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Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures
Microbiol. Res. (2002) 157, 13–17 http://www.urbanfischer.de/journals/microbiolres
Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures D. Samarˇzija1, S. Sikora2, S. Redˇzepovi´c2, N. Antunac1, J. Havranek1 1 2
Dairy Department Department of Microbiology, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia
Accepted: September 14, 2001
Abstract Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity. Key words: artisanal cultures – Lactococcus lactis subsp. cremoris – strain identification – RAPD analysis – PCR
Introduction Lactoccocus lactis subsp. cremoris strains have been used extensively in starter cultures for dairy product fermentations. Directly or indirectly, their metabolic products influence a wide variety of textures, flavours and the aroma of the final products. However, successful fermentation of dairy products depends on the use of the most effective strains and the number of available strains belonging to this subspecies is limited. Consequently, the variability among strains needed for specific fermentations is also limited (Chopin et al. 1976 ; Salama et al. 1995). Therefore, there is a need for Corresponding author: Dubravka Samarˇzija e-mail: [email protected] 0944-5013/02/157/01-013
$15.00/0
more L. lactis subsp. cremoris strains that could be used as starter cultures (Salama et al. 1991, 1993). Artisanal cultures and dairy products are potential sources of novel L. lactis subsp. cremoris strains (Paramte et al. 1997; Cogan et al. 1997; Weerkamp et al. 1996; Ayad et al. 1999). However, as most strains have similar physiological properties, it is very difficult to identify them by classical methods (Godon et al. 1992; Klijn et al. 1995). In recent years, 16S and 23S rRNA targeted probes were used to differentiate between L. lactis subsp. lactis and L. lactis subsp. cremoris (Beltz et al. 1990; Klijn et al. 1991, 1995; Schleifer et al. 1995; Salama et al. 1991, 1993; Weerkamp et al. 1996). However, ribosomal RNA-targeted probes are not suitable for the differentiation at the strain level (Salama et al. 1991; Neilan 1995; Beimfohr et al. 1997; Charteris et al. 1997). Therefore, a rapid and reliable molecular technique is required to differentiate novel isolates of L. lactis subsp. cremoris. Randomly Amplified Polymorphic DNA (RAPD) analysis (Welsh and McClelland 1990; Williams et al. 1990) has been employed to differentiate various bacterial species at the strain level even within the same serotype (Lawrence et al. 1993; Fani et al. 1993; Hansen and Winding 1997; Hilton et al. 1997). The application of RAPD analysis to lactic acid bacteria (LAB) is currently underway. Recently, RAPD has been used for typing of lactobacilli (Johansson et al. 1995; Cocconcelli et al. 1995 ; Cocconcelli et al. 1997 ; Tailliez et al. 1996; Quere et al. 1997; Klein et al. 1998), Leuconostoc spp. (Cibik et al. 2000) as well as to differentiate L. lactis strains (Cancilla et al. 1992; Erlandson and Batt 1997; Desmasures et al. 1998; Gaya et al. 1999). Microbiol. Res. 157 (2002) 1
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Fig. 1. RAPD profiles of L. lactis subsp. cremoris strains generated by using primers P2, P15, P16 and P17. Lanes: 1, reference strain B (NIZO 64B); 2, isolate C1; 3, isolate C6; 4, isolate C8; 5, isolate C9; 6, isolate C10; 7, isolate C11. Molecular weight markers: M, 1kb ladder; m, 20 bp ladder
established some 40 years ago by the late Prof. Dr. D. Sabadoˇs. Those cultures are undefined mixtures of mesophilic lactococci, that were isolated from artisanal dairy products in various part of Croatia. Strains C1, C6, C8, C9, C10 and C11, which had previously been determined to belong to L. lactis subsp. cremoris by classical methods, were chosen for this investigation. A proteolitc variant of L. lactis subsp. cremoris NIZO B64 (B) was used as reference strain. Fig. 2. Dendrogram of indigenous L. lactis subsp. cremoris strains derived from RAPD profiles
The aim of this study was to estimate the efficiency of the RAPD technique for discrimination closely related strains of L. lactis subsp. cremoris isolated from artisanal cultures.
Materials and methods Origin of the bacterial strains. The strains used in this study were derived from the culture collection of the Dairy Department, Faculty of Agriculture, Croatia, 14
Microbiol. Res. 157 (2002) 1
DNA preparation. Total genomic DNAs from all strains were isolated using standard phenol-chloroform-isoamyl alcohol extraction. DNA was ethanol precipitated in the presence of sodium acetate (0.3 mol l–1), washed in 70% ethanol, dried and redissolved in 150 µl TE buffer. Concentration and purity of DNA was estimated spectrophotometrically at 260 and 280 nm. Oligonucleotide primers. Four 10-mer primers (Microsynth, Balgach, Switzerland) were used in this study: P2: 5’GATCGGACGG 3’ (70% guanine + cytosine [G + C content]; P15: 5´CTGGGCACGA 3’ (70% G + C content); P16: 5´TCGCCAGCCA3’ (70% G + C content); P17: 5’CAGACAAGCC 3’ (60% G + C content). Primers P15, P16 and P17 were chosen for their ability to differentiate L. lactis subsp. lactis from L. lac-
tis subsp. cremoris (Erlandson and Batt 1997) while primer P2 was chosen arbitrarily. RAPD analysis. Amplification reactions were performed in a 25 µl reaction volume containing : 20 mmol l–1 Tris-HCl (pH 8,4), 50 mmol l–1 KCl, 2,5 mmol l–1 MgCl2, 200 µmol l–1 each of dATP, dCTP, dGTP and dTTP, 1 µmol l–1 primer, 30 ng genomic DNA and 1,5 U Taq DNA polymerase (Life Technologies, Switzerland). Amplification was performed in a Crocodile II thermocycler-version 1.2 (Appligen Inc; Pleasanton, CA) as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 36 °C for 30 s, and extension of the primer at 72 °C for 60 s. Finally, the mixture was held at 72°C for 7 min to allow complete extension of amplified products. Amplification products were electrophoresed on precast 6% poly(NAT) gels (Elchrom Scientific AG, Cham, Switzerland) for 2,5 h at 7 V cm–1 and at 20°C (Sikora et al. 1997). The gels were stained with ethidium bromide and visualized under u. v. illumination. Molecular size markers of a 1kb DNA ladder (Life Technologies, Switzerland) and 20/100 bp ladder (Gensura Laboratories, CA) were included in all gels. Data analysis. Banding patterns for each isolate, as well as for the reference strain, were recorded in a binary code: 1 (positive) and O (negative). Analysis of the binary scores was performed by using the biostatistical program, NTSYS-pc (Rohlf 1994). A similarity matrix was calculated using the simple matching coefficient and cluster analysis of the matrix data was performed using the unweighted pair-group method arithmetic average (UPGMA).
Results The RAPD patterns of six isolates C1, C6, C8, C9, C10, C11 and the reference strain of L. lactis subsp. cremoris NIZO B64 (B) obtained with four primers are shown in Fig. 1. Each primer resulted in a specific banding profile for all isolates. The number of DNA bands in these profiles ranged from seven to 17. Although all primers have generated many common bands, sufficient numbers of polymorphic bands were obtained to detect diversity among the test strains. Cluster analysis of the banding patterns revealed two tightly clustered groups (Fig. 2). The result was not unexpected as all isolates belong to the same subspecies (Godon et al. 1992). Group I consisted of the reference strain L. lactis subsp. cremoris NIZO B64 (B) and group II consisted of six very similar but not identical isolates C1, C6, C8, C9, C10 and C11. As the isolates were at least 76% similar to the reference strain (B), there was no doubt that they belong to L. lactis subsp. cremoris by genotypic criteria
(Tailliez et al. 1998; Desmasures et al. 1998; Mangin et al. 1999). Within group II it was possible to differentiate isolates C1 and C6 from the others. Although they differ considerably, C1 and C6 isolates are more similar to reference strain L. lactis subsp. cremoris NIZO B64 (B) than C8, C9, C10 and C11 isolates.
Discussion Lactococcus lactis is one of the most important groups of lactic acid bacteria that are used in the dairy industry. L. lactis subsp. cremoris seems to be the most important one, since the number of different strains in industrially prepared starter cultures are very few. On the other hand, the natural source of the L. lactis subsp. cremoris has still not been confirmed (Salama et al. 1995) and is subject of numerous controversies. Therefore, for the dairy industry the isolation of strains with different properties would be very useful. Results from our experiments indicate that some of C8, C9, C10 and C11 isolates obtained from artisanal cultures may possess certain novel characteristics that will be of potential value in dairy fermentations. This hypothesis was based on (i) their age and geographical location, (ii) the lack of mixing through last 40 years and (iii) unchanged cultivation conditions. However, due to population dynamics (Hugenholtz 1986) these organisms are likely to differ significantly from the strains that were originally isolated but it could be believed that enough differences still exist in comparison with commercial strains. The isolates within each mixture of mesophilic lactococci probably have developed their own individual eco-system (Cogan 1995; Cogan et al. 1997), which allowed us to believe that isolation of novel strains is possible. The identification of L. lactis subsp. cremoris strains from artisanal cultures by classical methods was found rather difficult because of the high level of phenotypic variability among isolates. The application of RAPD analysis clearly enabled differentiation among L. lactis at the subspecies level (data not shown). Furthermore, this study confirms that, by using RAPD analysis, it is possible to estimate diversity among L. lactis subsp. cremoris isolates also below the subspecies level. This was in agreement with earlier results (Desmasures et al. 1998; Gaya et al. 1999; Mangin et al. 1999). Erlandson and Batt (1997) found it more difficult to discriminate L. lactis subsp. cremoris strains. On the contrary, our results clearly showed that even very similar isolates (C8, C10 and C11) that might correspond to multiple isolates of the same strain, could be distinguished. Only different strains belonging to the same species give different amplification patterns (Cocconcelli, et al. 1995). However, in order to detect Microbiol. Res. 157 (2002) 1
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diversity among closely related strains and to obtain reproducible results by RAPD analysis primer selection, the magnesium chloride, primer and template concentrations as well as annealing temperature have to be carefully optimized (Wang et al. 1993; Voigt and Wöstemeyer 1995 ; Sikora et al. 1997 ; McEwan et al. 1998). As it is well known that artisanal cultures could be a rich source of strains with interesting properties, the most important characteristics of L. lactis subsp. cremoris strains C1, C6, C8, C9, C10 and C11 have to be further verified.
Acknowledgements The authors thank “Pliva” pharmaceutical company, Zagreb, Croatia for financial support and Dr. Krunoslav Kovacevic for his support and helpful advice during the experimental work.
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McEwan, N. R., Bakht, J., Cecchini, E., Coultart, A., Geri, C., McDonald, F. M., Rahman, N. N. A., Williamson, S. C. (1998): Examination of the pipetting variation between indviduals performing random amplified polymorphic DNA (RAPD) analysis. J. Microbiol. Meth. 22, 213–215. Parente, E., Rota, M. A., Ricciardi, A., Clementi, F. (1997): Characterisation of natural starter cultures used in the manufacture of pasta filata cheese in basilicata (Southern Italy). Int. Dairy J. 7, 775–783. Quere, F., Deschamps, A., Urdaci, M. C. (1997): DNA probe and PCR-specific reaction for Lactobacillus plantarum. J. Appl. Microbiol. 82, 783–790. Rohlf, F. J. (1990): NTSYS-pc numerical taxonomy and multivariate analysis system. Version 1.60. Exester Softer, Setauked, New York. Salama, M. S., Sandine, W. E., Giovannoni, S. J. (1991): Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp. cremoris. Appl. Environ. Microbiol. 57, 1313–1318. Salama, M. S., Sandine, W. E. Giovannoni, S. J. (1993): Isolation of Lactococcus lactis subsp. cremoris from nature by colony hybridization with rRNA probes. Appl. Environ. Microbiol. 59, 3941–3945. Salama, M. S., Musafija-Jeknic, T., Sandine, W. E., Giovannoni, S. J. (1995): An ecological study of lactic acid bacteria: isolation of new strains of Lactococcus including Lactococcus lactis subspecies cremoris. J. Dairy Sci. 78, 1004–1017. Schleifer, K-H., Erhmann, M., Beimfohr, C., Brockmann, E., Ludwig, W., Amann, R. (1995): Application of molecular methods for the classification and identification of lactic acid bacteria. Int. Dairy J. 5, 1081–1094.
Sikora, S., Redˇzepovi´c, S., Peji´c, I., Kozumplik, V. (1997): Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting. J. Appl. 82, 527–531. Tailliez, P., Quénée, P., Chopin, A. (1996) Estimation de la diversity parmi les souches de la collection CNRZ: application de la RAPD á un grupe de lactobacilles. Le Lait 76, 147–158. Tailliez, P., Tremblay, J., Ehrlich, S. D., Chopin, A. (1998): Molecular diversity and relationship whitin Lactococcus lactis, as revealed by Randomly Amplified Polymorphic DNA (RAPD). Syst. Appl. Microbiol. 21, 530–538. Voigt, K., Wöstemeyer, J. (1995) : The combination of Gilbert/Maxam chemical sequencing and dideoxynucleotide chain termination approach facilitates the construction of species specific PCR-primers based on diagnostic RAPD bands. Microbiol. Res. 150, 373–377. Wang, G., Whittam, T. S., Berg, C. M., Berg, D. E. (1993): RAPD (arbitrary primer) PCR is more sensitive than multilocus enzyme electrophoresis for distinguishing related bacterial strains. Nucleic Acids Res. 21, 5930–5933. Weerkamp, A. H., Klijn, N., Neeter, R., Smit, G. (1996): Properties of mesophilic lactic acid bacteria from raw milk and naturaly fermented raw milk products. Neth. Milk Dairy J. 50, 319–332. Welsh, J., McClelland, M. (1990): Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V. (1990) DNA polymorphisms amlified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535.
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Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures D. Samarˇzija1, S. Sikora2, S. Redˇzepovi´c2, N. Antunac1, J. Havranek1 1 2
Dairy Department Department of Microbiology, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia
Accepted: September 14, 2001
Abstract Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity. Key words: artisanal cultures – Lactococcus lactis subsp. cremoris – strain identification – RAPD analysis – PCR
Introduction Lactoccocus lactis subsp. cremoris strains have been used extensively in starter cultures for dairy product fermentations. Directly or indirectly, their metabolic products influence a wide variety of textures, flavours and the aroma of the final products. However, successful fermentation of dairy products depends on the use of the most effective strains and the number of available strains belonging to this subspecies is limited. Consequently, the variability among strains needed for specific fermentations is also limited (Chopin et al. 1976 ; Salama et al. 1995). Therefore, there is a need for Corresponding author: Dubravka Samarˇzija e-mail: [email protected] 0944-5013/02/157/01-013
$15.00/0
more L. lactis subsp. cremoris strains that could be used as starter cultures (Salama et al. 1991, 1993). Artisanal cultures and dairy products are potential sources of novel L. lactis subsp. cremoris strains (Paramte et al. 1997; Cogan et al. 1997; Weerkamp et al. 1996; Ayad et al. 1999). However, as most strains have similar physiological properties, it is very difficult to identify them by classical methods (Godon et al. 1992; Klijn et al. 1995). In recent years, 16S and 23S rRNA targeted probes were used to differentiate between L. lactis subsp. lactis and L. lactis subsp. cremoris (Beltz et al. 1990; Klijn et al. 1991, 1995; Schleifer et al. 1995; Salama et al. 1991, 1993; Weerkamp et al. 1996). However, ribosomal RNA-targeted probes are not suitable for the differentiation at the strain level (Salama et al. 1991; Neilan 1995; Beimfohr et al. 1997; Charteris et al. 1997). Therefore, a rapid and reliable molecular technique is required to differentiate novel isolates of L. lactis subsp. cremoris. Randomly Amplified Polymorphic DNA (RAPD) analysis (Welsh and McClelland 1990; Williams et al. 1990) has been employed to differentiate various bacterial species at the strain level even within the same serotype (Lawrence et al. 1993; Fani et al. 1993; Hansen and Winding 1997; Hilton et al. 1997). The application of RAPD analysis to lactic acid bacteria (LAB) is currently underway. Recently, RAPD has been used for typing of lactobacilli (Johansson et al. 1995; Cocconcelli et al. 1995 ; Cocconcelli et al. 1997 ; Tailliez et al. 1996; Quere et al. 1997; Klein et al. 1998), Leuconostoc spp. (Cibik et al. 2000) as well as to differentiate L. lactis strains (Cancilla et al. 1992; Erlandson and Batt 1997; Desmasures et al. 1998; Gaya et al. 1999). Microbiol. Res. 157 (2002) 1
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Fig. 1. RAPD profiles of L. lactis subsp. cremoris strains generated by using primers P2, P15, P16 and P17. Lanes: 1, reference strain B (NIZO 64B); 2, isolate C1; 3, isolate C6; 4, isolate C8; 5, isolate C9; 6, isolate C10; 7, isolate C11. Molecular weight markers: M, 1kb ladder; m, 20 bp ladder
established some 40 years ago by the late Prof. Dr. D. Sabadoˇs. Those cultures are undefined mixtures of mesophilic lactococci, that were isolated from artisanal dairy products in various part of Croatia. Strains C1, C6, C8, C9, C10 and C11, which had previously been determined to belong to L. lactis subsp. cremoris by classical methods, were chosen for this investigation. A proteolitc variant of L. lactis subsp. cremoris NIZO B64 (B) was used as reference strain. Fig. 2. Dendrogram of indigenous L. lactis subsp. cremoris strains derived from RAPD profiles
The aim of this study was to estimate the efficiency of the RAPD technique for discrimination closely related strains of L. lactis subsp. cremoris isolated from artisanal cultures.
Materials and methods Origin of the bacterial strains. The strains used in this study were derived from the culture collection of the Dairy Department, Faculty of Agriculture, Croatia, 14
Microbiol. Res. 157 (2002) 1
DNA preparation. Total genomic DNAs from all strains were isolated using standard phenol-chloroform-isoamyl alcohol extraction. DNA was ethanol precipitated in the presence of sodium acetate (0.3 mol l–1), washed in 70% ethanol, dried and redissolved in 150 µl TE buffer. Concentration and purity of DNA was estimated spectrophotometrically at 260 and 280 nm. Oligonucleotide primers. Four 10-mer primers (Microsynth, Balgach, Switzerland) were used in this study: P2: 5’GATCGGACGG 3’ (70% guanine + cytosine [G + C content]; P15: 5´CTGGGCACGA 3’ (70% G + C content); P16: 5´TCGCCAGCCA3’ (70% G + C content); P17: 5’CAGACAAGCC 3’ (60% G + C content). Primers P15, P16 and P17 were chosen for their ability to differentiate L. lactis subsp. lactis from L. lac-
tis subsp. cremoris (Erlandson and Batt 1997) while primer P2 was chosen arbitrarily. RAPD analysis. Amplification reactions were performed in a 25 µl reaction volume containing : 20 mmol l–1 Tris-HCl (pH 8,4), 50 mmol l–1 KCl, 2,5 mmol l–1 MgCl2, 200 µmol l–1 each of dATP, dCTP, dGTP and dTTP, 1 µmol l–1 primer, 30 ng genomic DNA and 1,5 U Taq DNA polymerase (Life Technologies, Switzerland). Amplification was performed in a Crocodile II thermocycler-version 1.2 (Appligen Inc; Pleasanton, CA) as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, primer annealing at 36 °C for 30 s, and extension of the primer at 72 °C for 60 s. Finally, the mixture was held at 72°C for 7 min to allow complete extension of amplified products. Amplification products were electrophoresed on precast 6% poly(NAT) gels (Elchrom Scientific AG, Cham, Switzerland) for 2,5 h at 7 V cm–1 and at 20°C (Sikora et al. 1997). The gels were stained with ethidium bromide and visualized under u. v. illumination. Molecular size markers of a 1kb DNA ladder (Life Technologies, Switzerland) and 20/100 bp ladder (Gensura Laboratories, CA) were included in all gels. Data analysis. Banding patterns for each isolate, as well as for the reference strain, were recorded in a binary code: 1 (positive) and O (negative). Analysis of the binary scores was performed by using the biostatistical program, NTSYS-pc (Rohlf 1994). A similarity matrix was calculated using the simple matching coefficient and cluster analysis of the matrix data was performed using the unweighted pair-group method arithmetic average (UPGMA).
Results The RAPD patterns of six isolates C1, C6, C8, C9, C10, C11 and the reference strain of L. lactis subsp. cremoris NIZO B64 (B) obtained with four primers are shown in Fig. 1. Each primer resulted in a specific banding profile for all isolates. The number of DNA bands in these profiles ranged from seven to 17. Although all primers have generated many common bands, sufficient numbers of polymorphic bands were obtained to detect diversity among the test strains. Cluster analysis of the banding patterns revealed two tightly clustered groups (Fig. 2). The result was not unexpected as all isolates belong to the same subspecies (Godon et al. 1992). Group I consisted of the reference strain L. lactis subsp. cremoris NIZO B64 (B) and group II consisted of six very similar but not identical isolates C1, C6, C8, C9, C10 and C11. As the isolates were at least 76% similar to the reference strain (B), there was no doubt that they belong to L. lactis subsp. cremoris by genotypic criteria
(Tailliez et al. 1998; Desmasures et al. 1998; Mangin et al. 1999). Within group II it was possible to differentiate isolates C1 and C6 from the others. Although they differ considerably, C1 and C6 isolates are more similar to reference strain L. lactis subsp. cremoris NIZO B64 (B) than C8, C9, C10 and C11 isolates.
Discussion Lactococcus lactis is one of the most important groups of lactic acid bacteria that are used in the dairy industry. L. lactis subsp. cremoris seems to be the most important one, since the number of different strains in industrially prepared starter cultures are very few. On the other hand, the natural source of the L. lactis subsp. cremoris has still not been confirmed (Salama et al. 1995) and is subject of numerous controversies. Therefore, for the dairy industry the isolation of strains with different properties would be very useful. Results from our experiments indicate that some of C8, C9, C10 and C11 isolates obtained from artisanal cultures may possess certain novel characteristics that will be of potential value in dairy fermentations. This hypothesis was based on (i) their age and geographical location, (ii) the lack of mixing through last 40 years and (iii) unchanged cultivation conditions. However, due to population dynamics (Hugenholtz 1986) these organisms are likely to differ significantly from the strains that were originally isolated but it could be believed that enough differences still exist in comparison with commercial strains. The isolates within each mixture of mesophilic lactococci probably have developed their own individual eco-system (Cogan 1995; Cogan et al. 1997), which allowed us to believe that isolation of novel strains is possible. The identification of L. lactis subsp. cremoris strains from artisanal cultures by classical methods was found rather difficult because of the high level of phenotypic variability among isolates. The application of RAPD analysis clearly enabled differentiation among L. lactis at the subspecies level (data not shown). Furthermore, this study confirms that, by using RAPD analysis, it is possible to estimate diversity among L. lactis subsp. cremoris isolates also below the subspecies level. This was in agreement with earlier results (Desmasures et al. 1998; Gaya et al. 1999; Mangin et al. 1999). Erlandson and Batt (1997) found it more difficult to discriminate L. lactis subsp. cremoris strains. On the contrary, our results clearly showed that even very similar isolates (C8, C10 and C11) that might correspond to multiple isolates of the same strain, could be distinguished. Only different strains belonging to the same species give different amplification patterns (Cocconcelli, et al. 1995). However, in order to detect Microbiol. Res. 157 (2002) 1
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diversity among closely related strains and to obtain reproducible results by RAPD analysis primer selection, the magnesium chloride, primer and template concentrations as well as annealing temperature have to be carefully optimized (Wang et al. 1993; Voigt and Wöstemeyer 1995 ; Sikora et al. 1997 ; McEwan et al. 1998). As it is well known that artisanal cultures could be a rich source of strains with interesting properties, the most important characteristics of L. lactis subsp. cremoris strains C1, C6, C8, C9, C10 and C11 have to be further verified.
Acknowledgements The authors thank “Pliva” pharmaceutical company, Zagreb, Croatia for financial support and Dr. Krunoslav Kovacevic for his support and helpful advice during the experimental work.
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