Buckwheat flour was incorporated into wheat flour at different levels (0, 20, 40, 60, 80, and 100 %) and the physicochemical, functional and antioxidant properties of the blended flour were studied. This study also investigated the effect of buckwheat on the retention of antioxidant properties of cookies during baking. The results showed significant variation in physicochemical and functional properties of the blended flour. The addition of buckwheat flour into wheat flour also increased the antioxidant properties of blended flour proportionally, but metal chelating properties decreased. The incorporation of buckwheat in wheat flour helped in better retention of antioxidant potential of cookies during baking process as buckwheat cookies (100 % buckwheat) showed greater percentage increase in antioxidant properties than control (100 % wheat). Quality characteristics of cookies such as hardness and spread ratio decreased, while as non-enzymatic browning (NEB) increased significantly with increase in the proportion of buckwheat flour in wheat flour. The Overall acceptability of cookies by sensory analysis was highest at 40 % level of blending. This study concluded that addition of buckwheat in wheat flour, may not only improve the physico-chemical and functional properties of the blended flour but may also enhance the nutraceutical potential of the product prepared from it.
Immobilization of the Aspergillus niger glucose oxidase in high yield was achieved using an immunoaffinity-based procedure. For this purpose IgGs, isolated from the sera of rabbits immunized with glucose oxidase, were favourably oriented by binding on to cobalt-charged iminodiacetate-Sepharose. Large amounts of glucose oxidase could be immobilized by incubating the IgG-bound matrix alternately with the enzyme and either intact IgG or F(ab)2 derived thereof, leading to the formation of multiple enzyme layers. After three incubation cycles using anti-(glucose oxidase) IgG, an 8-fold increase in the amount of enzyme immobilized was observed, while the increase was 11-fold when the F(ab)2 replaced intact IgG. The preparations obtained thus were highly active, as also indicated by the high effectiveness factor, eta. Immunoaffinity-layered immobilized preparations were markedly more resistant to inactivation induced by exposure to 60 degrees C, 4.0 M urea or storage at 4 degrees C. The preparations also exhibited a remarkable resistance against inactivation induced by the water-miscible organic solvents tetrahydrofuran, dioxan or acetone. Immobilized glucose oxidase preparations obtained using F(ab)2 were generally observed to be superior in stability compared with those immobilized with the help of intact IgG.
The formation of insoluble complexes of glycoenzymes with lectins and antibodies is one of the simplest methods of enzyme immobilization. Insoluble complexes of glucose oxidase were simply obtained by mixing the enzyme with concanavalin A or a specific polyclonal antibodies solution. The concanavalin A and immunocomplexes of glucose oxidase retained more than 80% of the original enzyme activity. Expression of very high enzyme activity in insoluble complexes suggested that these aggregates were quite porous and easily accessible to substrates. Insoluble complexes of glucose oxidase showed very high stability against denaturation induced by pH, temperature, urea and watermiscible organic solvents. Complexes of glucose oxidase obtained with concanavalin A and glycosyl-specific antiglucose oxidase polyclonal antibodies were quite comparable in stability while complexes prepared using polyclonal antibodies raised against the native glucose oxidase were slightly less stable. The complexes of glucose oxidase obtained with glycosyl-specific antiglucose oxidase polyclonal antibodies showed very high stability against inactivation mediated by exposure to water-miscible organic solvents. Insoluble complexes of glucose oxidase were cross-linked with glutaraldehyde to maintain their integrity in the presence of substrates. The cross-linking of complexes resulted in a slight decrease in enzyme activity but showed a pronounced enhancement in stability against various forms of denaturation.
The purified oligosaccharide chains of Aspergillus niger glucose oxidase were coupled to BSA with the help of the cross-linking reagent glutaraldehyde. The neoglycoconjugate thus obtained was purified by concanavalin A-Sepharose chromatography and characterized by SDS/PAGE. Immunization of rabbits with the neoglycoprotein raised the glycosyl-specific anti-(glucose oxidase) polyclonal antibodies. Antibodies were purified by (NH(4))(2)SO(4) precipitation, followed by DEAE-cellulose chromatography. The IgG-Sepharose was prepared by covalently coupling the purified polyclonal antibodies to the CNBr-activated Sepharose 4B. The large assembly of glucose oxidase was made on the IgG-Sepharose by alternate incubation of glucose oxidase and glycosyl-specific anti-(glucose oxidase) polyclonal IgG. The immunoaffinity-layered assembled preparations were highly active and, after six alternate binding cycles with enzyme and glycosyl-specific IgG, the amount of enzyme immobilized could be raised 30-fold. The K (m) value of immunoaffinity-layered glucose oxidase preparations remained unaltered, while the V (max) slightly decreased as compared with the soluble enzyme. A layer-by-layer immobilization of glucose oxidase resulted in significant improvement in stability against high temperature, 4.0 M urea and very high concentrations of water-miscible organic solvents such as acetone, dimethylformamide, dioxan and tetrahydrofuran.
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