Valorisation of stale bread into protein-rich biomass: an optimized <i>Rhizopus oryzae</i> fermentation strategy and characterization study

Bread is one of the most widely consumed and most wasted foods worldwide. It contains essential nutrients; bread provides an environment for microbial growth. This study aims to maximize biomass and protein production by Rhizopus oryzae through fermentation optimization. The RNA concentration of the biomass produced under optimal conditions was quantified. The cytotoxicity was evaluated in HT-29, HUVEC, and HepG2. A dilute acid-heat pre-treatment, conducted with 2.5% H2SO4 (v v-1) and 5% (w v-1) stale bread at 121°C for 2 min (4.83 g l-1 fermentable sugars, 0.86 mg 100 ml-1 HMF). Then, enzymatic hydrolysis of the pre-treated substrate increased fermentable sugar, with an optimum enzyme concentration of 0.05% and a hydrolysis time of 2 h. Under the optimized fermentation conditions (35°C, 3 days, 200 rpm, and pH 4.5), a biomass yield of 7.06 g l-1 and a protein content of 36.76% were obtained. The RNA content of biomass was determined to be within 0.24-0.35%. The absence of adverse effects on cellular proliferation and growth indicates the biocompatibility of the produced biomass. The results suggest that Rhizopus oryzae cultivated on stale bread is a promising candidate for functional food applications with enhanced protein content.