Study On Extraction, Purification And Stability Of Bromelain

This article was carried out to investigate the protease activity and protein content of pineapple pulp, skin, and stem. Pineapple stem had the higher activity than pulp and the skin in the same sample. Kaolin adsorption, ultrafiltration concentrated and organic solvent extraction and nano-TiO2 adsorption were studied for the purification of stem bromelain The stability of stem bromelain purified by nano-TiO2 adsorption was determined, and further purified by ion-exchange chromatography. Primarily results were as follows:1. The comparison of purification among different methods A 2.63 fold purification was obtained with the recovery of 64.3% treated by Kaolin adsorption,and those by ultrafiltration concentrated and nano-TiO2 adsorption were 1.34 fold,78.4% and 5.29 fold,64.7%;respectively. Comparison of the purification effect showed that: kaolin adsorption had disadvantages of complex processing, consumptive, excessive equipments, and low purification rate; Ultrafiltration concentrated and organic solvent extraction could not remove proteins of similar molecular weight with bromelain; Howe -ver,nano-TiO2 adsorption was the best purification method with simple, less-consumptive, and purified.2. Optimization of Nano-TiO2 adsorption process Orthogonal experiment of Nano-TiO2 adsorption process was researched based on single factor experiments. The optimized adsorption conditions of nano-TiO2 amount, pH, temperature, and adsorption time were 3.5%, 4.7, 15℃, 25min respectively. Under the controlled conditions, the bromelain adsorption rate was 98.2%, and the bromelain recovery was 79.3%, eluted 30min using citric acid buffer of 0.4mol/L(pH6.0). The eluate could be purified by two grade of ultrafiltration with 40KD and 20KD ultrafiltration membrane, with the pressure 2.5×105~3.0×105Pa, and the recovery of protease activity was 64.7%.3. Further separation and purification by Ion-exchange chromatogra -phy Further separation and purification of bromelain was operated by Ion-exchange chromatography (BIO-RAD's 158-0030 ion exchange resin). Using tris-phosphate buffer of 0.01mol/L (pH6.0) as the initial solution, and it was linear eluted by 0.1~1.5mol/L NaCl solution, the protease specific activity could reach to 6.4×103 U / mg .Pro, with purification rate 13.73 fold.4. Studies on the stability of bromelain The analysis of stem bromelain stability revealed that bromelain was stable at pH5.0-6.5, especially at pH6.0; Light was remarkable to the stable influence in the storing process of it.The bromelain activity under dark conditions was 13.1% higher than that in natural light conditions. The bromelain activity (S) decreased significantly with the increase of the storage temperature, and the linear equation was S = 119.5-1.411T, but it decreased rapidly above 70℃; And the bromelain's half-life was extended to 240 days with the effect of composites (cysteine, 0.005%; EDTA, 0.05%)...