Valorization of stale bread into protein-rich biomass: an optimized Rhizopus oryzae fermentation strategy and characterization study.

2026-03-14
Letters in applied microbiology 79(4)

PubMed: 41830919
DOI: 10.1093/lambio/ovag034

Study Design

Methods: Fermentation optimization of Rhizopus oryzae on stale bread; dilute acid-heat pretreatment with 2.5% H2SO4 and 5% stale bread at 121°C for 2 min, followed by enzymatic hydrolysis and optimized fermentation conditions (35°C, 3 days, 200 rpm, pH 4.5); cytotoxicity evaluated in HT-29, HUVEC, and HepG2.

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 pretreatment, 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 hydroxymethyl furfural). Then, enzymatic hydrolysis of the pretreated 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 R. oryzae cultivated on stale bread is a promising candidate for functional food applications with enhanced protein content.

Research Insights

Supplement	Dose	Health Outcome	Effect Type	Effect Size	Source
Rhizopus oryzae	—	Improved Cell Compatibility	Neutral	Small	View source The absence of adverse effects on cellular proliferation and growth indicates the biocompatibility of the produced biomass.