| Summary: | The enzymatic hydrolysis of wheat gluten was investigated using three commercial enzymes, a papain based preparation (P144), a papain preparation supplemented with microbial exopeptidases (P278) and microbial exopeptidases (P215). The rates and amounts of hydrolysis were measured by proton release in a pH stat reactor and by the TNBS (trinitrobenzene sulphonate) method. These methods although well established for soluble soya protein hydrolysis proved to be problematic with insoluble gluten. Several process variables of enzymatic hydrolysis, including enzyme type, gluten and enzyme concentrations, pH and temperature, were then investigated. It was found that the DH could be reduced by increasing the concentration of substrate e.g. P144, P278P and P215 using 0.5% w/v gluten gave 7%, 28%, 20% DH and using 5% w/v gluten gave 3%, 9% and 5% DH, respectively. The concentration of P144 was able to increase hydrolysis rate and DH while only hydrolysis rate was increased using P278P and P215. Both pH and temperature had significant effects on the hydrolysis rates and DH. These data and those from an investigation of the effects of hydrolysate additions, suggested that hydrolysis was inhibited by end-products. The prediction of hydrolysis as a function of gluten concentration was attempted using a Michaelis-Menten based kinetic model with end-product inhibition combined with first order enzyme decay. Relatively poor fits were obtained, suggesting a more complex model involving mixed inhibition was required. Characterisation of the enzyme hydrolysate using Gel Permeation and Reverse-Phased HPLC showed that the type of hydrolysate obtained was enzyme dependent. They consisted of small peptides between 3600 and 1000 MW that become increasingly less hydrophobic as the hydrolysis progresses. The data suggests that kinetics of hydrolysis were most probably dominated by the solubilisation/depolymerisation rather than any subsequent soluble hydrolytic reactions. The results indicate that enzyme-substrate interactions should be investigated in more detail. Using a membrane reactor it was possible to enhance hydrolysis by 20% over controls under diafiltration conditions. The variation of the membrane pore size produced hydrolysates of similar size suggesting that membrane selectively was poor, most probably due to surface fouling. The amount of hydrolysis using a 4000 MWCO was significantly lower than that observed with membranes of 9000 or 2500 MWCO. The results indicate that membrane reactors may have a role in the production of very high DH hydroysates and allow for more sophisticated use of enzymes and recovery methods, compared with simple pH controlled batch reactors.
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