Purification of antioxidant peptides from Malaysian fish sausage ( <i>Keropok Lekor</i> ) by-product hydrolysate: evaluating salt, solvent, and salt-mediated solvent precipitation with aqueous two-phase systems

The Malaysian fish sausage (<i>Keropok Lekor</i>, KL) is a fish snack made mainly from fish, sago flour, and seasonings. In KL production, the fish bones, innards, skins, and heads are usually removed, and only the flesh is used. The by-products are normally discarded, causing disposal problems and environmental pollution. The by-products can however be converted into high-value antioxidative peptides (APs) by microbial or enzymatic processing. After the bioconversion, the complicated, lengthy, and expensive bioseparation including filtration, precipitation, dialysis, and chromatography are commonly employed in purifying the APs. Thus, researchers are interested in developing alternative bioseparation technology. In this study, the feasibility of using precipitation techniques and aqueous two-phase system (ATPS) to isolate the APs from the protein hydrolysate produced from KL by-products by <i>Lactobacillus casei</i> fermentation was evaluated. Four types of precipitation were evaluated: (1) salt precipitation using ammonium sulfate ((NH₄)₂SO₄) and sodium chloride (NaCl), (2) solvent precipitation using ethanol and acetone, and (3) combination of salts and solvent precipitation, and (4) pH-shift precipitation. Amongst the precipitation techniques investigated, the pH-shift precipitation (pH 7.5) gave the highest purification fold (<i>P</i>_F,181.1). Both solvent and salt-solvent precipitations gave the lowest <i>P</i>_F (1.5 and 1.6, respectively). The salt precipitation (60%, w/v (NH₄)₂SO₄) achieved 2.0 for <i>P</i>_F. Besides, exceptionally high <i>P</i>_F (3,140.4) was obtained using ATPS (bottom phase of 15% (w/w) PEG2000 with 10% (w/w) phosphate system). Overall, the ATPS showed superior performance in enriching the AP compared to the precipitation techniques. The effectiveness of the AP purification may be increased by combining the ATPS with precipitation techniques. The study laid the groundwork for developing an effective bioprocessing, which is important to valorize the KL by-products to the high-value APs.