Jana Bezeková

SPECIES IDENTIFICATION OF ENTEROCOCCI BY BIOCHEMICAL TEST AND MOLECULAR-GENETIC METHODS

The aim of this study was comparison different methods of species identification of enterococci. One hundred and fifty three suspected colonies were isolated from milk and dairy products (cheeses from cows, ewes and goats milk). On the bases of their growth on BEA agar, microscopic characteristic, results of Gram staining, catalase test and PYRAtest was thirty four isolates assigned to the genus Enterococcus. These isolates were identified by commercial biochemical test EN-COCCUS. 52.9% of them were included in species E. faecalis, 29.4% in E. faecium, 14.7% in E. durans and 2.9% in E. group III. This group includes 3 species: E. durans, E. hirae, E. faecalis asaccharolytic var. Then 16S rRNA sequencing nucleotide of all isolates was realized. Results of sequencing were compared with NCBI database. Only 14.7% of isolates were in 100% accordance. One from them was species E. durans and others were designated as E. faecium. For 20.6% of detected isolates was in accordance with more reference strains. Other isolates were identical with reference strain on 99%. For verification of all results species-specific PCR was used and 52.9% isolates were identified as species E. faecalis, 32.4% as E. faecium and 14.7% as E. durans. Strains belonging to the species E. faecalis were identified the most reliable by all used methods.

Selected technological properties and antibiotic resistance of enterococci isolated from milk

The aim of this work was to determine counts of enterococci in raw cow milk, to isolate and identify them, to determinate their antibiotic resistance, ability of lactose fermentation, proteolytic and lipolytic activity in different conditions of cultivation. Counts of enterococci were determined after 48 ±2 h cultivation on Slanetz-Bartley agar at 37 ±1 °C. The counts of enterococci in raw cow milk fluctuated from 1.80 x 10 2 to 1.77 x 10 3 CFU.mL -1 with average value 7.25 x 10 2 CFU.mL -1 . Species identifications of enterococci isolates were performed using commercial EN-COCCUS test and confirmed by PCR. Majority of tested isolates (85.7%) was included to species E. faecalis . Antibiotic resistance was tested on Mueller-Hinton agar using following antimicrobial discs: vancomycin (VA) 30 µg.disc -1 , gentamicin (CN) 120 µg.disc -1 , erythromycin (E) 15 µg.disc -1 , tetracycline (TE) 30 µg.disc -1 , ampicillin (AMP) 10 µg.disc -1 , teicoplanin (TEC) 30 µg.disc -1 . From 13 isolates of enterococci, 1 strain was resistant to vancomycin, 1 strain to tetracycline and 1 to ampicillin, but higher prevalence of intermediate resistance of isolates was determined to tetracycline (5 strains). Ability of lactose fermentation was monitored by change of titratable acidity in UHT milk after 0, 18, 24, 40 and 48 h of cultivation at temperature 25, 30 and 37 °C. The tested strains of enterococci exhibit low milk acidifying ability. Production of proteolytic enzymes was evaluated after cultivation at temperature 7, 25 and 30 °C after 10 days on nutrient agar no. 2 with sterile skim milk (10% w/v) with pH 6.0 and 6.5. Proteolytic activity of tested enterococci strains varied depending on tested temperature and pH. Lipolytic activity was determined similarly like proteolytic activity but on tributyrin agar base with tributyrin (1% w/v). Lipolytic activity of isolated enterococci was very low. The tested strains produced halos with zone in range from 7 to 15 mm regardless of pH, cultivation time and temperature. Some of isolated and tested enterococci strains have shown suitable technological properties, but they have exhibited resistance to antibiotic. Normal 0 21 false false false EN-GB X-NONE X-NONE

THE TESTING OF SANITIZERS EFFICACY TO ENTEROCOCCI ADHERED ON GLASS SURFACES

The aim of this work was to test the ability of 6 strains of enterococci to adhere on glass surfaces in environment with different content of milk residues and then to evaluate efficacy of 2 commercial sanitizers (alkaline and acidic) used in milk production. Tested enterococci were isolated from milk, dairy products and from rinse water after sanitation milking machine. Suspension of enterococci (8 log CFU.mL -1 ) was prepared in phosphate buffered saline (PBS), PBS with content 0.1% and 1% of skimmed reconstituted milk. Glass plates were immersed into bacterial suspension for 1 h at 37 °C. The number of enterococci adhered on glass surface in PBS achieved an average value 3.47 log CFU.mm -2 , in PBS with 0.1% of milk 2.90 CFU.mm -2 , in PBS with 1% of milk 2.63 CFU.mm -2 . Differences between the tested files were not statistically significant (p >0.05). In the second part of work the glass plates with adhered enterococci were exposed to the effect of alkaline sanitizer (on basis of NaOH and NaClO), respectively acidic sanitizer (on basis of H3PO4). Sanitation solutions were prepared and tested according to manufacturer recommendations (concentration 0.25%, contact time 20 min, temperature 20 °C). Alkaline sanitation solution was 100% effective against all tested enterococci regardless to content of milk residues in environment. Acidic sanitation solution was 100% effective only against E. faecalisD (isolated from rinse water after sanitation). Average value of reduction of enterococci with acidic sanitation solution, which were on glass plates in environment PBS was 2.84 CFU.mm -2 , in PBS with 0.1% of milk was 2.45 CFU.mm -2 and in PBS with 1% of milk was 2.16 CFU.mm -2 . It can be concluded, that increase of milk residues in environment decrease the adhesion of enterococci on

The quality of processed cheeses and cheese analogues the same brand domestic and foreign production

Processed cheeses belong to Slovakia favorite dairy products. Processed cheeses are made from natural cheeses. In recent years the trend is to replace natural cheeses with other raw materials of non-dairy nature. The composition of the processed cheese analogues is not in many countries defined by legislation. The objective of this study was to determine and compare chemical properties (fat, dry matter, fat in dry matter, NaCl) two samples of processed cheeses (C, D - Vesela krava) and two samples cheese analogues (A, B - Kiri) the same brand domestic and foreign production. The evaluated was taste of processed cheeses and cheese analogues, too. Chemical analysis and sensory analysis were repeated four times. The results of chemical analysis shows that all rated samples processed cheeses made on Slovakia fulfilled demands declared (dry matter and fat in dry matter) as producers provided on the label. The most commonly fluctuate content of NaCl from 1 to 1.24 g.100g -1 . The higher coefficient of variation in the determination of NaCl (3.88%) was found in processed cheeses made in France. Processed cheese and cheese analogues made in France had not specified parameters for dry matter and fat in dry matter on the label. For production cheese analogues Kiri made in Slovakia was used different raw material than Kiri made in France. The taste of products was determined by descriptors - salty, slightly sweet, milky, buttery-creamy, fatty, sour, bitter, and unknown. The interesting that Kiri made in Slovakia had stronger milky and buttery-creamy taste than cheese analogue Kiri made in France. Significant differences were found in the slighty sweet taste of processed cheeses, the most points won processed cheese Vesela krava made in Slovakia.

SELECTED PROPERTIES OF LACTIC ACID BACTERIA ISOLATED FROM RAW COW'S MILK

For the identification of lactic acid bacteria derived from raw cows milk, 151 colonies were isolated. The grow conditions of lactic acid bacteria were at temperature 37 °C for 3 days on MRS medium. Based on microscopical preparation, negative catalase and Gram-positive test were 81 isolates confirmed as genus Lactobacillus. Out of these, 9 isolates were evaluated for acidifying activity in UHT milk at 25 °C, 30 °C and 37 °C at regular intervals during 24 hours. The average count of NSLAB lactobacilli in raw cows milk reached the value 1.54.104 KTJ.ml-1. It was found that all tested strains of lactobacilli did not cause significant changes of titratable acidity in milk at 25 °C and 30 °C. Only one strain significantly improved the titratable acidity of milk at 37 °C after 24 hours. The acidity reached the value from 7.5 °SH to 41.9 °SH. This strain was confirmed by PCR method as Lactobacillus helveticus.doi:10.5219/177