In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested for the immobilization of β-D-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of the enzyme and crosslinking with genipin. The particles were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The immobilization of the enzyme by crosslinking with genipin provided biocatalysts with satisfactory activity retention and thermal stability, comparable with the ones obtained with the traditional methodology of immobilization using glutaraldehyde. β-D-Galactosidase-chitosan-genipin particles were applied to galactooligosaccharides synthesis, evaluating the initial lactose concentration, pH and temperature, and yields of 30% were achieved. Moreover, excellent operational stability was obtained, since the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. Thus, the food grade chitosan-genipin particles seem to be a good alternative for application in food process.

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A new biocatalyst of lipase B from Candida antarctica (MCI-CALB) immobilized on styrene-divinylbenzene beads (MCI GEL CHP20P) was compared with the commercial Novozym 435 (immobilized lipase) in terms of their performances as biocatalysts for the esterification of acetic acid and n-butanol. The effects of experimental conditions on reaction rates differed for each biocatalyst, showing different optimal values for water content, temperature, and substrate molar ratio. MCI-CALB could be used at higher acid concentrations, up to 0.5 M, while Novozym 435 became inactivated at these acid concentrations. Although Novozym 435 exhibited 30% higher initial activity than MCI-CALB for the butyl acetate synthesis, the reaction course was much more linear using the new preparation, meaning that the MCI-CALB allows for higher productivities per cycle. Both preparations produced around 90% of yield conversions after only 2 h of reaction, using 10% (mass fraction) of enzyme. However, the main advantage of the new biocatalyst was the superior performance during reuse. While Novozym 435 was fully inactivated after only two batches, MCI-CALB could be reused for six consecutive cycles without any washings and keeping around 70% of its initial activity. It is proposed that this effect is due to the higher hydrophobicity of the new support, which does not retain water or acid in the enzyme environment. MCI-CALB has shown to be a very promising biocatalyst for the esterification of small-molecule acids and alcohols.

The enzymatic synthesis of fructooligosaccharides (FOS) was carried out using a partially purified β-fructofuranosidase from the commercial enzyme preparation Viscozyme L. Partial purification of β-fructofuranosidase from Viscozyme L was done by batch adsorption using ion-exchange resin DEAE-Sepharose, showing a 6-fold increase in specific activity. The biocatalyst was then covalently immobilized on glutaraldehyde-activated chitosan particles. Thermal stability of the biocatalyst was evaluated at 50 °C and 60 °C, being around 100 times higher at 60 °C when compared to the free enzyme. The immobilized biocatalyst was reused 50 times for FOS production (100 min per batch at 50 °C and pH 5.5) without significant loss of activity. The average yield (grams of FOS per grams of initial sucrose) was 55%. The immobilization process combined with partial purification method resulted in a derivative with activity of 1230 Ut/g, which is among the best for FOS production.

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The synthesis of pineapple flavour (butyl butyrate) catalysed by lipase from Rhizomucor miehei has been optimized using central composite design and response surface methodology. Initially, the best butyric acid concentration in the mixture was defined and found that 1 M butyric acid presented the highest initial reaction rate. The reaction parameters substrate molar ratio, enzyme content, and initial added water were evaluated in the central composite design with the reaction conversion yield as the dependent variable. The optimal conditions for butyl butyrate synthesis were found to be substrate molar ratio of 3.6:1 butanol:butyric acid; enzyme content of 6.5% of substrate mass fraction; added water 0.0% of substrate mass fraction. Under these conditions, over 90% of conversion was obtained in 16 h of reaction. Enzyme reuse was tested performing a treatment before each batch by washing the enzyme system with n-hexane, or simply reusing the biocatalyst in a new fresh reaction. Direct enzyme reuse caused a rapid decrease on the enzyme activity, while washings with n-hexane allowed the enzyme to be reused for six cycles keeping around 75% of its original activity.

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