Preparation Of Bagasse Nanocellulose Composite Hydrogel Beads And Immobilization Of Bromelain

Bromelain is widely used in the fields of food,medicine and cosmetics due to its good proteolytic activity and various physiological functions.However,the disadvantages,such as high purification cost,low stability and difficulty in recycling,limit its better application.Immobilization technology is one of the most effective methods to improve the stability of enzymes and realize the reuse of enzymes.The structure and properties of support materials are the core elements affecting the performance of immobilized enzymes.Nanocellulose is a potential carrier for immobilized enzymes due to its high aspect ratio,high porosity,and easy modification of surface hydroxyl groups.Bagasse is a by-product of sugarcane sugar production with high cellulose content,which is an ideal raw material for the preparation of nanocellulose.In this study,sugarcane bagasse nanocellulose（SBCNFs）was prepared from sugarcane bagasse through functional modification and structural characterization.Then,SBCNFs were used as nano-fillers to prepare two kinds of composite gel bead carriers for immobilization of bromelain by mixing with sodium alginate（SA）and sodium alginate-polyvinyl alcohol gel（SA-PVA）,respectively.The effect of reaction conditions on the immobilized load and enzyme activity recovery of the immobilized enzyme was investigated.The enzymatic properties of the free enzyme and the immobilized enzyme were compared to investigate the immobilization effect of the carrier on bromelain.Findings were as follows:（1）SBCNFs was prepared by carboxylation combined with high-pressure homogenization,and its structural properties were characterized.The results showed that SBCNFs were filamentous with a width of 3 to 10 nm and a length of micrometers.In the range of 1 to 3 μm,hydroxyl groups on the surface of the fibers were successfully oxidized to carboxyl groups,and the content of carboxyl group was 2.16 mmol·g^-1.Compared with sugarcane bagasse cellulose（SBC）,SBCNFs were more thermally stable and retained the cellulose type I structure,but the crystallinity index of SBCNFs changed from 32.62% to 71.37%.With the increase of SBCNFs suspension concentration,its viscosity increased and the shear thinning behavior enhanced.In the linear viscoelastic region（stress < 10%）,the storage modulus was greater than the loss modulus,and the elastic behavior was dominant.SBCNFs suspension showed solid properties with good thixotropy recovery.（2）SBCNFs-SA composite dry gel beads were prepared by drip method,and the immobilization effect of SBCNFs-SA as carrier on bromelain was investigated.The results showed that the diameter of SBCNFs-SA gel beads was about 2 mm.The addition of SBCNFs increased the roughness of the gel beads and showed a porous structure.After adding 0.6wt% SBCNFs,the equilibrium swelling ratio of SA gel beads was increased by about 20%,and SBCNFs-SA gel beads maintained stable spherical structure without disintegrating under acid-base conditions.The optimum conditions for immobilization of bromelsin were determined as follows: 0.4wt% SBCNFs in gel beads,2.0 mg·m L^-1 enzyme concentration,10 h immobilization time,pH of buffer solution at 7.0,and reaction temperature 25 ℃.Under these conditions,the solid load of SBCNFs-SA gel beads to bromersin was about 103.75 mg·g^-1,and the recovery rate was about 70.22%.The optimum reaction conditions of immobilized enzyme and free enzyme were similar.The stability of immobilized enzyme was better than that of free enzyme,and the recovery rate of immobilized enzyme was about 48.26% after repeated use for 10 times.（3）SA-SBCNFs-PVA double network gel beads were prepared by a two-step cross-linking reaction.1-ethyl-（3-dimethylaminopropyl）carbamyl diimide（EDC）/ n-hydroxysuccinimide（NHS）was used to activate the carboxyl group in the gel beads to covalently bind bromelsin,and the immobilization effect was investigated.The results showed that the addition of SBCNFs made the cross-section of the gel beads more porous.The optimal immobilization conditions were 0.6wt% SBCNFs,2.0 mg·m L^-1 initial enzyme concentration,pH of buffer solution at 7.0,1.5 h immobilization time,and 25 ℃ reaction temperature.Under these conditions,the carrier loading was about 138.21 mg·g^-1 and the enzyme recovery was about 84.21%.Compared with the free enzyme,the optimum reaction pH of immobilized enzyme changed from 7.5 to 7.0,and the optimum reaction temperature increased by about 5 ℃.Acid-base stability and thermal stability were significantly improved（P < 0.05）,the recovery rate of immobilized enzyme was about 62.16% after repeated use for 10 times.This technique solves the problem of weak adsorption and binding of bromelain and SBCNFs,and effectively improves the adsorption capacity of bromelain on the carrier and the recovery of enzyme activity.