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In this work, we studied the effect of some variables in the reaction between genipin and chitosan, to prepare a support for enzyme immobilization. Reaction temperature, pH, time and genipin concentration were evaluated for support activation and β-galactosidase from Aspergillus oryzae was used as the enzyme model. The immobilized enzyme was characterized by immobilization properties and kinetic parameters and by thermal and operational stability. It was found that support activation at alkaline pH (9.0), at 60 °C for 1 h, resulted in an activity recovery of 37.5%. The immobilized enzyme had a stabilization factor of 2.0 at 50 °C compared with the free enzyme. In batch process, the immobilized preparation kept 100% of its relative initial activity during 40 batches. This research shows, for the first time, a complete study about the parameters that affect the support activation process and the possible structure formed between genipin and chitosan.

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for β-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized β-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

A new support for the immobilization of β- d -galactosidase from Kluyveromyces lactis was developed, consisting of mesoporous silica/titania with a chitosan coating. This support presents a high available surface area and adequate pore size for optimizing the immobilization efficiency of the enzyme and, furthermore, maintaining its activity. The obtained supported biocatalyst was applied in enzyme hydrolytic activity tests with o-NPG, showing high activity 1223 Ug−1, excellent efficiency (74%), and activity recovery (54%). Tests of lactose hydrolysis in a continuous flow reactor showed that during 14 days operation, the biocatalyst maintained full enzymatic activity. In a batch system, after 15 cycles, it retained approximately 90% of its initial catalytic activity and attained full conversion of the lactose 100% (±12%). Additionally, with the use of the mesoporous silica/titania support, the biocatalyst presented no deformation and fragmentation, in both systems, demonstrating high operational stability and appropriate properties for applications in food manufacturing.

Milk whey (MW) and milk whey permeate (MWP) constitute the main part of co-products derived from milk processing. The use of the transgalactosylation activity of β-galactosidases is an excellent alternative to add value to these co-products since this is the route for the synthesis of galactooligosaccharides (GOS), a valuable prebiotic sugar. Hereby, we immobilized a β-galactosidase from Bacillus circulans onto glutaraldehyde-activated chitosan beads. The immobilized enzyme was characterized and the parameters affecting GOS synthesis (pH, temperature, and concentration of MW and MWP) were evaluated. The optimum pH and temperature of activity for the immobilized β-galactosidase were 50 °C and pH 6.0, respectively. The immobilization improves enzyme thermal stability by 2.9 times. In the batch process, the immobilized enzyme exhibited excellent operational stability at 40 °C, being able to hydrolyze 91% lactose after 17 cycles of reuse. Regarding GOS synthesis, the immobilized enzyme could be reused for 10 consecutive cycles retaining 74% of the initial activity at 50 °C. The maximum concentration of GOS achieved, using MW and MWP, was 159.4 g L−1 and 168.8 g L−1, respectively. Therefore, these results proved that it was possible to obtain, in high concentration, a useful and valuable prebiotic sugar from co-products of milk processing.

The effect of the degree of deacetylation (DD) on the characteristics of the support matrix formed through chitosan (CH) and genipin (GE) crosslinking was investigated by using CH with DD of 83, 94 and 96%. The rheological properties of CH dispersions were determined in order to study the influence of DD on the CH viscosity, gel network structures and gelation temperature (Tgel). The CH exhibited the shear-thinning behavior of pseudoplastic fluids. Tgel decreased with the increase of DD and the sol-gel transition temperature of CH-83, CH-94 and CH-96 were 75, 30 and 25 °C, respectively. The magnitude of viscous modulus (G’’) was higher than the elastic modulus (G’), revealing the formation of a weak gel structure. The CH dispersions have been used in the preparation of beads and then crosslinked with GE to form support matrices. Besides, to verify how it influences in a practical application, an enzymatic immobilization was carried out using a β- d -galactosidase (BG) as a model, which could be advantageously employed to promote hydrolysis of lactose. Scanning Electron Microscope images showed biocatalysts with multiple porous compartments. Fourier Transform Infrared Spectroscopy (FTIR) analyses confirmed chemical and physical interactions between CH, GE and BG. Thermogravimetric analysis (TGA) demonstrated that the DD of CH had a positive influence in the thermal degradation behavior of the biocatalysts up to 200 °C, which represents the temperature range used in most enzymatic reactions (up to 100 °C) in the industrial scale applications.