| The high purity Bromelain with the activity of 50-140×104u/g were extracted from the pineapple fruit by means of ultra-fine filter in this paper. The effects of photo, heat, pH, and the additives on the stability of Bromelain were studied by using chemical analyses, infrared spectroscopy and thermal analyses. The thermal inactivation point, the denatural enthalpy and the thermal inactivation curves of the bromelain of different water contents were obtained and the kinetic parameters of deactivation of Bromelain were also calculated. The results indicate that during the storage the effect of photo on the stability of Bromelain is obvious. The difference between the activity retention indexes of the bromelain stored for 10 days with and without light blocking is up to 10%. The Bromelain shows a relatively high stability at pH 5.0. With the rising of storage temperature, the activity retention index reduced obviously. When the liquid bromelain is incubated at the temperature ranging from 30 to 60℃, the Bromelain activity retention(R)and the storage temperature (T) follow to the relation of R=121.6-1.493T, When heating at 70℃, the descent in activity of powdered Bromelain is small, whereas heating above 70℃, the activity is decreased abruptly. The DSC results indicate that the equilibrium thermal denaturation point of Bromelain is about 85.2℃ and the denaturation enthalpy is 71.1J/g. The effect of moist on the equilibrium inactivation temperature is obvious. When the water content is ranging from 0 to 20%, the equilibrium denaturation point is decreased from 84.09 to 30.48℃ and is rebounded slowly. The IR spectrum of Bromelain heating at 80℃ shows no obvious difference comparing with that of unheated Bromelain, indicating that the primary structure of Bromelain is not changeable and the denaturation of Bromelain should be assigned to the variations in the secondary and tertiary structures. Therefore,the improvements of Half-cystine and Vitamin C on the stability of Bromelain were investigated and the results indicated that both the stabilization and protection of cysteine on Bromelain are superior to those of Vitamin C. The sodium benzoate can be used as the preservative for Bromelain and 0.05% dosage of sodium benzoate shows a certain protection on Bromelain. EDTA used to eliminate the effect of hazardous metals can improve the activity retention of Bromelain obviously. The macromolecular stabilizing agents such as p-cyclodextrin and B-1 can increase the protease activity retention index of Bromelain. A composite stabilizing system including cysteine, b-cyclodextrin and EDTA can improve the activity stability of bromelain obviously. The studies of the deactivation kinetics of Bromelain indicate that the thermal inactivation rate constant(K303) and deactivation half-life valve( r 303) of Bromelain at 30℃ are 0.2897/h and 2.39h,and those at 45℃ and 60℃ are K3180.5787/h, r 318=1.20h and KM =1.245/h, r 333=0.557h; respectively. The activation energies and frequency factors for 30-45℃ and 45-60℃ are as follows, AE, (30-45 ℃) =36.9 kJ/mol, A,=8. 39×10Yh ; AE2 (45-60℃) =22. 5 kJ/mol, A2=4. 23×103/h. By adding of B-1, the inactivation rate constant of Bromelain becomes small, the half-life is prolonged and the activation energy increases, indicating that the stability of Bromelain can be improved by B-1. |
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