Establishment of a rapid and effective plate chromogenic assay for screening of Aspergillus species with high β-fructofuranosidase activity for fructooligosaccharides production

Fructooligosaccharides (FOS) are commonly regarded as prebiotics and used as components of functional foods. Currently, the industrial sucrose-to-FOS biotransformation is mainly carried out using the microbial-derived beta-fructofuranosidases with transglycosylation activity as catalysts. Evaluation of the ability of a microorganism to produce beta-fructofuranosidase is commonly conducted by measuring enzyme activity. However, the traditional method requires several steps to identify strains with high beta-fructofuranosidase activity, which is not suitable for high-throughput screening. To facilitate screening of a large number of microbial cultures, this study developed a plate chromogenic assay method based on the glucose oxidase (GOD) - peroxidase (POD) bienzymatic system for screening of beta-fructofuranosidase-producing fungal strains and predicting their potential to produce FOS. This method used the amount of glucose released from sucrose as indicator to form clear pink halos around the microbial colonies with beta-fructofuranosidase activity. Cultivation conditions for the plate assay were optimized as cultivation time 5 h and spore inoculum concentration 10(8)/ml. Moreover, the method was applied to screening of an Aspergillus niger ATCC 20611 mutant library. The mutant All displaying the largest pink halo was screened out and its beta-fructofuranosidase activity was determined to be 1.65 fold than that of the parental strain. Thin layer chromatography (TLC) assay further indicated that A11 with the largest halo possessed the highest FOS synthesis ability. These results demonstrated the potential of this plate chromogyenic assay method in the rapid and effective identification of excellent FOS producers from a large number of strain samples.