Peptidases cisteínicas do fruto de noni (Morinda citrifolia L.) como agentes coagulantes de leite

Abstract

Milk-clotting is a basic step in the production of the whole cheese. The calf rennet, which has as the main constituent chymosin has been widely used for this purpose since it has high specificity for hydrolysis of the κ-casein. As a result of increased global cheese demand, along with a reduced supply of calf rennet, it has intensified the search for alternative sources. Chymosin produced by genetically modified microorganisms have proven to be an efficient substituent, however, attention has been focused on natural sources of peptidases which may promote different characteristics to the final product. Therefore this study aimed to characterize biochemically the peptidases of the pulp of the fruits of noni, and evaluate their technological potential and possible application in the hydrolysis of milk proteins and the production of cheese. For this, PPN obtained in sodium phosphate buffer 50 mM pH 7.0 was subjected: (i) proteolytic activity assays and (ii) milk-clotting by varying assay conditions and pretreatments PPN; (Iii) Hydrolysis of the total casein and κ-casein, over time analyzed by SDS-PAGE in gels of 15% polyacrylamide; (IV) Cheese production type "rennet". PPN peptidases have optimum pH activity around 6.0 and temperature 50 °C, inhibited by iodoacetamide and E-64. They showed low stability at alkaline pHs and temperatures above 40 °C after incubation for 30 min. Under the effect of divalent ions, proteolytic activity was inhibited by Mn, Co, Cu and Zn, however, it was not affected by Fe, Mg and Ca, either in the presence of NaCl, where there was even a significant increase in activity in all concentrations tested. The clotting activity, a reduction in the MCA (Milk-Clotting Activity) values when that activity occurred at concentrations above 60 mM CaCl2 and 0.1 M NaCl. PPN was able of to hydrolyse the three types of casein (α, β and κ). β- and κ- casein showed greater susceptibility to degradation. The products obtained from the hydrolysis of the κ-casein performed either by PPN as the commercial rennet (Coalhopar) had a mass of approximately 16 kDa, probably corresponding to the peptide para-κ-casein. The cheeses produced by PPN and commercial rennet have similar characteristics as the moisture values, fresh and dry mass, differing in clotting time. Coalhopar coagulated milk in about 3 min, while PPN extended to 8 min. PEN showed a yield (w:w) to about 79.02 g of noni pulp for the production a kilogram of cheese, which is equivalent to a mass of less than whole fruit.