Physico-chemical characteristics and acidic profile of PDO Pecorino Romano cheese: Seasonal variation

Small Ruminant Research 126 (2015) 73–79

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Physico-chemical characteristics and acidic profile of PDO Pecorino Romano cheese: Seasonal variation Margherita Addis ∗ , Myriam Fiori, Giovanni Riu, Massimo Pes, Enrico Salvatore, Antonio Pirisi Agris Sardegna, Agricultural Research Agency of Sardinia, Loc Bonassai, SS 291 Km 18.6, 07040 Olmedo, Italy

a r t i c l e

i n f o

Article history: Received 21 January 2015 Received in revised form 17 March 2015 Accepted 18 March 2015 Available online 26 March 2015 Keywords: PDO Pecorino Romano cheese Physico-chemical characteristics Acidic profile Seasonal variation

a b s t r a c t Pecorino Romano, a protected designation of origin (PDO) semi-cooked hard cheese, is the best known Italian dairy product obtained from ovine milk and one of the most exported Italian cheeses in the world. The aim of this work was to provide useful information on physico-chemical and nutritional characteristics of PDO Pecorino Romano cheese taking into account the variability related to the season of cheese manufacture. A total of 70 samples of Pecorino Romano cheese, with a ripening time ranged from 7 to 8 months, and manufactured at different times of the year (from March to June) were analyzed. The month of production affected cheese composition, and cheeses produced in late winter and spring were characterized by a less fat and salt content and a higher protein content with respect to those produced in early summer. Also the biochemical processes of proteolysis and lipolysis were influenced by the seasonality of productions. In particular cheeses produced at late winter were more proteolyzed and lipolyzed if compared with those manufactured at summer season. The nutritional value of cheese fat, associated in particular with linolenic, rumenic and vaccenic acids content, decreased with the progress of season probably due to variation in animal feed, pasture availability and fatty acids composition of grass lipids. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Sardinia island is the largest producer of Italian sheep’s milk and cheese. In particular, it is the region with the largest quantity of ewe milk collected annually, which represents about 70% of the totality of the Italian production. Most of milk represents the starting material for Pecorino Romano cheese making, which is the best known Italian dairy product obtained from ovine milk and one of the most exported Italian cheeses in the world (Pirisi and Pes, 2011). Pecorino Romano is a protected designation of origin (PDO) semi-cooked hard cheese, manufactured

∗ Corresponding author. Tel.: +39 0792842338; fax: +39 079389450. E-mail address: [email protected] (M. Addis). http://dx.doi.org/10.1016/j.smallrumres.2015.03.014 0921-4488/© 2015 Elsevier B.V. All rights reserved.

on an industrial scale. According to PDO specification (http://www.pecorinoromano.com/disciplinare.html, last access 07/07/2014), whole sheep milk is usually thermised at a temperature of 68 ◦ C for at least 15 s, and cooled down to coagulation temperature (37–39 ◦ C). Then, it is inoculated with a natural starter culture (scotta-innesto) obtained by fermentation of the residual whey coming from the Ricotta cheese manufacture. Milk is coagulated by addition of lamb paste rennet, obtained from locally reared animals. Paste rennet contains a balanced amount of milk-clotting and milk fat-hydrolysing enzymes, such as various lipases, that release free fatty acids (FFAs) during cheese ripening. After rennet coagulation, the coagulum is cut into small granules (about 2–4 mm in size) and the curd is stirred for 10 min and cooked by bringing the temperature to 45–46 ◦ C. Then, the curd is moved to a draining vat,

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pressed and cut into blocks, molded and branded. Pecorino Romano is dry salted for 4–5 times in rooms with a temperature of 10–12 ◦ C, over a period of about 70 days. The minimum ripening time is five months for the table cheese, and eight months for grated cheese (Pirisi et al., 2011). Despite its economic importance in Italian dairy sector, to the knowledge of the authors, to date very few studies have been carried out on Pecorino Romano cheese. The first studies on the physico-chemical characteristics of Pecorino Romano cheese date back to the late 60s to early 70s (Marcialis et al., 1968; Pettinau and Bottazzi, 1971). Lately, Addis et al. (2005a) studied the effect of different lamb paste rennets on chemical and sensory characteristics of Pecorino Romano cheese. Nudda et al. (2005) highlighted the seasonal variation in conjugated linoleic acid (CLA) and vaccenic acid (VA) concentrations of Pecorino Romano cheese after 24 h of production, but, the authors did not give any detail regarding the variation of the other physical and chemical characteristics of Pecorino Romano cheese with the progress of the production season. Tripaldi et al. (2014) focused their attention on the effect of reduced dry salting method on lipolytic and proteolytic parameters of Pecorino Romano cheese. The aim of this work was to provide useful information on physico-chemical and nutritional characteristics of PDO Pecorino Romano cheese, and particularly to take into account the variability related to the month of cheese manufacture. 2. Materials and methods 2.1. Samples A total of 70 samples of Pecorino Romano cheese were supplied by the “Consorzio per la Tutela del formaggio Pecorino Romano”. Cheeses belonged to different producing batches manufactured at different times of the year, in particular 10 from March, 30 from April, 20 from May and 10 from June production. The cheeses ripening time ranged from 7 to 8 months. 2.2. Cheese composition and nitrogen fractions Cheeses were analyzed for pH (pH meter 420 A, Orion, Boston, USA), dry matter (DM) (ISO 5534, 2004), fat (Soxhlet method, Soxhlet, 1879), total nitrogen (TN) (FIL-IDF, 1993:20B, part II), soluble nitrogen at pH 4.6 (pH 4.6 SN), soluble nitrogen in 12% trichloroacetic acid (TCA-SN), and soluble nitrogen in 10% phosphotungstic acid (PTA-SN) (Gripon et al., 1975). Water activity (aw ) was determined at 25 ◦ C by using a Novasina Aw Sprint instrument (Axair Ltd., Novasina Division, Switzerland). 2.3. Free fatty acids (FFAs) analysis The free fatty acids content was determined as previously described by Addis et al. (2005a). 2.4. Total fatty acids analysis Cheese fat extraction was based on the method described by Jiang et al. (1996). Aliquots (3 g) of cheese were transferred into a glass centrifuge tube, and 10 mL of deionized water and 18 mL of isopropanol were added. After vigorous shaking, 13 mL of hexane were added, and the mixture was homogenized using an Ultra–Turrax (T 25 Basic, IKA WERKE, Staufen Germany) for 3 min at 13.500 rpm. The mixture was then centrifuged at (1094 × g) for 10 min at 4 ◦ C, and the upper layer was transferred to a second glass test tube. The lower layer was extracted twice with 13 mL of hexane, and the supernatants were pooled with the previous hexane layer. The hexane layer was evaporated with a rotary evaporator at 30 ◦ C. The extracted fat was stored at −20 ◦ C until further analysis. Fatty acid

methyl esters (FAMEs) were obtained starting from 50 mg of cheese fat with a basic trans-methylation according to ISO 15884/FIL 182 method, and then analyzed through GC-FID. Individual FAMEs were identified on the basis of retention time, and the comparison with a standard mixture of 37 pure components. In addition, the identification of the isomers of the conjugated linoleic acid (CLA) has been accomplished by comparing the retention time of each chromatographic peak and those of a mixture of chromatographic standards (CLA 9cis, 11trans; CLA 10trans, 12cis; CLA 9cis, 11cis; CLA 9trans, 11trans). Furthermore, also a comparison of the chromatographic profile obtained with those described in literature (Kramer et al., 2004) has been taken into account for confirmatory purpose. The quantitative measurement of each fatty acid methyl ester was performed with a calibration curve using the internal standards Me-C5:0 (for FAMEs from C4:0 to C6:0), Me-C9:0 (for FAMEs from C8:0 to C10:0), Me-C13:0 (for FAMEs from C11:0 to C17:0), and Me-C19:0 (for FAMEs from C18:0 to C26:0). The concentration of each internal standard, that was added to the sample, was 100 mg g−1 of fat. 2.5. Statistical analysis The statistical package Minitab 16 (Minitab 16 Statistical Software (2010), State College, PA: Minitab, Inc.) was used for the statistical analysis. GLM (general linear model) analysis and Tukey test for multiple comparison of means were used for comparing cheeses produced at different times of the year. The model included the effects of month of cheese production (F, 4 levels: March, April, May and June).

3. Results and discussion Physico-chemical characteristics of PDO Pecorino Romano cheese, manufactured at different times of the year, are summarized in Table 1. pH values ranged from 5.07 to 5.31, and were comparable with those reported by Marcialis et al. (1968) but lower than those reported by Addis et al. (2005a) for Pecorino Romano cheese after 8 months of ripening. Cheeses manufactured in April and June had significant lower pH values when compared with those of March and May (P < 0.001), but this variation of pH values, although significant, was very small. Fat to dry matter ratio (43.4 ÷ 50.9%) and protein to dry matter ratio (33 ÷ 43%) were comparable with those reported by Addis et al. (2005a) in Pecorino Romano with a similar aging time. In general, fat to dry matter ratio tended to increase in cheeses produced in the summer season (June) compared with those produced during late-winter and spring season (March–April), while the protein to dry matter ratio tended to decrease proportionally. According to other authors, this trend was principally caused by the natural, seasonal-induced, variations in milk fat to protein ratio that occur with the season progress (Guinee et al., 2007). The increase of milk fat to protein ratio is linked, above all, to the change of fat content according to stage of lactation, diet and husbandry practices, and it is a typical phenomenon that occurs in sheep’s milk produced in Sardinia (Casu and Marcialis, 1966). In particular, at the final stage of lactation, there is a progressive decrease in the volume of produced milk resulting in an increase of content of some macro-components (fat and protein). Sheep’s milk produced in June and July is characterized by a higher fat content, generally not offset by an equal increase in protein content. Therefore, the fat to protein ratio usually ranges from 1.04 to 1.13 in March and from 1.03 to 1.09 in April, while in June and July exceeds the value of 1.20, ranging from 1.22 to 1.30 (average trend of the last 10 years, http://www.ara.sardegna.it/). Observations, borrowed from the experience, though show that, in several

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Table 1 Physico-chemical parameters of PDO Pecorino Romano cheese produced at different times of the year (means ± SD). Min ÷ max

Month of production March

pH Moisture (g 100 g−1 ) Fat/DM (%) Protein/DM (%) NaCl (g 100 g−1 ) Ash (g 100 g−1 ) Aw

5.07 ÷ 5.31 29 ÷ 35 43.4 ÷ 50.9 33 ÷ 43 4.5 ÷ 8.3 6.8 ÷ 10.8 0.815 ÷ 0.874

5.21a 32ab 47.3ab 39a 5.6b 8.2b 0.846c

P-value April

± ± ± ± ± ± ±

0.03 1 0.4 1 0.3 0.3 0.006

5.15b 32b 47.2b 39a 5.5b 8.1b 0.856b

May ± ± ± ± ± ± ±

0.04 1 1.9 1 0.5 0.5 0.011

5.21a 33a 48.4a 37b 5.3b 7.9b 0.863a

June ± ± ± ± ± ± ±

0.07 1 1.3 2 0.5 0.3 0.007

5.13b 32ab 49.0a 35c 6.5a 9.3a 0.837c

± 0.05 ±1 ± 0.9 ±1 ± 0.8 ± 0.7 ± 0.011

*** ** ** *** *** *** ***

DM, dry matter; Aw , water activity. Values within rows not sharing a common superscript are significantly different, ** P < 0.01; *** P < 0.001. A total of 70 samples of Pecorino Romano cheese were collected and in particular 10 from March, 30 from April, 20 from May and 10 from June productions.

dairy plants, the fat to proteins ratio in June and July often exceeds 1.35. Since, in the PDO specification, the milk standardization before cheese-making is not permitted, this disequilibrium affects the technological characteristics of milk determining substantial changes in the physico-chemical composition of resulting cheese. Pecorino Romano cheese is characterized by a high and variable salt content (from 3.5 to 6.5%) (Marcialis et al., 1968; Galistu et al., 1996) when compared with other Italian hard cheeses (Grana Padano, Parmigiano Reggiano, Fontina, Pecorino Toscano) where the salt content, on a wet basis, hardly exceeds 2.0% (Battistotti and Corradini, 1993). Pecorino Romano cheese manufactured in June was characterized by the highest NaCl content (P < 0.001). In general, at the same salting conditions, with increasing the fat content increased the level of salt in cheese. This fact was not due to the higher fat content per se, but rather to the concomitant decrease in the volume fraction of the protein matrix and the associated impedance to the movement of the hydrated Na+ and Cl− molecules. In particular, the extra path length due to the obstruction exerted by protein aggregates and the sieve effect of the pores of matrix on the diffusing species impede the diffusion of the migrating ions (Guinee and Sutherland, 2011). Because of the high incidence of the NaCl content on the inorganic matter of Pecorino Romano, the seasonal evolution of total ash in cheese follows closely that of the salt. Proteolysis in cheese was investigated by evaluating the evolution of proteolysis indices, expressed as a percentage of total nitrogen (Table 2). The pH 4.6 SN/TN ratio (12 ÷ 26%) was comparable with values obtained by Pettinau and Bottazzi (1971) and by Addis et al. (2005a) in Pecorino Romano cheeses after 6 and 8 months of ripening, respectively. This fraction contains numerous medium and small-size peptides, free amino acids and their degradation products derived mainly by rennet (chymosin and pepsin) and, to a lesser extent, by plasmin. The level of SN-TCA/NT (8 ÷ 21%, Table 2) was similar to that found by Addis et al. (2005a) in Pecorino Romano cheese after 8 months of ripening. Rennet enzymes are responsible for the production of a part of SN-TCA, but proteinases and peptidases from lactic acid bacteria make a more substantial contribution. The content of NS-PTA/NT (5 ÷ 14%, Table 2), comprising very small peptides and amino acids deriving mainly by the activity of microbial peptidases (Visser, 1977), was similar to values found previously (Addis et al.,

2005a). The levels of nitrogen fractions reported in this study confirmed that proteolysis in Pecorino Romano cheese is rather limited, as previously observed by Pettinau and Bottazzi (1971) and Galistu et al. (1995). All proteolytic parameters showed a decrease with the progress of the season of production; in fact cheeses manufactured in June were characterized by the lowest degree of proteolysis (P < 0.001). This behavior could be explained considering both the higher salt content and the lower water activity that characterize cheeses in summer season, and that could inhibit the activity of proteolytic enzymes. Fat content and composition exert a relevant influence on cheese quality. Cheese fat contributes to improve cheese texture, being homogeneously distributed within the protein matrix (Stampanoni and Noble, 1991). Furthermore, individual fatty acids are quite different among them and have different health effects as well. For example, conjugated linoleic acid isomers have potential positive effects on human health, and in particular the C18:2 9 cis 11 trans isomer (rumenic acid) has been reported to have anticancer, anti-atherogenic and anti-diabetic effects (Pariza, 2004). Conversely, some saturated and trans fatty acids (SFA and TFA) have been related with the onset of cardiovascular diseases in humans (Mensink et al., 2003; Willett, 2006). Data on milk fatty acids composition for PDO Pecorino Romano cheese production are shown in Table 3. Cheese fatty acids profile agrees, both qualitatively and quantitatively, with results previously reported for Sarda sheep milk (Addis et al., 2005b and Addis et al., 2007). A high variability characterizes the fatty acids profile of analyzed samples; saturated and unsaturated fatty acids (UFA) ranged from 63 to 73% and from 27 to 34% (of total FAMEs), respectively. Cheese fatty acids variability depends almost exclusively by milk fat composition, and not at all by cheese-making process (Lucas et al., 2006). The molecular characteristics of cheese fat components change with the time of the year (Perea et al., 2000). Pecorino Romano cheese produced in the early summer (June) was very different from that of spring (April–May) and late winter (March). In particular the cheese fat percentage of saturated short and medium chain fatty acids (C4:0 ÷ C14:0) was significantly higher in cheeses produced in late winter and in early spring when compared with those produced in late spring and early summer (P < 0.001). Conversely, long chain fatty acids (C16:0, C18:0 and C18:1) tended to increase significantly along the season (P < 0.001). This trend could be related to a change in the animal diet. The

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Table 2 Proteolysis indices of PDO Pecorino Romano cheese produced at different times of the year (means ± SD).

SN/TN (%) SN-TCA/TN (%) SN-PTA/TN (%)

Min ÷ max

Month of production March

April

May

June

12 ÷ 26 8 ÷ 21 5 ÷ 14

24a ± 2 19a ± 1 12a ± 1

19b ± 2 15b ± 2 9b ± 2

18b ± 3 14b ± 3 10b ± 2

14c ± 1 11c ± 1 6c ± 1

P-value

*** *** ***

Values within rows not sharing a common superscript are significantly different, *** P < 0.001. A total of 70 samples of Pecorino Romano cheese were collected and in particular 10 from March, 30 from April, 20 from May and 10 from June productions.

Sarda sheep production system depends on natural pasturing, and feed availability is strongly affected by seasonal and annual climatic variations (Carta et al., 1996). With the arrival of summer season, the decline of the quality of pasture might have induced a decrease in de novo synthesis of fatty acids with chain length less than 16 carbon atoms (Chilliard et al., 2001) and might have favored long-chain fatty acids, in particular C18:0 (stearic acid) and C18:1 (oleic acid), which are usually associated with the mobilization of stored fat (Chilliard et al., 2003). Dairy products are major dietary sources of rumenic acid and vaccenic acid (C18:1 11 trans). Rumenic acid originates from both ruminal biohydrogenation of dietary linoleic and linolenic acid and from endogenous synthesis in animal tissues (Bauman et al., 1999). The endogenous synthesis, starting from vaccenic acid via 9-desaturase enzyme in the mammary gland, is the major source of rumenic acid (Kay et al., 2004). The content of rumenic and vaccenic acids in Pecorino Romano cheese ranged from 1.3 to 2.9% and from 2.4 to 5.9% of total FAMEs, respectively (Table 3). These values were in agreement with data reported by Addis et al. (2007) and Addis et al. (2005b) for milk from Sarda sheep at pasture or fed with fresh forage, and with results published by Nudda et al. (2005) in Pecorino Romano cheese after 24 h of production. Several

studies conducted on ruminants nutrition confirmed that grazing helps to increase rumenic acid concentration in milk and cheese fat. In ewes’ milk, a higher CLA content has been correlated with increased amounts of legumes in the pastures, or also with more grass in natural pasture at an early stage of the season (Cabiddu et al., 2005). The CLA-enriching effect of pasture has been attributed both to effects on biohydrogenation pathway in the rumen and to the supply of linolenic acid precursor (Collomb et al., 2006). The results presented in this study highlighted a significant decrease in values of rumenic, vaccenic and linolenic acids of Pecorino Romano cheese during the season of production (P < 0.001), showing highest contents of these fatty acids in cheese produced at late winter and spring (March–April), intermediate values in cheese manufactured in May and lowest values in cheese made in early summer (June). These results confirm those found by Piredda et al. (2002), who reported that vaccenic and rumenic acid contents of milk and cheese decreased during the grazing season in ewes grazing sulla (Hedysarum coronarium) and annual ryegrass. This is consequent to the decline of quality of the pasture grass in terms of concentration of fatty acids precursors (in particular linolenic acid) (Cabiddu et al., 2005).

Table 3 Fatty acids content in PDO Pecorino Romano cheese produced at different times of the year (expressed as % of total fatty acid methyl esters; means ± SD). Min ÷ max

Month of production March

C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 9 cis C15:0 C16:0 C16:1 9 cis C17:0 C18:0 C18:1 11 trans C18:1 9 cis C18:2 9 cis 12 cis C18:3 9 cis 12 cis 15 cis CLA 9cis 11 trans SFA UFA

3.3 ÷ 4.7 1.7 ÷ 3.2 1.2 ÷ 2.9 4.9 ÷ 11.5 2.8 ÷ 5.7 9.2 ÷ 11.6 0.14 ÷ 0.27 1.03 ÷ 1.39 21.0 ÷ 27.8 0.56 ÷ 1.07 0.52 ÷ 0.81 10.4 ÷ 13.4 2.4 ÷ 5.9 16.7 ÷ 27.5 1.5 ÷ 3.4 0.6 ÷ 1.5 1.3 ÷ 2.9 63 ÷ 73 27 ÷ 34

4.1a 2.9a 2.4a 10.1a 4.8ab 9.9b 0.16c 1.13c 21.7c 0.64c 0.59c 11.4b 5.3a 18.6b 2.2b 1.4a 2.6a 69b 31b

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

P-value April

0.1 0.1 0.1 0.3 0.1 0.2 0.01 0.06 0.5 0.02 0.03 0.3 0.2 0.8 0.1 0.1 0.2 1 1

4.1a 2.9a 2.5a 10.5a 5.1a 10.7a 0.19b 1.22b 22.4c 0.66c 0.58c 10.9b 4.8a 17.6c 1.9c 1.3a 2.5a 71a 29c

May ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.2 0.3 0.1 0.4 0.02 0.09 0.6 0.04 0.04 0.4 0.6 0.5 0.1 0.1 0.1 1 1

3.8b 2.6b 2.0b 9.1b 4.6b 10.9a 0.21a 1.30a 23.9b 0.70b 0.67b 12.2a 3.6b 19.4b 2.2b 1.1b 1.8b 71a 29c

June ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.2 0.3 1.3 0.6 0.4 0.02 0.05 1.3 0.07 0.04 0.9 0.6 1.3 0.3 0.2 0.3 1 1

3.6b 1.9c 1.3c 5.7c 3.1c 9.7b 0.23a 1.23ab 25.5a 0.91a 0.72a 12.7a 2.8c 25.2a 3.1a 0.9c 1.4c 66c 34a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.7 0.2 0.3 0.01 0.05 1.2 0.09 0.06 0.6 0.3 1.4 0.2 0.1 0.1 1 1

*** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** ***

CLA 9cis 11trans, conjugated linoleic acid; SFA, saturated fatty acids; UFA, unsaturated fatty acids. Values within rows not sharing a common superscript are significantly different,*** P < 0.001. A total of 70 samples of Pecorino Romano cheese were collected and in particular 10 from March, 30 from April, 20 from May and 10 from June productions.

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Table 4 Free fatty acids content in PDO Pecorino Romano cheese produced at different times of the year (expressed as mmol kg−1 of cheese; means ± SD). Min ÷ max

Month of production March

C4:0 C6:0 C8:0 C10:0 SCFFAs C11:0 C12:0 C14:0 C15:0 C16:0 C16:1 cis MCFFAs C17:0 C18:0 C18:1 cis C18:2 cis C18:3 cis LCFFAs TCFFAs

3 ÷ 14 1÷5 0.7 ÷ 4.1 2 ÷ 10 8 ÷ 31 0.02 ÷ 0.13 0.7 ÷ 3.9 1.3 ÷ 7.8 0.13 ÷ 0.83 2.5 ÷ 15.6 0.14 ÷ 1.38 5 ÷ 30 0.06 ÷ 0.34 0.6 ÷ 3.5 2.0 ÷ 22.7 0.24 ÷ 2.83 0.12 ÷ 1.00 3.1 ÷ 30.4 16 ÷ 88

12a 4a 2.5a 6a 24a 0.08a 2.3a 2.9 0.25 4.5ab 0.26ab 10 0.10ab 1.2a 4.0ab 0.54ab 0.35a 6.2ab 40a

P-value April

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 1 0.3 1 3 0.01 0.3 0.3 0.03 0.4 0.02 1 0.01 0.1 0.3 0.04 0.03 0.4 4

6c 2c 1.4b 3b 13c 0.05b 1.5b 2.0 0.18 3.1b 0.18b 7 0.07b 0.8b 2.6b 0.30b 0.21b 4.0b 23b

May ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 1 0.3 1 3 0.01 0.2 0.3 0.02 0.4 0.02 1 0.01 0.1 0.4 0.04 0.04 0.6 4

9ab 3b 1.8b 4ab 18b 0.06b 1.8ab 2.4 0.24 4.1ab 0.24ab 9 0.09ab 1.0ab 3.5b 0.45b 0.23b 5.3b 32ab

June ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 2 0.8 2 8 0.03 0.9 1.0 0.10 1.6 0.09 4 0.03 0.3 1.0 0.12 0.09 1.5 13

7bc 2c 1.3b 3b 13bc 0.03c 1.3b 2.6 0.26 5.1a 0.39a 10 0.12a 1.2a 6.1a 0.84a 0.27ab 8.6a 31ab

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2 1 1.0 3 6 0.02 0.9 2.0 0.21 4.0 0.37 7 0.09 0.9 6.3 0.76 0.26 8.2 22

*** *** *** *** *** *** *** ns ns ** ** ns ** *** ** *** * ** ***

SCFFAs, short chain free fatty acids; MCFFAs, medium chain free fatty acids; LCFFAs, long chain free fatty acids; TFFAs, total free fatty acids. Values within rows not sharing a common superscript are significantly different, * P < 0.05; ** P < 0.01; *** P < 0.001. A total of 70 samples of Pecorino Romano cheese were collected and in particular 10 from March, 30 from April, 20 from May and 10 from June productions.

The quantitative and qualitative profile of free fatty acids released from cheese triglycerides during lipolysis process is considered to influence the flavor of cheeses either directly (Woo et al., 1984) or indirectly as precursors of methyl ketones, lactones, alkanes, esters, etc. (Urbach, 1991). Data on lipolysis in PDO Pecorino Romano cheese are shown in Table 4, where FFAs were grouped in three classes: short-chain FFAs (SCFFAs, C4:0 ÷ C10:0), mediumchain FFAs (MCFFAs, C12:0 ÷ C16:0) and long-chain FFAs (LCFFAs, C18:0 ÷ C18:3). The content of total free fatty acids (TFFAs) in Pecorino Romano cheese ranged from 16 to 88 mmol kg−1 of cheese (Table 4). SCFFAs resulted as the most abundant class (8 ÷ 31 mmol kg−1 of cheese, Table 4), presenting very high levels of butyric acid (C4:0) (3 ÷ 14 mmol kg−1 of cheese). Due to their low perception thresholds (Larráyoz et al., 1999) short chain fatty acids play an important role in the sensory characteristics of PDO Pecorino Romano cheese, in particular for piquant taste development. Among MCFFAs (5 ÷ 30 mmol kg−1 of cheese) and LCFFAs (3.1 ÷ 30.4 mmol kg−1 of cheese), C16:0 (2.5 ÷ 15.6 mmol kg−1 of cheese) and C18:1 9 cis (2.0 ÷ 22.7 mmol kg−1 of cheese) presented the highest values, respectively (Table 4). The values of free fatty acids obtained in this study were similar to those reported by Addis et al. (2005a) for Pecorino romano cheese after 8 months of ripening. The high relevance of short chain fatty acids and in particular of butyric acid is linked to use of lamb paste rennet in cheese-making process. Lamb paste rennet contains a rather complex lipolytic system, in which the most important enzyme is the pre-gastric lipase (PGL) (Nelson et al., 1977). The activity of PGL is high on triglycerides containing SCFA (O’Connor et al., 1993; De Caro et al., 1995), especially those containing butyric acid, which is predominantly esterified at sn-3 position of the glycerol backbone in sheep milk fat (Pitas and Jensen, 1970; Kim

and Lindsay, 1993), and therefore easily accessible to the enzymatic action. The content of total and individual fatty acids (Table 4), varied in a broad range probably due to the poor and not constant quality of the rennet used. Various factors, such as age and diet at which lambs are slaughtered, have been claimed to affect both the quantity and the quality of paste rennet enzymes (Addis et al., 2008). In the recent years there has been a great increase in production of paste rennet at industrial level, and abomasa derive from large slaughterhouses where lambs are kept on empty stomach for long time before being slaughtered. Moreover, nowadays lambs are weaned earlier than in the past to anticipate delivery of milk to dairies. These aspects can explain the qualitative decay of the activity of enzymes contained in paste rennet and in particular the high variability of PGL content. Consequently, the use of this rennet type in cheese-making could influence the characteristics of PDO Pecorino Romano cheese in an unpredictable and undesirable way (Addis et al., 2008). Statistical analysis indicated that SCFFAs content in cheeses manufactured in late winter was higher when compared with those produced in spring and early summer seasons, reaching the lowest values in April and June (P < 0.001). MCFFAs content was not affected by the period of production, whereas LCFFAs values tended to increase during season, with higher values obtained for Pecorino Romano cheese produced in June than that produced in April and May (P < 0.001). It has been reported, in the case of Idiazabal cheese, that the amount of individual free fatty acids changes from late winter to early summer productions (Chávarri et al., 1999). In fact, the major free fatty acids in March were C4:0, C10:0, C16:0 and C18:1 9 cis, whereas in summer, C4:0, C10:0 and C16:0 were present at lower concentration. The change in the free fatty acids

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profile of cheeses manufactured at different times of the year could be related both to the change of milk fatty acids profile and to the seasonal variation of endogenous (Chávarri et al., 1998) or microbial enzymatic activity that occurs in milk. 4. Conclusions The results reported in this work clearly demonstrated that the time of production during the year exerted a marked influence on physico-chemical and nutritional characteristics of Pecorino Romano cheese. Ovine milk destined to become Pecorino Romano cheese cannot be standardized (according to PDO specifications) for fat or protein content prior to cheese-making, thus the season of production influences cheese composition. In fact, cheeses produced in late winter and spring were characterized by a less fat and salt content with respect to those produced in early summer. The nutritional value of cheese fat, associated in particular with linolenic, rumenic and vaccenic acids content, decreased with the progress of lactation stage probably due to variation in animal feed, pasture availability and fatty acids composition of grass lipids. Also the proteolysis and lipolysis processes of cheese were influenced by the seasonality of productions. In particular cheeses produced at late winter were more proteolyzed and lipolyzed if compared with those manufactured at summer season. Further studies will be needed to verify the full impact of all these variations on the sensory characteristics of the cheese. In view of the above results, future changes of the product specification of Pecorino Romano cheese both by permitting milk standardization prior to cheese making or by restricting the manufacture period, could be considered valid ways to limit the variability of the chemical composition of the cheese according to the manufacture season. Conflict of interest The authors acknowledge that they have disclosed all and any actual or potential conflicts of interest with their work or partial benefits associated with it. Acknowledgements This research was supported by Regione Autonoma della Sardegna (Delib.G.R. n. 46/34 del 27/12/2010), within the program “Extraordinary measures of research and development for the benefit of farms and the processing and marketing enterprises”. The authors would like to thank Filomena Tavera for her help with the analytical determinations, and the Consorzio per la Tutela del formaggio Pecorino Romano for supplying cheese samples. References Addis, M., Piredda, G., Pes, M., Di Salvo, R., Scintu, M.F., Pirisi, A., 2005a. Effect of the use of three different lamb paste rennets on lipolysis of the PDO Pecorino Romano Cheese. Int. Dairy J. 15, 563–569. Addis, M., Cabiddu, A., Pinna, G., Decandia, M., Piredda, G., Pirisi, A., Molle, G., 2005b. Milk and cheese fatty acid composition in sheep

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