Novel semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus as starter cultures and with cook temperatures of 47, 50 or 53 degrees C. There was a progressive and significant degradation of both alpha(s1)- and beta-caseins during ripening of all cheeses. Increasing cook temperature significantly reduced degradation of alpha(s1)-casein during ripening, in the order 53<50<47 degrees C, as measured by densitometric analysis of urea-polyacrylamide gel electrophoresis (urea-PAGE) electrophoretograms. Mean levels of primary proteolysis, as measured by amounts of pH 4.6-soluble N, were also significantly reduced. A mathematical model, incorporating changes in pH and temperature during manufacture of the cheeses, predicted near to total inactivation of residual chymosin as a result of the cooking profiles used in cheesemaking. Increasing cook temperature did not completely inhibit primary proteolysis or hydrolysis of alpha(s1)-casein to alpha(s1)-casein (f24-199) during ripening, although these reactions were slowed. (c) 2006 Elsevier Ltd. All rights reserved.