International Journal of Food Microbiology 127 (2008) 168171

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Short communication

Grana Padano cheese whey starters: Microbial composition and strain distribution
Lia Rossetti a, Maria Emanuela Fornasari a, Monica Gatti b, Camilla Lazzi b,
Erasmo Neviani b, Giorgio Giraffa a,
a
b

Agriculture Research Council, Research Centre for Forage and Dairy Productions (CRA-FLC), Viale Piacenza 29, 26900 Lodi, Italy
Department of Genetics, Biology of Microorganisms, Anthropology, Evolution, University of Parma, Viale Usberti 11/A, 43100 Parma, Italy

A R T I C L E

I N F O

Article history:
Received 10 May 2008
Received in revised form 6 June 2008
Accepted 6 June 2008
Keywords:
Thermophilic lactic acid bacteria
Microbial identication
Microbial typing
Grana Padano cheese
Artisan whey starters

A B S T R A C T
The aim of this work was to evaluate the species composition and the genotypic strain heterogeneity of
dominant lactic acid bacteria (LAB) isolated from whey starter cultures used to manufacture Grana Padano
cheese. Twenty-four Grana Padano cheese whey starters collected from dairies located over a wide
geographic production area in the north of Italy were analyzed. Total thermophilic LAB streptococci and
lactobacilli were quantied by agar plate counting. Population structure of the dominant and metabolically
active LAB species present in the starters was proled by reverse transcriptase, length heterogeneity-PCR
(RTLHPCR), a culture-independent technique successfully applied to study whey starter ecosystems. The
dominant bacterial species were Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Streptococcus
thermophilus, and Lactobacillus fermentum. Diversity in the species composition allowed the whey cultures to
be grouped into four main typologies, the one containing L. helveticus, L. delbrueckii subsp. lactis, and S.
thermophilus being the most frequent one (45% of the cultures analyzed), followed by that containing only
the two lactobacilli (40%). Only a minor fraction of the cultures contained L. helveticus alone (4%) or all the
four LAB species (11%). Five hundred and twelve strains were isolated from the 24 cultures and identied by
M13-PCR ngerprinting coupled with 16S rRNA gene sequencing. Most of the strains were L. helveticus (190
strains; 37% of the total), L delbrueckii subsp. lactis (90 strains; 18%) and S. thermophilus (215 strains; 42%).
This result was in good agreement with the qualitative whey starter composition observed by RTLHPCR.
M13-PCR ngerprinting indicated a markedly low infra-species diversity, i.e. the same biotypes were often
found in more than one culture. The distribution of the biotypes into the different cultures was mainly dairy
plant-specic rather than correlated with the different production areas.
2008 Elsevier B.V. All rights reserved.

1. Introduction
Artisan starter cultures are used for the production of Italian hard
cheeses such as Grana Padano, Parmigiano Reggiano, and Provolone,
where they play an acknowledged role in the curd acidication and
achievement of the sensory characteristics of the cheese (Beresford
et al., 2001). Artisan cultures are still prepared in the traditional way by
removing some of the whey drained from the cheese vat at the end of
cheese-making. Nowadays, the cheese-making of Italian hard cheeses
includes a cooking step at 5055 C, which is usually applied before
whey drainage. The whey, which is still at 4852 C before drainage, is
then incubated at a controlled temperature of 4445 C until the pH
reaches a nal value of about 3.5 (Santarelli et al., 2008). The
concomitant pressure of both chemical and physical actions leads to
the selection of a characteristic microora, consisting of thermophilic,
aciduric, and moderately heat resistant lactic acid bacteria (LAB). It is
well established that whey starters for hard cooked cheeses are
Corresponding author. Research Unit: Dairy Productions, Via Lombardo 11, 26900
Lodi, Italy. Tel.: +39 0371 45011; fax: +39 0371 35579.
E-mail address: giorgio.giraffa@entecra.it (G. Giraffa).
0168-1605/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2008.06.005

dominated by a thermophilic LAB microbiota belonging to Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus
delbrueckii subsp. lactis, Lactobacillus fermentum, and Streptococcus
thermophilus (Parente and Cogan, 2004).
Apparently, the microbial composition and diversity of Grana Padano
cheese whey starters are modulated by the selectivity of the incubation
conditions of the drained whey, in strict synergy with small variations in
the technology applied (Neviani et al., 1995, Giraffa et al., 1998).
However, seasonal and geographical uctuations in the microbial
composition and acidication performance of the starters have often
been reported (Fortina et al.,1998; Giraffa et al., 2004). This suggests that
the biological richness of these cultures is related also to the microbial
composition of the drained whey, which in turn is related to that of the
raw milk. Several exhaustive studies concerning the microbial composition and diversity of the microbiota associated with the natural whey
starters for Grana cheese have been carried out (Giraffa et al.,1998, 2000;
Fortina et al., 1998; Gatti et al., 2004). Microbial diversity and population
structure of the LAB community present in these starters have been
described through length heterogeneity-PCR (LH-PCR), a cultureindependent technique (Lazzi et al., 2004). Nevertheless, recent
investigations suggest that a limited number of genotypically diverse

L. Rossetti et al. / International Journal of Food Microbiology 127 (2008) 168171

lactobacilli seem to dominate these cultures and that, in some instances,

the lack of cultivability of a portion of the thermophilic LAB microora
may affect the recovery of all the strains associated with the whey
starters (Cattivelli et al., 2002; Fornasari et al., 2006; Lazzi et al., 2007).
This seems to suggest that the actual microbial composition of the whey
starters is partially biased by ecological phenomena, and justies the
need for more updated knowledge about the thermophilic LAB
microora associated with these cultures.
The aim of this study was to further the knowledge on the
microbial composition and diversity of whey starter cultures for Grana
Padano cheese. To this end, 24 cultures collected over the last two
years from dairies located in a wide geographical production area
situated in the north of Italy were analyzed using both culturedependent and culture-independent methods. About 500 thermophilic LAB isolates were identied and characterized.
2. Materials and methods
2.1. Whey starters sampling
Twenty-four natural whey starters for Grana Padano cheese were
investigated. Samples named as starter 124, were collected, just
before being used, from small or medium size, local dairies located in
eight different provinces of Lombardia, Veneto, and Emilia Romagna
regions [1: Bergamo (BG); 26: Brescia (BS); 79: Cremona (CR); 10:
Lodi (LO); 1113: Mantova (MN); 1418: Piacenza (PC); 1921:
Vicenza (VI); 2224: Verona (VR)]. Culture samples were cooled at
46 C at the dairy plant and shipped under refrigerated transport to
the laboratory, where they were immediately analyzed.

169

variable regions of the 16S rRNA gene was reverse transcribed and
amplied using LH-PCR primers described previously (Lazzi et al., 2004).
One step RT-PCR was performed using the Gene Amp EZ rTth RNA PCR
Kit (Applera Italia, Monza, Italy) according to the instructions given by
the manufacturer. Segments of 16S rRNA gene were reverse transcribed
by an initial incubation at 60 C for 30 min. The resulting cDNA was
amplied by PCR using the following conditions: one cycle of 94 C for
1 min, 20 cycles of 94 C for 15 s, 49 C for 30 s, and 72 C for 30 s. Final
extension was carried out at 72 C for 7 min. Reactions were carried out
in a 9700 PCR system (Applied Biosystem, Foster City, CA). PCR without a
reverse transcription step was performed to verify the absence of
contaminant DNA. Bacterial composition of the 24 whey starters was
evaluated after separation of RT-PCR products by capillary electrophoresis on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Runs
were performed under denaturing conditions and LHPCR proles were
analyzed using Genescan software (version 3.1; Applied Biosystems).
The size, in basepairs, of the LHPCR fragments was estimated by
reference to the internal size standard using the Local Southern method
and no smoothing option.
2.3. Microbial counts and isolation of thermophilic LAB
Thermophilic lactobacilli were counted on whey agar medium
(WAM), according to Gatti et al. (2003) after anaerobic incubation at
42 C for 48 h. Thermophilic streptococci were counted on M17 agar
containing 7% (vol:vol) of sterile skimmed whey (M17-SSW) as
reported by Fornasari et al. (2006), after incubation at 37 C for
48 h. Both counts were performed in duplicate. A total of 512 colonies
from WAM and M17-SSW agar plates were selected by morphology,
isolated, and grown on appropriate media.

2.2. LHRTPCR analysis

2.4. Strain identication and typing
Length heterogeneityreverse transcriptasePCR (LHRTPCR) analysis was carried out on 24 whey starters. Total RNA was extracted using
Trizol Reagent (Invitrogen Srl, Milano, Italy) following the instructions
given by the supplier and stored at 80 C until use. Domain A of the

Thermophilic LAB isolates were preliminary identied by RAPDPCR ngerprinting according to Rossetti and Giraffa (2005). Genomic
DNA was extracted and used as a template in PCR ngerprinting

Table 1
Microbial analysis of Grana Padano cheese whey starters
Whey starters

Production
provincea (region)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Average

BG (Lombardia)
BS (Lombardia)
BS (Lombardia)
BS (Lombardia)
BS (Lombardia)
CR (Lombardia)
CR (Lombardia)
CR (Lombardia)
LO (Lombardia)
MN (Lombardia)
MN (Lombardia)
MN (Lombardia)
MN (Lombardia)
PC (Emilia-Romagna)
PC (Emilia-Romagna)
PC (Emilia-Romagna)
PC (Emilia-Romagna)
PC (Emilia-Romagna)
VI (Veneto)
VI (Veneto)
VI (Veneto)
VR (Veneto)
VR (Veneto)
VR (Veneto)

Microbial counts
(log CFU mL 1 SD)

Bacterial composition determined by RTLHPCR

WAM

M17-SSW

S. thermophilus

L. delbrueckii

L. helveticus

L. fermentum

9.01 0.08
8.70 0.01
9.74 0.01
9.10 0.07
8.93 0.01
9.80 0.02
8.81 0.12
8.67 0.05
8.61 0.30
9.90 0.01
9.65 0.06
9.42 0.04
9.48 0.06
9.39 0.03
8.73 0.03
9.84 0.01
8.58 0.03
9.11 0.12
9.01 0.02
8.47 0.10
8.80 0.01
8.80 0.04
9.31 0.01
9.11 0.03
9.12 0.44

6.99 0.07
6.08 0.11
7.41 0.03
7.00 0.03
7.85 0.01
8.41 0.08
5.80 0.11
7.61 0.01
6.39 0.06
5.99 0.01
7.51 0.02
7.38 0.01
7.67 0.01
5.27 0.04
4.19 0.16
6.28 0,01
4.59 0.16
4.95 0.02
3.65 0.26
2.00 0.07
3.00 0.17
7.58 0.04
7.92 0.09
7.64 0.02
6.22 1.72

bLOD
19%
9%
2%
4%
11%
bLOD
34%
5%
5%
3%
3%
11%
bLOD
13%
2%
10%
bLOD
2%
bLOD
bLOD
2%
bLOD
bLOD

11%
31%
27%
13%
24%
46%
6%
22%
25%
27%
30%
26%
45%
44%
28%
45%
32%
20%
14%
32%
22%
39%
27%
17%

89%
47%
64%
85%
72%
43%
94%
44%
70%
68%
67%
71%
44%
56%
60%
50%
58%
80%
85%
69%
78%
59%
73%
83%

bLOD
3%
bLOD
bLOD
bLOD
1%
bLOD
1%
bLOD
bLOD
bLOD
bLOD
bLOD
bLOD
bLOD
3%
bLOD
bLOD
bLOD
bLOD
bLOD
bLOD
bLOD
bLOD

Counts of thermophilic lactobacilli were performed in whey agar medium (WAM) and counts of thermophilic streptococci were performed in M17-SSW. Bacterial composition of the
same samples were determined by RTLHPCR and expressed in percentage of the species; the lower limit of detection (LOD) of the technique is 105CFU ml 1 (Lazzi et al., 2004).
a
BG: Bergamo; BS: Brescia; CR: Cremona; LO: Lodi; MN: Mantova; PC: Piacenza; VI: Vicenza; VR: Verona.

170

L. Rossetti et al. / International Journal of Food Microbiology 127 (2008) 168171

experiments using as a primer the M13 minisatellite core sequence

(Huey and Hall, 1989) with sequence 5'-GAGGGTGGCGGTTCT-3'. After
amplication, cluster analysis of the electrophoretic proles was
carried out by BioNumerics (Version 3.0; Applied Maths, BVBA, SintMartens-Latem, Belgium) and dendrograms were set at a similarity
level of 75%, which is the reproducibility level of the RAPD-PCR
method applied; RAPD-PCR ngerprints were compared to previously
implemented genotypic libraries to obtain a preliminary strain
identication on the basis of RAPD-PCR prole similarity (Rossetti
and Giraffa, 2005). On a reduced number of strains, including both
strains with atypical genotypic proles and representatives of each
genotypic cluster, the identication was rened by 16S rRNA gene
sequencing. The species assignment was performed through BlastN
(www.ncbi.nlm.nih.gov/BLAST) alignment of the obtained sequences
with the 16S rRNA gene sequences of LAB available from the EMBL
database. DNA extraction and sequencing were performed as
described previously (Rossetti and Giraffa, 2005).
3. Results and discussion
An overall picture of the dominant microora of twenty-four Grana
Padano cheese whey starters was determined by plate count and RT
LHPCR analysis (Table 1). The thermophilic lactobacilli and streptococci were evaluated using two different agar media containing whey
(WAM and M17-SSW), which were shown to be more suitable than
MRS and M17 to recover the rod-shaped and coccus-shaped (almost
exclusively as S. thermophilus) whey starter LAB, respectively (Gatti
et al., 2003; Fornasari et al., 2006). Thermophilic lactobacilli were
dominant in almost all the cultures. Their counts in WAM resulted
very similar and ranged from 8.58 to 9.90 log CFU mL 1, with an
average of 9.12 0.44 log CFU mL 1. The counts of thermophilic
streptococci ranged from 2.00 to 8.41 log CFU mL 1, with an average of
6.22 1.72 log CFU mL 1. M17-SSW counts enabled better differentiation of the cultures. The three cultures from plants 1921, coming from
the province of Vicenza, showed the lowest streptococcal counts
whereas the cultures from plants 35, 6, 1113, and 2224 coming
from the provinces of Brescia, Cremona, Mantova, and Verona
respectively, showed the highest ones. Differences in M17-SSW
counts between cultures appeared mostly plant-dependent and not
in relation to the different production areas (Table 1).
Length heterogeneityreverse transcriptasePCR (LHRTPCR) has
recently been applied to study the metabolically active microbial
populations recoverable from Grana Padano cheese whey starters
(Fornasari et al., 2006; Santarelli et al., 2008). RTLHPCR proles,
which are obtained from total RNA extracted from whey starters and
amplied by RTPCR, allow the discrimination of the dominant and
metabolically active bacterial species present in the LAB community.
The use of RTPCR is based on the evidence that active bacteria have
generally higher numbers of ribosomes than dead or dormant cells.
Since the areas under the peaks shown in the electropherograms are a
rough measure of the proportions of the species, their relative
estimation was also possible (Lazzi et al., 2004).
Length heterogeneityreverse transcriptasePCR (LHRTPCR)
revealed the presence of four metabolically active species, of which
L. helveticus and L. delbrueckii, were the two dominant ones, thus
conrming previous ndings (Lazzi et al., 2004; Fornasari et al., 2006;
Santarelli et al., 2008 The cultures were grouped into four typologies,
the one containing L. helveticus, L. delbrueckii, and S. thermophilus
being the most frequent one (45% of the cultures analyzed), followed
by that containing only the two lactobacilli (40%). Only a minor
fraction of the cultures contained L. helveticus alone (4%) or all the four
LAB species (11%) (Table 1).
A total of 286 and 226 isolates were picked up from WAM and
M17-SSW agar plates, respectively. A RAPDPCR ngerprinting was
performed to preliminary identify isolates and investigate the
microbial diversity beyond the species level, i.e. to establish the

number of genotypically different biotypes. Identication was possible

by grouping unknown DNA patterns within existing pattern groups
(each corresponding to a given taxonomic unit) using the Bionumerics
software. Even if this approach is generally considered as reliable,
giving N95% of correct identications (Rossetti and Giraffa, 2005), we
performed a further identication of representative biotypes within
the RAPDPCR pattern groups by 16S rRNA gene sequencing (data not
shown). Of the 512 isolates, 11 did not grow after plate isolation
and were discarded, 215 were identied as S. thermophilus, 190 as
L. helveticus, and 90 as L. delbrueckii subsp. lactis. The last six strains,
probably coming from whey contamination, belonged to Streptococcus
bovis (two strains), Streptoccocus mitis (two strains), and Staphylococcus epidermidis (two strains).
A good agreement in species proling between the cultureindependent and the culture-dependent results was observed. Moreover, there was a similar composition between whey starters used for
the production of cheeses produced by different technologies and in
very distant geographical areas, such as Grana Padano and Caciocavallo Silano, suggesting that technological rather than ecological
Table 2
Distribution of L. helveticus, L. delbrueckii subsp. lactis, and S. thermophilus biotypes in
whey starters
Species

Biotypes

Frequency of
biotypes within
the species (%)a

Biotype distribution in different

starters, labelled 1 to 24
(production provinceb)

L. helveticus

a
b
c
d

1.1
0.5
20.0
14.7

e
f
g
h

2.1
0.5
1.1
21.1

i
j
k
l
m

0.5
2.1
0.5
0.5
4.6

10.5

o
p

4.2
5.2

q
r

0.5
6.7

s
t
u
a

2.5
0.5
0.5
8.9

b
c

2.2
70.0

d
a
b
c

18.9
4.2
4.7
89.8

1.4

11 (MN)
16 (PC)
8 (CR); 9 (LO)
3 (BS); 7 (CR); 8 (CR); 16 (PC); 20 (VI);
23 (VR); 24 (VR)
16 (PC); 19 (VI); 20 (VI); 23 (VR)
23 (VR)
11 (MN)
5 (BS); 7 (CR); 8 (CR); 9 (LO);
12 (MN); 14 (PC); 16 (PC);
1921 (VI); 23 (VR)
21 (VI)
11 (MN); 16 (PC)
14 (PC)
4 (BS)
1 (BG); 10 (MN); 15 (PC); 16 (PC);
21 (VI); 23 (VR)
3 (BS); 6 (CR); 9 (LO); 11 (MN);
12 (MN); 16 (PC); 17 (PC); 24 (VR)
1 (BG); 4 (BS); 15 (PC), 18 (PC)
1 (BG); 7 (CR); 15 (PC); 18 (PC);
19 (VI); 24 (VR)
16 (PC)
3 (BS); 68 (CR); 1012 (MN);
17 (PC); 19 (VI)
4 (BS); 24 (VR)
5 (BS)
17 (PC)
9 (LO); 14 (PC), 15 (PC); 17 (PC);
24 (VR)
15 (PC); 17 (PC)
1 (BG); 35 (BS); 7
(CR); 1012 (MN); 1418 (PC);
1921 (VI); 23 (VR);
24 (VR)
8 (CR); 9 (LO)
2 (BS); 13 (MN)
8 (CR); 9 (LO)
1 (BG); 35 (BS); 68 (CR); 1013
(MN); 14 (PC); 1618 (PC);
2224 (VR)
3 (BS)

L. delbrueckii
subsp. lactis

S. thermophilus

The number of biotypes for each species was calculated by cluster analysis at a similarity
level of 75%.
a
The % within each species is calculated as follows: (number of isolates of each
biotype / number of isolates of all biotypes) 100.
b
BG: Bergamo; BS: Brescia; CR: Cremona; LO: Lodi; MN: Mantova; PC: Piacenza;
VI: Vicenza; VR: Verona.

L. Rossetti et al. / International Journal of Food Microbiology 127 (2008) 168171

pressures are determinant in selecting thermophilic LAB microbiota

associated with these cultures (Andrighetto et al., 2004; Ercolini et al.,
in press). The lack of L. fermentum isolates from cultures 2, 6, 8, and 16
where this species had been detected by RTLHPCR may be due to
either a lack of cultivability of this species or its low frequency in
the cultures (Table 1). Differently from Coppola et al. (1997) only
L. delbrueckii subsp. lactis was detected after colony isolation, thus
suggesting that L. delbrueckii subsp. bulgaricus is either absent or
uncultivable in Grana Padano cheese whey starters.
While L. helveticus and L. delbrueckii subsp. lactis were both
metabolically active (according to RTLHPCR) and cultivable (according to plate isolation), this was not always the case for S. thermophilus.
More specically, S. thermophilus was not detected by RTLHPCR in
eight whey starters, although in three of them (i.e. whey starters 1, 23
and 24) M17-SSW counts were well above the detection limit of the
technique, which is 105 CFU mL 1 (Lazzi et al., 2004). On the contrary,
S. thermophilus was detectable (and thus metabolically active) by RT
LHPCR in samples 15, 17 and 19, although the corresponding M17SSW counts resulted lower than the detection limit of the technique
(Table 1). A different viability and cultivability in media with different
composition and buffering ability could explain the variable trends in
S. thermophilus proling obtained by culture-independent and
culture-dependent approaches (van de Guchte et al., 2002; Fornasari
et al., 2006; Gatti et al., 2006). These data conrm once more the need
to apply a polyphasic approach taking into account both traditional,
culture-based and culture-independent methods to quantitatively and
qualitatively assess the composition of complex microbiota associated
with dairy ecosystems (Coppola et al., 2008).
Cluster analysis of RAPD-PCR proles showed that, at a similarity
level of 75%, L. helveticus, S. thermophilus, and L. delbrueckii subsp. lactis
strains were grouped into 21, four, and four genotypically different
biotypes, respectively (Table 2). More specically, a relatively wider
genotypic diversity emerged within L. helveticus although only four
biotypes (i.e. c, d, h, and n), which accounted for 66.3% of the isolates,
dominated over the remaining L. helveticus population. The better
adaptability of the aciduric L. helveticus to the stressing conditions
caused by the strongly acidic environment of the whey starter may
explain this higher strain diversity. Conversely, only few dominant
L. delbrueckii subsp. lactis and S. thermophilus biotypes were isolated, of
which two of them (i.e. biotypes c of both species) accounted for 70 and
89,9% of the isolates of the two species, respectively (Table 2).
Each culture was composed of two to ve different biotypes of L.
helveticus with the exception of culture 16, which was composed of eight
different biotypes. The most widespread L. helveticus biotypes were h,
r, n, and d, which were found in 11, nine, eight, and seven different
starters, respectively. Similarly, the most commonly found biotypes of
L. delbrueckii subsp. lactis and S. thermophilus were also the most
frequently found in the cultures, being present in 18 out of 24 samples.
However, not always the most abundant biotypes within a species
resulted also the most widespread in the cultures. This was the case of
biotype r, which only represented 7% of the L. helveticus biotypes but was
found in nine samples (Table 2). The qualitative distribution of the
biotypes into the different cultures was mainly dairy plant-specic
rather than correlated with the different production areas or provinces.
About half of the 21 L. helveticus biotypes appeared exclusively present
in some cultures; this was also the case of biotype d of S. thermophilus,
which was only present in the culture 3. We speculate that the dominant
LAB species of the whey starters are composed of sub-populations which
are differently modulated by the variability in the cheesemaking
parameters and/or the culture preparation.
This study allowed us to add further the knowledge on the
microbial composition, diversity, and species and strain distribution of
whey starter cultures for Grana Padano cheese recovered from dairies
located in a wide geographical production area. This extensive study
also gave a collection of different biotypes belonging to the dominant

171

LAB populations, which will be useful to preserve the characteristic

germoplasm of these natural cultures.
Acknowledgements
We are grateful to Dr. Angelo Stroppa who collected the whey starter
cultures analysed in this work. This research was realised in part through
the nancial support of the Consorzio per la Tutela del Formaggio Grana
Padano, Desenzano del Garda (Italy), which is administered by the
Agricultural Research Service of the Region Lombardy.
References
Andrighetto, C., Marcazzan, G., Lombardi, A., 2004. Use of RAPD-PCR and TGGE for the
evaluation of biodiversity of whey cultures for Grana Padano cheese. Letters in
Applied Microbiology 38, 400405.
Beresford, T.P., Fitzsimons, N.A., Brennan, N.L., Cogan, T., 2001. Recent advances in
cheese microbiology. International Dairy Journal 11, 259274.
Cattivelli, D., Parisi, M.G., Cappa, F., Cocconcelli, P.S., 2002. Ecology of lactic acid bacteria
from natural whey cultures. Book of abstracts, 7th Symposium on lactic acid
bacteria, September 1-5, 2002, Egmond aan Zee, The Netherlands. p. A46.
Coppola, R., Nanni, M., Iorizzo, M., Sorrentino, A., Sorrentino, E., Grazia, L., 1997. Survey
of lactic acid bacteria isolated during the advance stages of the ripening of
Parmigiano Reggiano cheese. Journal of Dairy Research 64, 305310.
Coppola, S., Blaiotta, G., Ercolini, D., 2008. Dairy products. In: Cocolin, L., Ercolini, D.
(Eds.), Molecular techniques in the microbial ecology of fermented foods. Springer
Science, New York, pp. 3190.
Ercolini, D., Frisso, G., Mauriello, G., Salvatore, F., Coppola, S., Microbial diversity in
natural whey cultures used for the production of Caciocavallo Silano PDO cheese.
International Journal of Food Microbiology (in press; available online 27 March 2008).
Fornasari, M.E., Rossetti, L., Carminati, D., Giraffa, G., 2006. Cultivability of Streptococcus
thermophilus in Grana Padano cheese whey starters. FEMS Microbiology Letters 257,
139144.
Fortina, M.G., Nicastro, G., Carminati, D., Neviani, E., Manachini, P.L., 1998. Lactobacillus
helveticus heterogeneity in natural cheese starters: the diversity in phenotypic
characteristics. Journal of Applied Microbiology 84, 7280.
Gatti, M., Lazzi, C., Rossetti, L., Mucchetti, G., Neviani, E., 2003. Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano
Reggiano cheese. Journal of Applied Microbiology 95, 463470.
Gatti, M., Trivisano, C., Fabrizi, E., Neviani, E., Gardini, F., 2004. Biodiversity among
Lactobacillus helveticus strains isolated from different natural whey starter cultures
as revealed by classication trees. Applied and Environmental Microbiology 70,
182190.
Gatti, M., Bernini, V., Lazzi, C., Neviani, E., 2006. Fluorescence microscopy for studying
the viability of micro-organisms in natural whey starters. Letters in Applied
Microbiology 42, 338343.
Giraffa, G., Rossetti, L., Mucchetti, G., Addeo, F., Neviani, E., 1998. Inuence of the
temperature gradient on the growth of thermophilic lactobacilli used as natural
starters in grana cheese. Journal of Dairy Science 81, 3136.
Giraffa, G., Gatti, M., Rossetti, L., Senini, L., Neviani, E., 2000. Molecular diversity within
Lactobacillus helveticus as revealed by genotypic characterization. Applied and
Environmental Microbiology 66, 12591265.
Giraffa, G., Andrighetto, C., Antonello, C., Gatti, M., Lazzi, C., Marcazzan, G., Lombardi, A.,
Neviani, E., 2004. Genotypic and phenotypic diversity of Lactobacillus delbrueckii
subsp. lactis strains of dairy origin. International Journal of Food Microbiology 91,
129139.
Huey, B., Hall, J., 1989. Hypervariable DNA ngerprinting in E. coli. Minisatellite probe
from bacteriophage M13. Journal of Bacteriology 171, 25282532.
Lazzi, C., Rossetti, L., Zago, M., Neviani, E., Giraffa, G., 2004. Evaluation of bacterial
communities belonging to natural whey starters for Grana Padano cheese by length
heterogeneity-PCR. Journal of Applied Microbiology 96, 481490.
Lazzi, C., Gatti, M., Bernini, V., De Dea Lindner, J., Neviani, E., 2007. Impiego di nuovi
terreni colturali a base di cagliata e di formaggio per il recupero e la differenziazione
della microora caratteristica di formaggi a lunga stagionatura. Scienza e Tecnica
Lattiero Casearia 58, 5569.
Neviani, E., Divizia, R., Abbiati, E., Gatti, M., 1995. Acidication activity of thermophilic
lactobacilli under the temperature gradient of Grana cheese making. Journal of
Dairy Science 78, 12481252.
Parente, E., Cogan, T.M., 2004. Starter cultures: general aspects. In: Fox, P.F., McSweeney,
P.L.H., Cogan, T.M., Guinee, T.P. (Eds.), Cheese Chemistry, Physics and Microbiology. Elsevier Academic Press, London, pp. 123148.
Rossetti, L., Giraffa, G., 2005. Rapid identication of dairy lactic acid bacteria by M13generated, RAPD-PCR ngerprint databases. Journal of Microbiological Methods 63,
135144.
Santarelli, M., Gatti, M., Lazzi, C., Bernini, V., Zapparoli, G.A., Neviani, E., 2008. Whey
starter for Grana Padano cheese: effect of technological parameters on viability and
composition of the microbial community. Journal of Dairy Science 91, 883891.
van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D., Maguin, E., 2002.
Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek 82, 187216.