Effect of Different Salting Technologies on the Chemical and Microbiological Characteristics of PDO Pecorino Siciliano Cheese
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Eur Food Res Technol (2011) 233:931–940 DOI 10.1007/s00217-011-1593-7 ORIGINAL PAPER Effect of different salting technologies on the chemical and microbiological characteristics of PDO Pecorino Siciliano cheese Massimo Todaro • Nicola Francesca • Stefano Reale • Giancarlo Moschetti • Fabrizio Vitale • Luca Settanni Received: 26 July 2011 / Revised: 21 September 2011 / Accepted: 27 September 2011 / Published online: 8 October 2011 Ó Springer-Verlag 2011 Abstract The present work was carried out to evaluate developing autochthonous starters to improve the micro- the effect of two salting technologies [dry salting (DS) and biological quality of PDO Pecorino Siciliano cheese. the combined dry-brine salting (DBS)] on the chemico- physical and microbiological characteristics of PDO Pec- Keywords Chemico-physical parameters Á Lactic acid orino Siciliano cheeses of different final weight (6 and bacteria Á Raw ewes’ milk cheese Á Salting process Á 12 kg). Dry matter was significantly influenced by both Spoilage microorganisms salting process and final size. Twelve kilogram cheeses treated by DBS showed higher protein content with higher soluble nitrogen per cent than 6 kg cheeses. Salt content Introduction was in the range 3.1–4.0% on dry matter. The colour did not show significant differences for any of the factors, but Any Italian hard cheese made from ewes’ milk is known as 12 kg cheeses subjected to DS showed higher yellow index ‘Pecorino’. Pecorino cheese is produced throughout Italy than the other cheeses. The resistance at 30% of strain was and the name often indicates the geographical origin. Out- influenced by cheese size, with 6 kg cheeses showing side the national borders, the most known Italian Pecorino higher resistances than 12 kg cheeses. All cheeses were cheese is ‘Pecorino Romano’, but there are other Pecorino dominated by coccus LAB, but pseudomonads and Enter- cheeses, which also enjoy a protected designation of origin obacteriaceae showed comparable levels of about 105 cfu/g. (PDO) status, that are being appreciated by foreigner con- Significant microbiological differences were evidenced sumers, in particular, ‘Pecorino Sardo’, ‘Pecorino Siciliano’ only for enterococci and yeasts concerning the final cheese and ‘Pecorino Toscano’. In general, Pecorino cheeses are size. Thirteen species of LAB, belonging to five genera characterized by a certain salty taste. (Enterococcus, Lactobacillus, Lactococcus, Pediococcus Pecorino Siciliano cheese is, probably, the oldest and Streptococcus), were identified, but several spoilage/ European cheese [3], the PDO disciplinary goes back to pathogenic species were also identified, especially Pseu- 1955 (GURI n. 295 of 12-22-1955) and provides the use of domonas putida, Citrobacter freundii and Stenotropho- entire ewe’s raw milk produced in Sicily, the use of tra- monas maltophilia. LAB isolates were preliminary ditional wooden equipment, the application of dry salting evaluated for their physiological characteristics in view of and a ripening period of at least 4 months. The dry salting technology is performed by manual aspersion of salt onto the cheese surface. However, this dry salting method pro- duces high variability on cheese salt content. M. Todaro Á N. Francesca Á G. Moschetti Á L. Settanni (&) DEMETRA Department, University of Palermo, PDO Pecorino Siciliano cheese is obtained without the Viale delle Scienze 4, 90128 Palermo, Italy addition of bacterial starters. Thus, the microflora acting e-mail: [email protected] during cheese making and ripening is indigenous, deriving from milk or the transformation environment and, for this S. Reale Á F. Vitale Istituto Zooprofilattico Sperimentale della Sicilia ‘Adelmo reason, it may be considered autochthonous. The presence Mirri’, Via G. Marinuzzi 3, 90129 Palermo, Italy of indigenous microorganisms provides characteristic 123 932 Eur Food Res Technol (2011) 233:931–940 features that link their presence to the typicality of a given 12 h for the 6 kg cheeses. For both cheese sizes, the cheese [18]. This is particularly true for the artisanal addition of a further 1% of dry salt per kg cheese was cheeses, which are produced in restricted areas, whose performed. territory and habits, as well as the pedoclimatic conditions During the first 2 months of ripening, all cheeses were and anthropogenic activities (not reproducible elsewhere), brushed weekly to remove moulds; the temperature and are the expression of history and tradition. relative humidity (RH) of the storage chamber were 16 °C After curdling of raw milk, several microbial groups e 85%, respectively. Afterwards, cheeses were kept for may be found in the fat-protein matrix obtained [21]. 3 months into a cave characterized by a temperature of During ripening, the complexity of the microbial structure 16 °C and 90% RH. Sixty cheeses, 30 of 12 kg and 30 of associated with curd diminishes, since lactic acid bacteria 6 kg, were produced in the three dairy factories. (LAB) produce organic acids, mainly lactic acid, deter- Three cheeses per size (6, and 12 kg), salting process mining the decrease in pH till 4.9–5.3 that, besides the (DS, DBS) and dairy factory (A, B, C), forming a total of presence of salt, low moisture, low temperatures and a 36 cheeses, were sampled after 5 months of ripening. deficiency of nutrients makes the environment stressing for Samples were collected as follows: 300 g were randomly many microorganisms [46]. In these conditions, the taken from different portions of cheese and subjected to microbiology of aged raw milk cheeses is typically repre- grating for chemical and microbiological analysis. sented by non-starter LAB (NSLAB) [43]. Several studies have been forwarded to the description of NSLAB com- Chemical and physical measurements munities in Italian raw milk ewes’ cheeses [12, 14, 41, 49]. However, some other microbial groups, such as Entero- Samples of cheeses were analysed for moisture, fat, protein bacteriaceae, have been reported in different Mediterra- (total nitrogen 9 6.38) and ash content according to IDF nean cheeses made from ewes’ raw milk and subjected to a Standards [30–33]. Salt content was determined by the short time of ripening, [23, 36]. The presence of Entero- Volhard method [2]. Cheeses colour was measured with a bacteriaceae is supposed to influence the final characteris- Minolta chroma meter (CR-300, Minolta, NJ 07446 USA), tics of cheese [7]. and results were expressed as lightness L*, redness a* and With the aim to standardize the salt content and to yellowness b* in the CIEL*a*b* system. Texture profile evaluate the microbiological quality of PDO Pecorino was determined at 21 °C. Each cylinder of cheese was Siciliano cheese, the influence of different salting meth- subjected to a load cell force of 90.9 kg and compression at odologies was evaluated on the chemico-physical charac- 30 and 40% using an Instron Universal Testing Machine teristics, microbiology, LAB and spoilage microbial (model 5000; Instron Corporation, Canton, MA). Analysis composition of cheeses of two different sizes. was performed in triplicate. Microbiological analysis Materials and methods Cheese samples were homogenized in sodium citrate (2% Cheese production and sample collection w/v) solution (cheese/diluent 1:9) by means of a stomacher (Laboratory Blender Stomacher 400, Seward, UK) for Milk employed for cheese productions was obtained from 2 min at the highest speed. Further decimal dilutions were sheep reared in three farms located in western Sicily prepared in Ringer’s solution (Oxoid, Milan, Italy). Cell (Agrigento and Trapani provinces). Cheese manufacturing suspensions were plated and incubated as follows: total was performed in three dairy factories (A, B and C) mesophilic count (TMC) on plate count agar (PCA) added annexed to the farms. Milk bulk comprised the milk from with 1 g/L skimmed milk (SkM), incubated aerobically at evening (kept refrigerated under slow stirring) and morning 30 °C for 72 h; total psychrotrophic counts (TPC) on PCA- milking. Cheeses were produced between 10 May and 25 SkM, incubated aerobically at 7 °C for 7 days; Entero- May (2010), following the disciplinary of PDO Pecorino bacteriaceae on violet red bile glucose agar (VRBGA), Siciliano cheese (Reg. CE n. 1107/96) at two different final incubated anaerobically at 37 °C for 24 h; enterococci on weights (6 and 12 kg). Two salting processes were applied kanamycin aesculin azide (KAA) agar, incubated aerobi- to the cheeses: dry salting (DS) and the combined dry-brine cally at 37 °C for 24 h; pseudomonads on Pseudomonas salting (DBS). DS technique involved the shedding of 3% agar base (PAB) supplemented with 10 mg/mL cetrimide of dry salt per kg of cheese, after 48 h from cheese making, fucidin, incubated aerobically at 20 °C for 48 h; positive and a further 1% of dry salt per kg of cheese after 15 days. coagulase staphylococci (PCS) on Baird Parker (BP) added DBS technique involved the immersion in brine after 48 h with R.P.F. supplement, incubated aerobically at 37 °C for from cheese making for 24 h for the 12 kg cheeses and for 48 h; rod LAB on de Man-Rogosa-Sharpe (MRS) agar, 123 Eur Food Res Technol (2011) 233:931–940 933 acidified at pH 5.4 with lactic acid (5 M), incubated by the manufacturer. Crude cell extracts were used as anaerobically at 30 °C for 48 h; coccus LAB on M17 agar, template for PCR. incubated anaerobically at 30 °C for 48; yeasts on yeast The isolates representative of each phenotypic group of glucose chloramphenicol (YGC) agar, incubated aerobi- LAB were identified by the amplification of the gene 16S/ cally at 25 °C for 48 h. Clostridial content was estimated 23S rRNA ITS (in the range 200–400 bp) performed as by the most probable number (MPN) technique using a described by White et al. [51], while the presumptive 3 9 3 scheme [21]. All media were purchased from Oxoid. spoilage microorganisms were identified by the 16S rRNA Microbiological counts were carried out in triplicate.