Kinetics of changes in plasmin activity and proteolysis on heating milk

Abstract

Heat-induced inactivation of bovine plasmin, denaturation of beta-lactoglobulin (beta-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of beta-casein was studied in a model system consisting of phosphocasein and beta-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of beta-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of beta-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65-80 degrees C was its interaction with beta-lg (kr at 60 degrees C, 0.0526; Ea, 176 KJ/mol). At the point of complete inactivation of plasmin approximately 45% of the beta-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of beta-lg with the casein micelles at 60 degrees C had a rate constant of 3.71 x 10-5 min-1 and an Ea of 259 KJ/mol; thermal denaturation of beta-lg was of much less importance, with a rate constant at 60 degrees C of the order of 1 x 10-10 min-1 and an Ea of 250 KJ/mol. On denaturation of all beta-lg in the system, a maximum of approximately 55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of beta-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of beta-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with beta-lg.

Selective determination of the heat-resistant proteolytic activity of bacterial origin in raw milk

“…Levels of inactivation of 98% (po0.0001), 92% (po0.001) and 94% (po0.0001) of plasmin activity took place for 0.01, 0.015 and 0.025 U mL À1 added plasmin, respectively. These results are in agreement with earlier findings of other researchers, who reported that heating milk for 10 min at 90 1C significantly reduced plasmin activity (Crudden, Oliveira, & Kelly, 2005;Grufferty & Fox, 1988;Kennedy & Kelly, 1997;Saint-Denis et al, 2001). Even after 10 days of incubation at 37 1C, there was no significant difference (p40.05) between the obtained inactivation percentages for the lowest plasmin concentrations (0.01 and 0.015 U mL À1 ).…”

Extreme high-temperature treatment of milk with respect to plasmin inactivation

“…Due to thermal processing free sulphydryl (S-H) groups will become available when b-lactoglobulin unfolds, which is the first stage of denaturation (De Jong, 1996). As a result of unfolding of b-lactoglobulin, the highly reactive S-H groups cause irreversible denaturation of plasmin (Crudden et al, 2005;Metwalli et al, 1998). The above implies that a certain degree of denaturation of b-lactoglobulin is necessary to increase the inactivation of plasmin and plasminogen.…”

“…Previous experiments showed that milk heated with the ISI had a degree of denaturation of b-lactoglobulin of o30%, whereas standard UHT-treatments of milk resulted in a degree of denaturation of 450% (Huijs et al, 2004). However, the effect of denatured b-lactoglobulin is usually not taken into account when predicting/designing UHTprocesses in relation to the inactivation of plasmin (Crudden et al, 2005). Another aspect with regard to plasmin activity is the storage temperature.…”

“…The inactivation kinetics of plasmin in milk have been described by Rollema and Poll (1986). It was shown that (partly) denatured b-lactoglobulin effects the rate of plasmin inactivation (Crudden, Oliveira, & Kelly, 2005). Due to thermal processing free sulphydryl (S-H) groups will become available when b-lactoglobulin unfolds, which is the first stage of denaturation (De Jong, 1996).…”

Kinetic studies of the thermal inactivation of plasmin in acid or sweet whey

“…By contrast, the rate of inactivation of plasmin in sweet whey was greater than the denaturation of b-lg, with $40% b-lg remaining native when all plasmin was inactivated. The unclear role of b-lg in the thermal inactivation of plasmin in whey systems is thus different from the interaction between the two species in milk during heating of milk; Crudden, Oliveira, and Kelly (2005) reported that the principal reaction contributing to the loss of plasmin activity during such heating was its interaction with b-lg.…”