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 to 1.77 x 10 CFU.mL with average value 7.25 x 10 CFU.mL. 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, gentamicin (CN) 120 μg.disc, erythromycin (E) 15 μg.disc, tetracycline (TE) 30 μg.disc, ampicillin (AMP) 10 μg.disc, teicoplanin (TEC) 30 μg.disc. 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.


INTRODUCTION
Enterococci are Gram-positive, non-sporeforming, catalase-negative, oxidase-negative, facultative anaerobic cocci that occur singly, in pairs, or in chains (Hollenbeck and Rice, 2012).Most enterococcal species are able to grow in the presence of 6.5% NaCl, 40% bile salts, at pH 9.6 (Ogier and Serror, 2008), at 10 and 45 °C and survive for at least 30 min at 60 °C (Domig et al., 2003).Enterococci are ubiquitous bacteria which occur in many different habitats such as in soil, surface water, ocean water, sewage, on plants and in the gastrointestinal tract of animals and humans.Based on their association with the gastrointestinal tract, enterococci often occur in foods of animal origin such as meat, fermented and cooked meat, as well as cheese (Franz et al., 2011).
Enterococci are normal components of the raw milk microbiota (Giménez-Pereira, 2005) and pasteurised milk microflora.Due to their psychrotrophic nature, their heat resistance and their adaptability to different substrates and growth conditions, count of enterococci can increase during milk refrigeration and survive after pasteurisation (Giraffa, 2003).
Presence of enterococci in dairy products can have conflicting effects, of either a risk as a foreign or intrusive flora indicating poor hygiene during milk handling and processing (if in excessive numbers), or as a benefit in contributing to produce unique traditional and emerging by-products, in protecting against diverse spoilers, and as probiotics (Giménez-Pereira, 2005).
On the other hand certain enterococcal strains are also successfully used as probiotics to improve human or animal health (Araújo and Ferreira, 2013).Enterococcus main characteristic is the ability to produce L-lactic acid (lactate) from hexoses by means of homofermentative lactic acid fermentation.Although the main product is lactate, they can also produce significant amounts of acetate, formate (Rea and Cogan, 2003).Acetate and the others are recognised as "flavour compounds" since they are important in determining the taste of many fermented dairy products (Battelli et al., 2011).Enterococci contribute to texture and aroma development of cheeses also thanks to their proteolytic and lipolytic activities (Martín-Platero et al., 2009).
The aim of this work was to determine counts of enterococci in raw cow milk from milk machines, to isolate and identify them, to determinate their antibiotic resistance, ability of lactose fermentation, proteolytic and lipolytic activity in different conditions of cultivation.

MATERIAL AND METHODOLOGY
Ten samples of raw cow milk were obtained from the milk machines.The counts of enterococci were determined by cultivation on Slanetz-Bartley agar (HiMedia Laboratories, India) at 37 ±1 °C after 48 ±2 h.Suspect colonies of enterococci (n = 38) were incubated on bile esculin azide agar (BEAA) (Biokar Diagnostic, France) at 37 ±1 °C for 24 ±2 h (STN 56 0100, 1970) for evaluation their hydrolysis.Then the isolates were identified using optical microscopy, catalase production and by PYR test (Lachema, Czech Republic).Species identification was performed using commercial EN-COCCUS test (Lachema, Czech Republic)  Then lactose fermentation, production of proteolytic and lipolytic enzymes was determined.Enterococci strains were incubated on glucose tryptone yeast extract agar (HiMedia Laboratories, India) at 37 °C for 24 ±2 h.The inoculum was prepared by suspending of enterococcal colonies in saline and by adjusting to equal 0.5 McFarland standard using Densilameter (Lachema, Czech Republic).Ability of lactose fermentation was monitored by change of titratable acidity in UHT milk (100 mL) with 1 mL of inoculum.It was cultivated at temperature 25, 30 and 37 °C.The titratable acidity was reported immediately after inoculation and after 18, 24, 40 and 48 h of cultivation.
The inoculated UHT milk after 48 h of cultivation at 7, 25 and 30 °C was used for detection of proteolytic activity of enterococci.It was determined using hole diffusion method on nutrient agar no. 2 (HiMedia Laboratories, India) with sterile skim milk (10% w/v), with pH value 6.0 and 6.5.The inoculated UHT milk (30 µL) was applied into the hole in medium.Production of proteolytic enzymes was evaluated after cultivation at temperature 7, 25 and 30 °C after 10 days.

RESULTS AND DISCUSSION
Enterococci are commonly found in raw milk with different flora reported in different countries, reflecting local practices and levels of hygiene.The counts of enterococci in our samples of raw cow milk ranged from 1.80 x 10 2 to 1.77 x 10 3 CFU.mL - with average value 7.25 x 10 2 CFU.mL -1 .
Sources tracking entry of enterococci into raw milk and subsequent transmission into processed products have indicated persistence of particular species of strain types, where the likely source of contamination of these is through milking process and processing equipment, as well Typical colonies of enterococci (n = 38) were isolated and cultivated on BEAA.The 42.11% and 26.32% of isolates showed strong and weak hydrolysis of esculin respectively, on selective medium.The remaining isolates did not hydrolyze esculin, so they were not included to genus of Enterococcus.Using PYRA test, 36.84% of total isolates was classified as enterococci.
The highest increase of titratable acidity was observed after 18 hour of cultivation at temperature 37 °C and the highest values of titratable acidity were observed after 40 hour of cultivation at both temperatures 30 and 37 °C.The lowest values of titratable acidity were reached after cultivation at 25 °C.
In Acidifying activity was weak, while interesting differences were found for proteolytic capability.The proteolytic system of LAB (including genera Enterococcus) is essential for the optimal growth in milk through the release of proteolytic enzymes.LAB have a complex system of proteases and peptidases, which allow them to use milk casein as a source of amino acids and nitrogen.Intra-and inter-specific variability in proteolysis is commonly reported for isolates from natural sources (Franciosi et al., 2009).Proteolytic activity of tested enterococci strains varied depending on tested temperature and pH value.In Figure 5 are shown proteolytic activities of strains no.98, 100 and 118.The lowest production of proteolytic enzymes was determined in strain no.98.No proteolytic activity was determined in strains no. 100 and 118 at 7 °C, in contrast with strain no.98.The highest values of proteolytic activity were determined in strain 100.

CONCLUSION
The results of this study demonstrated that raw cow milk is good source of autochthones enterococci.The tested indigenous strains of Enterococcus spp.showed interesting technological properties that could potentially be utilized further by the food industry especially in dairy technology (i.e.fermented dairy products and cheeses).Regarding the values of titratable acidity, the isolated strains are capable of producing a mild acid flavor to the fermented milk product and could be used as adjunct cultures.The proteolytic activity of enterococci as a selection criterion for the production of fermented milks may not be as crucial as it is for say, cheese production, but proteolytic strains could lead to the formation of peptides with bioactive properties during milk fermentation.The low lipolytic activity of tested enterococci can be considered as an advantage, because it may cause only a slight lysis of the milk fat without flavor change of final product.Furthermore, in order to assessment suitability of tested

Figure 1
Figure 1 PCR identification of enterococci strains.

Figure 2
Figure 2 Acidifying activity of strain No. 98 in dependence on time and temperature.

Figure 3
Figure 3 Acidifying activity of strain No. 100 in dependence on time and temperature.

Figure 4
Figure 4 Acidifying activity of strain No. 118 in dependence on time and temperature.
general, enterococci exhibit low milk acidifying ability.According to Morea et al. (1999) the pH of milk 24 hour after inoculation with strains of enterococci isolated from Mozzarella The poor acidifying capacity of enterococci isolated from food of dairy origin was confirmed also by Durlu-Ozkaya et al. (2001), Morandi et al. (2006), Serio et al. (2010) and Aspri et al. (2016).
Gardini et al. (2001) found out the maximum proteolysis of E. faecalis at an incubation temperature 32 -34 °C.The effect of pH value on this activity was rather weak (at least within the interval of values considered in this investigation).In a study of Serio et al. (2010) proteolytic activity was higher at 10 °C than at 30 °C, possibly due to the prolonged incubation time.Especially after 15 days, E. faecalis was the most active species.According to Aspri et al. (2016) 78% of tested enterococci have shown positive result for proteolytic activity after 4 days of cultivation at 37 °C.Lipolysis is an important process mainly in cheese ripening due to its role in the development of flavor and texture of the final product.Lipolytic activity of isolated enterococci was very low.Our strains produced halos with zone in range from 7 to 15 mm regardless of pH value, cultivation time and temperature.Limited reports exist on the lipolytic activity of enterococci with E. faecalis being the most lipolytic species, followed by E. faecium and E. durans (Giraffa, 2003).The results obtained in study of Aspri et al. (2016) confirmed that enterococci have generally low lipolytic activity, because none of the enterococci tested gave a zone.

Figure 5
Figure5Proteolytic activity of enterococci after 10 days of cultivation at different pH (6.0 and 6.5) and temperature.

Ruiz et al. (2016) E
).In the study of In this study was shown that strains E. faecalis and E. faecium were resistant the most frequently.Also in the results of Valenzuela et al. (2009), the strains E. faecalis isolated from milk and cheeses were the most frequent species of genus Enterococcus resistant to antibiotics.

Table 1
Evaluation of antibiotic resistance of enterococci isolated from raw cow milk according CLSI (2013).