Optimization of Solid-State Fermentation Conditions With Mixed Strains Using Box-Behnken Design for the Production of brewers’ Spent Grain Protein

As the most abundant by-product of the brewing industry, the valorization of proteins from brewers’ spent grains (BSG) is critical to reducing processing waste and meeting the growing demand for plant-derived protein globally. However, the applicability of brewers’ spent grain proteins in food manufacturing has been constrained by the high extraction cost and undesirable functionalities. In this research, BSG was used as the substrate to ferment with the mixed strains Bacillus subtilis, Saccharomyces cerevisiae, and Kluyveromyces marxianus and then subjected to protein extraction. The solid-state fermentation (SSF) conditions were optimized using single-factor tests together with Box-Behnken Design to 50% initial moisture content, 10% strain inoculum ratio, fermented at pH 5.8 under 32 °C for 6 days, with the highest soluble protein concentration of 0.678 ± 0.0047 µg/µL (equivalent to bovine serum albumin). Furthermore, proteins were extracted from unmalted raw barley grains and BSG control (with a yield of 0.338 ± 0.016 µg/µL and 0.352 ± 0.015 µg/µL, respectively) and then evaluated for their physiochemical characterizations as a comparison of solid-state fermented proteins. The physicochemical attributes of all the intact proteins were similar, except that the fermented samples showed a smaller molecular mass on the SDS-PAGE gel. In conclusion, these results indicated that SSF is a feasible strategy for the bioconversion of BSG into valuable food ingredients as it enhances protein extraction efficiency while reducing the production cost, thereby being beneficial to alleviate the associated environmental pollution and to broaden its possibilities for further integration into the circular economy.