Isolation, Characterization and Process Optimization of Resveratrol Production from Endophytic Fungi

Abstract

The present study focusses on the diversity of resveratrol producing fungal endophytes associated with different wine grape varieties grown in India. A total of 145 endophytic isolates were recovered of which 55 were from Pune region, 54 from Nashik while 26 were from Lucknow and 9 from Bengaluru. Only 30% isolates exhibited extracellular resveratrol production in the preliminary screening. However only 14 fungal endophytes exhibited significant resveratrol production in the range of 4.4g/L-89.1g/L in liquid cultures which was evaluated by HPLC analysis. Four potent resveratrol producing endophytes isolated were #12VVLPM; #4(P)VVLNM; #22(P) VVLPM and #19VVLPM. #12 VVLPM was identified to be Arcopilus aureus, while #22(P) VVLPM was identified to be Xylaria psidii. Both Arcopilus aureus and Xylaria psidii have so far not been reported to be resveratrol producers. The other two isolates viz. #4(P)VVLNM and #19VVLPM are generalists identified as Fusarium solani and Fusarium equiseti. Highest resveratrol production, 89.1 g/l was recorded in #12VVLPM identified as Arcopilus aureus. The fungal resveratrol exhibited a strong anti-oxidant activity in all the tested assays.The endophytic isolate #12VVLPM exhibited the best activity similar to the standards in the entire anti-oxidant assay performed during the present course of investigations. Yet another important result was the antimycotic activity against different plant as well as human pathogenic fungi tested. The highest inhibition was observed against B. cinerea (57.8± 0.8%) followed by Cercospora beticola (ATCC24888) by extracts of #12VVLPM while the least inhibition was observed in #22(P) VVLPM and #4(P) VVLNM against same pathogen. Among the resveratrol producing endophytic isolates #12VVLPM exhibited the best inhibitory activity against all the cultures tested in the panel. The fungal resveratrol was obtained in pure form ethnolic extract by eluting it in a column with mobile phase of MeOH: DCM :: 1.75:98.25. Fungal resveratrol (25 mg) was observed pure by TLC system 5% MeOH: DCM and concentrated by rotary evaporator. The homogeneity of the purified fungal resveratrol was confirmed by HPLC, which exhibited a single, symmetrical peak at Rt 3.36 min on C18 reverse phase column similar to that of standard resveratrol. For further confirmation of the presence of fungal resveratrol purified from the spent broth of Arcopilus aureus, 1H-NMR, 13C-NMR. MS and HR-MS, FTIR analysis was carried out which revealed the presence and purity of fungal resveratrol. Optimization of different physic-chemical and nutritional parameters affecting resveratrol production by ‘one-variable-at-a-time’ approach resulted in the production 1.22 fold increases of (109.61a ± 0.7 µg/ml resveratrol from 89.1a ± 0.08 µg/ml from initial yield of resveratrol. The interactions of the most influential factors obtained by one-variable-at-a-time method were further optimized using a statistical approach, response surface methodology (RSM). This finally resulted in 1.49 fold increase production of resveratrol (133.5 µg/ml) yield of resveratrol. After optimization of various physiological and nutritional parameters using one variable at a time and RSM using shake flasks, the optimized conditions were put to test for epigenetic modification. This finally resulted in 1.85 fold increase yield of resveratrol i.e. 165.0 µg/ml. The present study provides prime information on a novel source for the fermentative production of the billion dollar drug resveratrol by the endophytic fungus Arcopilus aureus. Fungal resveratrol was found to be as efficient as that of standard resveratrol which paves new way for exploitation of these bio actively potent endophytic fungi for industrial bioprocess. This study a step forward to proving that endophytic fungi are able to produce billion dollar drugs which are highly demandable due to its multifarious application and paves a new way for replacement of conventional sources of these molecules by serving as alternative.