Harnessing Triticale (× Triticosecale Wittmack) Malting for Amylase Enzyme Synthesis and Partial Purification

Abstract

Triticale malt is an underutilized cereal matrix with high amylolytic potential, which may be exploited for the recovery of plant-derived enzymes for food and biotechnological applications. This study aimed to obtain α-amylase- and β-amylase-enriched aqueous extracts from Bicentenario and Siglo XXI triticale malts and to partially purify these enzymes using ethanol/trisodium citrate aqueous two-phase systems (ATPS). Triticale grains were malted by germination for 5 days and dried at 50°C. Aqueous extraction was performed using ground malt and deionized water at a 1:10 ratio under different extraction times (30, 120, and 270 min) and temperatures (30°C and 40°C). The selected extracts were lyophilized and subjected to ATPS composed of ethanol and trisodium citrate. Enzyme activity was determined using the BETAMYL-3 method for β-amylase and the AOAC 2002.01, AACC 22-02.01, and ICC Standard No. 303 methods for α-amylase. Mild extraction conditions favored enzyme recovery, with the highest α-amylase activity observed in Siglo XXI extract at 30 min/30°C, increasing from 99.10 CU/g in malt to 507.42 CU/g in the aqueous extract. β-Amylase activity was also higher in Siglo XXI than in Bicentenario extracts. The 30% ethanol/18% trisodium citrate ATPS was the most effective system, promoting differential partitioning with α-amylase concentrated in the bottom phase (432.89 CU/mL) and β-amylase in the top phase (5.78 BU/mL). These results highlight the potential of triticale malt as a renewable source of amylolytic enzymes and support ethanol/trisodium citrate ATPS as a mild, sustainable strategy for obtaining amylase-enriched fractions, although further optimization is needed to improve β-amylase recovery and scalability.