An assessment of factors characterising the microbiology of Grana Trentino cheese,a Grana‐type cheese

In this study, microbiological aspects of Grana Trentino, a variant of Grana Padano cheese, were defined by plate counts, random amplified polymorphic DNA (RAPD) PCR genotying, 16S rRNA gene sequencing of bacterial isolates and PCR–denaturing gradient gel electrophoresis (PCR–DGGE). Results showed variability in monthly fluctuations of whey culture counts, differences in the diffusion of bacterial genotypes among producers and dairy plant‐specific microbial associations. Moreover, the presence of bacteria not previously reported in this cheese type was highlighted, including coagulase‐negative staphylococci and Lactobacillus sanfranciscensis‐like micro‐organisms.     

Optimization of conditions for profiling bacterial populations in food by culture-independent methods

In this study we used culture-independent methods to profile bacterial populations in food products. Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) were employed in order to identify bacterial species without the need of isolation and biochemical identification. The protocols used to extract the DNA, subsequently subjected to PCR amplification for DGGE, as well as the hybridization procedure for FISH, were optimised. Moreover, an extensive study on the primers and probes to be used for the direct detection and identification of microorganisms commonly found in food, was carried out. Meat and cheese samples, fresh or processed, were subjected to DGGE and FISH analysis and the results obtained highlighted how the processing in food industry is decreasing the bacterial biodiversity. Not only processed cheese or meat but also fermented products were dominated by only one or few species. Lactobacillus sakei, Lactobacillus curvatus and Brochothrix thermosphacta were the main species found in meat products, while in cheese(s) Lactococcus lactis, Streptococcus thermophilus and Leuconostoc spp. were repeatedly detected. The results obtained by the two culture-independent methods used always correlated well.     

Comparison of PCR‐DGGE and PCR‐SSCP analysis for bacterial flora of Japanese traditional fermented fish products,aji‐narezushi and iwashi‐nukazuke

BACKGROUND: The bacterial flora of two Japanese traditional fermented fish products, aji‐narezushi (salted and long‐fermented horse mackerel (Trachurus japonicas) with rice) and iwashi‐nukazuke (salted and long‐fermented sardine (Sardinops melanostica) with rice bran), was analysed using non‐culture‐based polymerase chain reaction (PCR) denaturing gradient gel electrophoresis (DGGE) and culture‐based PCR single‐strand conformation polymorphism (SSCP) methods. RESULTS: Viable plate counts in aji‐narezushi and iwashi‐nukazuke were about 6.3–6.6 and 5.7–6.9 log colony‐forming units g^?1 respectively. In the PCR‐DGGE analysis, Lactobacillus acidipiscis was detected as the predominant bacterium in two of three aji‐narezushi samples, while Lactobacillus versmoldensis was predominant in the third sample. By the PCR‐SSCP method, Lb. acidipiscis and Lactobacillus plantarum were isolated as the predominant bacteria, while Lb. versmoldensis was not detected. The predominant bacterium in two of three iwashi‐nukazuke samples was Tetragenococcus muriaticus, while Tetragenococcus halophilus was predominant in the third sample. CONCLUSION: The results suggest that the detection of some predominant lactic acid bacteria species in fermented fish by cultivation methods is difficult. Copyright © 2010 Society of Chemical Industry     

PCR–DGGE as a tool for characterizing dominant microbial populations in the Spanish blue-veined Cabrales cheese

The microbial populations of cheese milk and rennet extracts used in the production of traditional, Spanish, blue-veined Cabrales cheese were identified by PCR–DGGE analysis of the V3 region of the bacterial 16S rRNA gene and of the D1 region of the eukaryotic 26S rRNA genes. Ripe cheeses (60 days old) were examined in the same way. The results obtained by this culture-independent technique were compared to others previously obtained by conventional culturing methods. Rennet extracts were dominated by a number of Lactobacillus species, including Lb. plantarum, a non-starter lactic acid bacterium dominant during ripening. Lactococcus lactis was only found in one rennet extract. The cheese milk was clearly dominated by Lactococcus-like bacteria, with Lc. lactis in the greatest number. This bacterium was also dominant in the cheese samples (on both the surface and in the interior), in agreement with results obtained by culturing. The sequences of several bacterial DGGE bands from all samples showed less than 97% homology to known, cultured species. This indicates that unknown species are present in the Cabrales cheese environment and that culture-independent methods are needed to fully characterize this ecosystem.     

Novel two-dimensional DNA gel electrophoresis mapping for characterizing complex bacterial communities in environmental samples

Genomic DNA profiles such as denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), and single-strand conformation polymorphism (SSCP) have been commonly used to characterize bacterial communities in environmental samples. We recently developed a two-dimensional gel electrophoresis (2-DGE) method with a combination of chain-length polymorphism analysis (CLPA) and DGGE analysis, in order to improve the DNA resolution and resolve complex environmental DNA fragments produced by polymerase chain reaction (PCR) amplification. The 2-DGE method can generate high-resolution DNA separation maps on the basis of the lengths and composition polymorphisms of DNA sequences. It can thus facilitate detailed analyses between bacterial communities in complex environmental systems such as soil or water. For the present paper, we further developed two novel 2-DGE methods using a combination of CLPA and TTGE (or CLPA and SSCP) and here describe their potential application to the characterization of bacterial communities in nature using clustering analyses. The results show that DNA amplicons can undergo more detailed separation by the two new mapping than by their corresponding 1-DGE fingerprints. Our findings also suggest that these two new 2-DGE mapping techniques are more easily carried out than previously described DGGE-based 2-DGE mapping because they do not require a chemical denaturing gradient gel.     

Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses

Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene. Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure. The dominance of Streptococcus bovis and Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.     

The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process

A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp. in cheese samples suspected of late blowing. Strains of Clostridium spp. and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation. DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight. Moreover, volatile fatty acids were determined for comparison purposes. The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp. bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates. Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage. The sensitivity of the method is estimated to be 10(4) CFU/g.     

Analysis of the Bacterial Community in Zaopei During Production of Chinese Luzhou‐flavor Liquor

Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene analysis were carried out to analyze the bacterial community in Zaopei during production of Chinese Luzhou‐flavor liquor. Primers PRBA338F and PRUN518R were used for DGGE. Polymerase chain reaction (PCR) for clone analysis was preformed with primers EU27F and 1490R. The results by DGGE showed that with increasing fermentation time the diversity of bacteria in Zaopei decreased and after one week, only one bacterium phylotype was dominant. Gene clone libraries (16S rRNA) containing 55 clonal sequences were constructed. The bacterial diversity shift observed by DGGE was also shown by the clone library analysis. Bacteria closely related to Lactobacillus acetotolerans appeared to play a key role during Chinese liquor fermentation.     

Microbial diversity in natural whey cultures used for the production of Caciocavallo Silano PDO cheese

The microbial diversity of sixty-three Natural Whey Cultures (NWCs) for the manufacture of Caciocavallo Silano cheese PDO was studied. The NWCs were collected from different dairies covering the whole territory of PDO production including five different regions of southern Italy. The microbial species diversity was determined by direct DNA extraction from NWCs and Polymerase Chain Reaction (PCR) amplification of variable regions of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) and denaturing high performance liquid chromatography (DHPLC). DGGE and DHPLC fingerprinting yielded the same results in terms of number of bands/peaks and specific migration/retention time of the amplicons. The DHPLC technique was used in this study for the first time to assess a food-related mixed microbial community by a culture-independent approach and proved to be at least as effective as DGGE in profiling species diversity in NWCs. Cluster analysis of DGGE and DHPLC data revealed that the species-related groups of similarity were not dependent on the geographical origin of the NWCs. The presence of three groups of 10-14 NWCs at 100% of species similarity indicated that some species associations are very commonly occurring in the NWCs for Caciocavallo Silano cheese PDO. A RAPD-PCR analysis of the NWCs was also performed for the members of the above groups and it showed that, though characterized by the same species diversity, most of the identical NWCs included different biotypes. The sequences of DGGE bands and DHPLC peaks revealed the occurrence of mainly thermophilic lactic acid bacteria such as Lactobacillus delbrueckii, L. helveticus and Streptococcus thermophilus even though the mesophilic Lactococcus lactis also occurred in some NWCs. In conclusion, the results of this study indicate that the microbial diversity of NWCs used for the Caciocavallo Silano PDO cheese is not high, it is not dependent on the geographical origin and the same microbial species occur within the territory examined. The microbiota of fermented PDO products and its possible link with territory should be studied case by case in order to have useful evidences for the assessment of product quality and authenticity.     

A DGGE Marker‐Mediated Fast Monitoring of Bacterial Diversity and Comprehensive Identification of High‐Temperature Daqu Starter

Bacteria play an essential role in Daqu starter (Daqu) fermentation. The identification of Daqu bacteria was investigated by polymerase chain reaction (PCR) based denaturing gradient gel electrophoresis (DGGE) analysis of the highly variable V3 region of the 16S rRNA gene. Here, we define a novel DGGE marker for the quick identification of Daqu bacteria. A dynamic alteration of the bacterial populations at different stages of fermentation was determined through a 2‐y continuous monitoring. The physicochemical parameters of Daqu at different fermentation stages were investigated by weighing, NaOH titration, and HCl hydrolysis together with Fehling reagent methods. Furthermore, infrared spectral analysis using Fourier Transformed Infrared Spectroscopy was performed to determine physicochemical changes of Daqu. Therefore, our studies provide key insight for a comprehensive quality control of Daqu at different fermentation stages using the PCR‐DGGE analysis combined with the physicochemical measurement.     

Identification of Microbiota Present on the Surface of Taleggio Cheese Using PCR–DGGE and RAPD–PCR

Abstract: Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry‐salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR‐DGGE) and/or random amplification of polymorphic DNA (RAPD‐PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. Practical Application: Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.     

Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese

The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goat's milk without the addition of any bacterial starter culture. The presence of lactic acid bacteria (LAB) in Bukuljac cheese has been analyzed by using a polyphasic approach including microbiological and molecular methods such as rep-PCR with (GTG)5 primer. Lactobacillus paracasei subsp. paracasei represents a dominant strain in the microflora of analyzed cheese. Out of 55 Gram-positive and catalase-negative isolates, 48 belonged to L. paracasei subsp. paracasei species. Besides lactobacilli, five Lactococcus lactis subsp. lactis and two Enterococcus faecalis were found. Results of PCR-denaturing gradient gel electrophoresis (DGGE) of DNA extracted directly from the fresh cheese revealed the presence of Leuconostoc mesenteroides. Only lactobacilli showed a high proteolytic activity and hydrolyzed alpha(s1)- and beta-caseins. They are also producers of diacetyl. In addition, 34 out of 55 isolates, all determined as lactobacilli, showed the ability of auto-aggregation. Among 55 isolates, 50 also exhibited antimicrobial activity.     

Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE

The diversity and dynamics of the dominant microbial communities arising during the manufacture and ripening of four batches of naturally fermented Cabrales cheese were investigated by the PCR-DGGE culture-independent technique. Total microbial DNA was extracted from cheese milk, curd and cheese samples and used as template material in PCR experiments to amplify the V3 region of the bacterial 16S rRNA gene, plus the D1 region of the eukaryotic 26S rRNA gene. These regions were then analysed using DGGE. Eukaryotic and bacterial bands were identified by isolation, reamplification and sequencing. The results were compared to those obtained in a previous microbial characterization of the same four batches using classical culturing methods. Great variability was recorded between batches by the PCR-DGGE technique. This was also shown by culturing, and underlines the uniqueness of artisanal products. Lactocococcus lactis subsp. lactis was dominant from the cheese milk stage until the end of ripening, whereas populations of certain Lactobacillus species appeared during ripening. Populations of species never isolated by culturing were found to be numerous by the PCR-DGGE method, in particular Lactococcus garvieae and Lactococcus raffinolactis. Other, completely unknown lactococci were also detected. The dominant eukaryotic populations from day 15 onwards were those of Penicillium roqueforti and Geotrichum candidum.     

Changes in the composition of the bacterial flora on tray-packaged pork during chilled storage analyzed by PCR-DGGE and real-time PCR

In this study, a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to investigate the changes in the composition of the bacterial population of tray-packaged pork during chilled storage. Relative quantitative real-time PCR was further used to evaluate the predominant spoilage bacteria obtained from DGGE analysis for their relative amount to the total bacteria in meat samples. DGGE analysis of the V3 and V6-V8 regions of the 16S rRNA gene showed that Pseudomonas were the predominant bacterial species at the end of the monitoring period. Real-time PCR expressed as the ΔΔC(T) method showed that the average 2(-ΔΔC)(T) values increased continually during the storage period from less than 0.001 at day 0 to 4.438 at the end of the monitoring, which indicated that the proportions of Pseudomonas within the total bacteria in meat samples increased. Both methods confirmed that Pseudomonas was the predominant spoilage bacteria. Practical Application: This study uses new techniques to identify bacteria in fresh retail pork and to follow changes in the bacterial population during 12 d refrigerated storage. Pseudomonads were found to increase with storage time, becoming the dominant flora after 12 d.     

Effect of Penicillium roqueforti and incorporation of whey cheese on volatile profiles and sensory characteristics of mould‐ripened Civil cheese

In this study, four different types of mould‐ripened Civil cheese were manufactured. A defined (nontoxigenic) strain of a Penicillium roqueforti (SC 509) was used as secondary starter for the manufacture of mould‐ripened Civil cheese with and without addition of the whey cheese Lor; in parallel, secondary starter‐free counterparts were manufactured. A total of 83 compounds were identified. Ketones, alcohols and esters were the principal classes of volatile components. Principal component analysis of the headspace volatiles grouped cheeses by age and type. P. roqueforti inoculated cheese was clearly separated from the other cheeses at 180 days of ripening, and these cheeses were characterised with high levels of ketones (e.g., 2‐butanone, 2‐heptanone). Differences in the panel scores between the cheese samples were not significant during the first stage of ripening (up to 60 days); as ripening proceeded, these differences were become evident and P. roqueforti inoculated cheeses received higher scores than others. Addition of Lor in the manufacture of mould‐ripened Civil cheese caused lower points by the sensory panel, and the cheese inoculated with P. roqueforti and Lor‐free was the best type of mould‐ripened Civil cheese. The results showed that the use of P. roqueforti in the manufacture of mould‐ripened Civil cheese has significant impact on the volatile profiles and sensory attributes.     

Changes in the bacterial communities of vacuum-packaged pork during chilled storage analyzed by PCR–DGGE

In this study, PCR-denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial communities of vacuum-packaged pork during chilled storage. Eight kinds of lactic acid bacteria (LAB) were identified from the strains isolated from MRS plates by PCR–DGGE of the V3 region, and Lactobacillus sakei was the representative isolate at the end of the monitoring. By means of the direct meat analysis of PCR–DGGE, LAB increased gradually and Carnobacterium sp./Car. divergens, Lactobacillus sakei and Lactococcus sp./Lc. piscium, became the predominant bacteria at the end of the storage. The results of Lactobacillus-specific PCR and DGGE showed that different Lactobacillus populations were present at different storage periods and Lb. sakei became the predominant bacteria in the end. In conclusion, the PCR–DGGE technique as a culture-independent method is applicable to monitoring bacterial population dynamics in vacuum-packaged pork.     

Characterization of archaeal community in Luzhou‐flavour pit mud

The aim of this study was to investigate the archaeal community in different ages of pit mud by a combined fluorescence in situ hybridization (FISH) and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) method. Four probes were used to detect the major methanogenic archaea by FISH experiments, and the results showed that orders Methanosarcinales, Methanobacteriales, Methanomicrobiales and Methanococcales were detected in the various ages (50, 100 and 300 year) of pit mud, except for the 1 year‐old pit and the 100 year‐old sample, which exhibited the highest numbers of archaea. The amounts of the four methanogenic archaea significantly increased from the 50 to 100 year‐old pit mud and slightly decreased in the 300 year‐old pit mud. Results of PCR‐DGGE analysis suggest that all archaeal 16S rRNA gene sequences fall into the phylum Euryarchaeota, and Methanobacteriales and Methanomicrobiales dominated in low‐age (1 and 50 year) and old age (100 and 300 year) of pit mud, respectively. Analysis of the community diversity based on the DGGE profiles showed that the 100 and 300 year samples exhibited similar diversity indices compared with the 1 and 50 year samples. This is the first report about the archaeal community structure in different ages of pit mud determined by both FISH and PCR‐DGGE analysis. Copyright © 2015 The Institute of Brewing & Distilling     

Microbiological characterisation of Robiola di Roccaverano cheese using PCR-DGGE

The aim of this study was the microbiological characterisation of a typical Italian cheese “Robiola di Roccaverano” with Protected Designation of Origin (PDO). Fresh and ripened robiola samples were collected from four artisanal and one industrial producer. Artisanal producers used raw goat's milk and natural fermentation, whilst the industrial producer used mixed cow–goat's milk and selected starters. The microbial communities were monitored during different seasons and ripening times with PCR–DGGE and culture-dependent methods.The cluster analysis showed that the DGGE bacterial patterns were related to the different manufacturing and climatic conditions, revealing the occurrence of species associated to Robiola di Roccaverano. The DGGE profiles of yeasts were affected by ripening in summer season.Moreover, the results obtained allowed the identification of microbial species such as Lactococcus lactis subsp. lactis, Geotricum spp. and Kluyveromyces lactis that are related to the production of this typical cheese.     

用DGGE技术构建白酒酿造微生物指纹图谱的初步研究

利用PCR-DGGE技术及Quantity One软件分析对比了大曲、酒醅不同部位的微生物群落结构.通过从原始样品中直接提取DNA,并对全长16s rDNA及其V3区片断扩增,变性梯度凝胶电泳及软件分析等,发现不同样品之间的微生物群落存在一定联系及差异性.通过图谱与系统发育树的结合,能给不同样品赋予特定的分子标签.显示DGGE技术能为微生物菌落结构提供直观图谱,是研究自然传统发酵食品及其它天然微生态样品的先进方法.