Fermentation of brewer’s spent grains by Pleurotus ostreatus: process optimization by response surface methodology

Abstract

To achieve high-value utilization of brewer’s spent grains and to produce protein-rich food ingredients, brewer’s spent grains were upcycled by a liquid fungal fermentation and the fermentation conditions were optimized. Brewer’s spent grains (BSG) represent the most abundant by-product of the brewing industry. Although BSG are food-grade, their direct use in food is limited because of sensory changes of the products. In this study, screening experiments revealed that black beer spent grains could be efficiently upcycled by submerged fermentation with the edible fungus Pleurotus ostreatus. The conditions of the fermentation of BSG with P. ostreatus were optimized using response surface methodology, including the parameters substrate concentration, inoculum volume, initial pH value, and temperature. As no separation between BSG and mycelium was possible after the fermentation, ergosterol was used as a biomarker to determine the fungal growth. As optimum conditions, a BSG concentration of 17 g L^{− 1} dry matter, an inoculum volume of 4.4% (v/v), an initial pH of 9.3 and a temperature of 30 °C were identified. The fermentation with P. ostreatus increased the true protein content of the biomass compared to the spent grains and reduced the total fat content. The biological value was increased from 88 to 94 (reference standard is whole chicken egg with a biological value of 100). Tryptophan and lysine were limiting in the non-fermented spent grains, while after fermentation, the chemical score of lysine increased from 78 to 92, and tryptophan wasn’t limiting anymore. Fermentation by P. ostreatus enhanced the nutritional value of BSG, and the fermentation conditions were optimized by using response surface methodology.

Keywords

Brewer’s spent grains / Submerged fermentation / Pleurotus ostreatus / Response surface methodology

References

[1] Abdullah N, Ismail R, Johari NMK, Annuar MSM (2013) Production of liquid spawn of an edible grey oyster mushroom, Pleurotus pulmonarius (Fr.) Quél by submerged fermentation and sporophore yield on rubber wood sawdust. Sci Hortic 161:65–69. https://doi.org/10.1016/j.scienta.2013.06.026

[2] Ahlborn J, Stephan A, Meckel T, Maheshwari G, Rühl M, Zorn H. Upcycling of food industry side streams by basidiomycetes for production of a vegan protein source. Int J Recycl Org Waste Agricult, 2019, 8(1): 447-455

[3] Bakratsas G, Polydera A, Nilson O, Kossatz L, Xiros C, Katapodis P, Stamatis H. Single-cell protein production by Pleurotus ostreatus in submerged fermentation. Sustain Food Technol, 2023, 1(3): 377-389

[4] Bakratsas G, Antoniadis K, Athanasiou PE, Katapodis P, Stamatis H. Laccase and biomass production via submerged cultivation of Pleurotus ostreatus using wine lees. Biomass, 2024, 4(1): 1-22

[5] Belardi I, De Francesco G, Alfeo V, Bravi E, Sileoni V, Marconi O, Marrocchi A. Advances in the valorization of brewing by-products. Food Chem, 2025, 465: 141882

[6] Berger RG, Bordewick S, Krahe N-K, Ersoy F. Mycelium vs. Fruiting bodies of edible Fungi—A comparison of metabolites. Microorganisms, 2022, 10(7): 1379

[7] Bianco A, Budroni M, Zara S, Mannazzu I, Fancello F, Zara G. The role of microorganisms on biotransformation of brewers’ spent grain. Appl Microbiol Biotechnol, 2020, 104(20): 8661-8678

[8] Bickel Haase T, Klis V, Hammer AK, Pinto Lopez C, Verheyen C, Naumann-Gola S, Zorn H (2024) Fermentation of cocoa pod husks with Pleurotus salmoneo-stramineus for food applications. Food sci nutr 12(4):2551–2566. https://doi.org/10.1002/fsn3.3937

[9] Birhanli E, Yesilada O. Enhanced production of laccase in repeated-batch cultures of Funalia trogii and Trametes versicolor. Biochem Eng J, 2010, 52(1): 33-37

[10] Chaudhary G, Singh LK, Ghosh S. Alkaline pretreatment methods followed by acid hydrolysis of saccharum spontaneum for bioethanol production. Biores Technol, 2012, 124: 111-118

[11] Corrêa RCG, Brugnari T, Bracht A, Peralta RM, Ferreira ICFR. Biotechnological, nutritional and therapeutic uses of Pleurotus spp. (Oyster mushroom) related with its chemical composition: A review on the past decade findings. Trends Food Sci Technol, 2016, 50: 103-117

[12] Dexter Y, Cooke RC. Fatty acids, sterols and carotenoids of the psychrophile Mucor strictus and some mesophilic Mucor species. Trans Br Mycol Soc, 1984, 83(3): 455-461

[13] Dimou DM, Georgala A, Komaitis M, Aggelis G. Mycelial fatty acid composition of Pleurotus spp. And its application in the intrageneric differentiation. Mycol Res, 2002, 106(8): 925-929

[14] FAOThe state of food and agriculture 2019, 2019RomeFAO

[15] FAO, IFAD, UNICEF, WFP, WHO (2024) The State of Food Security and Nutrition in the World 2024. FAO; IFAD; UNICEF; WFP; WHO; Rome

[16] Farcas AC, Socaci SA, Dulf FV, Tofană M, Mudura E, Diaconeasa Z. Volatile profile, fatty acids composition and total phenolics content of brewers’ spent grain by-product with potential use in the development of new functional foods. J Cereal Sci, 2015, 64: 34-42

[17] Furlan SA, Virmond LJ, Miers DA, Bonatti M, Gern RMM, Jonas R. Mushroom strains able to grow at high temperatures and low pH values. World J Microbiol Biotechnol, 1997, 13(6): 689-692

[18] Guan A, Wang M, Gong Y, Huang T, Du Y, Zong S. Optimization of selenium biofortification by liquid fermentation based on 2,4-dichlorophenoxyacetic acid and its effect on nutritional value of Pleurotus ostreatus. J Food Compos Anal, 2025, 137: 106850

[19] Hadar Y, Cohen-Arazi E. Chemical composition of the edible mushroom Pleurotus ostreatus produced by fermentation. Appl Environ Microbiol, 1986, 51(6): 1352-1354

[20] Hamza A, Khalad A, Kumar DS. Enhanced production of mycelium biomass and exopolysaccharides of Pleurotus ostreatus by integrating response surface methodology and artificial neural network. Bioresour Technol, 2024, 399: 130577

[21] Jennison MW, Richberg CG, Krikszens AE. Physiology of Wood-rotting basidiomycetes: II. Nutritive composition of mycelium grown in submerged culture. Appl Microbiol, 1957, 5(2): 87-95

[22] Jonathan SG, Adebayo-Tayo BC, Egbomuche RC, Popoola OO. Optimization of growth conditions for mycelial yield and exopolysaccharride production by Pleurotus ostreatus cultivated in Nigeria. Afr J Microbiol Res, 2011, 5(15): 2130-2138

[23] Kalač P. A review of chemical composition and nutritional value of wild-growing and cultivated mushrooms. J Sci Food Agric, 2013, 93(2): 209-218

[24] Kanauchi O, Mitsuyama K, Araki Y. Development of a functional germinated barley foodstuff from brewer’s spent grain for the treatment of ulcerative colitis. J Am Soc Brew Chem, 2001, 59(2): 59-62

[25] Kissell LT, Prentice N. Protein and fiber enrichment of cookie flour with brewer’s spent grain. Cereal Chem, 1979, 56(4): 261-266

[26] Kjeldahl J. Neue methode Zur bestimmung des stickstoffs in organischen Körpern. Fresenius Z F Anal Chemie, 1883, 22(1): 366-382

[27] Klamer M, Bååth E. Estimation of conversion factors for fungal biomass determination in compost using ergosterol and PLFA 18:2ω6,9. Soil Biol Biochem, 2004, 36(1): 57-65

[28] Koutrotsios G, Mountzouris KC, Chatzipavlidis I, Zervakis GI. Bioconversion of lignocellulosic residues by Agrocybe cylindracea and Pleurotus ostreatus mushroom fungi – Assessment of their effect on the final product and spent substrate properties. Food Chem, 2014, 161: 127-135

[29] Lisci S, Tronci S, Grosso M, Hajrizaj R, Sibono L, Karring H, Gerganov A, Maschietti M, Errico M. Valorizing brewer’s spent grain: A sequential pathway of supercritical extraction, hydrolysis, and fermentation. Chem Eng Sci, 2024, 285: 119620

[30] Mandeel QA, Al-Laith AA, Mohamed SA. Cultivation of oyster mushrooms (Pleurotus spp.) on various lignocellulosic wastes. World J Microbiol Biotechnol, 2005, 21(4): 601-607

[31] Mansoldo FRP, Firpo R, Cardoso Vda, Queiroz S, Cedrola GN, Godoy SML, de Vermelho MG. New method for rapid identification and quantification of fungal biomass using ergosterol autofluorescence. Talanta, 2020, 219: 121238

[32] Manter K. Quantification of Phaeocryptopus gaeumannii colonization in Douglas-fir needles by ergosterol analysis. Pathol, 2001, 31(4): 229-240

[33] Manu-Tawiah W, Martin AM. Chemical composition of Pleurotus ostreatus mycelial biomass. Food Microbiol, 1987, 4(4): 303-310

[34] Mussatto SI. Brewer’s spent grain: a valuable feedstock for industrial applications. J Sci Food Agric, 2014, 94(7): 1264-1275

[35] Mussatto SI, Roberto IC. Acid hydrolysis and fermentation of brewer’s spent grain to produce xylitol. J Sci Food Agric, 2005, 85(14): 2453-2460

[36] Mussatto SI, Dragone G, Roberto IC. Brewers’ spent grain: generation, characteristics and potential applications. J Cereal Sci, 2006, 43(1): 1-14

[37] Mussatto SI, Fernandes M, Dragone G, Mancilha IM, Roberto IC. Brewer’s spent grain as Raw material for lactic acid production by Lactobacillus delbrueckii. Biotechnol Lett, 2007, 29(12): 1973-1976

[38] Niemi P, Tamminen T, Smeds A, Viljanen K, Ohra-aho T, Holopainen-Mantila U, Faulds CB, Poutanen K, Buchert J. Characterization of lipids and lignans in brewer’s spent grain and its enzymatically extracted fraction. J Agric Food Chem, 2012, 60(39): 9910-9917

[39] Ortiz I, Torreiro Y, Molina G, Maroño M, Sánchez JM. A feasible application of circular economy: spent grain energy recovery in the beer industry. Waste Biomass Valor, 2019, 10(12): 3809-3819

[40] Palmquist DL, Jenkins TC. Challenges with fats and fatty acid methods. J Anim Sci, 2003, 81(12): 3250-3254

[41] Papaspyridi L-M, Katapodis P, Gonou-Zagou Z, Kapsanaki-Gotsi E, Christakopoulos P. Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture. Biochem Eng J, 2010, 50(3): 131-138

[42] Prentice N, Refsguard JM. Enzymic hydrolysis of brewers’ spent grain. J Am Soc Brew Chem, 1978, 36(4): 196-200

[43] Rajarathnam S, Bano Z, Miles PG. Pleurotus mushrooms. Part I A. morphology, life cycle, taxonomy, breeding, and cultivation. Crit Rev Food Sci Nutr, 1987, 26(2): 157-223

[44] Resconi A, Bellezza Oddon S, Ferrocino I, Loiotine Z, Caimi C, Gasco L, Biasato I. Effects of brewery by-products on growth performance, bioconversion efficiency, nutritional profile, and microbiota and mycobiota of black soldier fly larvae. Anim, 2024, 18(9): 101288

[45] Ritota M, Manzi P. Pleurotus spp. Cultivation on different Agri-Food By-Products: example of biotechnological application. Sustainability, 2019, 11(18): 5049

[46] Robertson JA, I’Anson KJA, Treimo J, Faulds CB, Brocklehurst TF, Eijsink VGH, Waldron KW. Profiling brewers’ spent grain for composition and microbial ecology at the site of production. LWT - Food Sci Technol, 2010, 43(6): 890-896

[47] Santos M, Jiménez JJ, Bartolomé B, Gómez-Cordovés C, del Nozal MJ. Variability of brewer’s spent grain within a brewery. Food Chem, 2003, 80(1): 17-21

[48] Singh J, Suhag M, Dhaka A. Augmented digestion of lignocellulose by steam explosion, acid and alkaline pretreatment methods: A review. Carbohydr Polym, 2015, 117(6): 624-631

[49] Steudler S, Bley T. Biomass Estimation during macro-scale solid-state fermentation of basidiomycetes using established and novel approaches. Bioprocess Biosyst Eng, 2015, 38(7): 1313-1323

[50] Tshinyangu KK. Effect of grass hay substrate on nutritional value of pleurotus ostreatus var. Columbinus. Nahrung, 1996, 40(2): 79-83

[51] Valverde ME, Hernández-Pérez T, Paredes-López O. Edible mushrooms: improving human health and promoting quality life. Int J Microbiol, 2015, 2015(1): 376387

[52] Wan Mohtar WAAQI, Ab. Latif N, Harvey LM, McNeil B. Production of exopolysaccharide by Ganoderma lucidum in a repeated-batch fermentation. Biocatal Agric Biotechnol, 2016, 6: 91-101

[53] Wang D, Sakoda A, Suzuki M. Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain. Bioresour Technol, 2001, 78(3): 293-300

[54] Wilkinson S, Smart KA, Cook DJ. Optimisation of alkaline reagent based chemical pre-treatment of brewers spent grains for bioethanol production. Ind Crops Prod, 2014, 62: 219-227

[55] Zhu J, Wan C, Li Y. Enhanced solid-state anaerobic digestion of corn Stover by alkaline pretreatment. Biores Technol, 2010, 101: 7523-7528

Funding

Justus-Liebig-Universität Gießen (3114)

RIGHTS & PERMISSIONS

The Author(s)