Abstract
The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.
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References
Altschul SF, Madden TL, Schaver AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Aquilanti L, Silvestri G, Zannini E, Osimani A, Santarelli S, Clementi F (2007) Phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy. J Appl Microbiol 103:948–960
Ayad EHE, Verheul A, de Jong C, Wouters JTM, Smith G (1999) Flavour forming abilities and amino acids requirements of Lactococcus lactis strains isolated from artisanal and non-dairy origin. Int Dairy J 9:725–735
Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrilch SD, Sorokin A (2001) The complete genome sequence of lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 11:731–753
Buist G, Kok J, Leenhouts KJ, Dabrowska M, Venema G, Haandrikman AJ (1995) Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muraminidase needed for cell separation. J Bacteriol 177:1554–1563
Cogan TM (1995) Flavor production by dairy starter cultures. J Appl Bacteriol Symp Suppl 79:49S–64S
Corroler D, Mangin I, Desmasures N, Gueguen M (1998) An ecological study of Lactococci isolated from raw milk in the Camembert cheese registered designation of origin area. J Appl Environ Microbiol 64:4729–4735
Godon JJ, Delorme C, Dusko Ehrlich S, Renault P (1992) Divergence of genomic sequences between Lactococcus lactis ssp lactis and Lactococcus lactis ssp cremoris. J Appl Environ Microbiol 58:4045–4047
Ho SA, Hoyle JA, Lewis FA, Seker AD, Cross D, Mapstone NP, Dixon MF, Wyatt JI, Tompkins DS, Taylor GR, Quirke P (1991) Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals. J Clin Microbiol 29:2543–2549
Hugenholtz J, Kleerebezem M, Starrenburg M, Delcour J, de Vos W, Hols P (2000) Lactococcus lactis as a cell factory for high-level diacetyl production. J Appl Environ Microbiol 66:4112–4114
Klijn N, Weerkamp AH, De Vos WM (1995) Detection and characterization of lactose-utilizing Lactococcus species in natural ecosystems. J Appl Environ Microbiol 61:788–792
Mirhosaini M, Nahvi I, Kasra Kermanshahi R, Tavassoli M (2006) Isolation of Bacteriocin producing Lactococcus lactis strain from dairy products. Int J Dairy Sci 1:51–56
Nomura M, Kimoto H, Someya Y, Suzuki I (1999) Novel characteristic for distinguishing Lactococcus lactis ssp. lactis from ssp. cremoris. Int J Systemic Bacteriol 49:163–166
Nomura M, Kobayashi M, Okamoto T (2002) Rapid PCR-based method which can determine both phenotype and genotype of Lactococcus lactis subspecies. J Appl Environ Microbiol 68:2209–2213
Prodelalova J, Spanova A, Rittich B (2005) Application of PCR, rep-PCR and RAPD techniques for typing of Lactococcus lactis strains. Folia Microbiol 50:150–154
Salama MS, Jeknic TM, Sandine WE, Giovannoni SJ (1995) An ecological study of lactic acid bacteria: Isolation of new strains of Lactococcus including Lactococcus lactis ssp cremoris. J Dairy Sci 78:1004–1017
Temmerman R, Huys G, Swings J (2004) Identification of lactic acid bacteria: culture-dependent and culture-independent methods. Trends Food Sci Technol 15:348–359
Ward LJH, Brown JCS, Davey GP (1998) Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequences. FEMS Microbiol Lett 166:15–20
Wouters JTM, Ayad EHE, Hugenholtz J, Smit G (2002) Microbes from raw milk for fermented dairy products. Int Dairy J 12:91–109
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The authors acknowledge the financial support provided by the ICAR-AMAAS (Indian Council of Agricultural Research-Application of Microorganisms in Agriculture and Allied Sectors).
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Khemariya, P., Singh, S., Nath, G. et al. Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris . Ann Microbiol 63, 109–115 (2013). https://doi.org/10.1007/s13213-012-0451-6
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DOI: https://doi.org/10.1007/s13213-012-0451-6