Development of a Triticale-Based Amylolytic Biocatalyst for Starch Hydrolysis With Applications in Brewing Wort Sugar Enhancement

Abstract

This study aimed to develop a biocatalyst derived from triticale (X Triticosecale Wittmack) malt germinated for 5–8 days, by extracting α-amylase, β-amylase, and amyloglucosidase through aqueous methods, purifying them using aqueous two-phase systems (ATPSs), and co-immobilizing the enzymes on a gelatin support cross-linked with CaCl₂. After 7 days of germination, the enzyme extracts showed maximum activities of 549.6 CU/g for α-amylase, 54.8 BU/g for β-amylase, and 0.11 U/g for amyloglucosidase. The type 3 ATPS (30% ethanol/18% citrate) enabled recovery yields of 97% for α-amylase and 68.6% for β-amylase, with purification factors of 2.8 and 1.1, respectively. The purified enzymes exhibited optimal catalytic activity at 70°C and pH 5–6 (α-amylase) and at 60°C and pH 6–7 (β-amylase). Kinetic parameters indicated high substrate affinity (K_m < 200 mg/mL), with improved values after immobilization (α-amylase: K_m 118.2 mg/mL, V_max 15.6 mg/min; β-amylase: K_m 101.9 mg/mL, V_max 23.1 mg/min). The cross-linked gelatin support demonstrated a water absorption capacity of 469% and a solubility of 61% after 24 h, with immobilization efficiencies exceeding 100%. In barley wort mashing trials, the immobilized enzyme consortium increased the release of reducing sugars by 11.8 g/L in the first cycle and maintained an additional 5.8 g/L after five reuse cycles. These findings demonstrate the potential of a robust, stable, and reusable biocatalyst for industrial applications in brewing processes and starch hydrolysis.

Keywords

amylase immobilization / ATPS / triticale / α-amylase / β-amylase

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2025 The Author(s). Food Bioengineering published by John Wiley & Sons Australia, Ltd. on behalf of State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology.