Optimization of Extraction Parameters and Simultaneous Determination of Free Amino Acids in Milk Powder

Three variables(operating parameters) affecting the extraction efficiency of free amino acids from milk powder, solid-to-solvent ratio, zinc sulfate concentration(for protein precipitation), and centrifugation time, were optimized by response surface analysis(RSA) based on a Box-Behnken design. Simultaneous determination of mixed free amino acids and free amino acids in milk powder samples was performed by visible spectrophotometry combined with partial least square(PLS). The results showed that under the optimal conditions: solid-to-solvent ratio, 1:3.02(g/mL); zinc sulfate concentration, 10.23%; and centrifugation time, 6.79 min, the extraction yield was 170 μg/g. The complexation of copper sulfate with glycine, glutamic acid, valine and phenylalanine resulted in highly overlapping absorption spectra when mixed together, which made it difficult to simultaneously determine the individual components. The simultaneous determination of mixed free amino acids using PLS demonstrated that the recovery rate was 98.2%–99.2% and the relative prediction error(RPE) was 1.7%–2.9%.