Analysis of enzymically digested food proteins by Sephadex-gel filtration

Abstract

1. Portions of a preparation of freeze-dried cod fillet were subjected to different conditions of heat treatment and were then digested in vitro with pronase (an enzyme preparation from Streptomyces griseus), or successively with pepsin, pancreatin and erepsin, or with pepsin and papain. The digestions were conducted under ‘static’ conditions in flasks, and also in Sephadex gel G 10 in such a manner that the reaction products were continuously removed from the site of action of the digesting enzymes. The different digests were then passed in turn through a calibrated column of Sephadex gel G25 and each was resolved into three fractions containing ‘soluble protein’, ‘peptide’ and ‘free amino acids’. The distribution of several amino acids in these different fractions was determined and the results were assessed in relation to the availabilities of these amino acids in the original test proteins as measured in microbiological and chemical tests and, in some instances, in growth tests with rats. 2. The different enzyme systems gave broadly similar results. With increasing severity of heating, the ‘ free amino acids’ component in the digests became increasingly deficient in several amino acids relative to their content in the original unheated meal, and notably in lysine and the sulphur-containing amino acids. This evidence for a marked differential effect of heating in retarding the enzymic release in vitro of several amino acids was consistent with the results of feeding tests with rats, and of microbiological and chemical assays, which showed corresponding differences in the proportions of lysine, methionine and isoleucine made unavailable by the heat treatment. In severely heated meal more of the lysine, methionine and isoleucine was released by enzymes in vitro than was biologically available to the rat. With increasing severity of heat treatment, the biological availabilities of these amino acids for the rat fell more sharply than did the digestibility of the nitrogen, indicating that amino acids absorbed from the heated meals were relatively poorly retained.