Identification of non-starter lactic

acid bacteria
isolated
from
Pico
cheese
Marina F. P. Domingos-Lopes*, Susana Ribeiro, Mrcia C. Costa, Maria de
Lurdes Enes Dapkevicius, Clia C. G. Silva
CITA-A, Department of Agricultural Sciences, University of the Azores, Angra do
Herosmo, Portugal
*marinadomingos@uac.pt

Introduction

Artisanal, 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

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).

Methods
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 30C were used for DNA extraction by MOBIO UltraClean TM 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. 15. 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
Fig. 2 Proportions of isolates that
not shown). Identification to genus and species level by classic
were ascribed to the same genus
morphological/biochemical methods and by 16S rRNA gene sequencing did not
and to the same species by both
methods.
always
agree.
Most
isolates
(55%)
were
assigned
to
different
genera,
and
only
1Kb
1Kb
plus
11 12 13assigned
14 15
2 3the
6 7 8 9 10
4 5isolates
plus species (Fig. 2).
35%1 of
were
to the same
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Results

DN
A

PCR
products
~ 1388bp

1500
pb

Fig. 1 Identification of bacterial idolates from Pico cheese by morpho-biochemical

and by molecular biology methods

Fig. 3 Confirmation of the DNA quality extracted from 15 of the

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

Conclusions
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
References
cultures.
Further studies will be needed to fully explore the potential
[1] McSweeney, P.L.H: (2004), Int. J. Dairy. Technol., 57, 127144
application
of these
strains
[2] Randazzo, C.L.,
Vaughan,
E.E. & Caggia, C. (2006), Int. J. Food Microbiol., 109,
18
[3] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990), J. Mol.
Biol., 215, 403410

Fig. 4 PCR products of19 LAB: 1- L2C21K8; 2- L3A21E8; 3- L3A21R3;

4- L3A21R8; 5- L3B1M3; 6- L3B21M3; 7- L3B21M5; 8- L3B21M7; 9L3B1E2; 10- L3B21R1; 11- L3B21R7; 12- L3B21K7; 13- L3C1M4; 14L3C21M7; 15- L3C1E5; 16- L3C1E7; 17- L3C21E6; 18- L3C21E8; 19L3C21R3; Molecular Marker 1Kb plus

Fig.5 Partial 16S rRNA gene

sequences obtained
from isolate
L2A21M3b with the primer 1409R

Significance of study
This is the first effort towards the
Cheese (PDO) by culture-dependent
natural microbial population present
loss of microbial biodiversity while
purposes.

Acknowledgements

microbiological characterization of Pico

methods. Increasing information on the
in typical foods can help preventing the
providing novel bacteria for industrial

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