Margita Čanigová

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.

Honey characteristics after extraction and half-year storage

The aim of the study was to analyze the fresh honey after extracting and after half-year storage at room temperature. Overall, we analyzed 10 samples of rape ( Brassica napus ) honey coming from district Vranov nad Toplou located in the eastern Slovakia. The analysis consisted of the evaluation of the physico-chemical parameters (water content, free acidity and electrical conductivity) and microbiological evaluation (total plate count (TPC), counts of coliform bacteria, lactic acid bacteria, sporulating microorganisms and microscopic fungi). Water content, free acidity and electrical conductivity were measured according to IHC (2009), namely these parameters were detected by refractometer, titration and conductometer, respectively. We used dilution plating method for microbiological analysis. Fresh rape honey contained 18.3 ±1.0% of water. Free acidity of fresh rape honey was 12.7 ±2.0 meq.kg -1 and electrical conductivity was 0.14 mS.cm -1 . After half a year of storage, water content and electrical conductivity decreased nonsignificantly and free acidity increased nonsignificantly. Stored honey samples meet the requirements of Decree 41/2012 and 106/2012. From microbiological point of view, fresh rape honey showed relatively high microbial counts. Mean values of TPC, sporulating microorganisms, lactic acid bacteria and yeasts exceeded 2.00 log cfu/g. All spotted microbial groups decreased in the stored honey comparing with the fresh honey. We found significant ( p ˂0.01) differences of TPC, lactic acid bacteria and yeasts comparing the fresh and stored honey samples. Evaluating microbiological parameters, one sample of stored honey did not meet the requirements of Codex Alimentarius SR (2014). TPC exceeded the limit value. Based on the results we can conclude that all samples meet the requirements for good quality honey. Microbial counts in the honey decreased gradually. Probably, various microorganisms have important role in creation of the honey from the nectar of plants, but non-sporulating microorganisms die in the ripe honey.

Sensory quality, colour and oxidative stability of cured cooked ham with propolis extract.

The effect of 0.06% propolis ethanol extract on the sensory quality, colour and oxidative stability of cured cooked ham was evaluated. Half of the experimentally processed hams treated with 0.06% ethanolic extract of propolis with ascorbic acid (EEP), only with ascorbic acid (AA) and control samples (C) were sliced and vacuum packaged. Samples were kept at 4 °C 21 days (sliced) respectively 20, 50 and 100 days (unsliced). The results revealed that all samples were characterized without any significant colour discrepancies. In general, the thiobarbituric acid value (mg malondialdehyde/kg) increased gradually in all samples examined, with a significantly lower (P <0.05) level for treated samples than for controls. The significantly lowest (P <0.05) sensory parameters in comparison to unsliced hams were observed in sliced hams packaged in vacuum. Sliced hams with EEP were characterized with significantly lowest (P <0.05) intensity of characteristic aroma. Undesirable taste was detected in control sliced hams after storage period. Significantly (P <0.05) more desirable taste of sliced hams was observed in those with only ascorbic acid in comparison with EEP. In our study was demonstrated that 0.06% ethanol extract of propolis positive affected oxidation stability and not negative affected others technological (pH, colour) and sensory characteristics of poultry meat product - cured cooked ham. Normal 0 21 false false false CS JA X-NONE

ENTEROCOCCI AND THEIR ABILITY LIVE OUT ACTIVITY OF SANITATION DETERGENTS

We evaluated the effect of temperature decrease of sanitation solutions (35 °C) in condition of organic load (1% reconstituted powdered milk) and varying hardness of the water used for solution preparation (0 °, 15 °, 30 ° and 45 °) on the ability to randomly selected strains of enterococci survive exposure to acidic and alkaline sanitation solution (0.5% concentration, contact time 15 minutes) in model experiments. Increasing water hardness also increases the number surviving enterococci. Presence of organic loads and lower temperatures decreased the sanitation effect of the test solutions. The tested strains showed different tolerances to applied sanitation solutions. We found a weaker powerful of acid sanitation solution on base phosphoric acid after its application. doi: 10.5219/166

PRESENCE OF ENTEROCOCCI IN COW MILK AND THEIR ANTIBIOTIC RESISTANCE

Enterococci represent an important part of contaminate microflora in raw milk and dairy products. They constitute significant part of nosocomial pathogens with a remarkable capacity of expressing resistance to several antimicrobial agents. We aimed to assess occurrence and antibiotic resistance of enterococci in the raw milk samples and pasteurized milk samples. In this study total bacterial count, psychrotrophic count and count of enterococci were determine in raw milk cistern samples, storage tank milk samples and milk samples after pasteurization. A collection of 46 enterococcal isolates were identified and screened for their antibiotic resistance. Isolates of E. faecalis were dominant in raw milk samples (56.5 %). Sensitive to teicoplanine (30 mcg/disk) were 97.9 % of enterococcal isolates and 15.2 % isolates were resistant to vankomycin (30 mcg/disk).

Changes in chosen properties of soft cheeses with chilli pepper during storage

The aim of this study was to evaluate chosen physicochemical and sensory properties of soft cheeses with addition of chilli peppers Fatalii. These samples were packed into plastic vacuum packages and analysed during 14 days of storage at cooling temperature (6 ±1 °C). Within the physicochemical properties, dry matter content, fat content, moisture in fat-free-substance, fat in dry matter and pH values were determined. Physicochemical analyzes, except pH value measure, were carried out only on the 1th day following the cheeses production. Textural properties hardness and stickiness were measured by the texture analyser. Within the sensory properties, consistency (hard, spreadable and friable) and taste (salty and spicy) were evaluated. Measurements of pH value, textural and sensory analysis were carried out on the 1st, 7th and 14th day of storage. All of cheese samples were classified as full-fat (FDM 48.21%) soft (MFFS 71.92%) cheeses. Their pH values decreased during 14 days of storage. The hardness of cheese samples decreased (p ˂0.05), while their stickiness statistic significantly increased during storage. From the view of sensory evaluation, the hard consistency of cheeses statistic significantly decreased and the spreadable consistency increased (p ˂0.05) during storage. The changes of friable consistency were no statistic significant. During storage of cheese samples were observed statistic significantly increase in the spicy taste and statistic significantly decrease in the salty taste.

Effect of thyme and oregano aqueous tea infusions on the lipid oxidation and sensory characteristics of frankfurkters sausages

Lipid oxidation is one of the main limiting factors for the quality and acceptability of frankfurkter type of sausages. The antioxidant activity has been measured using a TBA assay. Dried oregano ( Origanum vulgare ) and thyme ( Thymus vulgare ) were purchased from a local store and kept in dark until the use. Aqueous extracts were prepared by one-step extraction with 3 g of each pulverized plant. Samples of freshly cooked sausages were evaluated by a 6 member semi-trained panel of laboratory co-workers. Panelists evaluate appearance overall texture, flavour, aroma and overall acceptability on a 6 point hedonic scale. In this work the antimicrobial and antioxidant effect of Thymus vulgare and Origanum vulgare significantly reduced ( p <0.05) lipid oxidation in frankfurkters sausages on 10th day of aerobic storage. The organoleptic changes in sausages had no negative effect on the sensory value of cooked sausages. Results obtained in this work indicated the technical viability of using the oregano and thyme aqueous tea infusions in relative low concentration, which is possible to enlarge the shelf-life of fresh sausages with the desired slight alteration of the original taste parameters. Results indicate that thyme and oregano aqueous tea infusions compare to essential oils can be cheaper alternative incorporate into pork frankfurkters as natural antioxidants. Normal 0 21 false false false EN-GB X-NONE X-NONE

Effect of thyme and oregano aqueous tea infusions on the microbiological characteristics of sausages

In this work the antimicrobial effect of Thymus vulgare and Origanum vulgare aqueous tea infusion on the total mesophilic bacterial count (TVC), psychrotrophic bacteria count (PBC) and enterococci count in the heat treated meat product - sausages were evaluated. To prepare 1 kg of sausage in experimental groups were used 10 cm 3 of Thymus vulgare resp. Origanum vulgare aqueous tea infusions. It was found that value of TVC and PBC in the experimental groups of sausages with Thymus vulgare addition after 7 days of storage (4 °C) were 2.78 resp. 2.14 log cfu.g -1 and with the Origanum vulgare addition were 2.49 resp. 1.90 log cfu.g -1 . The value of TVC and PBC in the control group of sausage were 3.13 resp. 2.72 log cfu.g -1 . During 10 days of storage (4 °C) the TVC and PBC in the sausages with Thymus vulgare addition increase and reached the value 4.81 resp. 3.52 log cfu.g -1 . In the sausages with the Origanum vulgare addition TVC and PBC after 10 days of storage reached the value 3.67 resp. 1.60 log cfu.g -1 . The value of TVC and PBC in the control group of sausage after 10 days of storage were 6.47 resp. 5.47 log cfu.g -1 . Counts of enterococci in control and experimental groups of sausages during 10 days of storage were not detected. Thyme and origanum aqueous tea infusions suppressed the development of TVC and PBC compare to control samples. Normal 0 21 false false false EN-GB X-NONE X-NONE

Effect of spices commercial mixture with GDL on the quality of fermented dry-cured sausages

The main fermented meat products are fermented sausages in which lactic acid bacteria (LAB) are the essential agents of the ripening process. Their application as starter organisms ensures the dominance of the starter during the whole ripening process. However, when no starter cultures are used, direct addition of acids like a glucono-delta-lactone (GdL) is preferred. The goal of this study was to determine the influence of commercial spices mixtur (containing GdL) on selected technological parameters of fermented dry-cured sausages - Danube sausage in comparison with currently available conventional spices. Comparison was evaluated also with addition of starter cultures. Determinations of technological (value of pH, water activity, color) and microbiological properties (count of Lactobacillus spp., Enterobacteriaceae family, yeasts and moulds) were realized after 24 hours, 5 and 30 days. The sensory analysis of sausages was carried out after 30 days of ripening process. In sausages with the addition of commercial spice mixture in combination with starter culture were determined the lowest values of pH and a w at the end of ripening process (30 days). Bacteria of Enterobacteriaceae family were occurred in the samples with the addition of currently available conventional spices at the beginning of ripening, but after 5 days of ripening were bacteria of this family not detected. The counts of yeasts in analyzed samples were not detected. Counts of LAB at the end of ripening proccess (30 days) were lower in coparison with result obtained after 5 days; however their count was comparable with count determined at the beginig of the ripening. Our results show, that the combination of starter culture and commercial spice mixture containig GdL may cause excesive sour taste and sensory defect of dry fermented meat products.

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