General Observations

It is generally accepted that proteolysis is a major event during cheese ripening. Casein breakdown involves the action of many enzymes from various sources; among them, in digenous milk proteinase (plasmin), chymosin and other milk-clotting enzymes, protein-ases and peptidases from the starter microorganisms and NSLAB, and enzymes from adjunct cultures, yeasts, and molds.

The extent of proteolysis in Feta cheese is, in general, lower than in most hard and semihard cheese varieties. During the 2-month minimum ripening time for Feta [according to the Greek Food Code (4)], the water-soluble nitrogen, expressed as % of the total N (WSN%TN) ranges from 12 to 20 (13,16,20,29); this depends somewhat on the residual rennet, the starter and nonstarter flora, and the time-temperature interaction during ripening. However, the WSN%TN, a parameter generally accepted as a ''ripening index,'' does not reflect the products of casein hydrolysis. The water extract of a 3-day-old Feta also includes milk serum proteins, mainly h-lactoglobulin (h-Lg) and a-lactalbumin (aLa). These proteins elute late from reverse-phase high performance liquid chromatography (RP-HPLC) columns and form large peaks (about the 25% of the total area) at the end of the chromatogram (30,31).

When the cheese is placed in brine, another process starts that also influences the N content of the water extract: the diffusion of cheese constituents into the brine (and, eventually, of salt into the cheese) until equilibrium is reached. Along the storage period, small and medium-sized nitrogenous compounds move into the brine. The solubility of casein breakdown products in 5% brine has been reported (32). Milk serum proteins, being also water soluble, follow in the same way. It was found that in chromatograms produced by RP-HPLC, the area corresponding to a-La and h-Lg decreases by about 40% (31). Therefore, it was proposed to substitute for WSN%TN as a ripening index another parameter, more closely related to the release of casein breakdown products. A colori-metric procedure based on the reaction of 2,4,6-trinitrobenzenesulfonic acid (TNBS) with free amino groups was found to give useful information on the degree of ripening of Feta cheese (33). In mature Feta samples, the correlation coefficient between WSN%TN and absorbance at 420 nm (A420) was higher than in fresh samples, as the contribution of the whey proteins to the WSN was reduced (29).

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