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 ¿-amylase- and ß-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 ß-amylase and the AOAC 2002.01, AACC 22-02.01, and ICC Standard No. 303 methods for ¿-amylase. Mild extraction conditions favored enzyme recovery, with the highest ¿-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. ß-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 ¿-amylase concentrated in the bottom phase (432.89 CU/mL) and ß-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 ß-amylase recovery and scalability.
