Screening of optimum conditions for extraction of total polysaccharides from persimmon fruits by papain deproteinization and ultra- filtration membranes methods

XIE Ling;ZHENG Linxi;XIA Zhilan;ZHONG Xiaohong;Horticulture&Landscape College,Hunan Agricultural University;

Total polysaccharides from persimmon fruit(TPsPF);;Enzymatic deproteinization;;Papain;;Ul tra-filtration membranes;;Molecular weight cut-off(MWCO)

【Objective】TPs PF(total polysaccharides from persimmon fruits) is known to have a variety of pharmacological activities. Extraction of the TPs PF by traditional chemical reagents would potentially have some toxic risk. The extraction of the TPs PF by biological and physical methods would be valuable for the reason of the safety of the extracts. Therefore, papain deproteinization and ultra-filtration membranes were employed for extracting the TPs PF in present study.【Methods】The flesh of persimmon was defated by chloroform and methanol(3∶1) at 60 ℃, and the crude TPs PF was extracted under reflux with alcohol. Then, different conditions of papain deproteinization by using removal rate of protein and retention rate of TPs PF as indexes. The effect of the enzyme content, temperature, processing time and p H value were investigated using the method of single factor test. After the treatments of papain deproteinization and decolorization of active carbon, three kinds of membranes with different molecular weight cut-off(MWCO) were used to filter the TPs PF. The TPs PF of 10-200 ku were enriched by using two membranes with MWCO(200 ku, 10 ku), meanwhile the effect of operating pressure, the polysaccharide concentration and temperature on ultra-filtration membrane flux and the retention rate of TPs PF were studied and theoptimal conditions were selected.【Results】According to the results of retention rate of TPs PF and removal rate of protein, the conditions of papain deproteinization process were optimized as follows: enzyme content of 1.0%, at 60 ℃, p H 6(or 7) and 1.5 h. When enzyme content was 1.0%, the highest retention rate of TPs PF was 92% and removal rate of protein was about 50%. The removal rate of protein reached the peak when enzyme content was 1.5%. Regarding the cost, we preferred to use 1.0% of enzyme. When the temperature varied from 30 ℃ to 60 ℃,there were no apparent change of the retention rate of the TPs PF and the removal rate of protein. However, the retention rate of TPs PF and the removal rate of protein decreased as the temperature increased to over 60 ℃. The processing time had significant effect on the retention rate of the TPs PF and The p H value had significant effect on the removal rate of protein in the process of enzymatic deproteinization. Among four factors, The p H value was the most effective factor on the removal rate of protein and the processing time was the most influencing factor on the retention rate of the TPs PF. The average retention rate of the TPs PF was 85.33%(RSD 2.7%), the average removal rate of protein was 76.29%(RSD 2.9%)and the average yield rate of the TPs PF was(136.37±6.90) mg·g-1in repeat experiment under optimized parameters. Furthermore, The ultra-filtration conditions of the TPs PF were as follows: the TPs PF of 10-200 ku was concentrated by ultra-filtration membranes with 10 ku and 200 ku under the pressure of 0.3 MPa, at 30 ℃ and the polysaccharide concentration was 20 g·L-1. When operating pressure varied from 0.15 to 0.3 MPa, the membrane flux increased apparently as the pressure increased. But when the pressure had reached to 0.35, the membrane flux decreased sharply. This indicated that the operating pressure had a important impact on the membrane flux rather than the retention rate of the TPs PF. It was confirmed that the membrane flux decreased as the polysaccharide concentration increased so that the initial concentration 20 g·L- 1was the best choice. Meanwhile the temperature had a significant impact on the membrane flux and the retention rate of the TPs PF. The membrane flux and the retention rate of the TPs PF gradually increased with the rise of the temperature(20-30 ℃). As temperature was between 30 ℃ and 45 ℃, the membrane flux changed only a bit and the retention rate of the TPs PF drope down sharply. The retention rate of TPs PF was(45.82±2.37)% in 5 parallel tests by ultra-filtration. We finally analyzed the loss proportion of the TPs PF in different stages during the course of refining and purification. The loss proportion of the TPs PF in different stages were ultra-filtration stage>depigmentation and salting-out stage>enzymatic deproteinization stage>other stage. The lareger loss of the TPs PF during depigmentation and salting-out stage might be due to the adsorption of active carbon to polysaccharides.【Conclusion】Compared with Sevage method, enzymatic deproteinization of papain had higher retention rate of the TPs PF and was more convenient. It is most important that it rarely destroy the activity of the target component. The extraction of the TPs PF via papain deproteinization and ultra-filtration membranes was simple, efficient and, more important, mild with regards to maintaining the biological activity of the extracts.