With either Na caseinate or skim-milk powder, 1% (w/v) additions of pepsin, chymotrypsin or Alcalase and an incubation time of 24 h at 37°C gave the best hydrolysates for subsequent plastein production. In spite of the fact that peptide species which were produced by treatment with the 3 enzymes were entirely different, they were all capable of producing qualitatively similar amounts of plastein when concentrated and further incubated with proteinase, suggesting that the identity of peptide components was relatively unimportant. αs1-, β- and κ- caseins, Na-caseinate and skim-milk powder all led to plastein products with broadly similar properties, confirming the comparative unimportance of peptide composition. This also indicated that for most practical applications fractionation of initial protein mixtures (as in many food products) would not be justified. Optimum peptide concentration for plastein formation with pepsin and caseinate hydrolysates was 20–35% (w/v) with an optimum pH range of 4–5; the best peptide molecular weight varied between 400 and 800 and the optimum temperature was either 37°C for 24 h or 50°C for 4–6 h. Higher temperatures (70°C) gave more rapid plastein formation but poorer yields. Lower temperatures (e.g. 20°C) gave similar yields, but incubation times required to be extended to at least 48 h. The same proteinase should be used for plastein synthesis as that used in the initial hydrolysis stage, or else further hydrolysis (over hydrolysis) could occur during the plastein formation stage causing lower yields due to differences in the specificity of the 2 enzymes. Even under ideal conditions plastein yields never exceeded 27% of the weight of peptide taken and at the present time it seems unlikely that the plastein reaction will have anything more than curiosity value. Economics must preclude its application in low-cost, high volume situations such as generally exist within the food industry.
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