Extraction and partial characterization of a coagulant preparation from Silybum marianum flowers. Its action on bovine caseinate

Abstract

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.

Onopordum acanthium L. (Asteraceae) flowers as coagulating agent for cheesemaking

“…Subsequent studies on those species (Esteves, Lucey, Wang, & Pires, 2003;Faro, Veríssimo, Lin, Tang, & Pires, 1995;Llorente et al, 2004) confirmed the presence of aspartic proteases in the extracts and similar results were obtained when other species belonging to the same taxonomic group (tribe Cardueae, family Asteraceae) were analyzed as potential milk coagulants (Tamer, 1993;Vairo Cavalli, Claver, Priolo, and Natalucci (2005)). …”

“…4), which is the lowest value reported for proteases from species belonging to the tribe Cardueae. Optimum proteolysis was achieved around pH 3.8 for Silybum marianum (Vairo Cavalli et al, 2005), proteinases extracted from Centaurea calcitrapa flowers express maximum activity between pH 4.0 and 5.0 (Domingos et al, 1998), maximum proteolytic activity was recorded at pH 5 for C. scolymus (Llorente et al, 2004), cardosins A and B isolated from flowers of C. cardunculus exhibit maximal activity between pH 5.0 and 5.5 (Faro et al, 1995), while higher peak activity in Australian cardoon (C. Cardunculus) was reached at pH 6.0 (Chen, Zhao, & Agboola, 2003), but in this case casein (not haemoglobin) was used as substrate. Maximum pH value for O. acanthium flowers is even lower than those obtained for calf, pig and lamb chymosins (Foltman & Szecsi, 1998).…”

“…The enzyme extract was obtained according to Vairo Cavalli et al (2005). The upper portions (stigmas and styles) of fresh flowers from O. acanthium (10 g) were ground under liquid nitrogen, homogenized at a ratio of 1 g per 3 mL of 0.1 mol/L citric acidesodium citrate buffer (pH 3.0) containing 1.0 mmol/L EDTA, and stirred for 30 min.…”

Chemical and productive properties of two Sardinian milk thistle (Silybum marianum (L.) Gaertn.) populations as sources of nutrients and antioxidants

“…It is grown also as an ornamental plant (Bhattacharya 2011) and as a tolerant species for soils polluted by heavy metals (Rio-Celestino et al 2006;Perrino et al 2014). In addition, is considered as a new source of plant rennet for aspartic peptidases present in its flowers (Vairo Cavalli et al 2005). Probably due to the plurality of biological activities from its secondary metabolites, milk thistle is also the most studied plant for the treatment of liver disease and this is documented by the huge increase of papers on this topic, over 800 publications, in the last 5 years (Alemardan et al 2013).…”

Callus culture for biomass production of milk thistle as a potential source of milk clotting peptidases

“…(Asteraceae), (Vairo Cavalli et al 2005) and the use of its flowers and leaves for the production of small-scale Serpa cheese has been reported (Cabral et al 1984).…”