Characterization of psychrotrophic bacterial communities in modified atmosphere-packed meat with terminal restriction fragment length polymorphism

Characterization of psychrotrophic lactic acid bacteria (LAB) and Brochothrix thermosphacta communities is needed to understand the microbial ecology of spoilage of modified atmosphere-packed (MAP) meats. To overcome the limitations of the currently used methods for the characterization of psychrotrophic bacterial communities in meat, we developed a culture-independent, 16S rRNA gene-targeted terminal restriction fragment length polymorphism (T-RFLP) method. An identification library consisting of 100 Gram-positive and 30 Gram-negative meat-associated bacterial strains was set up to identify the terminal restriction fragments derived from the communities. The taxonomic resolution level of the T-RFLP method was in between genus and species within the investigated LAB strains and within family and genus within the investigated Gram-negative strains. The established library was applied to identify the members of bacterial communities in MAP minced meat at the end of the shelf life. The T-RFLP results and plate counts on Man-Rogosa-Sharpe, Violet Red Bile Glucose, and Streptomycin sulfate thallium acetate actidione agars indicated that LAB and B. thermosphacta predominated in meat. The bacterial taxa associated with the T-RFLP results were compared to those identified among plate-grown LAB isolates by numerical ribopattern analysis. Both methods agreed that Leuconostoc spp. and Carnobacterium spp. prevailed in the LAB community in minced meat followed by Lactobacillus algidus, Lactococcus spp. and Weissella spp. Colony identification revealed that Leuconostoc gasicomitatum, L. gelidum, Carnobacterium divergens and C. maltaromaticum were the predominant LAB species. The T-RFLP results were shown to correlate with viable counts of Leuconostoc spp. and B. thermosphacta. The T-RFLP method was found to be a useful tool enabling rapid and high-throughput characterization of psychrotrophic bacteria prevailing in MAP meat.     

利用16S rDNA部分序列聚类分析鉴定乳球菌

在表型鉴定的基础上，应用已发表的16SrDNA序列设计引物，扩增L．sp．F001菌株16S rDNA基因，并进行测序，将结果与同属及非同属的乳酸菌进行同源性比较。结果表明，L.sp．F001菌株与同属的乳酸乳球菌同源性为99％上，尤其与乳酸乳球菌叶蝉亚种NC-DO2181T同源性为100％，与不同属的肉明串珠菌、漫游球菌、嗜盐四联球菌同源性分别为77．0％，82．1％，78．2％。结论：菌株L.sp．F001为乳酸乳球菌。     

Grape berry bacterial microbiota: Impact of the ripening process and the farming system

Wine grapes are a primary source of microbial communities that play a prominent role in the quality of grapes prior to harvesting, as well as in the winemaking process. This study investigated the dynamics and diversity of the epiphytic bacteria on the grape berry surface during maturation. The quantitative and qualitative effects of conventional and organic farming systems on this microbial community were investigated, using both cultivation-dependent and independent approaches. Analyses of grape berry bacterial microbiota revealed changes in the size and structure of the population during the berry ripening process, with levels rising gradually and reaching their highest value when the berries were over ripe. As the season progressed to maturity, Gram-negative bacterial communities (mostly Pseudomonas spp.) declined whereas Gram-positive communities (mostly Micrococcus spp.) increased. The 16S rRNA gene sequences of cultured isolates were analysed and over 44 species were identified from 21 genera in the Proteobacteria, Actinobacteria, and Firmicutes phyla. Copper concentrations originating from phytosanitary treatments varied according to the vineyard and farming system. A negative correlation between copper concentrations and cell densities provided clear evidence that copper inhibited bacterial communities. The bacterial community structure was analysed by targeting the 16S rRNA genes, using PCR-DGGE on cultivable populations and T-RFLP on whole communities in cell suspension. The results suggest that the farming system has a clear impact on the bacterial community structure.     

Spoilage of value-added, high-oxygen modified-atmosphere packaged raw beef steaks by Leuconostoc gasicomitatum and Leuconostoc gelidum

Moisture-enhancing and marinating of meats are commonly used by the meat industry to add value to raw, retail products. Recently in Finland, certain value-added beef steak products have proven to be unusually susceptible to microbial spoilage leading to untoward quality deteriorations during producer-defined shelf-life. This study was conducted to evaluate the role of lactic acid bacteria (LAB) in the premature spoilage of value-added beef packaged under high-oxygen modified atmospheres. Spoilage was characterised by green discolouration and a buttery off-odour. The predominant LAB in eight packages of spoiled, marinated or moisture-enhanced beef steaks were identified by reference to a 16 and 23S rRNA gene restriction fragment length polymorphism pattern (ribotype) database. Leuconostoc gasicomitatum, Leuconostoc gelidum, Lactobacillus algidus, Lactobacillus sakei and Carnobacterium divergens were found to predominate in the LAB populations at numbers above 10(8) CFU/g. Inoculation of moisture-enhanced steaks with LAB strains and strain mixtures originating from the spoiled products demonstrated the spoilage potential of L. gasicomitatum and L. gelidum isolates. These two species produced green surface discolouration and buttery off-odours similar to these found in the spoiled, commercial products.     

DGGE and real-time PCR analysis of lactic acid bacteria in bacterial communities of the phyllosphere of lettuce

Food associated indigenous microbial communities exert antagonistic effects on pathogens and may routinely deliver health relevant microorganisms to the GI tract. By using molecular, culture independent methods including PCR-DGGE of 16S rDNA-coding regions and real-time PCR (RT-PCR) as well as BIOLOG metabolic fingerprinting, microbial communities on lettuce were analyzed in samples from fields, from supermarkets and soil. Amplified 16S rRNA gene sequences (57.7%) could be assigned to species previously reported as typical for the phyllosphere including Pantoea agglomerans, Pseudomonas flavescens, Moraxella spp., and Mycobacterium spp. 71.8% of the sequences obtained represented so far undescribed taxa. Principal component analysis of BIOLOG metabolic profiles indicated a seasonal variation in the lettuce phyllosphere microbial community structure. Various lactic acid bacteria were detected including several Lactobacillus and Leuconostoc species in particular on lettuce from organic farming. By RT-PCR lactobacilli were found with a range of abundances from 1x10(4 )to 1x10(5 )copies/g lettuce. Considering the importance of salad in many diets lettuce may contribute to a constant supply with LAB.     

Bacterial community structure in kimchi, a Korean fermented vegetable food, as revealed by 16S rRNA gene analysis

Kimchi is a traditional Korean food fermented from a variety of vegetables. We elucidated the microbial community structure of five commercially produced kimchis made from Chinese cabbage by examining culture-independent 16S rRNA gene clone libraries. Most of the clones (347 out of 348) belonged to lactic acid bacteria and included several species of the genera Lactobacillus, Leuconostoc and Weissella. Weissella koreensis was found in all the samples and predominated in three of them (42.6-82%). Leuconostoc gelidum, Leuconostoc gasicomitatum and Lactobacillus sakei were common in the remaining kimchi clone libraries (>34%). The composition of bacterial phylotypes in kimchi varied between samples. Our approach revealed different community structures from those reported in previous culture-dependent studies based on phenotypic identification methods. The culture-independent method used here proved to be efficient and accurate and showed that the bacterial communities in kimchi differ from those in other fermented vegetable foods.     

Arcobacter spp. enumeration in poultry meat using a combined PCR-ELISA assay

A rapid assay for enumeration of Arcobacter spp. in chicken meat was developed by using the polymerase chain reaction (PCR) coupled to an enzyme-linked immunosorbent assay (ELISA). Following a short selective enrichment of poultry samples, bacterial DNA was extracted and amplified using digoxigenin-labelled primers specific for 16S rRNA sequences of Arcobacter spp. Amplified fragments were heat denatured before being quantified by an ELISA. In this technique, a biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-PCR products. A peroxidase antidigoxigenin conjugate was added to the plate and, in the presence of substrate, PCR products were quantitated based on an optical density reading. Distinct absorbance differences were obtained when assaying poultry samples containing Arcobacter spp. in the range 10-10(4) CFU/g.     

Leuconostoc gasicomitatum is the dominating lactic acid bacterium in retail modified-atmosphere-packaged marinated broiler meat strips on sell-by-day

Lactic acid bacteria (LAB) in retail, modified-atmosphere-packaged (MAP), marinated broiler meat strips on sell-by-day were mainly identified as Leuconostoc gasicomitatum. A total of 32 packages, three to five packages of seven differently marinated broiler meat products, were studied at the end of the producer-defined shelf life (at 6 degrees C, 7-9 days depending on the manufacturer). Prior to the microbiological analyses, appearance and smell of the product was checked and pH measured. Bacteria were cultured on MRS and Tomato Juice Agar (TJA), Rogosa SL agar (SLA), Plate Count Agar (PCA) and Streptomycin Thallium Acetate Agar (STAA) for the enumeration of LAB, lactobacilli, total bacterial count and Brochothrix thermosphacta, respectively. The average CFU/g of the 32 packages was 2.3 x 10(8) on PCA. The highest bacterial average, 3.1 x 10(8), was recovered on TJA, the corresponding CFU/g averages on MRS and SLA being 2.3 x 10(8) and 1.3 x 10(8), respectively. Despite the high LAB numbers detected, radical spoilage changes such as unpleasant odor, slime production and formation of gas were not seen. B. thermosphacta did not form a significant part of the bacterial population since none of the levels exceeded the spoilage threshold level of 10(5) CFU/g reported in previous studies for this organism. In order to characterize the dominating LAB population, as many as 85, 85 and 88 colonies from MRS, TJA and SLA, respectively, were randomly picked and cultured pure. LAB were identified to species level using a 16 and 23S rDNA HindIiI RFLP (ribotyping) database. Fifty-six of the 170 isolates picked from the non-selective LAB media (MRS and TJA) were identified as L. gasicomitatum, followed by Carnobacterium divergens (41 isolates), Lactobacillus sakei and Lactobacillus curvatus subsp. melibiosus (31 isolates) and L. curvatus subsp. curvatus (20 isolates) species. SLA proved not to be completely selective for lactobacilli because the growth of Leuconostoc spp. was not inhibited, Carnobacterium spp. were the only species not detected on SLA.     

Lactic acid bacteria in marinades used for modified atmosphere packaged broiler chicken meat products

Lactic acid bacteria (LAB) in some marinades commonly used in Finland for modified atmosphere packaged poultry meat products were enumerated and identified to determine whether the marinades contained LAB species that cause meat spoilage. The concentrations of LAB in 51 marinade samples ranged from less than 100 to 8.0 x 10(5) CFU/ml. Seventeen of the samples produced LAB growth only after enrichment, and in five samples no growth was detected either by direct culturing or enrichment. Eighty-eight randomly selected isolates, 51 from the enumerated plates and 37 from enriched samples, were identified using a database of 16S and 23S rRNA gene HindIII restriction fragment length polymorphism patterns of over 300 type and references LAB strains as operational taxonomic units in numerical analyses. The predominating LAB in the enumerated samples was Lactobacillus plantarum (25 of 51 isolates). Eleven isolates were identified as Lactobacillus paracasei subsp. paracasei, and nine were Lactobacillus parabuchneri. None of these species are considered specific spoilage LAB in marinated modified atmosphere packaged poultry meat products nor have they been reported to dominate in unspoiled late-shelf-life products. These results indicate that even though marinades may contain high numbers of LAB, they are not necessarily sources of specific meat spoilage LAB. Therefore, risks associated with meat quality are not predicted by quantitative enumeration of LAB in marinades.     

Autoinducer-2-like activity in lactic acid bacteria isolated from minced beef packaged under modified atmospheres

Fifteen fingerprints (assigned to Leuconostoc spp., Leuconostoc mesenteroides, Weissella viridescens, Leuconostoc citreum, and Lactobacillus sakei) of 89 lactic acid bacteria (LAB) isolated from minced beef stored under modified atmospheres at various temperatures were screened for their ability to exhibit autoinducer-2 (AI-2)-like activity under certain growth conditions. Cellfree meat extracts (CFME) were collected at the same time as the LAB isolates and tested for the presence of AI-2-like molecules. All bioassays were conducted using the Vibrio harveyi BAA-1117 (sensor 1(-), sensor 2(+)) biosensor strain. The possible inhibitory effect of meat extracts on the activity of the biosensor strain was also evaluated. AI-2-like activity was observed for Leuconostoc spp. isolates, but none of the L. sakei strains produced detectable AI-2-like activity. The AI-2-like activity was evident mainly associated with the Leuconostoc sp. B 233 strain, which was the dominant isolate recovered from storage at 10 and 15°C and at the initial and middle stages of storage at chill temperatures (0 and 5°C). The tested CFME samples displayed low AI-2-like activity and inhibited AI-2 activity regardless of the indigenous bacterial populations. The LAB isolated during meat spoilage exhibited AI-2-like activity, whereas the LAB strains retrieved depended on storage time and temperature. The production of AI-2-like molecules may affect the dominance of different bacterial strains during storage. The results provide a basis for further research concerning the effect of storage temperature on the expression of genes encoding AI-2 activity and on the diversity of the ephemeral bacterial population.     

Composition of the bacterial population of refrigerated beef, identified with direct 16S rRNA gene analysis and pure culture technique

The composition of the dominating population of freshly cut beef, and beef stored at 4 degrees C for 8 d, was studied by direct analysis of the 16S rRNA gene (PCR amplification, cloning and sequencing) and compared with pure culture technique where the isolates picked from the viable plate count were identified by sequencing of the 16S rRNA gene. The composition of the bacterial population was recorded at two different time points, at the start when the viable plate count of the meat was 4 x 10(2) colony forming unit (cfu) per cm(2) and when it was 5 x 10(7) cfu per cm(2). Direct gene analysis by PCR amplification generated 30 clones, and 79 isolates were picked from the plate count, and identified by 16S rRNA gene sequencing. At the low initial bacterial load of the beef, the two sampling strategies showed variations in the composition of species. Direct 16S rRNA gene analysis revealed a domination of Bacillus-like sequences while no such sequences were found in isolates from the viable plate count. Instead the population of the plate count was dominated by Chryseobacterium spp. In contrast, the two sampling strategies matched on the multiplying beef population, where both methods indicated Pseudomonas spp. as the dominating group (99% of the population-sequences), irrespectively of sampling strategy. Pseudomonas panacis/Pseudomons brennerii was the dominating taxon (99% similarity to type strain), but sequences with highest similarity to Pseudomonas lundensis (99%), Pseudomonas beteli (99%) and Pseudomonas koreensis (100%) were also found.     

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.     

The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by pyrosequencing and quantitative real time (qRT)-PCR

The purpose of this work was to study the bacterial communities in raw milk and in Danish raw milk cheeses using pyrosequencing of tagged amplicons of the V3 and V4 regions of the 16S rDNA and cDNA. Furthermore, the effects of acidification and ripening starter cultures, cooking temperatures and rate of acidification on survival of added Escherichia coli, Listeria innocua and Staphylococcus aureus in cheeses at different stages of ripening were studied by pyrosequencing and quantitative real time (qRT)-PCR. A high diversity of bacterial species was detected in raw milk. Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei and Lactobacillus rhamnosus were the main bacteria detected in raw milk and cheeses. Bacteria belonging to the genera Brevibacterium, Staphylococcus, Escherichia, Weissella, Leuconostoc, Pediococcus were also detected in both 16S rDNA and cDNA obtained from raw milk and cheeses. E. coli, which was added to milk used for production of some cheeses, was detected in both DNA and RNA extracted from cheeses at different stages of ripening showing the highest percentage of the total sequence reads at 7 days of ripening and decreased again in the later ripening stages. Growth of E. coli in cheeses appeared to be affected by the cooking temperature and the rate of acidification but not by the ripening starter cultures applied or the indigenous microbiota of raw milk. Growth of L. innocua and S. aureus added to milks was inhibited in all cheeses at different stages of ripening. The use of 16S rRNA gene pyrosequencing and qRT-PCR allows a deeper understanding of the behavior of indigenous microbiota, starter cultures and pathogenic bacteria in raw milk and cheeses.     

Phylogenetic diversity of microbial communities associated with the crude-oil, large-insoluble-particle and formation-water components of the reservoir fluid from a non-flooded high-temperature petroleum reservoir

The diversity of microbial communities associated with non-water-flooded high-temperature reservoir of the Niibori oilfield was characterized. Analysis of saturated hydrocarbons revealed that n-alkanes in crude oil from the reservoir were selectively depleted, suggesting that crude oil might be mildly biodegraded in the reservoir. To examine if any specific microorganism(s) preferentially attached to the crude oil or the other components (large insoluble particles and formation water) of the reservoir fluid, 16S rRNA gene clone libraries were constructed from each component of the reservoir fluid. The clones in the archaeal libraries (414 clones in total) represented 16 phylotypes, many of which were closely related to methanogens. The bacterial libraries (700 clones in total) were composed of 49 phylotypes belonging to one of 16 phylum-level groupings, with Firmicutes containing the greatest diversity of the phylotypes. In the crude-oil- and large-insoluble-particle-associated communities, a Methanosaeta-related phylotype dominated the archaeal sequences, whereas hydrogenotrophic methanogens occupied a major portion of sequences in the library of the formation-water-associated community. The crude-oil associated bacterial community showed the largest diversity, containing 35 phylotypes, 16 of which were not detected in the other bacterial communities. Thus, although the populations associated with the reservoir-fluid components largely shared common phylogenetic context, a specific fraction of microbial species preferentially attached to the crude oil and insoluble particles.     

一种基于多重实时荧光聚合酶链式反应熔解曲线分析的肉及肉制品掺假鉴别方法

为实现肉及肉制品掺假快速鉴别,分别以猪、牛线粒体DNA的COXⅠ,绵羊、山羊、狗、狐狸、貉线粒体DNA的16S rRNA及鸡、鸭线粒体DNA的12S rRNA基因为靶位点,设计扩增产物熔解温度(T_m值)具有显著性差异的特异性引物,建立一种用于快速鉴别肉或肉制品中猪、牛、绵羊、山羊、鸡、鸭、狗、狐、貉9种源性成分的5重实时荧光聚合酶链式反应熔解曲线分析方法,通过特异性、灵敏度及市售样品的检测,对该方法进行检验和评价。结果表明:方法具有良好的特异性及灵敏度,单物种DNA检出限为0.001~1 ng,多物种混合DNA检出限均为0.1 ng,通过市售样品检测表明该方法可用于实际样品(包括生鲜样品和熟制样品)掺假的快速鉴别。     

Behaviour of Brochothrix thermosphacta in presence of other meat spoilage microbial groups

The microbial flora of fresh meat stored aerobically at 5 degrees C up to spoilage was enumerated and collected in order to have mixed spoilage bacterial groups to be used in competition tests against Brochothrix thermosphacta. The bacterial groups collected as bulk colonies were identified by PCR-DGGE followed by partial 16S rDNA sequencing. The predominant bacteria associated with the spoilage of the refrigerated beef were B. thermosphacta, Pseudomonas spp, Enterobacteriaceae and lactic acid bacteria (LAB). The interactions between B. thermosphacta and the other spoilage microbial groups were studied in vitro at 5 degrees C. The results showed that a decrease of the growth of B. thermosphacta was evidenced in presence of LAB at 5 degrees C while the bacterium is the dominant organism when inoculated with mixtures of Pseudomonas spp., LAB and Enterobacteriaceae. A better understanding of bacterial meat spoilage interactions may lead to improved quality of fresh meat stored in refrigerated conditions.     

肉类调理食品中细菌多样性的分析

肉类调理食品方便快捷,营养美味,深受消费者青睐,但其丰富的营养物质容易造成微生物的滋生,影响食品品质和消费者健康。高通量测序技术可以全面、准确的研究肉类调理食品中微生物群落结构,为微生物防控和标准化生产提供理论基础。本文通过均质提取法对肉类调理食品中微生物基因组进行提取,并以16S rRNA可变区V3-V4作为测序片段,分析细菌多样性。实验表明,4种肉类调理食品细菌多样性均较高,菌群结构具有一定的相似性,炸鸡块、骨肉相连和冷冻牛排的优势菌群是Anderseniella属,而羊肉串优势菌群是不动杆菌属,此外肉类调理食品中还存在芽胞杆菌属、梭菌属、泛菌属、假单胞菌属、嗜冷杆菌属和乳球菌属等。本论文明确了使用均质提取法对肉类调理食品中微生物进行16S rRNA V3-V4区测序分析,为后续大规模菌群研究提供思路和方法。     

A role for Dehalobacter spp. in the reductive dehalogenation of dichlorobenzenes and monochlorobenzene

Previously, we demonstrated the reductive dehalogenation of dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and MCB to benzene in sediment microcosms derived from a chlorobenzene-contaminated site. In this study, enrichment cultures were established for each DCB isomer and each produced MCB and trace amounts of benzene as end products. MCB dehalogenation activity could only be transferred in sediment microcosms. The 1,2-DCB-dehalogenating culture was studied the most intensively. Whereas Dehalococcoides spp. were not detected in any of the microcosms or cultures, Dehalobacter spp. were detected in 16S rRNA gene clone libraries from 1,2-DCB enrichment cultures, and by PCR using Dehalobacter-specific primers in 1,3-DCB and 1,4-DCB enrichments and MCB-dehalogenating microcosms. Quantitative PCR showed Dehalobacter 16S rRNA gene copies increased up to 3 orders of magnitude upon dehalogenation of DCBs or MCB, and that nearly all of bacterial 16S rRNA genes in a 1,2-DCB-dehalogenating culture belonged to Dehalobacter spp. Dehalobacter 16S rRNA genes from DCB enrichment cultures and MCB-dehalogenating microcosms showed considerable diversity, implying that 16S rRNA sequences do not predict dehalogenation-spectra of Dehalobacter spp. These studies support a role for Dehalobacter spp. in the reductive dehalogenation of DCBs and MCB, and this genus should be considered for its potential impact on chlorobenzene fate at contaminated sites.     

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.     

应用Illumina MiSeq测序技术比较风干肉中细菌多样性和微生物安全性

为了解风干肉制品中细菌的多样性及其微生物的安全性,采用Illumina MiSeq高通量测序技术,分析风干肉制品中细菌16S rRNA V4区基因序列,进而比较不同风干肉微生物的群落结构组成及多样性差异。结果显示,14 个样品共获得466 975 条有效序列,975 个操作分类单元。多样性分析表明,自然发酵风干肉中具有高度的微生物群落多样性。微生物群落组成分析表明,自然发酵样品中厚壁菌门（Firmicutes,39%）、变形菌门（Proteobacteria,40%）、拟杆菌门（Bacteroidetes,14%）为优势菌门,所占比例都超过了10%,人工调控样品中Firmicutes（92%）为优势菌门。在自然发酵和人工调控肉制品中分别鉴定出241 个和102 个细菌属,细菌多样性在属的水平上差异显著（P＜0.05）。研究结果加深了对风干肉细菌群落组成和多样性的认识,为保证风干肉制品的质量安全、提高风干肉制品的品质、优化风干肉的生产工艺提供理论依据。