Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase‐positive cocci (GCC) population in sucuk, a traditional Turkish dry‐fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA‐PCR (RAPD‐PCR) and repetitive extragenic palindrome‐PCR (rep‐PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real‐time PCR DNA melting curve analysis and high‐resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species‐specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep‐PCR and/or PCR‐RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.     

Characterization and event‐specific quantitative detection of DAS‐59122‐7 maize insert with the application of plasmidic reference material

BACKGROUND: With the development of genetically modified organisms (GMOs), event‐specific qualitative and quantitative polymerase chain reaction (PCR) detection methods have become the internationally agreed standard. RESULTS: The flanking regions of DAS‐59122‐7 maize were characterized by inverse PCR (I‐PCR). In the qualitative PCR assay, a duplex PCR was established with the event‐specific and taxon‐specific primers, and the limit of detection (LOD) was 1 g kg^?1 (approximates to 38 haploid genome copies). In the quantitative TaqMan® real‐time PCR assay, a plasmidic reference material was constructed by recombinant PCR and standard curves were set up. By using the plasmidic reference material, we obtained standard curves with good linearity and relatively high efficiency. The results indicated the usability of the plasmid as standard material. CONCLUSION: From above results, we believe that the developed event‐specific qualitative and quantitative PCR systems for DAS‐59122‐7 maize in this study are acceptable and suitable for DAS‐59122‐7 maize detection. Copyright © 2008 Society of Chemical Industry     

Microbiological Analyses of Dry and Slurry Yeasts for Brewing

Classical microbiological methods in association with molecular methods (DNA amplification, Temperature Gradient Gel Electrophoresis (TGGE) and Denaturing Gradient Gel Electrophoresis (DGGE) were used. These methods, developed to rapidly analyze microbial communities on the basis of sequence‐specific separation of DNA amplicons, allowed the detection of DNA differences in the amplicons tested and the identification of the strains analyzed by the comparison of unknown sequences with sequences of known species. TGGE allowed the comparison of the different Saccharomyces cerevisiae strains used in brewing while DGGE allowed the identification of lactic acid bacteria (LAB) in beer. These methods are a reliable tool for fast comparison of strains of Saccharomyces cerevisiae collected from different craft breweries where they were used as starters to check the presence of possible yeast contaminants in the brewing process and for rapid LAB identification.     

PCR‐TTGE and RAPD‐PCR Techniques to Analyze Saccharomyces cerevisiae and Saccharomyces carlsbergensis Isolated from Craft Beers

In the Friuli Venezia Giulia region (North East of Italy) the production of craft beers has been increasing constantly. Usually microbreweries use yeasts supplied by Italian or foreign industrial breweries for beer production. Yeast species are often not known, moreover the vitality, the viability, the physiological state and the number of generation are not known. To improve the quality of the final product it is important to evaluate the quality of the yeast strain used and the lactic acid bacteria contamination. Various molecular methods have been developed to compare genetic characteristics of yeast strains used in beer and wine production. The methods proposed in this work, PCR‐TTGE and RAPD‐PCR techniques, allow the comparison of specific DNA sequences to identify and/or characterize yeast strains. The molecular methods are faster than traditional methods and they allowed the identification of the strains analysed as S. cerevisiae and the intraspecies differentiation among yeast strains tested within 8 h after cell growth.     

PCR‐RFLP and DAMD‐PCR genotyping for Salvia species

BACKGROUND: Identification of genotypes in Salvia is complicated owing to the morphological similarity and common occurrence of natural hybridisation within Salvia species. Species‐ and genotype‐specific DNA markers are very useful for plant identification, breeding and preservation programmes and can also provide a general overview on the prediction of plant essential oil yield. RESULTS: Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) was used for identification of species‐specific chloroplast and mitochondrial organelle DNA markers, and directed amplification of minisatellite DNA polymerase chain reaction (DAMD‐PCR) was used for genotyping of plant materials. Application of PCR‐RFLP resulted in species‐specific DNA markers, and use of DAMD‐PCR resulted in reproducible DNA patterns that are useful in Salvia genetic studies. Multivariate cluster analysis and principal coordinate analysis indicated that there were relationships between DNA marker patterns and essential oil yields at the species level. CONCLUSION: Results showed that genetic variations in Salvia are wide, and DNA patterns of relatedness among plant species appeared to correlate with essential oil yields. Further studies are required to confirm the application of PCR‐RFLP and DAMD‐PCR markers for selection of Salvia species with higher essential oil yield. Copyright © 2008 Society of Chemical Industry     

Cider Apple Native Microbiota Characterization by PCR‐DGGE

The surface microbiota of different recognized apple varieties used to elaborate a Protected Designation of Origin cider was studied to analyse the microbial diversity and its potential link to the microorganisms involved in the cider fermentation process. The V3 region of the bacterial 16S gene and the D1 domain of the eukaryotic 26S gene were amplified by polymerase chain reaction (PCR) and analysed by denaturing gradient gel electrophoresis (DGGE). The most intense bands found in the DGGE profiles were sequenced and compared with those in the GenBank database. The profiles showed a high microbial diversity, but little variation was found among the varieties. Identification of the bands showed that the usual species associated with an apple juice fermentation were not found, suggesting that the microorganisms responsible for spontaneous fermentation come from the equipment and the production environment. Copyright © 2015 The Institute of Brewing & Distilling     

Determination of lactic microflora of kefir grains and kefir beverage by using culture-dependent and culture-independent methods

Abstract: In this study, we investigated the bacterial compositions of kefir grains and kefir beverages collected from different regions of Turkey by using culture‐independent and culture‐dependent methods. In the culture‐independent detection, 10 different species of bacteria were detected in total by using the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis of the 16S rRNA gene V3 region. Among these species, Lactobacillus kefiranofaciens was the most dominant one in the kefir grains, while Lactococcus lactis was found to be significantly prevalent in the kefir beverages. In the culture‐dependent detection, the primary differentiation and grouping of the isolates from kefir beverages and kefir grains were performed using repetitive sequence‐based PCR (rep‐PCR) fingerprinting, and the results were validated by 16S rDNA full‐length sequencing. According to the results of culture‐dependent methods, the most frequently isolated species were L. lactis, Leuconostoc mesenteroides, and Lactobacillus kefiri, respectively. Only 3 species, which are L. lactis, Lactobacillus acidophilus, and Streptococcus thermophilus, were detected with both culture‐dependent and culture‐independent methods. This study showed that the combination of both methods is necessary for a detailed and reliable investigation of microbial communities in kefir grains and kefir beverages. Practical Application: Due to their artisan‐ and region‐dependent microflora, kefir products can be a source of interesting lactic acid bacteria, either new taxa or strains with specific functional properties, which might be used for the development of new starter cultures and innovative food products. Therefore, an increasing demand exists for new strains that show desirable effects on the product characteristics Artisan dairy products are a candidate source of such microorganisms. For this reason, in this study, the bacterial compositions of kefir grains and kefir beverages obtained from different regions of Turkey were studied using culture‐dependent and culture‐independent molecular methods.     

Ferulic acid reduced histamine levels in the smoked horsemeat sausage

This study assessed the effect of ferulic acid on the accumulation of histamine in traditional Xinjiang smoked horsemeat sausage. The dynamics of histamine‐producing microorganisms were monitored by PCR‐denaturing gradient gel electrophoresis (DGGE) with specific primer pairs, and histidine and histamine levels were measured by high‐performance liquid chromatography (HPLC). Relative parameters including microbiological growth, pH and moisture content were also evaluated. PCR‐DGGE provided a rapid and convenient method for detecting and identifying histamine‐producing bacteria. The results showed that ferulic acid reduced the histamine concentration and inhibited the growth of histamine‐producing bacteria during fermentation and ripening. Additionally, the inhibitory effect of ferulic acid was stronger than that of the starter culture. Therefore, ferulic acid appears to be a useful additive for manufacturing fermented sausages.     

Detection of chicken and turkey meat in meat mixtures by using real-time PCR assays

In this study, TaqMan-based real-time Polymerase Chain Reaction (PCR) techniques were developed for the detection of chicken and turkey meat in raw and heat-treated meat mixtures. Primers and TaqMan probe sets were designed to amplify 86 bp and 136 bp fragments for the chicken and turkey species, respectively, on the mitochondrial NADH dehydrogenase subunit 2 gene. In the results, it was possible to detect each species at the level of 0.1 pg template DNA with the TaqMan probe technique without any cross-reactivity with nontarget species (bovine, ovine, donkey, pork, and horse) while the detection level was 1 pg template DNA using conventional PCR. The TaqMan probe assays used in this study allowed the detection of as little as 0.001% level of both species in the experimental meat mixtures, prepared by mixing chicken and turkey meat with beef at different levels (0.001% to 10%). In conclusion, TaqMan probe assays developed in this research are promising tools in the specific identification and sensitive quantification of meat species even in the case of heat-treated meat products, and suitable for a rapid, automated, and routine analysis.     

Prolactin gene polymorphism in Nili‐Ravi buffaloes in relation to Sahiwal and Achai Cattle

In this study, prolactin gene polymorphism was investigated in Nili‐ Ravi buffaloes, Sahiwal and Achai cattle breeds, 100 per group, using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) technique. Only genotype GG was observed in the case of Nili‐Ravi buffaloes. In Sahiwal and Achai cattle, three genotypes were found, AA, AG and GG: the frequency of these genotypes were 72%, 18% and 10% in Sahiwal cattle and 44%, 34% and 22% in Achai cattle, respectively. The frequency of genotype AA was found to be higher in both cattle breeds. Results of chi‐square test at P < 0.05 revealed that animals of Achai cattle were in Hardy–Weinberg equilibrium, whereas Sahiwal cattle were found to be deviating.     

Selection of Dominant NSLAB from a Mature Traditional Cheese According to their Technological Properties and in vitro Intestinal Challenges

Abstract: Isolates (47) of lactobacilli from 5 different productions of Melichloro cheese were examined for potential use as adjunct cultures. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of whole‐cell proteins classified 29 isolates as L. paraplantarum and 18 as L. paracasei subsp. paracasei. Randomly amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) analysis differentiated the L. paraplantarum and L. paracasei subsp. paracasei isolates at strain level and both, RAPD analysis and whole‐cell protein profiling provided useful information about the diversity of nonstarter lactic acid bacteria (NSLAB) in the different cheese productions. The isolates were slow acidifiers and about 70% of them degraded, preferentially α_s‐casein. The amounts of amino acids accumulated in the milk increased with the incubation time. A similar enzyme profile was exhibited by strains of both species, except for α‐mannosidase and α‐fucosidase, which were not detected in the L. paracasei subsp. paracasei strains. All strains grew in the presence of bile at 0.3% and the majority was able to withstand pH 2.5 and pancreatin at 0.1%. Moreover, all strains reduced cholesterol in vitro, with higher removal ability recorded for strains of L. paraplantarum. A narrow spectrum of antibacterial activity was recorded for 88% of the strains. Selected isolates with appropriate technological and interesting in vitro intestinal challenges could be used as adjuncts and deserve further studies. Practical Application: Strains selected by this study could be used as adjuncts to make the Melichloro cheese. Their contribution to cheese flavor is then going to be studied to select the most appropriate. Of course these strains have to be also studied for their probiotic potential, to say that we have a probiotic food.     

Genotyping of the kappa‐casein and beta‐lactoglobulin genes in Anatolian water buffalo by PCR‐RFLP

The objective of this study was to identify allele and genotype frequencies of the κ‐CN and β‐LG genes in Anatolian water buffalo. A total of 126 water buffalos from Turkey were genotyped using the PCR‐RFLP method. For gene κ‐CN, only B allele and BB genotype were observed. And for gene β‐LG, two types of alleles (A and B) and three types of genotypes were observed. The genotype frequencies of AA, AB and BB of β‐LG in Anatolian water buffalo were 0.254, 0.698 and 0.048, respectively. Surprisingly, the frequency of allele A was higher than that of allele B in contrast to world buffalo breeds.     

Traceback Investigation for Salmonella Contamination at Egg Processing Plants in South Korea: Prevalence,Antibiotic Resistance,and Epidemiological Tracing by Rep‐PCR Fingerprinting

We conducted a survey of Salmonella from 8 egg‐breaking plants and a farm to determine the prevalence and the source of the bacteria. The contents of 2400 shell eggs (20 eggs per pool), 75 pasteurized liquid egg products, and 120 unpasteurized liquid egg products from 8 egg‐breaking plants in South Korea were examined. In liquid egg samples, 4 Salmonella‐positive samples from 120 unpasteurized ones (3.3%) and 5 positive samples from 75 pasteurized ones (6.7%) were identified; no eggs were positive for Salmonella among shell egg samples. To trace the source of Salmonella, we revisited the 2 Salmonella‐positive plants (plants A and C). We investigated the equipment and environments of the plants and a henhouse (farm A) that supplied shell eggs to plant A, and collected additional liquid eggs and shell eggs from plants A and C. All Salmonella isolates from plant A and the associated farm A, except for a single Typhimurium strain from farm A, were serotyped as Bareilly. Three serovars, including one Bareilly, four Tennessee, and one Richmond, were isolated from plant C. Most Salmonella isolates were susceptible to tested antibiotics. To identify differences between isolates, molecular subtyping by using the automated rep‐PCR system was conducted. All Salmonella Bareilly (S. Bareilly) strains from plant A exhibited high similarity, indicating possible contamination by Salmonella strains from the henhouse A. Meanwhile, 2 S. Bareilly strains from plant C, one from liquid egg at the 1st visit and the other from container at the 2nd visit, exhibited identical antibiotic resistance and similar subtyping pattern, but clearly discriminated from the ones of plant A.     

Discrimination of probiotic Lactobacillus strains for poultry by repetitive sequenced‐based PCR fingerprinting

BACKGROUND: Four repetitive element sequence‐based polymerase chain reaction (rep‐PCR) methods, namely repetitive extragenic palindromic PCR (REP‐PCR), enterobacterial repetitive intergenic consensus PCR (ERIC‐PCR), polytrinucleotide (GTG)₅‐PCR and BOX‐PCR, were evaluated for the molecular differentiation of 12 probiotic Lactobacillus strains previously isolated from the gastrointestinal tract of chickens and used as a multistrain probiotic. This study represents the first analysis of the comparative efficacy of these four rep‐PCR methods and their combination (composite rep‐PCR) in the molecular typing of Lactobacillus strains based on a discriminatory index (D). RESULTS: Species‐specific and strain‐specific profiles were observed from rep‐PCR. From the numerical analysis of composite rep‐PCR, BOX‐PCR, (GTG)₅‐PCR, REP‐PCR and ERIC‐PCR, D values of 0.9118, 0.9044, 0.8897, 0.8750 and 0.8529 respectively were obtained. Composite rep‐PCR analysis was the most discriminative method, with eight Lactobacillus strains, namely L. brevis ATCC 14869^T, L. reuteri C 10, L. reuteri ATCC 23272^T, L. gallinarum ATCC 33199^T, L. salivarius ATCC 11741^T, L. salivarius I 24, L. panis JCM 11053^T and L. panis C 17, being differentiated at the strain level. CONCLUSION: Composite rep‐PCR analysis is potentially a useful fingerprinting method to discriminate probiotic Lactobacillus strains isolated from the gastrointestinal tract of chickens. Copyright © 2011 Society of Chemical Industry