Comparison of culture-dependent and culture-independent techniques in the detection of lactic acid bacteria biodiversity and dynamics throughout the ripening process: The case of Turkish artisanal Tulum cheese produced in the Anamur region.

Our objective was to analyze the diversity of the microbiota over 180 d of ripening of eight batches of artisanal goatskin Tulum cheeses by culture-dependent and culture-independent (PCR-DGGE) methods. V3 region of the bacterial 16S rRNA gene was amplified with the PCR after direct DNA isolation from the cheese samples. Nine different species and five genera were determined by culturing, while 11 species were identified in the PCR-DGGE technique. This diversity revealed the uniqueness of artisanal cheese varieties. The dominant genera in all the cheese samples were composed of Enterococcus species. The culture-dependent method revealed five genera (Enterococcus,Bacillus,Lactococcus,Lactobacillus, Sphingomonas) while three genera (Enterococcus, Streptococcus, Lactococcus) were detected in the culture-independent method. It was concluded that combining the two methods is important for characterizing the whole microbiota of the Tulum cheese varieties produced in the Anamur region.

The Role of Selected Lactic Acid Bacteria on Organic Acid Accumulation during Wet and Spray-Dried Fish-based Silages. Contributions to the Winning Combination of Microbial Food Safety and Environmental Sustainability.

Organic acid contents of acidified and fermented fish silages made from gibel carp (Caracius gibelio) and klunzinger's ponyfish (Equulites klunzingeri) fishes, and from fish processing residues or by-products, were determined and studied. The silages were undertaken in wet and spray-dried fish-based raw-materials for 3 weeks at room temperature (ca. 25 °C). Selected lactic acid bacteria (LAB) of Enterococcus gallinarum, Lactobacillus brevis, Lactobacillus plantarum, Pediococcus acidilactici, and Streptococcus spp. were employed to produce fermented fish-based silages, while acidified silage was prepared resorting to the addition of formic acid (3%, v/v). Lactic and propionic acids were the dominant produced organic acids, while succinic acid was formed at the smallest amounts in fermented silages. In the acidified silage, lactic and formic acids were produced in amounts higher than 800 and 1000 mg organic acid/100g sample, respectively. Among the fermented fish-based silages, LAB strains unfolded considerable ability to presumptively produce propionic acid in gibel carp silage (>2370 mg organic acid/100g sample). Spray-dried fermented silages displayed significantly higher organic acid content than wet silages. Propionic acid accumulation was found at the highest levels in gibel carp silage fermented with L. plantarum (6335.40 mg propionic acid/100g sample). This research effort pointed out the good capability of various selected lactic acid bacteria strains to produce significant amounts of organic acids-especially lactic, acetic, and propionic acids-during the fermentation of fish-based silages. In terms of food safety and quality, such a production of relatively high amounts of organic acids in wet and spray-dried fish-based silages clearly indicated their suitableness to be used for animal feed.

Development and application of RTi-PCR method for common food pathogen presence and quantity in beef, sheep and chicken meat.

Two different RTi-PCR protocol were designed and quantifications were validated by using various amounts of DNA. Multiplex AGR1 and AGR2 RTi-PCR amplification reactions quantified successfully for Clostridium perfringens, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella enterica. Using the validated multiplex RTi-PCR reactions, the presence and quantification of pathogens were investigated in 40 beef, 41 sheep and 30 chicken meat samples. Results showed that the existence of C. perfringens, E. faecalis and S. aureus was 79%, 86% and 94%, respectively in the samples. Presence of E. coli O157:H7 and S. enterica were 90% and 91% in meat samples. The results showed that many meat samples were contaminated by examined five pathogens. Therefore, it is considered that these samples may pose a potential risk to the human health since same equipment are used for different animals in the slaughterhouse. This neglect increases the amount of pathogenic contamination.

Genetic Polymorphism of Manganese Superoxide Dismutase in Behçet's Disease.

OBJECTIVES: This study aims to investigate the genetic association between single nucleotide mutation in mitochondrial manganese superoxide dismutase and a Behçet's disease (BD) population by using molecular techniques. PATIENTS AND METHODS: Ninety-three BD patients (45 males, 48 females; mean age 33.15±8.99 years; range 17 to 65 years) and 125 controls (58 males, 67 females; mean age 28.33±7.31 years; range 18 to 62 years) were genotyped by polymerase chain reaction-restriction fragment length polymorphism method. The genotypic distributions in BD patients and controls were consistent with the Hardy-Weinberg equilibrium. RESULTS: Significant differences were observed between BD patients and controls in terms of genotypic distribution. Frequencies of alanine (Ala)/Ala, Ala/valine (Val), and Val/Val were 14.0% (n=13), 45.2% (n=42), and 40.9% (n=38) in BD patients and 21.6% (n=27), 53.6% (n=67), and 24.8% (n=31) in controls, respectively (p=0.033). CONCLUSION: The Val/Val genotype of the manganese superoxide dismutase gene is associated with the physiopathology of BD in a group of Turkish patients.

Cloning and Overexpression of the als, pflA, and adhB Genes in Streptococcus thermophilus and Their Effects on Metabolite Formation.

Streptococcus thermophilus is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with Lactobacillus delbrueckii subsp. bulgaricus. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of als, encoding α-acetolactate synthase (Als), the pflA, encoding pyruvate-formate lyase activating enzyme (PflA), and the adhB which encodes alcohol dehydrogenase (AdhB) of S. thermophilus NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the lacA promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the als gene. The level of formate increased slightly by overexpressing the pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level.

Proteomics analysis of the Flp regulon in Lactococcus lactis subsp. cremoris.

Lactococcus lactis subsp. cremoris MG1363 genome sequence was completed and encodes two flp genes flpA and flpB. Research carried out has suggested that the flpB proteins are transcriptional regulators that respond to the environmental oxygen level. A variety of flp deletion mutant strains with single and double mutation were created. Wild-type (MG1363) and its flp(-) derivatives were compared by 2DE to identify changes in protein intensity under different aerobic/anaerobic growth conditions. In total, 416 ± 20 and 444 ± 32 protein spots were quantified from anaerobic and aerobic cells, respectively, on pH 4-7 gels. Forty-five protein spots that changed were excised from 2DE gel, digested with trypsin and identified from their MALDI-TOF MS Peptide Mass Fingerprint. A variety of proteins were affected by the flp mutations and oxygen level. Some proteins were controlled by FlpA and FlpB independently and some required both FlpA and B for regulation. The identified proteins that are regulated by the Flp proteins can be grouped by biochemical function. These groups are oxidative stress, electron transfer, sugars, cell wall, ABC transporters, arginine metabolism, and pyrimidine biosynthesis pathway.

Biogenic amines formation in Streptococcus thermophilus isolated from home-made natural yogurt.

Twelve different biogenic amines formation in 58 isolates of Streptococcus thermophilus from home-made natural yogurt were investigated in histidine (HDB) and lysine decarboxylase broth (LDB). All S. thermophilus isolates had an ability to produce twelve different biogenic amines in HDB and LDB. Most of the S. thermophilus isolates formed low amounts of histamine (1-50 mg/L) from histidine. Apart from one isolate, S. thermophilus produced tyramine at low (47 isolates) and medium (10 isolates) levels. The amount of each specific biogenic amine produced by S. thermophilus was generally lower than 100 mg L(-1). Also, the presence of hdcA gene was investigated using PCR technique and relation between gene and histamine production was conducted in S. thermophilus isolates. This study showed that most of the S. thermophilus isolates have the ability to form biogenic amines, especially histamine, and tyramine, which is an important consideration when selecting strains as starter cultures.

The function of lactic acid bacteria and brine solutions on biogenic amine formation by foodborne pathogens in trout fillets.

The influences of lactic acid bacteria and brine solutions on the biogenic amine formation by Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enterococus faecalis, Pseudomonas aeruginosa, Listeria monocytogenes, Aeromonas hydrophila and Salmonella paratyphi A in fermented trout fillets were investigated. Fish fillets were divided into four groups, group 1 without any lactic acid bacteria inoculation, group 2 and group 3 with different salt concentration inoculated with lactic acid bacteria and food-borne pathogens, and group 4 inoculated with lactic acid bacteria and food-borne pathogens without a salt solution. The histamine content in trout fillets in group 4 was found to be more than 10mg/100g, while the other groups contained less than 7.5mg/100g. The highest tyramine production was found for group 1 and group 3, ranging from 3 to 18mg/100g. Lactic acid bacteria did not seem to play an important role on biogenic amine production by food borne pathogens, while adding brine solution on fillets has inhibitory effects on some of the biogenic amines.

Regulation of flpA, flpB and rcfA promoters in Lactococcus lactis.

E. coli fumarate nitrate reductase (FNR) binds to conserved FNR sites to regulate transcription under anaerobic condition. L. lactis subsp. cremoris MG1363 strain contains two FNR-like proteins (FlpA and B) encoded by flpA and flpB genes and the rcfA gene-encoded RcfA in L. lactis subsp. lactis IL1403 strain. Potential FNR-binding sites were located upstream of these genes. The flpA promoter is expressed in MG1363 anaerobically and aerobically. The flpB and rcfA promoters have typical class II FNR-dependent promoters and are activated anaerobically in MG1363 and IL1403, respectively. Despite their strong homology, the Flp and RcfA proteins cannot substitute for each other and control these promoters in the heterologous strains. The flpA and flpB promoters require FlpA and FlpB for activation in the MG1363 background. This was confirmed by expressing FlpB under nisin control in flp mutants and monitoring flpA promoter expression. In flpB- backgrounds, both FlpA and FlpB were required for flpA promoter expression. FlpB could not complement for the lack of FlpA protein in flpA- backgrounds.

Molecular study on cloned endoglucanase gene from rumen bacterium.

An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 alpha-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC.