Reframing the substrate as an active process component in fungal solid-state fermentation of foods

Reframing the substrate as an active process component in fungal solid-state fermentation of foods | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Perspective Reframing the substrate as an active process component in fungal solid-state fermentation of foods Simon Müller, Carole Zermatten, Till Germerdonk, Patrick A. Rühs This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8618445/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Apr, 2026 Read the published version in npj Science of Food → Version 1 posted 9 You are reading this latest preprint version Abstract Fungal solid-state fermentation can improve the nutritional quality of plant-based foods, yet most processes rely on substrates whose properties are inherited from raw materials rather than intentionally designed. This perspective addresses this limitation by reframing the substrate as an active process component. Integrating biochemical, mechanical, and architectural principles into substrate design enables the development of engineered substrates that promote efficient nutrient bioconversion in fermented foods. Biological sciences/Biochemistry Biological sciences/Biotechnology Physical sciences/Engineering Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Main Plant-based foods are increasingly recognized for their environmental benefits, yet their protein digestibility and mineral bioavailability are often limited due to restricted enzymatic accessibility and the presence of anti-nutritional factors (ANFs) 1 , 2 . Improving the nutritional quality of plant raw materials is therefore a key challenge for transitioning to sustainable, health-promoting food systems. Fungal solid-state fermentation (SSF) offers a biotechnological route to transform plant materials into foods with improved nutritional quality 3 . Filamentous fungi can improve protein digestibility, balance amino acid profiles, degrade ANFs, and generate beneficial secondary metabolites 4 . SSF has long been used in traditional fermented foods such as tempeh, miso, and koji, and is increasingly applied in modern bioprocessing to valorize plant proteins and side-stream materials 5 – 7 . To date, research has primarily focused on optimizing fungal strains, nutrient formulations, and bioreactor design to improve fungal growth and process performance. Despite progress in process control and bioreactor engineering, most SSF processes still rely on particulate substrates such as grains, legumes, or their processing fractions, whose biological origin determines their structure and mechanical properties. These particulate materials limit oxygen diffusion, restrict nutrient accessibility, and have fixed mechanical properties 8 , 9 . As a result, fungal growth and enzymatic activity are restricted to surface layers, resulting in heterogeneous bioconversion. While bioreactor innovations and aeration strategies can mitigate these limitations, they do not address the core issue: the substrate remains a passive medium rather than a designed component of the process. In this perspective, we propose three design principles that redefine the substrate as an engineered component of the SSF process (Fig. 1 ): (1) biochemical affinity between strain and substrate shapes nutrient bioconversion; (2) substrate mechanics regulate hyphal colonization; and (3) substrate architecture governs mass and heat transfer and enables volumetric growth. Together, these principles guide the design of engineered substrates that enhance the efficiency of nutrient bioconversion in plant-based foods. (1) Biochemical affinity between strain and substrate shapes nutrient bioconversion Biochemical affinity between a fungal strain and substrate composition governs the efficiency of nutrient bioconversion in solid-state fermentation (Fig. 2 ) 2 . Filamentous fungi acquire nutrients through absorptive heterotrophy. They secrete extracellular enzymes that break down complex biomolecules into soluble compounds 10 . The absorbed nutrients fuel their metabolism for biomass formation and secondary metabolite production. Changes in nutrient availability are sensed by the fungus and trigger shifts in gene expression and enzyme secretion, creating a feedback loop between substrate composition and fungal metabolic responses (Fig. 2 a) 11 . This bidirectional interaction allows fungi to exploit diverse plant materials and agricultural side streams as a nutrient source 5 , 10 , 12 . Three groups of enzymatic activities are central to improving the nutritional quality of plant substrates: proteolytic, phytase, and cellulolytic activity. Enzymatic bioconversion. Proteolytic enzymes hydrolyse proteins into short peptides or free amino acids, improving their digestibility (Fig. 2 b) 2 , 13 . Fungal metabolism also affects proteins through amino acid recycling: fungi assimilate amino acids from the substrate and re-assemble them into fungal proteins 14 . The resulting mycoproteins often display a more balanced amino acid profile, improving the digestible indispensable amino acid score (DIAAS) and compensating for limiting amino acids in plant-based diets 14 , 15 . Mineral bioavailability in plant raw materials is often limited by ANFs such as phytates, tannins, oxalates, and lectins that form insoluble complexes with minerals or bind to the gut epithelium, reducing absorption efficiency 16 , 17 . Mineral bioavailability can be enhanced through the hydrolysis of those ANFs by fungal enzymes 2 . For instance, phytases degrade phytates and liberate the bound iron, zinc, and calcium ions, improving their bioavailability (Fig. 2 b) 18 . Carbohydrate-active enzymes like cellulase and laccase break down complex polysaccharides, fibers, and lignin-associated components, releasing nutrients that would otherwise remain inaccessible (Fig. 2 b) 12 . This enzymatic activity improves the digestibility of fiber-rich plant materials, such as agricultural side streams. Moreover, many plant raw materials contain oligosaccharides such as raffinose and stachyose that humans cannot enzymatically digest. These compounds are instead fermented by the gut microbiota, often resulting in gas production. Oligosaccharidases secreted by filamentous fungi hydrolyse these undesirable components and reduce digestive discomfort 19 , 20 . Strain-substrate affinity. Fungal strains possess distinct enzymatic repertoires shaped by their genetic makeup and ecological specialization 21 . For example, proteolytic strains thrive on high protein, cellulolytic strains on high fiber, and strains with phytase activity on phytate-rich substrates (Fig. 2 ) 12 . Nutrient availability further regulates gene expression and enzyme secretion, meaning that a strain can adapt its enzymatic profile to different substrates. This inherent diversity and versatility in enzyme secretion suggest a broad potential for selecting fungal strains to match different bioconversion objectives 10 , 22 . Despite extensive understanding of fungal metabolism, modern SSF food processes rarely treat strain selection and substrate composition as intentional design parameters. Substrates are typically derived from traditional raw materials and used with minimal modification, while strain selection often reflects historical precedent rather than mechanistic matching. Traditional fermented foods emerged from empirical pairings between local substrates and fungal strains, refined over generations 23 . However, the effects of fermentation on protein digestibility, amino acid profiles, and ANFs reduction are highly strain specific. Nutritional outcomes of SSF vary widely, underscoring the importance of strain selection for achieving targeted nutritional improvements 2 . Systematic strain-substrate pairing remains limited because interactions in solid substrates are difficult to characterize, and conventional analytical methods often require destructive or extraction-intensive sample preparation 5 . Advances in high-throughput characterization of solid substrates, enzyme activities, and fungal metabolic responses, combined with data-driven modelling approaches, can enable rational strain-substrate selection. These tools will enable the prediction of combinations that activate specific metabolic pathways. This shift would move strain-substrate pairings from an empirical practice to design-driven fermentation for efficient bioconversion and targeted nutritional improvements. (2) Substrate mechanics regulate hyphal colonization The mechanical properties of the substrate determine how fungi attach, penetrate, and spread during SSF, directly influencing the efficiency and depth of nutrient bioconversion. Fungal hyphae grow by generating internal turgor pressure that drives tip extension against the surrounding material (Fig. 3 a) 24 . At the growing tip, localized remodeling of the cell wall allows turgor pressure to expand the wall, while new polysaccharides are inserted to support continuous growth 25 . Whether these forces result in invasive or surface-restricted growth depends on the substrate’s mechanical resistance to deformation. A given fungal strain has a stiffness threshold above which hyphal penetration is strongly reduced, reflecting the maximum pressure that the hyphal tip can exert 26 . Above this threshold, fungi shift to surface growth and rely on enzymatic degradation to soften the substrate 27 . Despite this clear coupling between substrate mechanics and fungal colonization, mechanical properties remain poorly quantified and are rarely designed intentionally in SSF 28 – 30 . Colonization across different substrate mechanics . Fungal colonization depends on the balance between hyphal forces and the mechanical resistance of the substrate. This balance determines whether growth is invasive, surface-restricted, or enabled over time through enzymatic softening (Fig. 3 b). In very soft and low-yield stress substrates such as slurries or low-concentration hydrogels, hyphae encounter minimal mechanical resistance. This allows formation of penetrative hyphae, but with limited structural support from the substrate 28 . Collapse or fluidization of the substrate reduces the structural advantages of SSF and shifts the system toward submerged-like fermentation 31 . In addition to bulk mechanical resistance, interfacial properties influence early hyphal attachment and surface growth. Filamentous fungi secrete hydrophobins, which are small, amphipathic surface proteins that lower interfacial tension and promote adhesion at air-substrate boundaries 32 – 34 . These proteins facilitate attachment and aerial hyphae formation, particularly on soft or weakly structured materials, but do not reduce the mechanical force required for substrate penetration, which remains governed by bulk mechanics. Viscoelastic solid materials, including concentrated hydrogels, solid foams, and porous biopolymer scaffolds, deform locally while retaining their macroscopic structure 29 , 35 . This allows hyphal penetration while maintaining substrate integrity. Hydrogel substrates based on different polysaccharides or proteins have been shown to impact radial extension rate and penetrative growth: higher network strength generally promotes faster radial extension and denser surface growth, whereas softer gels support deeper penetration into the substrate 29 , 36 . Stiff and predominantly elastic substrates such as high-concentration agar gels or cooked legumes, resist deformation under hyphal pressure 30 . Growth is largely confined to the air-substrate interface, forming dense aerial mycelium but limited internal colonization 30 . Hard and brittle materials, including dry lignocellulosic substrates such as wood, bran particles, or untreated plant fibers, present mechanical barriers that hyphae cannot overcome directly 37 . Hyphae primarily grow across the surface or within pre-existing pores or cracks 38 . Internal colonization depends on gradual enzymatic softening that enables deeper colonization over time. Early growth is therefore restricted to the surface and bioconversion is slow and spatially heterogeneous 39 . Effects of mechanics on enzymatic diffusion . Mechanical properties influence not only hyphal colonization, but also the movement of secreted enzymes within the substrate. Because enzymes move mainly through aqueous domains, their transport depends on the presence of water-filled pores, water activity, and substrate microstructure 40 , 41 . As water activity and the size of water-filled pores decreases, diffusion pathways become restricted and enzyme mobility decreases 42 . In wood-decay systems, for example, enzymes can advance ahead of the hyphal front only when sufficient moisture creates interconnected liquid domains 43 . Although enzyme transport has not been quantified specifically in conventional SSF substrates, a similar pattern is expected: connected aqueous domains allow enzyme diffusion, whereas dry or densely packed regions prevent it. As fermentation progresses, enzymatic degradation can locally soften the substrate and alter enzyme diffusion pathways, creating a dynamic interplay between substrate mechanics, enzyme mobility, and hyphal colonization. Toward designed substrate mechanics . Because substrate mechanics are rarely tuned in SSF, hyphal colonization and bioconversion efficiency are governed by the inherent mechanical properties of the raw materials rather than by intentional design. Future research should focus on identifying and controlling key mechanical properties, such as yield stress, viscoelasticity, and stiffness, that promote invasive growth while preserving structural integrity. These properties can be adjusted through particle packing, water content, and biopolymer concentration. In addition, the dynamic evolution of substrate mechanics during fermentation should be considered, as enzymatic degradation progressively alters material structure and influences fungal growth. Treating substrate mechanics as a design variable would enable predictable colonization patterns and more efficient nutrient bioconversion throughout the fermented material. (3) Substrate architecture governs mass and heat transfer Efficient nutrient bioconversion in SSF largely depends on the mass and heat transfer properties of the fungi’s growth environment. Oxygen diffusion, nutrient accessibility, and heat dissipation depend on the spatial organization of pores and on how they are connected to one another. Substrate architecture is therefore a key design parameter for enabling volumetric fungal activity and bioconversion throughout the fermented material (Fig. 4 ). Limitations of conventional substrate materials . Most SSF processes rely on particulate substrates such as grains, legumes, or milling fractions (Fig. 4 a) 9 , 44 . Their architecture is fixed by biological origin and random particle packing, resulting in limited porosity and low accessible surface area. Consequently, heterogeneous distribution of oxygen, nutrients, and metabolic heat develop within the substrate, confining fungal growth largely to the air-substrate interface and leaving deeper regions underutilized 9 , 45 . Efforts to mitigate these transport limitations have primarily focused on process interventions such as forced aeration, thin-layer configurations, intermittent mixing, and bioreactor innovations such as rotating drums, packed beds, or tray fermenters 6 , 44 , 46 . While these methods partially improve transport limitations, they do not modify the substrate's architecture. Because mass and heat transfer directly depend on substrate structure, particulate substrates inherently limit volumetric colonization and bioconversion efficiency. Architectural parameters that govern mass and heat transfer . Transport phenomena within a substrate are determined by its porous structure, including porosity, pore size distribution, surface-to-volume ratio, and the extent to which pores form continuous transport pathways (pore connectivity) 47 . Increasing the surface-to-volume ratio increases the accessible area for fungal attachment and enzymatic activity, which can accelerate nutrient bioconversion 48 , 49 . However, increasing porosity to achieve higher surface accessibility simultaneously reduces nutrient density, limiting the amount of substrate available to sustain metabolic activity 47 . This imposes a fundamental trade-off between surface accessibility and nutrient availability. An intermediate architecture would balance colonization area with nutrient availability, enabling uniform fungal growth and complete substrate utilization. Substrate architecture also affects the stability of the fermentation process by governing how effectively mass and heat can be exchanged throughout the substrate volume. Dense and low porosity substrates limit oxygen supply and CO 2 removal, while porous structures facilitate gas exchange 50 . Pore structure similarly influences heat transport: fungal metabolism produces heat, and insufficient removal of metabolic heat can inhibit growth or inactivate enzymes 8 , 51 . Because these constraints arise from substrate structure rather than operating conditions, they cannot be fully resolved by process control alone (aeration or cooling). Control over substrate architecture is therefore essential to achieve sufficient oxygen supply, heat dissipation, and metabolic activity throughout the substrate. Engineering substrate architecture. Because the architecture of particulate substrates is fixed, control over mass and heat transfer remains inherently limited. To support fungal metabolism throughout the entire substrate volume, materials with controllable architecture are needed (Fig. 4 b). One approach is the use of engineered porous scaffolds 35 . Scaffold porosity, pore size distribution, and connectivity can be designed to meet the transport and metabolic requirements of the fungal system. Increasing the scaffold’s porosity and accessible surface area improves gas exchange, heat removal, and fungal attachment. This shifts growth from isolated pores to continuous, volumetric growth. However, excessive porosity comes at the expense of reduced nutrient density. Establishing quantitative relationships between scaffold architecture, fungal growth, and bioconversion will be essential for developing optimal scaffold architectures. By decoupling mass and heat transfer from constraints imposed by raw materials, engineered substrate architectures enable more complete nutrient bioconversion than conventional particulate substrates. (4) Porous scaffolds as engineered substrates for solid-state fermentation Achieving high bioconversion efficiency in SSF requires substrates that integrate (1) biochemical affinity between fungal strain and substrate, (2) mechanical regulation of hyphal colonization, and (3) effective mass and heat transfer throughout the material. Conventional particulate substrates provide limited control over these properties. Engineered porous scaffolds enable rational design of substrate composition, mechanics, and architecture (Fig. 5 ), offering a platform for volumetric fungal colonization and efficient nutrient bioconversion. This section outlines key material considerations and fabrication strategies for constructing scaffolds for SSF. Material selection. Material choice defines biochemical affinity, mechanical properties, and processability into a porous structure (Fig. 5 a). Scaffold materials must provide stable attachment sites for hyphae, withstand stresses imposed during colonization, and contain an internal pore network that ensures oxygen transport, heat removal, and metabolic activity. To be processed into a porous structure, materials must behave as liquids during structuring to allow pore templating and form self-supporting, solid-like structures during fermentation. These requirements can be met by using concentrated viscoelastic suspensions of milled substrate particles that can be structured and subsequently stabilized. Suspension properties, such as solid loading, particle size, pH, ionic strength, and the presence of gelling biopolymers, should be tuned to balance flowability during structuring with mechanical stability in the final scaffold. Food-grade gelling biopolymers and intrinsic gelling mechanisms of plant-based materials can be used to achieve this balance. Polysaccharides such as alginate, agar, carrageenan, guar gum, and pectin form gel networks at low concentrations and allow tuning of viscoelasticity and stiffness 52 , 53 . In addition, the intrinsic gelling properties of plant substrates can be exploited for scaffold stabilization: starch-rich substrates can be solidified through thermogelation 54 , pectin-rich substrates form gels via calcium crosslinking or sugar/acid-induced gelation 55 , 56 , and protein-rich substrates such as soy, pea, or lupin can be stabilized through heat- or pH-induced gelation 57 – 59 . Gluten-containing materials provide cohesive elastic networks formed by glutenin and gliadin to maintain mechanical integrity 60 , 61 . Fabrication strategies. Scaffold fabrication must process viscous food suspensions into porous structures with defined architecture. Two complementary fabrication strategies can achieve this: additive manufacturing (3D printing), which enables deterministic control over pore architecture, and template structuring, in which porosity forms through the removal or incorporation of a secondary phase (Fig. 5 b). Extrusion-based 3D printing constructs scaffolds layer by layer with predefined pore size, spacing, and geometry 62 . This high level of control enables reproducibility and facilitates quantitative structure-function studies linking pore parameters to fungal colonization and bioconversion 37 , 63 , 64 . With recent advances in additive manufacturing, the use of food-grade and low-cost materials such as protein or polysaccharide bioinks has become increasingly more feasible 65 . However, the high level of precision comes at the expense of scalability, since current 3D printing methods operate in batch mode with limited throughput and relatively high processing costs 66 , 67 . Template-based approaches generate porosity by dissolving, removing, or expanding a secondary phase within the material. Methods such as porogen leaching 68 , emulsion templating 69 and foaming 70 create porous structures with high surface-to-volume ratios. While architectural precision is lower than in 3D printing, template structuring methods are compatible with established large-scale food processing operations. Their effectiveness depends on how suspension rheology and processing parameters interact to define the resulting scaffold structure. Together, material selection and fabrication strategies enable porous scaffolds that integrate biochemical strain-substrate affinity, mechanical regulation of growth, and controlled mass and heat transfer to support volumetric fungal activity. Implementing these design principles provides a route toward more uniform and efficient nutrient bioconversion in SSF. Conclusion This perspective establishes three substrate design principles for fungal SSF: (1) biochemical affinity between strain and substrate shapes nutrient bioconversion; (2) substrate mechanics regulate hyphal colonization; and (3) substrate architecture governs mass and heat transfer and enables volumetric growth. Treating substrate biochemistry, mechanics, and architecture as controllable design variables rather than fixed properties of raw materials can enable efficient bioconversion and improve the nutritional properties of fermented food materials. This approach links substrate properties directly to fungal physiology and nutritional outcomes through physical and biochemical mechanisms. Engineered porous scaffolds illustrate how these principles can be implemented in practice. Defined substrate composition allows targeted pairing with specific fungal strains; controlled mechanics regulate hyphal penetration; and designed architecture ensures efficient mass and heat transfer for uniform metabolic activity. Together, these features promote deeper colonization and more homogeneous bioconversion than conventional particulate substrates. Applying substrate design principles to microbial fermentation provides a general strategy for improving the nutritional value of plant raw materials. Extending process control to the substrate itself creates opportunities for scalable nutritional enhancement that are not achievable solely through operating conditions. Adopting this approach could support the development of more robust, scalable fermentation processes while improving nutritional quality and reducing the environmental footprint. Declarations Acknowledgements This study was funded by Nestlé, Bühler, and Givaudan via the ETH Foundation, grant number 2025-FS-406 - FungPow. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Author information Affiliation: Institute of Food, Nutrition and Health, ETH Zürich, Zürich, Switzerland Simon Müller, Carole Zermatten, Till Germerdonk & Patrick A. Rühs Contribution: S.M. and C.Z. contributed equally to this work. S.M., C.Z., T.G and P.R. conceptualized the work. S.M., C.Z. and T.G. wrote the original draft. S.M. created the figures. S.M., C.Z., and P.R. revised the final version of the paper. All authors reviewed the final version of the manuscript. Corresponding author: Patrick A. Rühs | [email protected] Competing interests: The authors declare no competing interests. References Grossmann, L. & McClements, D. J. The science of plant-based foods: approaches to create nutritious and sustainable plant-based cheese analogs. Trends Food Sci. Technol. 118, 207–229 (2021). Feng, X., Ng, K., Ajlouni, S., Zhang, P. & Fang, Z. Effect of solid-state fermentation on plant-sourced proteins: a review. Food Rev. Int. 40, 2580–2617 (2024). Boukid, F. et al. Fermentation for designing innovative plant-based meat and dairy alternatives. Foods 12, 1005 (2023). Steinkraus, K. H. Nutritional significance of fermented foods. Food Res. Int. 27, 259–267 (1994). Canoy, T. S. et al. Solid-state fermented plant foods as new protein sources. Annu. Rev. Food Sci. Technol. 15, 189–210 (2024). Singhania, R. R., Patel, A. K., Soccol, C. R. & Pandey, A. Recent advances in solid-state fermentation. Biochem. Eng. J. 44, 13–18 (2009). Oiza, N. et al. Solid-state fermentation from organic wastes: a new generation of bioproducts. Processes 10, 10 (2022). Raghavarao, K. S. M. S., Ranganathan, T. V. & Karanth, N. G. Some engineering aspects of solid-state fermentation. Biochem. Eng. J. 13, 127–135 (2003). Chen, H. Principles and application of solid-state fermentation carried out on inert support materials (adsorbed carrier solid-state fermentation). In Modern Solid State Fermentation 243–305 (Springer, 2013). doi: 10.1007/978-94-007-6043-1_6 . Lange, L., Agger, J. W. & Meyer, A. S. Fungal biotechnology: unlocking the full potential of fungi for a more sustainable world. In Grand Challenges in Biology and Biotechnology 3–32 (Springer, 2020). doi: 10.1007/978-3-030-29541-7_1 . Tomer, A. et al. Role of fungi in bioremediation and environmental sustainability. In Role of Microbes in Environmental Sustainability 187–200 (Springer, 2021). doi: 10.1007/978-3-030-54422-5_8 . Cohen, R. & Hadar, Y. The roles of fungi in agricultural waste conversion. In Fungi in Bioremediation 305–334 (Cambridge Univ. Press, 2001). doi: 10.1017/CBO9780511541780.012 . Samtiya, M., Aluko, R. E., Puniya, A. K. & Dhewa, T. Enhancing micronutrients bioavailability through fermentation of plant-based foods: a concise review. Fermentation 7, 63 (2021). Zwinkels, J., Wolkers-Rooijackers, J. & Smid, E. J. Solid-state fungal fermentation transforms low-quality plant-based foods into products with improved protein quality. LWT 184, 114979 (2023). Li, K., Qiao, K., Xiong, J., Guo, H. & Zhang, Y. Nutritional values and bio-functional properties of fungal proteins: applications in foods as a sustainable source. Foods 12, 4388 (2023). Nwe, N. & Stevens, W. F. Effect of urea on fungal chitosan production in solid substrate fermentation. Process Biochem. 39, 1639–1642 (2004). Raimbault, M. & Alazard, D. Culture method to study fungal growth in solid fermentation. Eur. J. Appl. Microbiol. Biotechnol. 9, 199–209 (1980). Hong, K.-J., Lee, C.-H. & Kim, S. W. Aspergillus oryzae GB-107 fermentation improves nutritional quality of food soybeans and feed soybean meals. J. Med. Food 7, 430–435 (2004). Liu, L. et al. Traditional fermented soybean products: processing, flavor formation, nutritional and biological activities. Crit. Rev. Food Sci. Nutr. 62, 1971–1989 (2022). Urminská, D., Haring, N., Fábry, V. & Urminská, J. Fermentable oligosaccharides, disaccharides, monosaccharides and polyols and their role in food digestion. J. Microbiol. Biotechnol. Food Sci. 11, e5521 (2022). Ghorai, S. et al. Fungal biotechnology in food and feed processing. Food Res. Int. 42, 577–587 (2009). Nielsen, J. Modelling the growth of filamentous fungi. Adv. Biochem. Eng. Biotechnol. 46, 187–223 (1992). Arrigan, D., Kothe, C. I., Oliverio, A., Evans, J. D. & Wolfe, B. E. Novel fermentations integrate traditional practice and rational design of fermented-food microbiomes. Curr. Biol. 34, R1094–R1108 (2024). Money, N. P. Biomechanics of invasive hyphal growth. In Biology of the Fungal Cell 237–249 (Springer, 2007). doi: 10.1007/978-3-540-70618-2_10 . Chevalier, L. et al. Cell wall dynamics stabilize tip growth in a filamentous fungus. PLoS Biol. 21, e3001981 (2023). Howard, R. J., Ferrari, M. A., Roach, D. H. & Money, N. P. Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc. Natl Acad. Sci. U.S.A. 88, 11281–11284 (1991). Ryder, L. S., Sprakel, J. & Talbot, N. J. Mechanobiology of fungal invasion. Curr. Biol. 35, R485–R490 (2025). Wobill, C., Azzari, P., Fischer, P. & Rühs, P. A. Host material viscoelasticity determines wrinkling of fungal films. ACS Biomater. Sci. Eng. 10, 6241–6249 (2024). Hofer, S., Nussbaum, N., Fischer, P. & Rühs, P. A. Strain, substrate, and matrix selection for controlled growth of wood-fungi. Soft Matter (2025); doi: 10.1039/D5SM00749F . Yang, L., Hu, X. & Qin, Z. Stiff substrate increases mycelium growth rate on surface. MRS Bull. 49, 1205–1216 (2024). Papadaki, A. et al. Upgrading grape pomace through Pleurotus spp. cultivation for the production of enzymes and fruiting bodies. Microorganisms 7, 207 (2019). Szilvay, G. R. et al. Self-assembled hydrophobin protein films at the air–water interface: structural analysis and molecular engineering. Biochemistry 46, 2345–2354 (2007). Quarantin, A. et al. Different hydrophobins of Fusarium graminearum are involved in hyphal growth, attachment, water–air interface penetration and plant infection. Front. Microbiol. 10, 449566 (2019). Wösten, H. A. B. & De Vocht, M. L. Hydrophobins, the fungal coat unravelled. Biochim. Biophys. Acta Biomembr. 1469, 79–86 (2000). Nussbaum, N., Repond, N., Gandia, A., Fischer, P. & Rühs, P. A. Mycelial growth of wood fungus Ganoderma sessile in porous scaffolds. Mater. Today Bio 35, 102282 (2025). Nussbaum, N. et al. Quantifying fungal growth in 3D: an ergosterol-based method to distinguish growth modes. Mater. Adv. 6, 7261–7272 (2025). Sharma, D. & Le Ferrand, H. 3D printed gyroid scaffolds enabling strong and thermally insulating mycelium-bound composites for greener infrastructures. Nat. Commun. 16, 5775 (2025). Goodell, B. et al. Fungal degradation of wood: emerging data, new insights and changing perceptions. Coatings 10, 1–19 (2020). Schwarze, F. W. M. R. Wood decay under the microscope. Fungal Biol. Rev. 21, 133–170 (2007). Bonnin, E. et al. Mobility of pectin methylesterase in pectin/cellulose gels is enhanced by the presence of cellulose and by its catalytic capacity. Sci. Rep. 9, 12551 (2019). Amsden, B. Solute diffusion within hydrogels: mechanisms and models. Macromolecules 31, 8382–8395 (1998). Guber, A., Blagodatskaya, E. & Kravchenko, A. Are enzymes transported in soils by water fluxes? Soil Biol. Biochem. 168, 108633 (2022). Zelinka, S. L. et al. The role of chemical transport in the decay resistance of modified wood. In Proc. 8th Eur. Conf. Wood Modification 35–43 (Aalto Univ., 2015). Mitchell, D. A., Berovič, M. & Krieger, N. Solid-state fermentation bioreactor fundamentals: introduction and overview. In Solid-State Fermentation Bioreactors: Fundamentals of Design and Operation (Springer, 2006). Rahardjo, Y. S. P., Sie, S., Weber, F. J., Tramper, J. & Rinzema, A. Effect of low oxygen concentrations on growth and α-amylase production of Aspergillus oryzae in model solid-state fermentation systems. Biomol. Eng. 21, 163–172 (2005). Ashok, A., Doriya, K., Rao, D. R. M. & Kumar, D. S. Design of solid-state bioreactor for industrial applications: an overview of conventional bioreactors. Biocatal. Agric. Biotechnol. 9, 11–18 (2017). Wang, L., Liu, Y. & Chen, H. Z. Advances in porous characteristics of the solid matrix in solid-state fermentation. In Current Developments in Biotechnology and Bioengineering: Current Advances in Solid-State Fermentation 19–29 (Elsevier, 2018). doi: 10.1016/B978-0-444-63990-5.00002-5 . Rahardjo, Y. S. P. Fungal mats in solid-state fermentation . (Wageningen University, Wageningen, The Netherlands, 2005). doi: 10.18174/121648 . Oostra, J. et al. Intra-particle oxygen diffusion limitation in solid-state fermentation. Biotechnol. Bioeng. 75, 13–24 (2001). Rahardjo, Y. S. P., Tramper, J. & Rinzema, A. Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives. Biotechnol. Adv. 24, 161–179 (2006). Vauris, A., Valcauda, S., Husson, F. & De Coninck, J. A novel method to assess heat transfer and impact of relevant physicochemical parameters for the scaling up of solid-state fermentation systems. Biotechnol. Rep. 36, e00764 (2022). Martau, G. A., Mihai, M. & Vodnar, D. C. The use of chitosan, alginate and pectin in the biomedical and food sector: biocompatibility, bioadhesiveness and biodegradability. Polymers 11, 1837 (2019). Imeson, A. Food Stabilisers, Thickeners and Gelling Agents . (Wiley-Blackwell, 2009). Ai, Y. & Jane, J. L. Gelatinization and rheological properties of starch. Starch/Stärke 67, 213–224 (2015). Thakur, B. R., Singh, R. K. & Handa, A. K. Chemistry and uses of pectin: a review. Crit. Rev. Food Sci. Nutr. 37, 47–73 (1997). Inbaraj, S. et al. Pectin hydrogels: gel-forming behaviors, mechanisms and food applications. Gels 9, 732 (2023). Nicolai, T. & Chassenieux, C. Heat-induced gelation of plant globulins. Curr. Opin. Food Sci. 27, 18–22 (2019). Hettiarachchy, N., Kannan, A., Schäfer, C. & Wagner, G. Gelling of plant-based proteins. In Product Design and Engineering: Formulation of Gels and Pastes 221–245 (Wiley, 2013). doi: 10.1002/9783527654741.ch8 . Hui, D. et al. A review of gelation of plant proteins and their influencing factors. Sustain. Food Proteins 3, e70037 (2025). Blomfeldt, T. O. J., Kuktaite, R., Johansson, E. & Hedenqvist, M. S. Mechanical properties and network structure of wheat gluten foams. Biomacromolecules 12, 1707–1715 (2011). Blomfeldt, T. O. J. et al. Novel foams based on freeze-dried renewable vital wheat gluten. Macromol. Mater. Eng. 295, 796–801 (2010). Vaezi, M., Zhong, G., Kalami, H. & Yang, S. Extrusion-based 3D printing technologies for 3D scaffold engineering. In Functional 3D Tissue Engineering Scaffolds 235–254 (Elsevier, 2018). doi: 10.1016/B978-0-08-100979-6.00010-0 . Nussbaum, N. Impact of host material on fungal growth: investigating nutrient density and physical properties . (ETH Zürich, Zürich, Switzerland, 2025). doi: 10.3929/ETHZ-C-000785036 . Gantenbein, S. et al. Three-dimensional printing of mycelium hydrogels into living complex materials. Nat. Mater. 22, 128–134 (2022). Zhang, J. Y., Pandya, J. K., McClements, D. J., Lu, J. & Kinchla, A. J. Advancements in 3D food printing: a comprehensive overview of properties and opportunities. Crit. Rev. Food Sci. Nutr. 62, 4752–4768 (2022). Le-Bail, A., Maniglia, B. C. & Le-Bail, P. Recent advances and future perspective in additive manufacturing of foods based on 3D printing. Curr. Opin. Food Sci. 35, 54–64 (2020). Nachal, N., Moses, J. A., Karthik, P. & Anandharamakrishnan, C. Applications of 3D printing in food processing. Food Eng. Rev. 11, 123–141 (2019). Hong, Y., Zhou, J. & Yao, D. Porogen templating processes: an overview. J. Manuf. Sci. Eng. 136, 010801 (2014). Zhang, H. & Cooper, A. I. Synthesis and applications of emulsion-templated porous materials. Chem. Soc. Rev. 34, 793–803 (2005). Dehghani, F. & Annabi, N. Engineering porous scaffolds using gas-based techniques. Curr. Opin. Biotechnol. 22, 661–666 (2011). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Apr, 2026 Read the published version in npj Science of Food → Version 1 posted Editorial decision: Revision requested 17 Feb, 2026 Reviews received at journal 17 Feb, 2026 Reviews received at journal 10 Feb, 2026 Reviewers agreed at journal 27 Jan, 2026 Reviewers agreed at journal 26 Jan, 2026 Reviewers invited by journal 22 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Submission checks completed at journal 20 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8618445","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Perspective","associatedPublications":[],"authors":[{"id":578530214,"identity":"7f6e4c26-7ef9-4966-a935-a92f7def1f7b","order_by":0,"name":"Simon Müller","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Müller","suffix":""},{"id":578530215,"identity":"9bbe909f-1118-496a-8c01-b7bb99f10528","order_by":1,"name":"Carole Zermatten","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"prefix":"","firstName":"Carole","middleName":"","lastName":"Zermatten","suffix":""},{"id":578530216,"identity":"833300e5-f019-4c9f-91be-29664a7f4550","order_by":2,"name":"Till Germerdonk","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"prefix":"","firstName":"Till","middleName":"","lastName":"Germerdonk","suffix":""},{"id":578530217,"identity":"5025ba1d-8942-446b-b138-636154f2d309","order_by":3,"name":"Patrick A. Rühs","email":"data:image/png;base64,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","orcid":"","institution":"ETH Zurich","correspondingAuthor":true,"prefix":"","firstName":"Patrick","middleName":"A.","lastName":"Rühs","suffix":""}],"badges":[],"createdAt":"2026-01-16 11:44:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8618445/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8618445/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41538-026-00827-2","type":"published","date":"2026-04-04T15:58:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":101203606,"identity":"9ef159d1-c66b-44c3-b19b-6a562b148ef5","added_by":"auto","created_at":"2026-01-27 09:40:12","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1813586,"visible":true,"origin":"","legend":"","description":"","filename":"SSFPerspective.docx","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/2263cda094580d232a562482.docx"},{"id":100995488,"identity":"90b4d39e-eefd-44b3-8418-0d8b59a6f473","added_by":"auto","created_at":"2026-01-23 15:19:16","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4791,"visible":true,"origin":"","legend":"","description":"","filename":"dff59c16ac8a4f74a985390a898b4391.json","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/2b5aa83b769cb40b0bc1dc49.json"},{"id":101204254,"identity":"83817234-d4b9-4934-965c-dbd71465c143","added_by":"auto","created_at":"2026-01-27 09:42:11","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":119995,"visible":true,"origin":"","legend":"","description":"","filename":"dff59c16ac8a4f74a985390a898b43911enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/96d704147bb001ed3a4cee8e.xml"},{"id":101203953,"identity":"d5d776f8-9452-4714-b5f4-11678087b507","added_by":"auto","created_at":"2026-01-27 09:41:08","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":252045,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/6e1cceb1eef1695ffa20eafc.png"},{"id":100995494,"identity":"49edf940-2c1d-4940-bf31-79550c44cb81","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159609,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/841d37cc5f07d3cc992dfcf7.png"},{"id":100995496,"identity":"0f8ad6d8-3818-4a11-b89d-4bc42dea7106","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":219005,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/c31542ef0c6814fdffc388cd.png"},{"id":100995497,"identity":"6b6016a4-00c6-4d08-bb97-0a21663e3515","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":321918,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/03c5520d29ba1a56b4788993.png"},{"id":101203219,"identity":"8de8a295-67dd-4251-a6c1-87581c94c8b7","added_by":"auto","created_at":"2026-01-27 09:39:06","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":259594,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/3108084aaa21d064e781c71e.png"},{"id":100995503,"identity":"4593fb50-c252-42fc-9df5-a20881afbe22","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60210,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/a7909d63a62ccc7eaf56e261.png"},{"id":101203860,"identity":"bb839004-63f1-47af-a8c4-d1338da6462a","added_by":"auto","created_at":"2026-01-27 09:40:48","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":48264,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/fd57d3692102febe7e2d1fa9.png"},{"id":100995500,"identity":"a5289485-334f-45e6-b1a2-4cffcb96f5e0","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":52134,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/5bba1340ad0c060b84c0bc76.png"},{"id":100995505,"identity":"36868be6-06fa-42a5-ae35-629f4b5fd70f","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100949,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/27c7d90162d94353ca0f2ada.png"},{"id":100995501,"identity":"094737e4-fe29-4c0f-b9c8-5b615b4ad6a0","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67868,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/d68579bdd0fc6161cfcdef82.png"},{"id":100995504,"identity":"c553904b-bcb8-4738-934b-aebca744adb0","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118288,"visible":true,"origin":"","legend":"","description":"","filename":"dff59c16ac8a4f74a985390a898b43911structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/a0933f5f8a843012c98726d4.xml"},{"id":100995506,"identity":"31f5e179-bf0a-4652-b748-e9b61846d48c","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":130204,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/65a9c354ca06d347091fd058.html"},{"id":101203628,"identity":"eb623a0b-932c-4955-a035-ec4982602fd9","added_by":"auto","created_at":"2026-01-27 09:40:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate design framework for fungal solid-state fermentation of foods.\u003c/strong\u003e Conventional particulate substrates have fixed biochemical composition, mechanical properties, and architecture, resulting in heterogeneous bioconversion and limited nutrient availability. Treating the substrate as a designable process component introduces three substrate design principles: (1) biochemical affinity between strain and substrate shapes nutrient bioconversion; (2) substrate mechanics regulate hyphal colonization; and (3) substrate architecture governs mass and heat transfer. Intentional design of these properties enables the development of engineered substrates that support volumetric bioconversion and improved nutritional properties. Figure was created with BioRender.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/1aa01a8ee264105fc22abe71.png"},{"id":100995486,"identity":"867e8eee-c89c-4d37-937c-e232811ea7a4","added_by":"auto","created_at":"2026-01-23 15:19:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":159609,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStrain-substrate affinity shapes nutrient bioconversion in fungal solid-state fermentation. \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eFungal strains secrete strain-specific extracellular enzymes that degrade substrate components, releasing nutrients that trigger metabolic responses and feedback on enzyme production. (b)\u003cstrong\u003e \u003c/strong\u003eDistinct enzymatic repertoires of fungal strains interact with substrate composition to achieve targeted nutrient transformations. Proteolytic strains acting on protein-rich substrates improve protein digestibility, phytase-active strains on phytate-rich substrates enhance mineral bioavailability, and cellulolytic strains on fiber-rich substrates enable fiber valorization. Figure was created with BioRender.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/4375823c65ef1784f1d0d276.png"},{"id":100995489,"identity":"2afe0111-eb1f-4e7b-808b-3979b714324c","added_by":"auto","created_at":"2026-01-23 15:19:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":219005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate mechanics regulate fungal colonization patterns in solid-state fermentation. \u003c/strong\u003e(a) Fungal hyphae extend by generating turgor pressure at the growing tip, resulting in penetration or surface growth depending on the mechanical resistance of the substrate. (b) Substrates with different mechanical properties result in distinct colonization modes. Soft, deformable substrates may collapse under growth conditions, whereas viscoelastic solids support invasive hyphal growth while maintaining structural integrity. Increasing stiffness and elasticity favor predominantly aerial growth, while hard and brittle substrates resist penetration, resulting in surface-restricted colonization. Figure was created with BioRender.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/a97897c52023357d300826f0.png"},{"id":101203605,"identity":"76154b53-217a-48b8-825d-a07d5a023b40","added_by":"auto","created_at":"2026-01-27 09:40:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":321918,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubstrate architecture governs mass and heat transfer during fungal solid-state fermentation.\u003c/strong\u003e (a) Conventional particulate substrates have limited porosity and surface area, restricting fungal growth to the air-substrate interface. (b) Engineered substrate architectures with increased porosity and interfacial area improve gas exchange, heat dissipation, and nutrient accessibility, enabling volumetric fungal growth throughout the substrate. Figure was created with BioRender.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/3c8ee0094c7873588137c534.png"},{"id":101203745,"identity":"6a561334-c481-42b2-a011-728fb395e6ff","added_by":"auto","created_at":"2026-01-27 09:40:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":259594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMaterial selection and fabrication strategies for porous scaffolds in fungal solid-state fermentation. \u003c/strong\u003e(a) Substrate materials are formulated as concentrated viscoelastic suspensions that can be structured and subsequently solidified to lock in the desired architecture. (b) Porous scaffolds can be fabricated using complementary strategies with trade-offs between precision and scalability. Template-based methods such as porogen leaching, emulsion templating, and foaming offer higher scalability, whereas 3D printing provides greater control over scaffold architecture. Figure was created with BioRender.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/31ca1b4b60386536bc5a556b.png"},{"id":106343500,"identity":"de90d450-fe14-4156-a51b-e0d376082cd2","added_by":"auto","created_at":"2026-04-07 16:06:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1926823,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8618445/v1/b966e45d-6740-4946-bea5-94e5c1417365.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reframing the substrate as an active process component in fungal solid-state fermentation of foods","fulltext":[{"header":"Main","content":"\u003cp\u003ePlant-based foods are increasingly recognized for their environmental benefits, yet their protein digestibility and mineral bioavailability are often limited due to restricted enzymatic accessibility and the presence of anti-nutritional factors (ANFs)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Improving the nutritional quality of plant raw materials is therefore a key challenge for transitioning to sustainable, health-promoting food systems.\u003c/p\u003e \u003cp\u003eFungal solid-state fermentation (SSF) offers a biotechnological route to transform plant materials into foods with improved nutritional quality\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Filamentous fungi can improve protein digestibility, balance amino acid profiles, degrade ANFs, and generate beneficial secondary metabolites\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. SSF has long been used in traditional fermented foods such as tempeh, miso, and koji, and is increasingly applied in modern bioprocessing to valorize plant proteins and side-stream materials\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. To date, research has primarily focused on optimizing fungal strains, nutrient formulations, and bioreactor design to improve fungal growth and process performance.\u003c/p\u003e \u003cp\u003eDespite progress in process control and bioreactor engineering, most SSF processes still rely on particulate substrates such as grains, legumes, or their processing fractions, whose biological origin determines their structure and mechanical properties. These particulate materials limit oxygen diffusion, restrict nutrient accessibility, and have fixed mechanical properties\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. As a result, fungal growth and enzymatic activity are restricted to surface layers, resulting in heterogeneous bioconversion. While bioreactor innovations and aeration strategies can mitigate these limitations, they do not address the core issue: the substrate remains a passive medium rather than a designed component of the process.\u003c/p\u003e \u003cp\u003eIn this perspective, we propose three design principles that redefine the substrate as an engineered component of the SSF process (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e): (1) biochemical affinity between strain and substrate shapes nutrient bioconversion; (2) substrate mechanics regulate hyphal colonization; and (3) substrate architecture governs mass and heat transfer and enables volumetric growth. Together, these principles guide the design of engineered substrates that enhance the efficiency of nutrient bioconversion in plant-based foods.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"(1) Biochemical affinity between strain and substrate shapes nutrient bioconversion","content":"\u003cp\u003eBiochemical affinity between a fungal strain and substrate composition governs the efficiency of nutrient bioconversion in solid-state fermentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Filamentous fungi acquire nutrients through absorptive heterotrophy. They secrete extracellular enzymes that break down complex biomolecules into soluble compounds\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The absorbed nutrients fuel their metabolism for biomass formation and secondary metabolite production. Changes in nutrient availability are sensed by the fungus and trigger shifts in gene expression and enzyme secretion, creating a feedback loop between substrate composition and fungal metabolic responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This bidirectional interaction allows fungi to exploit diverse plant materials and agricultural side streams as a nutrient source\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Three groups of enzymatic activities are central to improving the nutritional quality of plant substrates: proteolytic, phytase, and cellulolytic activity.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEnzymatic bioconversion.\u003c/em\u003e Proteolytic enzymes hydrolyse proteins into short peptides or free amino acids, improving their digestibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Fungal metabolism also affects proteins through amino acid recycling: fungi assimilate amino acids from the substrate and re-assemble them into fungal proteins\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The resulting mycoproteins often display a more balanced amino acid profile, improving the digestible indispensable amino acid score (DIAAS) and compensating for limiting amino acids in plant-based diets\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Mineral bioavailability in plant raw materials is often limited by ANFs such as phytates, tannins, oxalates, and lectins that form insoluble complexes with minerals or bind to the gut epithelium, reducing absorption efficiency\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Mineral bioavailability can be enhanced through the hydrolysis of those ANFs by fungal enzymes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. For instance, phytases degrade phytates and liberate the bound iron, zinc, and calcium ions, improving their bioavailability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Carbohydrate-active enzymes like cellulase and laccase break down complex polysaccharides, fibers, and lignin-associated components, releasing nutrients that would otherwise remain inaccessible (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. This enzymatic activity improves the digestibility of fiber-rich plant materials, such as agricultural side streams. Moreover, many plant raw materials contain oligosaccharides such as raffinose and stachyose that humans cannot enzymatically digest. These compounds are instead fermented by the gut microbiota, often resulting in gas production. Oligosaccharidases secreted by filamentous fungi hydrolyse these undesirable components and reduce digestive discomfort\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStrain-substrate affinity.\u003c/em\u003e Fungal strains possess distinct enzymatic repertoires shaped by their genetic makeup and ecological specialization\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. For example, proteolytic strains thrive on high protein, cellulolytic strains on high fiber, and strains with phytase activity on phytate-rich substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Nutrient availability further regulates gene expression and enzyme secretion, meaning that a strain can adapt its enzymatic profile to different substrates. This inherent diversity and versatility in enzyme secretion suggest a broad potential for selecting fungal strains to match different bioconversion objectives\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Despite extensive understanding of fungal metabolism, modern SSF food processes rarely treat strain selection and substrate composition as intentional design parameters. Substrates are typically derived from traditional raw materials and used with minimal modification, while strain selection often reflects historical precedent rather than mechanistic matching. Traditional fermented foods emerged from empirical pairings between local substrates and fungal strains, refined over generations\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, the effects of fermentation on protein digestibility, amino acid profiles, and ANFs reduction are highly strain specific. Nutritional outcomes of SSF vary widely, underscoring the importance of strain selection for achieving targeted nutritional improvements\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Systematic strain-substrate pairing remains limited because interactions in solid substrates are difficult to characterize, and conventional analytical methods often require destructive or extraction-intensive sample preparation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Advances in high-throughput characterization of solid substrates, enzyme activities, and fungal metabolic responses, combined with data-driven modelling approaches, can enable rational strain-substrate selection. These tools will enable the prediction of combinations that activate specific metabolic pathways. This shift would move strain-substrate pairings from an empirical practice to design-driven fermentation for efficient bioconversion and targeted nutritional improvements.\u003c/p\u003e"},{"header":"(2) Substrate mechanics regulate hyphal colonization","content":"\u003cp\u003eThe mechanical properties of the substrate determine how fungi attach, penetrate, and spread during SSF, directly influencing the efficiency and depth of nutrient bioconversion. Fungal hyphae grow by generating internal turgor pressure that drives tip extension against the surrounding material (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. At the growing tip, localized remodeling of the cell wall allows turgor pressure to expand the wall, while new polysaccharides are inserted to support continuous growth\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Whether these forces result in invasive or surface-restricted growth depends on the substrate’s mechanical resistance to deformation. A given fungal strain has a stiffness threshold above which hyphal penetration is strongly reduced, reflecting the maximum pressure that the hyphal tip can exert\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Above this threshold, fungi shift to surface growth and rely on enzymatic degradation to soften the substrate\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Despite this clear coupling between substrate mechanics and fungal colonization, mechanical properties remain poorly quantified and are rarely designed intentionally in SSF\u003csup\u003e\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e–\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e \u003cem\u003eColonization across different substrate mechanics\u003c/em\u003e. Fungal colonization depends on the balance between hyphal forces and the mechanical resistance of the substrate. This balance determines whether growth is invasive, surface-restricted, or enabled over time through enzymatic softening (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). In very soft and low-yield stress substrates such as slurries or low-concentration hydrogels, hyphae encounter minimal mechanical resistance. This allows formation of penetrative hyphae, but with limited structural support from the substrate\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Collapse or fluidization of the substrate reduces the structural advantages of SSF and shifts the system toward submerged-like fermentation\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In addition to bulk mechanical resistance, interfacial properties influence early hyphal attachment and surface growth. Filamentous fungi secrete hydrophobins, which are small, amphipathic surface proteins that lower interfacial tension and promote adhesion at air-substrate boundaries\u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e–\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. These proteins facilitate attachment and aerial hyphae formation, particularly on soft or weakly structured materials, but do not reduce the mechanical force required for substrate penetration, which remains governed by bulk mechanics. Viscoelastic solid materials, including concentrated hydrogels, solid foams, and porous biopolymer scaffolds, deform locally while retaining their macroscopic structure\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. This allows hyphal penetration while maintaining substrate integrity. Hydrogel substrates based on different polysaccharides or proteins have been shown to impact radial extension rate and penetrative growth: higher network strength generally promotes faster radial extension and denser surface growth, whereas softer gels support deeper penetration into the substrate\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Stiff and predominantly elastic substrates such as high-concentration agar gels or cooked legumes, resist deformation under hyphal pressure\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Growth is largely confined to the air-substrate interface, forming dense aerial mycelium but limited internal colonization\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Hard and brittle materials, including dry lignocellulosic substrates such as wood, bran particles, or untreated plant fibers, present mechanical barriers that hyphae cannot overcome directly\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Hyphae primarily grow across the surface or within pre-existing pores or cracks\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Internal colonization depends on gradual enzymatic softening that enables deeper colonization over time. Early growth is therefore restricted to the surface and bioconversion is slow and spatially heterogeneous\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e \u003cem\u003eEffects of mechanics on enzymatic diffusion\u003c/em\u003e. Mechanical properties influence not only hyphal colonization, but also the movement of secreted enzymes within the substrate. Because enzymes move mainly through aqueous domains, their transport depends on the presence of water-filled pores, water activity, and substrate microstructure\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. As water activity and the size of water-filled pores decreases, diffusion pathways become restricted and enzyme mobility decreases\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. In wood-decay systems, for example, enzymes can advance ahead of the hyphal front only when sufficient moisture creates interconnected liquid domains\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Although enzyme transport has not been quantified specifically in conventional SSF substrates, a similar pattern is expected: connected aqueous domains allow enzyme diffusion, whereas dry or densely packed regions prevent it. As fermentation progresses, enzymatic degradation can locally soften the substrate and alter enzyme diffusion pathways, creating a dynamic interplay between substrate mechanics, enzyme mobility, and hyphal colonization.\u003c/p\u003e\u003cp\u003e \u003cem\u003eToward designed substrate mechanics\u003c/em\u003e. Because substrate mechanics are rarely tuned in SSF, hyphal colonization and bioconversion efficiency are governed by the inherent mechanical properties of the raw materials rather than by intentional design. Future research should focus on identifying and controlling key mechanical properties, such as yield stress, viscoelasticity, and stiffness, that promote invasive growth while preserving structural integrity. These properties can be adjusted through particle packing, water content, and biopolymer concentration. In addition, the dynamic evolution of substrate mechanics during fermentation should be considered, as enzymatic degradation progressively alters material structure and influences fungal growth. Treating substrate mechanics as a design variable would enable predictable colonization patterns and more efficient nutrient bioconversion throughout the fermented material.\u003c/p\u003e"},{"header":"(3) Substrate architecture governs mass and heat transfer","content":"\u003cp\u003eEfficient nutrient bioconversion in SSF largely depends on the mass and heat transfer properties of the fungi\u0026rsquo;s growth environment. Oxygen diffusion, nutrient accessibility, and heat dissipation depend on the spatial organization of pores and on how they are connected to one another. Substrate architecture is therefore a key design parameter for enabling volumetric fungal activity and bioconversion throughout the fermented material (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLimitations of conventional substrate materials\u003c/em\u003e. Most SSF processes rely on particulate substrates such as grains, legumes, or milling fractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Their architecture is fixed by biological origin and random particle packing, resulting in limited porosity and low accessible surface area. Consequently, heterogeneous distribution of oxygen, nutrients, and metabolic heat develop within the substrate, confining fungal growth largely to the air-substrate interface and leaving deeper regions underutilized\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Efforts to mitigate these transport limitations have primarily focused on process interventions such as forced aeration, thin-layer configurations, intermittent mixing, and bioreactor innovations such as rotating drums, packed beds, or tray fermenters\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. While these methods partially improve transport limitations, they do not modify the substrate's architecture. Because mass and heat transfer directly depend on substrate structure, particulate substrates inherently limit volumetric colonization and bioconversion efficiency.\u003c/p\u003e \u003cp\u003e \u003cem\u003eArchitectural parameters that govern mass and heat transfer\u003c/em\u003e. Transport phenomena within a substrate are determined by its porous structure, including porosity, pore size distribution, surface-to-volume ratio, and the extent to which pores form continuous transport pathways (pore connectivity)\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Increasing the surface-to-volume ratio increases the accessible area for fungal attachment and enzymatic activity, which can accelerate nutrient bioconversion\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. However, increasing porosity to achieve higher surface accessibility simultaneously reduces nutrient density, limiting the amount of substrate available to sustain metabolic activity\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. This imposes a fundamental trade-off between surface accessibility and nutrient availability. An intermediate architecture would balance colonization area with nutrient availability, enabling uniform fungal growth and complete substrate utilization. Substrate architecture also affects the stability of the fermentation process by governing how effectively mass and heat can be exchanged throughout the substrate volume. Dense and low porosity substrates limit oxygen supply and CO\u003csub\u003e2\u003c/sub\u003e removal, while porous structures facilitate gas exchange\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Pore structure similarly influences heat transport: fungal metabolism produces heat, and insufficient removal of metabolic heat can inhibit growth or inactivate enzymes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Because these constraints arise from substrate structure rather than operating conditions, they cannot be fully resolved by process control alone (aeration or cooling). Control over substrate architecture is therefore essential to achieve sufficient oxygen supply, heat dissipation, and metabolic activity throughout the substrate.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEngineering substrate architecture.\u003c/em\u003e Because the architecture of particulate substrates is fixed, control over mass and heat transfer remains inherently limited. To support fungal metabolism throughout the entire substrate volume, materials with controllable architecture are needed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). One approach is the use of engineered porous scaffolds\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Scaffold porosity, pore size distribution, and connectivity can be designed to meet the transport and metabolic requirements of the fungal system. Increasing the scaffold\u0026rsquo;s porosity and accessible surface area improves gas exchange, heat removal, and fungal attachment. This shifts growth from isolated pores to continuous, volumetric growth. However, excessive porosity comes at the expense of reduced nutrient density. Establishing quantitative relationships between scaffold architecture, fungal growth, and bioconversion will be essential for developing optimal scaffold architectures. By decoupling mass and heat transfer from constraints imposed by raw materials, engineered substrate architectures enable more complete nutrient bioconversion than conventional particulate substrates.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"(4) Porous scaffolds as engineered substrates for solid-state fermentation","content":"\u003cp\u003eAchieving high bioconversion efficiency in SSF requires substrates that integrate (1) biochemical affinity between fungal strain and substrate, (2) mechanical regulation of hyphal colonization, and (3) effective mass and heat transfer throughout the material. Conventional particulate substrates provide limited control over these properties. Engineered porous scaffolds enable rational design of substrate composition, mechanics, and architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), offering a platform for volumetric fungal colonization and efficient nutrient bioconversion. This section outlines key material considerations and fabrication strategies for constructing scaffolds for SSF.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMaterial selection.\u003c/em\u003e Material choice defines biochemical affinity, mechanical properties, and processability into a porous structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Scaffold materials must provide stable attachment sites for hyphae, withstand stresses imposed during colonization, and contain an internal pore network that ensures oxygen transport, heat removal, and metabolic activity. To be processed into a porous structure, materials must behave as liquids during structuring to allow pore templating and form self-supporting, solid-like structures during fermentation. These requirements can be met by using concentrated viscoelastic suspensions of milled substrate particles that can be structured and subsequently stabilized. Suspension properties, such as solid loading, particle size, pH, ionic strength, and the presence of gelling biopolymers, should be tuned to balance flowability during structuring with mechanical stability in the final scaffold. Food-grade gelling biopolymers and intrinsic gelling mechanisms of plant-based materials can be used to achieve this balance. Polysaccharides such as alginate, agar, carrageenan, guar gum, and pectin form gel networks at low concentrations and allow tuning of viscoelasticity and stiffness\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In addition, the intrinsic gelling properties of plant substrates can be exploited for scaffold stabilization: starch-rich substrates can be solidified through thermogelation\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, pectin-rich substrates form gels via calcium crosslinking or sugar/acid-induced gelation\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, and protein-rich substrates such as soy, pea, or lupin can be stabilized through heat- or pH-induced gelation\u003csup\u003e\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Gluten-containing materials provide cohesive elastic networks formed by glutenin and gliadin to maintain mechanical integrity\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eFabrication strategies.\u003c/em\u003e Scaffold fabrication must process viscous food suspensions into porous structures with defined architecture. Two complementary fabrication strategies can achieve this: additive manufacturing (3D printing), which enables deterministic control over pore architecture, and template structuring, in which porosity forms through the removal or incorporation of a secondary phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Extrusion-based 3D printing constructs scaffolds layer by layer with predefined pore size, spacing, and geometry\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This high level of control enables reproducibility and facilitates quantitative structure-function studies linking pore parameters to fungal colonization and bioconversion\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. With recent advances in additive manufacturing, the use of food-grade and low-cost materials such as protein or polysaccharide bioinks has become increasingly more feasible\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. However, the high level of precision comes at the expense of scalability, since current 3D printing methods operate in batch mode with limited throughput and relatively high processing costs\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Template-based approaches generate porosity by dissolving, removing, or expanding a secondary phase within the material. Methods such as porogen leaching\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, emulsion templating\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e and foaming\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e create porous structures with high surface-to-volume ratios. While architectural precision is lower than in 3D printing, template structuring methods are compatible with established large-scale food processing operations. Their effectiveness depends on how suspension rheology and processing parameters interact to define the resulting scaffold structure.\u003c/p\u003e \u003cp\u003eTogether, material selection and fabrication strategies enable porous scaffolds that integrate biochemical strain-substrate affinity, mechanical regulation of growth, and controlled mass and heat transfer to support volumetric fungal activity. Implementing these design principles provides a route toward more uniform and efficient nutrient bioconversion in SSF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis perspective establishes three substrate design principles for fungal SSF: (1) biochemical affinity between strain and substrate shapes nutrient bioconversion; (2) substrate mechanics regulate hyphal colonization; and (3) substrate architecture governs mass and heat transfer and enables volumetric growth. Treating substrate biochemistry, mechanics, and architecture as controllable design variables rather than fixed properties of raw materials can enable efficient bioconversion and improve the nutritional properties of fermented food materials. This approach links substrate properties directly to fungal physiology and nutritional outcomes through physical and biochemical mechanisms.\u003c/p\u003e \u003cp\u003eEngineered porous scaffolds illustrate how these principles can be implemented in practice. Defined substrate composition allows targeted pairing with specific fungal strains; controlled mechanics regulate hyphal penetration; and designed architecture ensures efficient mass and heat transfer for uniform metabolic activity. Together, these features promote deeper colonization and more homogeneous bioconversion than conventional particulate substrates.\u003c/p\u003e \u003cp\u003eApplying substrate design principles to microbial fermentation provides a general strategy for improving the nutritional value of plant raw materials. Extending process control to the substrate itself creates opportunities for scalable nutritional enhancement that are not achievable solely through operating conditions. Adopting this approach could support the development of more robust, scalable fermentation processes while improving nutritional quality and reducing the environmental footprint.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Nestl\u0026eacute;, B\u0026uuml;hler, and Givaudan via the ETH Foundation, grant number 2025-FS-406 - FungPow. The funders played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAffiliation:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInstitute of Food, Nutrition and Health, ETH Z\u0026uuml;rich, Z\u0026uuml;rich, Switzerland\u003cbr\u003e\u0026nbsp;Simon M\u0026uuml;ller, Carole Zermatten, Till Germerdonk \u0026amp; Patrick A. R\u0026uuml;hs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.M. and C.Z. contributed equally to this work. S.M., C.Z., T.G and P.R. conceptualized the work. S.M., C.Z. and T.G. wrote the original draft. S.M. created the figures. S.M., C.Z., and P.R. revised the final version of the paper. All authors reviewed the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatrick A. R\u0026uuml;hs | [email protected]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGrossmann, L. \u0026amp; McClements, D. J. The science of plant-based foods: approaches to create nutritious and sustainable plant-based cheese analogs. \u003cem\u003eTrends Food Sci. Technol.\u003c/em\u003e 118, 207\u0026ndash;229 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFeng, X., Ng, K., Ajlouni, S., Zhang, P. \u0026amp; Fang, Z. Effect of solid-state fermentation on plant-sourced proteins: a review. \u003cem\u003eFood Rev. Int.\u003c/em\u003e 40, 2580\u0026ndash;2617 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoukid, F. \u003cem\u003eet al.\u003c/em\u003e Fermentation for designing innovative plant-based meat and dairy alternatives. \u003cem\u003eFoods\u003c/em\u003e 12, 1005 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinkraus, K. H. Nutritional significance of fermented foods. \u003cem\u003eFood Res. Int.\u003c/em\u003e 27, 259\u0026ndash;267 (1994).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCanoy, T. S. \u003cem\u003eet al.\u003c/em\u003e Solid-state fermented plant foods as new protein sources. \u003cem\u003eAnnu. Rev. Food Sci. Technol.\u003c/em\u003e 15, 189\u0026ndash;210 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinghania, R. R., Patel, A. K., Soccol, C. R. \u0026amp; Pandey, A. Recent advances in solid-state fermentation. \u003cem\u003eBiochem. Eng. J.\u003c/em\u003e 44, 13\u0026ndash;18 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOiza, N. \u003cem\u003eet al.\u003c/em\u003e Solid-state fermentation from organic wastes: a new generation of bioproducts. \u003cem\u003eProcesses\u003c/em\u003e 10, 10 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaghavarao, K. S. M. S., Ranganathan, T. V. \u0026amp; Karanth, N. G. Some engineering aspects of solid-state fermentation. \u003cem\u003eBiochem. Eng. J.\u003c/em\u003e 13, 127\u0026ndash;135 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, H. Principles and application of solid-state fermentation carried out on inert support materials (adsorbed carrier solid-state fermentation). In \u003cem\u003eModern Solid State Fermentation\u003c/em\u003e 243\u0026ndash;305 (Springer, 2013). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-94-007-6043-1_6\u003c/span\u003e\u003cspan address=\"10.1007/978-94-007-6043-1_6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLange, L., Agger, J. W. \u0026amp; Meyer, A. S. Fungal biotechnology: unlocking the full potential of fungi for a more sustainable world. In \u003cem\u003eGrand Challenges in Biology and Biotechnology\u003c/em\u003e 3\u0026ndash;32 (Springer, 2020). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-030-29541-7_1\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-29541-7_1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomer, A. \u003cem\u003eet al.\u003c/em\u003e Role of fungi in bioremediation and environmental sustainability. In \u003cem\u003eRole of Microbes in Environmental Sustainability\u003c/em\u003e 187\u0026ndash;200 (Springer, 2021). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-030-54422-5_8\u003c/span\u003e\u003cspan address=\"10.1007/978-3-030-54422-5_8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen, R. \u0026amp; Hadar, Y. The roles of fungi in agricultural waste conversion. In \u003cem\u003eFungi in Bioremediation\u003c/em\u003e 305\u0026ndash;334 (Cambridge Univ. Press, 2001). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1017/CBO9780511541780.012\u003c/span\u003e\u003cspan address=\"10.1017/CBO9780511541780.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSamtiya, M., Aluko, R. E., Puniya, A. K. \u0026amp; Dhewa, T. Enhancing micronutrients bioavailability through fermentation of plant-based foods: a concise review. \u003cem\u003eFermentation\u003c/em\u003e 7, 63 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZwinkels, J., Wolkers-Rooijackers, J. \u0026amp; Smid, E. J. Solid-state fungal fermentation transforms low-quality plant-based foods into products with improved protein quality. \u003cem\u003eLWT\u003c/em\u003e 184, 114979 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, K., Qiao, K., Xiong, J., Guo, H. \u0026amp; Zhang, Y. Nutritional values and bio-functional properties of fungal proteins: applications in foods as a sustainable source. \u003cem\u003eFoods\u003c/em\u003e 12, 4388 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNwe, N. \u0026amp; Stevens, W. F. Effect of urea on fungal chitosan production in solid substrate fermentation. \u003cem\u003eProcess Biochem.\u003c/em\u003e 39, 1639\u0026ndash;1642 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaimbault, M. \u0026amp; Alazard, D. Culture method to study fungal growth in solid fermentation. \u003cem\u003eEur. J. Appl. Microbiol. Biotechnol.\u003c/em\u003e 9, 199\u0026ndash;209 (1980).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong, K.-J., Lee, C.-H. \u0026amp; Kim, S. W. \u003cem\u003eAspergillus oryzae\u003c/em\u003e GB-107 fermentation improves nutritional quality of food soybeans and feed soybean meals. \u003cem\u003eJ. Med. Food\u003c/em\u003e 7, 430\u0026ndash;435 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, L. \u003cem\u003eet al.\u003c/em\u003e Traditional fermented soybean products: processing, flavor formation, nutritional and biological activities. \u003cem\u003eCrit. Rev. Food Sci. Nutr.\u003c/em\u003e 62, 1971\u0026ndash;1989 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrminsk\u0026aacute;, D., Haring, N., F\u0026aacute;bry, V. \u0026amp; Urminsk\u0026aacute;, J. Fermentable oligosaccharides, disaccharides, monosaccharides and polyols and their role in food digestion. \u003cem\u003eJ. Microbiol. Biotechnol. Food Sci.\u003c/em\u003e 11, e5521 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhorai, S. \u003cem\u003eet al.\u003c/em\u003e Fungal biotechnology in food and feed processing. \u003cem\u003eFood Res. Int.\u003c/em\u003e 42, 577\u0026ndash;587 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNielsen, J. Modelling the growth of filamentous fungi. \u003cem\u003eAdv. Biochem. Eng. Biotechnol.\u003c/em\u003e 46, 187\u0026ndash;223 (1992).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArrigan, D., Kothe, C. I., Oliverio, A., Evans, J. D. \u0026amp; Wolfe, B. E. Novel fermentations integrate traditional practice and rational design of fermented-food microbiomes. \u003cem\u003eCurr. Biol.\u003c/em\u003e 34, R1094\u0026ndash;R1108 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoney, N. P. Biomechanics of invasive hyphal growth. In \u003cem\u003eBiology of the Fungal Cell\u003c/em\u003e 237\u0026ndash;249 (Springer, 2007). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/978-3-540-70618-2_10\u003c/span\u003e\u003cspan address=\"10.1007/978-3-540-70618-2_10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChevalier, L. \u003cem\u003eet al.\u003c/em\u003e Cell wall dynamics stabilize tip growth in a filamentous fungus. \u003cem\u003ePLoS Biol.\u003c/em\u003e 21, e3001981 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoward, R. J., Ferrari, M. A., Roach, D. H. \u0026amp; Money, N. P. Penetration of hard substrates by a fungus employing enormous turgor pressures. \u003cem\u003eProc. Natl Acad. Sci. U.S.A.\u003c/em\u003e 88, 11281\u0026ndash;11284 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyder, L. S., Sprakel, J. \u0026amp; Talbot, N. J. Mechanobiology of fungal invasion. \u003cem\u003eCurr. Biol.\u003c/em\u003e 35, R485\u0026ndash;R490 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWobill, C., Azzari, P., Fischer, P. \u0026amp; R\u0026uuml;hs, P. A. Host material viscoelasticity determines wrinkling of fungal films. \u003cem\u003eACS Biomater. Sci. Eng.\u003c/em\u003e 10, 6241\u0026ndash;6249 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofer, S., Nussbaum, N., Fischer, P. \u0026amp; R\u0026uuml;hs, P. A. Strain, substrate, and matrix selection for controlled growth of wood-fungi. \u003cem\u003eSoft Matter\u003c/em\u003e (2025); doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/D5SM00749F\u003c/span\u003e\u003cspan address=\"10.1039/D5SM00749F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, L., Hu, X. \u0026amp; Qin, Z. Stiff substrate increases mycelium growth rate on surface. \u003cem\u003eMRS Bull.\u003c/em\u003e 49, 1205\u0026ndash;1216 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePapadaki, A. \u003cem\u003eet al.\u003c/em\u003e Upgrading grape pomace through \u003cem\u003ePleurotus\u003c/em\u003e spp. cultivation for the production of enzymes and fruiting bodies. \u003cem\u003eMicroorganisms\u003c/em\u003e 7, 207 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzilvay, G. R. \u003cem\u003eet al.\u003c/em\u003e Self-assembled hydrophobin protein films at the air\u0026ndash;water interface: structural analysis and molecular engineering. \u003cem\u003eBiochemistry\u003c/em\u003e 46, 2345\u0026ndash;2354 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuarantin, A. \u003cem\u003eet al.\u003c/em\u003e Different hydrophobins of \u003cem\u003eFusarium graminearum\u003c/em\u003e are involved in hyphal growth, attachment, water\u0026ndash;air interface penetration and plant infection. \u003cem\u003eFront. Microbiol.\u003c/em\u003e 10, 449566 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026ouml;sten, H. A. B. \u0026amp; De Vocht, M. L. Hydrophobins, the fungal coat unravelled. \u003cem\u003eBiochim. Biophys. Acta Biomembr.\u003c/em\u003e 1469, 79\u0026ndash;86 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNussbaum, N., Repond, N., Gandia, A., Fischer, P. \u0026amp; R\u0026uuml;hs, P. A. Mycelial growth of wood fungus \u003cem\u003eGanoderma sessile\u003c/em\u003e in porous scaffolds. \u003cem\u003eMater. Today Bio\u003c/em\u003e 35, 102282 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNussbaum, N. \u003cem\u003eet al.\u003c/em\u003e Quantifying fungal growth in 3D: an ergosterol-based method to distinguish growth modes. \u003cem\u003eMater. Adv.\u003c/em\u003e 6, 7261\u0026ndash;7272 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSharma, D. \u0026amp; Le Ferrand, H. 3D printed gyroid scaffolds enabling strong and thermally insulating mycelium-bound composites for greener infrastructures. \u003cem\u003eNat. Commun.\u003c/em\u003e 16, 5775 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodell, B. \u003cem\u003eet al.\u003c/em\u003e Fungal degradation of wood: emerging data, new insights and changing perceptions. \u003cem\u003eCoatings\u003c/em\u003e 10, 1\u0026ndash;19 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwarze, F. W. M. R. Wood decay under the microscope. \u003cem\u003eFungal Biol. Rev.\u003c/em\u003e 21, 133\u0026ndash;170 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonnin, E. \u003cem\u003eet al.\u003c/em\u003e Mobility of pectin methylesterase in pectin/cellulose gels is enhanced by the presence of cellulose and by its catalytic capacity. \u003cem\u003eSci. Rep.\u003c/em\u003e 9, 12551 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmsden, B. Solute diffusion within hydrogels: mechanisms and models. \u003cem\u003eMacromolecules\u003c/em\u003e 31, 8382\u0026ndash;8395 (1998).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuber, A., Blagodatskaya, E. \u0026amp; Kravchenko, A. Are enzymes transported in soils by water fluxes? \u003cem\u003eSoil Biol. Biochem.\u003c/em\u003e 168, 108633 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZelinka, S. L. \u003cem\u003eet al.\u003c/em\u003e The role of chemical transport in the decay resistance of modified wood. In \u003cem\u003eProc. 8th Eur. Conf. Wood Modification\u003c/em\u003e 35\u0026ndash;43 (Aalto Univ., 2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchell, D. A., Berovič, M. \u0026amp; Krieger, N. Solid-state fermentation bioreactor fundamentals: introduction and overview. In \u003cem\u003eSolid-State Fermentation Bioreactors: Fundamentals of Design and Operation\u003c/em\u003e (Springer, 2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahardjo, Y. S. P., Sie, S., Weber, F. J., Tramper, J. \u0026amp; Rinzema, A. Effect of low oxygen concentrations on growth and α-amylase production of \u003cem\u003eAspergillus oryzae\u003c/em\u003e in model solid-state fermentation systems. \u003cem\u003eBiomol. Eng.\u003c/em\u003e 21, 163\u0026ndash;172 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshok, A., Doriya, K., Rao, D. R. M. \u0026amp; Kumar, D. S. Design of solid-state bioreactor for industrial applications: an overview of conventional bioreactors. \u003cem\u003eBiocatal. Agric. Biotechnol.\u003c/em\u003e 9, 11\u0026ndash;18 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, L., Liu, Y. \u0026amp; Chen, H. Z. Advances in porous characteristics of the solid matrix in solid-state fermentation. In \u003cem\u003eCurrent Developments in Biotechnology and Bioengineering: Current Advances in Solid-State Fermentation\u003c/em\u003e 19\u0026ndash;29 (Elsevier, 2018). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/B978-0-444-63990-5.00002-5\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-444-63990-5.00002-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahardjo, Y. S. P. \u003cem\u003eFungal mats in solid-state fermentation\u003c/em\u003e. (Wageningen University, Wageningen, The Netherlands, 2005). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18174/121648\u003c/span\u003e\u003cspan address=\"10.18174/121648\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOostra, J. \u003cem\u003eet al.\u003c/em\u003e Intra-particle oxygen diffusion limitation in solid-state fermentation. \u003cem\u003eBiotechnol. Bioeng.\u003c/em\u003e 75, 13\u0026ndash;24 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRahardjo, Y. S. P., Tramper, J. \u0026amp; Rinzema, A. Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives. \u003cem\u003eBiotechnol. Adv.\u003c/em\u003e 24, 161\u0026ndash;179 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVauris, A., Valcauda, S., Husson, F. \u0026amp; De Coninck, J. A novel method to assess heat transfer and impact of relevant physicochemical parameters for the scaling up of solid-state fermentation systems. \u003cem\u003eBiotechnol. Rep.\u003c/em\u003e 36, e00764 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartau, G. A., Mihai, M. \u0026amp; Vodnar, D. C. The use of chitosan, alginate and pectin in the biomedical and food sector: biocompatibility, bioadhesiveness and biodegradability. \u003cem\u003ePolymers\u003c/em\u003e 11, 1837 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImeson, A. \u003cem\u003eFood Stabilisers, Thickeners and Gelling Agents\u003c/em\u003e. (Wiley-Blackwell, 2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAi, Y. \u0026amp; Jane, J. L. Gelatinization and rheological properties of starch. \u003cem\u003eStarch/St\u0026auml;rke\u003c/em\u003e 67, 213\u0026ndash;224 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThakur, B. R., Singh, R. K. \u0026amp; Handa, A. K. Chemistry and uses of pectin: a review. \u003cem\u003eCrit. Rev. Food Sci. Nutr.\u003c/em\u003e 37, 47\u0026ndash;73 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInbaraj, S. \u003cem\u003eet al.\u003c/em\u003e Pectin hydrogels: gel-forming behaviors, mechanisms and food applications. \u003cem\u003eGels\u003c/em\u003e 9, 732 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNicolai, T. \u0026amp; Chassenieux, C. Heat-induced gelation of plant globulins. \u003cem\u003eCurr. Opin. Food Sci.\u003c/em\u003e 27, 18\u0026ndash;22 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHettiarachchy, N., Kannan, A., Sch\u0026auml;fer, C. \u0026amp; Wagner, G. Gelling of plant-based proteins. In \u003cem\u003eProduct Design and Engineering: Formulation of Gels and Pastes\u003c/em\u003e 221\u0026ndash;245 (Wiley, 2013). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/9783527654741.ch8\u003c/span\u003e\u003cspan address=\"10.1002/9783527654741.ch8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHui, D. \u003cem\u003eet al.\u003c/em\u003e A review of gelation of plant proteins and their influencing factors. \u003cem\u003eSustain. Food Proteins\u003c/em\u003e 3, e70037 (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlomfeldt, T. O. J., Kuktaite, R., Johansson, E. \u0026amp; Hedenqvist, M. S. Mechanical properties and network structure of wheat gluten foams. \u003cem\u003eBiomacromolecules\u003c/em\u003e 12, 1707\u0026ndash;1715 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlomfeldt, T. O. J. \u003cem\u003eet al.\u003c/em\u003e Novel foams based on freeze-dried renewable vital wheat gluten. \u003cem\u003eMacromol. Mater. Eng.\u003c/em\u003e 295, 796\u0026ndash;801 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVaezi, M., Zhong, G., Kalami, H. \u0026amp; Yang, S. Extrusion-based 3D printing technologies for 3D scaffold engineering. In \u003cem\u003eFunctional 3D Tissue Engineering Scaffolds\u003c/em\u003e 235\u0026ndash;254 (Elsevier, 2018). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/B978-0-08-100979-6.00010-0\u003c/span\u003e\u003cspan address=\"10.1016/B978-0-08-100979-6.00010-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNussbaum, N. \u003cem\u003eImpact of host material on fungal growth: investigating nutrient density and physical properties\u003c/em\u003e. (ETH Z\u0026uuml;rich, Z\u0026uuml;rich, Switzerland, 2025). doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3929/ETHZ-C-000785036\u003c/span\u003e\u003cspan address=\"10.3929/ETHZ-C-000785036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGantenbein, S. \u003cem\u003eet al.\u003c/em\u003e Three-dimensional printing of mycelium hydrogels into living complex materials. \u003cem\u003eNat. Mater.\u003c/em\u003e 22, 128\u0026ndash;134 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, J. Y., Pandya, J. K., McClements, D. J., Lu, J. \u0026amp; Kinchla, A. J. Advancements in 3D food printing: a comprehensive overview of properties and opportunities. \u003cem\u003eCrit. Rev. Food Sci. Nutr.\u003c/em\u003e 62, 4752\u0026ndash;4768 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe-Bail, A., Maniglia, B. C. \u0026amp; Le-Bail, P. Recent advances and future perspective in additive manufacturing of foods based on 3D printing. \u003cem\u003eCurr. Opin. Food Sci.\u003c/em\u003e 35, 54\u0026ndash;64 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNachal, N., Moses, J. A., Karthik, P. \u0026amp; Anandharamakrishnan, C. Applications of 3D printing in food processing. \u003cem\u003eFood Eng. Rev.\u003c/em\u003e 11, 123\u0026ndash;141 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong, Y., Zhou, J. \u0026amp; Yao, D. Porogen templating processes: an overview. \u003cem\u003eJ. Manuf. Sci. Eng.\u003c/em\u003e 136, 010801 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H. \u0026amp; Cooper, A. I. Synthesis and applications of emulsion-templated porous materials. \u003cem\u003eChem. Soc. Rev.\u003c/em\u003e 34, 793\u0026ndash;803 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDehghani, F. \u0026amp; Annabi, N. Engineering porous scaffolds using gas-based techniques. \u003cem\u003eCurr. Opin. Biotechnol.\u003c/em\u003e 22, 661\u0026ndash;666 (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8618445/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8618445/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFungal solid-state fermentation can improve the nutritional quality of plant-based foods, yet most processes rely on substrates whose properties are inherited from raw materials rather than intentionally designed. This perspective addresses this limitation by reframing the substrate as an active process component. Integrating biochemical, mechanical, and architectural principles into substrate design enables the development of engineered substrates that promote efficient nutrient bioconversion in fermented foods.\u003c/p\u003e","manuscriptTitle":"Reframing the substrate as an active process component in fungal solid-state fermentation of foods","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 15:19:11","doi":"10.21203/rs.3.rs-8618445/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-18T04:14:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-17T20:11:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-10T23:43:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"247083512035192956187537980408830499513","date":"2026-01-27T12:26:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299807154262386194140010239440704006100","date":"2026-01-26T05:30:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-22T06:23:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-21T04:35:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T04:32:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Science of Food","date":"2026-01-16T10:55:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"201db56c-35cb-47fa-8bb3-fbd69e2dcd4e","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":61555414,"name":"Biological sciences/Biochemistry"},{"id":61555415,"name":"Biological sciences/Biotechnology"},{"id":61555416,"name":"Physical sciences/Engineering"},{"id":61555417,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-07T16:02:44+00:00","versionOfRecord":{"articleIdentity":"rs-8618445","link":"https://doi.org/10.1038/s41538-026-00827-2","journal":{"identity":"npj-science-of-food","isVorOnly":false,"title":"npj Science of Food"},"publishedOn":"2026-04-04 15:58:34","publishedOnDateReadable":"April 4th, 2026"},"versionCreatedAt":"2026-01-23 15:19:11","video":"","vorDoi":"10.1038/s41538-026-00827-2","vorDoiUrl":"https://doi.org/10.1038/s41538-026-00827-2","workflowStages":[]},"version":"v1","identity":"rs-8618445","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8618445","identity":"rs-8618445","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}