Grana Padano cheese whey starters: Microbial composition and strain distribution

International Journal of Food Microbiology 127 (2008) 168–171

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 identification 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 quantified by agar plate counting. Population structure of the dominant and metabolically active LAB species present in the starters was profiled by reverse transcriptase, length heterogeneity-PCR (RT–LH–PCR), 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 identified by M13-PCR fingerprinting 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 RT–LH–PCR. M13-PCR fingerprinting 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-specific 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 acidification 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 50–55 °C, which is usually applied before whey drainage. The whey, which is still at 48–52 °C before drainage, is then incubated at a controlled temperature of 44–45 °C until the pH reaches a final 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 microflora, 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: [email protected] (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 fluctuations in the microbial composition and acidification 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) 168–171

lactobacilli seem to dominate these cultures and that, in some instances, the lack of cultivability of a portion of the thermophilic LAB microflora 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 justifies the need for more updated knowledge about the thermophilic LAB microflora 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 identified 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 1–24, 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); 2–6: Brescia (BS); 7–9: Cremona (CR); 10: Lodi (LO); 11–13: Mantova (MN); 14–18: Piacenza (PC); 19–21: Vicenza (VI); 22–24: Verona (VR)]. Culture samples were cooled at 4–6 °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 amplified 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 amplified 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 LH–PCR profiles were analyzed using Genescan software (version 3.1; Applied Biosystems). The size, in basepairs, of the LH–PCR 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. LH–RT–PCR analysis 2.4. Strain identification and typing Length heterogeneity–reverse transcriptase–PCR (LH–RT–PCR) 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 identified by RAPDPCR fingerprinting according to Rossetti and Giraffa (2005). Genomic DNA was extracted and used as a template in PCR fingerprinting

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 RT–LH–PCR

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 RT–LH–PCR 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) 168–171

experiments using as a primer the M13 minisatellite core sequence (Huey and Hall, 1989) with sequence 5'-GAGGGTGGCGGTTCT-3'. After amplification, cluster analysis of the electrophoretic profiles 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 fingerprints were compared to previously implemented genotypic libraries to obtain a preliminary strain identification on the basis of RAPD-PCR profile similarity (Rossetti and Giraffa, 2005). On a reduced number of strains, including both strains with atypical genotypic profiles and representatives of each genotypic cluster, the identification was refined 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 microflora of twenty-four Grana Padano cheese whey starters was determined by plate count and RT– LH–PCR 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 19–21, coming from the province of Vicenza, showed the lowest streptococcal counts whereas the cultures from plants 3–5, 6, 11–13, and 22–24 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 heterogeneity–reverse transcriptase–PCR (LH–RT–PCR) 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). RT–LH–PCR profiles, which are obtained from total RNA extracted from whey starters and amplified by RT–PCR, allow the discrimination of the dominant and metabolically active bacterial species present in the LAB community. The use of RT–PCR 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 heterogeneity–reverse transcriptase–PCR (LH–RT–PCR) revealed the presence of four metabolically active species, of which L. helveticus and L. delbrueckii, were the two dominant ones, thus confirming previous findings (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 RAPD–PCR fingerprinting 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. Identification 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 identifications (Rossetti and Giraffa, 2005), we performed a further identification of representative biotypes within the RAPD–PCR 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 identified 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 profiling 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

n

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

d

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); 19–21 (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); 6–8 (CR); 10–12 (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); 3–5 (BS); 7 (CR); 10–12 (MN); 14–18 (PC); 19–21 (VI); 23 (VR); 24 (VR) 8 (CR); 9 (LO) 2 (BS); 13 (MN) 8 (CR); 9 (LO) 1 (BG); 3–5 (BS); 6–8 (CR); 10–13 (MN); 14 (PC); 16–18 (PC); 22–24 (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) 168–171

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 RT–LH–PCR 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 RT–LH–PCR) and cultivable (according to plate isolation), this was not always the case for S. thermophilus. More specifically, S. thermophilus was not detected by RT–LH–PCR 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– LH–PCR 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 profiling obtained by culture-independent and culture-dependent approaches (van de Guchte et al., 2002; Fornasari et al., 2006; Gatti et al., 2006). These data confirm 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 profiles 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 specifically, 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 five 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-specific 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 financial 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, 400–405. Beresford, T.P., Fitzsimons, N.A., Brennan, N.L., Cogan, T., 2001. Recent advances in cheese microbiology. International Dairy Journal 11, 259–274. 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, 305–310. 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. 31–90. 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, 139–144. 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, 72–80. 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, 463–470. 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 classification trees. Applied and Environmental Microbiology 70, 182–190. 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, 338–343. Giraffa, G., Rossetti, L., Mucchetti, G., Addeo, F., Neviani, E., 1998. Influence of the temperature gradient on the growth of thermophilic lactobacilli used as natural starters in grana cheese. Journal of Dairy Science 81, 31–36. 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, 1259–1265. 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, 129–139. Huey, B., Hall, J., 1989. Hypervariable DNA fingerprinting in E. coli. Minisatellite probe from bacteriophage M13. Journal of Bacteriology 171, 2528–2532. 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, 481–490. 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 microflora caratteristica di formaggi a lunga stagionatura. Scienza e Tecnica Lattiero Casearia 58, 55–69. Neviani, E., Divizia, R., Abbiati, E., Gatti, M., 1995. Acidification activity of thermophilic lactobacilli under the temperature gradient of Grana cheese making. Journal of Dairy Science 78, 1248–1252. 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. 123–148. Rossetti, L., Giraffa, G., 2005. Rapid identification of dairy lactic acid bacteria by M13generated, RAPD-PCR fingerprint databases. Journal of Microbiological Methods 63, 135–144. 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, 883–891. 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, 187–216.