Abstract: A total of 58 Village cheese samples produced from raw milk without using starter culture in Aydin province have been used for isolation of Lactococcus lactis isolates. For this purpose, a typical colony from each cheese sample representing lactococcus phenotype was confirmed by PCR using specific primers designed for conserved 16S rDNA regions. The number of bacteria growing on M17 agar plates containing nalidixic acid ranged from 1.1x10⁷-1.7x10⁹ cfu g^-1 suggesting that lactococci is widespread in Village cheeses. Catalase activity, microscopy, growing at 10 and 45°C and in the presence of 2, 4 and 6.5% of NaCl were used for phenotypical characterization of isolates. By phenotypical characterization 49 isolates represented lactococcus profile. All isolates were then analyzed by PCR and 29 of them were identified as L. lactis sp. lactis, while only 4 were L. lactis sp. cremoris. About 16 isolates exhibiting lactococcal profile with phenotypic tests did not give any amplification band with the tested primers however, 2 isolates having atypical phenotype were identified as sp. lactis by PCR. The results of the present study suggest that Lactococcus is the widespread in the flora of village cheeses and would have important role in the formation of desired flavour and textural properties.

INTRODUCTION

Lactococci is Gram positive, non-motile, catalase negative cocci belonging to lactic acid bacteria group. With an optimum growth temperature of 30°C, they can survive at 10°C but not able to grow at 45°C. They are generally members of Group N in the Lancefield serologic classification (Schleifer et al., 1985). This genus comprises of five species; L. lactis, L. garvieae, L. piscium, L. plantarum and L. raffinolactis and recently a new species named L. chungangensis took place in the nomenclature (Cho et al., 2008).

Among these species, L. lactis sp. lactis and L. lactis sp. cremoris are frequently used as starter cultures in the dairy industry, notably in cheese technology (Cogan and Hill, 1993). Understanding the importance of the microfloras in the production of traditional cheeses requires discrimination of strains in a mixed population. Classical identification methods, such as physiological and biochemical tests can not be efficient in some case to differentiate organisms at the species and subspecies levels. The L. lactis sp. lactis can be discriminated from the L. lactis sp. cremoris by arginine hydrolysis and differences in their growth characteristics at pH 9.2, 40°C and at 4% saline concentration in the medium (Salama et al., 1995). Molecular approaches developed in the last decades facilitated the characterization of bacterial species (Pu et al., 2002). Specific primers designed to differentiate L. lactis sp. lactis and cremoris subspecies have been using by a simple PCR assay (Beimfohr et al., 1997; Ward et al., 1998; Garde et al., 1999; Basaran et al., 2001).

It is generally accepted that cheeses made from raw milk have more intense flavour than cheeses made from pasteurised milk (Beuvier et al., 1997; Demarigny et al., 1997; Albenzino et al., 2001). Combination of starter and Non Starter Lactic Acid Bacteria (NSLAB) originating from raw milk may have role in the formation of typical flavour of these cheeses (Moreno et al., 2006). Thus, the biodiversity of LAB involved in cheese production is considered as a key factor for quality of artisanal products. Village cheese is a widespread traditionally produced cheese variety in Turkey. Since it is produced from raw milk and consumed freshly, attentions had been mainly paid on the pathogenic microorganisms and food borne diseases related to the consumption of this cheese (Gumussoy and Gonulalan, 2005; Little et al., 2008). Nevertheless, although Village cheese is preference because of its desired aroma and sensory characteristics there are no detailed data in the literature on its lactic acid flora and mainly lactococcal profile. The aim of this study is to isolate Lactococcus bacteria from Village cheeses and identify isolates by phenotypic tests and PCR at the subspecies level. The isolates may be used in the further studies for better understanding the role of lactococcci in Village cheeses.

MATERIALS AND METHODS

Cheese sampling: About 58 village cheese samples produced from raw milk were collected from various markets and bazaars in Aydin province and transported to the laboratory for analysis.

Microbiological analysis: About 10 g of cheese was homogenized in 90 mL of 2% sodium citrate using a BagMixers after which 10-fold serial dilutions were prepared in peptone bacteriological solution (0.1% bacteriological peptone and 0.85% NaCl). A volume of 1 mL of appropriate dilutions was used for double layer inoculation on the M17 agar (Merck 1.15108) supplemented with nalidixic acid (40 mg L^-1, Sigma N4382) and incubated at 30°C for 48 h. After incubation, a typical colony (white-cream colored, 2-3 mm in diameter) was purified twice by plating on M17 agar and M17 broth (Merck 1.15029). The colonies were then tested for morphology by microscopic examination, Gram staining, catalase production, growth at 10 and 45°C and in the presence of 2, 4 and 6.5% of NaCl. Isolates were stored in M17 broth containing 20% (v/v) glycerol at -80°C.

DNA extraction: The isolates were allowed to grow in liquid M17 lactose medium overnight at 30°C and centrifuged in 2 mL Eppendorf tubes for 5 min at 10.000 rps (Eppendorf mini plus). The supernatant was discarded and the bacterial pellet was washed twice with cold (+4°C) distilled water and re-centrifuged. The DNA was isolated using the genomic DNA isolation kit according to the protocol provided by the manufacturer (Fermentas, FE-K0512). DNA from L. lactis sp. lactis NRRL-B 1821, L. lactis sp. lactis CECT 4432 and L. lactis sp. cremoris NRRL-B 634 obtained from Izmir Institute of Technology were used as positive control.

PCR identification: In order to amplify the 16S rDNA gene, the oligonucleotide primers described by Pu et al. (2002) LacreR (19-GGGATCATCTTTGAGTGAT), LacF (19-GTACTTGTACCGACTGGAT) and CreF (19-GTGCTTGCACCGATTTGAA), for the specific amplification of L. lactis sp. lactis, L. lactis sp. cremoris, respectively were used. PCR reactions were carried out by using a Perkin Elmer Thermocycler (Model 3600) in a final volume of 50 mL containing 1.0 μL of template DNA, 5 mL of 10xPCR buffer, 25 mM MgCl2, 25 mM deoxynucleoside triphosphates, each primer at a concentration of 10 pmol μL^-1 and 5 U of Taq polymerase (Fermentas, FE-EP0602). The amplification cycle was as follows: an initial denaturation of 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 40 sec, annealing at 58°C for 40 sec and extension at 72°C for 1 min (Pu et al., 2002). PCR products were runned by electrophoresis on %1 (w/vol) agarose gels (Fermentas, Basica Le, HS-8012) in TAE buffer (Fermentas, FE-B49) at 90 V for the first 5 min and at 60 V for the remaining 60-75 min (ThermoEC 250-90). Gels were stained by 0.03 μL mL^-1 of ethidium bromide (Fermentas, ZD-A1152). Pictures of the gels were digitally captured using the Spectroline TC-312E/F UV trans-illuminator, camera Pulnix TM-7ETX and Sony printer.

RESULTS AND DISCUSSION

About 55 microscopically coccal formed isolates were selected from 58 cheese samples (Table 1). Special attention has been paid to select only one typical colony from each of cheese sample. The counts of bacteria grown on M17 plates demonstrated variations and ranged from 1.1x10⁷ -1.7x10⁹ cfu g^-1 implying that lactococcal flora is widespread in Village cheeses. Bacteria from three colonies were morphologically in rod forms. Out of other 55 isolates, 49 displayed typical lactococcal profile by their ability to grow at 10°C but not at 45°C and 6.5% of NaCl. All the isolates were then subjected to molecular identification at the subspecies level by using lactis and cremoris specific primers. Whilst 29 isolates (~57%) were identified as L. lactis sp. lactis, 4 isolates (~7%) identified as L. lactis sp. cremoris by PCR. Although, 16 isolates displayed lactococcus profile by phenotypic tests, any amplification band either with lactis or cremoris primers were observed. Interestingly, two isolates (isolates 4 and 7) growing in media containing 6.5% of NaCl, thus they did not represent lactococcal profile by phenotype but were identified as L. lactis sp. lactis by PCR.

Table 1:	Phenotypic characteristics and PCR results of the isolates from village cheeses
NI: Not Identified

The differentiation between L. lactis sp. lactis and L. lactis sp. cremoris is important due to the difference in their characteristics that they possess during the production of cheese. For a long time, the differentiation between the subspecies lactis and cremoris has been carried out based on the ammonia production from arginine test.

Fig. 1:	The specific multi-copy PCR product regions of 161 bp lenght in various strains

However, this test was reported not to give reliable results at all times (Garde et al., 1999).

The study reported here shows that the PCR methodology can be used in efficient differentiation of the two subspecies of the L. lactis isolates from the Village cheeses that are traditionally produced without using starter cultures (Fig. 1).

In the present study, 49 isolates were phenotypically identified as lactococci. By PCR assay, 29 of these isolates were confirmed as L. lactis sp. lactis and 4 isolates as L. lactis sp. cremoris. Bacteria that are members of this species can be confused with others, especially when they are categorized based on their phenotypic characteristics (Deasy et al., 2000). The strains with negative responses might have belonged to other groups of microorganisms of the Lactococcus genus.

This shows that the combination of different biochemical and molecular techniques may efficiently be used in the identification lactococcal isolates. Isolates exhibiting atypical profile were also reported by other researchers (Salama et al., 1993; Klijn et al., 1995; Ward et al., 1998; Mangin et al., 1999; Fortina et al., 2003; Bulut et al., 2005; Nomura et al., 2006; Ouzari et al., 2006). When the two sub-species of interest were taken into consideration, L. lactis sp. lactis was found to be more dominant in the Village cheese flora than L. lactis sp. cremoris. L. lactis sp. cremoris is generally a rare subspecies isolated from cheeses. Researchers have concluded that L. lactis sp. lactis was more stable in environmental conditions in comparison to L. lactis sp. cremoris which has less chance of survival in a non-dairy environment (Klijn et al., 1995; Corroler et al., 1998). From this perspective, the crowded presence of L. lactis sp. lactis in the environment of cheese production resulted in its dominance over L. lactis sp. cremoris in natural cheese flora.

CONCLUSION

The results of the current study revealed that lactoccocci are widespread in traditionally made Village cheese but their role in the maturation process and their contribution to flavour development are required to be elucidated. Isolates obtained by the present study may constitute a basis for the future experiments. Cheese manufacturing assays by using identified isolates would be much more interesting to evaluate their role in the ripening process.