Food Microbiology 63 (2017) 178e190

Contents lists available at ScienceDirect

Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Genetic diversity, safety and technological characterization of lactic

acid bacteria isolated from artisanal Pico cheese
M.F.P. Domingos-Lopes a, C. Stanton b, P.R. Ross c, M.L.E. Dapkevicius a, C.C.G. Silva a, *
a ~o e Tecnologia Agra
Centro de Investigaa ria e do Ambiente dos Aores (CITA-A), Universidade dos Aores, Angra do Herosmo, Portugal
b
Teagasc Moorepark Food Research Centre, Fermoy, Cork, Ireland
c
APC Microbiome Institute, University College Cork, Cork, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: A total of 114 lactic acid bacteria were isolated at one and 21 days of ripening from a traditional raw cow's
Received 3 June 2016 milk cheese without the addition of starter culture, produced by three artisanal cheese-makers in Azores
Received in revised form Island (Pico, Portugal). Identication to species and strain level was accomplished by16S rRNA gene and
18 November 2016
PFGE analysis. Carbohydrate utilization proles were obtained with the relevant API kits. Isolates were
Accepted 20 November 2016
Available online 23 November 2016
evaluated according to safety and technological criteria. The most frequently observed genus identied
by 16S rRNA sequencing analysis was Enterococcus, whereas API system mostly identied Lactobacillus.
The highest percentages of antibiotic resistance were to nalidixic acid (95%), and aminoglycosides (64
Keywords:
Lactic acid bacteria
e87%). All isolates were sensitive to several beta-lactam antibiotics and negative for histamine and
Cheese DNase production. Gelatinase activity was detected in 49.1% of isolates, 43% were able to degrade casein
Artisanal and 93% were a-hemolytic. Most enterococci presented virulence genes, such as gelE, asaI, ace. Diacetyl
Safety production was found to be species dependent and one strain (Leu. citreum) produced exopoly-
Adjunct culture saccharides. Selected strains were further studied for technological application and were found to be
slow acid producers in milk and experimental cheeses, a desirable trait for adjunct cultures. Two strains
were selected on the basis of technological and safety application as adjunct cultures in cheese pro-
duction and presented the best cheese aroma and avor in consumer preference tests. This is the rst
effort to characterize Pico cheese LAB isolates for potential application as adjunct cultures; the results
suggest the potential of two strains to improve the quality of this traditional raw milk product.
2016 Elsevier Ltd. All rights reserved.

1. Introduction Community with the attribution of the Protected Denomination

Due to their unique taste, artisanal cheeses produced through
spontaneous fermentation of unpasteurized milk receive a great
deal of attention from consumers around the world. Pico cheese is
an artisanal semi-hard cow's milk cheese produced in Azores Island
(Pico, Portugal) and is made on small scale using traditional prac-
tices involving the addition of animal rennet and salt to raw milk
and no starter addition. It has a very at cylindrical shape
(16e17 cm wide and 2e3 cm height), a distinct mild avour and a
very short maturation time (21 days). Pico cheese has its place in
the niche products that have been recognized by the European
* Corresponding author. Centro de Investiga~ ao e Tecnologia Agr aria e do
Ambiente dos Aores (CITA-A), Departamento de Cie ^ncias Agra
rias, Universidade
dos Aores, Rua Capita ~o Joa 
~o DAvila, 9700-042 Angra do Herosmo, Terceira,
Portugal.
E-mail address: celia.cg.silva@uac.pt (C.C.G. Silva).
Origin status (European Union, 2006).
In the traditional Pico PDO all the phases of processing are
manual. The manufacturing process starts with ltration of raw
cow milk through a cheesecloth and addition of calf rennet. Coag-
ulation is allowed to proceed for 45e60 min at 26e30
C after
which the coagulum is cut, the whey is drained off and the curd is
wrapped and manually pressed from both sides, in order to pro-
duce a smooth appearance. Cheese salting is done by adding salt to
each face of the cheese. On the following day after manufacture,
cheeses are placed in cold storage at 10e14
C and relative hu-
midity of 80e85%, for ripening during 20 days.
The lactic acid bacteria (LAB) belong to a large family of
fermentative Gram-positive, non-sporulating, micro-aerophilic
bacteria that produce lactic acid as the main fermentation prod-
uct from glucose, and they occur naturally as indigenous microbiota
in raw milk. Their presence contributes to the large differences in
the organoleptic, biochemical and avour characteristics of

http://dx.doi.org/10.1016/j.fm.2016.11.014
0740-0020/ 2016 Elsevier Ltd. All rights reserved.
 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
artisanal dairy products (Bao et al., 2012; Colombo et al., 2009;
Moraes et al., 2013; Randazzo et al., 2006). There have been many
reports describing the microbiota of different raw milk cheeses
made by artisanal manufacturing (Casalta et al., 2009; Ostlie et al.,
2005; Ouadghiri et al., 2005). Lactobacilli generally dominate the
non-starter lactic acid bacteria (NSLAB) population in most cheeses
(Bouton et al., 1998; Fitzsimons et al., 1999; Swearingen et al.,
2001). However, Enterococcus faecalis was found to be the pre-
dominant species detected in some artisanal cheeses (Franz et al.,
1999; Giraffa et al., 1997; Nieto-Arribas et al., 2011). Other LAB
species, Lactococcus lactis, Enterococcus faecium, Leuconostoc mes-
enteroides subsp. mesenteroides and Leu. mesenteroides subsp. dex-
tranicum, have also been found in the microbiota of artisanal raw
ewe's milk cheeses (Manolopoulou et al., 2003; Nieto-Arribas et al.,
2010; Terzic-Vidojevic et al., 2009).
To analyze and rapidly identify microbial communities, con-
ventional phenotypic, biochemical and physiological tests have
been used on cheese isolates (Fortina et al., 2003; Moraes et al.,
2013). Although these conventional methods have proven to be
useful and an indispensable tool for LAB characterization, they are
not fully reliable due to the similar nutritional and growth re-
quirements of LAB (Colombo et al., 2009; Fortina et al., 2003; Ostlie
et al., 2005). In addition, phenotypic methods are limited in terms
of both discriminating ability and accuracy, so distinguishing
strains always requires DNA-based techniques. Identication by the
16S rDNA gene sequence analyses has proven useful for differen-
tiating a wide range of LAB at species level (Bao et al., 2012;
Colombo et al., 2009; Fortina et al., 2003; Moraes et al., 2013).
Moreover, the combination of phenotypic and molecular methods
has become the preferred approach for determining and analyzing
the species composition of targeted microbial communities
(Kesmen et al., 2012; Ostlie et al., 2005).
Numerous studies have shown that autochthonous microor-
ganisms in fermented foods may improve safety, technological and
sensory properties, and also shelf-life of dairy products (Abriouel
et al., 2008; Bao et al., 2012; Moraes et al., 2013). However, the
demand of the dairy industry for more uniform and standardized
products led to a growing concern for the loss of typical indigenous
microbial populations present in artisanal cheeses. While artisanal
Pico cheese has important organoleptic features that must be
preserved, limited data exist on the composition of this cheese
microbiota (Riquelme et al., 2015).
The microbiological characterization of isolated LAB from Pico
cheese constitutes an important step towards the preservation of
the autochthonous bacterial heritage that provides specic and
unique characteristics to this cheese. In addition, safety assess-
ments with regard to virulence traits and antibiotic resistance
remain essential in the selection of LAB for application in food
production. Therefore, the objective of present study was to iden-
tify and characterize LAB isolated from Pico cheese in order to select
appropriate candidates for use as adjunct cultures. Strains that
were considered safe after this rst screening and exhibited good
technology characteristics were further investigated for acid pro-
duction, growth dynamics in the cheese environment and desirable
sensory characteristics in experimental fresh cheeses.
2. Materials and methods
2.1. Isolation of LAB
A total of 12 artisanal Pico cheeses from two production batches,
three artisanal cheese-makers and two ripening days (1 and 21)
were collected from the processing plants, during the summer
season. Cheese samples (25 g) were diluted in 225 ml of buffered
peptone water (Biokar, Beauvais, France) and homogenized with a
179
Stomacher Lab-Blender 400 (Seward Medical, London, UK). Serial
dilutions in sterile peptone water were prepared and plated in
duplicate on the adequate media for the isolation of ten colonies
from each countable dilution. The media used to obtain pure cul-
tures were as follows: presumptive enterococci - KF Agar (Biokar,
Beauvais, France), aerobic incubation at 43
C, for 48 h; presump-
tive leuconostocs - MSE Agar (Biokar), aerobic incubation at 21
C,
for 4 days; presumptive lactococci - M17 Agar (Biokar), aerobic
incubation at 30
C, 72 h; and presumptive lactobacilli - Rogosa
Agar (Merck), under anaerobic conditions using an AnaeroGen kit
(Oxoid, Milan, Italy), at 30
C, for 5 days.
All the isolates matching the basic traits of the LAB group, non-
spore forming, Gram-positive, catalase and oxidase-negative, were
considered for identication. The isolates were routinely propa-
gated in MRS broth (AES, Bruz, France), under aerobic conditions
and stored at 20
C and 80
C in liquid medium (MRS broth)
supplemented with 30% (vol/vol) glycerol as a cryoprotectant.
2.2. Identication of isolates
2.2.1. Phenotypic identication
Overnight cultures of isolates grown on MRS agar were sub-
mitted to Gram staining and tested for catalase production and
oxidase activity as described by Kozaki et al. (1992). All the isolates
matched to the basic traits of the LAB group, non-spore forming,
Gram-positive, catalase and oxidase-negative were considered for
identication. The phenotypic characteristics of 114 isolates were
studied using the API 50 CHL (75 isolates from MSE, M17 and
Rogosa Agar) and API 20 Strep (39 isolates from KF Agar) kits
(BioMe rieux, Marcy-lEtoile, France), following manufacturer's
instructions.
2.2.2. PCR amplication and sequence of 16S rRNA gene
Single colonies were sub-cultured, under aerobic conditions, for
24 h in MRS broth at 30
C and aliquots of 1 mL were transferred to
microcentrifuge tubes and centrifuged at 14,000 g for 2 min. The
cell pellet was submitted to total genomic DNA extraction using the
UltraClean Microbial DNA Isolation Kit (MoBio, Carlsbad, CA, USA)
according to the supplier's specications, and stored at 20
C.
Fragments of 1363e1388 bp of the eubacterial 16S rRNA sequence
coding region were amplied by polymerase chain reaction (PCR)
using the universal bacterial primers 46F (50 -GCYTAAYA-
CATGCAAGTCG-30 ) and 1409R (50 -GTGACGGGCRGTGTGTRCAA-30 )
(Northup et al., 2010) for LAB isolates and positive control Escher-
ichia coli ATCC 25922. The PCR reaction was performed in a nal
20 ml reaction volume containing 1  Taq Polymerase reaction
buffer, 1.8 mM of MgCl2, 0.25 mM of dNTP (Applied Biosystem,
Foster City, Ca., USA), 0.38 pmol of each primer, 1U Taq DNA poli-
merase (Fermentas, Life Technologies, Waltham, MA) and 25 ng of
DNA template. The amplication program consisted of an initial
denaturing step at 94
C for 3 min followed by 25 cycles of dena-
turation at 94
C for 30 s, annealing at 54
C for 30 s and elongation
at 72
C for 90 s and one cycle of nal extension at 72
C for 10 min,
performed in a thermocycler (TProfessional, Biometra, Germany).
The 16S rDNA amplicons were analyzed by electrophoresis on 1.5%
(wt/vol) agarose gel with 5 ml of SYBR Safe DNA gel stain (Invi-
trogen, Life technologies, USA) at 120 V for 45 min in 1  TAE buffer
(2 mol/L Tris base, 1 mol/L acetic acid, 0.05 mol/L EDTA pH 8.0) and
visualized by UV light. The molecular marker used was Gene
Ruler 1 kb plus DNA Ladder (Fermentas, Life Sciences).
The PCR products were puried by gel ltration and sequenced
with both primers Foward (46F) and Reverse (1409R) on an ABI
PRISM 3730XL Sequencer (Applied Biosystems, Foster City, USA)
by StabVida (Investiga~ ao e Servios em Cie ^ncias Biologicas, Lda,
Portugal).
180 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
2.2.3. Phylogenetic analysis
The partial retrieved 16S rRNA gene sequences were edited and
corrected manually with BioEdit Sequence Alignment Editor 7.1.11
software (www.mbio.ncsu.edu/BioEdit/bioedit.html). The
consensus sequences were obtained from two reads of the 16S rRNA
gene of the isolated strains by the program CAP3 Sequence As-
sembly Program and the identities of the isolates were determined
through the aligned in the GeneBank DNA database using the Basic
Local Alignment Search Tool - BLAST algorithm (http://www.ncbi.
nlm.nih.gov/BLAST/) and the Ribosomal Database Project (RDP)
(http://rdp.cme.msu.edu/) to determine the closest known relative
species on the basis of 16S rRNA gene homology (Altschul et al.,
1990). Only the sequences demonstrating the highest similarity in
terms of closest relative species and 99e100% percent of homology
were considered to belong to the same species.
Nucleotide sequence accession numbers: The 16S rRNA gene se-
quences of isolates have been deposited in the GenBank under
accession numbers KF193420eKF193427, KM079353eKM079361,
KM096813eKM096829, KM103931eKM103934, KM201349e
KM201363, KU311978eKU312038.
2.2.4. Pulse eld gel electrophoresis (PFGE) prole analysis
High-molecular-weight DNA was isolated from 1 ml of over-
night culture of Enterococcus, Leuconostoc and Lactococcus in MRS
broth or in MRS broth containing 20 mM threonine for Lactobacillus
and Leuconostoc citreum (L3C1E7 isolate). PFGE was performed as
describe by Simpson et al. (2002).
Briey, cells were harvested and washed twice with cell sus-
pension buffer (10 mM Tris-HCl, 1 M NaCl, pH 7.6). The pellet was
resuspended in 200 ml of the same buffer, for lactobacilli and Leu.
Citreum, and 300 ml for other isolates. The solution was vigorously
vortexed and the cell suspension was mixed with an equal volume
of 2% (wt/vol) low-melting-point agarose (Bio-Rad Laboratories,
Richmond, California) in 0.125 M EDTA (pH 7.6) and dispensed into
plug molds (agarose blocks with the DNA, Bio-Rad Laboratories,
Richmond, California), 10 mm long  5 mm wide  1 mm deep, and
left to solidify (in duplicates for each isolate) at room temperature.
Cells were lysed in situ, up to three plugs per strain, by gentle
shaking in 1 mL of a 10 mg/ml lysozyme (L-7651; Sigma-Aldrich)
solution in EC buffer (1 M NaCl, 6 mM Tris-HCl, 100 mM EDTA, 1%
(wt/vol) sarkosyl, pH 7.6) for enterococci, leuconostocs and lacto-
bacilli or in 1 ml of a 10 mg/ml lysozyme solution in EC buffer
containing mutanolysin (20 units/ml) for lactobacilli and Leu. Cit-
reum. Cells were incubated overnight at 37
C, then the solution
was replaced with 1 ml of proteinase K solution (P-6556; Sigma-
Aldrich) (0.5 mg/ml in 0.5 M EDTA, 1% sarkosyl, pH 8.0) for over-
night incubation at 55
C with gentle shaking. The lysis step was
repeated twice for lactobacilli and Leu. Citreum. The plugs were
washed twice in 1 ml of 1 mM PMSF (phenylmethylsulfonyl uo-
ride) in TE 10/1 (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) for 1 h at
37
C and then stored in 1 ml TE 10/100 (10 mM Tris-HCl, 100 mM
EDTA) at 4
C until required. For the macro-restriction of the
genomic DNA, a small slice (1 mm long by 2 mm wide) of the plugs
was cut from the agarose blocks (in quadruplicate) with sterile
coverslips and washed three times for 30 min each, at room tem-
perature with gentle shaking in 1 mL TE 10/0.1 (10 mM Tris-HCl,
0.1 mM EDTA, pH 8.0). Each slice was pre-incubated with 100 ml
of 1 restriction buffer 4 (New England Biolabs) for 30 min at 4
C
and then replaced with 100 ml of 1  fresh buffer 4 containing
1  BSA and 4 U of the following restriction enzymes: SmaI (New
England Biolabs, Hitchin, United Kingdom) for enterococci, Leuco-
nostoc and lactobacilli and 2 U of AscI (New England Biolabs,
Hitchin, United Kingdom) for lactobacilli and Leu. Citreum,
following incubation for 24 h at 25
C. After digestion, slices were
loaded into the wells of a 200 ml 1% PFGE pulsed-eld grade
agarose gel (Bio-Rad Laboratories) prepare in 0.5  Tris-borate
buffer (Sigma) and sealed with 1% agarose. The gels were run in
0.5  Tris-borate buffer (Sigma) using a CHEF-DR II PFGE appa-
ratus (Bio-Rad Laboratories) at 1 V (6 V/cm) for 16 h at 14
C with
the pulse ramped from 1 to 20 s. Gels were stained with ethidium
bromide (0.5 mg/mL) for 1 h, then distained in water, visualized
under UV light and photographed using Alpha Imaging System. The
sizes of PFGE fragments were estimated by using a standard
ranging from 2 to 194 kb (low-range PFGE marker, New England
Biolabs).
Conversion, normalization and further analysis of PFGE patterns
were performed using the Dice and UPGMA cluster analysis with
the software BioNumerics program (version 4.0, Applied Maths,
Kortrijk, Belgium). For data analysis, a Pearson's coefcient of
similarity between patterns was computed and used to construct a
UPGMA dendrogram for the 101 LAB isolates, from 2 batches, one
for each species. Isolates that showed similar patterns with an 80%
similarity were considered to be closely related and were thus
grouped as belonging to the same strain (Maluping et al., 2005;
Rodriguez et al., 2006; Tenover et al., 1995).
2.3. Technological characterization
2.3.1. Proteolytic and lipolytic activities
Extracellular proteolytic activity was determined by streaking
LAB suspensions onto the surface of PCA (AES, France) medium
supplemented with 10% (wt/vol) skim milk powder (Oxoid, En-
gland). Isolates were incubated at 30
C and after 72 h plates were
ooded with 1% HCl. Proteolytic activity was indicated by a clear
zone around the colonies (Franciosi et al., 2009).
Lipolytic activity was evaluated by streaking LAB cultures onto
the dried surface plates of Tributyrin Agar (Merck, Germany), with
incubation at 30
C for 72 h. Lipolytic activity was detected by a
clear zone surrounding the growth (Hantsis-Zacharov and Halpern,
2007).
2.3.2. Diacetyl and exopolysaccharide (EPS) production
Diacetyl production was determined according to Ribeiro et al.
(2014). Briey, LAB were inoculated in UHT milk and combined
with 0.5 ml of a-naphthol (1% w/v) (Merck, Germany) and KOH
(16% w/v) and incubated at 30
C for 10 min. Diacetyl production
was indicated by the formation of a red ring at the top of the tubes.
Each determination was performed in duplicate.
Screening for EPS production of the 114 isolates was performed
in MRS and modied MRS agar (in which the glucose present in the
original formulation was replaced by 5 and 10% lactose and sucrose,
on a w/v basis), incubated aerobically at 30
C for Lactococcus,
Leuconostoc and Enterococcus, and anaerobically at 37
C, for con-
trols and Lactobacillus for 72 h. After incubation, possible EPS pro-
duction was assessed based on the presence of a mucoid or ropy
colony using the loop touch test described by London et al. (2014).
In this test we used a positive and negative control for a ropy EPS
production, a b-glucaneproducing Lactobacillus paracasei National
Food Biotechnology Center (NFBC) 338 expressing the glycosyl-
transferase (Gtf) gene from Pediococcus parvulus 2.6 (GTF) - (338
Gtf) as positive control and a noneb-glucan-producing isogenic
control strain Lactobacillus paracasei NFBC 338 (PNZ) - (338 PNZ) as
negative control, from the Teagasc collection.
2.3.3. Enzyme proling
A total of overnight cultures, of 114 isolates, in MRS agar were
further characterized in terms of their enzymatic activity prole
using the API-ZYM system (Biomerieux, Craponne, France).
Suspensions with 5e6 MacFarland turbidity in API Suspension
Medium were prepared and inoculated in the microtubes of the
 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
API-ZYM strip. Incubation and reading were made according to the
manufacturer's instructions. The results were graded in an arbitrary
scale, from 0 (no activity) to 40 (40 or more nmole of hydrolyzed
substrate).
2.3.4. Milk acidication
Selected strains were grown overnight in MRS broth at 30
C
prior to testing. Tubes containing 10 ml of skim milk or UHT milk
were inoculated (1% v/v) with revitalized isolates and incubated at
30
C. The pH was measured at 0, 2, 4, 6, 12, 24 and 48 h using a pH
meter (WTW Inolab pH Level 1, Germany). All experiments were
performed in triplicate.
2.4. Safety evaluation
2.4.1. Haemolysis
For evaluation of haemolytic activity, Tryptose Blood Agar
(Merck, Germany) plates enriched with sheep blood were exam-
ined for signs of b-haemolysis (clear zones around colonies), a-
haemolysis (green zones around colonies) or g-haemolysis (no
zones around colonies) (Asteri et al., 2009).
2.4.2. Histamine, DNase and gelatinase production
Isolates were tested for their ability to produce histamine using
a differential medium according to Joosten and Northolt (1989).
Colonies surrounded by a purple halo were considered positive for
histidine decarboxylation.
For detection of Dnase, bacterial isolates were cultured in
duplicate on Dnase agar (Sigma-Aldrich, EUA) at 37
C for 48 h.
Appearance of clear pink zones around the colonies, after ooding
the plates with 1N HCl, was considered to be a positive indication of
Dnase production (Gupta and Malik, 2007).
Gelatinase activity was tested using gelatine agar plates con-
taining 30 g l1 gelatin (Biolife, Italy), 5 g l1 peptone (Fluka, EUA),
3 g l1 yeast extract (AES, France) and 17 g l1 agar (AES, France), at
37
C for 48 h. After ooding the plates with saturated ammonium
sulphate (Merck, Germany), the presence of a clear zone around the
colonies was considered to be a positive indication of gelatinase
production (Terzic-Vidojevic et al., 2009). Duplicates were prepared
for each isolate under study.
2.4.3. Susceptibility to antibiotics
The 114 isolates identied were tested for resistance to antibi-
otics by the disc diffusion method, according to Fortina et al. (2008).
Antibiotic discs (Oxoid, England) were used to determine the sus-
ceptibility of the strains to 22 antibiotics: amoxicillin/clavulanic
acid (20/10 mg/disc), chloramphenicol (30 mg/disc), carbenicillin
(100 mg/disc), ceftazidime (30 mg/disc), gentamicin (10 mg/disc),
ceftriaxone (30 mg/disc), cefotaxime (30 mg/disc), clindamycin
(2 mg/disc), erythromycin (15 mg/disc), kanamycin (30 mg/disc),
cephalotin (30 mg/disc), nalidixic acid (30 mg/disc), netilmicin
(30 mg/disc), ooxacin (5 mg/disc), penicillin (10 IU/disc), piper-
acillin (100 mg/disc), rifampicin (5 mg/disc), streptomycin (10 mg/
disc), trimethoprim/sulfamethoxazole (1.25/23.75 mg/disc), tetra-
cycline (30 mg/disc), tobramycin (10 mg/disc) and vancomycin
(30 mg/disc). Analyses were done in duplicate. The discs were
placed onto the surface of inoculated Mueller-Hinton (AES, France)
agar plates seeded with LAB strains that had been previously grown
in MRS broth for 24 h - 48 h at 30
C. After incubation at 30
C for
24 h, the diameter of inhibition halos around the disks was
measured with a digital calliper (Absolute Digimatic Caliper,
Mitutoyo, USA), to assess the susceptibility or resistance of the
examined isolates. Each isolate was classied as sensitive (S), in-
termediate (I) or resistant (R) according to the inhibition zone di-
ameters in agreement with the Clinical and Laboratory Standards
181
Institute tables (CLSI, 2014).
2.4.4. Presence of genes encoding virulence potential
The strains were tested for the presence of virulence genes: gelE
(gelatinase), asa1 (aggregation substance), esp (enterococcal sur-
face protein), cylA (cytolisin), efaA (endocarditis antigen), ace
(adhesion of collagen), antibiotic resistance genes: vanA and vanB
(both related to vancomycin resistance), and genes for amino acid
decarboxylases: hdc (histidine decarboxylase), as reported previ-
ously by Martin-Platero et al. (2009), Rivas et al. (2005) and
Vankerckhoven et al. (2004). The amplied products were sepa-
rated by electrophoresis on 2% (w/v) agarose gels in 1  TAE buffer.
Primers and fragment sizes were used as detailed previously
(Ribeiro et al., 2014). Genomic DNA from two cheese E. faecalis
isolates (E. faecalis L2B21K3, E. faecalis L3A21M8, Ribeiro et al.,
2014) and food E. faecium isolates (E. faecium C1434, E. faecium
C1231, Lopez et al., 2009) were used as positive controls in the
corresponding PCR reactions.
2.5. Application of selected strains to cheese making
2.5.1. Manufacture of fresh cheese
Cow's raw milk (3.5% fat, wt/wt) obtained from the Azores
University farm (Chegalvorada, Angra do Heroismo, Portugal) was
pasteurized at 73
C for 16 s in a shaking water bath (Julabo, Model
SW22, Germany) and then cooled in wet ice. Calcium chloride
(0.2 g/L; Merck) and NaCl (10 g/L) were then added to the milk. In
each trial, milk warmed at 32
C was distributed in ve 0.5 L vats
and individually inoculated with each LAB culture (1%). Rennet
(LMF 1/15,000, 0.2 g/L) was then added to the milk and incubated at
32
C for aprox. 40 min. Control cheese was made without any
inoculum. Once the coagulum was sufciently rm, it was cut into
1e2 cm cubes and heated at 37
C for 25 min. Whey was drained off
and curds were distributed into perforated sterile stain steel cir-
cular cheese containers (6.5 cm in diameter). Cheeses were stored
under refrigeration (4
C) inside appropriated plastic boxes with a
mesh covered with sterilized cheese cloth allowing whey drainage.
2.5.2. Analysis of the experimental fresh cheeses
Cheeses were sampled in duplicate (two different cheeses of the
same trial) for pH, titratable acidity and LAB counts in the begin-
ning of storage (time 0) and after 0, 6, 12, 24, 48 and 72 h of
storage at 4
C.
Cheese pH was measured directly with a pH meter (WTW Inolab
pH Level 1, Germany). Titratable acidity was determined by direct
titration of 4 g of fresh cheese dissolved in warm water, according to
AOAC method # 920.124 (AOAC, 1995).
For microbiological analyses, 10 g of cheese was transferred into
a stomacher (400 Circulator, Seward, United Kingdom) containing
90 mL of sterile 0.1% (wt/vol) buffered peptone water (AES). Further
dilutions were made from this original dilution and the quanti-
cation of microbial counts was carried out using the pour plate
technique. The lactobacilli were enumerated on MRS agar (Biokar)
and incubated under aerobic conditions at 30
C for 72 h, whereas
the Leuconostoc strain was enumerated on - MSE Agar (Biokar),
with aerobic incubation at 21
C, for 4 days. Three independent
experiments were performed and each experiment was conducted
in duplicate.
2.5.3. Sensory analysis
To evaluate the inuence of the selected strains in the nal
sensorial characteristics of fresh cheese, cheeses made with
selected strains were subjected to a panel evaluation.
Sensory analysis was performed on fresh (2 days old) cheese by
a non-trained panel of tasters comprising 70 participants from both
182 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
genders (33 male and 37 female), with ages ranging from 18 to 66
years old. The attributes judged were acidity, salty taste, rmness,
aroma and general acceptability. Cheese scoring was conducted on
a one to ve scale (in which 1 stands for absence and 5 for presence
at a strong level). General acceptability was assessed by the same 5-
point scale explaining how much the panellist liked a product,
ranging from 1-totally dislike to 5-like very much. Prior to
assessment, each cheese was divided into various portions and
equilibrated at room temperature. Panellists were exposed to each
sample on an individual plastic dish, and were asked to assess the
specic attributes.
2.6. Statistical analysis
Hierarchical cluster analysis and principal component analysis
were used to group the strains according to their different prop-
erties. Principal component analysis was done to group isolates
based on carbohydrate fermentation prole from API50 CHL and
API20 STREP, and enzyme prole from API-ZYM system. A matrix
was generated based on results of the API reading after 48 h
(negative and positive). Carbohydrates that were not fermented by
all isolates, or were fermented by one or two isolates were not
included in the matrix.
Cheese pH and titratable acidity was analyzed using ANOVA
repeated measures, with strain the between subject factor and time
as the within subject variable. When differences were statistically
signicant (P < 0.05), post hoc multiple comparisons were deter-
mined by the PLSD test. Values from each trial were determined
from means of duplicate data. Each cheese trial was repeated three
times and the nal results represent the average of each set of
experiments. Sensory evaluations were statistically analyzed using
non-parametric Friedman test. Post hoc multiple comparisons were
determined by Wilcoxon signed-rank tests conducted with a Bon-
ferroni correction. Differences were considered statistically signif-
icant at P < 0.05.
Statistical analyses were performed with SPSS software package
(IBM SPSS Statistics 20, IBM Corporation, New York, USA).
3. Results and discussion
3.1. Phylogenetic analysis
3.1.1. Genotypic characterization of isolates
On basis of phylogenetical analysis, 114 isolates were success-
fully identied at the species level, with more than 99% similarity
and belonged to four genera (Enterococcus, Lactobacillus, Lacto-
coccus and Leuconostoc). The Enterococcus genus was dominant in
samples from all cheese-makers at the two ripening days (1 and
21). These results differ considerably from a recent study applying
high-throughput DNA sequencing for identication the bacterial
population of Pico cheese (Riquelme et al., 2015). Indeed, this study
revealed that Lactococcus was the predominant genus detected in
Pico cheese microbiota, while the percentage of recovered reads
from Enterococcus genus was very low (average of 2%). This
discrepancy of results may indicate a bias of the LAB isolation
method, since Enterococcus spp. were isolated from all media used
in our study (KF, MSE, M17 and Rogosa Agar). Some selectivity was,
however, found for other genera: Lactococcus was only isolated
from M17 Agar, Lactobacillus was predominant in Rogosa Agar
(54.55%), and Leuconostoc had a higher proportion of isolates in
MSE agar (60%); enterococci isolates were most predominant in KF
(39.3%), M17 (28.6%) and MSE (22.6%) media. Similar studies have
showed that enterococci represent a considerable proportion of
isolates in traditional cheeses (Franz et al., 1999; Giraffa et al., 1997;
Losio et al., 2015; Nieto-Arribas et al., 2011). Enterococci are
recognized as an essential part of the natural microbiota of many
dairy products. In addition, the highest recovery of enterococci in
some cheeses also corroborates the difculties of growing and se-
lection of lactobacilli and lactococci (Nikolic et al., 2008; Terzic-
Vidojevic et al., 2007). Moreover, some enterococci are bacte-
riocin producers resulting in a better chance to compete and
outweigh other species (Ribeiro et al., 2014).
The results from 16S-rRNA gene sequence indicated that
E. faecalis was the dominant species among isolates. From a total of
114 isolates, 73.7% belonged to the Enterococcus genus: 81
E. faecalis, one E. italicus and two E. pseudoavium. Twenty-two
isolates (19.3%) were identied in the Lactobacillus genus: 15 Lb.
paracasei subsp paracasei, ve Lb. plantarum, one Lb. paraplantarum
and one Lb. otakiensis. Three isolates were assigned to Lactococcus
genus: two Lc. lactis and one Lc. garvieae; and ve were assigned to
the Leuconostoc genus: four Leu. mesenteroides and one Leu. citreum.
Within the LAB group, Enterococcus is the most controversial
genus. The use of enterococci in food has been questioned, due to
the associated potential health problems. Despite these consider-
ations, they were found at high densities in many raw milk cheeses
and may have benecial effects on aroma and avor development
during ripening of many cheese types (Abriouel et al., 2008; Fuka
et al., 2010; Marino et al., 2003; Moreno et al., 2006; Ogier and
Serror, 2008).
The dominant Lactobacillus species found in Pico cheese isolates
were Lb. paracasei ssp. paracasei and Lb. plantarum, similarly to
other studies on cheeses originated from Northern Europe
(Fitzsimons et al., 2001; Ostlie et al., 2005), Spain (Ortigosa et al.,
2006), Italy (Bonomo and Salzano, 2012; Marino et al., 2003) and
the Balkans (Nikolic et al., 2008; Terzic-Vidojevic et al., 2007). These
typical starters and NSLAB are industrially important food-grade
organisms, as they have the generally recognized as safe (GRAS)
status (Colombo et al., 2009). Apart from their acidifying ability,
they may also contribute to the avor development in fermented
foods by producing aromatic compounds via proteolytic activity
(Aquilanti et al., 2006). The occurrence of Lc. lactis subsp. lactis as
the predominant species, before and during the maturation in
cheese has also been reported for a number of artisanal dairy
products (El-Baradei et al., 2008; Randazzo et al., 2006). Other
lactococci rarely associated with dairy products, such as Lc. garvieae
(Aquilanti et al., 2006), were also found in our study. The Leuco-
nostoc species found in Pico cheese were Leuc. mesenteroides and
Leuc. citreum. The presence of Leuconostoc in raw milk cheese va-
rieties is reported by several authors (Bonomo and Salzano, 2012;
Hemme and Foucaud-Scheunemann, 2004). Leuconostoc species
constitute an advantage to the dairy industry, since they may be
important to enhance the avor in certain cheese varieties and may
produce polysaccharides that act as agents for texture and stabili-
zation (Pedersen et al., 2013).
3.1.2. Phenotypic identication
The API system employed was able to classify 10.5% of the iso-
lates at an excellent ID level, 35.1% at a very good ID, 29% at a good
ID, 5.3% at an acceptable ID, 11.4% at a very good and good ID to the
genus and 7% were identify with doubtful prole, identication not
valid and low discrimination. This phenotypic characterization
based on sugar fermentation assays resulted in identication of 64
Lactobacillus (56.1%), ve Lactococcus (4.4%), four Leuconostoc
(3.5%), 35 Enterococcus (30.7%) and one Streptococcus (0.9%). Five
isolates were not identied (4.4%).
Among the Lactobacillus genus, one isolate was identied as
Lactobacillus brevis (0.9%), one as Lactobacillus curvatus (0.9%), 41 as
Lactobacillus paracasei subsp. paracasei (36%), 10 as Lactobacillus
plantarum (8.8%), 2 as Lactobacillus rhamnosus (1.8%) and 9 as
Lactobacillus genus (7.9%). For the enterococci genus, one isolate
 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
was identied as Enterococcus avium (0.9%), one as Enterococcus
durans (0.9%), 31 as Enterococcus faecalis (27.2%) and two as
Enterococcus faecium (1.8%). Among the Leuconostoc genus, three
isolates were identied as Leuconostoc lactis (2.6%) and one as
Leuconostoc citreum (0.9%). Finally, for the Lactococcus genus, one
isolate was identied as Lactococcus lactis (0.9%) and four as Lac-
tococcus lactis subsp. lactis (3.5%). The identication at the species
level was not achieved in 4.4% of the isolates in the biochemical
method (data not shown).
The API system and 16S rRNA gene sequencing provided
different patterns of genera and species identication for the LAB
isolates. Indeed, isolates that were identied to Lactobacillus genus
by phenotypic tests were identied into Enterococcus genus by 16S
rRNA gene sequencing, and vice-versa. The phenotypic test did not
match the 16S rDNA gene sequencing for the majority of the iso-
lates tested. From 114 strains identied, only 32.5% of the isolates
were assigned to the same genus and species, 48.2% were assigned
to different genera, 7% corresponded to the same genus but
different species while the comparison was not possible in 12.3%
(data not shown).
Enterococcus faecalis, Lactobacillus paracasei subsp. paracasei and
Lactobacillus plantarum were the most common species, although
they were found in different proportions, according to the method
of identication. Nevertheless, by sequencing of 16S rDNA gene, the
most frequent genus observed was Enterococcus, whereas API sys-
tem mostly identied Lactobacillus spp. Other researchers have also
observed a large discrepancy between API data and 16S rRNA gene
sequencing, not just for LAB but also for many other bacteria
(Gomes et al., 2008; Moraes et al., 2013; Velasco et al., 2004).
One of the main reasons for the discrepancy between the
biochemical and genotypic data can be attributed to the loss or gain
of plasmids. Since some genes needed for fermentation of sugars
are encoded by plasmids, variation in plasmid content may result in
metabolic inconsistencies (Ahrne  et al., 1989). However, due to the
stability of genomic DNA whose sequences are not dependent on
cultural conditions or handling, genotypic techniques are un-
doubtedly faster and more effective for species identication
(Nigatu, 2000; Ostlie et al., 2005).
3.1.3. Pulse eld gel electrophoresis (PFGE) prole analysis
In an effort to identify Pico LAB isolates at the species and strain
level, pulse eld gel electrophoresis (PFGE) analysis was conducted
to produce phylogenetic trees. Various molecular techniques have
been developed over the past decade for the identication and
classication of the bacteria at the strain level. The molecular
methods based on DNA restriction fragments of rare cutting such as
PFGE have been applied extensively for the identication and
genotyping of LAB and bidobacteria from food or the human
gastrointestinal tract (McCartney, 2002).
The analysis of the 73 Enterococcus faecalis by using the re-
striction enzyme SmaI, discriminated 40 different genotypes
dened at a minimum similarity level of 80% (Maluping et al., 2005;
Rodriguez et al., 2006; Tenover et al., 1995) (Supplementary
Fig. S1a). Two strains of Leu. Mesenteroides, out of three isolates,
were also differentiated (Supplementary Fig. S1b). The application
of PFGE-AscI macro restriction analysis to a total of 14 Lactobacillus
paracasei subsp. Paracasei isolates resulted in 11 different nger-
prints (Supplementary Fig. S1c) and ve Lab. Plantarum resulted in
ve different strains (Supplementary Fig. S1d). This result high-
lights a high degree of variability within the Lactobacillus. Overall,
the data obtained showed the presence of different biotypes within
the same species, demonstrating the strong selective effect of the
harsh conditions of the cheese manufacturing on the indigenous
microbiota. These conditions can determine the selection of specic
microbial populations and also of particular strains of each bacterial
183
group (Bonomo and Salzano, 2013).
3.2. Technological characterization
3.2.1. Carbohydrate fermentation proling
In order to analyze the carbohydrate fermentation proles of all
isolates (114), principal component analysis (PCA) was done on the
basis of API-50 and API-20 systems. When principal component
analysis of the API-50 results was undertaken, four distinct clusters
A, B, C and D with 3, 2, 6 and 62 isolates, respectively, and one
outlier (isolate 18, Lb. paracasei), were obtained (Fig. 1a).
Principal component 1 (PC-1) accounted for 27% of variation
(Fig. 1b) and was mainly responsible for grouping of isolates ac-
cording to utilization of the following sugars: monosaccharides
(ribose, mannose, sorbose, D-tagatose), disaccharides and tri-
saccharides (cellobiose, gentobiose and melezitose), glycosides
(amygdalin, arbutin, esculin, salicin) and glycerol, on the right; and
two aldopentoses on the left (L-arabinose and D-xylose). Principal
component 2 (PC-2) accounted for 11% of variation and was
responsible for grouping of isolates according to utilization of more
complex sugars and polysaccharides (sorbose, melibiose, turanose,
rafnose, starch, glycogen, a-methyl-D-mannoside and D-arabitol)
on the upper part of the plot; and D-tagatose and glycerol on the
lower part of the plot.
Cluster A (Fig. 1a) was composed of one Lb. plantarum (isolate
10) and two E. faecalis (43 and 53, identied as Lb. plantarum by the
API system) all of which utilized ribose, galactose, glucose, fructose,
mannose, mannitol, sorbose, N-acetyglucosamine, amygdaline,
arbutine, esculine, salicine, cellobiose, maltose, lactose, melibiose,
sucrose, trehalose, rafnose, gentibiose, D-arabitol and gluconate.
These sugars loaded highly on the positive dimension of PC1 and
PC2 (Fig. 1b). Cluster B included also three isolates (isolates 5, 12
and 15 identied as Leu. citreum, Lb. plantarum and Lb. otakiensis,
respectively) which were characterized by fermenting L-arabinose,
loaded highly on the negative dimension of PC1 (Fig. 1b). Some
isolates (Leu. citreum, Lb. plantarum) also utilized fructose, manose,
N-acetyglucosamine, sucrose and trehalose. Cluster C contained six
isolates with diverse sugar fermentation proles: two Leu. mesen-
teroides (3 and 4), Lb. paraplantarum (14), Lb. paracasei (19) and two
E. faecalis (31 and 64). All of them utilized galactose, glucose,
fructose, N-acetyglucosamine, trehalose, maltose (except strain 14)
lactose (except strain 19) and sucrose (except strains 14 and 31).
Four isolates (3, 4, 19 and 64) also utilized D-xylose, mannose,
maltose and sucrose and isolate 31 also used glycerol, mannitol,
sorbitol and esculin. Cluster D was the larger group and was
dominated by isolates identied by 16S rRNA sequencing analysis
as E. faecalis (isolates 25e30, 32e42, 44e52, 54e63, 65e75). This
cluster also included isolates identied as Leu. mesenteroides (1, 2),
Lc. lactis (6, 7), Lc. garvieae (8), Lb. plantarum (9, 11, 13) and Lb.
paracasei (16, 17, 20e24). Cluster D was composed of strains that
fermented ribose (except isolate 16), galactose, glucose, fructose,
mannose, N-acetyglucosamine, arbutin (except isolate 22), esculin,
salicin, cellobiose (except isolate 54), maltose (except isolate 74),
lactose (except isolates 60 and 74), trehalose (except isolates 25 and
49) and gentobiose (except isolates 32 and 74). Further, most iso-
lates also utilized mannitol, sorbitol, amygdalin, sucrose, melezi-
tose and D-tagatose. Isolate 18 (Lb. paracasei) was an outlier as it
was capable to ferment a great variety of sugars (L-arabinose,
ribose, galactose, glucose, fructose, mannose, mannitol, N-acety-
glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose,
maltose, lactose, melibiose, sucrose, trehalose, rafnose, starch,
glycogen, gentobiose, D-arabitol and gluconate).
Principal component analysis was also done for the API 20 Strep
results (Fig. 1c, d). All isolates tested positive for acetoin production,
b-glucosidase, lactose, and trehalose, negative for a-galactosidase,
184 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190

Fig. 1. (a) Principal component analysis scores plot showing clusters of the isolates formed according to their sugar fermentation proles from API-50. Isolates were labelled as
following: Leu. mesenteroides (1e4), Leu. citreum (5), Lc. lactis (6,7), Lc. garvieae (8), Lb. plantarum (9e13), Lb. paraplantarum (14), Lb. otakiensis (15), Lb. paracasei (16e24) and
E. faecalis (25e75). (b) Principal component analysis loadings plot showing the fermented sugars that categorized the isolates into different groups from API-50. (c) Principal
component analysis scores plot showing clusters of the isolates formed according to their sugar fermentation proles from API-20. Isolates were labelled as following: Lb. paracasei
(1e6), E. faecalis (7e37) and E. pseudoavium (38,39). (d) Principal component analysis loadings plot showing the fermented sugars that categorized the isolates into different groups
from API-20. (e) Principal component analysis scores plot showing clusters of the isolates formed according to enzymatic activities for the 114 isolates in the APIZYM gallery system.
(f) Principal component analysis loadings plot showing the enzymatic activities that categorized the isolates into different groups.
 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
b-glucuronidase, alkaline phosphatase, inulin (except isolates 2 and
28) and glycogen, and were not included in the analysis. Two axes
accounted for 57% of the total variation and separate the 39 isolates
into three distinct groups (A, B and C) and three outliers (isolates 5,
6 and 39, Fig. 1c).
PC-1 accounted for 36% of variation and distinguished the iso-
lates according to the presence of b-galactosidase and the utiliza-
tion of L-arabinose and rafnose on the right; hippurate hydrolysis
and fermentation of mannitol and sorbitol on the left. PC-2
explained 21% of the variation and was dened by arginine dehy-
drolase activity and starch fermentation on the upper part of the
plot, and ribose fermentation, on the lower part of the plot (Fig. 1d).
Cluster A was dominated by E. faecalis (isolates 7e21, 23e37),
but also included one Lb. paracasei (1) and one E. pseudoavium (38).
All of them were b-glucosidase (ESC), pyrrolidonyl arylamidase
(PYRA), leucine arylamidase (LAP) and arginine dehydrolase (ADH)
positive, and fermented ribose (except isolate 37), mannitol, sor-
bitol, lactose, trehalose and starch. Most of isolates in this cluster
also tested positive for hippurate hydrolysis. Cluster B included only
two isolates identied as Lb. paracasei (3) and E. faecalis (22) that
loaded highly on the positive dimension of PC-2 (Fig. 1d) as they
tested negative for hippurate hydrolysis, positive for arginine
dehydrolase and leucine arylamidase, and fermented L-arabinose,
mannitol, sorbitol and starch. Cluster C was composed of two Lb.
paracasei isolates (2 and 4) characterized by being hippurate hy-
drolysis negative, b-galactosidase and leucine arylamidase positive,
and fermenting ribose, L-arabinose, rafnose and starch. Isolate 5
(Lb. paracasei) was characterized by the absence of b-galactosidase
and arginine dehydrolase activities and utilization of few sugars
(ribose and mannitol, apart from lactose and trehalose). Isolate 6
(Lb. paracasei) was placed close to clusters A and B since it was b-
galactosidase and arginine dehydrolase positive and utilizes starch.
Isolate 39 was identied as E. pseudoavium and tested negative for
hippurate hydrolysis, b-galactosidase, leucine arylamidase, L-arab-
inose, sorbitol, rafnose and starch.
3.2.2. Enzyme proling
The pattern of enzymatic activities for the 114 isolates in the
APIZYM gallery system was analyzed by PCA. Fig. 1e, f shows the
score plot of the rst two principal components (PC-1 and PC-2).
These two PCs accounted for 57% of the total variance. PC-1
explained 38% of the total variation and distinguished the isolates
according to the activities of alkaline phosphatase, lipase (C14),
cystine arylamidase, trypsin, a-galactosidase, a-glucosidase, N-
acetyl-b-glucosaminidase and a-fucosidase activities (located in
the negative side), and valine arylamidase, b-glucoronidase, b-
glucosidase and a-mannosidase activities (located in the positive
side). PC-2 (20% of the total variation) differentiated the strains
according to esterase (C4 and C8), leucine arylamidase and
chymotrypsin activities (upper side), b-galactosidase, N-acetyl-b-
glucosaminidase and valine arylamidase activities (lower side). In
addition, three groups of strains were identied (Fig. 1e). Cluster A
was composed of Leu. mesenteroides (1), Lb. paraplantarum (14), Lb.
paracasei (23, 24) and E. faecalis (66, 67, 73, 101). These isolates were
characterized by the absence of alkaline phosphatase, esterase C4
(except 1), esterase C8, lipase C14, trypsin, a-chymotrypsin, a-
galactosidase (except 1), b-glucuronidase, a-mannosidase and a-
fucosidase activities. Cluster B include 17 isolates identied as Leu.
mesenteroides (3), Lb. paracasei (23, 24), Lb. otakiensis (30) and
E. faecalis (74, 76, 78, 79, 81, 82, 90e92, 95, 107, 108 and 110) and
were capable of synthesizing a wide spectrum of extracellular en-
zymes. They presented high or moderate activity for all enzymes
tested with exception of the isolates in parenthesis (8 for trypsin;
23, 24, 90 and 91 for a-galactosidase; 24 and 91 for b-galactosidase;
24, 91 and 107 for b-glucuronidase; 23, 90 and 110 for a-
185
mannosidase and 81 for a-fucosidase activities). Cluster B diverge
from other clusters by displaying high activities of valine arylami-
dase, a-mannosidase, b-glucoronidase and b-glucosidase, loading
on the positive side of PC-1 axe (Fig. 1e). Enzymes as b-glucor-
onidase and b-glucosidase deserve special attention as they are
known to be potential mediators of colon carcinogenesis (Nowak
and Slizewska, 2014). Cluster C contained the higher number of
isolates (89), which comprise Leu. mesenteroides (2, 4), Leu. citreum
(5), Lc. lactis (6, 7), Lc. garvieae (8), Lb. plantarum (9e13), Lb. para-
casei (15e17, 19, 20, 22, 25e29), E. faecalis (31e65, 68e72, 75, 77, 80,
83e89, 93,94, 96e100, 102e106, 109, 111), E. italicus (112) and
E. pseudoavium (113, 114). Most of isolates in this cluster revealed
lower or absent activity for lipase (C14), trypsin, a,b-galactosidase,
b-glucuronidase, a,b-glucosidase, N-acetyl-b-glucosaminidase, a-
mannosidase and a-fucosidase. In contrast, most of the strains
showed high activity for esterase (C4 and C8), leucine, valine and
cystine arylamidase, a-chymotrypsin, acid phosphatase and
naphthol-AS-BI-phosphohydrolase. Interestingly, cluster C holding
the highest number of isolates also express the desirable enzymatic
features for the ripening and production of cheese. The enzymatic
characteristics of LAB used in cheese production are one of the
factors that most affect cheese avor (McSweeney, 2004). All iso-
lates in cluster B and about half of isolates from cluster C express
acid phosphatase, an essential enzyme for the hydrolysis of phos-
phopeptides prevalent in cheese ripening (Fox and McSweeney,
1996). Isolates in cluster C showed higher technologic capacity as
they exhibited high esteraseelipase (C4 and C8) and aminopepti-
dase (leucine, valine and cystine arylamidase) activities. Esterases
increase short chain free fatty acids which are responsible for
piquancy avors of cheese (Abeijon et al., 2006). Aminopeptidases
release amino acids in the cheese environment and have also been
linked to avor formation. These amino acids can be metabolized
by bacterial enzymes to further products, playing a vital role in
cheese avor (Abeijon et al., 2006; Moreno et al., 2006).
3.2.3. Proteolytic and lipolytic activities
Technological properties, such as extracellular proteolytic and
lipolytic activities are important traits aiding the ripening, texture
and aroma development of cheeses. Near half of isolates identied
as Leu. mesenteroides, Lc. lactis, Lb. paracasei, E. faecalis and
E. pseudoavium, were able to degrade casein, but none of Leu. cit-
reum, Lb. plantarum, Lb. paraplantarum and E. italicus showed pro-
teolytic activity (Supplementary Table S1). These results were
similar to those produced by several LAB strains isolated from
traditional cheeses (Giraffa, 2003; Hemme and Foucaud-
Scheunemann, 2004; Moreno et al., 2006). Also, Bonomo and
Salzano (2013) reported mediumehigh proteolytic activity in 41%
of Leu. mesenteroides and 46% of Lb. paracasei isolated from Peco-
rino di Filiano cheese. In contrast, lipolytic activity was found 30% of
isolates, one Leuconostoc, two lactobacilli and 31 enterococci (data
not shown). Therefore, enterococci are the major genera contrib-
uting to cheese lipolysis, in agreement with other authors (review
by Giraffa, 2003). Low level of lipolysis is important for cheese
production, as a balanced concentration of free fatty acids and
proteolysis products have an important contribution to cheese
avour (Papanikolaou et al., 2012).
3.2.4. Diacetyl and exopolysaccharide (EPS) production
A large number of isolates examined (87%) were characterized
as diacetyl producers (Supplementary Table S1). Nevertheless, the
ability to produce diacetyl from citrate was found to be species and
strain dependent, as 60% of Leuconostoc, 33% of lactococci, 82% of
lactobacilli and 92% of enterococci produced diacetyl. The produc-
tion of diacetyl was observed in most Lb. paracasei subsp. paracasei,
Lb. plantarum and E. faecalis strains, and this result was in
186 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
agreement with other authors (Bonomo and Salzano, 2013; Giraffa,
2003; Hemme and Foucaud-Scheunemann, 2004; Moreno et al.,
2006; Zuo et al., 2014). Twenty-six isolates were regarded as high-
level diacetyl producers (one Lc. lactis, one Lb. plantarum, ve Lb.
paracasei and 19 E. faecalis), 45 isolates produced medium levels of
diacetyl, whereas 28 isolates produced low levels of diacetyl.
Diacetyl is responsible for the distinct aroma properties and quality
of fermented dairy products (Marilley and Casey, 2004). Being the
predominant genera with high diacetyl production, lactobacilli and
enterococci may play an important role in the development of the
distinctive organoleptic properties of this traditional cheese. As
regards leuconostoc strains, they produce low or none diacetyl
from citrate. These results were in agreement with those of Nieto-
Arribas et al. (2010), which suggested that leuconostoc strains were
slow diacetyl producers.
Regarding exopolysaccharide (EPS) production, only one isolate
(Leu. citreum) was positive for EPS phenotype. Several Leuconostoc
(Leu. mesenteroides and Leu. citreum) strains are known to produce
exopolysaccharides, which are primarily dextrans (Maina et al.,
2008). Yet, EPS production is sucrose-dependent, being unlikely
that Leu. citreum produces any EPS in the typical cheese. Never-
theless, this strain may have a great commercial potential as EPS
can improve the appearance, stability and rheological properties of
several dairy products. Moreover, EPS production by LAB received
increasing attention due to their immunogenic properties (Patel
et al., 2010).
3.3. Safety evaluation
3.3.1. Phenotypic tests
Gelatinase, DNase and histamine production, and hemolytic
activity was investigated in all isolates. All isolates tested negative
for histamine and Dnase production, but gelatinase activity was
detected in 54 isolates (47%) (Supplementary Table S2). Gelatinase
activity was high among enterococci, as 64% of the Enterococcus
isolates tested positive for gelatinase production (Supplementary
Table S2). One Lactococcus (L. lactis e L3A21M1) and one Lactoba-
cillus (Lb. plantarum e L3C1E8) also produced gelatinase, while
none of the Leuconostoc strains were gelatinase producers
(Supplementary Table S2). Gelatinase hydrolyses collagen and their
presence in enterococci may initiate and propagate the inamma-
tory processes associated to this genus.
For the hemolysis, 93% of the isolates were a-hemolytic, 3.5%
presented b hemolysis and 3.5% g hemolysis. The b phenotype was
found in two strains of Lb. paracasei subsp. Paracasei (L3B21R1 and
L3B21R2) and two Enterococcus (E. faecalis e L3A21E8 and
E. pseudoavium e L3C21K2). The g hemolysis was found in two Leu.
Mesenteroides (strains L3A21M4 and L3C21R7), one Lb. plantarum
(L3A21R6), and one E. faecalis (L3B21R5).
3.3.2. Susceptibility to antibiotics
All isolates were sensitive to b-lactam antibiotics (amoxicillin/
clavulanic acid, carbenicillin, penicillin and piperacillin) and only
two enterococci were resistant to chloramphenicol
(Supplementary Fig. 2A). Among all isolates, the highest percent-
ages of resistance were to nalidixic acid (95%), and aminoglycosides
such as kanamycin (87%), streptomycin (85%), tobramycin (83%),
gentamicin (68%) and netilmicin (64%). Frequent resistance of LAB
to aminoglycosides and nalidixic acid has been reported by various
studies (Casado Munoz et al., 2014; Pesavento et al., 2014; Jaouani
et al., 2015; Sharma et al., 2016). These results conrm other
studies showing the widespread incidence of antibiotic resistance
in dairy food isolates resulting, in part, from the extensive use of
antibiotics over the past few years in both animals and humans
(Pesavento et al., 2014; Sharma et al., 2014).
Among the enterococci, a high percentage of isolates were also
resistant to clindamycin (85%), and cephalosporins such as cefta-
zidime (65%), ceftriaxone (63%) and cefotaxime (29%). Tetracycline
resistance was found only among enterococci (25%) and a low
percentage of enterococci were resistant to rifampicin (17%),
erythromycin (6%), trimethoprim/sulfamethoxazole (4%), cefalotin
(2%), ooxacin (2%) and chloramphenicol (2%). In contrast, no
resistance to other clinically important antibiotics such as amoxi-
cillin/clavulanic acid, penicillins or vancomycin was found among
these enterococci. The presence of antibiotic-resistant enterococci
in food opens the question of the transmission of genetic de-
terminants to the commensal gut bacteria. Nevertheless, entero-
cocci are known to be intrinsically resistant to cephalosporins, b-
lactams and aminoglycosides. Regarding acquired antibiotic resis-
tance, vancomycin-resistant enterococci (VRE) are of most serious
concern as vancomycin is considered a last resort medication for
the treatment of serious, life-threatening infections. Several studies
showed the occurrence of vancomycin-resistant enterococci in food
of animal origin (Carasi et al., 2014; Courvalin, 2006; Giraffa, 2002;
Hammad et al., 2015). Noteworthy, all the 84 Enterococcus isolates
were sensitive to vancomycin. This is remarkable, since recent
studies indicated a high incidence of vancomycin-resistance phe-
notypes among Enterococcus faecalis isolated from traditional
cheeses (Porto et al., 2016; Yuksel et al., 2015).
Nearly half of lactobacilli were also found to be resistant to
cephalosporins (40e50%).
On the contrary, all lactobacilli isolates were sensitive to
amoxicillin/clavulanic acid, chloramphenicol, carbenicillin, eryth-
romycin, penicillin, piperacillin, rifampicin and tetracycline. Our
results are in accordance with those obtained by Sharma et al.
(2016) in Lactobacillus isolated from commercial probiotic prepa-
rations. Although tetracycline resistance has been reported to be
the most common acquired resistance in food isolates of Lactoba-
cillus, none of isolates in our study were found to be sensitive
against tetracycline. Instead, six Lactobacillus isolates were found to
be resistant to vancomycin. Nevertheless, resistance to vancomycin
in this genus is intrinsic, presenting no safety concerns in food
(Sharma et al., 2016). Leuconostoc and Lactococcus isolates were also
sensitive to most of antibiotics tested, with exception of cephalo-
sporins, aminoglycosides and nalidixic acid. Three Leuconostoc
were also found to be resistant to vancomycin, but this genus rarely
causes diseases in humans due to the intrinsic nature of resistance
(Casado Munoz et al., 2014). Intrinsic resistance to some antibiotics
might be considered an advantage of these genera, as they can play
a probiotic role in human gastrointestinal tract during antibiotic
therapy (Sharma et al., 2016). The results obtained in the present
study with regard to the susceptibility of Lactobacillus, Leuconostoc
and Lactococcus isolates to different antibiotics partially agree with
those obtained by other authors, and conrmed the safety nature of
these species concerning antibiotic resistance.
3.3.3. Virulence genes
The possible presence of genes responsible for virulent potential
was investigated for all strains by specic PCR assays
(Supplementary Fig. 2B). All enterococci isolates presented genes
for virulence factors, such as production of gelatinase (gelE, 77%),
aggregation substance (asa1, 63%), endocarditis antigen (efaA, 99%)
and adhesion of collagen (ace, 94%). Genes for cytolisin (cylA), and
enterococcal surface protein (esp) were present in 32e33% of
Enterococcus isolates and few (5%) harbour genes for resistance to
vancomycin (vanA, vanB) and histidine decarboxylase (hdc2).
Despite the high frequency of isolation of enterococci strains car-
rying virulence genes, it is worth stressing that these are a part of
the spontaneous microbiota present in other traditional cheeses,
such as Manchego and Brazilian cheeses, which are consumed
 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190 187

without any apparent risk for consumers (Moraes et al., 2012; favorable inuence in the avor of fermented foods.
Nieto-Arribas et al., 2011). Nevertheless, the presence of virulence The selected strains were further assessed for technological
genes in enterococci may increase the risk of genetic transfer to applications; milk acidication and avor formation in experi-
pathogenic bacteria, since these genes are usually located in con- mental cheeses (fresh cheeses), assessed by a taste panel. The re-
jugative plasmids (Eaton and Gasson, 2001). sults for the acidifying activity up to 48 h of growth in UHT milk and
The gene encoding for endocarditis antigen (efaA) was also skim milk are shown in Table 1. All strains were classied as slow
present in most lactobacilli (68.2% of isolates), Lactococcus (3 out of acid producers, since none of the strains reduced the milk pH by
3 isolates) and Leuconostoc (4 out of 5 isolates). Rare cases of
endocarditis associated to these organisms have been reported, but
this infection usually develops in predisposing patients with
anatomical alterations of the heart valves (Zechini et al., 2006).
Other virulence genes were also present in few lactobacilli (gelE,
asa1, esp, cylA, ace, vanA and vanB) and Leuconostoc (cylA, asa1, hdc2
and ace) strains. However, these species are commonly founded in
microbial communities of several traditional dairy foods and have a
long history of apparent safe use as starter cultures for dairy, wine
and vegetable fermentations (EFSA, 2007). Six strains, one Leuco-
nostoc mesenteroides (L3C21R7) and ve Lactobacillus paracasei
subsp. paracasei (L2A1K8, L2B21R1a, L2B21R3, L3B1M2, L3C21M6)
were negative for all virulence genes tested. In this regard, these
strains are good candidates for safe use as starter/adjunct cultures
in food fermentations.

3.4. Technological application

The selection of LAB for technological applications is a complex

process involving the evaluation of some technological features,
favorable enzymatic activities, as well the attribution of Qualied
Presumption of Safety (QPS) status. Since all Enterococcus strains
harbored at least one virulence factor, they were considered un-
suitability for food applications, as their use may represent a risk for
consumers. The safety assessment of other strains with QPS status
(Lactobacillus, Lactococcus and Leuconostoc) conrmed the selection
of 6 strains. This selection was based on absence of any gene
encoding virulence factors, the susceptibility to antibiotics (pre-
senting exclusively intrinsic resistance) and absence of hemolysis
(beta-hemolysis), DNAse, gelatinase and histamine production.
These six strains were identied as Leuconostoc mesenteroides
(isolate 4 - L3C21R7) and Lactobacillus paracasei subsp. paracasei
(isolates 16, 18, 19, 22 and 28, strains L2A1K8, L2B21R1, L2B21R3,
L3B1M2, L3C21M6, respectively). After examination of enzymatic
activities, two Lb. paracasei strains were excluded (isolates 18 -
L2B21R1 and 22 - L3B1M2), as they presented high b-glucor-
onidase, b-glucosidase and N-acetyl-b-glucosaminidase activities.
The activity of these enzymes may rise a safety concern for appli-
cation in foods, as they are known to transform many pro-
mutagens and pro-carcinogens to their mutagenic and carcino-
genic forms (Chadwick et al., 1992). Therefore, only four strains
were classied as safe and also presented the best enzymatic
characteristics: high esterase, lipase, aminopeptidase, acid phos- Fig. 2. Changes in lactic acid bacteria (A), pH (B) and titratable acidity (C) in fresh
cheeses stored at 4
C. Viable counts (CFU/g cheese), pH and titratable acidity (g lactic
phatase and proteolytic activities (isolates 4 - L3C21R7, 16 - L2A1K8, acid/100 g cheese) were monitored in control cheeses () and cheeses inoculated with
19 - L2B21R3, and 28 - L3C21M6). Moreover, the Lb. paracasei Leu. mesenteroides L3C21R7 (), Lb. paracasei subsp. paracasei L2A1K8 (,), Lb. para-
strains (isolates 16, 19 and 28) were high diacetyl producers, being a casei subsp. paracasei L2B21R3 () and Lb. paracasei subsp. paracasei L3C21M6 ().

Table 1
Acidifying activity of four selected strains (Leu. mesenteroides - L3C21R7 and Lb. paracasei subsp. paracasei -L2A1K8, L2B21R3 and L3C21M6) in skim and UHT milk.

Species D pH in skim milk D pH in UHT milk

2h 4h 6h 8h 10 h 12 h 24 h 48 h 2h 4h 6h 8h 10 h 12 h 24 h 48 h

Leu. mesenteroides
L3C21R7 0.01 0.07 0.13 0.17 0.27 0.37 1.01 1.42 0.08 0.20 0.22 0.37 0.54 0.69 1.20 1.70
Lb. paracasei subsp. paracasei
L2A1K8 0.00 0.04 0.10 0.20 0.26 0.34 1.20 2.14 0.03 0.07 0.12 0.22 0.29 0.35 1.26 2.59
L2B21R3 0.02 0.03 0.06 0.08 0.09 0.12 0.22 1.18 0.03 0.04 0.05 0.05 0.07 0.10 0.35 2.35
L3C21M6 0.05 0.13 0.16 0.22 0.30 0.41 1.25 2.05 0.08 0.18 0.23 0.41 0.64 0.86 2.15 2.63
188 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
more than 1 unit (ranging between 0.06 and 0.37), after 6 h.
Although, none of these strains could be considered as starter
cultures, they could harbour the adequate technological charac-
teristics for selection as adjunct cultures.
Results obtained for cell counts, pH and titratable acidity in fresh
cheeses made with the selected four lactic cultures (L3C21R7,
L2A1K8, L2B21R3 and L3C21M6) and control (no bacteria) are
indicated in Fig. 2. No detectable counts were found on control
cheeses. All strains grew well in fresh cheese samples, increasing
1.5 to 2 log after 72 h, reaching 109e1010 CFU/g. The values for pH of
control cheeses were consistently higher (P < 0.05) compared to
cheeses inoculated with the strains. However, acid production
(expressed as percentage of lactic acid) was low and did not differ
signicantly (P > 0.05) among control cheeses and inoculated
cheeses. Furthermore, the strains did not differ (P > 0.05) in respect
of pH and acid production and no signicant (P > 0.05) changes
were found on cheese pH and titratable acidity along time (72 h).
The low acidication prole exhibited by these strains was a
desirable feature for using these LAB as adjunct cultures in cheese
production, as over acidication of the curd adversely affected
cheese yield, rheology and the sensory acceptability (Gobbetti et al.,
2015).
The sensory evaluation of the experimental fresh cheeses
inoculated with the selected strains is presented in Table 2. Tasters
attributed low cheese scores for acidity (1.91e2.11), salty taste
(2.30e2.40) and aroma (2.07e2.76) and average scores for rmness
(2.97e3.31) and general acceptability (3.42e3.96). No signicant
differences (P > 0.05) were found among cheeses for acidity, salty
taste and rmness (Table 2). This is not surprising since the strains
used were slow acid producers. These results are in accordance
with similar studies using fresh cheeses (Coelho et al., 2014).
Traditionally fresh cheeses were characterized by their soft paste
and mild avor, as they were made from pasteurized milk without
addition of a starter culture (Silva et al., 2015). Therefore, this
cheese type is a valuable experimental system for studying po-
tential starter/adjunct cultures, since any changes in aroma/avor
are easily perceived. Aroma of cheeses made with two strains of Lb.
paracasei subsp. paracasei (strains L2B21R3 and L3C21M6) were
signicantly (P < 0.05) higher when compared to control (Table 2).
These two strains were also high diacetyl producers, while the
other two strains produced medium levels (L2A1K8) or did not
produced diacetyl (L3C21R7). The production of diacetyl by LAB
results on the typical butter avour/aroma in dairy products such as
milk, cheese and yoghurt (Marilley and Casey, 2004). Therefore,
diacetyl is an important avor compound for the aroma perceived
by tasters. The high aroma of cheese inoculated with strain
L2B21R3 may result also from the high esterase activity expressed
by this strain, as low levels of free fatty acids were known to
contribute to cheese aroma and avor (Papanikolaou et al., 2012).
General acceptability of fresh cheeses results from the
conjunction of desire acidity, aroma, texture and avor, and best
Table 2
Sensory evaluation of fresh cheeses without bacteria (control) and inoculated with selected strains (L3C21R7, L2A1K8, L2B21R3 and L3C21M6). Mean data SEM from 70
tasters based on a 5-point scale (1 stands for absence and 5 for presence at a strong level).
Cheeses Acidity Salty taste
Control 2.10 0.13 2.37 0.13
Leu. mesenteroides
L3C21R7 1.99 0.13 2.36 0.14
Lb. paracasei subsp. paracasei
L2A1K8 1.91 0.13 2.30 0.12
L2B21R3 2.11 0.13 2.40 0.13
L3C21M6 2.01 0.12 2.37 0.12
aec
Values within each column not followed by the same letter are signicantly different (P < 0.05).
describes the nest attributes for a good adjunct culture. The
organoleptic results of experimental cheeses demonstrated that Lb.
paracasei subsp. paracasei L3C21M6, was the preferred strain
assessed by the taste panel (Table 2). This strain presented high
alkaline phosphatase, lipase (C14), leucine and valine arylamidase,
trypsin, a-glucosidase and a-mannosidase activities (40 nmole),
and medium activities of esterase (C4 and C8), cystine arylamidase,
a-chymotrypsin, acid phosphatase and naphthol-AS-BI-
phosphohydrolase. In addition, this strain displayed extracellular
proteolytic activity and strong production of diacetyl from citrate.
From the results presented in this study, both strains L3C21M6
and L2B21R3 have good potential to be used as adjunct cultures for
the manufacturing of cheese, due to the production of pleasant
aroma/avors in a short time (2 days) by favorable enzymatic ac-
tivities. Adjunct cultures with desired properties are of particular
interest to get reproducible and enhanced cheese quality. The
strains used in the present study were isolated from a cheese with a
short maturation time, since Pico cheese does not usually exceed 20
days of maturation. The distinctive intense avor of Pico cheese
results from high proteolytic and esterase/lipase activities in com-
bination with strong diacetyl production (unpublished results).
These two Lactobacillus strains may play an important role in the
development of the sensorial properties typical of this artisanal
cheese.
4. Conclusion
Artisanal cheeses may harbour a rich source of diverse lactic
acid bacteria (LAB) with interesting functional properties. In the
present study, LAB from traditional Pico cheese were isolated,
identied and characterized in order to get better knowledge of the
indigenous bacterial population responsible for this cheese unique
avour. The species isolated in Pico cheese were, in decreasing
order of importance, E. faecalis, Lb. paracasei, Lb. plantarum, Leu.
mesenteroides, Lc. lactis, E. pseudoavium E. italicus, Lb. para-
plantarum, Lb. otakiensis, Lc. garvieae and Leu. citreum. These LAB
are essential for developing the diversity and typical features of
Pico cheese variety.
A high variability of some technologically relevant traits was
found among isolates and could be the basis for the selection of
specic strains to be used as adjunct cultures in the production of
traditional cheeses. Selected strains with b-galactosidase, ester-
aseelipase and aminopeptidase activities, moderate proteolytic
activity, high diacetyl production and also lacking b-glucoronidase
and b-glucosidase activities, have potential to be used as adjunct
cultures in cheese-making. In addition, the selected specic strains
needed to meet safety criteria by the absence of potential patho-
genic factors. Indeed, no isolates showed high-level resistance to
clinically important antibiotics and only a low percentage of strains
produced b-haemolytic reaction (3.5%). Remarkably, all enterococci
were susceptible to vancomycin whereas few Lactobacillus and
Firmness Aroma General acceptability
3.00 0.11 2.07 0.11a 3.54 0.12a
3.13 0.13 2.29 0.14ab 3.42 0.14a
3.31 0.12 2.24 0.13ab 3.63 0.12a
3.13 0.12 2.76 0.15c 3.75 0.14ab
2.97 0.13 2.41 0.12bc 3.96 0.11b
 M.F.P. Domingos-Lopes et al. / Food Microbiology 63 (2017) 178e190
Leuconostoc strains presented intrinsic resistance to this antibiotic.
Nevertheless, some virulence genes were prevalent in different
strains isolated from this cheese, which may pose a food safety
concern, especially among Enterococcus sp.
Among several strains posing no safety concerns, two Lactoba-
cillus strains were selected on the basis of several technological
parameters and organoleptic evaluation. These strains early inu-
enced cheese avor and may be safely applied as adjunct cultures
for cheese manufacture, in particular, Pico cheese. According to the
present study, these strains (Lb. paracasei subsp. paracasei L2B21R3
and L3C21M6) are promising candidates for application as adjunct
cultures in cheese production.
Acknowledgment
This work is funded by National founds from FCT e Fundaa ~o
^ncia e a Tecnologia under the project PTDC/AGR-ALI/
para a Cie
104385/2008. The doctoral fellowship of MFP Domingos-Lopes was
nanced by FRCT - Fundo Regional para a Cie ^ncia e Tecnologia
(M3.1.2/F/009/2011). Authors also acknowledge Dr. Mary C. Rea, of
TEAGASC e Food Research Center, for her useful advice and
knowledge in PFGE analysis.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.fm.2016.11.014.
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