Response Surface MethodologyBased Optimization of Rusip Fermentation Conditions and Characterization of Bioactive Peptide Fractions with Antioxidant and Antimicrobial Properties

Abstrak

Optimization of raw materials and processing conditions in rusip, a traditional fermented anchovy, has the potential to enhance its bioactive properties. This study consisted of two stages, including optimization of rusip processing parameters (salt, sugar, and fermentation time) using Response Surface Methodology (RSM) based on a Central Composite Design to enhance bioactivity, and physicochemical and microbiological characterization, and bioactivity evaluation of the rusip optimized formulation, including ultrafiltration-based fractionation of its water-soluble extract. The rusip optimized formulation was determined as 5% sugar, 18% salt, and 12 days of fermentation with antioxidant activity of 60.50% (DPPH assay) and inhibition zones of 4.13 mm against Escherichia coli and 3.27 mm against Staphylococcus aureus (agar well diffusion assay), with model validation ranging from 95.86% to 98.03%. Subsequently, the optimized rusip formula was analyzed for physicochemical and microbiological characteristics. The water-soluble extract of rusip optimized formulation was separated using an ultrafiltration membrane, which showed that the <3kDa fraction demonstrated the highest antioxidant (46.09%) and antimicrobial activity (4.9 mm against E. coli and 4.7 mm against S. aureus). Proteolytic enzyme sensitivity analysis showed that exposure to enzymes significantly reduced and eliminated the bioactivity of the rusip fraction, thus indicating the compounds responsible for the bioactivity were proteins or peptides. Qualitative HPLC profiling showed the <3 kDa fraction exhibited an intense peak at 214 nm with early retention time, suggesting the presence of abundant low-molecular-weight peptide compounds. This study provides a scientifically optimized formulation and production method for rusip, revealing its potential as a functional food with natural antioxidant and antimicrobial peptides.