Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Phenotypic, genotypic and technological characterization of

predominant lactic acid bacteria in Pecorino cheese from
central Italy
L. Aquilanti, G. Silvestri, E. Zannini, A. Osimani, S. Santarelli and F. Clementi
Department of Food Science, Polytechnic University of Marche, Via Brecce Bianche (Monte Dago), Ancona, Italy

Keywords
acidification, amplified ribosomal DNA
restriction analysis (ARDRA), autolysis, lactic
acid bacteria, Pecorino cheese, proteolysis,
RAPD.
Correspondence
Gloria Silvestri, Department of Food Science,
Polytechnic University of Marche Via Brecce
Bianche (Monte Dago) 60131 Ancona, Italy.
E-mail: anmicro@univpm.it
2007 ⁄ 0061: received 15 January 2007,
revised 7 May 2007 and accepted 14 June
2007
doi:10.1111/j.1365-2672.2007.03513.x
Abstract
Aims: Identification and biotyping of lactic acid bacteria (LAB) isolated from
raw-milk Pecorino cheese manufactured in the Marche region (central Italy)
for selection of suitable starter cultures or adjuncts.
Methods and Results: Preliminary characterization with morphological and
biochemical assays were undertaken for 112 Gram-positive and catalase-
negative isolates. Unequivocal identification of the isolates was obtained
through restriction analysis of the amplified 16S rRNA gene and sequencing
of 360–380 bp amplicons. Fifty-nine isolates belonging to LAB species gener-
ally recognized as safe and potentially utilized as starters or flavour-producing
adjuncts were preselected and tested for their acidifying, proteolitic and
autolytic activities. Fifty-five of these isolates were also subject to RAPD
(randomly amplified polymorphic DNA) fingerprinting and unweighted pair-
group method with arithmetic averages (UPGMA) cluster analysis for the
estimation of genotypic intra-species variation. As a result, in Pecorino
cheese, a heterogeneous lactic acid bacteria population, which includes strains
with metabolic characteristics of technological interest, was characterized.
Conclusions: The polyphasic approach proposed allows the bacterial ecology of
Pecorino cheese to be investigated and allows to assess the potential role of
autochthonous LAB strains for the dairy industry.
Significance and Impact of the Study: The great economic importance of Peco-
rino cheese encouraged a deeper knowledge of its microbiota, which is known
to influence the peculiar sensory properties of this cheese, also in view of its
exploitation.

depending on the length of ripening, from 1 to 2 months

Introduction
Pecorino is a typical Italian ewes’ milk cheese whose
features largely depend on local and regional practices
(De Angelis et al. 2001). In the Marche region (central
Italy), its manufacture still follows a strict traditional
technology. Raw or, rarely, pasteurized whole milk is
coagulated by the addition of artisan ovine rennet. Wild
herbs (basil, sweet marjoram, green figs, bramble and
bugloss sprouts, cloves, nutmeg, black pepper, wild thyme)
may be added to the rennet to aromatize the curd, which is
manually broken up into hazelnut- or rice-sized pieces,
to up to 1 year. The curd is generally heated to 45–48C,
transferred onto moulds and manually pressed to drain
the whey. Moulded cheeses are repeatedly dusted with salt
or immerged in brine. Ripening is carried out at a mod-
erate temperature (10–15C), with frequent turning and
cleaning of the moulded cheeses with water and tepid
whey. In fully ripened cheeses (after 1 year or more), the
rind may be rubbed with olive oil. Moulded cheeses are
cylindrical and 1–3 kg in weight; the rind is thin and
yellowish-white, while the inner part is straw yellow and
characterized by rare and small eye-like spots and a soft,

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948 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
L. Aquilanti et al. Lactic acid bacteria from Pecorino cheese

semi-hard or hard texture, depending on the length of Materials and methods

ripening. The taste is savoury and slightly aromatic.
The great economic importance of Pecorino, which is Reference strains
in great demand by consumers, has stimulated an interest
Forty-two reference strains (Table 1) were used as con-
in obtaining a deeper knowledge of this cheese. Accord-
trols in the phenotypic and molecular analyses. They were
ingly, compositional and microbiological traits of Peco-
purchased from: (i) Deutsche Sammlung von Mikrorgan-
rino cheeses produced in different Italian geographical
ismen und Zellkulturen (DSMZ, Braunschweig, Germany,
areas have been described more recently, such as Pecorino
http://www.dsmz.de/); (ii) the American Type Culture
Sardo (Cosentino et al. 2001; Manca et al. 2001; Mannu
Collection (ATCC, Manassas, USA, http://www.atcc.org/);
and Paba 2002), Pecorino Siciliano (Randazzo et al.
(iii) the National Collections of Industrial and Marine
2006), Pecorino del Salento (Cappello et al. 2001), Peco-
Bacteria (NCIMB, Aberdeen, Scotland, UK, http://
rino Crotonese (Gardini et al. 2006), Pecorino abruzzese
www.ncimb.co.uk/); and (iv) the Northern Regional
(Chaves-Lopez et al. 2006), Pecorino Romano, Pecorino
Research Laboratory ARS Culture Collection (NRRL,
Toscano and Pecorino Umbro (De Angelis et al. 2001).
Peoria, Illinois, USA, http://nrrl.ncaur.usda.gov/). All of
However, as far as we know, none of the ewes’ milk
the strains were revitalized as indicated by the culture
cheeses manufactured in the Marche region have been
suppliers.
previously characterized, except for the Fossa (pit) cheese
(Avellini et al. 1999; Gobbetti et al. 1999), a typical prod-
uct made from a mixture of cows’ and sheeps’ milk, Cheese-making and sampling
which is aged for about 3 months in pits dug into tuffa-
Raw whole milk, curd and Pecorino cheese at different
ceous rock.
stages of ripening (7, 21, 28, 42, 90 and 120 days) were
At the farmhouse level, Pecorino cheese is commonly
sampled in three family-run dairy farms in the Marche
produced from raw milk without the addition of selected
region (Central Italy): two (F1 and F2) are located in the
starter cultures, and therefore the acidification and the
district of Ancona and the third (F3) in the district of
ripening process depend entirely on the autochthonous
Macerata. Although the cheese-making techniques
lactic acid bacteria (LAB) population that originates from
adopted in these three dairy farms vary somewhat, the
the raw milk and the dairy environment. As has been pre-
steps and conditions for producing these Pecorino cheeses
viously shown (De Angelis et al. 2001), this resident mic-
adhere strongly to tradition. The ewes’ milk from the
robiota is mainly composed of mesophilic lactobacilli that
Sarda breed sheep was collected twice a day; the milk
largely contribute the determination of the typical flavour
from the evening milking was cooled to 4C and mixed
and texture of these traditional cheeses. Accordingly, it is
with the morning-collected milk. The milk was then fil-
of great importance to preserve the pool of genetically
tered and warmed up to about 37C. After coagulation
diverse dairy strains that are present in environments that
with lamb rennet, which had been prepared beforehand
have not yet been affected by the introduction of selected
by working the lamb abomasum together with milk, salt,
industrial strains (Desmasures et al. 1998). Moreover,
cinnamon and nutmeg, the curd was broken up while
interest in the uniqueness of such traditional cheeses has
warming up to 42–45C for about 5 min. After removal
led to increased efforts towards the characterization of the
of the whey, the curd was transferred into perforated
wild strains involved in their production (Mannu et al.
moulds of 14–20 cm in diameter and 6–10 cm in depth,
2000).
pressed to drain the whey and held at 20–25C for
On the basis of these assumptions, the LAB isolates
4–10 h (F1 and F2) or at 40C for 1 h (F3). Salting of
from Pecorino cheeses manufactured in three family-run
the moulded cheese was performed in 20% NaCl brine
dairies in the Marche region were characterized to assess
for 12 h (F2 and F3) or for 24 h (F1). In all of the three
the biodiversity within this wild population and to select
farms, the inoculum was provided by bacterial cells pres-
suitable starter cultures or adjuncts to be used by the
ent in the milk. The ripening period was performed at
local dairy industry for the production of Pecorino
room temperature (about 25C) (F2, F3) or at 10–15C
cheese. Accordingly, the isolates were preliminary
(F1) and ranges from a minimum of 4–6 weeks (F3) up
grouped on the basis of their phenotypic features and
to 12–20 weeks (F1, F2).
subsequently identified by using the amplified ribosomal
All of the samples were taken from one batch: the
DNA restriction analysis (ARDRA) technique. A pres-
raw milk was sampled just before the renneting, while
elected pool of isolates was finally subjected to the
the curd was sampled immediately after the moulding.
RAPD typing and the assessment of the acidifying, pro-
The samples were chilled to 4C and analysed within
teolytic and autolytic activities.
24 h.

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960 949
 Table 1 Theoretical and experimental profiles obtained by ARDRA using the AluI, FokI and HaeIII enzymes

950
Restriction fragments (in bp)

Species 16S rRNA gene sequence GenBank acc. no. Strain AluI FokI HaeIII

Lactobacillus casei ssp. casei AF526388; AY196978 NCIMB4114; NRRL1922 115–220 123–224 316
Lact. casei ssp. rhamnosus M58815; AF243146 – 115–220 123–224 316
Lact. paracasei AY196966; AY735405 NRRL4560; DSM4905; DSM5622 114–218 122–222 314
Lact. zeae D86516; AF429522 DSM20011; DSM20178 114–212 122–237 308
Lact. pentosus D79211 DSM20199; DSM20314 114–252 122–244 315
Lact. plantarum M58827; X52653 DSM2601; DSM20174 51–115–201 119–244 315
Lact. paraplantarum AJ306297; AB191250 DSM10667; DSM10641 114–251 122–243 313
Lact. brevis AB024299; M58810 DSM20054; DSM1267; DSM2647 51–314 121–244 271
Lact. acidophilus n.a. DSM9126 115–123 123–129 160
Lactic acid bacteria from Pecorino cheese

AF375937; AF375895; AF375886 DSM20079 104–114 122–129 135

Lact. rhamnosus AF429477; AF429485; AF429478 DSM20021 114–210 122–235 103–203
Lact. curvatus AJ270951; AM113777 DSM20019 71–299 122–248 319
Lact. farciminis AJ417499; M58817 DSM20184 50–69–92–132 119–204 316
Lact. delbrueckii ssp. bulgaricus AJ414694; AJ414693 DSM20080 51–115–164 123–202 267
Lact. delbrueckii ssp. delbrueckii X52654 – 51–115–153 123–229 256
AY050172; AY773949 52–114–164 112–122–129 268
Lact. delbrueckii ssp. lactis M58823 – 51–115–126 98–123–129 89–178
AJ414695 52–104–164 87–122–129 268
Lact. delbrueckii ssp. indicus AY421720 – 114–164 101–122–129 257
Lact. sakei AY204893; AB183697 DSM20017 72–272 94–250 321
Lact. helveticus AF213704; AF213705; AF213704 – 114–199 115–122–129 79–207
Lact. fermentum AF522394; AF302116 DSM20052 114–261 122–232 55–225
Lact. parabuchneri AY026751; AJ970317 DSM5708 115–261 123–253 280
Lact. buchneri M58811; AB205055 DSM20057 51–115–211 123–254 281
Lact. reuteri X76328; AF429540; AF429537 – 115–261 122–232 51–56–269
Lact. salivarius AY137588 – 355 355 259
Lact. crispatus AF257097; AF257096 – 115–189 105–123–129 79–226
Lact. gallinarum AJ417737 – 115–197 113–123–129 79–234
AJ242968 114–219 54–60–122–130 79–236
Lact. gasseri AF519171; M58820 – 51–115–206 123–249 320
Lact. halotolerans M23037; AB022926 – 75–148–156 358 327
Lact. johnsonii AJ002515 – 115–206 123–231 302
Lact. parakefiri AY026750 – 115–253 123–245 223
Lactococcus lactis ssp. lactis AB100803 DSM20729; ATCC11454; DSM20481 115–226 81–260 290
L. lactis ssp. cremoris AB100802 DSM4645; DSM20069 115–227 341 289
Lactococcus plantarum X54259 – 73–86–147 87–261 296
L. garvieae X54262 – 63–115–173 123–228 299
AB012306 113–237 350 300
L. raffinolactis X54261 – 66–84–86–115 166–189 303
Leuconostoc mesenteroides ssp. lactis AB023968; AY026048 DSM20202 332 332 236
L. Aquilanti et al.

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
ª 2007 The Authors
 L. Aquilanti et al. Lactic acid bacteria from Pecorino cheese

Lactic acid bacteria isolation

55–237
HaeIII

235
235
238
254

350
297
298
325
288
305
275
294
275
275
Ten grams of each sample was homogenized in 90 ml of
a sterile 2% sodium citrate solution in a Stomacher appa-
ratus (400 Circulator, PBI International, Milan, Italy) at
260 rev min)1 for 1 min. The homogenates were serially

88–92–168
diluted in a sterile peptone–saline solution (1 gl)1 pep-

123–233
123–233

123–238
123–223
123–238
123–238
tone and 8Æ5 gl)1 NaCl). Aliquots (100 ll) of each dilu-
Restriction fragments (in bp)

FokI

331
331
334
329
358
329

349

357
tion were streaked on MRS agar (Oxoid, Basingstoke,
UK) and M17 agar (Oxoid). The MRS plates were incu-
bated at 37C for 72–96 h under anaerobic conditions
62–86–86–114
62–86–86–114

108–114–155
57–115–187
67–115–179
54–115–177
67–117–179
(Anaerogen, Oxoid), while the M17 plates were incubated
at 22C for 72 h and at 45C for 48 h, for the isolation of
115–262

115–246
mesophilic lactococci and Streptococcus termophilus
331
331
334
351
358
350
AluI

respectively. Plates with 30 to 300 colonies were selected

for LAB isolation. For each sampling point, one to five
colonies were randomly picked from the countable M17
DSM20240; DSM20343; DSM20200

and MRS agar plates and streaked out three times on the
same media used for the isolation to check for purity.
Colony morphology, Gram and catalase reactions were
checked, and the isolates were stored at )80C in a mix-
NCIMB8189; DSM20346

DSM20479; DSM20617

DSM20333; DSM20238

ture of MRS broth and glycerol (3 : 2 v ⁄ v).

Preliminary differentiation of isolates

DSM20484

The isolates were preliminary clustered on the basis of the

Strain

acid production from carbohydrates (cellobiose, galactose,

–
–
–

–
–
–
–
–
–

maltose, mannitol, melibiose, ribose, sucrose, trehalose,

arabinose, glycerol, fructose, glucose, lactose, mannose,
16S rRNA gene sequence GenBank acc. no.

melezitose, raffinose, rhamnose, d-xylose), the hydrolysis

of esculin and arginine, the tolerance to 2Æ0, 4Æ0 and 6Æ5%
AY188354; AB161183; DQ176426

n.a., GenBank nucleotide sequence not available; –, reference strain not available.

NaCl, the d- or l-lactic acid and dextran production as

AJ249891; AJ249539; AJ249535

previously described (Aquilanti et al. 2007). Data were

converted into a binary data matrix, where 1 coded for
fermentation ⁄ growth and 0 for absence of fermenta-
AB023244; AB023246
AB023242; AB122035

AB023240; AB015642
AF111948; AY165007

AJ276355; DQ411813
AJ420803; DQ411814
AB002481; AF396922
AF515228;AB120032

tion ⁄ growth. A similarity data matrix was calculated on

Y18161; AJ420804

the basis of the Nei and Li coefficient (Nei and Li 1979)

and used for the unweighted pair-group method with
AB023247

AB022925

AJ305321

AJ276354

arithmetic averages (UPGMA) clustering of the cultures.

Amplified ribosomal DNA restriction analysis

Theoretical analysis
Leuc. mesenteroides ssp. mesenteroides
Leuc. mesenteroides ssp. dextranicum

To develop a reliable method for the genotype-based

Leuconostoc pseudomesenteroides
Leuc. mesenteroides ssp. cremoris

identification of the isolates, a wide comparative in silico

study was preliminary carried out by examining 94 com-
Streptococcus thermophilus

plete 16S rRNA gene sequences from Lactobacillus, Lacto-

Pediococcus. acidilactici

coccus, Pediococcus, Streptococcus, Weissella, Leuconostoc,

Leuconostoc carnosus
Leuconostoc citreum

Enterococcus durans
Table 1 Continued

Carnobacterium and Enterococcus strains, as retrieved from

Weissella hellenica
Ped. pentosaceus

the GenBank DNA database (http://www.ncbi.nlm.nih.

E. casselliflavus
Strep. bovis

gov ⁄ ) (Table 1). All of the sequences were aligned with

E. faecium
E. faecalis

ClustalW (http://www.ebi.ac.uk/clustalw/) and subjected

Species

to theoretical restriction mapping with the TACG applica-

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960 951
Lactic acid bacteria from Pecorino cheese
tion (http://www.medkem.gu.se/cutter), by comparing the
restriction profiles obtained with 53 commercially avail-
able endonucleases.
Experimental analysis
The bacterial DNA was extracted from MRS broth cul-
tures by the method of de Los Reyes-Gavilan et al.
(1992). The quantity and purity of the DNA were
assessed by optical reading at 260 nm and 280 nm respec-
tively, as described by Sambrook et al. (1989). A fragment
of about 360–380 bp of the 16S rRNA gene was amplified
by PCR with the Y1 and Y2 primers described by Young
et al. (1991). The amplification reaction was performed in
a total volume of 50 ll containing 10 ng DNA and
the following mix components: 1 · reaction buffer
(DiaTech, Jesi, Italy), 0Æ6 U Taq polymerase (DiaTech),
0Æ2 mmol l)1 dNTPs, and 1Æ5 mmol l)1 MgCl2. Initial
denaturation was performed at 94C for 3 min, followed
by 30 cycles of: 94C for 45 s, 55C for 45 s and 72C for
60 s, and final extension at 72C for 7 min. The PCR was
carried out in a Gene Amp 9700 thermocycler (Applied
Biosystem, Foster City, USA). The 16S rDNA amplicons
were analysed on 1% agarose (Bio-Rad Laboratories,
Richmond, USA) gel, using a 1-kb ladder (M-medical,
Milan, Italy) as a molecular weight standard. Gels were
run at 3 V cm)1 (constant voltage) in 0Æ5 · TBE
(45 mmol l)1 Tris, 45 mmol l)1 boric acid, 1 mmol l)1
EDTA) and stained with 0Æ5 lg ml)1 ethidium bromide.
Amplicons were digested at 37C for 4 h with AluI,
FokI and HaeIII (Roche Diagnostics, Milan, Italy).
Restriction patterns were analysed on 3% agarose gels
using a 50-bp ladder (Amersham Biosciences GE Health-
care, Milan, Italy) as a molecular weight standard. Digital
images were viewed and captured using the Image Master
VDS (Amersham Biosciences) equipped with the Multi-
Analyst system (Bio-Rad) and stored as TIFF files.
Preliminary identification of the isolates was performed
by comparing their restriction patterns with those
obtained from the reference strains listed in Table 1. The
diagnosis of species was verified by sequencing of the 16S
rRNA gene amplicon from a few selected isolates.
The PCR products were previously purified using
micro-columns (GFX Purification Kit, Amersham Bio-
sciences), according to the manufacturer’s instructions
and sent to MWG Biotech (Milan, Italy) for sequencing.
The identities of the isolates were determined through a
search of the GenBank DNA database using the blast
algorithm (Altschul et al. 1990). The isolate ascribed to
Lactobacillus plantarum sensu latu after sequencing of the
16S rRNA gene was further subjected to the recA multi-
plex PCR assay described by Torriani et al. (2001) for a
finer discrimination at the species level.
952 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
L. Aquilanti et al.
Technological characterization
The acidifying activity, proteolysis and tendency to lysis
of the isolates ascribed to taxa, which are generally
utilized as starter or flavour-producing adjuncts (Lacto-
bacillus, Lactococcus, Pediococcus, Carnobacterium, Weissella,
Streptococcus), were assessed as previously described
(Aquilanti et al. 2007).
Statistical analysis
Three repetitions of each technological assay were per-
formed. The arithmetic means and standard deviations
were calculated. To highlight the significant intra-species
differences in the autolytic, proteolytic and acidifying
activities within a preselected subset of isolates, a one-way
analysis of variance (anova), along with the Tukey Kra-
mer honestly significant difference (HSD), was carried
out using the statistica software package (version 5Æ1,
StatSoft Inc., Tulsa, OK). The following variables were
considered: VAR1, species, VAR2, acidifying activity at
8 h incubation; VAR3 release of glycine; and VAR4,
extent of autolysis.
Randomly amplified polymorphic DNA analysis
The RAPD analysis was performed with the primers:
5¢-AGCAGCGTGG-3¢ (Cocconcelli et al. 1995) and 5¢-GA
GGGTGGCGGTTCT-3¢ (Huey and Hall 1989).
When the first primer was used, the amplifications
were performed in 25 ll reaction volumes, containing
160 mmol l)1 (NH4)2SO4, 67 mmol l)1 Tris–HCl (pH
8Æ8), 0Æ01% Tween-20, 200 lmol l)1 of each of dATP,
dGTP, dCTP and dTTP, 3 mmol l)1 MgCl2, 1 lmol l)1
primer, 2 ng DNA and 2 U Taq DNA polymerase (Euro-
Taq, Euroclone, Milan, Italy), using the following condi-
tions: initial denaturation at 94C for 2 min, followed by
35 cycles of denaturation at 94C for 1 min, annealing at
42C for 1 min, elongation at 72C for 90 s, and a final
elongation step at 72C for 10 min.
When the second primer was used, the amplifications
were performed in 25 ll reaction volumes, containing
160 mmol l)1 (NH4)2SO4, 67 mmol l)1 Tris–HCl (pH
8Æ8), 0Æ01% Tween-20, 200 lmol l)1 of each of dATP,
dGTP, dCTP and dTTP, 3 mmol l)1 MgCl2, 2 lmol l)1
primer, 62Æ5 ng DNA and 0Æ625 U Taq DNA polymer-
ase (EuroTaq, Euroclone, Milan, Italy), using the fol-
lowing conditions: initial denaturation at 94C for
2 min, followed by 40 cycles of denaturation at 94C
for 1 min, annealing at 45C for 20 s, elongation at
72C for 2 min, and a final elongation step at 72C for
10 min.
ª 2007 The Authors
L. Aquilanti et al.
The PCR products were separated by electrophoresis
on 1Æ5% (w ⁄ v) agarose gels in TBE [Tris-borate-EDTA
(pH 8)] buffer. A 50 bp DNA ladder (Amersham Phar-
macia, Uppsala, Sweden) was used for the molecular
weight standards. The DNA fragments were stained with
ethidium bromide and viewed under UV light, at
254 nm. The band patterns were normalized and further
processed with the GelCompar 4Æ0 software (Applied
Maths, Kortrijk, Belgium). Hierarchical cluster analysis
(UPGMA) was carried out on the matrix of similarity
data obtained using the formula of Nei and Li (1979) and
the ntsys.pc package, version 1Æ8 (Rohlf 1993). An 85%
similarity was arbitrarily selected as a threshold for the
definition of the homogeneous RAPD-based clusters. The
reproducibility of the RAPD fingerprints was determined
by triplicate loading on agarose gels of independent, trip-
licate reaction mixtures prepared from 16 reference
strains, belonging to the species Lactobacillus casei ssp.
casei, Lactobacillus. zeae, Lactobacillus. brevis, Lactococcus
lactis ssp. lactis, L. lactis ssp. cremoris, Strep. thermophilus
and Pediococcus acidilactici (Table 1).
Results
Isolation and preliminary phenotype-based
characterization
One-hundred and twelve Gram-positive and catalase-neg-
ative bacteria were isolated from raw milk, curd and Pec-
orino cheese. These isolates and the reference strains
listed in Table 1 were preliminary clustered on the basis
of their phenotypic features. The phenetic classification of
the isolates, which was performed by correlating each iso-
late to the closest reference strain, resulted in the identifi-
cation of 24 isolates as Enterococcus faecium, 19 as E.
faecalis, 21 as L. lactis, 10 as Lact. plantarum, two as Lac-
tobacillus paracasei, two as Lactobacillus fermentum, one
as Lactobacillus rhamnosus and two as Lactobacillus spp.
For the remaining 31 isolates, an unambiguous diagnosis
of species could not be achieved.
Theoretical ARDRA
First, the evaluation of the theoretical patterns obtained
with 53 commercially available endonucleases led to the
selection of a cost-effective set of three enzymes, namely
FokI, HaeIII and AluI, which were highly discriminative.
When these three enzymes were tested, the GenBank
DNA sequences derived from reference strains ascribed to
the same species generated identical theoretical patterns
(Table 1), with the exception of those belonging to Lacto-
bacillus delbrueckii ssp. lactis, Lactobacillus delbrueckii
ssp. delbrueckii, Lactobacillus gallinarum, and Lactococcus
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
Lactic acid bacteria from Pecorino cheese
garvieae; the virtual digestion of these last led to the gen-
eration of strain-specific profiles. Moreover, the combined
use of the three endonuclases allowed the virtual discrimi-
nation of the large majority of the species under study,
with the exception of five groups of taxa: (i) Lact. para-
plantarum, Lact. pentosus; (ii) Lact. casei ssp. casei, Lact.
casei ssp. rhamnosus, Lact. paracasei; (iii) Lact. delbrueckii
ssp. bulgaricus, Lact. delbrueckii ssp. delbrueckii, Lact. del-
brueckii ssp. lactis; (iv) Leuconostoc mesenteroides ssp. lac-
tis, Leuc. mesenteroides ssp. dextranicum, Leuc.
mesenteroides ssp. mesentaroides, Leuc. mesenteroides ssp.
cremoris; and (v) E. faecium, E. durans.
Experimental ARDRA
The reliability and reproducibility of the theoretical
restrictions were experimentally verified on 42 reference
strains purchased from international culture collections
(Table 1). As expected, fragments of approximately 360–
380 bp were obtained, corresponding to a portion of the
16S rRNA gene. Restriction fragments smaller than 50 bp
were not considered, as they were not reproducibly visu-
alized. For all but one reference strain (Lactobacillus
acidophilus DSM9126), the experimental patterns were
comparable to those obtained through the in silico simu-
lation (Table 1).
A restriction pattern database, including both the theo-
retical and experimental profiles, was built up and used
for the assignment into species of the isolates (Table 1).
In greater detail, this database included both the profiles
obtained experimentally from the reference strains pur-
chased from international culture collections and the pro-
files produced during the in silico trials, which were
preliminary carried out onto the nucleotide sequences
retrieved from the GenBank. When the restriction pat-
terns obtained from a given reference strain were identical
to those generated theoretically from the corresponding
GenBank sequence(s), the only set of theoretical patterns
was reported; on the other hand, when the two
approaches (theoretical and experimental) led to dissimi-
lar restriction patterns, both these set of profiles were
reported (Table 1).
Each AluI, HaeIII and FokI pattern defined a haplotype,
while the combination of the three haplotypes defined a
phylotype. All the 112 isolates were identified by compar-
ing their phylotypes with those obtained from the refer-
ence strains and the GenBank sequences (Table 1). One
representative isolate within each phylotype was selected
for the sequence analysis of the PCR amplicons.
This approach allowed 38 and 74 isolates to be identi-
fied at the genus and the species level, respectively. In
more detail, 31 isolates were ascribed to the genus Entero-
coccus, six to Weissella and one to Carnobacterium, while
953
Lactic acid bacteria from Pecorino cheese
the isolates identified at the species level were ascribed to
Lactobacillus brevis (n = 22), Lactobacillus zeae (n = 3),
Lact. casei sensu latu (n = 3), Lact. plantarum sensu latu
(n = 1), Lact. salivarius (n = 1), Lact. parabuchneri
(n = 1), L. lactis ssp. lactis (n = 5), L. lactis ssp. cremoris
(n = 6), Strep. thermophilus (n = 2), Pediococcus acidilac-
tici (n = 6), Pediococcus pentosaceus (n = 1), Carnobacteri-
um maltaromicus (n = 2), Carnobacterium divergens
(n = 1), E. faecalis (n = 18) and E. durans (n = 2).
By comparing the phenotype- and genotype-based
diagnoses, it emerged that all of the cultures preliminary
recognized as enterococci on the basis of their phenotypic
features were confirmed as belonging to this genus, except
for two isolates, which were ascribed to the genera Lacto-
coccus and Pediococcus. A completely different picture
emerged for the remaining isolates. Indeed, the pheno-
type-based diagnoses were correct at the genus level for
only eight lactobacilli and at the species level for only
three L. lactis isolates.
With regard to the isolation source, while the entero-
cocci were recovered in all of our samples, Lact. brevis
was the only species found in 12- and 20-week-ripened
Pecorino cheese, whereas Carnobacterium maltaromicus
was only isolated from raw milk and curd samples.
Technological characterization
The acidifying, proteolitic and autolytic activities of a
preselected pool (59 isolates) of LAB ascribed to species
(Lactobacillus spp., Lactococcus spp., Pediococcus spp. Car-
nobacterium spp., Weissella spp. and Streptococcus thermo-
philus) generally recognized as safe and potentially
utilized as starters or flavour-producing adjuncts were
investigated (Table 2). In table 2, the tolerance of these
isolates to 2, 4, and 6Æ5% NaCl is also reported.
As concerns the acidifying activities, three classes were
defined (Roushdy 1999): class I included the isolates that
lowered the pH to ‡ 6Æ0, while class II and class III com-
prised the isolates that reached 6Æ0 > pH ‡ 5Æ5 and
pH < 5Æ5 respectively. After 8 h of fermentation, only
eight isolates, which were classified as fast acid producers,
fell into class III: five of these (isolates 156, 212, 55, 23,
220) were lactococci, while the other three belonged to
Strep. thermophilus (isolate 59), C. maltaromicus (isolate
15) and Weissella spp. (isolate 17). Although the majority
of the remaining isolates were initially slow, for most of
them the acid production was enhanced later, and after
24 h, 40 further isolates fell into class III.
When the degree of caseinolytic breakdown in crude
cell-free extracts was investigated, the ranges of the prote-
olytic activities were: 113Æ61–410Æ37 lg glycine ml)1 for
the bacilli ascribed to Lactobacillus, Carnobacterium and
Weissella; 144Æ53–292Æ91 lg glycine ml)1 for the lactococci
954 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
L. Aquilanti et al.
and Strep. thermophilus; and 88Æ88–231Æ08 lg glycine ml)1
for the pediococci.
From the one-way anova, a high degree of diversity
was revealed within each taxon considered, except for
Lact. casei, the isolates of which were characterized by
acidifying, proteolitic and autolytic activities that were
not significantly different (Table 2a).
The ability of the 59 preselected isolates to spontane-
ously lyses in a saline solution was expressed as percent-
age decreases in the OD650 nm after 24 h incubation in a
lyses buffer. The results obtained showed levels of autoly-
sis ranging from about 10% to 47%. The highest values
(>40%) were detected for the three isolates ascribed to
Lact. casei (isolate 4, 30 and 198) and the isolate Lact.
brevis 1G, while the lowest percentages of autolysis
(£10%) were showed by two isolates (55, 155) belonging
to L. lactis ssp. lactis.
RAPD analysis
Fifty-five of the preselected pool of isolates, which were
ascribed to taxa including more than one isolate, under-
went molecular typing using the RAPD technique. Hierar-
chical cluster analysis of the RAPD profiles produced the
dendrogram shown in Fig. 1. An 85% similarity was arbi-
trarily chosen as the threshold for the grouping of the
isolates. Twenty-five clusters were defined. Nine of them
include more than one strain, while 16 of them contain
only one strain. In more detail, it was possible to define
six clusters for L. lactis ssp. cremoris, five for Ped. acidilac-
tici, three for Lact. brevis and Weissella ssp., two for
L. lactis ssp. lactis, Strep. thermophilus and C. maltaromicus,
and one for Lact. zeae and Lact. casei. A low RAPD pat-
tern variability was found within Lact. brevis, although
this species accounted for a high number of isolates. In
contrast, a unique RAPD profile was seen for each of the
isolates ascribed to L. lactis ssp. cremoris, Strep. thermo-
philus and C. maltaromicus.
Discussion
The uniqueness of traditional, artisan, raw-milk cheeses
manufactured without the addition of commercial starter
cultures has led to increased efforts towards the identifi-
cation and typing of dairy strains, as well as to the char-
acterization of their technological properties (Mannu
et al. 2000). These cheeses are characterized by the pres-
ence of wild bacterial populations, which are mainly com-
posed of mesophilic lactobacilli (De Angelis et al. 2001),
enterococci (Giraffa 2003) and lactococci (Mannu et al.
2000). One of the problems in identifying these micro-
organisms is the analogous nutritional requirements and
their growth under similar environmental conditions
ª 2007 The Authors
L. Aquilanti et al. Lactic acid bacteria from Pecorino cheese

Table 2 Technological characterization of the LAB isolated from the Pecorino cheese. The data are expressed as mean values ± standard devia-
tions. Within each data set referred to each LAB species, the values labelled with the same letter(s) are not significantly different (P < 0Æ05)

NaCl pH Release of
Resistance glycine Autolysis
Species Isolate Farm Source (%) 8h 24 h (lg mL)1) (%)

Lactobacillus 1G F1 90-d-cheese 6Æ5 5Æ75 ± 0Æ07 c 5Æ63 ± 0Æ04 b 410Æ37 ± 4Æ37 a 45Æ56 ± 2Æ11 a
brevis 2G 6Æ5 6Æ25 ± 0Æ03 a 5Æ62 ± 0Æ03 b 119Æ80 ± 4Æ37 d 21Æ11 ± 2Æ41 b
3G 6Æ5 6Æ24 ± 0Æ04 ab 6Æ10 ± 0Æ01 a 187Æ80 ± 4Æ37 cd 34Æ41 ± 1Æ52 ab
4G 6Æ5 6Æ27 ± 0Æ01 a 6Æ14 ± 0Æ01 a 178Æ53 ± 17Æ49 cd 22Æ00 ± 2Æ45 b
5G 6Æ5 5Æ94 ± 0Æ04 b 5Æ16 ± 0Æ08 bcd 212Æ53 ± 30Æ60 cd 31Æ54 ± 5Æ14 ab
6G 6Æ5 5Æ59 ± 0Æ02 c 4Æ97 ± 0Æ05 cd 206Æ35 ± 4Æ37 cd 27Æ43 ± 0Æ63 b
7G 6Æ5 6Æ24 ± 0Æ02 ab 5Æ56 ± 0Æ03 b 135Æ25 ± 8Æ74 d 20Æ32 ± 0Æ45 b
8G 6Æ5 6Æ22 ± 0Æ04 ab 5Æ37 ± 0Æ04 b 252Æ72 ± 8Æ74 bc 24Æ15 ± 0Æ59 b
10G 6Æ5 6Æ02 ± 0Æ01 b 4Æ68 ± 0Æ04 de 265Æ08 ± 34Æ97 bc 22Æ51 ± 2Æ65 b
11G 6Æ5 6Æ01 ± 0Æ01 b 4Æ45 ± 0Æ02 ef 218Æ71 ± 4Æ37 cd 19Æ01 ± 1Æ39 b
12G 6Æ5 6Æ25 ± 0Æ02 a 6Æ12 ± 0Æ01 a 234Æ17 ± 8Æ74 bcd 20Æ27 ± 2Æ27 b
13G 6Æ5 6Æ27 ± 0Æ01 a 6Æ15 ± 0Æ01 a 193Æ99 ± 4Æ37 cd 23Æ61 ± 1Æ96 b
15G 6Æ5 5Æ71 ± 0Æ10 c 4Æ28 ± 0Æ15 f 237Æ26 ± 21Æ85 cd 21Æ00 ± 1Æ14 b
18G 120-d-cheese 6Æ5 5Æ81 ± 0Æ19 bc 5Æ05 ± 0Æ09 cde 364Æ00 ± 8Æ74 a 18Æ53 ± 6Æ25 b
22G 6Æ5 5Æ82 ± 0Æ18 bc 5Æ17 ± 0Æ12 bcd 206Æ35 ± 4Æ37 cd 22Æ10 ± 1Æ80 b
23G 6Æ5 5Æ67 ± 0Æ11 c 4Æ44 ± 0Æ11 ef 178Æ53 ± 0Æ00 cd 24Æ08 ± 2Æ94 b
25G 6Æ5 5Æ66 ± 0Æ04 c 5Æ36 ± 0Æ22 b 212Æ53 ± 4Æ37 cd 17Æ74 ± 2Æ76 b
26G 6Æ5 5Æ61 ± 0Æ10 c 4Æ82 ± 0Æ13 cde 150Æ71 ± 4Æ37 d 24Æ82 ± 8Æ58 ab
27G 6Æ5 5Æ66 ± 0Æ04 c 5Æ30 ± 0Æ13 bc 215Æ62 ± 8Æ74 cd 19Æ66 ± 2Æ84 b
28G 6Æ5 6Æ21 ± 0Æ08 ab 5Æ34 ± 0Æ05 b 218Æ72 ± 4Æ37 cd 20Æ57 ± 1Æ65 b
29G 6Æ5 5Æ79 ± 0Æ11 bc 5Æ32 ± 0Æ03 b 292Æ91 ± 4Æ37 bc 20Æ45 ± 0Æ63 b
30G 6Æ5 5Æ72 ± 0Æ11 c 4Æ94 ± 0Æ01 cde 193Æ98 ± 4Æ37 cd 25Æ39 ± 4Æ10 b
Lact. zeae 3 F3 Milk 6Æ5 6Æ01 ± 0Æ01 c 5Æ18 ± 0Æ01 b 206Æ35 ± 13Æ12 a 21Æ02 ± 0Æ71 b
10 Curd 6Æ5 6Æ04 ± 0Æ01 b 5Æ42 ± 0Æ08 a 175Æ44 ± 4Æ37 b 17Æ38 ± 2Æ54 b
200 42-d-cheese 6Æ5 6Æ08 ± 0Æ01 a 5Æ37 ± 0Æ05 a 209Æ44 ± 8Æ74 a 33Æ88 ± 1Æ58 a
Lact. casei 4 F3 Milk 6Æ5 6Æ01 ± 0Æ05 a 5Æ51 ± 0Æ08 a 140Æ25 ± 15Æ25 a 44Æ03 ± 1Æ05 a
30 7-d-cheese 6Æ5 5Æ99 ± 0Æ06 a 5Æ50 ± 0Æ01 a 136Æ08 ± 10Æ52 a 47Æ00 ± 2Æ00 a
198 42-d-cheese 6Æ5 6Æ09 ± 0Æ01 a 5Æ48 ± 0Æ70 a 146Æ07 ± 17Æ05 a 46Æ59 ± 1Æ59 a
Lact. plantarum 39 F3 7-d-cheese 6Æ5 6Æ09 ± 0Æ10 n.d. 5Æ79 ± 0Æ13 n.d. 113Æ61 ± 4Æ37 n.d. 20Æ52 ± 0Æ79 n.d.
Lact. parabuchneri 103 F3 21-d-cheese 6Æ5 5Æ62 ± 0Æ06 n.d. 4Æ48 ± 0Æ07 n.d. 141Æ43 ± 17Æ48 n.d. 34Æ97 ± 3Æ00 n.d.
Weisella spp. 93G F2 Milk 6Æ5 5Æ55 ± 0Æ07 d 4Æ86 ± 0Æ06 c 166Æ16 ± 8Æ74 a 14Æ52 ± 0Æ95 d
94G 6Æ5 6Æ06 ± 0Æ08 abc 5Æ08 ± 0Æ03 b 243Æ45 ± 13Æ12 a 28Æ99 ± 0Æ25 a
96G 6Æ5 5Æ78 ± 0Æ03 cd 5Æ13 ± 0Æ04 b 268Æ18 ± 4Æ37 a 23Æ89 ± 1Æ57 b
100G 6Æ5 5Æ81 ± 0Æ16 bc 5Æ17 ± 0Æ04 b 187Æ80 ± 4Æ37 a 19Æ02 ± 1Æ39 c
17 F3 Curd 4 4Æ96 ± 0Æ04 e 4Æ77 ± 0Æ05 c 150Æ71 ± 4Æ37 a 15Æ50 ± 0Æ71 d
62 21-d-cheese 2 6Æ08 ± 0Æ11 ab 5Æ71 ± 0Æ09 a 187Æ80 ± 4Æ37 a 22Æ68 ± 1Æ07 b
Carnobacterium 5 F3 Milk 6Æ5 5Æ64 ± 0Æ05 a 4Æ84 ± 0Æ04 a 193Æ99 ± 4Æ37 b 28Æ00 ± 1Æ52 a
maltaromicus 15 Curd 4 4Æ95 ± 0Æ07 b 4Æ62 ± 0Æ04 b 200Æ17 ± 4Æ37 a 12Æ04 ± 3Æ17 b
C. divergens 224 F3 42-d-cheese 6Æ5 6Æ05 ± 0Æ07 n.d. 5Æ45 ± 0Æ04 n.d. 240Æ35 ± 17Æ49 n.d. 36Æ84 ± 0Æ67 n.d.
Lactococcus lactis 155 F3 7-d-cheese 6Æ5 5Æ50 ± 0Æ05 a 4Æ35 ± 0Æ05 d 170Æ02 ± 3Æ98 c 10Æ01 ± 0Æ29 b
ssp. lactis 156 4 4Æ81 ± 0Æ01 c 4Æ54 ± 0Æ02 c 166Æ19 ± 8Æ74 c 29Æ88 ± 3Æ10 a
157 4 5Æ58 ± 0Æ11 a 4Æ98 ± 0Æ04 a 240Æ35 ± 0Æ00 b 25Æ03 ± 0Æ71 a
212 14-d-cheese 4 4Æ98 ± 0Æ03 b 4Æ68 ± 0Æ09 b 292Æ91 ± 4Æ37 a 25Æ35 ± 1Æ71 a
55 21-d-cheese 4 5Æ48 ± 0Æ03 a 4Æ30 ± 0Æ01 d 163Æ07 ± 4Æ37 c 9Æ76 ± 0Æ34 b
L. lactis ssp. 20 F3 7-d-cheese 4 5Æ70 ± 0Æ01 b 4Æ66 ± 0Æ06 a 240Æ35 ± 8Æ74 ab 23Æ55 ± 11Æ36 a
cremoris 23 2 4Æ86 ± 0Æ06 c 4Æ59 ± 0Æ04 a 144Æ53 ± 4Æ37 c 20Æ71 ± 3Æ35 a
152 4 5Æ99 ± 0Æ01 a 4Æ59 ± 0Æ12 a 212Æ53 ± 4Æ38 b 16Æ93 ± 0Æ75 a
216 42-d-cheese 6Æ5 5Æ92 ± 0Æ07 a 5Æ09 ± 0Æ76 a 261Æ99 ± 4Æ35 a 25Æ61 ± 0Æ87 a
217 4 5Æ75 ± 0Æ06 b 4Æ90 ± 0Æ21 a 203Æ26 ± 17Æ49 b 25Æ83 ± 1Æ18 a
220 6Æ5 4Æ79 ± 0Æ02 c 4Æ64 ± 0Æ08 a 231Æ08 ± 13Æ12 b 23Æ16 ± 2Æ61 a
Streptococcus 16 F3 Curd 6Æ5 5Æ75 ± 0Æ07 a 4Æ99 ± 0Æ01 a 147Æ62 ± 8Æ74 b 15Æ03 ± 0Æ31 a
thermophilus 59 21-d-cheese 4 4Æ53 ± 0Æ04 b 4Æ05 ± 0Æ06 b 215Æ62 ± 0Æ00 a 16Æ42 ± 1Æ74 a

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960 955
Lactic acid bacteria from Pecorino cheese L. Aquilanti et al.

Table 2 Continued

NaCl pH Release of
Resistance glycine Autolysis
Species Isolate Farm Source (%) 8h 24 h (lg mL)1) (%)

Pediococcus 9 F3 Milk 4 5Æ83 ± 0Æ04 c 5Æ31 ± 0Æ01 a 88Æ88 ± 4Æ37 d 36Æ94 ± 1Æ51 a
acidilactici 132 6Æ5 5Æ79 ± 0Æ01 c 5Æ42 ± 0Æ14 a 181Æ62 ± 13Æ12 c 14Æ64 ± 0Æ51 bc
18 Curd 4 6Æ25 ± 0Æ04 a 5Æ48 ± 0Æ04 a 95Æ07 ± 4Æ37 d 29Æ84 ± 1Æ14 ab
19 7-d-cheese 4 5Æ95 ± 0Æ07 b 5Æ31 ± 0Æ01 a 206Æ35 ± 13Æ12 b 27Æ52 ± 7Æ49 abc
24 2 5Æ78 ± 0Æ03 c 4Æ97 ± 0Æ66 ab 231Æ08 ± 4Æ37 a 18Æ06 ± 9Æ31 bc
196 42-d-cheese 6Æ5 5Æ51 ± 0Æ01 d 4Æ37 ± 0Æ01 b 181Æ62 ± 4Æ37 c 21Æ67 ± 1Æ98 bc
Ped. 177 F3 42-d-cheese 6Æ5 5Æ61 ± 0Æ15 n.d. 4Æ53 ± 0Æ04 n.d. 197Æ07 ± 26Æ23 n.d. 21Æ89 ± 0Æ46 n.d.
pentosaceus

(Vandamme et al. 1996). For this reason, over the last
decade, several molecular techniques have been developed
and applied for their reliable identification, and a variety
of genotype-based methods is now available. One of these
is ARDRA, which has been successfully used for the dis-
crimination of LAB from a wide variety of food and envi-
ronments (Ben Amor et al. 2007).
Our results confirm the suitability of this molecular
technique for the generation of genus- and species-spe-
cific restriction patterns within the LAB groups, from
which we have obtained useful information on the com-
position of the microbiota involved in the Pecorino
cheese-making process. Nevertheless, the production of
atypical profiles not comparable to those obtained from
reference strains or nucleotide sequences deposited into
databases still represents an occasional drawback of the
method. The generation of these profiles is generally
caused by single mutations in the 16S rRNA gene or to a
strain-dependent variability in the large-subunit rDNA
sequence. When theoretical patterns are concerned, the
obtaining of atypical profiles might also be due to errors
in the sequences retrieved from published databases (Ro-
das et al. 2003). Finally, a further drawback of the AR-
DRA method is the impossibility of discriminating strictly
phylogenetically related species and subspecies, which has
already been highlighted in other taxonomic studies,
based on the comparative analysis of 16S rRNA gene
sequences (Rodas et al. 2003).
With regard to the biodiversity seen in Pecorino cheese,
it is widely acknowledged that enterococci constitute a
notable part of the bacterial population of artisan raw-
milk cheeses (Giraffa 2003). As previously shown, their
recovery in raw milk could be due to faecal contamina-
tion, either directly or indirectly through contaminated
water sources, milking equipment and bulk storage tanks
(Gelsomino et al. 2001), while their isolation from curd
and ripened cheeses can be ascribed to their tolerance to
high-salt content and low pH, as well as to their adapt-
ability to a wide range of different substrates and growth
conditions (Giraffa 2003). The peculiar cheese-making
technique used for the manufacture of Pecorino cheese,
which includes heating the curd to about 42C, could also
contribute to the selection of these thermally resistant
micro-organisms. Despite their remarkable abundance in
raw-milk cheeses, the use of these micro-organisms as
starter strains or adjuncts in the dairy industry is still
debated due to their feasible role as potential pathogens
and ⁄ or reservoirs of transferable antibiotic-resistance
genes (Giraffa et al. 1997).
Besides the enterococci, a large number of different
LAB species is shown here to participate in the Pecorino
cheese-making process. The species Lact. brevis, Lact.
plantarum and Lact. casei, as well as their closely related
species Lact. paracasei and Lact. zeae, have been previ-
ously detected in other ewes’ raw-milk cheeses (Avellini
et al. 1999; Cappello et al. 2001; De Angelis et al. 2001;
Coda et al. 2006; Randazzo et al. 2006). Lact. plantarum
and Lact. paracasei have recently been shown to enhance
flavour intensity when used as adjunct cultures during
cheese manufacturing (Antonsson et al. 2003), while Lact.
brevis has been hypothesized to impact on cheese ripen-
ing, because of its potential to metabolise amino acids via
non-transaminating reactions and endogenous transami-
nation (Liu et al. 2003).
The contribution of the Carnobacterium species to
cheese ripening has not been fully elucidated yet. Not-
withstanding, the species C. divergens and C. maltaromi-
cus have been described as members of the dominant
bacterial community in mozzarella cheese (Morea et al.
1999), and C. maltaromicus was found in mould-ripened
soft cheeses (Milliere et al. 1994; Herbin et al. 1997);
none of these have hitherto been reported in ewe’s milk
cheeses.
With regard to the technological characterization, for
almost all of the isolates, the tendency to lyses under star-
vation conditions to release glycine through the catabo-
lism of caseins, and to acidify the skimmed milk medium
appeared as strain-dependent characters, as they varied

ª 2007 The Authors

956 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
L. Aquilanti et al. Lactic acid bacteria from Pecorino cheese

primer
L. lactis ssp. cremoris 5´-GAGGGTGGCGGTTCT-3´ 5´-AGCAGCGTGG-3´
152
212
L. lactis ssp. lactis 55
155
156
157
Lact. casei 4
30
198
62
94G
Weisella spp. 96G
100G
93G
L. lactis ssp. cremoris 20
220
L. lactis ssp. cremoris 217
23
216
1G
3G
4G
25G
27G
6G
13G
7G
8G
18G
22G
28G
29G
26G
2G
12G
15G
Lact. brevis 23G
30G
5G
11G
10G
P. acidilactici
196
132
Lact. zeae 3
10
200
9
P. acidilactici 19
24
S. thermophylus
18
16
Weisella spp. 59
17
C. maltaromicus 5
15
0·20 0·40 0·60 0·80 1·00

Figure 1 Clustering of 55 preselected LAB isolated from raw milk, curd and Pecorino cheese on the basis of UPGMA-dendrograms from RAPD
profiles. An 85% similarity was arbitrarily chosen as a discriminating threshold to define the homogenous clusters. Each internode labelled with
( ) includes isolates ascribed to the same species.

significantly among strains within the same species. This 2004), Lactococcus (Ouzari et al. 2002) and Pediococcus
finding is in good agreement with the other studies on (Mora et al. 2003).
dairy isolates ascribed to Leuconostoc (Cibik and Chapot- With respect to the salt tolerance, our lactobacilli strains
Chartier 2000), Lactobacillus (Cibik and Chapot-Chartier were more resistant to sodium chloride than both mesophilic

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960 957
Lactic acid bacteria from Pecorino cheese
and thermophilic cocci. This evidence is in good agreement
with what reported by Korkeala et al. (1992), about the effect
of sodium chloride on growth of lactic acid bacteria.
For the autolytic phenotype, four isolates (1G, 4, 30,
198) were identified as undergoing a high level of auto-
lysis (>40%) in the system described; the characteristics
of one of these strains, the isolate Lact. brevis 1G that
showed both high proteolysis (glycine > 350 lg ml)1)
and extensive autolysis make it of particular interest to
the dairy industry. This isolate was also characterized by
a high salt resistance (6Æ5% NaCl), as well as the ability
to ferment galactose and to produce d- and l-lactic
acid.
The impact of cell lyses of the dairy starter strains on the
proteolysis rate has been acknowledged (Crow et al. 1995),
as well as its possible contribution to the decrease in bitter-
ness through the hydrolysis of large hydrophobic peptides
(Lepeuple et al. 1998). Thus, autolysis of LAB strains used
as starters in cheese-making is supposed to accelerate
ripening and affect flavour development (Chapot-Chartier
et al. 1994; Crow et al. 1995). Moreover, Bourdat et al.
(2002) indicated that amino acid catabolism leading to
aroma formation can also be stimulated by autolysis. As
confirmation, the use of a highly autolytic Lactobacillus
strain as an adjunct culture for the production of cheddar
cheese has greatly contributed to the increase in its proteo-
lysis and the improvement of its texture (Sallami et al.
2004). Further studies on the role of non-starter meso-
philic lactobacilli as ripening agents clearly highlighted
their crucial contribution to the cheese ripening at low
temperatures (8–10C) (Oberg and Broadbent 1993).
On the basis of acid production, the isolates were
divided into three groups, and only those in the one group
that lowered the pH to £5Æ0 after 8 h fermentation were
classified as fast acid producers. As the ability to lower pH
at a high and predictable rate and to contribute to second-
ary proteolysis during cheese ripening are essential in
cheese starter strains (Fox et al. 1990), we identified three
isolates (L. lactis ssp. lactis 212, L. lactis ssp. cremoris 220,
Strep. thermophilus 59) that proved to be both fast acid
producers and highly proteolytic for suitable starter cul-
tures for the production of uniformly high-quality
Pecorino cheese. All these isolates were also able to ferment
galactose, to produce l- lactic acid and to tolerate high salt
content (at least 4%). This latter feature contributes to
make them good candidates as starter cultures for the
manufacture of Pecorino cheese, as NaCl-tolerant micro-
organisms can act more actively during the ripening
process, after brining or dry salting of the cheese moulds.
Due to the critical role of proteolysis in the cheese
flavour and body development, the characterization of
this activity in cultures candidates as starters or adjuncts
for the cheese-making industry is of great importance
958 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 948–960
L. Aquilanti et al.
(Oberg and Broadbent 1993). The screening of the prote-
olytic ability of our isolates with the o-phthaldialdehyde
test allowed a considerable variation in the concentration
of glycine released to be observed, except for the strains
belonging to Weissella spp. which exhibited a much smal-
ler range of proteolysis. In more detail, a five-fold differ-
ence in total proteolysis between the highest and lowest
strains was recorded, thus confirming the strain-depen-
dant diversity of the proteolytic enzymes of LAB (Oberg
and Broadbent 1993).
By comparing the proteolytic and acidifying activities
of our tested isolates, it was not always possible to high-
light a strict correlation between these two features. This
finding is in agreement with the data reported by other
authors (Fortina et al. 1998; Durlu-Ozkaya et al. 2001).
We found likewise a few isolates with high acidifying
(pH < 5Æ0 after 24 h) and moderate or low proteolytic
(glycine released < 200 lg ml)1) activities, as well as an
isolate (Lact. brevis 1G) that shows very low acidification
(pH > 5Æ5 after 24 h) but very high proteolysis (glycine
released > 350 lg ml)1).
As previously shown, the RAPD technique can be used
successfully for the typing of dairy LAB strains (Coccon-
celli et al. 1995; De Angelis et al. 2001). With a few rare
exceptions (isolates 152 and 17), the isolates ascribed to
the same taxon were clustered together, because of the
presence of species-specific RAPD patterns. Except for
Lact. brevis, this typing technique allowed the highlighting
of a high genotypic polymorphism within each of the
nine LAB species assayed. Different numbers of strains
within the same species were found to participate in the
cheese-making process, ranging from a minimum of one
(Lact. casei and Lact. zeae) to a maximum of six (L. lactis
ssp. cremoris). In a few cases, the isolates that showed
identical RAPD profiles were also found to be character-
ized by autolytic, proteolytic and acidifying activities that
are not statistically different. This is the case for the three
isolates (4, 30, 198) ascribed to Lact. casei, which are
therefore considered as clones.
By using a combination of phenotypic and molecular
techniques, an effective species and strain differentiation
was achieved, and the dominant LAB species and biotypes
in these Pecorino cheeses from the Marche region were
defined. From the evaluation of the microbial
communities investigated, a remarkable heterogeneity
emerged, which can probably be ascribed to the use of
raw milk with no added starter cultures. The detection of
such microbial variability underlines once more the
importance of protecting the natural microbiota of
traditional cheeses that are produced according to local
practices, and possibly to encourage the exploitation of
new selected strains showing promising cheese-making
characteristics.
ª 2007 The Authors
L. Aquilanti et al.
Acknowledgments
This work was supported by the Italian ‘‘Ministero delle
Politiche Agricole e Forestali’’, special grant: ‘‘Valorizzazi-
one e salvaguardia della microflora caratteristica delle
produzioni casearie italiane’’, Publication no. 14. The
authors wish to thank Caseificio Gneddu (Ancona, Italy),
Caseificio Marchese (Macerata, Italy) and Caseificio
Chessa (Montecarotto, Ancona, Italy) for supplying the
raw milk, curd and Pecorino cheese samples.
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