PCR-DGGE研究热鲜肉贮藏过程中的菌相变化

应用传统微生物培养和聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)方法研究热鲜肉分别在5、15、25、30℃贮藏过程中的菌相变化。将热鲜肉贮藏于一定温度,每隔适当时间取出,测定菌落总数,并提取细菌DNA,进行PCR-DGGE分析。细菌计数结果表明,热鲜肉贮藏于5、15、25、30℃时,菌落总数分别在约14d、75h、19h和16h达到最小腐败量7.2(lg(CFU/g))。PCR-DGGE结果表明,热鲜肉在不同温度下贮藏时,贮藏末期优势腐败菌并不一致。在贮藏过程中主要优势腐败菌有巨大球菌属、克雷伯氏菌属、假单胞菌属、柠檬酸细菌属、不动杆菌属和埃希氏菌属。     

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。     

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.     

Lactobacillus pentosus dominates spontaneous fermentation of Italian table olives

Culture-dependent and -independent approaches were applied to identify the bacterial species involved in Italian table olive fermentation. Bacterial identification showed that Lactobacillus pentosus was the dominant species although the presence of Lactobacillus plantarum, Lactobacillus casei, Enterococcus durans, Lactobacillus fermentum and Lactobacillus helveticus was observed. Rep-PCR allowed to obtain strain-specific profiles and to establish a correlation with table olive environment. PCR-DGGE (Denaturing Gradient Gel Electrophoresis) confirmed the heterogeneity of bacterial community structure in fermented table olives as well as the prevalence of L. pentosus. The strains were characterized on the basis of technological properties (NaCl tolerance, β-glucosidase activity and the ability to grow in synthetic brine and in presence of 1 g/100 mL oleuropein). L. pentosus showed a high capacity of adaptation to the different conditions characterizing the olive ecosystem. This species showed the highest percentage of strains able to grow in presence of 10 g/100 mL NaCl, oleuropein and in the synthetic brine. Moreover, all the strains belonging to L. pentosus and L. plantarum species showed a β-glucosidase activity. This study allowed both to identify the main species and strains associated to Italian table olives and to obtain a lactic acid bacteria collection to apply as starter culture in the process of olive fermentation.     

Study on the Change Rules of Parameters of Luzhou-flavor Liquor in Pits and Their Correlations （V）.Fungus

利用PCR-DGGE技术研究了洋河酒醅真菌群落结构及其发酵过程中的变化规律。结果表明,所有酒醅样品均出现9～18条较清晰的条带,其中5号、6号、7号、8号、11号条带在所有样品中均有检出,且优势度较高;所有样品生物多样性指数都在1.57～2.41之间。发酵前期,多样性指数总体呈先降低并保持平稳的趋势,发酵第30天时达到最小值,第40天有所回升,之后出现下降并保持相对平稳;相似性指数除了第30天和第40天为0.46外,其他相邻时间内样品间的相似性指数均在0.53～0.92之间。表明在发酵过程中,相邻发酵时间酒醅样品真菌群落间的相似性较好。     

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.     

Effect of organic loading rate and solids retention time on microbial population during bio-hydrogen production by dark fermentation in large lab-scale

The experimental investigation aimed at the study of the microbial population during the continuous operation of a complete mixed reactor in large lab-scale (30 L) by variation of the Organic Loading Rate (OLR) and Sludge Retention Time (SRT) ranging from 10 g sucrose/(L?d) to 30 g/(L?d) and from 12 h to 48 h respectively. H₂ yield reached to 1.72 mol H₂/mol hexose for HRT = 1.6 d and OLR = 20 g sucrose/(L?d). In each phase the dominant microbial genera were identified by sequencing after a Polymerase Chain Reaction (PCR) with universal primers for the domains of Archaea and Eubacteria and specific for Clostridium species and genetic material isolation by Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The phylogenetic analyses showed that hydrogen producing Clostridium species could be affiliated in all experimental phases. Other dominant genera were affiliated mainly to Ethanoligenes harbinense and uncultured Prevotella and Selonomonas species. Bio-hydrogen production was associated to a mixed butyric/ethanol type fermentation facilitated mainly by Clostridium tyrobutyricum and E. harbinense in the presence of lactate as intermediate metabolic product.     

Characterisation of the dominant bacterial population in modified atmosphere packaged farmed halibut (Hippoglossus hippoglossus) based on 16S rDNA-DGGE

It is not well understood why Atlantic halibut (Hippoglossus hippoglossus) has longer shelf-life than most other white fish species. Our approach was to examine the microbiological diversity of the spoilage microbiota during modified atmosphere (MA) packaging of farmed Atlantic halibut. Portions were packaged with gas mixtures of CO(2):N(2) and CO(2):O(2) (50%:50%) and with air as a reference. The packages were stored at 4 degrees C and samples were taken 6 times during the 23 days of storage. Analyses with molecular techniques (PCR-DGGE) determined profiles of the bacterial populations in the various samples and sequencing detected the bacterial species present. In addition, samples were analysed for microbial, chemical and sensory parameters. The shelf-life was 10-13 days when stored in air and between 13 and 20 days for MA packages, with oxygen-enriched packages suggested as the better gas mixture, based on microbial growth and sensory scores. From sequence analyses of the bacterial population Photobacterium phosphoreum and Pseudomonas spp. were found to dominate in the halibut. Brochothrix thermosphacta was found in most samples at the end of the storage period. Shewanella putrefaciens was found sporadically and in low concentrations based on microbial methods, but not detected by PCR-DGGE.