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CN = casein; WSN%TN = water-soluble N% total N; TCA-SN%TN = 12% (w/v) trichloroacetic acid-soluble

Source: Data compiled from Refs. 14, 16, 20, and 31.

CN = casein; WSN%TN = water-soluble N% total N; TCA-SN%TN = 12% (w/v) trichloroacetic acid-soluble

Source: Data compiled from Refs. 14, 16, 20, and 31.

Electrophoretograms of Feta on urea polyacrylamide gels (Fig. 1) reveal the degradation of casein during ripening. The breakdown of as1-casein starts very early. Many zones with an electrophoretic mobility higher than as1-casein are present in the 4-day sample; of these, the zones corresponding to the peptides produced by the action of chymosin (e.g., as1-I-casein) are the most intense. Hydrolysis of h-casein starts later, generating peptides with lower electrophoretic mobility than the parent protein (g-caseins region). It is worth noticing that the intensity of all zones decreases during ripening, as proteins and large peptides are hydrolyzed into smaller fragments not able to be fixed on the gel. Only the production of the peptides with slow (g-caseins region) and intermediate mobility (zone X of unknown identity) increases with time.

The results of many investigations (14,16,20,31) have showed that rennet plays an important role in Feta cheese ripening. From investigations (34,35) it appears that the extended as1-casein hydrolysis during early ripening is due to the residual rennet; because of the low pH and high moisture of the Feta curd, residual rennet is higher than for other cheese varieties (36-38). Additionally, the environmental conditions favor chymosin activity. Although chymosin is active on both as1- and h-casein, h-casein degradation is strongly retarded by the presence of salt (39). On the other hand, the activity of plasmin is relatively low, due to the low pH and the high NaCl content. The role of plasmin is mainly important in the ripening of cheeses in which rennet enzymes have been destroyed by high cooking temperature. This is not the case, however, with Feta. As a result, as1-casein is the main substrate of the proteolytic activity; 40-50% is hydrolyzed in 15-20 days (14,20), whereas the percentage of hydrolyzed h-casein is less than 10%. The later breakdown of

Figure 1 Electrophoretogram of Feta cheese at various ages (4, 20, 60, 180, 360 days). TC, total casein. (From Ref. 14.)

as1-casein could be attributed to the synergistic action of the residual rennet and the cell-bound proteinases of starter bacteria.

The RP-HPLC profile of the water-soluble fraction confirms the significant role of residual rennet in Feta cheese ripening (Fig. 2). Most of the identified peptides of that fraction originate from the N-terminal half of the as1-casein. Cleavage of Phe23-Val24, Phe32-Arg33, Leu98-Leu99, Leu101-Lys102, and Leu109-Glu110 bonds could be attributed to chymosin action. h-Casein is the main substrate for plasmin activity, which is reflected in the production of g-caseins from its C-terminal part. However, the two identified peptides originating from h-casein in Feta cheese extract apparently result from the cleavage of Leu190-Tyr191 and Ile205-Leu206 bonds by chymosin (30). Although the isolation of k-casein peptides from cheese has not been reported, a peptide corresponding to k-casein (f 96-105) was also detected in the water-soluble fraction of Feta cheese. It presumably originated from para-n-casein and its formation could be the result of the action of lactococcal proteinase at Met95-Ala96, which exhibits the characteristics of a susceptible cleavage site for such an enzyme (40).

Casein Hplc Profile

Figure 2 Reversed-phase HPLC profile of the water-soluble fraction of 6-month-old Feta showing the peaks collected and identified. Eluent A was 1 mL of trifluoroacetic acid (TFA)/L of deionized water. Eluent B was 0.9 mL of TFA, 399.1 mL of deionized water, and 600 mL of acetonitrile/L. Gradient: 0 to 10 min, eluent A; 10 to 90 min, 0 to 80% eluent B; 90 to 100 min, 100% eluent B. The flow rate was 0.8 mL/min. The absorbance of the eluate was monitored at 214 nm. Peak numbers correspond to the following compounds: 1, Tyr; 2, Phe; 3, as1-CN (f 4-14) and as1-CN (f 40-49); 4, as1-CN (f 1-14); 5, h-CN (f 164-180), as1-CN (f 102-109) and as1-CN (f 24-30); 5A, n-CN (f 96-105) and as1-CN (f 91-98); 6, as1-CN (f 24-32); 7, h-CN (f 191-205); 8, a-LA; 9, h-LG (f 16-?); 10, h-LG. (From Ref. 30, courtesy of the Journal of Dairy Science.)

Figure 2 Reversed-phase HPLC profile of the water-soluble fraction of 6-month-old Feta showing the peaks collected and identified. Eluent A was 1 mL of trifluoroacetic acid (TFA)/L of deionized water. Eluent B was 0.9 mL of TFA, 399.1 mL of deionized water, and 600 mL of acetonitrile/L. Gradient: 0 to 10 min, eluent A; 10 to 90 min, 0 to 80% eluent B; 90 to 100 min, 100% eluent B. The flow rate was 0.8 mL/min. The absorbance of the eluate was monitored at 214 nm. Peak numbers correspond to the following compounds: 1, Tyr; 2, Phe; 3, as1-CN (f 4-14) and as1-CN (f 40-49); 4, as1-CN (f 1-14); 5, h-CN (f 164-180), as1-CN (f 102-109) and as1-CN (f 24-30); 5A, n-CN (f 96-105) and as1-CN (f 91-98); 6, as1-CN (f 24-32); 7, h-CN (f 191-205); 8, a-LA; 9, h-LG (f 16-?); 10, h-LG. (From Ref. 30, courtesy of the Journal of Dairy Science.)

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