Preparation, characterization and optimization of cross-linked fructosyltransferase aggregates for the production of prebiotic fructooligosaccharides

In this study, the preparation, characterization, and evaluation of the cross-linked enzyme aggregates (CLEAs) of fructosyltransferase isolated from the mutant-type Aureobasidium pullulans NAC8 for the production of fructooligosaccharides (FOS) with prebiotic properties were investigated. The reaction conditions were optimized statistically by using response surface methodology. CLEAs were characterized by using both photo-microscopy and scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX). The secondary structure of the protein was predicted by using Fourier transform infrared spectroscopy (FTIR). FOSs produced as a result of the biotransformation of sucrose was quantified by using a chemometric analysis of FTIR spectral data. The prepared FOSs were investigated for the prebiotic effects under anaerobic conditions using a standard strain of Lactobacillus sp. Following were the statistically determined, optimum conditions for the production of FOSs: 5% (v/v) glutaraldehyde, pH 5.5, and a temperature of 32°C. The values of R², F value, adjusted precision, and coefficient of variation were found to be 0.94, 11.50, 9.34, and 35.34%, respectively. Validation of the model resulted in the reusability of the CLEAs in six reaction cycles as it retained 70% of the residual activity. The SEM-EDX and photomicrographic analysis showed that the sizes of CLEAs varied, which ranged between 10 and 40 μm. Aggregation leads to the formation of a higher number of beta sheets in the CLEAs than that in the free enzyme. The total FOSs produced by the CLEAs was around 57% (w/v). A. pullulans FOS supplemented to the medium had a stimulatory effect on the growth of bi dobacteria compared with the control medium. The growth curves of Lactobacillus sp. reached the plateau phase afteR²4 h of cultivation with FOS. The CLEA of fructosyltransferase catalyzed the biocatalysis of sucrose to FOSs, a process with a biotechnological importance as prebiotics.