永川毛霉型豆豉在发酵过程中微生物总量与区系变化规律

采用巢式聚合酶链式反应配合变性梯度凝胶电泳技术对永川豆豉发酵过程中微生物区系生态演化进行解析。结果表明：永川豆豉在制曲过程中霉菌和细菌呈对数增长,进入后发酵阶段菌落总数快速下降,并保持在较低水平。永川豆豉在制曲前期有多种乳酸菌生长,后期乳酸菌受霉菌增长抑制,种类减少。在后发酵阶段奥德赛芽孢杆菌（Bacillus odysseyi）、乳酸杆菌（Lactobacillus oligofermentans）和乳酸杆菌（Lactobacillus lindneri）是优势菌群。同时在制曲初期也发现了费格森埃希菌（Escherichia fergusonii）等杂菌生长。永川豆豉制曲阶段优势霉菌是总状毛霉（Mucor racemosus）,同时伴有有性根霉（Rhizopus sexualis）、匍枝根霉（Rhizopus stolonifer）、大孢联轭霉（Syzygites megalocarpus）、米根霉（Rhizopus oryzae）的生长,后发酵阶段有接合酵母（Zygosaccharomyces sp.）的参与。     

PCR-DGGE技术分析腌制麻竹笋中微生物多样性

采用聚合酶链式反应-变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE）技术对盐质量浓度5 g/100 mL和19 g/100 mL腌制麻竹笋的微生物区系进行研究。结果表明,经DNA提取、巢式PCR、DGGE电泳和克隆测序后,从低盐质量浓度（5 g/100 mL）腌制笋中分离出4 条明显的亮带,经鉴定分别为食窦魏斯氏菌（Weissella cibaria）、乳球菌属（Lactococcus sp.）、魏斯氏菌属（Weissella sp.）和乳酸乳球菌（Lactococcus lactis）;从高盐质量浓度（19 g/100 mL）腌制笋中分离出5 条明显的亮带,经鉴定分别为绿色气球菌（Aerococcus viridans）、赖氨酸芽孢杆菌属（Lysinibacillus sp.）、未得到培养的细菌（unculturedbacterium）、厌氧芽孢杆菌属（Anoxybacillus sp.）和芽孢杆菌属（Bacillus sp.）;低盐腌制笋的优势菌多为益生菌,而高盐腌制笋的优势菌则多为抗性较强的菌。基于16S rDNA的PCR-DGGE技术为分析腌制麻竹笋中微生物多样性提供了一条可靠、快速的有效途径。     

Yeast diversity during tapping and fermentation of palm wine from Cameroon

In the present study, we have investigated the occurrence of yeast flora during tapping and fermentation of palm wine from Cameroon. The yeast diversity was investigated using both traditional culture-dependent and culture-independent methods. Moreover, to characterize the isolates of the predominant yeast species (Saccharomyces cerevisiae) at the strain level, primers specific for δ sequences and minisatellites of genes encoding the cell wall were used. The results confirm the broad quantitative presence of yeast, lactic acid bacteria and acetic acid bacteria during the palm wine tapping process, and highlight a reduced diversity of yeast species using both dependent and independent methods. Together with the predominant species S. cerevisiae, during the tapping of the palm wine the other species found were Saccharomycodes ludwigii and Zygosaccharomyces bailii. In addition, denaturing gradient gel electrophoresis (DGGE) analysis detected Hanseniaspora uvarum, Candida parapsilopsis, Candida fermentati and Pichia fermentans. In contrast to the progressive simplification of yeast diversity at the species level, the molecular characterization of the S. cerevisiae isolates at the strain level showed a wide intraspecies biodiversity during the different steps of the tapping process. Indeed, 15 different biotypes were detected using a combination of three primer pairs, which were well distributed in all of the samples collected during the tapping process, indicating that a multistarter fermentation takes place in this particular natural, semi-continuous fermentation process.     

Description of the microflora of sourdoughs by culture-dependent and culture-independent methods

Four types of sourdoughs (L, C, B, Q) from artisanal bakeries in Northern Italy were studied using culture-dependent and culture-independent methods. In all samples, the yeast numbers ranged from 160 to 10⁷ cfu/g, and the numbers of lactic acid bacteria (LAB) ranged from 10³ to 10⁹ cfu/g. The isolated LAB were sequenced, and a similarity was noted between two samples (C, Q), both in terms of the species that were present and in terms of the percentage of isolates. In these two samples, Lactobacillus plantarum accounted for 73% and 89% of the bacteria, and Lactobacillus brevis represented 27% and 11%. In the third sample (B), however, the dominant LAB isolate was Lb. brevis (73%), while Lb. plantarum accounted for only 27%. The fourth sourdough (L) was completely different from the others. In this sample, the most prominent isolate was Weisella cibaria (56%), followed by Lb. plantarum (36%) and Pediococcus pentosaceus (8%). In three out of four samples (L, C and Q), all of the yeasts isolated were identified as Saccharomyces cerevisiae, yet only Candida humilis (90%) and Candida milleri (10%) were isolated in the fourth sample (B). The microbial ecology of the sourdoughs was also examined with direct methods. The results obtained by culture-independent methods and DGGE analysis underline a partial correspondence between the DNA and RNA analysis. These results demonstrate the importance of using a combined analytical approach to explore the microbial communities of sourdoughs.     

Lactic acid bacteria ecology of three traditional fermented sausages produced in the North of Italy as determined by molecular methods

In this study the bacterial biodiversity during the maturation process of three traditional sausages produced in the North of Italy (Salame bergamasco, Salame cremonese and Salame mantovano) was investigated by using culture-dependent and -independent methods. Eleven plants, in the three provinces considered here, were selected because starter cultures were never used in the production. The bacterial ecology, as determined by plate counts, was dominated by lactic acid bacteria (LAB), with minor contribution of coagulase negative cocci and yeasts. After molecular identification of 486 LAB strains, the species more frequently isolated were Lactobacillus sakei and Lactobacillus curvatus. This evidence was also confirmed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE). All the samples analyzed were characterized by the constant presence of L. sakei and L. curvatus bands. A richer biodiversity was only detected at the beginning of maturation. The results obtained by the molecular characterization of the L. sakei and L. curvatus and by the cluster analysis of the DGGE profiles highlighted a plant-specific population, rather than a geographic characterization of the products, underlining how the environmental and processing conditions are able to select specific microbiota responsible for the main transformations during the fermentation and ripening of the sausages.     

变性梯度凝胶电泳法初步解析茯砖茶渥堆发酵过程中细菌群落结构

为解析茯砖茶渥堆发酵过程中细菌群落结构和种类,对渥堆过程中不同时间段细菌16S rDNA 的V3可变区进行扩增,对细菌变性梯度凝胶电泳（denaturing gradient gel electrophoresis,DGGE）图谱中条带进行克隆、测序和序列比对。结果表明：黑毛茶在渥堆过程中以渥堆24 h为分界点,前后各自细菌群落结构相似,但前后的差异较大;从16S rDNA 的V3可变区比对结果证明黑毛茶渥堆过程中有诺卡氏菌属、新鞘脂菌属、短波单胞菌属、韦龙氏假单胞菌属、突那梭菌属、克雷伯氏菌属、乳杆菌属及不可培养的ε-变形菌、腐败螺旋菌属、黏球菌属、根瘤菌属和未知分类地位的不可培养细菌6 种。采用DGGE指纹图谱能更全面、更真实地反映黑毛茶渥堆发酵过程中细菌群落的结构和多样性变化。     

Microbial community dynamics of a blue-veined raw milk cheese from the United Kingdom

A commercial blue-veined cheese made from unpasteurized milk was examined by conventional culturing and PCR denaturing gradient gel electrophoresis analysis of the bacterial community 16S rRNA genes using 3 primer sets, V3, V4V5, and V6V8. Genomic DNA for amplification was extracted directly from raw milk, starter culture, cheese at different stages of production, fully ripened cheese, and from the cultured cells grown on various media. The outer rind was sampled separately from the inner white core and blue veins. A diverse microbiota containing Lactococcus lactis ssp. lactis, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus gallinarum, Staphylococcus devriesei, Microbacterium sp., Sphingobacterium sp., Mycetocola sp., Brevundimonas sp., Enterococcus faecalis, Proteus sp., and Kocuria sp. was detected in the raw milk using culturing methods, but only Lactococcus lactis ssp. lactis, Lactobacillus plantarum, and Enterococcus faecalis survived to the final cheese and were detected both in the core and the rind. Using PCR denaturing gradient gel electrophoresis analysis of the cheese process samples, Staphylococcus equorum and Enterococcus durans were found in the rind of prepiercing samples but not in the core and veins; after piercing, these species were found in all parts of the cheese but survived only in the rind when the cheese was fully ripened. Brevibacterium sp., Halomonas sp., Acinetobacter sp., Alkalibacterium sp., and Corynebacterium casei were identified only by PCR denaturing gradient gel electrophoresis and not cultured from the samples. Brevibacterium sp. was initially identified in the cheese postpiercing (core and veins), Halomonas sp. was found in the matured cheese (rind), and Acinetobacter sp., Alkalibacterium sp., and Corynebacterium casei were also found in the prepiercing samples (rind) and then found through the subsequent process stages. The work suggests that in this raw milk cheese, a limited community from the milk survive to the final cheese, with salt addition and handling contributing to the final cheese consortium.     

Characterizing Soy Sauce Moromi Manufactured by High‐Salt Dilute‐State and Low‐Salt Solid‐State Fermentation Using Multiphase Analyzing Methods

Present study was to characterize the physiochemical properties, free amino acids (FAAs), volatiles and microbial communities of various moromi, respectively sampled from different stages of high‐salt dilute‐state (HSDS) and low‐salt solid‐state (LSSS) fermentation, using multiphase analyzing methods. Phospholipid fatty acids (PLFA) analysis indicated that Gram‐positive bacteria were dominant bacteria and fungi were principal microbes. For DGGE analysis, dominant microbes in moromi were mainly fell into Weissella, Tetragenococcus, Candida, Pichia, and Zygosaccharomyces. During fermentation, the dominant microbes shifted from nonhalophilic and less acid‐tolerant species to halophilic and acid‐tolerant species. Total of 15 FAAs and 44 volatiles were identified in moromi, mainly Glu, Asp, Tyr, and acids, alcohols, esters, aldehydes, respectively. Odor activity values analysis suggested that the final moromi of LSSS fermentation had more complicated odors than that of HSDS fermentation. Conclusively, technological parameters, microbial communities, raw materials and fermentation process may result in the discrepancy of HSDS and LSSS moromi.     

利用DGGE分析窖泥复合功能菌液的细菌群落结构

采用变性梯度凝胶电泳(DGGE)分析窖泥复合功能菌液中各原料(窖泥、酒糟、大曲粉、黄水、己酸菌)对其细菌群落结构的影响。群落结构相似性分析结果显示,从窖泥复合功能菌液原配方中去除酒糟、己酸菌和黄水后进行培养,菌液细菌群落结构与窖泥复合功能菌液的相似性指数(Sc)仅为0.34;多样性指数分析结果显示,去除酒糟或己酸菌,细菌多样性指数(H)显著下降(p＜0.05),去除己酸菌的窖泥复合功能菌液多样性指数(H)下降最为明显。结果表明,窖泥复合功能菌液中各组分对菌液的细菌群落结构存在一定影响,其中以己酸菌的影响最大。该研究为改良优化窖泥复合功能菌液的配方提供理论依据。