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Changes in the concentrations of the free fatty acids (FFA) containing an even number of carbon atoms in Serra cheese were monitored using a two-way factorial design over a ripening period of 35 days throughout the cheesemaking season. Both time variables had statistically significant effects at the 5% level on the concentrations of FFA. The concentration of stearic acid increased significantly to 351 mg kg−1 (fat basis) only during the first week of ripening. Concentrations of palmitic and oleic acids increased significantly during all stages of ripening and approximately doubled during the 35-day ripening period to reach levels of 425 and 453 mg kg−1, respectively. Concentrations of butyric, caprylic, lauric and myristic acids were approximately constant during the first week and increased significantly thereafter up to 285, 17, 94 and 59 mg kg−1, respectively, at 35 days. The concentrations of caprylic and myristic acids increased five-fold in 35 days, the concentration of butyric acid increased three-fold, and the concentration of lauric acid almost doubled during the same period. The concentrations of caproic, capric, linoleic and linolenic acids increased significantly to 48, 121, 188 and 126 mg kg−1, respectively, only after 21 days of ripening. The concentration of linoleic acid doubled in that time period, while increases in the concentrations of caproic, capric and linolenic acids were much smaller. The concentrations of butyric, caproic and palmitic acids in cheeses manufactured in February were statistically lower than those in cheeses manufactured in May. The principal FFAs throughout ripening were, according to chain length and degree of saturation, butyric (short-chain), capric (medium-chain), palmitic and stearic acids (saturated long-chain), and oleic (unsaturated long-chain). Saturated and unsaturated long-chain FFA were present at the highest concentration at all stages of ripening. It is concluded that lipolysis in Serra cheese proceeds slowly to total FFA concentration of 2167 mg kg−1 (fat basis) by 35 days of ripening.

An intracellular hydrolase able to cleave Image -lysine-p-nitroanilide was purified from Lactobacillus plantarum strain ESB5004 via two steps of precipitation with ammonium sulfate (at 30 and 50% (w/v)), followed by hydrophobic interaction and ion-exchange chromatographies. The aminopeptidase was purified up to 11-fold, with a final yield of ca. 1%. Its native molecular weight is ca. 70 kDa, and it is apparently composed of two subunits, the molecular weight of which is 34 kDa. The enzyme was assayed using a wide variety of p-nitroanilide (pNA) derivatives as substrates: it hydrolyzed preferentially pNA adducts of hydrophobic and basic amino acid residues; no hydrolysis was in particular observed of Glu-pNA, Gly-pNA or Pro-pNA. The enzyme activity was removed by the metal-chelating agent EDTA, thus suggesting that it is a metallo-enzyme; however, the EDTA-inhibited enzyme was reactivated in the presence of Co2+. Optimal aminopeptidase activity was obtained at 28 °C (pH 7.0) and pH 6.5 (37 °C). The enzyme was inhibited by 10 mM CaCl2 or MgCl2.

Summary The individual and interactive effects of four production factors (amount of vegetable rennet, temperature of coagulation, pressing and salting of the fresh cheese) and two ripening factors (temperature and relative humidity) on microbiological, physico-chemical, biochemical, textural and sensory characteristics of Serra cheese were simultaneously studied using a 2vi6-1 factorial design. Highly significant effects of salting and ripening relative humidity upon the characteristics of the cheese were detected. Addition of salt to the surface of the fresh cheese reduced microbial growth, water activity, moisture and lactic acid contents, proteolysis, lipolysis, aroma and softness of the cheese. Conversely, increase of the relative humidity during ripening increased these charactereristics. Pressing had no statistically significant effect on cheese characteristics.

Seasonal changes of the ash content and mineral concentrations in Serra cheese were studied over a typical 35-day ripening period. Statistically significant differences (at the 5% level) exist between the ash content and the concentrations of Na, K, Ca, P, Mg and Zn in cheeses during ripening. The highest concentrations of Na was obtained in cheese ripened for 7 days, whereas the concentrations of K, Ca, P, Ng and Zn decreased significantly during ripening. For 35-day-old cheeses, concentrations of Na, K and Cu were lowest and concentration of P was highest for cheeses manufactured in May. The concentration of Ca was lowest for cheeses manufactured in February. On average, the most concentrated minerals (in g kg-1 of total solids, TS) in 35-day-old Serra cheese were Na (18·56), Ca (9·70) and P (7·92) and, at a lower level, K (1·70) and Mg (0·96). Only trace levels (in mg kgTS-1) of Zn (94·33), Cu (2·26) and Mn (1·25) were detected. A high mineral nutrition quality was thus ascribed to 35-day-old Serra cheese based on the average nutritional densities: 4·8 for Ca, 4·0 for P, 1·1 for Mg, 3·4 for Na, 2·4 for Zn, 0·4 for Cu, 0·2 for Mn and 0·2 for K.

In order to optimize the process of cheesemaking of Serra cheese, the most famous traditional Portuguese cheese manufactured with raw ewes' milk at farm level, several cheeses were produced according to a second-order composite factorial design using temperature of coagulation, amount of plant rennet added and amount of salt added to fresh cheese as the manipulated technological variables. Sensory, microbiological, physicochemical and biochemical analyses were carried out for all experimental cheeses and for two cheeses manufactured by traditional practices. The amount of salt added to fresh cheese affected significantly the sensory, microbiological and biochemical characteristics of 60 day-old Serra cheese, whereas the amount of plant rennet added and the coagulation temperature affected significantly only the sensory and microbiological attributes. None of the three technological parameters at the levels tested played a statistical role at the 5% significance level in the chemical composition of the cheese. It was found that 0.3 g of plant rennet added per litre of milk, a coagulation temperature of 28 °C, and 0.05 g of salt added per cm2 of fresh cheese surface provide the best quality of cheese in terms of highest sensory scores and lowest numbers of viable enterococci and coliforms.

This paper comprehensively reviews fundamental and applied aspects of the manufacture of Serra cheese, its composition, the biochemical reactions that take place during coagulation and ripening, and the microbial ecology. Serra cheese is the most traditional cheese manufactured in Portugal. Aspects that make it unique are 1) its manufacture by the coagulation of raw ewe milk using a vegetable rennet (cardoon flower) and 2) its final buttery texture and flavor. The wide variation of the final quality of this "Appelation d'Origine Controllee" cheese has been explained by the intrinsic variabilities of raw materials, cheese-making practices, and maturation. The available studies pertaining to Serra cheese have indicated that 1) the coagulant activity of cardoon flower extract on ewe milk is higher and is more affected by pH and salt concentration than that of animal rennet, 2) the best coagulation temperature is 27 to 29°C, 3) the best ripening conditions are 8°C and 90% relative humidity, 4) the lactic acid fermentation results mainly from the action of lactic acid-type Streptococcus, Leuconostoc , and Lactobacillus species, 5) a yellow to reddish surface viscous material (mainly composed of yeasts) is important in the ripening process, 6) maturation is essentially surface-driven, and 7) the low temperatures prevailing during winter help to control the extensive microbial contamination that occurs from the poor sanitary conditions during cheese manufacture. These points are described and critically discussed in light of the principles of dairy science.