New Genetic Determinants for qPCR Identification and the Enumeration of Selected Lactic Acid Bacteria in Raw-Milk Cheese.

Lactic acid bacteria (LAB) play an important role in the ripening of cheeses and contribute to the development of the desired profile of aroma and flavor compounds. Therefore, it is very important to monitor the dynamics of bacterial proliferation in order to obtain an accurate and reliable number of their cells at each stage of cheese ripening. This work aimed to identify and conduct a quantitative assessment of the selected species of autochthonous lactic acid bacteria from raw cow's milk cheese by the development of primers and probe pairs based on the uniqueness of the genetic determinants with which the target microorganisms can be identified. For that purpose, we applied real-time quantitative PCR (qPCR) protocols to quantify Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis subsp. cremoris cells in cheese directly after production and over three-month and six-month ripening periods. While L. lactis subsp. cremoris shows good acidification ability and the ability to produce antimicrobial compounds, L. delbrueckii subsp. bulgaricus has good proteolytic ability and produces exo-polysaccharides, and S. thermophilus takes part in the formation of the diacetyl flavor compound by metabolizing citrate to develop aroma, they all play an important role in the cheese ripening. The proposed qPCR protocols are very sensitive and reliable methods for a precise enumeration of L. delbrueckii subsp. bulgaricus, S. thermophilus, and L. lactis subsp. cremoris in cheese samples.

Determining the prevalence of inv-positive and ail-positive Yersinia enterocolitica in pig tonsils using PCR and culture methods.

BACKGROUND: Yersiniosis is believed to be the third most common intestinal zoonosis in the European Union, after campylobacteriosis and salmonellosis. Yersinia enterocolitica is the most common species responsible for human infections. Pigs are regarded as the biggest reservoir of pathogenic Y. enterocolitica strains, which are mainly isolated from pig tonsils. The aim of this paper is to examine the prevalence of inv-positive and ail-positive Y. enterocolitica in pigs which were slaughtered in a Polish abattoir. METHODS: Real-time PCR and culture methods were used to assess the prevalence of patho- genic Y. enterocolitica strains in pig tonsils. Real-time PCR was applied to detect inv-positive and ail-positive Y. enterocolitica. Y. enterocolitica was also isolated by applying direct plating, unselective (tryptic soy broth) and selective (irgasan-ticarcillin-potassium chlorate bouillon) enrichment. RESULTS: A total of 180 pigs were studied, of which 85% and 32% respectively were found to be infected with inv-positive and ail-positive Y. enterocolitica. The 92 inv-positive and ail-positive isolates, from 57 culture- positive tonsils, underwent bio- and serotyping. The most common was bioserotype 4/O:3, which was found in 53 (93%) out of 57 culture-positive tonsils. Strains of bioserotypes 2/O:5, 2/O:9 and 2/O:5.27 occurred in significantly lower numbers. CONCLUSIONS: The prevalence of inv-positive and ail-positive Y. enterocolitica was found to be high in the ton- sils of slaughtered pigs, using real-time PCR. The real-time PCR method for the detection and identification of pathogenic Y. enterocolitica is sensitive and specific, which has been verified by specificity and sensitivity tests using the pure cultures. Serotypes were distinguished from each other using PCR serotyping. The PCR method was essential in forming our conclusions.

Inhibitory properties of lithium, sodium and potassium o-, m- and p-coumarates against Escherichia coli O157:H7.

BACKGROUND: The aim of this paper was to assess the inhibitory properties of salts of phenolic acids against Escherichia coli O157:H7 ATCC 8739. Escherichia coli O157:H7 is a pathogen which is able to produce verotoxins provoking hemorrhagic diarrhea in humans. There is a strong need for the effective natural methods eliminating E. coli O157:H7 from food. Methodology. The following salts were tested: sodium, potassium and lithium salts of ortho-coumaric, meta-coumaric and para-coumaric acids. The 1%, 2%, 3%, 4% and 5% water solutions of each substance were prepared. Agar-well diffusion method was applied. Petri dishes were incubated at 35°C for 24 h. At the end of the incubation period, inhibition zones which appeared on the medium Petri dishes were calculated in millimeters. Results. It was found that lithium salt of o-coumaric acid, potassium salt of o-coumaric acid, lithium salt of m-coumaric acid and sodium salt of m-coumaric acid were most effective towards E. coli O157:H7, while potassium salt of m-coumaric acid, a sodium salt of p-coumaric acid were slightly less effective and lithium salt of p-coumaric acid did not possess any antimicrobial activity. Conclusion. The salts of phenolic acids having various structural features showed different characteristics towards foodborne pathogens. Such findings indicate that phenolic acids and their salts may be a potential bio-alternative for chemical food pres. METHODS: nd. The aim of this paper was to assess the inhibitory properties of salts of phenolic acids against Escherichia coli O157:H7 ATCC 8739. Escherichia coli O157:H7 is a pathogen which is able to produce verotoxins provoking hemorrhagic diarrhea in humans. There is a strong need for the effective natural methods eliminating E. coli O157:H7 from food. Methodology. The following salts were tested: sodium, potassium and lithium salts of ortho-coumaric, meta-coumaric and para-coumaric acids. The 1%, 2%, 3%, 4% and 5% water solutions of each substance were prepared. Agar-well diffusion method was applied. Petri dishes were incubated at 35°C for 24 h. At the end of the incubation period, inhibition zones which appeared on the medium Petri dishes were calculated in millimeters. RESULTS: It was found that lithium salt of o-coumaric acid, potassium salt of o-coumaric acid, lithium salt of m-coumaric acid and sodium salt of m-coumaric acid were most effective towards E. coli O157:H7, while potassium salt of m-coumaric acid, a sodium salt of p-coumaric acid were slightly less effective and lithium salt of p-coumaric acid did not possess any antimicrobial activity. CONCLUSIONS: The salts of phenolic acids having various structural features showed different characteristics towards foodborne pathogens. Such findings indicate that phenolic acids and their salts may be a potential bio-alternative for chemical food preservation.