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.     

应用PCR-DGGE技术分析酱香型白酒酒曲细菌多样性

利用PCR-DGGE技术研究酱香型白酒制曲过程中的细菌菌群结构及其消长规律,鉴定出不同样品的优势茵群。结果表明,酱香型大曲间的细茵组成存在明显差异,母曲与出仓曲的相似性系数仅为O．29。随着曲药的发酵,细菌多样性下降,优势茵群变化明显。其中,芽孢杆菌属（Bacillus）、明串珠菌属（Leuconostoc）、片球菌属（Pedio-cocuss）、魏斯氏菌属（Weissella）、棒状杆菌属（Corynebacterium）、高温放线茵属（Thermoactinomyces）和乳酸杆菌属（Lactobacillus）是母曲和翻仓曲的优势菌群,而出仓曲的主要类群为芽孢杆菌属（Bacillus）和乳酸杆菌属（Lactobacillus）。另外,还发现了在白酒曲药中不曾报道的种群和不能培养的细茵：Uncultured Propionibacteriaceae bacterium、Uncultured Weissellasp、Uncultured cyanobacterium,该技术克服了传统分离培养的缺点。     

How to Determine the Geographical Origin of Seafood?

Traceability of seafood is a much needed service for the seafood industry. Current ways of tracing seafood are minimal while tracing of shellfish is nearly nonexistent. Tracing fish and shellfish are necessary for indicating where the fish and shellfish were fished from, farmed and packed from. This study reviews history of traceability of aquaculture and analytical approaches to verify the origin of seafood. It then describes the new molecular technique of the traceability by using PCR-DGGE to discriminate the geographical origin of fish (cases studies of Pangasius fish from Viet Nam and Sea bass fish from France) by analysis the DNA fragments of microorganisms (bacteria) on fish. This method is based on the assumption that the microbial communities of food are specific to a geographic area.     

Comparison of the bacterial species diversity of spontaneous cocoa bean fermentations carried out at selected farms in Ivory Coast and Brazil

To compare the spontaneous cocoa bean fermentation process carried out in different cocoa-producing regions, heap and box (one Ivorian farm) and box (two Brazilian farms) fermentations were carried out. All fermentations were studied through a multiphasic approach. In general, the temperature inside the fermenting mass increased throughout all fermentations and reached end-values of 42-48 °C. The main end-products of pulp carbohydrate catabolism were ethanol, lactic acid, acetic acid, and/or mannitol. In the case of the fermentations on the selected Ivorian farm, the species diversity of lactic acid bacteria (LAB) and acetic acid bacteria (AAB) was restricted. Lactobacillus fermentum and Leuconostoc pseudomesenteroides were the predominant LAB species, due to their ethanol and acid tolerance and citrate consumption. The levels of mannitol, ascribed to growth of L. fermentum, were fermentation-dependent. Also, enterobacterial species, such as Erwinia soli and Pantoea sp., were among the predominating microbiota during the early stages of both heap and box fermentations in Ivory Coast, which could be responsible for gluconic acid production. Consumption of gluconic acid at the initial phases of the Ivorian fermentations could be due to yeast growth. A wider microbial species diversity throughout the fermentation process was seen in the case of the box fermentations on the selected Brazilian farms, which differed, amongst other factors, regarding pod/bean selection on these farms as compared to fermentations on the selected Ivorian farm. This microbiota included Lactobacillus plantarum, Lactobacillus durianis, L. fermentum, Lactobacillus mali, Lactobacillus nagelii, L. pseudomesenteroides, and Pediococcus acidilactici, as well as Bacillus subtilis that was present at late fermentation, when the temperature inside the fermenting mass reached values higher than 50 °C. Moreover, AAB seemed to dominate the Brazilian box fermentations studied, explaining higher acetic acid concentrations in the pulp and the beans. To conclude, it turned out that the species diversity and community dynamics, influenced by local operational practices, in particular pod/bean selection, impact the quality of fermented cocoa beans.     

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.     

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。     

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。     

污泥发酵液对A2O脱氮除磷和微生物的影响

为研究剩余污泥发酵液作碳源对微生物群落结构的影响,将发酵液与市政污水按流量比1∶35回用于厌氧-缺氧-好氧反应器,在室温下运行90 d．聚类分析表明,发酵液明显改变了微生物群落结构,5~30 d和45~90 d的微生物属于不同的聚集区; 微生物多样性分析表明,发酵液使Shannon-Wiener指数从2.6升高到3.1,系统运行稳定性增强; PCR-DGGE分析表明,发酵液对微生物群落具有一定的选择性,氨氧化菌Nitrosomonas sp.、硝化菌Betaproteobacteria和 Nitrospira sp.、反硝化菌Comamonas sp.和聚磷菌Gammaproteobacteria得到富集,TN和TP去除率从64.5％和52.4％提高到84.7％和94.3％．