Development of new meat analogues from filamentous fungi cultivated on oenological by-products: A quality perspective

Development of new meat analogues from filamentous fungi cultivated on oenological by-products: A quality perspective Luziana Hoxha a,b , Ivana Sucic c , Mohammad J. Taherzadeh b,* , Matteo Marangon a a Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro, PD, Italy b Swedish Centre for Resource Recovery, University of Borås, 50190 Borås, Sweden c Brenntag S.p.A., 35127 Padova, Italy ARTICLE INFO Keywords: Edible filamentous fungi Grape marc Wine lees Meat analogues Quality attributes ABSTRACT Interest in the link between diet, health, and sustainable nutrition has grown, driving exploration of alternative proteins. Edible filamentous fungi offer a promising high-quality protein source for meat analogues. This study explores Neurospora intermedia biomass, cultivated on grape marc and wine lees produced via submerged fermentation in a demo-scale bubble column reactor, to create clean-label, vegan, and gluten-free meat analogue balls (MABs). MABs were formulated with 21.4 % (w/w) fungal protein and compared with pea-based textured vegetable protein. Protein sources were evaluated for techno-functional properties, and MABs were assessed for cooking characteristics, nutritional value, color, texture, microstructure, and sensory attributes. Fungal proteins exhibited high water (5.92–6.51 g/g) and oil (up to 5.81 g/g) absorption capacities. Fungi-based MABs had a cooking efficiency of 86.5–87.6 % and crude protein content up to 37 % on a dry basis. Texture profile analysis showed improved cohesiveness (0.63–2.23), and springiness (0.55–2.45) compared to those made from peabased texturized proteins. Scanning electron microscopy confirmed a fibrous microstructure. Sensory evaluation under blind conditions by untrained panelists highlighted juiciness, a fibrous texture, and a fungior meataroma mimicry. These results support the potential of Neurospora intermedia biomass grown on oenological byproducts as a nutritious, functional protein source for next-generation meat analogues. The study has strong potential to contribute to the circular bioeconomy fields with broader sustainability implications. 1. Introduction With growing awareness of the environmental and health impacts of meat consumption, sustainable protein alternatives have emerged as a promising solution. These options generate up to 250 times fewer emissions than animal-based products (da Silva, Mateus, de Freitas, & Fernandes, 2024). The shift toward alternative proteins is also fueled by global protein shortages, ethical and dietary restrictions (e.g., halal, kosher, vegan, vegetarian), climate change, and economic and resource challenges (Akcan, ¨ Onel, & Ergezer, 2024; Alexander et al., 2017). One of the rapidly growing sectors in the food industry is the meat analogue sector, which incorporates a variety of alternative protein sources. Meat analogues, also known as imitation meat, meat substitutes, mimic meat, or meat replacements, are designed to replicate the taste, texture, and appearance of conventional animal-based meat (Ahmad et al., 2022). These products are available in both raw and precooked formats, including minced meat, strips, chunks, chicken-like blocks, ground beef alternatives, steaks, ham, mortadella, sausages, burgers, patties, frankfurters, nuggets, meatballs, spreads, and even seafood alternatives (Kyriakopoulou, Keppler, & van der Goot, 2021). The broad spectrum of alternative protein sources includes plantbased proteins, single-cell proteins (SCPs) derived from microbes such as bacteria, yeast, fungi, as well as microalgae, seaweed, edible insects, animal stem cells, and precision fermentation products (Alexander et al., 2017; Boukid & Gagaoua, 2022; Hashempour-Baltork, Khosravi-Darani, Hosseini, Farshi, & Reihani, 2020). Among these, microbial proteins, used as whole biomass or extracted protein, have garnered significant attention for their potential in food applications (Gnaim, Dyer, & Ledesma-Amaro, 2025; Wikandari, Tanugraha, Yastanto, Manikharda, & Teixeira, 2023). Mycoprotein-based products, rich in protein and fiber and low in fat, are linked to health benefits, including reduced energy intake and improved appetite regulation (Derbyshire & Delange, 2021). These products are particularly appealing to health-conscious consumers and those following vegetarian, vegan, or flexitarian diets (T. * Corresponding author. E-mail address: [email protected] (M.J. Taherzadeh). Contents lists available at ScienceDirect Innovative Food Science and Emerging Technologies journal homepage: www.elsevier.com/locate/ifset https://doi.org/10.1016/j.ifset.2025.104409 Received 15 August 2025; Received in revised form 18 November 2025; Accepted 5 December 2025 Innovative Food Science and Emerging Technologies 108 (2026) 104409 Available online 6 December 2025 1466-8564/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). Finnigan, Mach, & Edlin, 2024). A key priority in alternative protein production is applying circular economy principles by upcycling agri-food byproducts. Many of these byproducts, such as rice bran, spent brewery grain, wheat straw, potato pulp, apple pomace, banana, and citrus wastes, pineapple peel, molasses, cheese whey, prawn shells, pea byproducts, and mixed-food industry residues, have been valorized for protein production via fungal fermentation (Landeta-Salgado et al., 2024; Majumder, Miatur, Saha, & Hossain, 2024; Ritala, H¨ akkinen, Toivari, & Wiebe, 2017; Salvatore, Leue-Rüegg, Beretta, & Müller, 2024; Upcraft et al., 2021). Particularly, wine and distillery byproducts, which have a significant environmental impact and contribute to the global carbon footprint, can be transformed into protein-rich fungal biomass through fungal fermentation (Hoxha, Taherzadeh, & Marangon, 2025). However, fungal products derived from agri-food side-streams bioconversion must demonstrate a verified safety profile to gain approval in Europe, emphasizing the importance of thorough risk assessment (Ballester, Roqu´ e, Ricci-Cabello, Rotger, & Malih, 2023). Beyond safety, consumer perception and acceptance are critical when introducing fungal biomass into the human diet, particularly from side-stream sources (Chezan, Flannery, & Patel, 2022). Several fungal-derived products are already commercially available for food applications (Souza et al., 2025). Among them, mycoproteins are receiving growing attention, particularly used in meat alternatives such as burgers, mince, and marinated pieces (Salvatore et al., 2024). Currently, mycoproteins intended for human consumption are primarily produced from filamentous fungi of the genera Fusarium, Aspergillus, and Rhizopus (T. Finnigan et al., 2024; Gnaim et al., 2025). However, ongoing research continues to explore new fungal species with the potential to upcycle agri-food by-products without depending on synthetic glucose sources (Upcraft et al., 2021). One promising specie is Neurospora intermedia, a fast-growing edible filamentous fungus that has demonstrated significant potential for converting waste into food (Gmoser et al., 2020; Hellwig, Gmoser, Lundin, Taherzadeh, & Rousta, 2020; Maini Rekdal et al., 2023, 2024; Starzynska-Janiszewska, Stodolak, Dulinski, Mickowska, & Sabat, 2017). Traditionally, N. intermedia has been used in Indonesia for producing the meat substitute Oncom. Due to the absence of consumption history in the EU prior to May 1997, N. intermedia is likely considered a novel food under EU regulations and would require thorough assessment both for its quality and safety authorization before it can be authorized for commercialization in the region (European Commission, 2015). Clinical trials have been essential in demonstrating digestibility and safety before wider release. Mycoprotein is recognized as a safe food ingredient (Miller & Dwyer, 2001), and no evidence suggests adverse effects from Neurospora in humans or animals (Perkins & Davis, 2000). Furthermore, recent research supports its promising safety profile and its potential to expand gastronomic applications (Maini Rekdal et al., 2023, 2024). Given the growing popularity of mycoprotein-based foods as sustainable alternatives to animal-derived proteins, this study aimed to explore the use of edible Neurospora intermedia biomass, cultivated on grape marc and wine lees through submerged fermentation in a demonstration-scale bioreactor, as a novel protein ingredient in vegan and gluten-free meat analogue formulations. To the best of the authors’ knowledge, this represents the first investigation of this edible fungal biomass grown on oenological by-products for such applications. A comprehensive evaluation of key quality attributes was carried out on the resulting meat analogue balls (MABs), and their performance was benchmarked against a widely used commercial plant-based protein, dried pea-based texturized vegetable protein (TVP), using standardized formulations to assess relative suitability and potential advantages. More broadly, the study aimed to contribute to the advancement of alternative proteins within a circular bioeconomy framework by valorizing underutilized winemaking and distillery by-products. 2. Materials and methods 2.1. Oenological by-products and filamentous fungus strain Oenological by-products were collected from Acquavite distillery in Vazzola, Italy: grape marc (GM) in November 2023 and wine lees (WL) in March 2024. The GM was derived from grapes grown in the Veneto region grapes, primarily Glera skins (~80 %), and was processed through seed removal, drying, and milling after fermentation and distillation. The WL originated from the subsequent wine produced from the same regional sources, and consisted of a mixture of gross and fine lees from Prosecco production. For cultivation, a 4 % (w/v) GM liquor, obtained via hydrothermal pretreatment at 121 ◦C for 20 min, was used as liquid cultivation media. The WL were centrifuged, and the resulting liquid fraction was diluted to 50 % (v/v) prior to being used as cultivation media. Both GM and WL media were supplemented with 5 g/L yeast extract and adjusted to pH 5 using 5 M NaOH (Sigma-Aldrich, Darmstadt, Germany). The edible filamentous fungus Neurospora intermedia (CBS 131.92) was used, sourced from the Westerdijk Fungal Biodiversity Institute (Utrecht, Netherlands). Fungal cultivations were conducted in a 1300 L bubble column bioreactor (Knislinge Mekaniska Verkstad AB, Kristianstad, Sweden) with 1000 L working volume. The procedures for fungal biomass cultivation and harvesting, followed those described in our previous work (Hoxha, Lennartsson, & Taherzadeh, 2025). Approximately 1 L pre-culture of N. intermedia spores was prepared in GM or WL media in baffled Erlenmeyer flasks and incubated at 35 ◦C with shaking at 115 rpm for 24 h in waterbath (Grant OLS-Aqua Pro, UK). The preculture was transferred to a 26 L BCR (Bioengineering, Wald, Switzerland) containing 20 L of the cultivation media and incubated at 35 ◦C with 1 vvm (volume of air per volume of liquid per minute) aeration for 24 h. The resulting biomass suspension was transferred to the 1300 L BCR, where cultivation was incubated at 35 ◦C with 1 vvm aeration for 24 h. Harvested fungal biomass was washed three times with tap water, to remove residual cultivation medium (Campos, Wahalathanthrige, Russell, Harrison, & Strong, 2025), and pressed using a 12 L juice press (Bauhaus, Belp, Switzerland), then freeze-dried at 0.022 mbar with a condenser temperature of −84 ◦C (Labconco FreeZone, Thermo Fisher Scientific Inc.). Biomass yield was determined gravimetrically, resulting in 3.290 ±0.001 g/L for GM (Hoxha, Lennartsson, & Taherzadeh, 2025) and 4.33 ±0.14 g/L for WL (unpublished data). 2.2. Preparation of meat analogue balls In this study, meat analogue balls (MABs) were prepared using 21.4 % (w/w) of three alternative protein sources: freeze-dried Neurospora intermedia biomass grown on grape marc (GM) and wine lees (WL), and dried pea-based textured vegetable protein (TVP). This inclusion level was determined through preliminary trials evaluating texture, as well as moisture absorption and retention, and was subsequently optimized to maintain juiciness, and prevent dryness (data not shown). Mycoprotein was benchmarked against pea TVP, a widely used ingredient in commercial meat analogues and the closest nutritional match to mycoprotein-based products (Loˇ znjak ˇ Svarc et al., 2022). Pea-based TVP is increasingly favored due to the demand for soy-free protein alternatives, known as a common allergen (Meinlschmidt, Schweiggert-Weisz, Brode, & Eisner, 2016), associated with GMO-related concerns, presence of anti-nutritional factors that can reduce digestibility, and may negatively influence consumer taste perceptions (Jafarzadeh et al., 2024). The MABs preparation flow diagram is illustrated in Fig. 1. Protein ingredients were ground using a high-shear thermo-mixer (HotmixPro Gastro Master) at 3000 rpm for 30 s, then transferred to a planetary mixer (Hobart N50) for rehydration with an infusion solution containing 42.0 % (w/w) cold water (4 ◦C) and 2.65 % (w/w) clean-label spices, flavorings, and colorants (Brenntag S.p.A., Padova, Italy; Table S1). L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 2 Additionally, 0.80 % salt was added. Once the infusion solution was fully absorbed by the protein base, 20.7 % (w/w) pre-emulsion was added. The pre-emulsion was prepared by dispersing 7 % methyl cellulose in 80 % cold water (4 ◦C) and mixing at 1800 rpm for 40 s using a high-shear thermo-mixer (HotmixPro Gastro Master), followed by the addition of 13 % sunflower oil and mixing for 1 min at 1800 rpm to obtain a semi-solid consistency. It was stored at 4 ◦C and used within 24 h. To improve gel strength and optimize texture, the formulation included 6.5 % (w/w) stabilizing solution (glutinous rice flour and bamboo fiber) alongside methylcellulose, which together acted synergistically to enhance water binding (Liu et al., 2023). Sunflower oil (5.75 %, w/w) was added to improve juiciness and replicate the fat content of conventional meat. The resulting meat analogue dough was manually shaped into ~20 g balls. Samples were placed on aluminum trays and stored at 4 ◦C until analysis or cooking. Each batch, prepared in triplicate, was cooked in a commercial hot air fryer (COSORI, model CAF-R903) at 180 ◦C for 5 min (Bakhsh et al., 2021). Compared to conventional frying, air frying is known to limit thermal oxidation, reduce acrylamide and polar compound formation, maintain color and texture, preserve fatty acids, essential amino acids, and phenolic compounds, and enhance protein digestibility (Baskaya-Sezer, 2025). Within 24 h of preparation, cooking efficiency, moisture content, color, pH, water activity (a w ), and texture were measured. Samples were freeze-dried for proximate composition, mineral content, and microstructure analysis. 2.3. Analysis 2.3.1. Proximate and mineral analysis Proximate and mineral analysis of raw and cooked products were performed following AOAC (2016) methods. Moisture (AOAC 934.01), ash (AOAC 942.05), crude protein (Kjeldahl method, AOAC 2001.11) using a Kjeltec™ 8400 system with a nitrogen conversion factor of 6.25, and crude fat (AOAC 2003.05) by diethyl ether extraction were determined. Minerals, including phosphorus, zinc, copper, magnesium, iron, calcium, and manganese were analyzed by Inductively Coupled Plasma–Optical Emission Spectroscopy (ICP-OES) according to AOAC 985.01, 2006.03, and 2011.14, with quantification based on calibration curves from certified standards. Carbohydrate content was calculated by difference (De Angelis et al., 2020) and energy values estimated using Atwater factors (European Commission, 2025). For the proximate analyses three meat analogue balls were randomly selected from the same Fig. 1. A) Flow diagram illustrating the processing of meat analogue balls (MABs) formulated with alternative protein sources, including pea-based textured vegetable protein (TVP) and Neurospora intermedia biomass cultivated on grape marc (GM) or wine lees (WL). B) Schematic illustration of the cooking process for MABs, cooked at 180 ◦C for 5 min using a commercial hot air fryer. L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 3 dough formulated, and mean value of three replicates (n =3) was calculated. 2.3.2. Physico-chemical properties Color, pH, and water activity (a w ) were measured in both raw and cooked meat analogue balls. Color parameters (L*, a*, b*) were recorded using a portable colorimeter (CR-410, Konica-Minolta) calibrated with standard illuminant D65. Multiple surface points were averaged to obtain L* (brightness), a* (green–red), and b* (blue–yellow) values. pH was measured in triplicate using a calibrated portable pH meter (HANNA Checker Plus) by inserting the probe into the product center. Water activity was measured at 25 ◦C with a water activity meter (LabMaster.aw, Novasina AG). 2.3.3. Techno-functional properties of protein ingredients and cooking characteristics of MABs Water absorption capacity (WAC), oil absorption capacity (OAC), and rehydration capacity (RHC) of protein ingredients were assessed as per Hong, Shen, and Li (2022). For WAC, 0.6 g of ground TVP or fungal biomass was mixed with 10 mL of Milli-Q water in a pre-weighed tube, vortexed, left at room temperature for 5 min, centrifuged (3000 ×g, 30 min), drained, and reweighed. For OAC, 1 g of sample was treated similarly but rested for 30 min, with excess oil drained postcentrifugation. For RHC, protein ingredients were soaked in water (1:15 w/v) for 2 h, then drained for 1 h. Calculations were based on the Eqs. 1 and 2: WAC or OAC (g g)=m2−m1−m0 m0 (1) where m₀ is the mass of the dry protein sample (g), m₁ is the mass of the empty container (g), and m₂ is the mass of the container with the protein sample and absorbed liquid (water or oil) (g). RHC (g g)=weight after rehydration −weight before rehydration weight before rehydration (2) WAC and RHC were expressed as grams of water per gram of protein sample, and OAC as grams of oil per gram of protein sample. All tests were in triplicate (n =3). Cooking characteristics of meat analogues balls (MABs) including cooking yield, moisture and fat retention, and diameter reduction were analyzed following Akcan et al. (2024) (Eqs. 3 to 6): Cooking yield (%) = (Cooked Weight (g) ) (Raw Weight (g) ) ×100 (6) 2.3.4. Texture profile analysis of MABs Texture profile analysis (TPA) was conducted on individual MABs (~20 g each, n =3). Raw MABs, packed in partial vacuum, stored at 4 ◦C were analyzed within 24 h of preparation. Cooked MABs (at 180 ◦C for 5 min) were allowed to rest for 30 s at room temperature (RT) before TPA analysis. All measurements were performed at RT using a TA.XTplus Texture Analyzer (Stable Micro Systems, UK) equipped with a 50 kg load cell and an HDP/PFS flat probe, operated via Exponent software (v6.1.3.0). Each sample underwent a double compression test adapted from Sun et al. (2022): pre-test speed 5 mm/s, test speed 1 mm/s. A 40 % strain was selected as produced meaningful structural changes and reproducible TPA properties values (hardness, cohesiveness, adhesiveness, springiness, and resilience), whereas higher strain levels caused sample rupture (data not shown). Texture attributes were measured in triplicate. 2.3.5. Microstructure analysis Microstructure analysis of freeze-dried samples was performed using Environmental Scanning Electron Microscopy (ESEM; FEI Quanta 200, USA) in low vacuum mode (0.5 Torr) at 20 kV with a tungsten filament. Samples (~10 mm ×5 mm ×5 mm) were mounted on adhesive-coated aluminum stubs. Elemental microanalyses were conducted via Energy Dispersive X-ray Spectroscopy (EDS) using an Oxford Xplore 30 detector and AZtec 6.0 software (Oxford Instruments, UK). Prior to establishing the final protocol using ESEM in low vacuum mode, both untreated and freeze-dried samples were evaluated, and no significant microstructural differences were observed (data not shown). Although low vacuum mode allows observation of hydrated samples, the required time to reach the low vacuum working conditions for multiple samples together was considerably longer. To improve efficiency and ensure consistency, freeze-dried samples were used, avoiding further treatment. 2.4. Sensory evaluation Fifty-two untrained panelists (26 female, 26 male; aged 18–70) participated in the sensory evaluation. Under blind, non-tasting conditions, they rated appearance, odor, texture, and overall liking on a 0–5 scale. Three types of meat analogue balls, one formulated with textured vegetable protein (TVP) and two with fungal biomass grown on wine lees (WL) and grape marc (GM), were air-fried and served hot. Samples were presented individually in sensory booths, identified by randomized three-digit codes and evaluated at room temperature (ISO 8589, 2007). Demographic data (age, gender, nationality) were also collected. For the sensory evaluation each type of cooked meat analogue ball was presented to each panelist, and mean values were calculated (n =52). 2.5. Statistical analysis Results are presented as mean ±SD (n =3, unless otherwise specified). Statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test. Pearson correlation analysis assessed relationships between variables. Significance was set at p <0.05. All analyses were performed using OriginPro 2025 (OriginLab, USA). Diameter reduction (%) = Raw sample diameter (mm) − Cooked Sample diameter (mm) Raw sample diameter (mm)×100 (3) Fat Retention (%) = (Cooked Weight (g) × %Fat in Cooked Sample ) (Raw Weight (g) × %Fat in Raw Sample )×100 (4) Moisture Retention (%) = (Cooked Weight (g) × %Moisture in Cooked Sample ) (Raw Weight (g) × %Moisture in Raw Sample )×100 (5) L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 4 Fig. 2. Main composition including A) proximate composition, B) essential minerals and C) color values of raw and cooked meat analogue balls (MABs) prepared with three alternative protein sources: TVP formulated with pea-based texturized vegetable protein; WL formulated with N. intermedia biomass cultivated on wine lees; GM formulated with N. intermedia biomass cultivated on grape marc. Mean ±standard deviation (n =3), with different superscript letters for the same parameter indicating significant differences (p <0.05) among MABs samples according to Tukey’s test. L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 5 3. Results and discussions 3.1. Nutritional quality of meat analogue balls This study evaluated key quality attributes to guide the screening and optimization of new meat analogue balls (MABs) formulations, minimally processed, and with clean-label ingredients, designed to meet growing consumer demand for healthy diets. 3.1.1. Proximate composition of MABs Proximate composition and mineral content for raw and cooked MABs are presented in Fig. 2 (A-C). Protein-rich raw materials are crucial for developing the fibrous structure characteristic of meat analogues (Bulgaru et al., 2025). Meat analogues typically contain 4–25 % alternative proteins from legumes, grains, mushrooms, or mycoprotein (Kyriakopoulou et al., 2021). In this study, 21.4 % (w/w) of protein ingredient was used, either as pea-based TVP (65 % crude protein, unpublished data) or Neurospora intermedia biomass grown on wine lees (61.4 % crude protein, unpublished data) and grape marc (45.4 % crude protein as per Hoxha, Lennartsson, and Taherzadeh (2025). Crude protein content in raw MABs ranged 30.7–35.0 % dry basis (db). The GM sample was statistically different (p <0.05), whereas no significant difference was observed between the TVP and WL samples. After cooking, crude protein increased to 36.98–38.62 % db. Cooked TVP and GM samples were statistically different (p <0.05), while WL sample, did not differ significantly from either samples (Fig. 2A). Additionally, 100 g of dried sample (equivalent to a 204–216 g serving of the cooked product) could provide over 71 % of the recommended daily protein intake (50 g/day) of an average adult (2000 kcal) according to Regulation (EU) No 1169/2011 (European Commission, 2025), meeting the adult requirement of 0.8 g per kg of body weight (WHO, 2007). Fungi-based products in this study offer protein content comparable to leading commercial meat analogues such as Beyond Meat™ (15.7 g per 100 g serving) (USDA, 2022) and Quorn™ (19.1 g per 100 g serving) (USDA, 2017). Unlike commercial meat analogues which contain egg white a known allergen e.g., Quorn™ (USDA, 2017), the fungi-based MABs in this study are formulated with allergen-free ingredients. Therefore, these fungi-based MABs offer a broader consumer appeal, including individuals with dietary restrictions such as vegans. However, further studies are needed to evaluate their safety, toxicological and allergenic potential, and consumer acceptance, although no adverse effects of Neurospora have been reported in humans or animals (Maini Rekdal et al., 2024; Perkins & Davis, 2000). Moreover, fungi-based MABs offer sustainability benefits, as fungal protein is produced by upcycling byproducts from the wine and distillery industries. In general, alternative proteins are more cost-competitive and sustainable than animal-based ones (Munialo, Baeghbali, & Acharya, 2025), while significantly reducing greenhouse gas emissions (by 89–93 %) and land use (by 97 %) (Saget et al., 2021). In light of the study’s limitation regarding the absence of direct comparisons with animal-based meat, future research should benchmark composition, texture, cooking loss, color, and microstructure to more accurately evaluate how closely these analogues mimic conventional meatballs’ nutritional, structural, and sensory properties. Additionally, future research should explore N. intermedia potential to upcycle other side streams, creation of new food formulations with diverse protein sources, and provide comprehensive comparisons with both conventional meatballs and other novel alternatives. Crude fat content in raw MABs ranged 21.9–22.8 % (db), with no significant differences between samples, as expected given that the identical recipe was used. Generally, fat values fell within the typical range for meat analogues containing pre-emulsions (Kyriakopoulou et al., 2021). Pre-emulsion is a functional ingredient, which can influence the nutritional composition, along with texture, pH, color, waterholding capacity, and cooking loss without significantly altering sensory quality (Kothuri et al., 2025). After cooking, fat content of MABs decreased to 19.8–20.8 % db, with significant differences observed between WL and GM samples, while TVP fell between these groups (p < 0.05). These differences may be attributed to the type of protein ingredient used. Specifically, MABs formulated with fungal biomass grown on grape marc showed higher oil retention capacity (91.7 ±2.46 %) compared to those made with fungal biomass grown on wine lees (86.9 ±4.38 %) (Fig. 2A), as further discussed in the following sections. In this study, MABs contained comparable fat levels with meat analogues of Beyond Meat™ (17.4 g per 100 g serving) (USDA, 2022), but significantly higher than Quorn™ (2.21 g per 100 g serving) (USDA, 2017). The product’s fat content exceeds 28 % of the nutrient reference value (70 g/day) of an average adult intake references (2000 kcal/day) (European Commission, 2025). Fat correlated strongly with energy and ash content (r >0.82, p <0.01; Table S2a), indicating that fat-rich ingredients and seasonings (e.g., salt or flavorings) elevate calories and modulate mineral content through interactions with other components, particularly with proteins. Ash content varied by protein source, with fungi-based products showing significantly higher levels (5.11–5.73 % db) than TVP-based products (4.24 % db). After cooking, ash content decreased to 3.37–3.65 % db, due to thermal processing, which can alter food matrix, influence mineral speciation and bioavailability (Barciela-Alonso & Bermejo-Barrera, 2015). Cooked TVP and GM samples did not differ significantly, while the WL was intermediate (p <0.05). Overall, cooking led to a reduction in ash and fat levels, but increased protein content (Fig. S2), partially reflecting trends reported by Akcan et al. (2024), who observed proportional increases in protein, fat, and ash contents after cooking. The product’s total carbohydrates (up to 41 % db; Fig. 2A) and energy (up to 493 kcal/100 g db; Table S3) exceed 14.1 % and 24 % of the nutrient reference value (260 g/day and 2000 kcal/day) of an average adult intake references (2000 kcal/day), respectively (European Commission, 2025). Although dietary fiber was not measured in this study, mycoprotein is classified as ‘high fiber’ by the European Commission, providing ≥6 g per 100 g (European Parliament and Council, 2014), owing to its glucan-rich fungal cell walls. Its dietary fiber profile comprises approximately 12 % soluble and 88 % insoluble fibers, with a minor fraction of chitin and a predominance of glucans (Majumder et al., 2024). 3.1.2. Essential mineral content of MABs The essential mineral content analysis of the MABs revealed significant variation influenced by both the protein source and the cooking process (Fig. 2B), affecting nutritional quality. The fungi-based products demonstrated higher levels of essential minerals compared to those formulated with the TVP. These findings suggest that fungal protein derived from oenological by-products has potential for the development of functional meat alternatives targeting micronutrient deficiencies. Phosphorus (P) content was highest in raw WL products (1488.2 mg/ 100 g, db), followed by GM (798.9 mg/100 g, db) and TVP (620.5 mg/ 100 g, db). Cooking led to a notable decrease in P content, particularly in WL and TVP products (p <0.05). Although GM products experienced a slight reduction after cooking (724.5 mg/100 g, db), they still met the recommended intake of 700 mg/day of an average adult (2000 kcal) (European Commission, 2025). Calcium (Ca) was most abundant in raw GM products (391.2 mg/ 100 g, db), followed by TVP and WL. However, Ca levels declined significantly after cooking, and no significant differences were observed between TVP-based and fungi-based MABs post-cooking (p <0.05). Magnesium (Mg) content was highest in raw WL products (139.73 mg/ 100 g, db). Cooking caused a significant reduction in Mg levels across all MABs, GM cooked products had the highest levels (p <0.05). Iron (Fe) content was significantly higher in GM products, in both raw and cooked forms. Post-cooking was observed a decrease in Fe levels and no significant differences observed among products. Zinc (Zn) was most abundant in fungi-based MABs, particularly raw L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 6 WL products had significantly highest value (20.6 mg/100 g, db) (p < 0.05). However, Zn decreased substantially upon cooking, and significant differences were observed across samples (p <0.05). Finally, copper (Cu) and manganese (Mn) levels were highest in raw GM products (8.68 and 3.74 mg/100 g, db), followed by WL and TVP (p <0.05). Both minerals decreased significantly after cooking, with no significant differences among the cooked fungi-based MABs (p <0.05). The observed variations in mineral content (Fig. S2) after cooking are likely due to differences in how minerals are bound within the food matrix and their susceptibility to leaching, oxidation, or other physicochemical heat-induced changes, including the release of metal ions from proteins due to the reduction of available carboxylic groups (G´ omez, Iba˜ nez, & Beriain, 2019). Cooking can also lead to moisture loss, protein unfolding, fiber softening, and cellular disruption, all of which influence mineral retention (Vu, Zhou, & McClements, 2022). These findings highlight the need for further research into mineral accessibility, as well as further optimization of cooking methods. Generally, fungi-based MABs may meaningfully contribute to the recommended daily intake of essential minerals of an average adult (2000 kcal) (European Commission, 2025), with cooked products containing 823.63–936.24 mg/100 g db of total essential mineral content, offering a nutritional advantage over TVP-based MABs, which provided 700.77 mg/100 g db (Table S3). 3.2. Physico-chemical properties of meat analogue balls: Color, pH, and water activity Color is a critical factor in consumer acceptance and product selection, which varies based on the characteristics of added ingredients (Akcan et al., 2024). To mimic meat, this study’s MABs formulation incorporated 0.3 % (w/w) beetroot extract, an ingredient, which is used by leading commercial brands of meat alternatives such as Beyond Meat™ (USDA, 2022) and Impossible Foods™ (Kyriakopoulou et al., 2021). The L* (brightness) values ranged 40.88–54.72 in raw MABs and increased after cooking to 43.97–59.66, consistently higher in TVP products (Fig. 2C). Color is affected by both the intrinsic color of ingredients and processing factors (Corrˆ ea, da, Ferreira, & Guerra, 2023). Brightness is primarily influenced by water and fat content, and the inclusion of beetroot extract in the formulations is supposed to further enhance L* values (Gamarra-Castillo, Echeverry-Monta˜ na, MarbelloSantrich, Hern´ andez-Carri´ on, & Restrepo, 2022). Significant differences in color parameters were observed between TVP-based and fungi-based MABs (p <0.05). TVP samples exhibited significantly higher a* (redness) and b* (yellowness) values compared to fungi-based MABs (Fig. 2C). Raw products with higher fat content showed significantly lower red and yellow tones (p <0.05). Furthermore, increased ash levels in raw products were significantly correlated with decreased brightness, redness, and yellowness values (p <0.01, Table S2b). Color changes after cooking varied by product type: brightness (L*) increased in TVP and GM samples but decreased in the WL sample; redness (a*) and yellowness (b*) increased in TVP and WL samples but decreased in the GM sample. Color changes may be influenced by beetroot extract, which is known to lose red pigmentation and develop more yellow tones when heated, contributing to brownish hues (Gamarra-Castillo et al., 2022). Additionally, the protein sources also influenced color, as demonstrated by strong positive correlations with color parameters in cooked products (r >0.75, p <0.01, Table S2c), further contributing to the replication of a cooked meat-like appearance (Tan, Zhang, & McClements, 2023). pH, known to influence color development, showed a strong positive correlation with b* (yellowness) values in both MABs raw (r =0.72, Table S2b) and cooked (r =0.68, Table S2c) (p <0.05). This suggests that the product’s pH, formulated with beetroot extract colorant and the TVP or fungal protein sources, may affect pigment stability and interactions within meat analogue matrix. Acidulants such as citric, acetic, and lactic acids are commonly used to adjust pH in meat analogues, but must be carefully applied due to their impact both on flavor and protein structure (Kyriakopoulou et al., 2021). The pH is a key quality indicator of meat, closely associated with product freshness and protein stability (Taraseviˇ cien˙ e et al., 2022). In this study, raw meat analogue balls (MABs) formulated with pea-based TVP and fungal biomass exhibited pH values ranging 5.70–6.06 (Table S3), which fall within the typical range reported for meat analogues (G´ omez et al., 2019). GM products had a significantly lower pH compared to TVP and WL (p <0.05). The pH levels increased slightly after cooking in TVP and WL products, which may be due to the formation of basic Maillard reaction products, the loss of volatile organic acids, the concentration of alkaline peptides, or the thermal breakdown of proteins and carbohydrates (Fiorentini, Kinchla, & Nolden, 2020). Conversely, the cooked GM products showed a decrease in pH (5.61), possibly due to the concentration of intrinsic organic acids resulting from moisture loss or the release of acidic degradation products during heating. Additionally, ingredients used to improve water-holding capacity may contribute to increased pH levels (Kothuri et al., 2025). Water activity (a w ) values ranged from 0.935 to 0.910 in raw products, decreasing slightly after cooking to 0.905–0.913 (Table S3). These relatively high a w levels may increase the risk of microbial growth and shorten shelf life, emphasizing the need for effective preservation strategies. Understanding both pH and water activity (a w ), among other factors, is essential for managing the Maillard reaction. While this reaction enhances flavor and color, it can reduce the nutritional value by modifying essential amino acids like lysine, binding key minerals such as copper, zinc, and iron, and promoting protein crosslinking through the formation of advanced glycation end products (AGEs) and related compounds (Zhang, Ames, Smith, Baynes, & Metz, 2009). Due to its complexity, the Maillard reaction influences sensory attributes, nutritional quality, and product stability during storage and distribution of the final products (El Hosry et al., 2025). 3.3. Techno-functional properties of protein ingredients and cooking characteristics of MABs The techno-functional properties of the three protein sources utilized in this study, along with cooking characteristics and water holding capacity of the meat analogue balls (MABs) are presented in Table 1. In meat alternatives, water typically comprises 50–80 % of the product, plays a vital role by influencing density, enabling biochemical reactions such as protein cross-linking, and acting as a medium for energy transfer (Gamarra-Castillo et al., 2022). Water absorption capacity (WAC) can serve as a quality indicator of protein ingredients, contributing to a preferable texture in meat analogues (Mandliya, Pratap-Singh, Vishwakarma, Dalbhagat, & Mishra, 2022). In the present study, WAC was significantly higher in fungal proteins (5.92 to 6.51 g/g), compared to the TVP (4.22 g/g). Therefore, since methylcellulose was included at the same level in all formulations, the high juiciness observed in WL samples can likely be attributed to the greater water absorption capacity (WAC) of fungal protein. The strong negative correlation between WAC and color parameters (L*, a*, b*) observed in both raw and cooked MABs (r <−0.78, p <0.05, Table S2b and S2c), suggests the presence of higher levels of pigments, polyphenols, or Maillard products, polar compounds that bind water and may reduce brightness by darkening the MABs. Oil absorption capacity (OAC) is considered an indicator of flavor and mouthfeel due to its role in oil entrapment within meat analogue matrix (Mandliya et al., 2022). To enhance juiciness, tenderness, and mouthfeel, commonly are added oils such as coconut, palm, sunflower, canola, and corn (Kyriakopoulou et al., 2021). In the present study, in products formulation was used sunflower oil, also was used to assess OAC. The OAC varied significantly among proteins sources, with the lowest observed in the WL sample (0.17 g/g), followed by TVP (1.78 g/ g), and the highest in the GM sample (5.81 g/g) (p <0.05). Since protein L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 7 functions. Including water and oil absorption capacities, depend on factors like amino acid composition, chemical structure, and environmental conditions (e.g., temperature, pH, and ionic strength) (Ahmad et al., 2022). Future studies should explore such parameters and how molecular and structural characteristics influence protein functionality in fungi-based meat analogues. Rehydration capacity (RHC) refers to the ability of protein ingredients to retain water when rehydrated in excess water (Mattila, Marhendraswari, Nikinmaa, & Sozer, 2025). In this study RHC was significantly higher in the WL protein (1.36 g/g), whereas no significant differences were observed between the TVP (0.57 g/g) and GM (0.46 g/ g) samples. A significant negative correlation between RHC and OAC (r = − 0.78, p <0.05, Table S2) suggests that differences in product structure and the balance between hydrophilic and hydrophobic sites, may influence water or oil absorption capacities. Additionally, water availability in the MABs matrix and the extent of interactions with peabased TVP or fungal protein, methylcellulose, glutinous rice flour, bamboo fiber, is known to have an influence in the functional properties of proteins (Kyriakopoulou et al., 2021). Water holding capacity (WHC) reflects the ability of meat analogues to retain water during rehydration and contribute to the formation of a protein gel network, thereby influencing product juiciness (Mandliya et al., 2022). In the present study, WHC values showed slight variation among MABs, with the highest observed in TVP-based formulation (88.94 %), while fungi-based formulations showed slightly lower values: 86.90 % for WL and 86.26 % for GM. This difference may be attributed to structural disintegration during rehydration, influenced by cell structure and porosity, which aligns with the study of Mandliya et al. (2022), who reported reduced WHC when mycelium was used. Parameters like cooking efficiency (CE), moisture retention (MR), oil retention (OR), and changes in diameter (DR) are crucial for understanding meat analogues behavior during cooking, related closely to its quality and sensory properties (Akcan et al., 2024). Moisture and oil retention are critical for achieving desirable texture, juiciness, and flavor in meat analogues (Akcan et al., 2024; Kyriakopoulou et al., 2021). In the present study, MR was highest in TVP-based MABs (82.9 %), followed by GM (79.8 %) and lowest in WL (77.7 %), with a significant differences were observed between TVP and WL samples (p < 0.05). Additionally, OR ranged from 86.9 % to 91.7 %, with no significant differences among samples. Notably, both MR and OR values of MABs formulated with all protein sources trialled exceeded those reported for beef meatballs by Akcan et al. (2024). Cooking losses from liquid release and evaporation significantly impact cooking efficiency, influenced by temperature, time, sample size, and notably, the cooking method. In the present study, air frying was employed, a method known to reduce moisture loss compared to traditional frying (T´ ellez-Morales, Rodríguez-Miranda, & AguilarGaray, 2024). CE ranged 86.5–90.1 %, with TVP products showing the highest CE value (90.11 %), followed by GM (87.64 %) and WL the lowest (86.54 %) (p <0.05). Product appearance, including size changes, is a key quality factor influencing consumer preference and requires careful monitoring (Akcan et al., 2024). Diameter reduction (DR) was greatest in TVP-based meat analogue balls (5.82 %), followed by GM (3.57 %) and WL (0.11 %) samples (p <0.05). DR had a strong positive correlation with MR (r = 0.88, p <0.01, Table S2c), which agrees with the study of Akcan et al. (2024), who reported that lower DR corresponds to a higher moisture retention. In addition, fungal protein sources with higher WAC were associated with lower DR, particularly WL-based MABs, as indicated by a strong negative correlation (r = − 0.83, p <0.01, Table S2c). Fungibased MABs showed the lowest DR (0.11 % for WL, 3.57 % for GM), suggesting less structural collapse and potentially lower protein denaturation compared to TVP-based MABs. Filamentous hyphae can form a highly crosslinked, fibrous network that provides mechanical stability and elasticity, and when associated with polysaccharides, which act as natural plasticizers, they help mitigate protein unfolding and aggregation under heat (Finnigan, Theobald, & Bajka, 2025). MR showed a strong positive correlation with both DR and CE (r = 0.92, p <0.01, Table S2c), highlighting its critical role in cooking performance. Since protein denaturation, along with water and fat loss, contributes to a reduction in CE and DR (Akcan et al., 2024), these findings support the hypothesis that fungal proteins, particularly WL, may be more heat stable under cooking conditions. Additionally, variations in CE, DR, and MR reflect differences in structural organization and molecular interactions driven by ingredient composition, protein source, and cooking process (Kothuri et al., 2025). 3.4. Texture profile analysis of raw and cooked meat analogue balls Texture is key to consumer acceptance in meat analogue products and guides product development, formulation, and quality control. Texture Profile Analysis (TPA) is widely used for evaluating meat products and meat substitutes (Schreuders, Schlangen, Kyriakopoulou, Boom, & Van Der Goot, 2021). In this study TPA was performed before and after cooking of MABs, to provide a comprehensive evaluation of the texture attributes, presented in Fig. 3. The mouthfeel characteristics (hardness, springiness, cohesiveness) can be predicted by TPA, which can vary depending by ingredient composition, protein source, and cooking process (Kothuri et al., 2025; Nwosisi, Nandwani, & Ravi, 2019). Hardness, measured as the peak force in the first compression, relates to the strength of gel to withstand compression, while the sensory-wise it represents the maximum force required to compress the food between the molar teeth (Chandra & Shamasundar, 2015). In this study, raw MABs hardness values ranged from 0.47 to 0.51 kg with no statistically significant differences among the samples. Cooking significantly increased hardness, especially in GM (3.10 kg), followed by TVP (1.19 kg) and WL (0.91 kg) (p <0.05). Sensory evaluation reflected this trend, with GM rated highest (4.02 ±0.94), then TVP (3.35 ±0.81), and WL lowest (1.56 ±0.75) (Table 2). This difference is likely attributed to the Table 1 Techno-functional properties of alternative protein sources: pea-based texturized vegetable protein (TVP) and protein-rich Neurospora intermedia biomass cultivated on grape marc (GM) and wine lees (WL), and cooking characteristics of meat analogue balls (MABs) prepared from these protein sources. Protein source Techno-functional of alternative protein sources WAC (g/g)* OAC (g/g)* RHC (g/ g)* TVP 4.22 ±0.20 b 1.78 ±0.12 b 0.57 ± 0.11 b WL 6.51 ±0.83 a 0.17 ±0.02 c 1.36 ± 0.15 a GM 5.92 ±0.43 a 5.81 ±0.49 a 0.46 ± 0.062 b MABs Water holding capacity and cooking characteristics of MABs WHC (%)** DR (%)*** OR (%)*** MR (%)*** CE (%)*** TVP 88.94 ± 1.20 a 5.82 ±0 a 90.6 ± 3.18 a 82.9 ± 0.69 a 90.11 ± 0.46 a WL 86.90 ± 1.35 a 0.11 ± 0.20 c 86.9 ± 4.38 a 77.7 ± 2.13 b 86.54 ± 0.31 c GM 86.26 ± 0.94 a 3.57 ±0 b 91.7 ± 2.46 a 79.8 ± 0.49 ab 87.64 ± 0.16 b Mean ±standard deviation (n =3), with different superscript letters in the same column indicating significant differences (p <0.05) among MABs samples according to Tukey’s test. TVP: Pea-based texturized vegetable protein and TVPbased MABs; WL: N. intermedia biomass grown on wine lees and WL-based MABs; GM: N. intermedia biomass grown on grape marc and GM-based MABs. WAC: Water Absorption Capacity; OAC: Oil Absorption Capacity; RHC: Rehydration Capacity, all measured on *protein sources. WHC: Water Holding Capacity, measured on **raw MABs. DR: Diameter Reduction; OR: Oil Retention; MR: Moisture Retention; and CE: Cooking Efficiency, calculated based on measurements *** before and after cooking of MABs. L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 8 protein sources, binders, and beetroot extracts, as well as their interactions, which are known to significantly influence hardness (Gamarra-Castillo et al., 2022). Notably, in cooked GM products, protein appeared to contribute to the formation of a stronger gel network compared to TVP and WL samples, resulting in a denser structure, likely due to aggregation and cross-linking (Gravelle, Marangoni, & Barbut, 2017). It was hypothesized that the protein-to-macromolecules (fiber) ratio plays a critical role in hardness, with MABs tending to be harder when formulations contain less crude protein and higher fiber content. Additionally, the observed correlations between hardness and crude protein as well as between WAC and color, are hypothesized to reflect the role of fungal cell wall polysaccharides such as chitin and β-glucans in water and fat binding, as well as in texture formation (Kyanko, Canel, Ludemann, Pose, & Wagner, 2013). The greater hardness observed, particularly in GM products, may be attributed to their lower crude protein content and likely higher proportion of dietary fibers from filamentous fungi biomass, such as β-glucan, chitin, and chitosan (Colosimo et al., 2021). The significant negative correlation between crude protein content and hardness (r = − 0.73, p <0.05, Table S2c) further supports this observation. Gibis, Pribek, Kutzli, and Weiss (2021) also reported that higher protein levels are associated with increased porosity in heated fibers. However, further research is needed to confirm these relationships. Fat content had a significant positive correlation with hardness (r =0.76, p <0.05, Table S2c). Conversely, GM products showed a strong negative correlation between fat and hardness (r = − 1.00, p <0.05, Table S2d), suggesting that oil proportion influences hardness. This relationship, along with the influence of oil type and its interactions with other ingredients, warrants further investigation. Additionally, hardness may be affected by the cooking method. In this study air-frying was utilized, which is known to influence hardness by lowering starch gelatinization degree and promote crust formation (T´ ellez-Morales et al., 2024). Cohesiveness is defined as the ratio of the positive force area during the second compression to that of the first. In this study, raw GM products exhibited the highest cohesiveness (2.23), followed by WL (2.07), and TVP (1.19), suggesting that fungi-based MABs have superior internal bonding strength and resistance to structural breakdown. However, post-cooking, cohesiveness significantly dropped in GM (0.63) likely due to the development of a denser and more brittle texture. In contrast, TVP (1.24) and WL (1.45) retained relatively higher cohesiveness (p <0.05), potentially indicating better protein network formation and a more stable internal structure, compared to GM cooked products. The protein source plays a key role in MABs consistency and structural integrity, particularly in WL, providing MABs with enhanced resistance to disintegration under mechanical stress (Chandra & Shamasundar, 2015). Adhesiveness is defined as the negative force area during the first bite. It represents the work needed to overcome the attractive forces between the food surface and any surface that comes into contact with food (Chandra & Shamasundar, 2015). In this study, all raw MBAs showed no significant differences in adhesiveness values (−0.037 to −0.020 kg⋅s). After cooking, adhesiveness slightly decreased across all samples (−0.006 to −0.002 kg⋅s), again with no significant differences. This reduction was associated with increased hardness and decreased cohesiveness (r = − 0.72 and r =0.75, p <0.05, Table S2c), likely resulting from protein denaturation, matrix expansion, crust formation, and cooking loss. Springiness is related to the relative height reduction between the 2 compressions, i.e., how well it returns to its original shape between the end of the first bite and the start of the second (Chandra & Shamasundar, 2015). In this study, raw MABs showed springiness values ranging from 1.22 to 2.45, with the highest observed in GM (2.45) and WL (2.24), and the lowest in TVP (1.22). No significant differences were found. The higher values in fungi-based MABs suggest greater elasticity. Upon cooking, springiness decreased in fungi-based products, WL dropped to 1.59 and GM was significantly reduced to 0.55 (p <0.05). This reduced springiness in cooked fungi-based MABs was strongly associated with higher hardness (r = − 0.97, p <0.01), and lower cohesiveness (r =1.00, p <0.01) (Table S2c), indicating a firmer, less elastic texture and reduced ability of the matrix to recover its shape after deformation. Conversely, TVP showed a slight increase in springiness after cooking (1.34), which along with WL, maintained relatively higher values, suggesting that these MABs may require more mastication effort (Chandra & Shamasundar, 2015). Resilience is a measurement of how the sample recovers from deformation, given by the ratio of work done during the decompression Fig. 3. Texture properties of raw and cooked meat analogue balls (MABs) prepared with alternative protein sources: pea-based texturized vegetable protein (TVP) and protein-rich Neurospora intermedia biomass grown on grape marc (GM) and wine lees (WL). Texture properties: hardness, cohesiveness, adhesiveness, springiness, and resilience) values are presented as mean ±standard deviation (n =3). Different superscript letters within each raw or cooked MAB groups per each texture property indicate significant differences among samples (p <0.05), as determined by Tukey’s test. L. Hoxha et al. Innovative Food Science and Emerging Technologies 108 (2026) 104409 9