Characterization of peptides produced by the action of psychrotrophic proteinases on κ-casein

Abstract

Psychrotrophic bacteria, capable of growing in milk during refrigerated storage before processing, produce thermostable proteinases that strongly influence the keeping quality of long life dairy products (Cousin, 1982). This is the case with UHT milk, where the activity of residual or reactivated bacterial proteolytic enzymes is linked to flavour deterioration and gelation. Furthermore, since most of these enzymes are acid proteinases with a broad specificity and particularly active against κ-casein (Fairbairn & Law, 1986), their hydrolysis products can interfere with detecting the fraudulent addition of rennet whey (López-Fandiño et al. 1993a, b; Recio et al. 1996, 2000). This is usually based upon detecting, by reversed-phase (RP-) HPLC (Olieman & van Riel, 1989; Van Riel & Olieman, 1995a) or capillary electrophoresis (CE) (Van Riel & Olieman, 1995b), caseinmacropeptide (CMP), the 106–169 fragment of κ-casein released by chymosin during the initial stages of cheesemaking (for review, see López-Fandiño & Olano, 1999). Nevertheless, it is not yet clear whether degradation of κ-casein by psychrotrophic enzymes gives rise to hydrolysis products that are identical to those produced by chymosin or just indistinguishable by CE (Van Riel & Olieman, 1995b; Recio et al. 1996).

In the present investigation, we studied CMP-like degradation products produced by the action of extracellular psychrotrophic proteinases on κ-casein by RP-HPLC and CE. Electrospray ionization–mass spectrometry (ESI–MS) was used for peptide identification. Characterization of breakdown products specific to psychrotrophic enzymes may help the investigation of their residual activity remaining in milk after processing, as well as provide an indication of the suitability of CMP as an indicator of adulteration of UHT milk with whey solids.