In this study, the general goal is to optimize the production of cell-free culture extract (CFC) containing bioactive peptides by co-cultivation of Limosilactobacillus fermentum and Saccharomyces cerevisiae in whey sludge bed and to investigate its biological properties. Temperature, time, and type of inoculation were considered as variables to optimize wet biomass production using the response surface method (RSM), and tests of ACE inhibitory activity, antioxidant activity, antimicrobial activity, proteolytic activity, and cell viability were performed on CFC prepared from the optimal treatment. The results of the central composite design (CCD) showed that the highest wet biomass production occurred at 34.86 °C with simultaneous inoculation for 43.1 h. The results obtained from CFC tests show the highest antimicrobial activity against Staphylococcus aureus and the highest DPPH radical activity of about 79.89 %. The value of IC50 in cytotoxicity assay against HT-29 cancer cells was 80.17 mg/mL. All rights reserved, Elsevier.