A Taguchi robust design method with an L9 orthogonal array and larger-the-better characteristics was implemented to optimize experimental conditions for the hydrolysis of raw anchovy using a pressure-assisted enzymatic reaction method. The degree of hydrolysis (DH), nitrogen recovery (NR) and yield were considered as the response parameters. Pressure, reaction temperature, reaction time, and mixed enzyme amount were chosen as control parameters. As a result of the Taguchi analysis in this study, the pressure was found to be the most influential parameter on DH and NR. The amount of mixed enzyme in the reaction also had a significant effect on DH and NR. Meanwhile, the optimum values were confirmed to be similar at 95% confidence and 5% significance level through analysis of variance (ANOVA). Furthermore, new hydrolysates at optimum conditions and control hydrolysates at atmospheric pressure were compared in terms of the DH, resulting in the improvement of DH by more than 52.6%.

Choline is important an organic compound for normal membrane function, acetylcholine synthesis, lipid transport, and methyl metabolism. In biological tissues and foods, there are multiple choline compounds that contribute to choline content. There are so many analytical methods for choline determination, such as radioisotopic, high-performance liquid chromatography, and gas chromatography/mass spectrometry. However, these existing methods are expensive, unmanageable, and time-consuming. In this study, we modified enzymatic method, which is applicable for the determination of choline in milk and infant formulas, and applied to bovine serum and muscle. The calibration curves were linear with higher correlation coefficients than 0.994 . Recoveries obtained by calibration curves from the spiked bovine serum and muscle samples varied between 70.6 and 85.2%. The method may be suitable for use as a routine method in the determination of choline for biological tissue and food samples.