Sequential enzymatic pretreatment and Saccharomyces boulardii fermentation of cactus cladode biomass for bioactive food ingredients and cell culture serum alternatives.

Novel biomass valorization technologies are critical for sustainable food production systems that prioritize health-promoting functional properties of foods. Sequential enzymatic delignification and saccharification (DL + SC) of cactus (Opuntia ficus-indica) cladode biomass (CCB), followed by Saccharomyces boulardii (SB) fermentation, was investigated for biomass processing. CCB delignification using Aspergillus laccases was optimized using a central composite design. At optimal conditions (35.95 °C, pH 5.55, 37.68 laccase Myceliophthora units/g dry CCB, 6 h 56 min reaction time), a 59.39 % reduction in acid-insoluble lignin concentration was achievable. Pretreated CCB was saccharified with Trichoderma reesei cellulase (10 FPU/g dry CCB, 50 °C, 48 h). DL + SC-pretreated CCB exhibited higher reducing sugar content than independent treatments, improving SB growth (68.11 % relative to yeast malt broth). Effects of pretreatment and fermentation of CCB on the bioactivities were evaluated in a benzo(a)pyrene-induced oxidative stress and DNA damage model of Caco-2 cells. Phenolic-rich extracts (50-100 µg/mL) of DL + SC-pretreated and fermented CCB significantly alleviated oxidative stress and mitigated DNA damage in Caco-2 cells compared to individual pretreatments. Protein isolated from DL + SC-pretreated and fermented CCB was evaluated as a candidate serum substitute and partially supported ILMC proliferation (54.65 ± 1.70 % relative to 10 % fetal bovine serum medium), compared with the fermented YM/SB control (62.18 ± 2.24 %). These findings demonstrate that DL + SC pretreatment of CCB combined with SB fermentation can produce bioactive food ingredients and candidate protein-rich growth supplements for cell culture applications. However, bioactivity and serum replacement evidence remains partial, in vitro, and restricted to a single cell line.