Optimization of Bacoside a Production in Cell Suspension Culture of Bacopa monnieri for Scale-Up using Stirred Tank Reactor

Abstract

Bacopa monnieri(Brahmi), an amphibious plant growing in tropical climate, has been reported to be a source of the secondary metabolite,bacoside A,known for the regeneration of nerve cells in the brain.As a result of its excessive demand in the pharmaceutical industry, cultivation of this plant has gained wide popularity.In order to augment the supply of this drug from natural origin,alternative plant cell culture techniques need to be developed.Field-grown plantlets of high-yielding variety of Bacopa monnieri were utilized in this study to develop high-yielding callus cultures for the mass production of bacoside A.The explant type was optimized for utmost callus biomass production. Substrate inhibition kinetics was studied to determine the inhibitory concentrations of major limiting substrates. Using response surface methodology,the phytohormone concentrations were optimized for the improved synthesis of biomass and bacoside A.The optimized factors were utilized to study the batch and repeated-batch cultivation of Bacopa monnieri in stirred tank bioreactor.A combination of bavistin treatment (0.5% solution for 5 minutes)and sodium hypochlorite treatment (1% solution for 5 minutes)was found to effectively sterilize the field-grown explants for invitro culture development.Rooting of the shoots was induced in MS medium containing 1 mg/l IAA in 7 days. Callus was induced from leaf explants in MS medium containing 0.5 mg/l NAA and 0.1 mg/l BAP within 10 days. Leaf explants were found to be the most ideal explant for callusinduction. 1.0 mg/l NAA and 0.1 mg/l BAP were the optimum concentration for the induction of callus from leaf explant; and 0.2 mg/l NAA and 1.2 mg/l BAP were the optimized concentrations for the maintenance of the callus. The growth of the cells in suspension culture was observed to get inhibited at 120 g/l sucrose (carbon source) and 1.25 g/l KH2PO4 (phosphate source). Using the optimized conditions,batch cultivation of Bacopa monnieri was carried out in a stirred tank reactor. 18.97 g/l DW biomass and 18.05 mg/g DW bacoside A was produced by using cell suspension as inoculum for batch cultivation in stirred tank reactor in 6 days, whereas, 3.04 g/l DW biomass and 27.36 mg/g DW bacoside A was produced by using pre-established callus culture as inoculum in the reactor in 9 days. Repeated-batch cultivation of Bacopa monnieri in
stirred tank reactor yielded 1.40 g/l DW biomass in 13 days. Hence this study outlines an economical alternative method for the propagation and conservation of Bacopa monnieri. Also, it provides commercially feasible method for the large scale production of bacoside A.