Characteristic Property of Low Bitterness in Protein Hydrolysates by a Novel Soybean Protease D3

ABSTRACT:  Enzymatic hydrolysis is 1 means of improving the functional properties of food protein; however, in most cases, bitter peptides are generated by such treatment, and the resulting product is therefore not acceptable as a food ingredient. We have already reported a novel cysteine protease, D3, purified from germinating soybean cotyledons. Because of its substrate specificities, most hydrophobic amino acid residues in the hydrolysate are presumed not to be located at the peptide termini. It was therefore expected that protein hydrolysate by protease D3 would taste less bitter than other enzymatic hydrolysates. The objective of this study was to demonstrate the low bitterness of protein hydrolysates by protease D3. For that purpose, soy protein and casein hydrolysates were prepared with treatment of protease D3, subtilisin, pepsin, trypsin, and thermolysin, respectively. The bitterness of these hydrolysates was evaluated by measuring points of subjective equality (PSE). The PSE value demonstrated that the protein hydrolysates by protease D3 were significantly less bitter than the other enzymatic hydrolysates, indicating that the products had a taste mild enough to be acceptable as a less-bitter peptide food ingredient. These results suggested that a prominent feature of protease D3 was its capacity to produce less-bitter peptides. Therefore, it is thought that protease D3 could be applied to produce protein hydrolysates for use as ingredients in a variety of food products.     

The Effective Methods in Refolding and Activation of Cathepsin L-like Soybean Protease D3

A novel cysteine protease D3, which was purified from germinating soybean cotyledons, showed high homology with cathepsin L and cathepsin K. In our previous study, because of the specificity of the enzyme, hydroly‐sates treated with D3 treatment showed a prominent property of less bitterness than other hydrolysates treated with commercially available proteases. However, active recombinant D3 prepared from Escherichia coli inclusion bodies was so intricate and less productive that it made further studies on this protease and hydrolysates difficult. In the concrete, the refolding process of the immature proD3 from inclusion bodies takes more than a day, and autocatalytic activation of refolded immature proD3 at low pH was difficult to control. In this study, we aimed to establish an efficient refolding and activating method of protease D3. In the refolding step, the procedures could be simplified by using a size‐exclusive column‐based method. In the activation step from immature proD3, we utilized another protease, subtilisin, rather than autocatalytic activation by D3 itself. After subtilisin treatment, the peptide having 12 amino acids‐length of N‐terminal pro sequence was initially cleaved, and residual proD3 showed only a half proteolytic activity of active D3. However, when the pH was shifted lower (pH4.5), D3 automatically changed to have the same proteolytic activity as active one, and this activated recombinant had the same N‐terminal sequence as purified D3 from germinating soybean cotyledons. By using this method, all preparation processes of D3 from inclusion bodies to active D3 could be completed within a few hours, and it became possible to carry out the investigation on hydrolysates on a large scale.