The flowers of cardoon (Asteraceae) are a rich source of aspartic peptidases which possess milk clotting activity-and are thus used in traditional cheesemaking in the Iberian Peninsula. This study was aimed at characterizing the enzymatic action of the aspartic peptidases present in flowers of Silybum marianum (L.) Gaertn. (Asteraceae), specifically upon degradation of caseins. The proteolytic activities toward Na-caseinates previously prepared from caprine and ovine milks were studied, in a comparative fashion, using urea-PAGE, tricine-SDS-PAGE, densitometry, electroblotting and sequencing. Caprine a s1-and b-caseins were degraded up to 68% and 40%, respectively, during 24 h of incubation. Only one important and well-defined band corresponding to a molecular weight of 14.4 kDa-i.e. a fragment of b-casein, was observed by 12 h of hydrolysis. By 24 h of incubation, ovine a sand b-caseins were degraded up to 76% and 19%, respectively. In what concerns specificity, the major cleavage site in ovine caseinate was Leu99-Arg100 in a s1-casein.

Asclepias fruticosa L. is a small shrub containing latex with proteolytic activity. The crude extract (latex diluted 1:250 and ultracentrifuged) contained 276 microg of protein/mL and the proteolytic activity reached 1.2 caseinolytic U/mL. This enzyme preparation was very stable even after 2 hours at 45 degrees C, but was quickly inactivated after 5 minutes at 80 degrees C. Chromatographic purification was achieved by FPLC using a cation exchanger (SP-Sepharose FF). Thus, a unique proteolitically active fraction could be isolated, being homogeneous by bidimensional electrophoresis and mass spectrometry (Mr = 23,652). The optimum pH range was achieved at 8.5-10.5. The enzyme activity was completely inhibited by specific cysteine peptidases inhibitors. Isoelectric focusing followed by zymogram showed the enzyme had a pI greater than 9.3. The N-terminus sequence (LPDSVDWREKGVVFPIRNQGK) shows a great deal of similarity to those of the other cysteine endopeptidases isolated from latices of Asclepiadaceae even when a high degree of homology could be observed with other plant cysteine endopeptidases.

No abstract

No abstract

No abstract

BACKGROUND The most common milk‐clotting enzymes in the cheese industry are recombinant chymosins. Food naturalness is a factor underpinning consumers' food choice. For consumers who avoid food with ingredients from genetically modified organisms (GMOs), the use of vegetable‐based rennet substitute in the cheese formulation may be a suitable solution. Artichokes that deviate from optimal products, when allowed to bloom due to flower protease composition, are excellent as raw material for vegetable rennet preparation. As enzymatic milk clotting exerts a significant impact on the characteristics of the final product, this product should be studied carefully. RESULTS Mature flowers from unharvested artichokes (Cynara scolymus cv. Francés) that did not meet aesthetic standards for commercialization were collected and used to prepare a flower extract. This extract, as a coagulant preparation, enabled the manufacture of cheeses with distinctive characteristics compared with cheeses prepared with chymosin. Rennet substitution did not affect the actual yield but led to significant changes in dry matter yield, humidity, water activity, protein content, and color, and conferred antioxidant activity to the cheeses. The rennet substitution promoted significant modifications in springiness, and in the microstructure of the cheese, with a more porous protein matrix and an increment in the size of the fat globules. Both formulations showed a similar microbiota evolution pattern with excellent microbiological quality and good sensory acceptance. CONCLUSIONS The rennet substitute studied here produced a cheese adapted to specific market segments that demand more natural and healthier products made with a commitment to the environment but well accepted by a general cheese consumer. © 2020 Society of Chemical Industry

In this article, we report the cloning of an aspartic protease (AP) from flowers of Arctium minus (Hill) Bernh. (Asteraceae) along with the use of depigmented aqueous flower extracts, as a source of APs, for the hydrolysis of whey proteins. The isolated cDNA encoded a protein product with 509 amino acids called arctiumisin, with the characteristic primary structure organization of typical plant APs. Bovine whey protein hydrolysates, obtained employing the enzyme extracts of A. minus flowers, displayed inhibitory angiotensin-converting enzyme (ACE) and antioxidant activities. Hydrolysates after 3 and 5 h of reaction (degree of hydrolysis 2.4 and 5.6, respectively) and the associated peptide fraction with molecular weight below 3 kDa were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-assisted laser desorption ionization/time of flight mass spectrometry, and reverse phase-high-performance liquid chromatography. The results obtained in this study demonstrate the viability of using proteases from A. minus to increase the antioxidant and inhibitory ACE capacity of whey proteins.