Coimmobilization of Naringinases on Silk Fibroin Nanoparticles and Its Application in Food Packaging

Bombyx mori silk fibroin is a macromolecular biopolymer with remarkable biocompatibility. It was degummed and subjected to a series of treatments, including dissolution and dialysis, to yield an aqueous solution of silk fibroin, which was introduced rapidly into excess acetone to produce crystalline silk fibroin nanoparticles (SFNs), which were conjugated covalently with naringinase using glutaraldehyde as the cross-linking reagent. The SFN naringinases are easily recovered by centrifugation and can be used repeatedly. Naringinase is a bienzyme consisting of α-L-rhamnosidase and flavonoid-β-glucosidase. The enzyme activity and its kinetics were similar to those of the native form, and the optimum reactive temperature for both is 55°C. In our study, centrifugation allowed the separation of enzyme and substrate; after eight cycles the SFN naringinases retained >70% residual activity. The highly efficient processing technology and the use of SFN as a novel vector for a bienzyme have great potential for research and the development of food processing such as the debittering of naringin-containing juices. 1. Introduction Naringinase is a bienzyme consisting of α-rhamnosidase (EC 3. 2.1.40) and flavonoid-β-glycosidase (EC 3.2.1.21) [1]. α-L-Rhamnosidase hydrolyses naringin into the flavonoid prunin, which is converted to naringenin (4,5,7-trihydroxyflavonone) by β-D-glucosidase. Naringinase catalyses the hydrolysis of naringin to naringenin, glucose, and rhamnose. On the one hand, the removal of naringin (the bitter orange substance) with specific debittering of the strong flavour caused no detriment to the nutritional value of orange juice. On the other hand, the enzyme-extracted single glycosides of naringenin degradation products can be better absorbed by the body and can be used as pharmaceutical raw materials. Many immobilized preparations of naringinase have been used successfully for debittering and for producing biochemicals such as rhamnose and prunin. Several authors have described the effects of immobilised naringinase. In early 1985, Manjón et al. covalently immobilized naringinase to glycophase-coated, controlled-pore glass. The efficient kinetic parameters shown by the most active and stable derivative enabled it to be used for the debittering of naringin-containing juices [2]. In another case naringinase of Penicillium sp. when entrapped in cellulose triacetate fibers and when it showed it had a higher Km values than in its soluble form [3]. In the third case naringinase was immobilizated in packaging films that had an increased catalytic