Effect of subcritical water extraction and precipitation methods on the functional properties, antioxidant activity, and ACE1 inhibition of palm kernel cake protein

Abstract

Protein is an essential macronutrient for human and animal nutrition. Plant protein sources can be classified into protein flours, protein concentrates, and protein isolates, each offering beneficial nutritional and functional properties. This study investigated the use of subcritical water (SCW) for extracting protein from palm kernel cake (PKC). SCW extraction was carried out at three temperatures (120, 150, and 180 °C) and two durations (15 and 30 min). These conditions were compared with a conventional alkali (2 M NaOH) extraction method. Two protein precipitation methods; isoelectric (pH 3.5) and cold ethanol (80 %), were investigated. Temperature and time significantly influenced protein yield. At 150 °C and 30 min, SCW extraction followed by cold ethanol precipitation resulted in the highest protein yield of 66.24 ± 4.22 %. Isoelectric precipitation under the same SCW condition gave only 1.33 ± 0.06 %. Conversely, the alkali method showed a higher protein yield (94.01 ± 3.99 %) via isoelectric precipitation. Cold ethanol precipitation with alkali extraction was less than 0.01 %. The protein content from SCW extraction was 42.01 ± 1.72 % (flour), while that from alkali extraction was 65.53 ± 1.15 % (concentrate). SCW protein flour showed higher antioxidant activity by DPPH (78.74 ± 2.33 μmol TE/g) and total phenolic content (190.43 ± 1.75 mg GAE/g). However, alkali protein concentrate had higher ABTS (87.88 ± 0.90 μmol TE/g) and reducing power (0.39 ± 0.05 μmol TE/g). SCW protein flour demonstrated the strongest ACE1 inhibition and favorable emulsifying, foaming, and absorption properties. These findings emphasize SCW extraction as an eco-friendly strategy for obtaining bioactive protein from PKC with potential applications in functional foods.