Identification of non-starter lactic acid bacteriaisolated from Pico cheese

This work aimed at the phenotypic and genotypic identification of lactic acid bacteria (LAB) isolates obtained from Pico cheese, a traditional raw milk Azorean cheese attributed with a protected designation origin (PDO).

Marina F. P. Domingos-Lopes*, Susana Ribeiro, Márcia C. Costa, Maria de Lurdes Enes Dapkevicius, Célia C. G. Silva

CITA-A, Department of Agricultural Sciences, University of the Azores, Angra do Heroísmo, Portugal*marinadomingos@uac.pt

To obtain preliminary information on non-starter LAB involved in ripening of Pico Cheese, a total of 113 LAB stains where isolated from 18 cheeses, obtained from 3 manufacturers, from 3 production batches and 2 ripening days (1 and 21). The LAB isolates were characterized and identified by using a combination of phenotypic (cell morphology, Gram staining, physiological and biochemical tests - API50CH) and genotypic methods (PCR-16S rRNA gene sequencing). Single colonies grown for 12 h in MRS broth at 30°C were used for DNA extraction by MOBIO UltraCleanTM Genomic DNA Isolation Kit. Fragments of 1388 bp of the eubacterial 16S rDNA gene were amplified by polymerase chain reaction (PCR) using the universal primers 46F and 1409R and the PCR product was purified and sequenced by both primers. Subsequently, the sequences were aligned to 16S rRNA gene sequences in the Genebank database using the BLAST algorithm [2]. Only the sequences demonstrating the highest similarity in terms of closest relative species and percent of homology (96% to 99%) were considered to belong to the same species.

Results are presented in Fig. 1–5. Classic, morpho-biochemical methods identified 14 species among the isolates under study, while 16S rRNA gene sequencing showed the presence of 10 species. Both employed methodologies showed that the most prevalent species found in our isolates were E. faecalis, Lb. paracasei and Lb. plantarum, albeit these species were represented as different percentages of the isolates, depending on the identification method. For instance, E. faecalis represented 72% of the identified isolates when 16S rRNA gene sequencing was employed, while it accounted for just 31% of the identified species when biochemical methods were used (Fig. 1). Identification to the species level was not accomplished in 14% of the isolates when classic, morpho-biochemical methods were used and in 9% of the isolates when using 16S rRNA gene sequencing (results not shown). Identification to genus and species level by classic morphological/biochemical methods and by 16S rRNA gene sequencing did not always agree. Most isolates (55%) were assigned to different genera, and only 35% of the isolates were assigned to the same species (Fig. 2).

The identification of LAB isolates from this traditional raw milk cheese is an important step towards the preservation of the autochthonous bacterial population, protecting its typical organoleptic characteristics. Alternatively, the finding of new LAB strains may provide novel cultures for the dairy industry to use as starters, adjuncts, protective or probiotic cultures. Further studies will be needed to fully explore the potential application of these strains

This is the first effort towards the microbiological characterization of Pico Cheese (PDO) by culture-dependent methods. Increasing information on the natural microbial population present in typical foods can help preventing the loss of microbial biodiversity while providing novel bacteria for industrial purposes.

References[1] McSweeney, P.L.H: (2004), Int. J. Dairy. Technol., 57, 127–144[2] Randazzo, C.L., Vaughan, E.E. & Caggia, C. (2006), Int. J. Food Microbiol., 109, 1–8[3] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990), J. Mol. Biol., 215, 403–410

AcknowledgementsThis work is financed by National founds from FCT – “Fundação para a Ciência e Tecnologia”, Project PTDC/AGR-ALI/104385/2008. M. Domingos-Lopes thanks the FRC – “Fundo Regional para a Ciência”, Azores, Portugal for the Ph.D. Grant M3.1.2/F/009/2011.

IntroductionArtisanal, raw milk cheeses such as Pico cheese are interesting ecosystems that harbour a still underexploited wealth of microbial species that may possess relevant technological properties. The metabolic activities of cheese microbiota, in particular lactic acid bacteria (LAB), are at the basis of ripening, a process during which several biochemical phenomena leading to the development of the characteristic sensorial properties take place [1]. Studying LAB biodiversity in Pico cheese may help to gain knowledge on its ripening process and may yield isolates with desirable technological properties for industrial applications. Culture-dependent methods remain important when studying cheese bacterial biodiversity. Morphological, physiological and molecular biology techniques are used to provide a deeper insight into the microbiota of these products [2].

Aims

Results

Methods

Conclusions Significance of study

1 2 3 4 5 6 7 8 9 10 1514131211 16 1917 18

1Kb plus

PCR products~ 1388bp

1500 pb

DNA

1Kb plus1 2 3 4 5 6 7 8 9 10 1514131211

Fig. 1 – Identification of bacterial idolates from Pico cheese by morpho-biochemical and by molecular biology methods

Fig. 2 – Proportions of isolates that were ascribed to the same genus and to the same species by both methods.

Fig.5 – Partial 16S rRNA gene sequences obtained from isolate L2A21M3b with the primer 1409R

Fig. 3 – Confirmation of the DNA quality extracted from 15 of the 111 isolates analyzed: 1- L2B1K6, 2- L2C21E8, 3- L2C21K8, 4- L3A21E8, 5- L3A21R3, 6- L3A21R8, 7- L3B1M3, 8- L3B21M3, 9- L3B21M5, 10- L3B21M7, 11- L3B1E2, 12- L3B21R1, 13- L3B21R7, 14- L3B21K7, 15- L3C1M4. Molecular Marker 1Kb plus.

Fig. 4 – PCR products of19 LAB: 1- L2C21K8; 2- L3A21E8; 3- L3A21R3; 4- L3A21R8; 5- L3B1M3; 6- L3B21M3; 7- L3B21M5; 8- L3B21M7; 9- L3B1E2; 10- L3B21R1; 11- L3B21R7; 12- L3B21K7; 13- L3C1M4; 14- L3C21M7; 15- L3C1E5; 16- L3C1E7; 17- L3C21E6; 18- L3C21E8; 19- L3C21R3; Molecular Marker 1Kb plus