时结合多元统计学等手段探讨汉中泡菜水中微生物结构的差异。结果表明,泡菜水样品中的细菌菌群主要隶属于24个门的553属,优势细菌门为硬壁菌门（Firmicutes）和变形菌门（Proteobacteria）,其平均相对含量分别为89.12%和7.89%;优势细菌属为片球菌属（Pediococcus）、乳杆菌属（Lactobacillus）、假单胞菌属（Pseudomonas）和明串珠菌属（Leuconostoc）,其平均相对含量分别为52.06%、31.40%、4.89%和1.83%。汉中地区的泡菜水样品可以划分为两个聚类,隶属于不同聚类泡菜水样品的α多样性指数无显著性差异（P＞0.05）,但细菌菌群结构存在较大差异（P＜0.05）,且片球菌属和乳杆菌属的相对含量存在显著性差异（P＜0.05）。同时,不同聚类的泡菜水中均存在一些低丰度物种,且均为一些较为独特的细菌类群。     

基于Illumina MiSeq高通量测序技术解析利川酱豆真菌多样性

该研究利用Illumina MiSeq高通量测序技术对湖北省恩施土家族苗族自治州利川市酱豆样品的真菌多样性和群落结构进行解析。结果表明,利川酱豆样品中的真菌菌群隶属于3个门、15个纲、27个目、34个科、45个属,核心真菌门为子囊菌门（Ascomycota）（96.79%）和担子菌门（Basidiomycota）（2.63%）;核心真菌属为毕赤酵母属（Pichia）（37.46%）、曲霉属（Aspergillus）（29.48%）、德巴利氏酵母属（Debaryomyces）（3.92%）、假丝酵母属（Candida）（2.86%）、Starmerella（2.62%）、节担菌属（Wallemia）（2.19%）和镰刀菌属（Fusarium）（1.03%）,且核心真菌之间的相关性均不显著（P＞0.05）。     

Identification of microorganisms producing lactic acid during solid‐state fermentation of Maotai flavour liquor

Lactic acid is the main acid produced during the Maotai liquor brewing process, influencing the quality of the base liquor and fermentation process. However, the microorganisms responsible for lactic acid production have not been identified. In this work, the dynamic changes in bacterial community structure in the Zaosha round (second sorghum feeding and fermentation) of the brewing process were analysed by 16S rRNA high‐throughput sequencing. Results show that lactic acid bacteria (LAB) and Bacillus spp. are the dominant bacteria in the brewing process, where Bacillus spp. are found in the early stage, whilst LAB are found throughout the brewing process. Furthermore, 10 types of LAB and five Bacillus spp. were isolated from Zaopei (a mixture of fermented grains including sorghum and wheat) by a culture‐dependent method. Lactobacillus panis accounts for 68% of the LAB, and Bacillus amyloliquefaciens for 54% of Bacillus spp. Solid‐state fermentation experiments were performed with L. panis and B. amyloliquefaciens and lactic acid production was consistent with the accumulation of lactic acid in Zaosha. The results showed that L. panis was the main producer of lactic acid in pits, while B. amyloliquefaciens plays an important role in the production of lactic acid in the early stages of fermentation. The approach used in this study may facilitate the identification of key microorganisms with specific functionality involved in other food and beverage fermentation processes. © 2018 The Institute of Brewing & Distilling     

Characterization and detection of toxoflavin‐producing Burkholderia in rice straws and Daqu for Chinese Maotai‐flavour liquor brewing

Rice straws are widely used in Daqu (starter) preparation for Maotai‐flavour liquor brewing to provide adequate thermal insulation, water retention and aeration. As the straws have direct contact with the Daqu during its preparation, the microorganisms carried by the straws can be transferred to the Daqu. In this study, yellow pigment‐producing strains were isolated from the rice straws using a dilution plate method. Most of these strains were characterized as toxoflavin‐producing Burkholderia spp. by 16S rRNA sequencing, toxA gene‐specific PCR and toxoflavin detection. The PCR assays revealed the existence of Burkholderia in the Daqu samples. The toxoflavin content in a number of Daqu samples was determined using HPLC. The highest observed content of toxoflavin in the Daqu was >8 mg/kg, close to its LD₅₀. Although no toxoflavin was detected in the distilled liquor, the safety problem regarding the use of rice straws in Daqu preparation is still noteworthy, since Burkholderia and toxoflavin can be transferred and can accumulate during the brewing process. The results can contribute to a further assessment of the contamination risk in liquor brewing caused by Burkholderia and to safer production in this traditional process. Copyright © 2015 The Institute of Brewing & Distilling     

基于MiSeq测序技术石花酒大曲中微生物多样性解析

该研究采用Illumina MiSeq高通量测序技术对襄阳市石花酒大曲中微生物多样性进行分析。结果表明,4个样品中优势细菌门（＞0.1%）为变形杆菌门（Proteobacteria）、厚壁菌门（Firmicutes）和放线菌门（Actinobacteria）,且均为核心菌门;优势细菌属（＞1%）为假单胞菌属（Pseudomonas）、芽孢杆菌属（Bacillus）、乳杆菌属（Lactobacillus）、链霉菌属（Streptomyces）、魏斯氏菌属（Weissella）、明串珠菌属（Leuconostoc）和Kroppenstedtia。优势真菌门（＞0.1%）为子囊菌门（Ascomycota）、担子菌门（Basidiomycota）和Mucoromycota,其中子囊菌门为核心菌门;优势真菌属（＞1%）为复膜孢酵母属（Saccharomycopsis）、红曲菌属（Monascus）、热子囊菌属（Thermoascus）、青霉菌属（Penicillium）、曲霉属（Aspergillus）和Leiothecium。样品中假单胞菌属和曲霉属呈现显著正相关（P＜0.05）。综上,4个石花酒大曲样品中共有大量的核心菌群,且样品中细菌与真菌有一定的相关性。