Enhanced Biosynthesis of Phenolics and Antioxidants by Marine Endophyte Penicillium rubens: Optimization and Bioactivity Evaluation

Marine-derived endophytic fungi have long been recognised as valuable sources of therapeutic metabolites. In the present study, the endophyte Penicillium rubens strain PrubVJ was studied for optimised production of bioactive compounds using Response Surface Methodology (RSM). Experimental data predicted that incubation time was identified as the most influential variable, with maximum protein yield (190 mg/L) observed at day 20, antioxidant activity (74%) at 10–15 days and total phenolics (1,724 ± 19 µg GAE g⁻¹ DW) at day 25. The optimal fermentation conditions (25°C, pH 3.6, 4 g/L salinity, 10 days incubation) with a desirability of 0.92, yielding 158 ± 1.325 mg/L protein, 1,798 ± 18 µg GAE g⁻¹ DW phenolics, and 84.9% antioxidant activity were predicted from the RSM model. Experimental validation under these conditions achieved a desirability of 0.92, with 190 mg/L protein, 1,724 µg phenolics, and 74% antioxidant activity, confirming the reliability and robustness of the model. The developed quadratic models exhibited strong statistical reliability with stochastic parameters such as F-value, p-value and other factors such as R ² , adjusted R ² and predicted R ² in all three responses was reported to be above 80%, validating model adequacy. Experimental validation at the predicted optimum achieved a desirability of 0.92, confirming excellent agreement between predicted and observed outcomes. Collectively, these findings establish P. rubens strain PrubVJ as a promising microbial platform, providing a reliable framework for optimising fungal fermentation. This study recommends the optimized strategy for large-scale pharmaceutical bioprocessing of fungal metabolites, to reduce production costs and accelerate the discovery of novel bioactive metabolites with pharmaceutical relevance, particularly for anticancer, antimicrobial, and antioxidant applications.