The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50–54°C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

High pressure (HP) processing has attracted considerable interest in dairy research in recent years; its effects on cheese have been reviewed by O'Reilly et al. (2001). HP treatment of cheese may influence several of its properties, e.g., for hard and semi-hard cheeses, such as Cheddar and Gouda, the most notable effects are on the texture and rate of ripening. HP treatment increased elasticity and improved organoleptic properties of Gouda cheese (Kolakowski et al. 1998). Messens et al. (2000) reported that, immediately after treatment at 400 MPa, Gouda cheese was less rigid and solid-like and more viscoelastic than untreated cheese. HP treatment also enhances the functional properties of Mozzarella cheese (Johnston & Darcy, 2000; O'Reilly et al. 2002).

Osteopontin (OPN) is an acidic phosphorylated glycoprotein found in many tissues and physiological fluids. Bovine OPN is a mature protein comprising 262 amino acids with a calculated molecular weight of 29 kDa. However, SDS-PAGE analysis reveals that the protein isolated from milk migrates to a molecular mass of 60 kDa (Sørensen & Petersen, 1993; Bayless et al. 1997). Bovine milk OPN is phosphorylated at 27 serine residues and one threonine residue (Sorensen et al. 1995); three O-glycosylated threonines were also identified, but no asparagine residues were glycosylated in spite of the presence of three putative N-glycosylation sites. In contrast, eight phosphates are recognized in bovine bone OPN (Salih et al. 1996), and 12 phosphoserines and one phosphothreonine are proposed in addition to five O-linked oligosaccharides and at most one N-linked oligosaccharide in the case of rat bone OPN (Prince et al. 1987). Thus, the possibility of tissue or species-specific differences in post-translational modification has been discussed.

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29–fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35·6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 °C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7·0. The ratio of milk clotting to proteolytic activity was 3·03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100±21 min and a z-value of 5.9±0.3 °C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.