Secondary proteolysis in Serra cheese during ripening and throughout the cheese-making season

Experimental Serra cheeses were manufactured from raw ewe’s milk and thistle flowers following a two-way factorial design. The content of nitrogen soluble in water (WSN), in 2% trichloroacetic acid (2% TCA-N), in 12% TCA (TCA-N) and in 5% phosphotungstic acid (5% PTA-N), and the pH and salt-in-moisture concentration were measured throughout the ripening period (sampling at 0, 7, 21 and 35 days) and the cheese-making season (sampling in November, February and May). Proteolysis in 35-day-old Serra cheese was quantitatively high [average values of 34.6% and 11.9% for WSN/TN (total nitrogen) and 2% TCA-N/TN, respectively], but qualitatively low (average values of 5.8% and 1.2% for 12% TCA-N/TN and 5% PTA-N/TN, respectively). The ratios WSN/TN and 2% TCA-N/TN were lowest for cheeses ripened in February, whereas the ratio 12% TCA-N/TN was highest for cheeses ripened in November. By 35 days of ripening, the average pH and salt-in-moisture concentration values were 5.2 and 4.8%, respectively. No correlation was found to occur between the measured pH or salt-in-moisture concentration and the values of soluble nitrogen fractions throughout the cheese-making season

Role of adventitious microflora in proteolysis and lipolysis of Serra cheese: preliminary screening

Six different types of bacteria (five strains of lactic acid bacterium and one strain of coliform) isolated from 35-day-old Serra cheese were assayed for proteolytic and lipolytic activities on milk agar and tributyrin agar, respectively. Peptidase and lipase activities were further studied via analytical assaying of free amino acids by spectrophotometry and of free fatty acids by HPLC in in vitro curdled milk (previously prepared from heat-sterilized ovine milk coagulated with thistle flower) subject to ripening for 21 days at 5 °C and 95% relative humidity. Leuconostoc mesenteroides ssp. mesenteroides/dextranicum and Lactococcus lactis ssp. lactis displayed significant proteinase and peptidase activities, where Leuconostoc lactis and Enterococcus faecium only exhibited peptidase activity. Leuc. mesenteroides ssp. mesenteroides/dextranicum, Ent. faecium, and L. lactis ssp. lactis only showed lipase activity on milk fat after a long incubation period. Short- and medium-chain fatty acid residues were released preferentially by microbial lipases, although long-chain fatty acids were also significantly released by lipases from Leuc. mesenteroides ssp. mesenteroides/ dextranicum. Lactobacillus paracasei ssp. paracasei and Hafnia alvei did not display measurable protease, peptidase, or lipase activities. In view of their hydrolase activities, it is suggested that L. lactis ssp. lactis and Leuconostoc spp. (with possible incorporation of Ent. faecium) are a potential mixed-strain starter for Serra cheesemaking

Changes of mineral concentrations in Serra Cheese during ripening and throughout the cheesemaking season

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

Changes in the major free fatty acids in Serra cheese throughout ripening

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 signtcantly 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 3.5-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

Changes of lactose, lactic acid, and acetic acid contents in Serra cheese during ripening

Changes in the quantities of lactose, lactic acid and acetic acid in Serra cheese were monitored using a triplicate two-way factorial design over a ripening period of 35 days (sampling at 0, 7, 21 and 35 days) throughout the cheesemaking season (sampling in November, February and June). The amount of lactose in total solids of cheese (TS) decreased slowly from 6.17% to 0.21% (w/wTS) as ripening time elapsed. As a result of sugar metabolism, the lactic acid content increased from 0.07% at day 0 to 2.10% (w/wTS) by 35 days, whereas the acetic acid content increased from 0.00% to 0.24% (w/wTS) during the first week. The lactose content was statistically correlated with the lactic acid content but not with the acetic acid content

The technology, chemistry, and microbiology of Serra Cheese: a review

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 Controllée” 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

Purification and characterization of an intracellular aminopeptidase from a wild strain of Lactobacillus plantarum isolated from traditional Serra da Estrela cheese

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

Influence of native lactic acid bacteria on the microbiological, biochemical and sensory profiles of Serra da Estrela cheese

Cheesemaking from batches of raw ewe’s milk was carried out via inoculation with wild strains of Lactococcus lactis subsp. lactis ESB110019 and Lactobacillus plantarum ESB5004 independently, or combined with each other. Those two strains had been isolated from the native microflora of typical Serra da Estrela cheese. One control batch was processed in parallel without addition of any starter. The evolution in viable counts of the main micro-organisms (viz. lactic acid bacteria, Enterobacteriaceae, staphylococci and yeasts), as well as in secondary proteolysis (WSN, 2% TCASN, 12% TCASN and 5% PTASN), was monitored throughout ripening time (over a 63-day period) in cheeses from each batch. The sensory features of the fully ripened cheeses were also assessed. Cheeses manufactured with starter showed significantly lower levels of viable Enterobacteriaceae than those manufactured without starter; viable counts of enterococci and staphylococci did significantly increase after addition of L. lactis or Lb. plantarum, respectively. Proteolysis in terms of WSN and 5% PTASN was not significantly affected by the lactic acid bacteria tested when compared to the control, but L. lactis played a significant role toward increasing the 2% TCASN content of cheeses; both strains led to a statistically significant increase of the 12% TCASN. The scores for flavor and texture of the control cheeses were somewhat above those for the experimental cheeses manufactured with starter