Biovalorisation of brewer’s spent grain by solid-state fermentation: xylanase production by Thermomyces lanuginosus and compositional upgrading of fermented biomass

Abstract

Brewer’s spent grain (BSG), the major solid by-product of the brewing industry, is a nutrient-rich lignocellulosic biomass whose efficient and reproducible valorisation is challenged by its recalcitrant structure and pronounced compositional heterogeneity and batch-to-batch variability, arising from differences in cereal type and cultivar, type and proportion of malts and adjuncts used, beer style, and malting and mashing conditions. Unlike previous BSG-based fungal solid-state fermentation (SSF) studies that have generally focused on either enzyme production or compositional modification of the fermented solid, this study integrates xylanase production, partial purification and hydrolytic evaluation with characterisation of the resulting fermented BSG within a single SSF-based valorisation framework. Xylanase activity reached a maximum of (16.47 ± 1.04) kU g⁻¹ dry matter (DM) after 10 days of SSF. The crude enzyme extract was freeze-dried and subsequently partially purified by ammonium sulphate precipitation followed by dialysis, resulting in a 2.91-fold increase in specific activity and an activity recovery of 39.48%. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis revealed a major protein band with an apparent molecular mass of approximately 22 kDa in the partially purified preparation. The crude and partially purified enzyme preparations were tested as biocatalysts in the hydrolysis of BSG, sugar beet pulp, and hemp oil cake, with hydrolytic performance depending on both the substrate and the enzyme preparation. SSF resulted in a 1.33-fold relative enrichment of crude protein on a dry-matter basis and reduced the relative lipid content. The concentrations of the analysed contaminants in both raw and fermented BSG were below the relevant (European Union) EU maximum, guidance, or indicative values. These findings represent a preliminary chemical safety assessment and do not establish the overall safety or nutritional suitability of fermented BSG as a feed ingredient. Overall, these findings support the potential of T. lanuginosus based SSF for BSG valorisation through two complementary routes: xylanase production and the generation of fermented biomass with an increased relative crude protein concentration.

Keywords

Brewer’s spent grain / Thermomyces lanuginosus / Solid-state fermentation / Xylanase / Protein- enriched animal feed

References

[1] AOACAOAC Official Method 942.05. : Official Methods of Analysis of AOAC INTERNATIONAL, 199516Arlington (VA)AOAC International

[2] AOACAOAC Official Method 945.16. Official Methods of Analysis of AOAC INTERNATIONAL, 200518Gaithersburg (MD)AOAC International

[3] Barrera-León M, Terán-Cabanillas E, Avena-Bustillos RDJ, Cárdenas-Torres FI, Amézquita-López BA, Gómez-Favela MA, Alemán-Hidalgo DM, Arias-Gastélum M. Transformation of Brewer’s Spent Grain Through Solid-State Fermentation: Implications for Nutrition and Health. Recycling, 2025, 105170.

[4] Behera SS, Ray RC. Solid state fermentation for production of microbial cellulases: recent advances and improvement strategies. Int J Biol Macromol, 2016, 86: 656-69.

[5] Botella C, Hernandez JE, Webb C. Dry weight model, capacitance and metabolic data as indicators of fungal biomass growth in solid state fermentation. Food Bioprod Process, 2019, 114: 144-53.

[6] Božinović M, Sokač T, Šalić A, Dukarić AM, Tišma M, Planinić M, Zelić B. Standardization of 3, 5-dinitrosalicylic acid (DNS) assay for measuring xylanase activity: detecting and solving problems. Croat J Food Sci Technol, 2023, 15215162.

[7] Bucić-Kojić A, Planinić M, Šibalić D, Tišma M. Release of fermentable sugars from corn silage: The effect of biological pretreatment. Chem Biochem Eng Q, 2023, 37424553.

[8] Castro LEN, Colpini LMS. All-around characterization of brewers’ spent grain. Eur Food Res Technol, 2021, 24712301321.

[9] Dahiya S, Rapoport A, Singh B. Biotechnological Potential of Lignocellulosic Biomass as Substrates for Fungal Xylanases and Its Bioconversion into Useful Products: A Review. Fermentation, 2024, 10282.

[10] dos Santos AL, e Silva AD, Morais NW, dos Santos AB. Brewery Spent Grain as sustainable source for value-added bioproducts: opportunities and new insights in the integrated lignocellulosic biorefinery concept. Ind Crops Prod, 2023, 206: 117685.

[11] EFSA. Panel on Contaminants in the Food Chain (CONTAM)Animal dietary exposure in the risk assessment of contaminants in feed. EFSA J, 2024, 227e8858.

[12] Eliopoulos C, Arapoglou D, Chorianopoulos N, Markou G, Haroutounian SA. Conversion of brewers’ spent grain into proteinaceous animal feed using solid state fermentation. Environ Sci Pollut Res, 2022, 2920295629.

[13] European CommissionDirective 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. Off J Eur Communities, 2002, L140: 10-22

[14] European CommissionRegulation (EC) 183/2005 of the European Parliament and of the Council of 12 January 2005 laying down requirements for feed hygiene. Off J Eur Union, 2005, L35: 1-22

[15] Farhan M, Hasani IW, Khafaga DSR, Ragab WM, Ahmed Kazi RN, Aatif M, Muteeb G, Fahim YA. Enzymes as catalysts in industrial biocatalysis: advances in engineering, applications, and sustainable integration. Catalysts, 2025, 159891.

[16] Gaffney M, Doyle S, Murphy R. Optimization of Xylanase Production by Thermomyces lanuginosus in Solid State Fermentation. Biosci Biotechnol Biochem, 2009, 731226404.

[17] Gauterio GV, da Silva LGG, Hübner T, da Rosa Ribeiro T, Kalil SJ. Xylooligosaccharides production by crude and partially purified xylanase from Aureobasidium pullulans: Biochemical and thermodynamic properties of the enzymes and their application in xylan hydrolysis. Process Biochem, 2021, 104: 161-70.

[18] Gerber S, Frost SC, Walker AM, Kaplan DL. Advancing solid-state fermentation with culinary fungi for nutrient-dense, minimally processed, whole-foods and resilient food systems—a narrative review. Front Nutr, 2026, 13: 1798618.

[19] Global Market Insights. Enzyme market size – industry analysis and forecast 2026–2035. 2026. https://www.gminsights.com/industry-analysis/enzymes-market Accessed 4 May 2026.

[20] Goering HK, Van Soest PJ. Forage fiber analyses (Apparatus, Reagents, Procedures and Some Application), 1970, Washington (DC): Agricultural Research Service

[21] Grand View Research. Europe enzymes market size and outlook 2026–2033. 2025. https://www.grandviewresearch.com/horizon/outlook/enzymes-market/europe. Accessed 24 Apr 2026.

[22] Green AA, Hughes WL. Colowick SP, Kaplan NO. Protein fractionation on the basis of solubility in aqueous solutions of salts and organic solvents. Methods Enzymol, 1955, New York: Academic, 67-901

[23] Hejna A, Aniśko-Michalak J, Skórczewska K, Barczewski M, Sulima P, Przyborowski JA, Cieśliński H, Marć M. Brewers’ Spent Grain from Different Types of Malt: A Comprehensive Evaluation of Appearance, Structure, Chemical Composition, Antimicrobial Activity, and Volatile Emissions. Molecules, 2025, 30132809.

[24] Hoque M, Devi KTR. Agro-industrial waste management: solid-state fermentation for biomass conversion and valorisation to value-added products. Syst Microbiol Biomanuf, 2025, 5: 990-9.

[25] Hu J, Arantes V, Saddler JN. The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: Is it an additive or synergistic effect? Biotechnol. Biofuels, 2011, 4: 36.

[26] Karp SG, de Oliveira Penha R, Soccol VT, Weber MZ, Fusaro T, Aguiar AB, Thuma SS, Biagini G, Guieseler BS, Manzoki MC, Soccol CR. Circular economy approaches in the production of alternative proteins. Syst Microbiol Biomanuf, 2026, 6243.

[27] Kim BS, Jeong BM, Kim D, Kim SR, Kim IJ. Biochemical Characterization of a GH11 Xylanase from Xylanase-Producing Trichoderma citrinoviride. Appl Biochem Biotechnol, 2025, 1976392945.

[28] Kocabaş DS, Güder S, Özben N. Purification strategies and properties of a low-molecular weight xylanase and its application in agricultural waste biomass hydrolysis. J Mol Catal B Enzym, 2015, 115: 66-75.

[29] Komarova MI, Semenova MV, Volkov PV, Shashkov IA, Rozhkova AM, Zorov IN, Kurzeev SA, Satrutdinov AD, Rubtsova EA, Sinitsyn AP. Efficient Hydrolysis of Sugar Beet Pulp Using Novel Enzyme Complexes. Agronomy, 2025, 151101.

[30] Leadbeater DR, Setchfield AT, Dowle AA, Bruce NC. Comparative exo-proteomics of solid and submerged state fermentation using the lignocellulose degrading Ascomycete Parascedosporium putredinis NO1. Bioresour Technol, 2025, 435: 132864.

[31] Liguori R, Pennacchio A, Vandenberghe LPS, De Chiaro A, Birolo L, Soccol CR, Faraco V. Screening of Fungal Strains for Cellulolytic and Xylanolytic Activities Production and Evaluation of Brewers’ Spent Grain as Substrate for Enzyme Production by Selected Fungi. Energies, 2021, 14154443.

[32] Liu M, Huang S, Yan P, Yin H, Yu J, Wu X, Wang L. Effective Degradation of Brewer Spent Grains by a Novel Thermostable GH10 Xylanase. Appl Biochem Biotechnol, 2024, 1968483748.

[33] Lock TJ, Mah SH, Lai ZW. Versatile Applications of Brewer’s Spent Grain: Solid-State Fermentation and Nutritional Added Value. Appl Biochem Biotechnol, 2024, 1968550832.

[34] Lojananan N, Cheirsilp B, Intasit R, Billateh A, Srinuanpan S, Suyotha W, Boonsawang P. Successive process for efficient biovalorization of Brewers’ spent grain to lignocellulolytic enzymes and lactic acid production through simultaneous saccharification and fermentation. Bioresour Technol, 2024, 397: 130490.

[35] Marinho GO, Nogueira EA, Pasin TM, Oliveira TBD, Roa JPB, Nelson DL. An Environmentally Safe Production of Xylanases by Fusarium sp. EA 1.3.1 Using Agroindustrial Residues: Biochemical Characterization and Potential Applications. Asian J Biochem Genet Mol Biol, 2023, 14: 11-26.

[36] Miller GL. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem, 1959, 3134268.

[37] Mitri S, Salameh SJ, Khelfa A, Leonard E, Maroun RG, Louka N, Koubaa M. Valorization of brewers’ spent grains: Pretreatments and fermentation, a review. Fermentation, 2022, 8250.

[38] Moran-Aguilar MG, Costa-Trigo I, Calderón-Santoyo M, Domínguez JM, Aguilar-Uscanga MG. Production of cellulases and xylanases in solid-state fermentation by different strains of Aspergillus niger using sugarcane bagasse and brewery spent grain. Biochem Eng J, 2021, 172: 108060.

[39] Naik AS, Brugiere M, Dunne K, Sorohan M, Grasa L, Javed B. Protease hydrolysed brewers’ spent grain bioactive extracts for applications in human health. Syst Microbiol Biomanufacturing, 2024, 43106275.

[40] Official Method AOACAOAC. 2001.11. In: Official Methods of Analysis of AOAC INTERNATIONAL. 18th ed. Gaithersburg (MD): AOAC International; 2005a.

[41] Oliveira AC, Amorim GM, Azevêdo JAG, Godoy MG, Freire DM. Solid-state fermentation of co-products from palm oil processing: production of lipase and xylanase and effects on chemical composition2018

[42] Osei ED, Naik AS, Kinsella G, Delaney T. Potential of Brewer’s spent grain bioactive fractions as functional ingredients for companion and farm animal foods – A review. Appl Food Res, 2025, 52101138.

[43] Pal A, Khanum F. Purification of xylanase from Aspergillus niger DFR-5: Individual and interactive effect of temperature and pH on its stability. Process Biochem, 2011, 46487987.

[44] Pandey A, Soccol CR, Mitchell D. New developments in solid state fermentation: I-bioprocesses and products. Process Biochem, 2000, 3510115369.

[45] Perwez M, Al Asheh S. Valorization of agro-industrial waste through solid-state fermentation: Mini review. Biotechnol Rep, 2025, 45: e00873.

[46] Planinić M, Zelić B, Čubel I, Bucić-Kojić A, Tišma M. Corn forage biological pretreatment by Trametes versicolor in a tray bioreactor. Waste Manag Res, 2016, 3488029.

[47] Raheja Y, Gaur P, Islam T, Chaurasia AK, Gaur VK, Chadha BS. Advancement in lignocellulolytic enzyme production: tailored strategies to overcome challenges in biomass hydrolysis. Syst Microbiol Biomanuf, 2025, 5394868.

[48] Rasmussen ML, Shrestha P, Khanal SK, Pometto ALIIIvan Leeuwen JH. Sequential saccharification of corn fiber and ethanol production by the brown rot fungus Gloeophyllum trabeum. Bioresour Technol, 2010, 10110352633.

[49] Ribarova F, Atanassova M, Marinova D. Total phenolics and flavonoids in Bulgarian fruits and vegetables. JU chem Metal, 2005, 40325560

[50] Rudjito RC, Jimenez-Quero A, Munoz MDCC, Kuil T, Olsson L, Stringer MA, Krogh KBRM, Eklöf J, Vilaplana F. Arabinoxylan source and xylanase specificity influence the production of oligosaccharides with prebiotic potential. Carbohydr Polym, 2023, 320: 121233.

[51] Samantaray B, Behera RR, Mishra RR, Thatoi H. In silico studies of fungal xylanase enzymes: structural and functional insight towards efficient biodegradation of lignocellulosic biomass. Syst Microbiol Biomanuf, 2024, 43117492.

[52] Šelo G, Planinić M, Tišma M, Grgić J, Perković G, Koceva Komlenić D, Bucić-Kojić A. A Comparative Study of the Influence of Various Fungal-Based Pretreatments of Grape Pomace on Phenolic Compounds Recovery. Foods, 2022, 11111665.

[53] Shrivastava S, Shukla P, Mukhopadhyay K. Purification and preliminary characterization of a xylanase from Thermomyces lanuginosus strain SS-8. 3 Biotech, 2011, 1: 255-9.

[54] Šibalić D, Šalić A, Tušek AJ, Sokač T, Brekalo K, Zelić B, Tran NN, Hessel V, Tišma M. Sustainable Production of Lipase from Thermomyces lanuginosus: Process Optimization and Enzyme Characterization. Ind Eng Chem Res, 2020, 59482114454.

[55] Silva LC, França IAC, Kupski L, Barros BCB, Madrona GS. Mixed-strain fermentation enhances protein content, phenolic composition, antioxidant capacity, and free amino acid content in brewer’s spent grain. Biocatal Agric Biotechnol. 2026;104178. https://doi.org/10.1016/j.bcab.2026.104178.

[56] Škerget M, Kotnik P, Hadolin M, Hraš AR, Simonič M, Knez Ž. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem, 2005, 8921918.

[57] Soltero-Sánchez C, Romero-Borbón E, Ortega-de la Rosa ND, Camacho-Ruiz MA, Córdova J. Enhancing Xylanase and Cellulase Production by Two Locally Isolated Fungal Strains Under Solid-State Fermentation of Water Hyacinth and Sugarcane Bagasse. Fermentation, 2025, 1110578.

[58] Sonia KG, Chadha BS, Saini HS. Sorghum straw for xylanase hyper-production by Thermomyces lanuginosus (D2W3) under solid-state fermentation. Bioresour Technol, 2005, 961415619.

[59] Statista. Beer production worldwide from 1998 to 2024 (in billion hectoliters). 2024. https://www.statista.com/statistics/270275/worldwide-beer-production/ Accessed 26 Feb 2026.

[60] Sulis DB, Lavoine N, Sederoff H, Jiang X, Marques BM, Lan K, Cofre-Vega C, Barrangou R, Wang JP. Advances in lignocellulosic feedstocks for bioenergy and bioproducts. Nat Commun, 2025, 1611244.

[61] Tišma M, Bucić-Kojić M, Planinić M. Bio-based products from lignocellulosic waste biomass: a state of the art. Chem Biochem Eng Q, 2021, 35213956.

[62] Tišma M, Žnidaršič-Plazl P, Šelo G, Tolj I, Šperanda M, Bucić-Kojić A, Planinić M. Trametes versicolor in lignocellulose-based bioeconomy: State of the art, challenges and opportunities. Bioresour Technol, 2021, 330: 124997.

[63] Wang J, Huang Z, Jiang Q, Roubik H, Xu Q, Gharsallaoui A, Cai M, Yang K, Sun P. Fungal solid-state fermentation of crops and their by-products to obtain protein resources: The next frontier of food industry. Trends Food Sci Technol, 2023, 138: 628-44.

[64] Yeboah MK, Söffker D. Consolidated bioprocessing of lignocellulosic biomass: A review of experimental advances and modeling approaches. Bioresour Bioprod, 2026, 214.

[65] Zeko-Pivač A, Tišma M, Žnidaršič-Plazl P, Kulisic B, Sakellaris G, Hao J, Planinić M. The potential of brewer’s spent grain in the circular bioeconomy: State of the art and future perspectives. Front Bioeng Biotechnol, 2022, 10: 870744.

[66] Zeko-Pivač A, Bošnjaković A, Planinić M, Parlov Vuković J, Novak P, Jednačak T, Tišma M. Improvement of the Nutraceutical Profile of Brewer’s Spent Grain after Treatment with Trametes versicolor. Microorganisms, 2022, 10112295.

[67] Zhang G, Li Z, Chen G, Zhang L, Cai W, Deng S, Zhang H, Wu L, Li H, Liu H. Purification and characterization of the low molecular weight xylanase from Bacillus cereus L-1. Braz J Microbiol, 2023, 54: 2951-9.

Funding

European Regional Development Fund(PK.1.1.12.0097)

Rights & permissions

Jiangnan University