CHARACTERISATION OF POLYGALACTURONASE PRODUCED BY ASPERGILLUS AWAMORI CICC 2040 ON SELECTED PRETREATED FRUIT PEELS AND ITS APPLICATION IN JUICE PROCESSING

Abstract

Utilisation of orange and plantain peels as substrates for production of polygalacturonase is increasing due to their high pectin content and availability. However, high ligno-cellulosic content of these peels limits their bio-conversion. Pretreatment of the peels for improved bio-conversion is sparsely reported. This study was therefore designed to evaluate effects of alkaline and microwave pretreatments of orange and plantain peels on the activity of polygalacturonase produced from Aspergillus awamori CICC 2040, and its applications in juice extraction and clarification. Orange (Citrus sinensis L.) and plantain (Musa paradisiaca Linn.) peels were oven dried at 60 oC for 48 h and milled into powders. The powders were subjected to alkaline and microwave pretreatments separately, using response surface methodology. Factors interacted for alkaline pretreatment included Particle Size, PS (<0.4250, 0.4250<PS<0.8025 and 0.8025<PS<1.1800 mm), NaOH molarity (0.010, 0.055 and 0.100 M) and time (1.0, 6.5 and 12.0 h), while those for microwave pretreatment were PS (<0.4250, 0.4250<PS<0.8025 and 0.8025<PS<1.1800 mm), microwave power (240, 480 and 720 W) and time (2.50, 6.25 and 10.00 min). These factors were interacted to determine combinations for Maximum Polygalacturonase Activity (MPA). Combinations that gave MPA from alkaline and microwave treatments were also interacted (combined treatment). Pretreated and untreated (control) powders were inoculated with 106 spore/mL Aspergillus awamori, incubated at 28 oC for 5 days, and crude polygalacturonase was extracted and its activity determined. Crude polygalacturonase was purified with activated charcoal (0.5%, w/v). Molecular weight, activation energy, catalytic and substrate affinity constants of crude and purified polygalacturonase and their activities at different pH (3-12) and temperature (25-75 oC) were determined. Applications of crude and purified polygalacturonase in mango juice extraction and apple juice clarification were evaluated and compared with a commercial polygalacturonase. Yield and Flow Behaviour Index (FBI) of mango juice, and colour intensity of apple juice were determined. Data were analysed using descriptive statistics and ANOVA at α0.05. iv Alkaline pretreatment combinations that gave MPA were <0.4250 mm, 0.100 M and 1.0h, and 0.8025<PS<1.1800 mm, 0.010 M and 1.0h for orange and plantain peels, respectively. Same microwave pretreatment combination, 0.8025<PS<1.1800 mm, 720 W and 10.00 min gave MPA for orange and plantain peels. Activity of crude polygalacturonase obtained for the control, alkaline, microwave, and combined alkaline and microwave pretreated samples were 25.17, 38.46, 26.21 and 42.05 U/mL; and 26.04, 38.82, 26.72 and 44.98 U/mL for orange and plantain peels, respectively. Molecular weight, activation energy, catalytic and substrate affinity constants for crude polygalacturonase were 20.00-96.20 kDa, 2.26-3.07 kJ/mol, 29.41-40.00 U/mL and 0.206-0.320 mg/L, respectively, and 30.00-31.00 kDa, 1.23-2.15 kJ/mol, 55.55-90.91 U/mL and 0.722-0.909 mg/mL for purified polygalacturonase. Optimum pH and temperature for crude polygalacturonase were 5.5 and 55oC, respectively, and 4.5 and 40oC for purified polygalacturonase. Yield of mango juice was 58.20-87.60% and FBI was 0.377-0.627. Colour intensity of apple juice was 37.89±0.04-42.61±0.01. Yield of mango juice and colour intensity of apple juice increased significantly following treatment with polygalacturonase. Alkaline and microwave pretreatments of orange and plantain peels enhanced the production of polygalacturonase by Aspergillus awamori. Polygalacturonase was effective for juice extraction and clarification