Optimisation of low temperature (8ᵒC) enzymatic hydrolysis of acid whey using design of experiments (DOE) for the generation of thermally stable whey protein hydrolysates

The impact of enzymatic hydrolysis at 8ᵒC of liquid whey protein concentrate (WPC35) derived from acid casein manufacture using Prolyve 1000®, a microbial protease preparation, under free-fall pH conditions on the physicochemical and the thermal stability properties of the resultant hydrolysates was investigated. The hydrolysis process was optimised using a design of experiments (DOE) approach. Eleven hydrolysates (H1–H11) were generated using 3 factors x 2 levels, i.e., enzyme:substrate (E:S) 0.25–1.00% (v/w), starting pH 7.5–9.0 and incubation time 10–96 h. Hydrolysate degree of hydrolysis (DH) values ranged from 0.75 (H10) to 4.74% (H6) which increased to 1.91 (H10) and 7.24% (H6) following subsequent thermal processing (mimicking the evaporation process) at 54ᵒC x 15 min and 64ᵒC x 10 min. The apparent viscosity (ηapp) of all hydrolysates was measured to assess their heat stability during heating at 85ᵒC for up to 20 min, this was shown to be E:S and pH-dependent. While the unhydrolysed samples formed a gel on heating at ~74-80ᵒC. Following DOE analysis, it was found that E:S had a significant impact on the DH, the extent of intact protein degradation (�g) and the thermal stability. The �g following processing at 64ᵒC showed a strong correlation (R2 = 0.924; p < 0.001) with ηapp, and thus this parameter may be used to predict the thermal stability of the hydrolysate samples. The optimised hydrolysis conditions for the generation of heat stable WPC35 hydrolysates during 8◦C incubation were achieved using an E:S = 0.625% at a starting pH = 8.65. Performance of protein hydrolysis reactions at low temperature has potential to enhance process sustainability.