Artichoke (Cynara scolymus L.) flower extract was assayed with the aim of replacing animal rennet in the manufacture of Gouda-type cheeses from bovine milk. Floral extract coagulated milk within a suitable time for use on an industrial scale, while the yield of cheese obtained was equal to that achieved with bovine abomasum. Five proteolytic fractions with milk-clotting activity were isolated in a two-step purification protocol, three belonging to the cardosin group. Cheeses made with C. scolymus proteases must be brined for a longer period (40 h) to prevent overproteolysis and avoid the development of a background flavor. The type of coagulant (bovine or vegetable) had no significant effect on the cheeses' chemical parameters analyzed throughout ripening, and no significant organoleptic differences were detected between those manufactured with C. scolymus or animal rennet. The results indicate that C. scolymus flower extract is suitable for replacing animal rennet in the production of Gouda-type cheeses.

An outstanding parameter in cheese making is the type of coagulant, which greatly influences the characteristics of the final products. Proteolysis is the most important set of biochemical changes during ripening of most cheeses, and is carried out, in different magnitude, by proteolytic agents originated in milk, rennet (or rennet substitute), and starter and non-starter micro-organisms (Silva & Malcata, 2000). The demand for alternative sources of milk coagulants, to replace the expensive and limited natural rennet supplies, has increased (Esteves et al. 2001). All commercial enzymes employed as milk coagulant are aspartic proteinases, which are most active at acidic pH and preferentially cleave peptide bonds between residues with hydrophobic side-chains (Silva & Malcata, 1999). Because of the presence of aspartic proteinases, aqueous crude extracts from flowers of Cynara cardunculus (Veríssimo et al. 1995, 1996), Cynara humilis, and/or Cynara scolymus are traditionally employed in the Iberian Peninsula as vegetable rennet for cheesemaking (Reis et al. 2000). Milk clotting activity was also proved in flowers of Centaurea calcitrapa and Onopordum turcicum (Tamer, 1993; Domingos et al. 1998). All these species are included within the Asteraceae family and furthermore in the same tribe: Cardueae Cass.= Cynareae Less. (Ariza Espinar & Delucchi, 1998). When a potential rennet substitute is studied, it is particularly important to evaluate adequately the degradation patterns of the caseins because of their effects on yield, consistency, and flavour of the final cheese (Fox, 1989). It is important to guarantee a well-balanced breakdown of curd proteins (caseins) in order to avoid formation of undesired attributes in cheese such as low viscosity and high bitterness (Visser, 1993). One of the most frequently used methods to monitor proteolytic processes on caseins is urea-polyacrylamide gel electrophoresis. On the other hand, tricine-SDS polyacrylamide gel electrophoresis improves the separation, identification and quantification of casein hydrolysates because it allows the visualization of large and small peptides (Pardo & Natalucci, 2001), with the additional advantage of allowing the estimation of molecular masses. Both methods are then suitable to characterize the performance of vegetable rennet in different ways. This preliminary study had the following objectives: the partial characterization of (i) the aspartic proteolytic activity present in flowers of Silybum marianum (L.) Gaertn. (Asteraceae); and (ii) the hydrolytic profile of bovine caseins.

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Typical aspartic proteinases from plants of the Astereaceae family like cardosins and cyprosins are well-known milk-clotting enzymes. Their effectiveness in cheesemaking has encouraged several studies on other Astereaceae plant species for identification of new vegetable rennets. Here we report on the cloning, expression and characterization of a novel aspartic proteinase precursor from the flowers of Cirsium vulgare (Savi) Ten. The isolated cDNA encoded a protein product with 509 amino acids, termed cirsin, with the characteristic primary structure organization of plant typical aspartic proteinases. The pro form of cirsin was expressed in Escherichia coli and shown to be active without autocatalytically cleaving its pro domain. This contrasts with the acid-triggered autoactivation by pro-segment removal described for several recombinant plant typical aspartic proteinases. Recombinant procirsin displayed all typical proteolytic features of aspartic proteinases as optimum acidic pH, inhibition by pepstatin, cleavage between hydrophobic amino acids and strict dependence on two catalytic Asp residues for activity. Procirsin also displayed a high specificity towards κ-casein and milk-clotting activity, suggesting it might be an effective vegetable rennet. The findings herein described provide additional evidences for the existence of different structural arrangements among plant typical aspartic proteinases.