Microbial Transformation Of Piceid In Polygonum Cuspidatum And Separation Of The Target Product

Dry caudex of Polygonum cuspidatum Sieb.et Zucc.(Huzhang in Chinese) is a Chinese traditional herbal medicine,which has been used for dispelling wind and dampness, eliminating stasis to activate blood circulation,relieving cough and reducing sputum. Resveratrol,the most valuable component in Huzhang,is widely used in food additive,health products and cosmetic industries due to its various pharmaceutical properties such as antimicrobial,anti-inflammatory,anti-virus,antioxidant and anticancer activities.However, its content is very low,which increases its production cost.Piceid,the glucoside of resveratrol, has similar pharmaceutical functions,and its content is much higher,however,its bioavailability is much lower than resveratrol.In general,aglycone is acquired by hydrolysis of its glucoside,and acid or alkaline is often used as the hydrolytic reagent.However,the hydrolytic reaction is always carried out under extreme conditions such as high temperature and high pressure,which not only requires high equipment characteristic but also causes pollution.In contrast,biotransformation only requires mild conditions,simple procedures, lower cost and results in less pollution.In this paper,biotransformation of piceid in P. cuspidatum to resveratrol was performed by Aspergillus oryzae and its separation and purification was studied.A food-grade microorganism A.oryzae with highβ-glucosidase activity was firstly selected.Properties of the enzyme were studied and biotransformation of piceid in raw herb of P.cuspidatum by the crude enzyme was investigated.The results indicated that the optimum pH of theβ-glucosidase is 4.8 and the optimum temperature is 50℃.Concentration of resveratrol increased to 2.9 times higher after 12h transformation of raw herb by the crudeβ-glucosidase,with the percent conversion of piceid 100%.Based on the above results,biotransformation of piceid in raw herb of P.cuspidatum to resveratrol by A.oryzae was investigated,and mechanism of the bioprocess was discussed. Percent extraction of resveratrol reached 1.36%after 46h shake flask cultivation,3.6 times higher than that by microwave-assisted extraction of raw herb.The same results were obtained by enzymatic hydrolysis of raw herb for 12h with commercial cellulase,but the high cost of the enzyme limits its industrial application.Seventy nine percent of piceid was hydrolyzed to resveratrol after acid hydrolysis of raw herb for 20min,while 46.7%of resveratrol was destroyed during the process,so acid hydrolysis is not a feasible strategy. Compared with enzymatic and acid hydrolysis,biotransformation only requires mild conditions as well as simple procedures and results in high yield,so it has great potential for industrial application.Therefore,fermentation in a 5L bioreactor was carried out by the acclimated strain based on the shake flask cultivation,and the operation conditions were optimized.After 20h fermentation at 37℃and air flow rate of 1.5vvm,temperature was increased to 50℃and maintained for 2h.The concentration of resveratrol was 3.4 times higher,reaching the level of the reported spontaneously fermentation for 72h,with the percent conversion of piceid 105%. The investigation about the mechanism of transformation and release of resveratrol and piceid indicates that they exist in herb cells in a free state.Biotransformation of piceid to resveratrol and hydrolysis of cellulose in the cell walls to release the two effective constituents are simultaneously conducted during fermentation.Microwave-assisted aqueous two-phase extraction(MAATPE) was then investigated with ethanol/ammonium sulphate to obtain effective constituents,including piceid,resveratrol and emodin from both raw herb and fermentation broths,and the partition behavior of the products was studied.The aqueous two-phase system consisting of 25%(w/w) ethanol and 21%(w/w) ammonium sulphate was selected to obtain equal yield of piceid,and 1.1 and 1.9 times higher yields of resveratrol and emodin,respectively,from raw herb of P.cuspidatum than that by microwave-assisted extraction(MAE) and heat reflux extraction.Yields of resveratrol and emodin from fermentation broths were 1.2 and 3.3 times higher,respectively, than that by MAE,and 1.6 and 4.2 times higher,respectively,than that by stirring extraction at room temperature.Contents of resveratrol and emodin in the extract of fermentation broths were 1.3 and 2.3 times higher,respectively,than that by MAE,and 1.4 and 2.6 times higher, respectively,than that by stirring extraction at room temperature,while contents of glucose and polysaccharides in the extract were 27%and 79%,respectively,of that by MAE and stirring extraction at room temperature.MAATPE possesses advantages over both MAE and aqueous two-phase extraction,and integrates the two processes into a single step to get higher yields at lower cost.Therefore,it is a potential method for the extraction and purification of target products.Macroporous resin and polyamide column chromatography were finally combined to purify resveratrol from the fermentation broths,and the adsorption dynamics of resveratrol was evaluated.The results show that apparent static adsorptive capacities of resveratrol on macroporous resin and polyamide are 11.70mg/g and 57.9mg/g,respectively,and apparent dynamic adsorptive capacities are 12.47mg/g and 58.98mg/g,respectively.Both of the adsorption isotherms correlate well with the Langmuir type.After macroporous resin and polyamide two-step column chromatography,the purity of resveratrol reached 95.8%,with the yield of 69.8%.Emodin in the culture was also recovered,with the purity of 18.6%and the yield of 84.2%.This method is proved potential for industrial application due to its simple procedure,lower cost as well as high purity and yield of resveratrol.