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J. Dairy Sci. 85:1390–1397  American Dairy Science Association, 2002.

Microbial Community Dynamics during the Scamorza Altamurana Cheese Natural Fermentation F. Baruzzi,* A. Matarante,* M. Morea,* and P. S. Cocconcelli†,‡ *Istituto Tossine e Micotossine da Parassiti Vegetali, CNR, Bari, †Istituto di Microbiologia, Universita` Cattolica del Sacro Cuore, Piacenza, ‡Centro Ricerche Biotecnologiche, Universita` Cattolica del Sacro Cuore, Cremona, Italy

ABSTRACT The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products. (Key words: lactic acid bacteria, natural microbial community, stretched cheese, proteolytic activity)

Received January 23, 2001. Accepted November 19, 2001. Corresponding author: F. Baruzzi; e-mail: [email protected].

Abbreviation key: LAB = lactic acid bacteria, RAPD = randomly amplified polymorphic DNA. INTRODUCTION The pasteurization of milk, which plays a fundamental role in the control of pathogenic bacteria, results in a significant reduction of the natural bacterial populations involved in cheese making. Although the use of selected starter cultures in large-scale industrial processes makes it possible to process millions of liters of milk into cheese in a controlled way, it reduces the biodiversity in the dairy microflora (Salama et al., 1993). However, some traditional dairy products are still fermented utilizing unselected backslopping starters, resulting in a wide range of products with different flavors, consistencies, and microbiological quality (Cogan et al., 1997). The market for these food products could expand since they are regarded as a premium type for their organoleptic characteristics; however, the use of selected starter cultures, indispensable for the upscaling of the manufacturing process, usually diminishes the unique features of the original cheese (Caplice and Fitzgerald, 1999). In Southern Italy, milk is mainly processed into stretched cheeses. Backslopping is still widespread in the making of dairy products; as in the case of Mozzarella cheese made from unpasteurized cows’ milk obtained by traditional processing whose natural microflora was found to be composed of 13 rod-shaped isolates and 25 Gram-positive cocci strains (Morea et al., 1998, 1999). In the last few years various molecular typing methods such as RFLP, pulsed-field gel electrophoresis, ribotyping, and PCR-derived techniques, randomly amplified polymorphic DNA (RAPD), repetitive extragenic palindromic, and enterobacterial repetitive intergenic consensus PCR have been used to distinguish between lactic acid bacteria (LAB) strains. Recently, RAPD analysis has been used to estimate the diversity among

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several genera of bacteria such as Lactobacillus, Enterococcus, Lactococcus, and Staphylococcus isolated from many sources (Cocconcelli et al., 1995; Corroler et al., 1998; Morea et al., 1998; 1999; Rebecchi et al., 1998) and 16S rDNA sequence analysis for the taxonomic identification of biotypes. To understand whether natural whey could be used as a selected starter in typical Southern Italian stretched cheese making, the microflora involved in Scamorza Altamurana cheese processing and ripening was studied. As pasteurized milk is normally used to make this cheese, natural whey microflora plays a major role in two different fermentation steps: the first occurs during saltless curd fermentation at room temperature for 18 to 24 h, the second during a brief ripening at controlled temperature and humidity after stretching and salting. In this work, a polyphasic approach, based on microbial enumeration on culture media and “two-step RAPD-PCR” (Baruzzi et al., 2000), was successfully used to characterize the growth and the changes of whey lactic microflora during curd and cheese fermentation. The taxonomic identification of isolates was carried out by 16S rDNA sequence analysis combined with some physiological tests and specific PCR protocols. The physiological characterization of the LAB contained in Scamorza Altamurana cheese, such as acid production, proteolysis, and citrate utilization was carried out to gain an insight into the role played by different strains in curd and cheese fermentation. MATERIALS AND METHODS Scamorza Altamurana Cheese Processing The Scamorza Altamurana was produced according to the procedure hereafter described. Liquid rennet was added to filtered pasteurized whole cow’s milk, inoculated with 1% (vol/vol) of the natural whey culture (pH 3.80) and heated up to 37 to 38°C. An unselected starter was obtained with the fresh whey derived from cheese making, heated up to 40 to 42°C, and allowed to cool for about 24 h until the next cheese processing. After about 30 min, the curd was cut by hand to peanut size. The curd fragments were heated up to 40°C, cut to halfsize, kept under whey for 2 h and drained. The curd was then transferred onto a steel surface, cut into large pieces several times, covered, and allowed to ripen at room temperature for 18 to 24 h. The curd at pH 5.32 was stretched in hot water (70 to 80°C), worked by hand to give the cheeses weighing approximately 300 g a flask-like shape, dipped in cold water to firm and salted in brine (27 to 30% NaCl) for 2 h. The cheese could either be eaten fresh or briefly ripened at 8 to 10°C and 75 to 80% relative humidity.

Cheese Sampling Samples were collected during one process of traditional Scamorza Altamurana making in a cheese factory located in Apulia, a region in the South of Italy. A second whey sample was taken after 7 mo to check the differences in natural lactic acid microflora. Samples of acidified whey, fermented curd before the stretching process, Scamorza cheese after shaping and salting (Scamorza T-0), and Scamorza cheese after 6 d of ripening (Scamorza T-6) were collected. Immediately after collection, the samples were frozen in liquid nitrogen. Once transferred to the laboratory, the curd and Scamorza cheese samples were minced in a food processor with 2% sodium citrate solution (1:9, wt/wt). All the samples were stored at −80°C. Microbiological Analysis of Cheese Samples The frozen samples were thawed and serially diluted in peptonate saline solution. The appropriate dilutions were plated in triplicate on different media: Rogosa and M17 agar for the enumeration of presumptive LAB, and violet red bile agar for coliforms. Rogosa and M17 plates were incubated under anaerobic conditions (Anaerocult A , Merck, Darmstadt, Germany), whereas VRBA was incubated under aerobic conditions. All the plates were kept at 30°C for 48 h. These growth conditions were chosen because curd fermentation and cheese ripening, the main fermentation steps, are carried out at room temperature and at 8 to 10°C, respectively. For each collection step, 40 randomly selected colonies of bacilli and cocci Gram-positive, catalase-negative isolated from Rogosa and M17 agar plates were cultured in MRS and M17 broths, respectively, incubated overnight at 30°C, frozen at −80°C and used for further characterization. All the media were obtained from Difco Laboratories (Detroit, MI). LAB Strain Typing PCR amplification was carried out in a Thermal Cycler 9700 (Perkin Elmer, Alameda, CA). Taq polymerase, deoxynucleoside triphosphates, and DNA molecular weight markers were purchased from Roche Diagnostics (Milano, Italy). Gel-filtration purified oligonucleotides were obtained from Sigma-Genosys Ltd (Cambs, UK). Strain typing was performed using the two step RAPD-PCR protocol previously described by Baruzzi et al., (2000), with some modifications. Cell lysis from presumptive cocci for PCR reactions was carried out using a synthetic resin (Gene Releaser, Bioventures, TN), as previously described (Morea et al., 1999), Journal of Dairy Science Vol. 85, No. 6, 2002

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whereas genomic DNA from presumptive lactobacilli was extracted with a Quantum Prep Aqua Pure Genomic DNA-Isolation kit (Bio-Rad Laboratories, S.r.l., Segrate, Milano, Italy). Amplification consisted of 10 cycles of the RAPD-PCR protocol of Cocconcelli et al., (1995) (first step) and 40 additional cycles of 30 s at 94°C, 30 s at 55°C, 30 s at 72°C (second step), and a final extension step consisting of 72°C for 5 min. Ten additional aspecific cycles were usually added to type the presumptive cocci. Taxonomic Identification of Strains All the strains were identified by amplification and sequencing of 16S rRNA genes as already described (Klijn et al., 1995; Cocconcelli et al., 1997). The DNA sequences were obtained using an ABI PRISM Big Dye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Monza, Italy) and both the forward and reverse primers for 16S rDNA were used. The reaction products were analyzed with an Applied Biosystem 310 automated DNA sequencer (PE Applied Biosystems). The software package of the University of Wisconsin Genetics Group was used for the analysis and comparison of DNA sequences. Taxonomic strain identification and Similarity-rank (S_ab) calculations were performed, comparing the 16S rDNA sequences of Scamorza cheese isolates with more than 30,000 unaligned sequences present in the Sequence Match of the Ribosomal Database Project II (RDP II), as described by Maidak et al. (2000). For the strains belonging to Lactobacillus delbrueckii, the subspecies classification was obtained by carbohydrate pattern fermentation, as described by Kandler and Weiss (1986), and the PCR protocol based on the presence of the proline iminopeptidase (pepIP) gene, as described by Torriani et al. (1999). Plasmid extraction (Anderson and McKay, 1983) was carried out for some Streptococcus thermophilus isolates. Physiological Characterization of Isolated Strains The acidifying capacity was determined by pH measurements after 24 h of growth in skimmed milk at 30°C. The proteolytic activity of strains was tested after 1 and 6 d of fermentation in skimmed milk, following the o-phthaldialdehyde method of Church et al. (1983). Streptococcus thermophilus C24W and Lactococcus lactis subsp. lactis H (Morea et al., 1999) were used as negative and positive controls, respectively. To improve the growth in milk, in the 6-d assay, 1 g/L yeast extract (Difco) was added. The reported values were calculated as the average of 10 readings at A340. Journal of Dairy Science Vol. 85, No. 6, 2002

Citrate utilization by isolated strains was detected using the medium of Kempler and McKay (1980). Citrate-positive colonies were blue after 48 h of incubation at appropriate temperatures under anaerobic conditions. RESULTS Bacterial Growth Kinetics Bacterial counts were performed on samples of whey, curd, and cheese collected during Scamorza Altamurana cheese processing and ripening. The viable counts of both bacilli and cocci obtained from Rogosa and M17 Agar plates, respectively, are shown in Figure 1. In the acid whey at pH 3.80 after 24 h of fermentation, the mesophilic microflora enumerated onto Rogosa was 1.2 × 106 cfu/ml, whereas mesophilic cocci, recovered from M17 plates, were 7.4 × 105 cfu/ml. In the curd, after 24 h of fermentation at room temperature, the dominant community was constituted by cocci with more than 1.0 × 109 cfu/g, whereas lactobacilli decreased to 7.3 × 105 cfu/g. During stretching, when the water temperature was close to 80°C, the lactic acid microflora dropped as a result of the high temperature treatment, reducing lactobacilli to 7.0 × 104 cfu/g and cocci to 8.9 × 105 cfu/ g. In Scamorza Altamurana cheese, after 6 d of ripening at 8 to 10°C with 75 to 80% relative humidity, both LAB communities increased to more than 8 log10. The coliforms, counted on VRBA plates, were only present in curd and Scamorza T-6 samples with values below the limits laid down by Council Directive 92/46/ EEC of 16 June 1992. Biotype Dynamics During Cheese Processing The application of the two-step RAPD-PCR protocol made it possible to follow the growth kinetics of 25 different strains deriving from 320 colonies. Some RAPD patterns of different LAB biotypes are shown in Figure 2. In the acid whey after 24 h of fermentation, nine different biotypes were identified; three of them were more than 3.0 × 105 cfu/ml, whereas the remaining ones were present at about 4 log10. In the microbial association of the curd, two new biotypes from M17 agar plates prevailed among the six new isolates recognized. After thermal treatment, six different profiles were identified; two (B15Z and C48W) derived from the whey and C6C from the curd. When Scamorza cheese was ripened for 6 d, seven new RAPD patterns were found together with C34C deriving from curd, and the abovementioned B15Z deriving from whey. After 7 mo from the first sampling, another whey sample was harvested to study the changes of whey microflora over time; RAPD analyses of the new isolates


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Figure 1. Growth dynamics of bacterial groups in Scamorza cheese. Bacterial counts on Rogosa (䊐) and M17 (⌬) at 30°C. (T-0, cheese after shaping and brining; T-6, cheese after six days ripening).

showed that the C48W and B15Z biotypes were still present. Taxonomic Characterization of Isolated Strains The taxonomic position of all the 25 biotypes was achieved by means of sequence analysis of at least 500 bp of the 5′ region of 16S rRNA gene. The S_ab values and current classification of strains from the samples of whey, curd, and cheese, collected during traditional Scamorza Altamurana cheese making, are given in Table 1. The sequences of C34C, C43Z, C22S and C13S strains gave the same S_ab values and sequence alignment with both Streptococcus macedonicus and Streptococcus waius, two new species recently described by Tsakalidou et al. (1998) and Flint et al. (1999), respectively. Awaiting further studies to give a clear definition of the species they belonging to, the strains were classified as S. macedonicus because this species was first described. The C11Z and C6C S. thermophilus strains isolated in curd and Scamorza cheese samples showed the same RAPD pattern but different colony morphologies and different growth behaviors in M17 and Elliker (Teuber and Geis, 1986) broths. Plasmid extraction showed extrachromosomal elements only from C6C culture. For these reasons, the S. thermophilus C11Z and C6C were regarded as different isogenic strains. The carbohydrate fermentation pattern and the presence of PCR products related to the proline iminopepti-

dase (pepIP) gene (Kandler and Weiss, 1986; Torriani et al., 1999) made it possible to define the subspecies of the L. delbrueckii strains. The LAB species present in the samples are shown in Figure 3. In whey, the strains were identified as L. delbrueckii, L. fermentum, L. gasseri, L. helveticus, S. thermophilus, and Weissella viridescens. The community in the curd was composed of three species of lactobacilli, L. delbrueckii, L. fermentum, and L. helveticus, and three species of streptococci, S. bovis, S. macedonicus, and S. thermophilus. Only some strains belonging to L. delbrueckii, S. macedonicus, and S. thermophilus species survived the thermal treatment in the stretching step. The above-mentioned species were also found in the last sample together with some new strains belonging to Enterococcus durans, L. fermentum, and L. paracasei subsp. paracasei species. Physiological Characterization of Isolated Strains All the LAB strains were tested for physiological features of interest in dairy processing, such as acidifying capacity, proteolytic activity, and citrate fermentation ability. The positive strains are shown in Table 2. The Streptococcus thermophilus strains were able to decrease pH values below 5.00 after 24 h of fermentation. All the strains were analyzed for their ability to degrade milk proteins in 24 h of growth. The proteolytic activity of the positive strains (A340 > 0.280) was confirmed after 6 d of fermentation. Two Lactobacillus Journal of Dairy Science Vol. 85, No. 6, 2002

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BARUZZI ET AL. Table 1. Taxonomic identification of lactic acid bacteria strains isolated during Scamorza Altamurana cheese processing as described in Materials and Methods. Item no.

Strain

Reference in RDP

S_ab1 Value

Isolated in

601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625

B21S B11W B26W B49C B15Z B63Z B7C B1S B2C B4W B24W B30W B8S B22S C15C C13S C22S C34C C43Z C11Z C24W C30S C48W C6C B12W

Enterococcus durans Lactobacillus helveticus L. helveticus L. helveticus L. delbrueckii subsp. bulgaricus L. delbrueckii subsp. delbrueckii L. delbrueckii subsp. bulgaricus L. fermentum L. fermentum L. fermentum L. gasseri L. gasseri L. paracasei subsp paracasei L. paracasei subsp paracasei Streptococcus bovis S. macedonicus S. macedonicus S. macedonicus S. macedonicus S. thermophilus S. thermophilus S. thermophilus S. thermophilus S. thermophilus Weissella viridescens

0.868 0.834 0.859 0.860 0.946 0.761 0.978 0.854 0.883 0.900 0.943 0.919 0.981 0.983 0.792 0.927 0.925 0.928 0.913 0.915 0.874 0.800 0.919 0.929 0.908

T-6 Whey Whey Curd Whey, T-0, T-6 T-0 Curd T-6 Curd Whey Whey Whey T-6 T-6 Curd T-6 T-6 Curd, T-6 T-0 T-0 Whey T-6 Whey, T-0 Curd, T-0 Whey

1

Similarity rank calculation.

helveticus strains, isolated from whey and curd samples, showed higher proteolytic activity in milk in comparison with that of L. paracasei and L. delbrueckii strains. Citrate-fermenting ability was found in one strain of Weissella viridescens, isolated from whey, and in one isolate of L. fermentum derived from the T-6 sample of Scamorza cheese. DISCUSSION

Figure 2. RAPD patterns of different LAB biotypes isolated from Rogosa plates. Lanes: 1, B26W; 2, B49C; 3, B11W; 4, B15Z; 5, B63Z; 6, DNA molecular weight marker VI (pBR328 DNA BgI I-Hinf I digested); 7, B7C. Journal of Dairy Science Vol. 85, No. 6, 2002

Scamorza cheese is a traditional Southern Italian stretched cheese processed like Mozzarella cheese, although the curd is usually fermented for a longer period of time, salting is done by dipping the shaped curd in brine after stretching, and Scamorza can either be eaten fresh or briefly ripened. For these reasons, Scamorza cheeses can vary considerably due to the differences in milk processing, curd fermentation, and cheese ripening. Scamorza Altamurana is characterized by long curd fermentation, the longest among the stretched cheeses. In traditional processing, natural whey constitutes the most important differentiating factor. The preservation of a stable microflora in an unselected whey starter is the indispensable requirement to obtain dairy products endowed with typical characteristics in largescale processing. The changes of natural whey microflora in the different steps of traditional Scamorza Altamurana cheese processing have been investigated


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Figure 3. Distribution of LAB species in Scamorza Altamurana samples (T-0, cheese after shaping and brining; T-6, cheese after six days ripening). 1, Streptococcus thermophilus; 2, Lactobacillus delbrueckii; 3, S. macedonicus; 4, L. fermentum; 5, L. helveticus; 6. L. gasseri; 7, Weissella viridescens; 3, S. bovis; 9, L. paracasei subsp. paracasei; 10, Enterococcus durans.

with the aim of evaluating the potentiality of its strains to become selected starters. The most important strain association was Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, commonly found in some dairy products such as Mozzarella cheese and yoghurt. The strains isolated from Scamorza Altamurana cheese seemed to act in a different way: S. thermophilus could be involved in curd acidification, whereas L. delbrueckii, endowed with proteolytic activity, could contribute to the organoleptic features by hydrolyzing milk proteins. During the Scamorza cheese processing and ripening, five isolates were assigned to the S. thermophilus species. In whey, strain C48W prevailed over all LAB strains, accounting for more than 40% of lactic microflora and more than 97% of cocci community. That it was found together with S. thermophilus C24W after 24 h of spontaneous fermentation of whey derived from cheese making could be due to its superior acid resistance compared with other S. thermophilus isolates. The S. thermophilus C6C and C30S, amounting to 9.6 × 108 and 3.5 × 108 cfu/g, were the dominant strains in curd and T-6 samples, respectively. In fresh Scamorza cheese, strains C11Z and C6C constituted about the 70% of the total lactic microflora surviving thermal treatment. Even if these strains showed the same RAPD pattern, their growth behavior both in M17 agar plates and in M17 and Elliker broths were different. Plasmid extraction showed extrachromosomal elements only from C6C culture. Some genetic studies on S. thermophilus demonstrated that its morphology was correlated to the presence or absence of plasmids (Mercenier, 1990; Som-

kuti et al., 1998; O’Sullivan et al., 1999), for these reasons, S. thermophilus C11Z and C6C were regarded as different isogenic strains. Three strains of L. delbrueckii were found in different samples: since pasteurized milk was used, most probably they were also present in whey, where only B15Z was isolated. They were classified as one L. delbrueckii subsp. delbrueckii and two proteolytic L. delbrueckii subsp. bulgaricus strains. The most important L. delbrueckii subsp. bulgaricus strain was B15Z present in whey (7% on Rogosa cell count), in fresh cheese (90%), after 6 d fermentation (33%) and most probably also in curd although the amount was not detectable. The proteolytic activity evaluated in skim milk after 24 h and after 6 d fermentation, together with its increase from 6.3 × 104 cfu/g (T-0) to 4.2 × 107 cfu/g (T-6), demonstrated that its activity begins in fresh Scamorza cheese and continues for 6 d, contributing to changes in organoleptic and nutritional characteristics of this fermented product. To study the changes in the microflora of whey over time, a sample was harvested after 7 mo from the first sampling. The S. thermophilus C48W and L. delbrueckii subsp. bulgaricus B15Z dominant strains were found at 90 and 10% of cocci and bacilli community, confirming their role as the main starter strains. The analysis of other LAB indicated that new strains belonging to L. fermentum and L. helveticus species appeared. These results seem to show that secondary lactobacilli species are stable in whey microflora even if changes occur in the recovery of strains. In a natural whey, which is used daily, some strains withstand environmental and technological changes Journal of Dairy Science Vol. 85, No. 6, 2002

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BARUZZI ET AL. Table 2. Phenotypic properties of some strains from Scamorza Altamurana cheese processing. Strain C15C C7S C24W C30S C44W C48W C6C

Reference in RDP

Physiological Features Milk acidification1

Streptococcus bovis S. macedonicus S. thermophilus S. thermophilus S. thermophilus S. thermophilus S. thermophilus Skim milk control

5.00 5.32 4.08 4.83 4.82 4.66 4.67 6.60 Proteolytic activity absorbance values2

B26W B49C B7C B15Z B22S B8S H C24W Citrate fermentation3 B1S B12W

Lactobacillus helveticus L. helveticus L. delbreuckii subsp. bulgaricus L. delbrueckii subsp. bulgaricus L.paracasei subsp. paracasei L. paracasei subsp. paracasei Lactococcus lactis subsp. lactis S. thermophilus Skim milk control

1d

6d

0.375 0.505 0.320 0.345 0.368 0.295 0.600 0.280 0.180

1.801 2.170 1.603 1.472 1.055 1.319 0.940 0.515 0.619 + +

L. fermentum Weissella viridescens

1

pH values after 24 h of growth in milk at 30°C. Proteolytic activity according to o-phthaldialdehyde absorbance values at 340 nm. 3 Citrate fermentation checked on Kempler and McKay agar plates. 2

constituting the core of an unselected starter and are supported by several other LAB strains that replace them over time. These natural microflora dynamics contribute to differentiate traditional cheeses from industrial ones. Many strains from Scamorza Altamurana cheese samples are able to hydrolyze milk proteins. The proteolytic strains changed during processing, amounting to 4.0 × 105 cfu/g (L. helveticus B26W and L. delbrueckii subsp. bulgaricus B15Z) in whey, 7.0 × 105 cfu/g (L. helveticus B49C and L. delbrueckii subsp. bulgaricus B7C) in curd, 6.3 × 104 cfu/g (L. delbrueckii subsp. bulgaricus B15Z) in T-0 and 9.2 × 107 cfu/g (the strain B15Z and L. paracasei subsp. paracasei B8S and B22S) in ripened cheese; therefore, to obtain a cheese such as traditionally produced Scamorza Altamurana, the selected or unselected starter should contain them. The proteolytic activity assessed at 1 and 6 d demonstrated that Lactobacillus strains are more important during ripening in comparison with Lactococcus lactis subsp. lactis H proteolytic strain present in fresh stretched cheese (Morea et al., 1999). L. helveticus strains B49C and B26W were the most proteolytic strains, followed by L. delbrueckii strains usually known to be good proteolytic lactobacilli (Sasaki et al., 1995). These findings, indicating that L. helvetiJournal of Dairy Science Vol. 85, No. 6, 2002

cus proteolytic strains are of major importance in ripened Scamorza cheese, suggest that their presence must be safeguarded in whey starter cultures. During Scamorza cheese processing, strains classified as S. macedonicus were frequently found, whereas some strains belonging to Enterococcus durans, L. gasseri, S. bovis, and Weissella viridescens were occasionally observed. Although, on the basis of physiological tests carried out, they did not show any interesting biotechnological applications, more in-depth studies should be performed on other phenotypic features, such as exopolysaccharide production and lipolytic activity, and on taxonomic identification, to understand their role in the making of this or other cheeses where the same natural starter microflora is utilized. CONCLUSIONS The growth dynamics of the natural microbial community involved in the fermentation of Scamorza Altamurana cheese was investigated using a polyphasic approach that identified 25 different LAB strains. All the streptococci were highly acidifying whereas lactobacilli were proteolytic. The strains Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z were present during all steps of the process


and were recovered in a subsequent whey microflora control in amount similar to those found in the previous whey sample. The permanence of these dominant strains in natural microflora is the first requirement for upscaling Scamorza Altamurana cheese processing using natural starters. The finding of six proteolytic strains, present in natural microflora during cheese making and ripening, leads to the supposition that they play a fundamental role in developing the organoleptic characteristics of Scamorza Altamurana cheese. The proteolytic strains L. helveticus B49C and L. delbrueckii subsp. bulgaricus B7C, found in curd, and L. paracasei subsp. paracasei B8S and B22S, isolated from ripened cheese, were not recovered in the whey sample; however, they should be safeguarded in any case to prevent the loss of the characteristics that contribute to the uniqueness of this typical cheese. ACKNOWLEDGMENTS This work was supported by the EC structural funds FESR no. 94.05.09.013: “Valorizzazione dei prodotti alimentari tipici mediterranei: ottimizzazione dei processi produttivi e di trasformazione e aspetti nutrizionali”. We thank A. Maiullari for his cooperation in supervising the manufacture and ripening of the cheese carried out at “Caseificio dei Colli Pugliesi” (Santeramo in Colle, Bari, Italy) dairy. We also thank G. Stea for his technical assistance in DNA sequencing. REFERENCES Anderson, D. G., and L. L. McKay. 1983. A simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46:549–552. Baruzzi, F., M. Morea, A. Matarante, and P. S. Cocconcelli. 2000. Changes in the Lactobacillus community during Ricotta forte cheese natural fermentation. J. Appl. Microbiol. 89:807–814. Caplice, E., and G. F. Fitzgerald. 1999. Food fermentations: role of microorganisms in food production and preservation. Int. J. Food Microbiol. 50:131–149. Church, F. C., H. E. Swaisgood, D. H. Porter, and G. L. Catignani. 1983. Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. J. Dairy Sci. 66:1219–1227. Cocconcelli, P. S., D. Porro, S. Galandini, and L. Senini. 1995. Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci. Lett. Appl. Microbiol. 21:376–379. Cocconcelli, P. S., M. G. Parisi, L. Senini, and V. Bottazzi. 1997. Use of RAPD and 16S rDNA sequencing for the study of Lactobacillus population in natural whey culture. Lett. Appl. Microbiol. 25:8–12.

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Journal of Dairy Science Vol. 85, No. 6, 2002