Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese

biology 114 (2007) 211–220www.elsevier.com/locate/ijfoodmicro

International Journal of Food Micro

Genotypic and phenotypic diversity of Lactococcus lactis isolates fromBatzos, a Greek PDO raw goat milk cheese

L. Psoni a, C. Kotzamanidis b, M. Yiangou b, N. Tzanetakis a, E. Litopoulou-Tzanetaki a,⁎

a Laboratory of Food Microbiology and Hygiene, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greeceb Department of Genetics, Development and Molecular Biology, School of Biology, Aristotle University of Thessaloniki, 54124, Thessaloniki, Greece

Received 10 March 2006; received in revised form 17 July 2006; accepted 19 September 2006

Abstract

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese madeone in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability anddistinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except onewere clustered together (at a similarity level of ∼50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of2–8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having uniqueplasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and thoseformed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling andPFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to theseason of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersiniaenterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of theisolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates fromwinter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the ∼67% of the isolates from summer cheese showed higher Leu-than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for αs-caseindegradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggestthat among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.© 2006 Elsevier B.V. All rights reserved.

Keywords: Lactococcus lactis; Cheese; Genotypic; Phenotypic; Heterogeneity

1. Introduction

Lactococci are an important group of lactic acid bacteria(LAB) and are used worldwide for the manufacture of fermenteddairy products, e.g. cheese. Large-scale industrial processes relyon a limited number of starter cultures (Marshall, 1991) and thereis a recognized demand for new or improved strains with theability to produce new flavour compounds, in order to replace andcomplement the starter strains currently used in industrialfermentations (Marshall, 1991; Forde and Fitzgerald, 1999).

⁎ Corresponding author. Tel.: +30 2310 473622; fax: +30 2130 473622.E-mail address: [email protected] (E. Litopoulou-Tzanetaki).

0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ijfoodmicro.2006.09.020

The evolution of lactic microflora in cheeses with a ProtectedDesignation of Origin (PDO) is of particular interest, because theyare often made from raw milk containing a large number ofadventitious microorganisms (Corroler et al., 1998). In the lastdecade much effort has been put into the biochemical, genetic andtechnological characterization of “wild” lactococci strains isolatedfrom traditional cheeses, especially those with a recognized PDO(Corroler et al., 1998; Gaya et al., 1999; Mannu et al., 2000;Sanchez et al., 2000; Mannu and Paba, 2002), but also from othersmade with natural or no starter cultures (Cogan et al., 1997;Desmasures et al., 1998; Delgado and Mayo, 2004).

Results on the microflora of Batzos, a raw goat milk PDOGreek cheese showed that in cheese throughout the wholelactation season Lactococcus lactis subsp. lactis was the most

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frequently isolated LAB species (Psoni et al., 2003) constituting69.0, 28.0 and 61.7% of the LAB isolates from fresh cheese madein winter, spring and summer, respectively. However, changesoccurring in the microflora composition have been studied bymeans of classic phenotypic identification methods, which havebeen repeatedly shown to be unreliable (Schleifer and Kilper-Bälz, 1987; Pot et al., 1994). Properties typical of the L. lactissubsp. lactis, such as growth at relatively high temperatures andNaCl concentrations, have also been reported for strainsgenetically identified as L. lactis subsp. cremoris (Salama et al.,1995) and strains originally classified as L. lactis subsp. lactis,were recognized as L. lactis subsp. cremoris with reliablemolecular methods (Godon et al., 1992). The development ofnew methods, involving various DNA-based typing techniques,has opened up new perspectives for typing strains from raw milkand from traditional PDO cheeses (Desmasures et al., 1998;Corroler et al., 1999; Mannu et al., 2000; Sanchez et al., 2000;Mannu and Paba, 2002; Delgado and Mayo, 2004). The use ofselected isolates as starters to make cheese from pasteurized milkis of particular importance for the preservation of the typical andunique organoleptic properties of artisanal cheeses (Bertozzi andPannari, 1993). These cheeses may, therefore, be interestingsources of lactococcal strains eventually responsible of formingparticular flavour compounds.

In this study 40 lactococcal isolates from raw goat milkBatzos cheese made throughout the whole lactation season(Psoni et al., 2003) were extensively characterized. Our purposewas to study their genotypic and phenotypic biodiversity and toselect strains distinguished for their acid and flavour formingcapabilities as well as their abilities to produce antibacterialsubstances. These properties would be taken into considerationin an appropriate starter for cheese production.

2. Materials and methods

2.1. Bacterial strains and culture conditions

A total of 40 isolates, phenotypically identified as L. lactissubsp. lactis (Psoni et al., 2003), coming from threemanufacturers of Batzos cheese made in the beginning ofJanuary (winter; 20 isolates), beginning of April (spring; 5isolates) and beginning of July (summer; 15 isolates) wereanalyzed. The following type and reference strains were usedfor identification purposes: L. lactis subsp. lactis LMG 6890from BCCM/LMG Bacteria Collection (Laboratory of Micro-biology, University of Gent, Gent, Belgium), L. lactis subsp.cremoris NCTC 1259 (NCTC, National Collection of TypeCultures, AFRC, Reading, England) and Lactococcus raffino-lactis DSM 20016 (DSM, German Collection of Microorgan-isms and Cell Cultures, Brawnschweig). Stock cultures oflactococci were kept in M17 broth plus glycerol (70:30) at−80 °C and grown in M17 broth. Food-borne pathogens usedfor testing the antibacterial activities of the strains were: E. coliO157:H7 EDL-932 (toxigenic strain, obtained from Prof.Genigeorgis, University of California, Davis California,USA), Staphylococcus aureus NCTC 9751, Yersinia enteroco-litica 0:9/4360 (supplied by the Pasteur Institute, Paris), Lis-

teria monocytogenes LMG 3568 and a Bacillus cereus strain(supplied by the School of Medicine, Aristotle University ofThessaloniki). The bacteriocin sensitive strain Enterococcusfaecalis Ef1 was kindly supplied by the Laboratory of DairyTechnology, Agricultural University of Athens. Culture of E.faecalis Ef1 was kept in MRS broth plus glycerol (70:30) at−80 °C and was subcultured in MRS Broth (pH 6.2) foractivation before use. The pathogens were maintained in BrainHeart Broth plus glycerol (70:30) at −80 °C and grown in BrainHeart Broth before use. All media were obtained from Merck(Darmstadt, Germany). The cultures were activated by twosuccessive transfers in the appropriate broth before use.

2.2. RAPD-PCR analysis

Strains were grown overnight in M17 broth. The DNA wasisolated by the method of Querol et al. (1992). RAPD-PCRreactions were performed with primers M13 and D8635, aspreviously described by Andrighetto et al. (2001). Amplifica-tion products were separated by electrophoresis on 1.5% (w/v)agarose gels in 0.5× TBE buffer (0.45 mM—Tris–HCl,0.45 mM—boric acid, 1 mM—EDTA, pH 8.3).

2.3. PFGE analysis

The preparation of intact genomic DNA in agarose blockswas performed by a slight modification of the method describedby Jacobsen et al. (1999). Restriction enzyme digestion wasperformed with 20 U of Sma I (New England Biolabs Inc.Hitchin, Hertfordshire, UK) according to the supplier'sinstructions. DNA fragments were resolved in 1% (w/v)PFGE certified agarose (Bio-Rad Laboratories Ltd, HemelHempstead, Hertfordshire, UK) in 0.25× TBE buffer by pulsed-field gel electrophoresis (PFGE) using the Rotaphor type Velectrophoresis unit (Biometra GmbH, Goettingen, Germany).To obtain optimal separation of fragments the gels were run at180 V with pulse time ramped from 2 to 15 s linear, field angle110° to 120° linear for 20 h at 22 °C. The agarose gels werestained with ethidium bromide (0.5 μg/ml) and visualized underUV light. The Low Range PFGE Marker (2.03–194.0 kbp; Bio-Rad), was included as size marker and normalization reference.

2.4. Statistical analysis

RAPD-PCR and PFGE profiles on photo-positives werescanned (AGFA scanner, AGFA GEVAERT N.V., Morstel,Belgium) into a computer and subsequently analyzed using theGel Compar software version 4.0 (Applied Maths, Kortrijk,Belgium). Calculation of the similarity of the band profile andgrouping of the RAPD-PCR and PFGE patterns was based onthe Dice correlation coefficient, and the unweighted pair groupmethod using arithmetic averages (UPGMA) cluster analysis.

2.5. Plasmid analysis

The extrachromosomal DNA of the test strains was extractedas described by Anderson and McKay (1983). The plasmids

Fig. 1. Dendrogram obtained by using RAPD profiles for Lactococcus isolates from two primers, followed by evaluation using the Dice coefficient (r) and theUPGMA. M1, M2 isolates from cheese made in winter; M4: isolates from cheese made in spring; M7: isolates from cheese made in summer; D, A, B, G: cheese at 12,30, 60, 90 days of ripening, respectively; no indication: one day-old cheese.

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were separated by agarose gel electrophoresis (0.8% agarosegels in 40 mM—Tris-acetate, 1 mM—EDTA, pH 8.0 buffer) at100 V. A molecular weight marker of 125–23,130 bp (λ DNA/Hind III Fragments, Invitrogen Ltd, Paisley, UK) was used todetermine the plasmid sizes.

2.6. Acidification ability and proteolytic activity

The acidifying activity in milk was tested by inoculating(1.0% v/v) 100 ml sterile reconstituted skim milk with anovernight culture, which had been previously activated by two

successive transfers in milk. The pH changes were measured bypH meter (glass electrode; HANNA Instruments, Padova, Italy)after 6, 16 and 24 h of incubation at 30 °C. Isolates lowering themilk pH by more than 1.25 pH units after 6 h of incubation wereconsidered to be fast acid producers (Cogan et al., 1997).

The proteolytic activity of the strains grown in milk wasmeasured by the o-PA (o-phthaldialdehyde) method (Churchet al., 1983) after 6, 16 and 24 h at 30 °C and the results wereexpressed in L-glycine equivalent. Their caseinolytic activitywas also estimated after 6 h, 16 h and 24 h in milk by UREA-PAGE, according to Andrews (1983). The gels were scanned

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and the peak areas were estimated by Gel Compar version 4.0(Applied Maths); the individual proteins were quantified bycomparison with the corresponding peak areas for the milk.

2.7. Peptidase activity of intact cells

The aminopeptidase activity of the strains was tested on thesubstrates L-lysine p-nitroanilide (Lys-pNA), L-leucine p-nitroanilide (Leu-pNA) and L-proline p-nitroanilide (Pro-pNa)(Sigma-Aldrich Ltd, Athens, Greece). The method suggested byRequena et al. (1991) as modified by Arizcum et al. (1997) wasused. A solution of 15.5 mM of the substrates in methanol wasprepared. The reaction mixture consisted of 200 μl of eachsolution, 0.4 ml of intact cells (washed twice with phosphatebuffer, pH 7.0, and subsequently diluted to a suspension at 4 ofMcFarland scale), and 3.6 ml of 50 mM sodium phosphatebuffer at pH 7.0. The mixtures in test tubes were incubated byshaking in a water bath at 30 °C for 1 h and the reaction wasthen quenched by adding 1 ml of 30% (v/v) acetic acid. Thecontent of the tubes was filtered through Whatman filter paper(Whatman International Limited, Maidstone, Kent, UK). Theamount of p-nitroanilide released was measured at 410 nm by aspectrophotometer (Shimadzu UV-120-02, Shimadzu BiotechCorporation, Newcastle, UK) and the results were expressed in

Fig. 2. Plasmid profiles of the Lactococcus isolates from Batzos cheese obtained throIII Fragments molecular weight marker of 125–23.130 bp, used as internal controlM1B4; 7, M1B9; 8, M1A6; 9, M1D1; 10, M1D2; 11, M1D3; 12, M28; 14, M43; 15,marker of 125–23.130 bp, used as internal control of electrophoretic conditions, laneM78; 12, M13; 13, M15; 14, M71; 15, M17; 16, M1A4; 17, M49; 18, M7D3; 19, M

aminopeptidase activity units, where 1 U is the increase of0.001 of absorbance in 1 min.

2.8. Antimicrobial activity

Activated strains grown for 16 h at 30 °C were screened forinhibitory activity by the method of Kekessy and Piquet (1970),which may detect the production of bacteriocins. Indicatorstrains were grown in the appropriate broth to an optical densityof ∼0.45 at 600 nm. The producer strains were inoculated byspot inoculation on the surface of M17 agar plates (4 strains foreach plate). After incubation (48 h, 37 °C) the agar wasdetached from the edges of the Petri dish with a sterile spatula.The plate was then inverted into the lid and the new sterilesurface was overlaid by the indicator strains inoculated (1% ofthe first decimal dilution) into an appropriate soft agar. Afterincubation, the zones of inhibition, if present, were measured.The growth of E. coli O157:H7 in the presence of L. lactissubsp. lactis strain M7D2 in 100 ml sterile reconstituted skimmilk was also assayed, using 1% inoculum for each. E. coli(1%) and L. lactis subsp. lactis (1%) were also grownindividually in 100 ml sterile reconstituted milk. During growththeir counts were determined in violet red bile agar and MRSagar, respectively.

ughout ripening during the whole lactation season. A) Lanes 1, 13 λ DNA/Hindof electrophoretic conditions, lane 2, MID7; 3, M1G3; 4, M1A8; 5, M1A9; 6,M4G2; 16, M4A9. B) Lanes 1, 11 λ DNA/Hind III Fragments molecular weight2, M11; 3, M19; 4, M110; 5, M29; 6, M710; 7, M77; 8, M7D2; 9, M7D9; 10,2D6; 20, M4A7.

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2.9. Methanethiol formation

Methanethiol production from L-methionine was detected asdescribed by Sharpe et al. (1977). Bacteria grown on M17 agarwere suspended in 0.05 M—Tris/HCl buffer, pH 8.0 and 1 mlsamples were incubated in rubber-stoppered test tubes with12.5 mM L-methionine and 0.25 mM 5,5′-dithiobis (2-nitrobenzoic acid) (DNTB) in a total volume of 5 ml, for 2 hat 30 °C. Controls consisting of either cell suspensions or L-methionine alone were assayed in parallel. Methanethiolproduction was indicated by the solution turning yellow.

3. Results and discussion

3.1. Genotypic diversity

RAPD-PCR, plasmid analysis and PFGE were used toexplore the genetic diversity of 40 lactococcal strains fromBatzos cheese. All strains were phenotypically characterized asL. lactis subsp. lactis (Psoni et al., 2003). None of the isolateswas able to hydrolyze citrate, as suggested by their negativeVoges Proskauer test (Psoni et al., 2003), and therefore, it wasassumed that no L. lactis subsp. lactis biovar diacetylactis waspresent in the cheese.

The different banding patterns obtained by RAPD-PCR areshown in Fig. 1. The lowest similarity level obtained byrepeated RAPD analysis of the same strain was 87.6%, henceonly clusters of isolates with a correlation coefficient below

Fig. 3. PFGE patterns of Sma I digests of genomic DNA of Lactococcu

87.6% were considered different (data not shown). Clusteranalysis of the banding patterns revealed two major clusters, Aand B, with 44.6% similarity. Cluster A grouped ninelactococcal isolates and the reference strain L. lactis subsp.cremoris NCTC 1259 at a similarity of 48.1%. Cluster Bcomprised 28 isolates and the reference strain L. lactis subsp.lactis LMG 6890 at a similarity level of 50.9%. Thus, althoughthe use of RAPD method for species assignation of lactococcihas previously revealed an excellent agreement with phenotypicgroups based on classical biochemical tests (Corroler et al.,1998; Tailliez et al., 1998; Mannu et al., 2000; Mannu and Paba,2002), our results support the idea that the RAPD method isgenerally not appropriate for identification (Bouton et al.,2002). RAPD-PCR can not detect minor changes in thechromosome and amplification bands are random and stronglydependent from the conditions of the method and the primerused (Farber, 1996). In cluster B, four groups of isolates weredistinguished with similarities ranging from 67.0 to 86.1%,while two strains (M4A7 and M7D9) formed unique patterns.There were also two minor clusters C and D of two isolateseach. Cluster C grouped two isolates which did not cluster withany reference strain. Strain M1D2 formed cluster D with the L.raffinolactis DSM 20016 reference strain at a similarity of53.3%. Groups A1, B1, B2 and B3 contained subgroups ofisolates with over 87.6% similarity, which probably corre-sponded to isolates of the same strain. These subgroups werecomposed of isolates (i) from cheese made in the same season atdifferent times of ripening (group A1, B1); (ii) from cheese of

s isolates from Batzos cheese. Test strains are explained in Fig. 1.

Table 1Acidification ability of 40 lactococci isolates from Batzos cheese throughoutripening during the whole lactation season

Table 2Hydrolysis of αs- and β-caseins by Lactococcus isolates

Strains 6 h 16 h 24 h

β- αs- β- αs- β- αs-

M11 4.14 27.06 36.52 54.43 36.52 54.43M110 18.91 44.51 28.64 48.74 28.64 53.42M13 25.84 29.91 27.21 42.37 27.21 42.37M15 21.05 28.82 21.05 29.43 44.33 29.72M17 17.51 9.38 32.61 15.93 40.01 31.69M19 12.16 22.38 22.79 29.24 34.06 36.16M28 9.99 53.64 27.70 57.81 39.46 72.95M29 13.55 66.36 13.55 66.36 35.28 74.27M1D1 16.37 36.04 33.40 56.42 71.16 65.33M1D2 75.64 46.75 97.60 87.87 100.00 100.00M1D3 58.40 67.14 94.76 91.56 97.82 96.31M1D7 68.62 94.73 71.28 97.36 91.44 97.36M2D6 6.90 31.32 17.26 39.93 65.80 69.04M1A4 65.34 96.49 75.56 97.36 89.57 98.24M1A6 36.99 49.77 45.00 57.95 45.91 59.30M1A8 31.44 32.22 38.11 45.73 40.54 52.08M1A9 35.25 42.35 40.59 42.72 41.91 53.31M1B4 40.94 70.88 47.35 71.88 58.12 72.86M1B9 45.14 63.87 58.91 81.40 67.20 96.72M1G3 74.06 99.80 83.14 100.00 83.27 100.00M43 25.32 50.63 42.01 63.41 45.67 68.74M49 17.50 39.96 25.67 43.98 37.28 72.36M4A7 23.79 47.46 28.24 47.46 34.87 87.46M4A9 45.43 69.95 55.84 69.95 69.93 87.67M4G2 47.20 55.80 50.23 56.39 70.88 78.90M71 26.68 68.99 46.70 82.57 50.80 97.94M710 31.42 60.87 39.49 69.45 49.81 82.98M77 20.50 60.55 20.72 63.82 40.61 78.24M78 21.04 56.76 53.90 77.57 75.86 87.97M7A2 35.86 53.99 35.86 58.16 45.50 85.78M7A3 22.20 42.90 27.58 8.68 42.32 66.67M7B7 0.00 8.36 10.13 71.74 17.16 71.74M7B8 23.97 45.73 29.55 44.67 37.06 98.55M7B9 5.09 30.31 6.01 30.31 36.40 57.44M7D2 28.73 59.04 29.89 66.55 35.21 66.55M7D3 23.11 13.89 99.31 98.32 100.00 100.00M7D4 11.16 15.49 38.56 50.89 38.61 79.52M7D9 39.25 67.80 46.00 83.01 72.56 100.00M7G1 7.06 25.24 29.55 42.04 66.57 58.66M7G5 24.64 68.29 24.64 68.29 26.31 69.79

The extent of hydrolysis (% reduction) was assessed via the ratio of intensities ofthe corresponding bands at 6 h, 16 h and 24 h to those of the initial time.

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the same season and the same time of ripening (group B3) or(iii) from cheese of different seasons and different times ofripening (group B2). Thus, 24 genotypes of lactococci in totalwere discriminated by RAPD-PCR. There were isolates of thesame strain found several times in the ripening cheese (forexample M1D7, M1A6, M1A9, M1A8, M1A4, M1B4 of groupA1 from 12-, 30- and 60-day old cheese) and others that werefound in the fresh cheese and they were not isolated again (forexample M13, M15, M17, M11 of B3 group of strains).

All 40 isolates were subjected to plasmid content analysisand the electrophoretic profiles were visually inspected andcompared. Plasmid profile analysis was not sufficient for typingall the isolates (Mannu et al., 2000). In eight isolates no plasmidwas detected by the isolation and separation conditionsemployed. Isolates displaying completely different plasmidprofiles were considered as different genotypes. Thus, nine outof the 32 isolates were found to have unique plasmid profile

patterns. The remaining 23 isolates were subdivided into sixgroups (Fig. 2), each containing isolates that shared the sameplasmid pattern. Strain M71 had at least four common plasmidswith strains M13, M15 and M17 and could be considered asderivative of the same parental strain. In all, 16 different strainsof lactococci were detected. It is well known that somebiotechnological properties of lactic acid bacteria, such asacidifying and proteolytic activities, are often plasmid mediated(Efstathiou and McKay, 1976; Morelli et al., 1986) and can beeasily lost.

The phenotypic identification of the 40 L. lactis subsp. lactisisolates was confirmed by PFGE profiling results for all but oneisolate. The PFGE analysis of the chromosomal macrorestric-tion patterns generated by Sma I digestion of the total bacterialDNA grouped 39 out of the 40 isolates and the L. lactis subsp.lactis LMG 6890 reference strain into one large cluster at a

Fig. 4. Aminopeptidase activities of whole cells of Lactococcus isolates from Batzos cheese obtained throughout ripening during the whole lactation season. Means oftwo trials (x±SD). Test strains are explained in Fig. 1.

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similarity level of 48.8% (Fig. 3). Strain M49 was grouped withthe L. raffinolactis DSM 20016 reference strain (similarity66.7%). Most of the L. lactis subsp. lactis groups obtained withPFGE did not fully overlap those obtained by RAPD techniqueexcept for the group of strains lacking plasmids (Bouton et al.,2002). For example, group A1 of RAPD-PCR contained eightstrains from cheese made in winter, seven of which constitutedPFGE subgroup Ib. Strain M1A4 of RAPD-PCR group A1 wasrelated to strain M1D2 according to PFGE results, but the latterstrain was related to L. raffinolactis by RAPD-PCR. Thedifferent exploration of DNA polymorphism by PFGE andRAPD-PCR could explain these discrepancies. PFGE is basedon restriction enzyme polymorphism and analyzes the wholechromosome, while RAPD analyzes the sequence and extent ofpolymorphism within regions amplified by primers.

Pulsed-field gel electrophoresis, however, confirmed theplasmid analysis results except for three isolates (M7D2,M1D1, M1D3). Isolates that lacked plasmids were only typedby PFGE. All these isolates were grouped together at asimilarity level of 75.3% (subgroup IIa). The strains (M1D2,M1A4, M28, M4A7, M4A9, M2D6 and M49) with uniqueplasmid patterns also formed unique PFGE patterns. The sixgroups of isolates formed by plasmid profile analysis were alsodistinguished as separate subgroups according with PFGE atsimilarities of 70.8% (subgroup Ib), 94.7% (subgroup Ic),84.6% (subgroup IIb), 88.3% (subgroup IId), 94.8% (subgroupIIe), and 75.8% (subgroup IIf). Strains M1D1 and M1D3 (100%similarity), each with a unique plasmid profile, formedsubgroup IIc. Strain M7D2, which shared the same plasmidprofile with the isolates M77, M78 and M7D9, formed a uniquePFGE macrorestriction pattern. The strains grouped in sub-

groups Ia, Ib, IIc and IIe were isolates from cheese made inwinter; subgroups Ic, IIa and IId contained isolates from cheesemade in summer; subgroup IIb contained isolates from springcheese; and IIf comprised isolates from winter and summer.Isolates obtained from cheese made in winter and spring wereclustered in group III. Isolates with identical PFGE patternswere regarded as being the same strain, while isolates havingPFGE patterns differing only in one band were assigned todifferent closely related types (Miranda et al., 1991; Mannu andPaba, 2002). In this respect, PFGE divided the 40 lactococcalisolates into 32 genotypes. Our results therefore confirmedPFGE as a highly discriminant strain typing technique (Giraffaet al., 2004).

In addition, these findings suggest that a high geneticheterogeneity exists within the Lactococcus isolates (Corroleret al., 1999; Mannu et al., 2000) obtained from the same cheesethroughout the whole lactation season. Furthermore, asestablished by RAPD-PCR and PFGE profiles, reference strainshad little relation with those studied in this work, indicating thatthey were clearly distinct (Corroler et al., 1998). It seems thatthe test strains have developed their own individual eco-systemand the isolation of novel strains is possible (Cogan et al.,1997). According to both, RAPD-PCR and PFGE results, thereare groups of strains highly correlated with the period ofisolation, found several times during ripening of Batzos cheese,as brine concentration is increased till up to ∼10% (Psoni et al.,2003). This suggests that these wild autochthonous strains werevery resistant to the environmental constraints in the maturingcheese (Mannu and Paba, 2002). This is considered as animportant factor in the protection of traditional raw milk cheesesand particularly the PDO ones (Bouton and Grappin, 1995;

Fig. 5. Proteolytic activities of Lactococcus isolates from Batzos cheese obtained throughout ripening during the whole lactation season (o-PA method; means of twotrials). Test strains are explained in Fig. 1.

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Corroler et al., 1998). Their typical characteristics are possiblygreatly related to the non-starter lactic acid bacteria grown tohigh levels throughout ripening (Psoni et al., 2003).

3.2. Phenotypic diversity

The evaluated rate as well as the intensity of acidificationpermitted us to estimate the acidifying ability of the test strains(Table 1). Two out of the 40 lactococcal isolates lowered themilk pH by more than 1.25 pH units after 6 h, which suggestsfast acidification ability (Cogan et al., 1997). The overallproportion of fast isolates was 5.0% similar to 8.2% reported byCogan et al. (1997) for isolates from raw milk and artisanaldairy products and lower than the 18.41% found by Gaya et al.(1999) for isolates from cheese. Mannu et al. (2000) alsodemonstrated the presence of a few rapid acid-producinglactococci in traditional Pecorino Sardo cheese. Concerning theintensity of acidification, i.e. the ability to reduce the pH of themilk at 24 h (Bouton et al., 2002), three main groups of strainswere observed: (I) high acidifying isolates showing a pHdecrease over 2 pH units (2.13 – 2.40; 42.5% of the isolates);(II) the group of medium acidifying activity, showing a pH dropranging between 1.5 and 2.0 pH units (1.55–1.98; 32.5% of theisolates); (III) low acidifying isolates (25% of the isolates)causing a pH decrease lower than 1.5 pH units (0.60–1.49).Groups I and III contained isolates obtained throughout thewhole lactation season. In group II of medium acidifying ability,isolates from winter and summer cheese were allocated. Thevariability in the acidifying activity indicates that this feature isstrain dependent (Sanchez et al., 2000). It is also noteworthy,that the majority of the high acidifying isolates were derivedfrom the fresh cheese. It is therefore tempting to speculate that

these isolates act as starters to drive the initial acidification ofthe curd (Mannu et al., 2000).

With respect to proteolytic and peptidase activities, biotypescharacterized by a very different casein breakdown as well aspeptidase activities were observed. Peptidase activity may berelated to the cell wall or to the outer surface of the cellmembrane or to a possible intracellular enzyme leakage(Requena et al., 1991). Another possibility might be the transferof oligopeptides into the cell and their degradation byintracellular aminopeptidases (Juillard et al., 1995). The valuesfor aminopeptidase activities of whole cells of the test strains forthree peptide derivatives (Fig. 4) suggest that the majority(75%) of the isolates from cheese made in winter exhibited lys-N leu-Npro-aminopeptidase activity. Perez et al. (2003) alsodetermined high lys- and low pro-aminopeptidase activity forisolates from cheese made in winter. On the other hand, leu-Nlys-aminopeptidase activity was determined for ∼67% of theisolates from cheese made in summer, which at the same timeexhibited, in general, higher pro-aminopeptidase activity thanthe isolates from cheese made in winter (mean activities: 1.14and 0.46 U, respectively). This property might be important forthe degradation of β-casein which is reach in proline (Broomeand Hickey, 1991).

As shown in Table 2, the majority (92.5%) of the isolatesdegraded, prefentially, αs-casein as also observed for other NSLABfrom traditional Greek cheeses (Mama et al., 2002; Bintsis et al.,2003; Psoni et al., 2006). After 24 h of incubationmore than 90% ofthe αs- and/or β-casein was degraded by 25% and 10% of theisolates, respectively. The products of casein degradation are freeamino acids, which support the growth of lactococci (Juillard et al.,1995) as well as low- and high-molecular weight peptides. Thelevels of amino groups accumulated in the milk increased from 6 to

219L. Psoni et al. / International Journal of Food Microbiology 114 (2007) 211–220

24 h for the 47.6% of the isolates, while 20.0% of them graduallydecreased the amounts accumulated at 6 h (Fig. 5). The behaviour of20% of the isolates was to increase the amino group level from 6 to16 h and then degraded them. It is now accepted that free aminoacids released in cheese are not solely responsible for the typicalcheese flavour, mainly because their levels are lower than their tastethreshold values with the exception of proline, which might thusplay a direct role in the taste of cheese (Kubickova andGrosh, 1998).

There were several patterns of antibacterial activities againsttarget strains (data not shown). L. lactis subsp. lactis LMG6890 was not inhibited by any strain. All strains affectedadversely the growth of E. coli O157:H7 and for the majority(87.5%) of them a complete inhibition (no growth of the targetstrain) was noted. Associative growth studies also showed (datanot shown) that when E. coli is grown in the presence of L.lactis strain M7D2 its growth is lower by 1 and 2 log10 cfu/g at8 h (pH 5.2) and 24 h (pH 4.38), respectively, as also observedby Elotmani et al. (2002) in a similar study. In addition, most(85%) of the strains exhibited antagonistic activity against Y.enterocolitica and for some of them an antibacterial activityagainst S. aureus (40% of the isolates), B. cereus (32.5%), L.monocytogenes (20%) and E. faecalis (5%) was noted. Thesefindings suggest that wild lactococci isolates from artisanaldairy products may exhibit antimicrobial activity (Ayad et al.,2002) and inhibit undesirable bacteria as well as bacteriapathogenic to humans (Maisnier-Patin et al., 1992).

The 90% of the test strains formed methanethiol frommethionine at various levels depending on the strain, assuggested by the intensity of the colour developed under theexperimental conditions (data not shown) and may contribute tothe cheese flavour (Engels and Visser, 1994). Cystathionine β-lyase, an enzyme that catalyses the conversion of methionine tomethanethiol, ammonia and α-ketoglutarate was purified fromlactococci and has been implicated in the generation ofmethanethiol in Cheddar cheese (Alting et al., 1995).

4. Conclusions

In conclusion, a wide genotypic and phenotypic heterogeneitywithin L. lactis subsp. lactis strains isolated from Batzos cheesethroughout ripening during the whole lactation season wasobserved. The genotypic analysis was effective in discriminatingstrains according to the season. In addition, our results suggestthat in natural dairy microbial populations, only a few strains withgood acidifying activity are probably present (Mannu et al., 2000;Cogan et al., 1997). Strain similarities and differences in theacidifying and proteolytic activities were determined indicatingthat the wild strains present in the cheese during ripening maycontribute to developing typical flavour in the traditional cheesethrough enzyme complexes (Desmasures et al., 1998; Mannuet al., 2000). Wild strains with antibacterial properties used asstarters, may control the growth of undesirable microorganisms.These strains could be used together with others with flavourforming capability in an appropriate starter for cheese manufac-ture. The analysis of bacterial populations of traditional raw milkcheeses may thus be a useful means of obtaining atypical isolateswith unusual properties, which can contribute to the quality and

the development of typical cheese taste and flavour (Mannu et al.,2000).

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