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

Abstract: 【Objective】TPsPF (total polysaccharides from persimmon fruits) is known to have a variety ofpharmacological activities. Extraction of the TPsPF by traditional chemical reagents would potentiallyhave some toxic risk. The extraction of the TPsPF by biological and physical methods would be valuablefor the reason of the safety of the extracts. Therefore, papain deproteinization and ultra-filtration mem⁃branes were employed for extracting the TPsPF in present study.【Methods】The flesh of persimmon wasdefated by chloroform and methanol (3∶1) at 60 ℃, and the crude TPsPF was extracted under reflux withalcohol. Then, different conditions of papain deproteinization by using removal rate of protein and reten⁃tion rate of TPsPF as indexes. The effect of the enzyme content, temperature, processing time and pH val⁃ue were investigated using the method of single factor test. After the treatments of papain deproteinizationand decolorization of active carbon, three kinds of membranes with different molecular weight cut-off(MWCO) were used to filter the TPsPF. The TPsPF of 10-200 ku were enriched by using two membraneswith MWCO (200 ku, 10 ku), meanwhile the effect of operating pressure, the polysaccharide concentrationand temperature on ultra-filtration membrane flux and the retention rate of TPsPF were studied and theoptimal conditions were selected.【Results】According to the results of retention rate of TPsPF and remov⁃al rate of protein, the conditions of papain deproteinization process were optimized as follows: enzyme con⁃tent of 1.0%, at 60 ℃, pH 6 (or 7) and 1.5 h. When enzyme content was 1.0%, the highest retention rate ofTPsPF was 92% and removal rate of protein was about 50%. The removal rate of protein reached the peakwhen enzyme content was 1.5%. Regarding the cost, we preferred to use 1.0% of enzyme. When the tem⁃perature varied from 30 ℃ to 60 ℃,there were no apparent change of the retention rate of the TPsPF andthe removal rate of protein. However, the retention rate of TPsPF and the removal rate of protein de⁃creased as the temperature increased to over 60 ℃. The processing time had significant effect on the retention rate of the TPsPF and The pH value had significant effect on the removal rate of protein in the processof enzymatic deproteinization. Among four factors, The pH value was the most effective factor on the re⁃moval rate of protein and the processing time was the most influencing factor on the retention rate of theTPsPF. The average retention rate of the TPsPF was 85.33% (RSD 2.7%), the average removal rate of pro⁃tein was 76.29% (RSD 2.9%)and the average yield rate of the TPsPF was (136.37±6.90) mg·g-1 in repeatexperiment under optimized parameters. Furthermore, The ultra-filtration conditions of the TPsPF were asfollows: theTPsPF of 10-200 ku was concentrated by ultra-filtration membranes with 10 ku and 200 kuunder the pressure of 0.3 MPa , at 30 ℃ and the polysaccharide concentration was 20 g·L-1. When operat⁃ing pressure varied from 0.15 to 0.3 MPa, the membrane flux increased apparently as the pressure in⁃creased. But when the pressure had reached to 0.35, the membrane flux decreased sharply. This indicatedthat the operating pressure had a important impact on the membrane flux rather than the retention rate ofthe TPsPF. It was confirmed that the membrane flux decreased as the polysaccharide concentration in⁃creased so that the initial concentration 20 g·L^-1 was the best choice. Meanwhile the temperature had asignificant impact on the membrane flux and the retention rate of the TPsPF. The membrane flux and theretention rate of the TPsPF gradually increased with the rise of the temperature (20-30 ℃). As tempera⁃ture was between 30 ℃ and 45 ℃, the membrane flux changed only a bit and the retention rate of the TP⁃sPF drope down sharply. The retention rate of TPsPF was (45.82±2.37)% in 5 parallel tests by ultra-filtra⁃tion. We finally analyzed the loss proportion of the TPsPF in different stages during the course of refiningand purification. The loss proportion of the TPsPF in different stages were ultra-filtration stage＞depig⁃mentation and salting-out stage＞enzymatic deproteinization stage＞other stage. The lareger loss of theTPsPF during depigmentation and salting-out stage might be due to the adsorption of active carbon topolysaccharides.【Conclusion】Compared with Sevage method, enzymatic deproteinization of papain hadhigher retention rate of the TPsPF and was more convenient. It is most important that it rarely destroy theactivity of the target component. The extraction of the TPsPF via papain deproteinization and ultra-filtra⁃tion membranes was simple, efficient and, more important, mild with regards to maintaining the biologicalactivity of the extracts.