Protein hydrolysis for amino acid analysis revisited

Several protein hydrolysis methods exist for amino acid composition analysis, yet their suitability for accurately quantifying individual amino acids in foods remains unclear. We compared six different hydrolysis methods for chromatography-based amino acid analysis of plant-based food matrices, including oat, pea, and pea aquafaba, using milk, bovine serum albumin, and β-lactoglobulin as animal protein references. Enzymatic hydrolysis resulted in superior amino acid recoveries compared to other hydrolysis methods, enabling the quantification of asparagine and glutamine and yielding 2-3 fold higher tryptophan recovery than alkaline hydrolysis. Microwave and enzymatic hydrolysis recovered 2-4 fold more methionine from plant-based matrices than the ISO-recommended method, which relies on pre-oxidation. However, enzymatic hydrolysis underestimated aspartic acid and glutamic acid content and is more expensive when analysing only a few samples. While no single hydrolysis method was ideal, we demonstrated that enzymatic hydrolysis enabled the simultaneous quantification of 18 amino acids from diverse protein matrices.