Proteolysis in model Portuguese cheeses: Effects of rennet and starter culture Cla ´udia I. Pereira a , Eliza O. Gomes b , Ana M.P. Gomes a , F. Xavier Malcata a, * a Escola Superior de Biotecnologia, Universidade Cato ´ lica Portuguesa, Rua Dr. Anto ´ nio Bernardino de Almeida, P-4200-072 Porto, Portugal b Polytech’ Montpellier, Place Euge `ne Bataillon, F-64000 Montpellier, France To shed further light onto the mechanisms of proteolysis that prevail throughout ripening of Portuguese cheeses, model cheeses were manufactured from bovine milk, following as much as possible traditional manufacture practices – using either animal or plant rennet. The individual role upon proteolysis of two (wild) strains of lactic acid bacteria – viz. Lactococcus lactis and Lactobacillus brevis, which are normally found to high viable numbers in said cheeses, was also considered, either as single or mixed cultures. Our experimental results confirmed the influence of rennet on the proteolysis extent, but not on proteolysis depth. On the other hand, the aforementioned strains clearly improved release of medium- and small-sized peptides, and contributed as well to the free amino acid pool in cheese. Keywords: Cheese ripening; Soluble nitrogen fractions; Casein hydrolysis; Cheese coagulant; Lactic acid bacteria Proteolysis has for long been regarded as the most important multi-step, biochemical event in cheese ripening (Fox, Law, McSweeney, & Wallace, 1993; Fox & McSwee- ney, 1996); in fact, it accounts for the development of a number of organoleptic features, encompassing both fla- vour and texture. The main vectors for proteolysis in cheese are proteinases indigenous to milk (plasmin and cathepsin D), included in the coagulant formulation (either from animal, plant or microbial origin), and released upon lysis by starter and non-starter bacteria. Each one plays a specific role, but the whole set, rather than each one indi- vidually, affects the final product. Portuguese cheeses bearing DOP status are traditionally manufactured from whole raw milk; the most usual cheese- making protocols encompass milking of animals by early morning, brief storage of milk at room temperature, clot- ting with plant rennet in the absence of any added starter culture, cutting and manual working of the curd, spontane- ous whey drainage, and ripening for a minimum of 30 days (Macedo, Malcata, & Oliveira, 1993). A broad group of microorganisms eventually gain access to the curd from the cheesemaking environment, and will consequently con- tribute to the ripening process. Lactic acid bacteria are lar- gely predominant in such a microbial group, and are believed to play a key role on the organoleptic characteris- tics of the final cheese. However, studies pertaining to model cheeses under strictly controlled conditions are scarce to date, so knowledge on the specific effect of each factor upon the final cheese is still poor. Clotting of milk is usually an enzyme-driven step during which rennet enzymes bring about cleavage of Met 105 – Phe 106 of j-casein, that is present on the surface of casein micelles in milk, surrounding a s - and b-caseins (which are more hydrophobic in nature). Animal rennet possesses a pair of enzymes: chymosin and pepsin. Plant rennet (viz. that extracted from dried flowers of the wild thistle, Cynara car- dunculus L.), which has been used for centuries in the manu- facture of traditional cheeses in Portugal (Freitas & Malcata, * Corresponding author. Tel.: +351 22 558 00 04; fax: +351 22 509 03 51. E-mail address: fxmalcata@esb.ucp.pt (F.X. Malcata).