The bakery industry generates substantial food waste, leading to global environment pollution and economic losses. However, these by-products contain valuable bioactive compounds, such as melanoidins, which are brown pigmented compounds with significant antioxidant, antimicrobial and anti-inflammatory properties, making them ideal functional ingredient. This study aimed to evaluate the optimal enzymatic extraction of melanoidins from bread crust using different proteolytic enzymes: pronase E, serine-endo-protease (SP), metalloenzyme protease (MP) and endoprotease papain enzyme (EP). While the yield of melanoidins from MP and EP was lower than that of pronase E, SP yield was significantly higher. No cytotoxicity of bioaccessible, gastrointestinal and colonic fractions was observed in the cell lines Caco-2, HUVEC and SHSY5Y. The antioxidant properties and ability to stabilize the hydroxyl radical were characterized in bioaccessible fractions of the four melanoidins extracts. Moreover, melanoidins positively influenced the intestinal microbiota by increasing beneficial bacteria like Bifidobacterium and Lactobacillus, while reducing non-beneficial bacteria such as Bacteroides spp. and Clostridium leptum, with particularly significant results for SP and EP enzyme extracts. In conclusion, SP and EP enzymes appear to be viable alternatives to pronase E for obtaining melanoidin extracts as functional ingredients. These enzymes offer potential cost advantages and contribute to the sustainable utilization of bakery by-product melanoidins in various food applications. Implementing these alternatives could help to reduce food waste, environmental pollution and economic losses in the bakery industry while promoting the use of valuable bioactive compounds.
