液熏罗非鱼片加工过程中微生物群落的PCR-DGGE 分析

为弄清液熏罗非鱼片加工过程中微生物污染的源头,以进一步防控产品的微生物污染提供依据。应用基于细菌16S rDNA 的PCR-DGGE(PCR-denaturing gradient gel electrophoresis,PCR- 变性梯度凝胶电泳)技术分析液熏罗非鱼片主要加工关键环节的微生物群落结构,提取样品中的细菌总DNA,对细菌的16S rDNA 的V6～V8 区段进行PCR 扩增后,进行变性梯度凝胶电泳(DGGE),对DGGE 图谱进行微生物多样性分析,对主要条带进行序列分析并构建系统发生树。结果表明：10 个条带所代表的优势种很可能来源于以下几个属：巨型球菌属(Macrococus)、微球菌属(Micrococus)、肠道细菌属(Enterobacter)、假单胞菌属(Pseudomonas)、弧菌属(Vibrio)、突柄杆菌属(Prosthecobacter)、布特菌属(Buttiauxella),其中肠道细菌属、微球菌属、假单胞菌属和弧菌属细菌都具有使产品腐败的潜能。本研究表明：液熏罗非鱼片中呈现微生物多样性,PCR-DGGE 技术可用于研究液熏罗非鱼片加工过程中的微生物群落结构及变化,且具有一定的优越性。     

PCR—DGGE技术及其在食品行业中的应用

现代分子生物学技术PCR-DGGE是一种分析微生物群落的有效工具,可以用于研究生态系统中微生物多样性和群落动态性.简要介绍了PCR-DGGE技术原理及其在食品行业中微生物生态学领域的应用.     

Bio-hydrogen production from thin stillage using conventional and acclimatized anaerobic digester sludge

To assess the viability of biohydrogen production from thin stillage, a comparative evaluation of anaerobic digester sludge (ADS) and acclimatized anaerobic digester sludge (AADS) for biohydrogen production over a wide range of S⁰/X⁰ ratio (0.5-8 gCOD/gVSS) was performed. A maximum hydrogen yield of 19.5 L H₂/L thin stillage was achieved for the AADS while tests with ADS achieved a maximum yield of only 7.5 L H₂/L thin stillage. The optimum range of S⁰/X⁰ ratio for hydrogen production was found to be 1 to 2 gCOD/gVSS using conventional ADS and 3 to 6 gCOD/gVSS using AADS. The biomass specific hydrogen production rate for the AADS was 3.5 times higher than rate for the ADS throughout the range of S⁰/X⁰ ratio examined in this study. The DGGE profiles of the 16S rDNA gene fragments confirmed the superior performance of the AADS over the ADS, showing that the widely known hydrogen producers Clostridium acetobutyricum, Klebsiella pneumonia, Clostridium butyricum and Clostridium pasteurianum were the predominant species.     

冰鲜鸡肉贮藏过程中微生物菌相变化分析

应用基于16S rDNA的PCR-DGGE(变性梯度凝胶电泳)技术对冰鲜鸡肉微生物多样性进行研究。分别取鸡胸肉和鸡腿肉贮藏0、3、5、7、9d的样品,提取样品总DNA,通过PCR扩增、变性梯度凝胶电泳,将16S rDNA(V6～V8区)的PCR扩增片段割胶测序确定样品中的微生物群落,与传统培养方法进行比较。传统培养方法表明：鸡胸肉和鸡腿肉在低温低氧分压条件下贮藏,导致腐败的优势菌相差不明显,且变化趋势相一致;DGGE图谱表明,初始污染数量较多的微生物不一定是腐败优势菌,能适应低温低氧分压的微生物最终成为腐败优势菌,且鸡胸肉和鸡腿肉的腐败优势菌有一定差异性。传统培养和DGGE割胶测序所得腐败优势菌的菌相不完全相同,综合两种研究方法,确定冰鲜鸡肉腐败优势菌为乳酸菌、大肠菌群、热杀索丝菌、腐败希瓦氏菌链菌和肉杆菌。     

应用PCR-DGGE方法研究甜酒曲中真菌多样性

提取6种甜酒酒曲的总DNA,利用真菌18S rRNA基因片段的通用引物NS1、Fung-GC,采用变性梯度凝胶电泳（PCR-DGGE）方法,进行不同甜酒酒曲中真菌多样性的分析。结果表明,6种甜酒酒曲中共分离出3属真菌和不可培养真菌,湖南韶山的甜酒曲中主要是扣囊复膜酵母属和根霉属的菌株;湖南湘潭甜酒曲中的真菌为酿酒酵母属、扣囊复膜酵母属和根霉属;湖南祁东、福建福州和浙江丽水甜酒曲中的真菌均为酿酒酵母属、扣囊复膜酵母属、根霉属和不可培养真菌;浙江兰溪甜酒曲中的真菌为根霉属和不可培养真菌。在传统的酒曲中的真菌主要是酿酒酵母属和扣囊复膜酵母属,此外不可培养真菌在甜酒酒曲中也起到非常重要的作用。     

Microbiota during fermentation of chum salmon (Oncorhynchus keta) sauce mash inoculated with halotolerant microbial starters: Analyses using the plate count method and PCR-denaturing gradient gel electrophoresis (DGGE)

Nine different combinations of mugi koji (barley steamed and molded with Aspergillus oryzae) and halotolerant microorganisms (HTMs), Zygosaccharomyces rouxii, Candida versatilis, and Tetragenococcus halophilus, were inoculated into chum salmon sauce mash under a non-aseptic condition used in industrial fish sauce production and fermented at 35 ± 2.5 °C for 84 days to elucidate the microbial dynamics (i.e., microbial count and microbiota) during fermentation. The viable count of halotolerant yeast (HTY) in fermented chum salmon sauce (FCSS) mash showed various time courses dependent on the combination of the starter microorganisms. Halotolerant lactic acid bacteria (HTL) were detected morphologically and physiologically only from FCSS mash inoculated with T. halophilus alone or with T. halophilus and C. versatilis during the first 28 days of fermentation. Only four fungal species, Z. rouxii, C. versatilis, Pichia guilliermondii, and A. oryzae, were detected throughout the fermentation by PCR-denaturing gradient gel electrophoresis (PCR-DGGE). In FCSS mash, dominant HTMs, especially eumycetes, were nonexistent. However, under the non-aseptic conditions, undesirable wild yeast such as P. guilliermondii grew fortuitously. Therefore, HTY inoculation into FCSS mash at the beginning of fermentation is effective in preventing the growth of wild yeast and the resultant unfavorable flavor.     

PCR-DGGE技术对中华开菲尔微生物菌群的分析

为了解中华开菲尔微生物菌群的结构特征,本论文运用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术对开菲尔菌株发酵过程中微生物菌群的结构变化进行了实验分析,结果表明:细菌菌群DGGE图谱上出现有三种不同迁移位置的斑带,而酵母菌群DGGE图谱上只有一条斑带;经过DNA序列的对比分析可知:细菌菌群分别为肠膜明串珠菌(Leuconostoc mesenteroides)、马乳样乳杆菌(Lactobacillus kefiranofaciens)和开菲尔乳杆菌(Lactobacillus kefir),它们的序列同源性都达到100%;酵母菌群为德尔布有孢圆酵母(Torulaspora delbrueckii),其序列同源性为99%。本论文首次报道了德尔布有孢圆酵母在开菲尔菌落中的存在。     

长距离输水过程中微生物安全的分子生物学分析

应用PCR-DGGE分子生物学方法对东江水源工程沿途水样中的微生物群落结构的动态变化以及种群多样性进行了研究。结果表明,采用分子生物学方法能够检测出水中多样性的细菌。相似性分析表明,采用涵管方式远距离引水,其沿途水中微生物群落结构变化并不是很大,优势菌大致相同;16SrDNA鉴定表明,分子生物学方法能够检测出传统人工培养方法不能检测出来的细菌,原水不含致病菌,这能够对原水输送工程微生物安全性进行更准确的评价。     

发酵乳活性双歧杆菌种类快速识别及定量研究

本文建立了发酵乳制品中双歧杆菌种类和数量快速测定方法。以发酵乳中双歧杆菌为靶标,采用PCR-DGGE技术快速识别双歧杆菌种类;采用Real-Tine PCR手段测定双歧杆菌数量。研究结果表明,PCR-DGGE能准确、快速鉴别双歧杆菌种类,检出限为105 CFU/g;Real-Tine PCR能准确、快速定量双歧杆菌,检出限为104 CFU/g。该方法可用于发酵乳中双歧杆菌的快速识别和定量。     

Culture-independent study of the diversity of microbial populations in brines during fermentation of naturally-fermented Aloreña green table olives

Aloreña table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The present study focused on Aloreña table olives manufactured by small and medium enterprises (SMEs) from Valle del Guadalhorce (Southern Spain) under three different conditions (cold storage, and ambient temperature fermentations in small vats and in large fermentation tanks). The microbial load of brines during fermentation was studied by plate counting, and the microbial diversity was determined by a culture-independent approach based on PCR-DGGE analysis. The viable microbial populations (total mesophilic counts, yeasts and molds, and lactic acid bacteria — LAB) changed in cell numbers during the course of fermentation. Great differences were also observed between cold, vat and tank fermentations and also from one SME to another. Yeasts seemed to be the predominant populations in cold-fermented olives, while LAB counts increased towards the end of vat and tank fermentations at ambient temperature. According to PCR-DGGE analysis, microbial populations in cold-fermented olives were composed mostly by Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. together with halophilic archaea (mainly by haloarchaeon/Halosarcina pallida and uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis) and yeasts (Saccharomyces cerevisiae and Candida cf. apicola). Vat-fermented olives stored at ambient temperature included a more diverse bacterial population: Gordonia sp./Pseudomonas sp., Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. and Thalassomonas agarivorans together with halophilic archaea and yeasts (mainly S. cerevisiae and C. cf. apicola, but also Pichia sp., and Pichia manshurica/Pichia galeiformis). Some LAB were detected towards the end of vat fermentations, including Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus. Only the tank fermentation showed a clear predominance of LAB populations (Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.) together with some halophilic archaea and a more selected yeast population (P. manshurica/P. galeiformis). The present study illustrates the complexity of the microbial populations in naturally-fermented Aloreña table olives.