Optimization of culture condition of potent strain Phormidium retzii for the optimal production of stigmasterol- an acetylcholinesterase inhibitor using Design of Experiments software

Despite the availability of synthetic acetylcholinesterase (AChE) inhibitors for Alzheimer’s Disease (AD), they often possess side effects raising urgent need for natural alternatives providing AChE inhibition with minimal adverse effects. Research has increasingly focused on bioactive products from microalgae, with Phormidium retzii showing promise as an AChE inhibitor, offering novel AD treatment. The research aimed to optimize culture conditions to produce stigmasterol by Phormidium retzii using Design of Experiments (DoE) software. P. retzii was cultivated at pilot scale, with biomass collected, shade-dried, subjected to extraction for AChE inhibition testing until the death phase. The extract containing stigmasterol from 11th day showed maximum inhibition (81.03±0.23%). Other extracts showed minimal or no inhibition, and the culture reached death phase on 17th day. Applying N-1 technique, key components of BG11 media for stigmasterol production were defined as sodium nitrate, magnesium sulphate, sodium carbonate and dipotassium hydrogen phosphate. Varying these components in different ratios yielded maximal AChE inhibition (79.43±0.81%) in media containing K2HPO4 (0.04 g/L), NaNO3 (1.5 g/L), MgSO4 (0.075 g/L) and Na2CO3 (0.06 g/L). The optimum light intensity was 2000 lux. Results were input into software, resulting eight experimental runs with ±1 values of each component. AChE inhibitions were determined for all runs and fed into the software. Optimum culture conditions for optimal stigmasterol production were determined as K2HPO4 (0.06 g/L), NaNO3 (2 g/L), MgSO4 (0.05 g/L) and Na2CO3 (0.04 g/L) at pH 8 with 2000 lux. Under these conditions, on 11th day, dried biomass was 1.22 g with AChE inhibition of 84.36±0.02%.