Zülal Kesmen

Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk

In this study, the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage. On the one hand, the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples. On the other hand, rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates, and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region. As a result of the PCR-DGGE analysis of all the samples, total 8 different lactic acid bacteria were identified, and Lactobacillus sakei, Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria. The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb. sakei, Lactobacillus plantarum, Lb. curvatus, Lactobacillus brevis, Lactobacillus farciminis and Lactobacillus alimentarius. However, Leuconostoc and Weisella were also detected as minor genera. Again, Lactococcus piscium, Weissella halotolerans, Staphylococcus succinus and the comigrated Staphylococcus piscifermentans/Staphylococcus condimenti/Staphylococcus carnosus group were detected only with the culture-independent method while Lb. plantarum, Leuconostoc mesenteroides and Leuconostoc citreum were identified only by using the culture-dependent method. In the results, it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products.

A novel method to differentiate bovine and porcine gelatins in food products: NanoUPLC-ESI-Q-TOF-MS E based data independent acquisition technique to detect marker peptides in gelatin

We presented a novel nanoUPLC-MS(E) workflow method that has potential to identify origin of gelatin in some dairy products; yoghurt, cheese and ice cream. In this study, the method was performed in two steps. In the first step, gelatin was extracted from these products before the MS-sample preparation. In the second step, tryptic gelatin peptides were separated and analyzed with ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (nanoUPLC-ESI-q-TOF-MS(E)). The novelty of this setup was that it functioned in a data independent acquisition mode and that alternate low and elevated collision energy was applied to acquire precursor and product ion information. This enabled accurate mass acquisition on the peptide level to identify the gelatin peptides. The marker peptides specific for porcine and bovine could be successfully detected in the gelatin added to the dairy products analyzed, revealing that the detection of marker peptides in the digested gelatin samples using nanoUPLC-ESI-q-TOF-MS(E) could be an effective method to differentiate porcine and bovine gelatin in the dairy products.

Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques.

Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria (AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the V7-V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The culture-independent GTG5-rep PCR fingerprinting was used in the preliminary characterization of AAB isolates and species-level identification was carried out by sequencing of the 16S rRNA gene, 16S-23S rDNA internally transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples obtained from apple while K. europaeus was identified as the dominant species, followed by Acetobacter indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis (HRM), were applied to acetic acid bacterial isolates for the first time. The target sequence of ITS region generated species-specific HRM profiles and Tm values allowed discrimination at species level.

Taqman-Based Duplex Real-Time Polymerase Chain Reaction Approach for the Detection and Quantification of Donkey and Pork Adulterations in Raw and Heat-Processed Meats

In this study, a rapid and highly specific TaqMan-based duplex real-time polymerase chain reaction method, based on the simultaneous amplification of fragments of the mitochondrial ND2 and ND5 genes, was developed and optimized for the identification and quantification of pork and donkey meats in raw and cooked binary donkey/beef and pork/beef mixtures. Accumulation of polymerase chain reaction products was monitored by measuring the fluorescent signals from FAM and HEX labeled probes specific for pork and donkey, respectively. As a consequence, target meat species could be detected at a level of 0.001% in raw and oven-cooked meat mixtures, and at a level of 0.01% in autoclaved meat mixtures. The result in this study indicated that the TaqMan-based duplex polymerase chain reaction assay could be successfully used in the identification and quantification of donkey and pork in adulteration studies with a high degree of specificity and sensitivity.

Characterization of butter spoiling yeasts and their inhibition by some spices.

This study was designed to identify the yeasts in packaged and unpackaged butters and screen antiyeast activity of spices, including marjoram (Origanum majorana L.), summer savory (Satureja hortensis L.), and black cumin (Nigella sativa L.) against the most dominant yeast species in the packaged and unpackaged butters. Mean total yeast populations were 5.40 log CFU/g in unpackaged butter samples and 2.22 log CFU/g in packaged butter samples, indicating better hygienic quality of packaged samples. Forty-nine yeast species were isolated and identified from butter samples with the most prevalent isolates belonging to genera Candida-C. kefyr, C. zeylanoides, and C. lambica-and with moderate number of isolates belonging to genera Cryptococcus, Rhodotorula, Saccharomyces, and Zygosaccharomyces. Black cumin exhibited the highest antiyeast activity against C. zeylanoides and C. lambica species, even inhibited these species, while summer savory inhibited C. kefyr. The results of this study revealed clear antimicrobial potential of black cumin against the yeast species isolated from butters. Marjoram, summer savory, and black cumin could be used as natural antimicrobial agents against spoilage yeasts in food preservation, especially in butter.

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.

Application of high-resolution melting analysis for differentiation of spoilage yeasts

A new method based on high resolution melting (HRM) analysis was developed for the differentiation and classification of the yeast species that cause food spoilage. A total 134 strains belonging to 21 different yeast species were examined to evaluate the discriminative power of HRM analysis. Two different highly variable DNA regions on the 26 rRNA gene were targeted to produce the HRM profiles of each strain. HRM-based grouping was compared and confirmed by (GTG)5 rep-PCR fingerprinting analysis. All of the yeast species belonging to the genera Pichia, Candida, Kazachstania, Kluyveromyces, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Ustilago, and Yarrowia, which were produced as species-specific HRM profiles, allowed discrimination at species and/or strain level. The HRM analysis of both target regions provided successful discrimination that correlated with rep-PCR fingerprinting analysis. Consequently, the HRM analysis has the potential for use in the rapid and accurate classification and typing of yeast species isolated from different foods to determine their sources and routes as well as to prevent contamination.

Assessment of Multi Fragment Melting Analysis System (MFMAS) for the Identification of Food-Borne Yeasts

Multi Fragment Melting Analysis System (MFMAS) is a novel approach that was developed for the species-level identification of microorganisms. It is a software-assisted system that performs concurrent melting analysis of 8 different DNA fragments to obtain a fingerprint of each strain analyzed. The identification is performed according to the comparison of these fingerprints with the fingerprints of known yeast species recorded in a database to obtain the best possible match. In this study, applicability of the yeast version of the MFMAS (MFMAS-yeast) was evaluated for the identification of food-associated yeast species. For this purpose, in this study, a total of 145 yeast strains originated from foods and beverages and 19 standard yeast strains were tested. The DNAs isolated from these yeast strains were analyzed by the MFMAS, and their species were successfully identified with a similarity rate of 95% or higher. It was shown that the strains belonged to 43 different yeast species that are widely found in the foods. A clear discrimination was also observed in the phylogenetically related species. In conclusion, it might be suggested that the MFMAS-yeast seems to be a highly promising approach for a rapid, accurate, and one-step identification of the yeasts isolated from food products and/or their processing environments.

Determination of Viable Salmonella Typhimurium Cells in Heat Treated Milk By PMA/Real-Time PCR Method

Geliş 24 Kasım 2017 Kabul 27 Aralık 2017 Gıdaların üretimi sırasında uygulanan farklı teknolojik işlemler bakteriler üzerinde öldürücü etki göstermesine rağmen, bu bakterilere ait DNA’lar belirli bir süre varlıklarını korudukları için realtime PCR tekniği ile tespit edildiklerinde yanlış pozitif sonuçlara neden olabilmektedirler. Realtime PCR tekniğinin bu eksikliğini gidermek amacıyla son yıllarda DNA ekstraksiyonu öncesinde, ölü hücrelere ait DNA’ların Propodium Monoazide (PMA) ile muamele edilerek ortamdan uzaklaştırılmasına dayanan yeni bir yöntem geliştirilmiştir. Bu çalışmada ise, ısıl işlem uygulanmış süt örneklerinde, S. Typhimurium’un canlı hücrelerinin tespiti için real-time PCR yöntemi, PMA uygulaması ile kombine edilmiştir. Bu amaçla S. Typhimurium ile inokule edilen süt örneklerine farklı sıcaklık (60, 65, 70 ve 75oC) ve sürelerde (15, 60, 300 ve 900 sn) ısıl işlem uygulanmış ve canlı bakteri sayısı, direkt real-time PCR, PMA/real-time PCR ve klasik kültüre alma yöntemleriyle karşılaştırmalı olarak belirlenmiştir. Sonuçta test edilen tüm sıcaklık derece ve sürelerinde PMA/realtime PCR yönteminin direkt real-time PCR tekniğinin aksine ölü hücrelerden kaynaklanan yanlış pozitif sonuçları belli bir dereceye kadar önlediği ancak kültürel sayım sonuçlarına kıyasla daha yüksek sonuçlar verdiği belirlenmiştir. Dolaysıyla PMA/real-time PCR yöntemi ile elde edilen yüksek pozitif sonuçları elemine etmek için, PMA uygulamasına ait koşulların optimizasyonuna yönelik daha ileri çalışmalara ihtiyaç olduğu sonucuna varılmıştır. Anahtar Kelimeler: Real-time PCR Propidium monoazide Canlı-ölü bakteri ayrımı S. Typhimurium Süt

Erratum to: Application of high-resolution melting analysis for differentiation of spoilage yeasts

In the article by Erdem et al. published in Journal of Microbiology 2016; 54, 618–625, the figure 1 should be corrected as below.