Valorisation of Orange (Citrus Sinensis L.) Processing Wastes For Pectinase Enzymes Production Using Aspergillus Brasiliensis

Abstract

Orange processing wastes are produced in large quantities about 45% to 50% of the fresh fruits, the wastes are not adequately utilised thus creating environmental challenges. This study focused on conversion of the wastes into pectinase enzymes by optimizing various process parameters, such as temperature, pH, and incubation time through solid-state fermentation using Aspergillus brasiliensis isolated from rotted orange peels. Enzymes evaluated on application in juice extraction and clarification. Aspergillus brasiliensis strain was isolated and screened using PDA media. The strain was selected for morphological identification and molecular characterisation and used for pectinase enzymes production through solid- state fermentation. The optimum enzymes production conditions were determined by varying temperature, pH and incubation time. Pectinase enzymes application was evaluated in juice extraction and clarification using 0 to 5% v/v concentration. For juice extraction, the volume of juice increased was determined compared to juice without pectinase enzymes, with juice clarity at 660 nm absorbance. The microorganism isolated from rotted orange peels was identified as A. brasiliensis using macroscopic, microscopic characteristics and molecular technique of transcribed sequence of ITS1 and ITS4 region for identification. Optimized conditions for maximum production of pectinase enzymes showed that parameters have a significant effect on enzyme production at P< 0.05 after data was analysed using Box Behnken Design. Based on enzyme activity the maximum pectinase enzyme activities of polygalacturonase, lyase and esterase were 44.58U/g, 16.53U/g and 74U/g respectively at a temperature of 35℃, pH 4 and 6 days of fermentation. Based on the clear zone diameter the maximum pectin hydrolysis was observed at 31℃, pH 4 and 7 days of fermentation, where the clear zone diameter was 12 mm. The produced pectinase enzymes applied in apple juice extraction and clarification, results showed that it has a significant effect on juice yield and juice clarity. The percentage of juice yield increased from 65 to 84.5% at 4% enzyme concentration, in juice clarity the absorbance was decreased from 0.365 to 0.040, the pH of juice was decreased from 3.55 to 3.35 and pectin content from o.45% to 0.040%, this was due to action of produced pectinase enzyme on pectin in the juice. The findings suggest that this approach could be useful for the industrial-scale production of pectinolytic enzymes for various applications. Further studies are required to characterize the enzyme for potential heterologous expression and its application in upcycling waste into valuable products using indigenous microorganisms through biotechnology.