In-vitro formation of cocoa-specific aroma precursors: aroma-related peptides generated from cocoa-seed protein by co-operation of an aspartic endoprotease and a carboxypeptidase

Abstract

Cocoa-specific aroma precursors were obtained when acetone-dry powder prepared from unfermented cocoa seeds was subjected to autolysis at pH 5·2. Hydrophobic free amino acids and hydrophilic peptides were preferentially generated under these conditions. When this mixture of proteolysis products was formulated and roasted in the presence of reducing sugars, cocoa and/or chocolate aroma was detected by two independent panels. Aroma precursors extracted and partially purified from fermented cocoa seeds also consisted predominantly of hydrophilic peptides and hydrophobic free amino acids. No cocoa-specific aroma precursors were obtained when the acetone-dry powder from unfermented cocoa seeds was incubated at pH 3·5. Few free amino acids were released under these conditions, but a large number of hydrophobic peptides were formed. When these hydrophobic peptides were digested with carboxypeptidase A from porcine pancreas, mixtures of hydrophilic peptides and hydrophobic free amino acids were generated, which were shown to contain cocoa-specific aroma precursors. No typical cocoa aroma was, however, obtained when synthetic mixtures of amino acids adapted to the spectrum of free amino acids present in fermented cocoa seeds (or aroma-precursor extracts) were roasted in the presence of reducing sugars. Our findings therefore indicate that the essential cocoa-specific aroma precursors are among the hydrophilic oligopeptides. Ungerminated cocoa seeds contain one predominant endoprotease (an aspartic endoprotease with a pH optimum at pH 3·5) and a carboxypeptidase activity (pH optimum: 5·8). Our findings indicate that the co-operative action of these two enzymes on cocoa-seed protein is required for the generation of the cocoa-specific aroma precursors.