Survey of Lactic Acid Bacteria During the Ripening of Caciocavallo Cheese Produced in Molise
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Lait 83 (2003) 211–222 © INRA, EDP Sciences, 2003 211 DOI: 10.1051/lait:2003011 Original article Survey of lactic acid bacteria during the ripening of Caciocavallo cheese produced in Molise Raffaele COPPOLA*, Mariantonietta SUCCI, Elena SORRENTINO, Massimo IORIZZO, Luigi GRAZIA Dipartimento di Scienze e Tecnologie Agroalimentari, Ambientali e Microbiologiche, Università degli Studi del Molise, Via De Sanctis, 86100 Campobasso, Italy (Received 5 January 2001; accepted 10 December 2002) Abstract – The microbiology of raw milk, traditionally-made Caciocavallo cheese from Molise was studied throughout ripening. Three different media (MRS agar, MRS agar at pH 5.4 and LBS) and four different incubation temperatures (15, 22, 28 and 45 °C) were used for lactobacilli, and M17 agar and Slanetz & Bartley medium were used for cocci with the aim of obtaining a complete view of the evolution of these microbial groups. The overall microbiological quality of the product was quite satisfactory; enterobacteria were already absent at 36 h following shaping. High counts of lactic acid bacteria were found in the cheese through the 3 months of ripening. The microbial population present from day 1 of ripening consisted essentially of mesophilic lactic acid bacteria, predominantly lactobacilli, which gradually obtained the advantage over the thermophilic strains. The predominant species were as follows: Lactobacillus paracasei subsp. paracasei, Lb. pentosus, Lb. coryneformis subsp. torquens, Lb. plantarum and Lb. brevis together with Lb. casei, Lb. mali, Lb. rhamnosus, Lb. sakei and Lb. coryneformis subsp. coryneformis. The thermophilic lactic acid bacteria could be ascribed to Lactobacillus helveticus, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. delbrueckii. Lactococci were either Lc. lactis subsp. lactis or Lc. plantarum and enterococci were E. faecalis or E. faecium. Caciocavallo / raw milk cheese / microbiology / ripening Résumé – Vue d’ensemble sur les bactéries lactiques pendant l’affinage du fromage Caciocavallo produit dans le Molise. Nous avons suivi les changements microbiologiques au cours de l’affinage du fromage “Caciocavallo” produit avec du lait cru dans des zones de production traditionnelle de la région Molise. Des niveaux élevés en bactéries lactiques ont été relevés jusqu’à environ 3 mois d’affinage. Trois milieux différents (MRS agar, MRS agar acidifié à pH 5,4 et LBS) incubés à quatre températures d’incubation (15, 22, 28 et 45 °C) ont été utilisés pour avoir une vue d’ensemble de la flore lactique du fromage. La qualité microbiologique globale du produit était satisfaisante. En effet, dès 36 heures on a constaté l’absence d’entérobactéries. Dès les premiers jours de la maturation la microflore lactique présente correspondait essentiellement à des bactéries lactiques mésophiles (surtout des lactobacilles) qui avaient pris graduellement le dessus sur les micro-organismes thermophiles. Parmi les lactobacilles mésophiles isolés, les espèces les plus représentées étaient : Lactobacillus paracasei subsp. paracasei, Lb. pentosus, Lb. coryneformis subsp. torquens, Lb. plantarum et Lb. brevis ainsi que Lb. casei, Lb. mali, Lb. rhamnosus, Lb. sakei * Correspondence and reprints Tel.: +390874404607; fax: +390874404652; e-mail: [email protected] 212 R. Coppola et al. et Lb. coryneformis subsp. coryneformis. Les bactéries lactiques thermophiles ont pu être identifiées comme Lb. helveticus, Lb. delbrueckii subsp. bulgaricus, Lb. delbrueckii subsp. lactis et Lb. delbrueckii subsp. delbrueckii, les lactocoques comme Lc. lactis subsp. lactis et Lc. plantarum et les entérocoques comme E. faecalis et E. faecium. Fromage au lait cru / Caciocavallo / microbiologie / affinage 1. INTRODUCTION that are generally grazed. The cheese is obtained by coagulation of whole raw cow Many cheese varieties, though probably milk, at a natural pH, to which natural unheard of outside their specific areas whey starter and liquid calf rennet is of production, are indeed made and added. It is cooked and manually stretched consumed within these well-defined loca- at about 85 °C and is salted but not sub- tions. As economic and environmental jected to a smoking process; it is ripened conditions change within a given region, either for a brief time (up to 3 months), a the art of making some cheeses can be lost, period characterised by proteolysis, or it is particularly when it is a farmhouse-type slowly ripened (up to 12 months), charac- cheese, as the manufacturing procedures terised by proteolytic-lipolytic activity. are not well described in the literature [14]. However, the greater part of the products is In southern Italy there is a wide variety consumed within 3 months of ripening. of fresh and ripened pasta filata cheeses. Preparation of the natural whey starter Among the many types, Caciocavallo dis- used in its production is fundamentally tinguishes itself as a ripened pasta filata analogous to what has been reported else- cheese manufactured by almost the same where. It is a culture of bacteria, most of technological processes as those employed which are acid producers, obtained from for Provolone cheese. Variations can be whey of the previous day’s cheese-making noted between the different types of and incubated at room temperature [25]. Caciocavallo produced in different areas. The microbiological characteristics of Caciocavallo from Molise, especially the the starter cultures used in the transforma- home-style varieties produced in various tion of milk to pasta filata cheese of both locations throughout the region, represents a short and long maturation have been stud- fine example of typical production, meriting ied in depth [3, 25]. However, little is protection and valorisation not only for its known about the microorganisms involved superb quality but also for its economic in the ripening stages of the cheeses. It has, value as it is widely consumed nationally in fact, been too often surmised that a very and is gradually becoming appreciated mature cheese product is affected essen- world-wide. In 1993, Italian legislation tially by the quality of the starter employed established the “Caciocavallo Silano” and only marginally by microbial activity denomination. In 1996 it was recognised as of the bacteria eventually present during a DOP (Protected Origin Denomination) the ripening period. Such conjectures were product under the auspices of the European based on the belief that the conditions of Community. These provisions, however, the cheese, after the initial stages of ripen- unified the different varieties to represent a ing, completely thwarted microbial growth single typical cheese. and survival. This theory was particularly The manufacturing technology of thought to be applicable to cooked cheeses, Caciocavallo from Molise has been and even more so, to stretched cheeses, described elsewhere [6]. Typical Molisan since it was based on the presumption that Caciocavallo is made locally throughout cooking and stretching temperatures dras- the year from milk produced by dairy cows tically reduce the microbial population. Lactic acid bacteria in Caciocavallo cheese 213 Recent studies carried out on Parmigiano (BBL, Cockeysville, MD, USA) after Reggiano cheese showed the presence of anaerobic incubation (BBL Gaspack Sys- noticeable counts of lactic acid bacteria tem) at 15 °C for 10 d, 22 °C for 72 h, until the end of the ripening period [4, 5, 28 °C for 72 h and 45 °C for 48 h. 18]. Thermophilic streptococci were counted Based on the reasons discussed above, in M17 agar (Oxoid) after incubation for we sought to investigate the presence of 72 h at 45 °C. Presumptive lactococci were lactic acid bacteria during ripening of counted in M17 agar (Oxoid) after incuba- Caciocavallo from Molise produced with tion for 72 h at 22 °C. Enterococci were traditional dairy-farm technologies in order counted in Slanetz & Bartley medium to obtain an overview of the density of (Oxoid) after incubation for 48 h at 37 °C. counts and to identify the particular species Enterobacteriaceae were counted in Violet of lactic acid bacteria present. Red Bile Glucose Agar (Oxoid) after incu- bation for 24 h at 37 °C. Five colonies showing different mor- 2. MATERIALS AND METHODS phologies were randomly picked from each 25–50 colony count plate of MRS, 2.1. Samples MRS pH 5.4 and LBS incubated at the four different temperatures and of M17 at 22 Three different cheese-making trials and 45 °C and Slanetz & Bartley medium. were monitored in a farmhouse-style dairy Purified strains were classified according plant in the province of Isernia. Based on information collected from a survey of the to the references [9, 13, 23]. entire manufacturing territory, this dairy All strains were maintained at 4 °C as factory was chosen for its “typical” techno- deep cultures in semisolid (0.45% agar) logical processes and use of raw milk for MRS agar supplemented with 0.6% cheese-making, which are representative CaCO3. All isolates were tested for mor- of the manufacturing techniques and con- phology, Gram-reaction and catalase. ditions of small establishments in the region. Whole Caciocavallo cheeses, taken 2.2.1. Lactobacilli from the same production run, were marked and ripened after shaping and salt- Gas production from glucose was ing. The various samples were collected determined in MRS broth without citrate 36 h after stretching and at 7, 14, 20, 30, 40 containing inverted vials (Durham). Amy- and 85 d of ripening. They were trans- gdalin, arabinose,