Low-molecular weight peptide mixture and method of producing same

A method of producing a low-molecular weight peptide mixture comprising a major proportion of dipeptides and tripeptides produced from a starting natural protein to which at least two desired amino acids are incorporated, said at least two desired amino acids being the ones whose contents in the starting protein are low, whereby said dipeptides and tripeptides have a modified amino acid composition different from an amino acid composition of said starting protein, comprising the steps of

• dissolving at least one alkaline protease selected from the group consisting of papain, bromelain and ficin, in a buffer solution adjusted to an optimum pH for the protease up to 10,
• adding at least one starting protein in a buffer solution having an alkaline pH up to 10 and a concentration of 10 to 60% by weight of the protein material and thoroughly mixing the solution,
• adding at least two ethyl esters of amino acids, said amino acids being selected from the group consisting of L-lysine, L-methionine, L-valine, L-isoleucine and L-leucine,
• the pH of said buffer solution being optimum for the incorporation of said amino acid in said starting protein in the presence of said alkaline protease in a plastein reaction,
• reacting said ester of at least two amino acids with said starting protein in a plastein reaction in the mixed solution having an alkaline pH whereby said at least two amino acids are covalently incorporated into said starting protein to produce a plastein reaction solution containing a modified protein,
• subjecting said plastein reaction solution to concentration using a reverse osmosis membrane having an NaCl rejection of not higher than 40% to thereby purify said reaction solution by removing inorganic salts, unreacted amino acid and amino acid ester, and other low-molecular weight impurities from the reaction solution, and forming an aqueous solution containing 3 to 20% by weight of said modified protein,
• hydrolyzing said modified protein in said purified aqueous solution by simultaneously or successively adding at least two acidic proteases having different specificity than said at least one alkaline protease, said acidic proteases being selected from the group consisting of pepsin, molsin and orientase, to said aqueous solution containing 3 to 20% by weight of said modified protein at an acidic pH, and maintaining a reaction temperature of 25.degree. to 60.degree. C. for from 8 to 72 hours to produce a low-molecular weight peptide mixture having an amino acid content which has different proportions of amino acids than does said starting protein, and
• separating from the hydrolyzed solution said low-molecular weight peptide mixture which comprises a major proportion of dipeptide and tripeptides and containing not more than 15% by weight of free amino acids and not more than 20% by weight of a high-molecular weight fraction of compounds having a molecular weight of not lower than 700 and removable by gel filtration.