| This paper attempted to co-immobilize tannase and β -glucosidase, aiming at applying in tea beverage to attain the double efficacies of haziness-removing and aroma-increasing, thus established the foundation for the processing of pure tea beverage. Choosing two kinds of carriers, alginate sodium and chitosan, and inspecting four different kinds of co-immobilized methods, the results showed that the best effect of co-immobilization rooted in choosing alginate sodium as the co-immobilized carrier and the co-immobilized method of crosslinking-embedding-crosslinking. Adopting alginate sodium as co-immobilized carrier and the co-immobilized method of crosslinking-embedding-crosslinking, we optimized the best co-immobilized conditions as follows: 2.5 ml of tannase and 2.5 ml of β-glucosidase joined 6.7 ml of 3.0 % alginate sodium at the rate of 0.75:1(v/v). After fully mixed, 2.5 % glutaraldehyde was added to make its final concentration in solution achieve 1.25 % and shaked 30 minutes on the oscillator, and then placed at 20 ℃ for 4 hours. It was poured into 2.0 % of CaCl2 solution with injector and the small spheroids were strained out. After changing CaCl2 solution, the small spheroids were stiffened for 2 hours in the refrigerator. The small spheroids were further strained out and crosslinked with 0.075 % glutaraldehyde at 10 ℃ for 3 hours. The created small spheroids were strained out again, washed with buffer and inhaled the water completely. When co-immobilized under the optimum conditions, the activity recoveries of tannase and β-glucosidase could reach 67.3 % and 46.0 %, respectively. The properties of co-immobilized tannase andβ-glucosidase were also studied in this paper. The results indicated that the optimum temperature of both kinds of enzymes decreased by 10 ℃, served as 35 ℃ and 45 ℃, respectively. There was no variation in the optimum pH of both kinds of enzymes, served as 5.0 and 6.0, which were identical with those of their free enzymes. Compared with their free enzymes, the stabilities to thermal, acid and alkali and chemical reagent as well as storing stability of co-immobilized tannase and β-glucosidase were markedly enhanced. The apparent Km of co-immobilized β-glucosidase was 1.97×10-3 mol/L, which was less than that of its free enzyme, whereas the apparent Km of co-immobilized tannase was 11.6×10-4 mol/L, higher than that of its free enzyme. The co-immobilized enzymes were applied in tea beverage to remove haziness and increase aroma. The results showed that after treated by co-immobilized enzymes, the total amount of essential oil in green tea, oolong tea and black tea was increased by 20.69 %, 10.30 % and 6.79 %, respectively. The content of non-estern catechins in green tea, black tea and oolong tea were increased by 52.17 %, 12.94 % and 8.83 %, respectively. The content of estern catechins in green tea, oolong tea and black tea decreased by 20.0 %, 16.68 % and 5.04 %, respectively. The anti-sediment effect of green tea infusion treated with co-immobilized enzymes was also studied. It indicated that the turbidity of green tea infusion treated without co-immobilized enzymes was higher than that treated with co-immobilized enzymes remarkably at lower temperature. After stored for three months, the pellucid degree of green tea infusion treated with co-immobilized enzymes was very high, but green tea infusion treated without co-immobilized enzymes had produced a few sediments after stored for 60 days.
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