INTRODUCTION

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Non-starter lactic acid bacteria (NSLAB) usually increase from a low number in fresh curd to dominate the microflora of mature cheese (36). In contrast to starters, NSLAB tolerate the hostile environment of cheese during ripening; this environment typically is characterized by 32 to 39% moisture, 4 to 6% salt in moisture, pH 4.9 to 5.3, 5 to 13°C, and a deficiency of nutrients (15, 48). Mesophilic lactobacilli predominate in the NSLAB community, although pediococci and micrococci may also be found (6, 11, 15). Lactobacillus casei subsp. casei, L. casei subsp. pseudoplantarum, Lactobacillus paracasei subsp. paracasei, and Lactobacillus plantarum are the most frequently isolated taxa, but other facultatively and obligately heterofermentative species of lactobacilli are also found (18, 28). Adventitious mesophilic lactobacilli are usually present because of postpasteurization contamination but may also constitute part of the raw milk microflora and survive pasteurization (48).

The role of NSLAB in ripening has not been resolved satisfactorily yet, although inclusion of adjunct cultures of some strains of NSLAB or use of raw milk during cheese manufacturing increases the level of free amino acids, peptides, and free fatty acids, which leads to enhanced flavor intensity and accelerates cheese ripening (4, 14, 31, 32, 34). A comparison of the proteolytic and lipolytic enzymes of starters and NSLAB by quadratic response surface methodology (19) showed greater adaptation to cheese-like conditions by several enzymes of mesophilic lactobacilli. Further application of the same mathematical methodology showed that adaptation of peptidase activities to cheese-like conditions depends on the species and strain of mesophilic lactobacilli (20). The relative abundance of certain species and, especially, the heterogeneity of NSLAB strains in cheese may determine the relationships between NSLAB and cheese flavor. Very few data on the typing of lactobacilli in food ecosystems are available (11).

Most of the Italian ewe cheeses are semihard or hard Pecorino-like cheeses. They are produced at industrial or semi-industrial plants, have different national and international markets, and are produced by different technologies, but they all have typical features which depend on local and regional traditions. The indigenous microbial contents of cheeses, which are selected by the raw milk and cheese-making environment and technology, could be considered some of the main factors in determining the typical cheese features. Although Italian ewe cheeses use thermophilic lactic acid bacterial starters, heterogeneous NSLAB constitute a great part of the indigenous microbial community. Studies of NSLAB diversity may be helpful for (i) differentiating cheeses, (ii) establishing the effects of selected technological parameters on specific differences in the microbial flora, (iii) developing a monitoring system for studying the microflora dynamics in mixed-population fermentations, (iv) evaluating the real contributions of species and strains to cheese ripening, and, in general, (v) obtaining information about the diversity of a large adventitious population. Such information could allow selection of the most suitable strains to introduce as adjunct starters in pasteurized milk cheeses in order to reproduce more closely the flavor of raw milk cheeses or to accelerate cheese ripening.

Randomly amplified polymorphic DNA (RAPD) analysis is a PCR-based method which requires a short time compared with other genetic methods, provides good levels of discrimination, and is applicable to large numbers of strains (49). Moreover, the resolving power of this method can be easily enhanced by increasing the number of primers used to randomly amplify the bacterial genome (46). RAPD analysis has been used to estimate the diversity of Lactobacillus strains in the Centre National de Recherches Zootechniques collection (46), to type strains of L. plantarum (31), Lactococcus lactis isolated from raw milk used to produce Camembert (33), and dairy propionibacteria (40), to establish the correct nomenclature and classification of strains of L. casei subsp. casei (10), and to study the population of NSLAB in mature commercial cheese (11).

Analysis of cell wall protein profiles has been used to study and compare several strains of lactobacilli (1, 38, 50) and to differentiate the thermophilic lactobacilli present in natural or selected starters used to produce several Italian cheeses (16). In particular, Gatti et al. (16) reported that extraction of cell wall proteins, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separation, has been found to be a reliable and rapid way to characterize large numbers of strains and to relate differences in cell wall protein profiles of strains to adaptation to different ecological niches and cheese technology.

To our knowledge, no previous studies have used phenotypic, genotypic, and cell wall protein analyses to characterize a large number of ewe cheese-related bacteria.

In this paper we describe phenotypic, genotypic (RAPD-PCR analysis), and cell wall protein characterization of NSLAB isolated from several Italian ewe cheeses.

	MATERIALS AND METHODS

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Cheese samples. Twelve ewe cheeses, corresponding to six different types of cheese, were supplied by the main Italian producers (Table 1). The names of the producers are not included in Table 1. The cheeses were mainly Pecorino-like cheeses which are produced in several regions in central and southern Italy. All of the cheeses were analyzed when they were ready for market. Standard procedures were used to analyze cheeses for fat (Soxhlet method using diethyl ether), protein (micro-Kjeldahl method), salt (24), and moisture (25). The pH was measured by using a cheese slurry prepared from 20 g of cheese in 12 g of water (2).

NSLAB isolation and enumeration. Twenty grams of cheese was diluted in 180 ml of a 2% sodium citrate solution and homogenized in a Stomacher Lab-Blender 400 (PBI International, Milan, Italy). Serial dilutions were made in 0.25× Ringer's solution and plated on Lactobacillus-selective agar (Becton Dickinson and Co., Cockeysville, Md.). After incubation under microaerophilic conditions for 72 h at 30°C, 20 isolated colonies were randomly selected from each of a duplicate set of countable Lactobacillus-selective agar plates for each cheese. The isolates were propagated in MRS broth (Difco Laboratories, Detroit, Mich.) and purified. All isolates were checked for the catalase reaction and examined microscopically before stock preparations were prepared.

Group differentiation of mesophilic Lactobacillus isolates. Four hundred gram-positive, catalase-negative, nonmotile, randomly isolated bacterial rods were first clustered on the basis of growth at 15 and 45°C, CO₂ production from glucose, NH₃ production from arginine, and esculin hydrolysis. About 30% of the isolates belonging to each group were then used for phenotypic, genotypic, and cell wall protein analyses.

Phenotypic characterization. In addition to the preliminary assays, presumptively mesophilic lactobacilli were characterized for sugar fermentation by using the API 50 CHL system (bioMerieux, Marcy-l'Etoile, France) as recommended by the manufacturer. The APILAB Plus version 4.0 program (bioMerieux) was used to analyze the fermentation profiles obtained with the identification strips. Additional assays, such as assays for growth responses with and without Tween 80 in the culture medium, isomers of lactate, and ethanol production, were also performed as described in the species descriptions published by Kandler and Weiss (29) and Hammes and Vogel (22). As described by Fitzsimons et al. (11), the pH difference in MRS broth lacking acetate, citrate, and Tween 80 and in MRS broth lacking acetate and citrate after incubation for 48 h at 30°C was used to determine whether Tween 80 was required for growth.

Genotypic characterization. Mesophilic Lactobacillus isolates were characterized genotypically by RAPD-PCR analysis. Genomic DNAs from identified strains were extracted as described by De Los Reyes-Gàvilan et al. (9) from 2-ml samples of overnight cultures growth in MRS broth at 30°C. The final concentration of lysozyme used for cell lysis was 2 mg/ml. The concentration and purity of DNA were assessed by determining the optical densities at 260 and 280 nm, as described by Sambrook et al. (41); the DNA concentration of each sample was adjusted to 25 ng/µl. One microliter of each DNA (25 ng/µl) in a 25-µl PCR mixture was sufficient to give reproducible results.

Cell wall protein characterization. Cell wall protein was extracted by using a slightly modified version of the method of Gatti et al. (16). Twenty-four-hour-old cells of mesophilic lactobacilli cultivated in MRS broth (Difco) were harvested, washed twice in 0.05 M Tris-HCl (pH 7.5) containing 0.1 M CaCl₂, and resuspended in 1 ml of the same buffer at an A₆₀₀ of 10.0. After centrifugation at 8,000 × g for 5 min, cell wall proteins were extracted from the pellets with 1.0 ml of extraction buffer (pH 8.0) containing 0.01 M EDTA, 0.01 M NaCl, and 2% (wt/vol) SDS. Suspensions were stored at room temperature for 60 min, heated at 100°C for 5 min, and centrifuged at 11,600 × g for 10 min at 4°C. The supernatants were analyzed by SDS-PAGE with a Phast system (Pharmacia, Uppsala, Sweden) and stained with Comassie blue (23). Densitometry of SDS-PAGE gels was performed with GelCompar 4.0 software (Applied Maths), and the R_f values of individual proteins were calculated to compare the protein profiles of the strains. The 70 kit molecular weight protein standard (molecular weight range, 14,300 to 66,000; 54 µg of total protein) in addition to -galactosidase (molecular weight, 116,000; 8 µg of protein) and ₂-macroglobulin (molecular weight, 170,000; 6 µg of protein) was used (Sigma Chemical Co., St. Louis, Mo.). The reproducibility of SDS-PAGE was estimated by loading two independent, triplicate cell wall protein extraction preparations from nine strains on two gels, and cluster analysis was performed as described above. The relative error (E) for each band in each gel was calculated as follows: E = [(R_f  R_fm)/R_fm] × 100, where R_f is the distance between the top of the separating gel and a protein band and R_fm is the mean R_f for the band obtained in different gels. Clustering of the resultant profiles was performed as described above for genotypic characterization.

	RESULTS

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Cheese characteristics. The main characteristics of the 12 ewe cheeses from which NSLAB were isolated are shown in Table 1. These cheeses are the most important Italian ewe cheeses, and most were supplied by different factories. Some cheeses were produced from raw milk, and others were produced from pasteurized milk; the curd was cooked for 2 min at 40 to 55°C except for Canestrato cheese (70 to 75°C for ca. 30 s). The cheeses were produced by using thermophilic natural lactic acid bacterial starters, and the ripening time ranged from 45 days to 10 months depending on the type of cheese (fresh, semihard, or hard).

Phenotypic characterization. Four hundred presumptive Lactobacillus isolates (gram-positive, catalase-negative, nonmotile rods) that were randomly isolated from the 12 Italian ewe cheeses were differentiated into preliminary groups based on growth at 15 and 45°C, CO₂ production from glucose, NH₃ production from arginine, and esculin hydrolysis (22, 45). About 30% of the isolates belonging to each of the preliminary groups (a total of 123 isolates) were characterized further.

Genotypic characterization. Primers P2, P3, P5, P6, P8, P9, and P10 did not give PCR products or gave very few bands, despite extended annealing times at a low temperature. Fitzsimons et al. (11) obtained species-specific bands when they used primer P2 for characterization of some Lactobacillus strains isolated from Cheddar cheese; nevertheless, in our PCR conditions, primer P2 gave only a limited number of non-species-specific bands. P1, P4, and P7 generated the greatest pattern diversity and were selected for genotypic characterization. The importance of using several primers to improve the RAPD resolving power, especially when RAPD patterns generated with single primers are very similar, has been described by other authors (47). The reproducibility of the RAPD fingerprints was assessed by comparing the PCR products obtained with primers P1, P4, and P7 and DNA prepared from three separate cultures of the same strain. Nine strains were studied, and the patterns for the same strain were 93% similar, indicating that the reproducibility of the technique under the conditions used was high (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Dendrogram, obtained from combined RAPD patterns with three primers, of Lactobacillus isolates from ewe cheeses and type strains. A cluster analysis was conducted with similarity estimates by using UPGMA.

View larger version (53K): [in this window] [in a new window]   FIG. 2.   RAPD patterns obtained with primers P1 (a), P4 (b), and P7 (c), showing the representative fingerprints of the 11 clusters. Lane 1, DNA molecular size standards (123-bp ladder DNA; Gibco BRL, Life Technologies); lanes 2 to 12, clusters 1 to 11, respectively.

Cell wall protein characterization. The reproducibility of SDS-PAGE was estimated by duplicate loading of independent, triplicate cell wall protein extracts from nine strains on two gels. The relative error for each band in each gel was less than 1% (21). Based on preliminary assays, the resolving power of SDS-PAGE was best when 12% acrylamide was used (data not shown).

View larger version (69K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE patterns of cell wall proteins from Lactobacillus isolates. Lane 1, standard proteins (see Materials and Methods); lane 2, L. fermentum 65H; lane 3, L. brevis 68E; lane 4, L. curvatus 1HD; lane 5, L. casei subsp. casei M16; lane 6, L. paracasei subsp. paracasei 4H3; lane 7, L. casei subsp. pseudoplantarum 115K; lane 8, L. plantarum 144; lane 9, L. pentosus 2HG; lane 10, L. rhamnosus 15H3.

View larger version (30K): [in this window] [in a new window]   FIG. 4.   Dendrogram, obtained from SDS-PAGE patterns of cell wall proteins, of Lactobacillus isolates from ewe cheeses and type strains. A cluster analysis was conducted with similarity estimates by using UPGMA.

	DISCUSSION

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NSLAB were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced in different factories. Pecorino Umbro, Pecorino Sardo, Pecorino Toscano, Pecorino Romano, Fossa, and Canestrato cheeses are the Italian ewe cheeses with the greatest market popularity (12, 17, 18) and differ with respect to several technological and chemical characteristics (Table 1). The concentrations of NSLAB cells in the cheeses ranged from 5.5 to 8.2 log CFU/g, values which correspond to the values usually found in cheeses during ripening (11, 13). Phenotypic characterization of 123 catalase-negative, nonmotile, rod-shaped isolates identified nine Lactobacillus species and subspecies which accounted for various percentages of the total. Most of the 12 cheeses contained four different species of NSLAB (Table 2). L. plantarum isolates (32% of the total) were found in 11 of the 12 ewe cheeses. L. paracasei subsp. paracasei isolates (12% of the total) were found in all the cheese types except Fossa cheese, but variations in the percentage depended on the factory of origin. Fossa cheese is a very typical cheese characterized by 3 months of ripening in an underground pit (Fossa) (18). L. paracasei subsp. paracasei has always been found at the highest concentrations in Cheddar cheeses (11, 28). The few L. casei subsp. pseudoplantarum isolates were found only in fresh ewe cheeses, and L. brevis isolates were found mainly in cheeses with very restrictive ripening conditions. In contrast, Pecorino Sardo cheeses ripened for different periods contained the same species after 45 days or 6 months of ripening (the only exception was L. rhamnosus). Other authors (11, 35) have observed changes in the composition of the NSLAB populations in Cheddar cheese which depended on the age of the cheese.

Although phenotypic tests provide some evidence of metabolic capabilities, there are some problems, such as a lack of reproducibility and a lack of discriminatory power. Designation of certain neotype strains based only on phenotypic characteristics gave confused results which were resolved only by using molecular techniques (10). In an effort to corroborate the biochemical classification, we also used genotypic and cell wall protein analyses. To our knowledge, this is the first study which combined characterization of NSLAB based on two different molecular techniques in addition to phenotypic analysis.

Other studies (11, 33, 40, 46, 47) have used RAPD analysis to differentiate strains belonging to a limited number of species of lactobacilli, lactococci, and propionibacteria. Fitzsimons et al. (11) were the first workers to use this technique to study NSLAB from Cheddar cheeses. The isolates from Cheddar cheeses were mainly L. paracasei subsp. paracasei, and only a few isolates of L. plantarum, L. curvatus, and L. brevis were found. A combination of two primers was used, and even if characteristic profiles were obtained with both primers, RAPD analysis with one of the two primers gave species-specific DNA bands. Fitzsimons et al. (11) concluded that their technique was useful for rapidly classifying large numbers of NSLAB occurring in cheese and allowing studies of this community. We used RAPD analysis to differentiate isolates belonging to nine NSLAB species or subspecies in addition to several phenotypically unidentified isolates. Although the combination of three primers used in this study (primers P1, P4, and P7) was useful for partially differentiating 10 type strains, we did not find a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered (Fig. 1 and 2 and Table 2).

The resolving power of RAPD analysis, however, is less than that of protein profiling since the latter technique resolved more the reference isolates at the strain level (5, 11, 37, 42). Indeed, we obtained a combination of cell wall protein profiles which were species specific and could resolve NSLAB in the presence of many species (Fig. 3 and 4 and Table 2). Only isolates that were phenotypically identified as L. plantarum and L. pentosus isolates or L. casei subsp. casei and L. paracasei subsp. paracasei isolates, were grouped together. Although grouped together, the last two species were correctly separated within the same cluster. Gatti et al. (16) found species-specific proteins for Lactobacillus helveticus and Lactobacillus delbrueckii, but within L. delbrueckii there was no discrimination between L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus when the same electrophoretic technique was used (16). Under our conditions, RAPD analysis did not discriminate between L. plantarum and L. pentosus, but L. paracasei subsp. paracasei isolates were separated from L. casei subsp. casei. As shown in the dendrogram in Fig. 4, the NSLAB species showed different degrees of overall similarity. When the variability of the two strains of L. rhamnosus was not included, the protein profiles of the L. plantarum-L. pentosus group, L. brevis, and the L. casei subsp. casei-L. paracasei subsp. paracasei group were the most heterogeneous. In spite of the lower resolving power of the method, the L. plantarum-L. pentosus group and L. brevis were also found to be the most heterogeneous groups when RAPD analysis was used (Fig. 1). Fitzsimons et al. (11) found the greatest strain diversity in Cheddar cheese for the subspecies L. paracasei subsp. paracasei. However, greater genotypic differentiation observed in a certain species could be the result of a higher number of strains used for comparison and, therefore, of the increased probability of encountering more distantly related taxonomic units (40).

Based on a level of homology greater than 77%, the limit used to differentiate species by protein profiling, and an arbitrarily range from 90 to 100%, strains of the same species which occur in different subclusters may be regarded as more dissimilar strains. Based on this criterion, Italian ewe cheeses (30 to 35 isolates were examined from each cheese) contained different numbers of strains, which ranged from 3 to 16. In Cheddar cheese the average number of strains per cheese was found to be seven (11). Except for Pecorino Romano cheese from factory I, all the cheeses produced from raw ewe milk contained a larger number of different strains (8 to 16 strains) than cheeses produced from pasteurized milk (3 to 7 strains). In particular, Fossa and Canestrato cheeses contained 13 and 16 strains, respectively, belonging to L. plantarum, L. fermentum, L. brevis, L. curvatus (only in Fossa cheese), and L. paracasei subsp. paracasei (only in Canestrato cheese) in addition to several unidentified strains. Based on RAPD analysis and considering the strains included in different clusters more dissimilar (Fig. 1 and Table 2), the microbial diversity of ewe cheeses seemed to decrease. Except for Pecorino Umbro AII, Pecorino Toscano CI, and Pecorino Romano DI, the Italian ewe cheeses contained four to seven NSLAB strains. Protein profiling showed that the same cheese produced in different factories (e.g., Pecorino Sardo and Pecorino Romano cheeses) contained not only some different species but also some strains which belonged to the same species and clustered differently. In contrast, isolates from the same cheese frequently grouped together in the same cluster (e.g., L. plantarum AC4, CL3, 440, and 422 from Pecorino Romano DII cheese; L. plantarum 4H5, 4H2, and 4H1 from Pecorino Sardo BIII cheese; and L. brevis 109 and F31 and L. brevis 1HA and 1HC from Canestrato and Pecorino Sardo BI cheeses, respectively). Also, the majority of strains isolated from Cheddar cheeses made in the same factory grouped together in the same cluster (11).

Based on characterization of a large number of NSLAB species by phenotypic, genotypic, and cell wall protein analyses, the following conclusions can be drawn: (i) although 10 primers were screened and 3 of them were used, RAPD analysis did not completely resolve classification of NSLAB; (ii) cell wall protein profiling seems to be a more appropriate tool for classifying NSLAB; (iii) in some cases, such as discrimination between L. casei subsp. casei and L. paracasei subsp. paracasei, RAPD analysis helped improve the resolving power of protein profiling; (iv) RAPD analysis and particularly protein profiling provided useful information about the diversity of NSLAB in cheeses; and (v) Italian ewe cheeses are characterized by a very heterogeneous NSLAB flora which is influenced by geographical and technological factors, which may be responsible for cheese diversity.

The results of the present work could represent a useful tool for nonrandom selection of NSLAB for use as adjunct cultures in pasteurized milk cheese making in order to improve and standardize product quality.