Comparison of methods for analysis of proteolysis by plasmin in milk

Abstract

Sensitive methods that are currently used to monitor proteolysis by plasmin in milk are limited due to their high cost and lack of standardisation for quality assurance in the various dairy laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and fluorescamine, were selected to assess their suitability for the detection of proteolysis in milk by plasmin. Commercial UHT milk was incubated with plasmin at 37°C for one week. Clarification was achieved by isoelectric precipitation (pH 4.6 soluble extracts) or 6% (final concentration) trichloroacetic acid (TCA). The pH 4.6 and 6% TCA soluble extracts of milk showed high correlations (R2 > 0.93) by the TNBS, fluorescamine and RP-HPLC methods, confirming increased proteolysis during storage. For gel electrophoresis, extensive proteolysis was confirmed by the disappearance of α- and β-casein bands on the seventh day, which was more evident in the highest plasmin concentration. This was accompanied by the appearance of α- and β-casein proteolysis products with higher intensities than on previous days, implying that more products had been formed as a result of casein breakdown. The fluorescamine method had a lower detection limit compared with the other methods, whereas gel electrophoresis was the best qualitative method for monitoring β-casein proteolysis products. Although HPLC was the most sensitive, the TNBS method is recommended for use in routine laboratory analysis on the basis of its accuracy, reliability and simplicity.

Age Gelation, Sedimentation, and Creaming in UHT Milk: A Review

“…Plasmin produces peptides that are soluble at pH 4.6 but not in 4% TCA, whereas exogenous enzymes produce peptides that are soluble under both conditions. Thus a sample with a high NCN but with a low NPN is likely to be hydrolyzed by plasmin, whereas a sample with high NCN and NPN is likely to be proteolyzed by exogenous enzymes or a combination of exogenous enzymes and plasmin (Chove et al., ; Datta & Deeth, ).…”

Preliminary Study on the Role of Ionic Calcium in Gelation and Proteolysis of UHT Milk

“…There was a poor correlation between the pH reduction caused by acid addition and that resulting from increasing temperature. Sediment formation was related to pH change at high temperature (Lewis, 2011). It was concluded from one study that the nature of the calcium-and heat-induced gelation or coagulation of milk is influenced, and can be manipulated, by a combination of factors including ionic calcium concentration, temperature, milk pH and milk solids level (Deeth & Lewis, 2015).…”

“…Datta and Deeth (2001) stated that although SHMP inhibited gelation, it did not affect proteolysis. It was documented that addition of 0.15 mg/L plasmin caused gelation of UHT milk in 90 days (Kohlmann et al, 1991), indicating the significance of minor proteolysis by plasmin prior to gelation (Chove et al, 2011). It was also revealed that neither gelation nor proteolysis (9 months at 20 °C ) was observed in a study where serine protease inhibitors were added to UHT skim milk to inhibit plasmin activity, implicating the role of plasmin in proteolysis preceding gelation (De Koning et al, 1985).…”

Detection of Proteolysis in Milk by Pseudomonas fluorescens Using Urea PAGE Method

“…In a study by Chove et al, (2011) analysis of proteolysis by plasmin enzyme on day 7, showed completely disappearance of both the α-and β-casein bands, indicating extensive proteolysis on these caseins. Microbial enzymes have been reported to show preference to hydrolyse κ-casein, which is usually found on the surface of the casein with the formation of para-κ-casein (Snoeren & Van Riel, 1979), followed by extensive hydrolysis which is non-specific (Law et al, 1977).…”