Brewer's spent grain (BSG), the primary byproduct of the brewing industry, is characterized by its high content of lignocellulosic fibers and proteins, making it a promising raw material to produce high-value bioproducts. In this context, solid-state fermentation (SSF) using filamentous fungi has emerged as a sustainable strategy to promote the partial degradation of the lignocellulosic matrix, thereby increasing the availability of proteins and bioactive compounds. The recovery efficiency of these compounds can be further enhanced through ultrasound-assisted extraction (UAE), a technology that provides higher extraction yields while reducing solvent consumption. Furthermore, the use of solvent mixtures may maximize the extraction of phenolic compounds. Therefore, the valorization of brewer's spent grain through the combination of solid-state fermentation and optimized extraction processes represents an innovative strategy for producing high-value ingredients, contributing to the development of functional foods, the advancement of the bioeconomy, and the consolidation of sustainable biorefineries.

OBJECTIVES: The overall objective of this study was to investigate the effect of solid-state fermentation (SSF), using a mixed culture of Rhizopus oryzae and Trichoderma virens, to enhance the nutritional potential of brewer's spent grain, and how to maximize the extraction of phenolic compounds of the fermented waste. Article 1 – This study aimed to optimize the solid-state fermentation conditions of brewer's spent grain using Rhizopus oryzae and Trichoderma virens to maximize the release of proteins, free amino acids, and phenolic compounds, as well as to evaluate the enzymatic activity and antioxidant capacity of the fermented substrate. Article 2 – This study aimed to investigate solid-state fermentation as a biological pretreatment strategy to enhance the ultrasound-assisted extraction of phenolic compounds from brewer's spent grain by optimizing solvent composition and extraction conditions, as well as to evaluate the phenolic profile and antioxidant activity of the resulting extracts.

MATERIALS AND METHODS: Article 1 – Brewer's spent grain was subjected to solid-state fermentation using Rhizopus oryzae, Trichoderma virens, and a mixed culture of both fungi. Fermentation conditions were optimized using a central composite rotational design to evaluate the effects of moisture content, temperature, and fungal proportion on protein content and antioxidant activity. The optimized conditions were subsequently applied in a kinetic study in which protein content, antioxidant activity (ABTS, DPPH, and FRAP), the phenolic compound profile by UHPLC-MS/MS, and the activities of hydrolytic enzymes (cellulase, endoglucanase, α-amylase, β-amylase, and protease) were determined. At the optimal experimental condition, the free amino acid composition was determined by high-performance liquid chromatography, and the structural changes in the substrate were evaluated by scanning electron microscopy. The results were subjected to statistical analysis to determine the optimal fermentation conditions and the correlations among the evaluated variables. Article 2 – Fermented brewer's spent grain obtained under the optimized solid-state fermentation conditions was used as the raw material for phenolic compound extraction. Initially, different solvents were evaluated for their extraction efficiency, and the most effective ones were selected for optimization using a simplex-centroid mixture design. Subsequently, the effects of ultrasonic power and extraction time on the yield of total phenolic compounds were investigated. The optimized condition was used to characterize the phenolic profile by UHPLC-MS/MS and to determine antioxidant activity using the ABTS and FRAP assays.

RESULTS AND DISCUSSION: Article 1 – Solid-state fermentation proved to be an efficient strategy for the valorization of brewer's spent grain, promoting significant changes in its nutritional and functional composition. In the first study, the optimal fermentation conditions were established at 73.0% moisture, 28.5 °C, and mixed inoculation with Rhizopus oryzae and Trichoderma virens, resulting in an increase in protein content from 20.82% to 23.60%. In addition, antioxidant activity increased by 484.4%, 625.0%, and 376.0%, as determined by the ABTS, DPPH, and FRAP assays, respectively. The results also revealed correlations among enzymatic activity, the release of free amino acids and phenolic compounds, and the increase in antioxidant activity, demonstrating that fermentation promoted biochemical modifications capable of simultaneously improving the nutritional and functional quality of brewer's spent grain. Article 2 – In the second study, the fermented material was used as a biological pretreatment to enhance the ultrasound-assisted extraction of total phenolic compounds (TPC). The evaluation of different solvents demonstrated that the water/acetone mixture at a 1:1 (v/v) ratio showed the highest efficiency for recovering total phenolic compounds compared with the individual solvents, resulting in a 174.0% increase in TPC content. Optimization of the extraction parameters demonstrated that lower ultrasonic power (255 W) favored the preservation of bioactive compounds, whereas longer extraction times 45  promoted higher phenolic compound yields and significant modifications in the phenolic profile of the extracts, as confirmed by UHPLC-MS/MS. These changes resulted in high antioxidant activity and demonstrated the potential of the obtained extracts for biotechnological applications. Collectively, the results demonstrate that integrating solid-state fermentation with ultrasound-assisted extraction constitutes a sustainable and efficient strategy for the valorization of brewer's spent grain, enabling the production of ingredients rich in bioactive compounds with high potential for application in the food, pharmaceutical, and cosmetic industries.

CONCLUSIONS: Therefore, solid-state fermentation using mixed cultures proved to be an efficient strategy for adding value to brewer's spent grain by promoting structural and biochemical modifications that enhanced the production of hydrolytic enzymes, increased the availability of bioactive compounds, and improved the nutritional potential of the substrate. The results demonstrated that optimizing the fermentation conditions, particularly the initial moisture content, plays a decisive role in maximizing the biotechnological utilization of this agro-industrial coproduct. Furthermore, the efficiency of total phenolic compound recovery was directly influenced by both solvent composition and the parameters of ultrasound-assisted extraction, highlighting the importance of integrated optimization of these processing steps. Thus, combining solid-state fermentation with a properly optimized extraction process represents a promising strategy for the valorization of brewer's spent grain, expanding its potential application in the production of functional ingredients, value-added foods, and sustainable biorefinery processes.