From Waste to Value: Investigating Mushroom Stems from Pleurotus ostreatus Grown on Mealworm Frass as a Nutritional Source for Aquaculture Feed

Academic Editor: Roberto Mancinelli Received: 6 June 2025 Revised: 7 July 2025 Accepted: 11 July 2025 Published: 16 July 2025 Citation: Hilali, S.; Stierlin, E.; Tello Martín, M.L.; Amaral, D.; Pérez-Clavijo, M.; Girão, M.; Carvalho, M.d.F.; Pérez Bonilla, A.M.; de Diego, S.; Ramírez, P.; et al. From Waste to Value: Investigating Mushroom Stems from Pleurotus ostreatus Grown on Mealworm Frass as a Nutritional Source for Aquaculture Feed. Sustainability 2025,17, 6496. https://doi.org/10.3390/ su17146496 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article From Waste to Value: Investigating Mushroom Stems from Pleurotus ostreatus Grown on Mealworm Frass as a Nutritional Source for Aquaculture Feed Soukaina Hilali 1,* , Emilie Stierlin 1, María Luisa Tello Martín 2, Diogo Amaral 3,4 , Margarita Pérez-Clavijo 2, Mariana Girão3, Maria de Fátima Carvalho 3,4 , Andrea María Pérez Bonilla 5, Sabas de Diego 5, Pablo Ramírez 5 and Rodrigo Ozorio 3,* 1Celabor, Research Center, Avenue du Parc 38, 4650 Chaineux, Belgium; [email protected] 2Mushroom Technological Research Center of La Rioja (CTICH), Carretera Calahorra, km 4, 26560 La Rioja, Spain; [email protected] (M.L.T.M.); [email protected] (M.P.-C.) 3 Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR LA), University of Porto, Terminal de Cruzeiros do Porto de Leixões, 4450-208 Matosinhos, Portugal; [email protected] (D.A.); [email protected] (M.G.); [email protected] (M.d.F.C.) 4 School of Medicine and Biomedical Sciences (ICBAS), University of Porto, Rua Jorge de Viterbo Ferreira, 228, 4050-313 Porto, Portugal 5Tebrio Group S.L, Ctra. Nacional 620 km 244, 37120 Salamanca, Spain; [email protected] (A.M.P.B.); [email protected] (S.d.D.); [email protected] (P.R.) *Correspondence: [email protected] (S.H.); [email protected] (R.O.) Abstract This study investigated mealworm frass as a sustainable substrate for Pleurotus ostreatus cultivation while valorizing mushroom stems as aquaculture feed. Mushrooms were grown on substrates containing 0–15% frass, and nutritional analyses were conducted on both fruiting bodies (for human consumption) and stems (for fish feed). Increasing frass levels significantly enhanced protein content, rising from 7.78% to 22.31% in stems and 24.74% to 30.99% in fruiting bodies. Lipid concentrations showed minor fluctuations while, in contrast, β -glucan content declined with high frass inclusion percentages. Essential amino acid levels peaked at 7.37% in stems (15% frass) and 8.08% in fruiting bodies (12.5% frass). Polyunsaturated fatty acids dominated the fatty acid profile, increasing with high frass levels. Mushroom bodies and stems were additionally investigated for their antimicrobial activity to determine whether they could offer protection against common fish and human pathogens. Antimicrobial assays revealed that dichloromethane extracts from stems grown on 12.5% and 15% frass exhibited inhibitory activity (inhibition zones of 10–11 mm) against Tenacibaculum maritimum, a microorganism that poses a significant threat to aquaculture. These findings highlight mealworm frass as a promising substrate for enhancing mushroom nutritional value while providing a sustainable, protein-rich feed ingredient for aquaculture. Keywords: oyster mushroom; insect frass; antimicrobial properties; amino acid profile; fatty acid profile 1. Introduction The growth of aquaculture underscores the urgent need for sustainable practices to address environmental challenges, while ensuring food security. In aquaculture, conventional feed ingredients such as fishmeal and soybean meal are associated with significant ecological impacts, including overfishing, habitat destruction, and deforestation [ 1 , 2 ]. As industry Sustainability 2025,17, 6496 https://doi.org/10.3390/su17146496 Sustainability 2025,17, 6496 2 of 23 strives to meet the growing demand for fish protein, there is a pressing need to develop sustainable feed alternatives that minimize these environmental burdens while supporting fish health, namely by adding functionalities such as antibacterial properties that contribute to disease prevention and reduce reliance on antibiotics, ultimately enhancing aquaculture sustainability [3]. The Pleurotus genus has shown significant promise as a functional feed additive and nutraceutical in both human health and aquaculture. Rich in bioactive compounds like β -glucans and polysaccharides [ 4 , 5 ], Pleurotus mushrooms possess cholesteroland glucose-lowering effects, immune-modulatory properties, and antiproliferative activity against cancer cells [ 6 ]. In aquaculture, dietary supplementation with Pleurotus extracts has positive effects on the growth rate (SGR), feed conversion ratio (FCR), lysozyme activity, phagocytosis, and disease resistance in species like rainbow trout, Amur catfish, Nile tilapia, and Pangasius catfish [7–10]. Insect frass, particularly produced by mealworms, has gained attention as an ecofriendly agricultural input due to its high content of organic matter and essential nutrients. Mealworm frass, a waste product obtained from mealworm farming, consists of the excrement generated by the mealworm larvae throughout their entire life cycle, which makes the total output at an industrial scale substantial. This, together with its nutrient-rich composition and a very low production cost, positions mealworm frass as an efficient and sustainable resource within circular farming systems [11]. Studies have shown that insect frass, particularly from species like Tenebrio molitor and Hermetia illucens, significantly improves plant growth, soil health, and nutrient uptake when used as a soil amendment. Its richness in essential macro and micronutrients, combined with beneficial microbial communities, promotes biomass production and induces plant defenses against pathogens [ 12 , 13 ]. Using it as a substrate for cultivating edible mushrooms represents a promising innovation, enhancing the valorization of insect farming by-products and opening new pathways for sustainable agricultural practices. According to Putri et al. [ 14 ], white oyster mushroom (Pleurotus ostreatus) cultivation with mealworm frass significantly enhances the nutritional profile of the harvested mushrooms. The addition of frass, alone or in combination with molasses, led to notable increases in ash, crude protein, and crude fat content, while reducing crude fiber. The highest protein content was observed in the frass-only treatment (23.4%), underscoring frass’s potential as a sustainable, nutrient-rich growth substrate. These findings position mealworm frass as a promising alternative substrate for optimizing the nutritional value of oyster mushrooms in commercial settings. Edible mushrooms themselves generate significant agricultural residues, such as stems, which are often discarded despite their valuable nutritional properties [ 15 ]. Transforming these stems into aquaculture feed ingredients offers a dual solution: reducing agricultural waste and providing a sustainable alternative to conventional feed sources. This study investigated the potential uses of mushroom stems from Pleurotus ostreatus, the second most widely cultivated and distributed edible mushroom worldwide, after the champignon (Agaricus bisporus), thanks to its remarkable adaptability [ 16 ]. The mushrooms used in this study were cultivated using mealworm frass, as an emerging ingredient for fish feed. The nutritional composition, safety, as well as the functional properties, namely in terms of antimicrobial potential, of these stems and fruiting body were assessed to ensure their viability for both human consumption and aquaculture applications. This research examined the effect of different inclusion levels of insect frass in mushroom substrates, on mushroom yield, quality, and aquaculture feed potential. The aim is to demonstrate how waste valorization can contribute to a more sustainable and resilient aquacultural feed production system. Sustainability 2025,17, 6496 3 of 23 2. Materials and Methods 2.1. Insect Frass The mealworm frass, commercially known as :oFrass ® , is a natural product, suitable for organic farming. It consists exclusively of the excrement of the yellow mealworm (Tenebrio molitor). The frass is separated from the insects and their rearing substrate by mechanical means using proprietary technology. The product has a sandy texture, brownish color, is odorless, and feels like dry granules to the touch. 2.2. Mushroom Cultivation The mealworm insect frass provided by TEBRIO (Salamanca, Spain), was used as an ingredient for mushroom production at different percentages (CONTROL = 0%, 2.5%, 5%, 7.5%, 10%, 12.5% and 15%) mixed with commercial mushroom substrate, supplied by the compost yard Micelios Fungisem S.A. (Calahorra, Spain). To prepare the commercial mushroom substrate, the wheat and corn straw are first chopped. The chopped straw is then moistened with water containing urea to ensure the appropriate nitrogen content. The chopped and moistened straw is left to drain for a few hours to eliminate excess moisture and initiate fermentation. After this conditioning, the mixture is placed in a fermentation tunnel, where it will remain for approximately 7 days. After fermentation, the mixture is transferred to another tunnel for pasteurization. This process eliminates potential competing microorganisms from the compost and leaves it ready for mycelium inoculation. Trials were in 2023 performed at CTICH facilities. CTICH has had experience in mushroom research since 2003. Ten replicates per percentage of inclusion were prepared. The different substrates were mixed, watered, placed in polypropylene bags, and sterilized in an autoclave at 121 ◦ C for 180 min. The already sterilized substrate was spawned with Pleurotus ostreatus mycelium (strain H9) at 1% rate. The bags were sealed with a heat sealer and taken to an incubation room where they remained for 12 days at 25 ◦ C. Once the mycelium had colonized the entire substrate, the bags were moved to a growing room at 16 ◦ C, with 90% humidity, and 800 ppm of CO 2 to force pinning. Mushroom were collected individually from each bag, weighed, and the stems were separated from the caps manually with a knife. Each fraction was chopped with a chopper (SAMMIC CK 38V; Gipuzkoa, Spain) and dried at 35 ◦ C in a dryer (Klarstein Master Jerky 550; Chal-Tec GmbH, Berlin, Germany). The dry material was ground in a centrifugal mill ULTRA ZM 200 (Retsch; GmbH, Haan, Germany). The mushroom powder obtained was then analyzed following the next protocols (Section 2.3). Samples of the commercial mushroom substrate, insect frass, and the different mixes were collected and the following elements and parameters were measured following AOAC guidelines [ 17 ]: total nitrogen, ashes, humidity, fibers, pH, conductivity, and organic content (Table 1). Table 1. Parameters of the commercial substrate and mealworm frass used in the trial. Sampe Humidity (%) Ashes (%s.m.s.) Nitrogen (%) pH Conductivity (mS/cm) Organic Matter (%) Lignin (%) Cellulose (%) Hemicellulose (%) Substrate 72.5 ±2.1 17.0 ±4.0 0.9 ±1.1 8.4 ±0.5 1.8 ±0.6 83.0 ±4.0 9.9 ±2.4 42.2 ±3.6 24.5 ±4.4 Insect frass 6.9 ±2.5 7.1 ±0.1 3.4 ±0.2 5.8 ±0.2 6.2 ±0.9 92.9 ±0.1 7.4 ±2.6 16.0 ±3.1 41.6 ±4.6 The parameters of the commercial substrate and mealworm frass used in the trial are presented in Table 1. Sustainability 2025,17, 6496 4 of 23 2.3. Nutritional Analysis 2.3.1. Protein Quantification The protein content was quantified by nitrogen determination using the Kjeldhal method. The sample is mineralized with concentrated sulfuric acid in the presence of a catalyst, converting organic nitrogen into ammonium sulphate. Ammonia is released by adding sodium hydroxide to the cooled mineralization. This ammonia is then distilled into an excess boric acid solution. The whole is titrated with a titrated solution of hydrochloric acid. The quantity of ammonia produced is used to determine the nitrogen content of the sample. The protein content is finally calculated by multiplying the nitrogen value by a conversion factor of 4.38, appropriate for mushrooms [18]. 2.3.2. Lipid Content The lipid content of the samples was determined using Soxhlet extraction. Approximately 5 g of dried and ground sample was placed in a Soxhlet cartridge and extracted with hexane as the solvent for 4 h under reflux. After extraction, the hexane solvent was evaporated using a rotary evaporator, and the remaining lipid residue was dried to a constant weight in a desiccator. 2.3.3. Glucan Determination The β -glucan content of mushroom samples was determined using the Megazyme β -Glucan Assay Kit (K-YBGL), following the manufacturer’s protocol (Megazyme ® ; IDA Business Park, Bray, Wicklow, Ireland). Briefly, approximately 200 mg of each sample was subjected to two separate hydrolysis procedures to quantify total glucans and α -glucans (100g for each hydrolysis). For α -glucan determination, 100 mg of the sample was hydrolyzed with 1.7 M sodium hydroxide to solubilize α -glucans and free glucose. The extract was then neutralized and treated with amyloglucosidase and invertase to enzymatically hydrolyze starch and sucrose into glucose. The released glucose was quantified using a glucose oxidase/peroxidase (GOPOD) assay. For total glucan determination, the remaining 100 mg of the sample was hydrolyzed using sulfuric acid, breaking down all glucan types ( α - and β -) into glucose, which was again quantified using the GOPOD assay. The β -glucan content was calculated by subtracting α -glucan and free glucose contributions, based on corresponding control assays, and expressed as a percentage of the dry weight of the sample. 2.3.4. Chitin Quantification For the chitin quantification, 1 g of samples and standards (chitin and glucosamine) was hydrolyzed using 25 mL of concentrated hydrochloric acid and 15mL of Milli-Q water to partially depolymerize chitin into glucosamine. Hydrolysis was conducted in a water bath at 90 ◦ C with continuous agitation (140 rpm) for 4 h. The hydrolysates were diluted with 50 mL Milli-Q water (ELGA LabWater, Veolia Water Technologies, High Wycombe, UK) and analyzed by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry UPLC-MS/MS (Waters Corporation, Milford, MA, USA) using an Acquity UPLC-DAD-MS/MS system (including TQD Acquity) equipped with a Waters Acquity UPLC HILIC column (1.7 µ m, 2.1 × 100 mm). Glucosamine was quantified by monitoring specific mass transitions and comparing peak areas to calibration curves generated from standard solutions. Sustainability 2025,17, 6496 5 of 23 The concentration of chitin was expressed as a percentage using the following equation: Chitin% = FD ×Cm 10000000 where FD represents the dilution factor (final volume in mL divided expressed in g), and C m is the concentration of glucosamine obtained from the UPLC-MS/MS analysis (in ppb). 2.4. Fatty Acid Profiling and Quantification by GC-FID First, an acid hydrolysis breaks the ester bonds of fatty acids in glycerides, glucoand phospholipids, and sterol esters. Fatty acids are extracted with n-hexane, then methylated with methanolic solution of BF3to form fatty acid methyl esters (FAMEs). FAMEs are measured by gas chromatography with flame ionization detection (GC-FID). The fatty acid profile was analyzed using a Shimadzu chromatograph, equipped with a split–splitless injector, a FI detector, and a Shimadzu autosampler. Both the injector and detector temperatures were set at 225 ◦ C. Separation was performed on a 100 m × 0.25 mm i.d. × 0.20 µ m HP-88 column. Helium served as the carrier gas at an internal pressure of 250 kPa. The column temperature was initially held at 120 ◦ C for 5 min, then increased to 240 ◦ C at a rate of 3 ◦ C/min and held for 10 min. The split ratio was 1:30, and the injection volume was 1 µ L. Fatty acids were identified by comparing the relative retention times of FAME peaks from samples with standards. A Supelco mixture of 37 FAMEs (standard CRM47885) purchased from Sigma-Aldrich (St. Louis, MO, USA) was used. 2.5. Amino Acid Profiling and Quantification by UPLC-DAD-MS The amino acids (AA) profile was determined according to Kairos amino acid kit (Waters, Milford, MA, USA). The samples were hydrolyzed prior to the UPLC analysis with 6N HCl. Reverse-phase chromatography was performed using a 2 µ L injection volume and a flow rate of 0.4 mL/min, in a high-performance liquid chromatography system (Waters, Milford, MA, USA). Separation was achieved using a Cortecs UPLC C18 column with dimensions of 2.1 mm ×150 mm, 1.6 µm (Waters, Milford, MA, USA) at 55 ◦C. 2.6. Heavy Metal Analysis Heavy metal concentrations in the samples were quantified using inductively coupled plasma mass spectrometry (ICP-MS) Thermo ICAP RQ. Prior to analysis, approximately 0.5 g of each dried and ground sample was subjected to acid digestion using a mixture of concentrated nitric acid (HNO 3 ) and hydrogen peroxide (H 2 O 2 ) in a microwave digestion system to ensure complete mineralization using Ultrawave ECR. The digested samples were diluted with deionized water to a final volume, filtered, and analyzed by ICP-MS. Calibration curves were prepared using certified standard solutions and the results were expressed as milligrams of heavy metal per kilogram of dry sample. 2.7. Preparation of Crude Extracts for Antimicrobial Analysis Three crude extracts from each mushroom biomass sample (14 samples: stems and fruiting bodies cultivated with 0%, 2.5%, 5%, 7.4%, 10%, 12.5%, 15% of mealworm frass) were prepared to evaluate its antimicrobial properties, following the process described by Girão et al., 2019 [ 19 ], with small adjustments to the protocol. All samples were freezedried, ground to a fine powder, and stored at − 20 ◦ C until further use. For each sample, 5 g of freeze-dried biomass was subjected to independent extractions with each of the following solvents, used separately and in order of decreasing polarity: deionized water, acetone/methanol (1:1, v/v), and dichloromethane. The biomass was placed in 100 mL Erlenmeyer flasks, followed by the addition of 30 mL of the appropriate solvents, and the flasks incubated for 30 min, at 200 rpm and room temperature. The organic or aqueous Sustainability 2025,17, 6496 6 of 23 layer was recovered and concentrated using a rotary evaporator, with the extraction process being repeated twice for enhanced yield. The recovered crude extract was weighed and dissolved in dimethyl sulfoxide (>99.9%, DMSO; Sigma-Aldrich, St. Louis, MO, USA) at 1 mg mL−1to be used for the antimicrobial assay. 2.8. Antimicrobial ASSAY The antimicrobial properties of the crude extracts were tested using the agar-based disk diffusion method, following a previously described method [ 20 ], against a panel of reference pathogenic strains relevant for aquaculture—Edwardsiella tarda DSM30052, Aeromonas hydrophila DSM3018, Pseudomonas anguilliseptica DSM1211, Yersinia ruckeri ATCC29473, Listonella (Vibrio)anguillarum ATCC19264, Tenacibaculum maritimum ATCC43397, and Lactococcus garvieae DSM 20684—and human health—Escherichia coli ATCC25922, Staphylococcus aureus ATCC29213, Salmonella enterica ATCC25241, and Candida albicans ATCC10231. Each reference microorganism was grown in the appropriate culture medium and incubation temperature (Table A1). For the bioassay, each strain was suspended in the respective liquid medium,D and its turbidity adjusted to 0.5 McFarland standard (OD 625 = 0.08–0.13) [ 21 ]. The suspensions were used to inoculate agar plates, and blank paper disks (6 mm in diameter) loaded with 15 µ L of each crude extract (1 mg mL −1 ) were placed on the surface of the inoculated medium. Positive control disks were inoculated with 15 µ L of enrofloxacin (1 mg mL −1 ; Sigma-Aldrich, MO, USA) for the bacterial strains, and 15 µ L of nystatin (1 mg mL −1 ; Sigma-Aldrich, MO, USA) for the yeast C. albicans, while negative control disks were inoculated with 15 µ L of DMSO. Each extract was tested in two independent experiments. Antimicrobial activity was determined by measuring the diameter of the inhibition halo formed around each disk, while negative control disks were inoculated with 15 µ L of DMSO. Each extract was tested in two independent experiments. Antimicrobial activity was determined by measuring the diameter of the inhibition halo formed around each disk. 2.9. Statistical Analysis All statistical analyses were performed using R software (version 4.4.0; R Core Team, 2024). A one-way ANOVA was used to assess the effect of inclusion level. When significant differences were detected (p< 0.05), Tukey’s HSD test was applied for multiple comparisons. Values in the same column followed by the same superscript letter are not significantly different according to Tukey’s multiple comparison test (p< 0.05). Different letters indicate statistically significant differences between groups. 3. Results 3.1. Mushroom Yield Due to Substrate Variation The average mushroom yield (expressed as kg of mushrooms/kg of substrate) for each treatment can be found in Figure 1. The yield from the control group was approximately 0.3 kg mushrooms per kg of substrate. The highest yield was achieved with the addition of 2.5% insect frass, averaging around 0.35 kg/kg substrate. However, at 5% insect frass, the yield notably dropped to 0.25 kg/kg substrate. As the inclusion level increased to 7.5%, the yield was not significantly different to the control level, averaging 0.3 kg/kg substrate. Similarly, the 10% frass group showed a yield of around 0.3 kg/kg substrate. A slight decrease in yield was observed at 12.5% frass, averaging just below 0.3 kg/kg substrate. The lowest yield occurred at 15% insect frass, where the yield dropped to 0.22 kg/kg substrate. Sustainability 2025,17, 6496 7 of 23 Figure 1. Mushroom yield (kg mushrooms/kg substrate) with different insect frass inclusion percentage (p-value ≤ 0.001). The a, b, c indicate statistically significant differences between groups based on Tukey′s HSD test. 3.2. Nutritional Composition of Mushroom Stems and Fruiting Body The nutritional composition of the mushroom fruiting bodies and stems are presented in Table 2. Protein concentrations increased significantly with higher frass levels in both the stems. It was observed to be the lowest at 0% frass inclusion (7.8%) and peaks at 15% frass inclusion (22.3%). For the fruiting body, protein levels start at 24.7% and steadily rise to 31.0% at 15% frass. However, according to the p-value obtained via the ANOVA to test the variation, is not statistically significant (p-value = 0.332). Table 2. Nutritional composition of PO fruiting body (PO.FB) and PO stems (PO.St) at different frass inclusion percentage. Inclusion Percentage Protein β-Glucan (%) Chitin (%) PO. St * PO. FB PO. St * PO. FB * PO. St PO. FB 0% 7.8 ±0.6 d24.7 ±0.5 a37.9 ±0.8 a26.4 ±0.4 a5.5 ±1.2 a6.1 ±0.5 a 2.5% 10.5 ±0.8 cd 26.5 ±0.7 a36.6 ±0.8 ab 19.2 ±0.3 b6.2 ±0.8 a6.5 ±0.7 a 5% 15.0 ±0.7 bc 27.8 ±1.7 a26.7 ±0.1 cd 14.9 ±0.6 c6.6 ±0.6 a6.9 ±0.5 a 7.5% 14.5 ±0.1 bc 30.5 ±1.0 a25.3 ±3.1 cd 16.1 ±0.4 c6.6 ±0.7 a6.8 ±0.3 a 10% 15.2 ±1.2 b30.6 ±2.2 a29.8 ±0.8 bc 15.1 ±0.4 c7.3 ±0.6 a7.3 ±0.6 a 12.5% 14.8 ±1.0 bc 29.1 ±3.8 a24.4 ±0.4 cd 14.2 ±0.2 c6.7 ±0.5 a7.1 ±0.5 a 15% 22.3 ±0.7 a31.0 ±2.1 a20.3 ±0.9 d14.3 ±0.8 c6.4 ±0.6 a6.4 ±0.5 a *p-value ≤0.001. In contrast to this, for protein, the β -glucan decreases in both stems and fruiting bodies as frass percentage increases. In the stems, beta-glucan concentration dropped from 37.9% at 0% frass to 20.3% at 15% frass. A similar decline occurred in the fruiting bodies, with beta-glucans going from 26.4% to 14.3% at the highest frass concentration. As for the chitin concentration, it remained relatively stable with different frass concentrations in the fruiting bodies. In the stems, chitin content increased slightly, from 5.5% at 0% frass inclusion to 7.3% at 10% frass inclusion, and then decreased to 6.4% at 15% frass inclusion. This variation is statistically significant with a p-value of 0.009. In both stems and fruiting bodies, lipid concentrations were also measured and the values slightly fluctuated with increasing frass levels. In the stems, lipid content starts at 1.6% with 0% frass inclusion and increases slightly to 2.1% at 15% frass inclusion. Similarly, Sustainability 2025,17, 6496 8 of 23 the fruiting body showed a lower increase in lipid content, from 3.4% at 0% to 3.9% at 5%, before reaching slightly higher frass levels (3.3% at 15%). 3.3. Amino Acid Composition Total amino acids, including essential and non-essential contents in the stems and fruiting bodies, are presented in Figure 2.P. ostreatus fruiting bodies and stems have a higher non-essential amino acid content than essential amino acids content. For the stems, total essential amino acids content is 2.44% in the 0% frass treatment and was highest at 7.37% in the 15% frass treatment. For the fruiting body, amino acids levels were at their lowest at 6.13% with 2.5% frass and highest at 8.08% in the 12.5% frass treatment. The amino acid profile is presented in more detail in Tables A2 and A3. Figure 2. Effect of the insect frass from mealworm on total amino acid contents (% DM) of P. ostreatus stems (A) and fruiting body (B). 3.4. Fatty Acid Composition Figure 3presents the effect of the mealworm frass on fatty acid contents of P. ostreatus stems and fruiting body (Figure 3B), specifically the concentration of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs). The data indicates that PUFAs are the dominant fatty acid class in both PO stems and fruiting bodies, significantly increasing with frass inclusion at 7.5% and then again at 15% for PO stems and slightly increasing at 10% frass concentrations for the fruiting body. SFAs in PO stems are consistently higher than MUFAs but lower than PUFAs, suggesting a balanced lipid profile. MUFA levels remain the lowest across all concentration with the lowest concentration around 7.5% inclusion for PO stems. The detailed fatty acid profile is presented in Tables A4 and A5. 3.5. Heavy Metal Content Analysis The data in Figure 4indicates varying concentrations of heavy metals in stems cultivated with mealworm frass at different levels (0–15%). For the fruit body cultivated with mealworm frass at different levels, the key toxic metals such as Cd, Pb, As, and Hg were also below detection levels. Essential minerals like Zn, Cu, Fe, and Mn increase with high frass levels and are present in beneficial amounts. Sustainability 2025,17, 6496 9 of 23 Figure 3. Effect of the mealworm frass on fatty acid contents of P. ostreatus stems (A) and fruiting body (B) expressed in percentages (%). (A) (B) Figure 4. Traces elements and potentially toxic metals concentration in the fruiting bodies (A) and stems (B) of P. ostreatus, expressed in mg/kg of DM. 3.6. Analysis of Antimicrobial Activity The 14 mushroom biomass samples analyzed in this study, comprising both stems and fruiting bodies, and cultivated using different percentages of insect frass, were extracted with three independent solvents: water, an acetone/methanol mixture (1:1, v/v), and dichloromethane. This process yielded 14 extracts per solvent, totaling 42 crude extracts, with extraction yields ranging from 1503.8 mg for the aqueous extract of the fruiting Sustainability 2025,17, 6496 16 of 23 The global expansion of aquaculture creates a pressing demand for sustainable feed solutions, presenting a significant opportunity for this approach. Governments and international organizations are increasingly promoting sustainable practices in aquaculture through policies, subsidies, and regulatory frameworks [ 59 ]. Such support can facilitate the adoption of novel feed. • Consumers preference for sustainability in aquaculture and support from governments and eco-friendly policies Consumer demand for sustainably is another promising opportunity. With growing awareness of environmental issues, consumers are more inclined to support products derived from eco-friendly practices. This trend could incentivize aquaculture producers to integrate mushroom-stem-based feed grown using mealworm frass into their operations. 4.2.4. Threats •Regulatory barriers and strict feed safety standards Several threats could hinder the implementation of this solution. Regulatory barriers remain a key concern, as the use of novel feed specifically by-products still must meet strict safety and quality standards [2]. •Strong competition from established feed ingredients The dominance of traditional feed ingredients and established supply chains. These established products benefit from robust supply chains and economies of scale, making it difficult for alternative feed solutions to compete without significant cost advantages or subsidies [60]. •Environmental risks and economic viability Environmental risks also deserve attention. While mealworm frass and mushroom stems are sustainable resources, improper handling or processing could lead to unintended ecological impacts, undermining the credibility of the approach. Economic viability is another potential obstacle, as fluctuations in the costs of insect farming, mushroom cultivation, and feed processing could affect profitability. 5. Conclusions The mushroom stems, typically considered a low-value byproduct, were shown to be nutritionally suitable for aquaculture feed, contributing to resource efficiency and reducing waste in the production process. The inclusion of mealworm frass in the mushroom cultivation substrate presents significant benefits and trade-offs in terms of yield, nutritional value, and potential aquaculture applications. Nutritionally, frass supplementation enhanced the protein content of both P. ostreatus fruiting bodies and stems, making them valuable protein sources for human and aquaculture consumption. The amino acid profile of the fruiting body improved with frass inclusion, particularly in non-essential amino acids, while the stems demonstrate a substantial increase in essential amino acids critical for fish growth. Beyond its nutritional potential, P. ostreatus grown using mealworm frass exhibited promising antimicrobial properties, with potential applications against fish pathogens such as T. maritimum. Further studies are required to characterize its bioactive compounds and explore its role in disease prevention within aquaculture. Overall, integrating frass into mushroom cultivation enhanced yield at a concentration of 7.5% to 12.5%, improving nutritional quality, and offering a sustainable approach for developing functional feed ingredients, supporting both human nutrition and aquaculture sustainability. Sustainability 2025,17, 6496 17 of 23 Author Contributions: Conceptualization: S.H. and E.S.; methodology: S.H., E.S., M.L.T.M., D.A. and M.G.; validation: M.P.-C., R.O., M.d.F.C. and S.H.; formal analysis: S.H., E.S. and D.A.; investigation: S.H., E.S., M.L.T.M., D.A. and M.G.; resources: M.L.T.M., M.P.-C., A.M.P.B., S.d.D. and P.R.; data curation: S.H., E.S., M.L.T.M., D.A. and M.G.; writing—original draft preparation: S.H., E.S. and D.A.; writing—review and editing: S.H., E.S., M.L.T.M., M.P.-C., D.A., M.G., M.d.F.C., S.d.D., P.R. and R.O.; supervision: M.d.F.C., S.H. and R.O.; project administration: S.H. and R.O. All authors have read and agreed to the published version of the manuscript. Funding: The authors are thankful for the support of the “SmartAqua4FuturE—SAFE” Project, funded by the European Union Horizon Europe Programme (HORIZON-CL6-2022-FARM2FORK01-05) under the grant agreement No. 101084549. This study was partially supported by national funds through Foundation for Science and Technology (FCT, Portugal) within the scope of UIDB/04423/2020 and UIDP/04423/2020. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The original contributions presented in this study are included in the article material. Further inquiries can be directed to the corresponding authors. Conflicts of Interest: Authors A.M.P.B., S.d.D. and P.R. were employed by the company TEBRIO, and declare no conflicts of interest. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as potential conflicts of interest. All the authors declare that this study received funding from the Horizon Europe Programme. Appendix A. Table A1. Culture conditions for the reference strains used in the antimicrobial assay. Strain Culture Medium Incubation Time (h) Incubation Temperature (◦C) Edwardsiella tarda DSM 30052 Tryptic soy 24 28 Aeromonas hydrophila DSM 3018 Tryptic soy 24 28 Pseudomonas anguilliseptica DSM 12111 Tryptic soy 48–72 25 Yersinia ruckeri ATCC 29473 Tryptic soy 24–48 28 Listonella (Vibrio) anguillarum ATCC 19264 Tryptic soy 48 28 Tenacibaculum maritimum ATCC 43397 Marine agar 24–48 25 Lactococcus garvieae DSM 20684 Tryptic soy yeast 24–48 28 Escherichia coli ATCC 25922 Mueller–Hinton 24 37 Staphylococcus aureus ATCC 29213 Mueller–Hinton 24 37 Salmonella enterica ATCC 25241 Mueller–Hinton 24 37 Candida albicans ATCC 10231 Sabouraud dextrose 24 37 Sustainability 2025,17, 6496 18 of 23 Appendix B. Amino Acid Content in P. ostreatus Stems and Fruiting Body Table A2. Amino acids content in the PO stems. (a) PO Stems 0% 2.5% 5% 7.5% 10% 12.5% 15% Alanine 0.46 0.68 0.93 0.82 0.84 1.03 1.52 Arginine 0.34 0.54 0.79 0.72 0.73 0.88 1.57 Aspartic acid LQ LQ 1.37 LQ 1.33 1.51 2.22 Cystine LQ 0.11 0.16 0.12 0.11 0.11 0.21 Glutamic acid LQ 1.51 2.04 1.89 1.98 2.22 3.10 Glycine 0.41 0.56 0.72 0.63 0.65 0.80 1.15 Histidine 0.16 0.21 0.29 0.27 0.30 0.34 0.49 Hydroxyproline LQ LQ LQ LQ LQ LQ LQ IsoLeucine 0.29 0.43 0.55 0.47 0.50 0.61 0.92 Leucine 0.44 0.66 0.88 0.75 0.78 0.98 1.48 Lysine 0.37 0.51 0.68 0.63 0.67 0.82 1.20 Methionine 0.08 0.11 0.17 0.13 0.14 0.17 0.31 Ornithine 0 0 0.08 LQ 0.16 0.15 0.17 Phenylalanine 0.36 0.48 0.60 0.53 0.56 0.65 0.92 Proline 0.36 0.49 0.60 0.54 0.56 0.66 0.94 Serine 0.31 0.45 0.63 0.53 0.57 0.68 1.00 Threonine 0.36 0.50 0.62 0.55 0.58 0.66 0.97 Tryptophan LQ LQ LQ LQ LQ LQ LQ Tyrosine 0.19 0.26 0.36 0.29 0.30 0.32 0.60 Valine 0.39 0.54 0.68 0.61 0.63 0.76 1.08 Total non-essential amino acids 2.07 4.59 7.68 5.53 7.24 8.36 12.49 Total essential amino acids 2.44 3.44 4.47 3.94 4.14 4.98 7.37 Total 4.51 8.04 12.16 9.48 11.38 13.34 19.86 Table A3. Amino acids content in the PO fruiting body. (b) PO Fruiting Body 0% 2.5% 5% 7.5% 10% 12.5% 15% Alanine 1.23 1.19 1.40 1.20 1.36 1.46 1.43 Arginine 1.20 1.49 1.81 1.75 1.94 2.14 1.71 Aspartic acid 1.93 1.85 2.19 1.84 2.17 2.39 2.22 Cystine 0.21 0.21 0.27 0.25 0.25 0.24 0.24 Glutamic acid 4.90 4.06 4.58 4.35 4.99 4.71 3.84 Glycine 1.01 0.97 1.08 0.97 1.07 1.20 1.13 Histidine 0.46 0.44 0.57 0.53 0.64 0.64 0.59 Hydroxyproline LQ LQ LQ LQ LQ LQ LQ IsoLeucine 0.77 0.74 0.95 0.80 0.87 0.91 0.90 Leucine 1.27 1.19 1.37 1.15 1.34 1.45 1.42 Lysine 1.03 1.03 1.32 1.21 1.32 1.45 1.34 Methionine 0.27 0.26 0.36 0.34 0.35 0.38 0.37 Ornithine 0.00 0.00 0.23 0.23 0.28 0.31 0.22 Phenylalanine 0.80 0.77 0.97 0.92 0.95 0.99 0.99 Proline 0.78 0.76 0.98 0.88 0.96 1.03 1.01 Serine 0.94 0.89 1.16 1.04 1.14 1.20 1.11 Threonine 0.88 0.82 1.03 0.96 1.04 1.09 1.02 Tryptophan LQ LQ LQ LQ LQ LQ LQ Tyrosine 0.55 0.52 0.66 0.65 0.70 0.81 0.77 Valine 0.91 0.88 1.11 1.03 1.10 1.17 1.16 Total non-essential amino acids 12.74 11.95 14.36 13.16 14.87 15.50 13.69 Total essential amino acids 6.41 6.13 7.67 6.93 7.63 8.08 7.78 Total 19.14 18.08 22.02 20.10 22.50 23.58 21.47 Sustainability 2025,17, 6496 19 of 23 Appendix C. Fatty Acid Profile in PO Stems and Fruiting Body Table A4. Fatty acid profile in the PO stems. (a) PO Stems 0.0% 2.5% 5.0% 7.5% 10.0% 12.5% 15.0% Myristic acid 0.00 0.00 0.00 0.12 0.11 0.11 0.14 Caproic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Pentadecanoic acid 1.47 1.53 1.45 1.93 1.55 1.44 1.47 Palmitic acid 7.16 7.72 7.82 0.93 8.19 8.83 10.16 Palmitoleic acid 0.00 0.00 0.00 0.11 0.07 0.09 0.25 Heptadecanoic acid 0.00 0.11 0.10 0.15 0.13 0.13 0.12 10-heptadecenoic acid 0.35 0.30 0.19 0.28 0.25 0.20 0.13 Stearic acid 0.94 0.72 0.70 0.84 0.69 0.71 0.83 Elaidic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Oleic acid 4.29 3.76 3.96 4.66 3.89 4.30 6.76 Linolelaidic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.48 Linoleic acid 37.81 42.45 45.10 53.22 46.17 49.32 54.06 Arachidic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 g-Linolenic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.06 Alpha-linolenic acid (ALA) 0.00 0.00 0.00 0.00 0.50 0.00 0.07 11-Eicosenic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.05 Eicosadienoic acid 0.00 0.00 0.00 0.00 0.10 0.00 0.06 Behenic acid 0.00 0.00 0.08 0.09 0.09 0.08 0.09 Gamma-Eicosatrienoic acid (DGLA) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Erucic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Tricosanoic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Lignoceric acid 0.12 0.13 0.14 0.21 0.16 0.16 0.20 Nervonic acid 0.12 0.16 0.20 0.23 0.18 0.20 0.38 Docosahexaenoic acid (DHA) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Table A5. Fatty acid profile in the PO fruiting body. (b) PO Fruiting Body 0.0% 2.5% 5.0% 7.5% 10.0% 12.5% 15.0% Myristic acid 0.08 0.09 0.10 0.10 0.09 0.10 0.10 Caproic acid 0.06 0.00 0.00 0.00 0.00 0.00 0.00 Pentadecanoic acid 1.93 1.73 1.63 1.81 1.83 1.58 1.26 Palmitic acid 8.57 8.80 8.71 9.14 8.65 8.82 8.96 Palmitoleic acid 0.11 0.15 0.17 0.17 0.12 0.15 0.16 Heptadecanoic acid 0.10 0.09 0.08 0.10 0.11 0.10 0.09 10-heptadecenoic acid 0.16 0.15 0.11 0.10 0.10 0.10 0.09 Stearic acid 1.16 1.06 0.95 0.95 0.84 0.78 0.92 Elaidic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Oleic acid 9.38 9.16 8.33 7.94 6.59 6.95 11.63 Linolelaidic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Linoleic acid 47.78 48.49 50.86 52.34 52.59 53.09 52.12 Arachidic acid 0.05 0.00 0.00 0.00 0.00 0.00 0.07 g-Linolenic acid 0.09 0.10 0.09 0.10 0.10 0.10 0.09 Alpha-linolenic acid (ALA) 0.26 0.04 0.23 0.48 0.06 0.10 0.55 11-Eicosenic acid 0.00 0.00 0.05 0.07 0.54 0.00 0.07 Eicosadienoic acid 0.00 0.04 0.19 0.00 0.06 0.12 0.08 Behenic acid 0.23 0.18 0.10 0.17 0.11 0.09 0.17 Gamma-Eicosatrienoic acid (DGLA) 0.17 0.14 0.09 0.23 0.33 0.16 0.00 Erucic acid 0.00 0.00 0.00 0.00 0.00 0.00 0.03 Tricosanoic acid 0.06 0.00 0.00 0.04 0.03 0.03 0.00 Lignoceric acid 0.58 0.51 0.32 0.39 0.38 0.32 0.25 Nervonic acid 0.33 0.29 0.27 0.26 0.23 0.24 0.24 Docosahexaenoic acid (DHA) 0.00 1.94 0.00 0.00 1.80 0.00 0.00 Sustainability 2025,17, 6496 20 of 23 Appendix D. Trace Elements Content in PO Stems and Fruiting Body Table A6. Trace elements content in PO fruiting body. Trace Metals (mg/kg DM%) Stems 0% Stems 2.5% Stems 5% Stems 7.5% Stems 10% Stems 12.5% Stems 15% Al ** 21.3 ±0 <20 <20 <20 <20 <20 <20 As ** <5 <5 <5 <5 <5 <5 <5 Ca ** 159.5 ±6.5 122 ±9 128.5 ±8.5 107.9 ±25.1 81.65 ±8.1 99.4 ±16.6 107.8 ±9.2 Cd ** <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 Co ** <1 <1 <1 <1 <1 <1 <1 Cr ** <2 <2 <2 <2 <2 <2 <2 Cu ** <10 <10 <10 <10 <10 12.2 ±1.9 13.6 ±0.15 Fe ** 72.85 ±4.15 67.5 ±4.1 74.6 ±0.8 66.65 ±0.15 64.7 ±00 62.85 ±3.95 61.5 ±1 Hg ** <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 K * 31,250 ±550 32,150 ±450 33,050 ±250 30,700 ±200 30,850 ±350 29,750 ±50 27,600 ±400 Mg * 1525 ±5 1670 ±20 1650 ±10 1515 ±15 1615 ±5 1445 ±25 1340 ±10 Mn * 9.7 ±0.1 11 ±0.2 11.8 ±0.1 11.1 ±00 12.75 ±0.15 12.8 ±1.2 11.55 ±0.05 Mo ** <1 <1 <1 <1 <1 <1 <1 Ni ** <1 <1 <1 <1 <1 <1 <1 P * 6580 ±30 9845 ±155 11,300 ±200 10,650 ±50 12,000 ±100 11,300 ±100 10,600 ±200 Pb ** <10 <10 <10 <10 <10 <10 <10 S * 2550 ±20 2535 ±75 2685 ±15 2620 ±20 2910 ±10 2850 ±10 3025 ±55 Se ** <10 <10 <10 <10 <10 <10 <10 Si * 63.15 ±0.75 36.95 ±14.15 25.6 ±7.3 21.65 ±2.25 38.35 ±0.9 27.8 ±6.3 44 ±6.3 Sn ** <5 <5 <5 <5 <5 <5 <5 V ** <2 <2 <2 <2 <2 <2 <2 Zn ** 84.2 ±2.4 82.15 ±2.35 78.7 ±0.4 73.7 ±1 82.65 ±0.15 95.45 ±11.55 74.3 ±1.9 *p-value < 0.05; ** p-value ≥0.05. Table A7. Trace element content in PO stems. Trace Metals (mg/kg DM%) Stems 0% Stems 2.5% Stems 5% Stems 7.5% Stems 10% Stems 12.5% Stems 15% Al * 40.45 ±7.05 43 ±9.8 60.9 ±4 45.75 ±3.55 32.7 ±1 26.8 ±3.9 <20 As ** <5 <5 <5 <5 <5 <5 <5 Ca * 410.5 ±21.5 616 ±60 777.5 ±55.5 536.5 ±2.5 325 ±9 253 ±6 248.5 ±48.5 Cd ** <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 Co ** <1 <1 <1 <1 <1 <1 <1 Cr ** <2 <2 <2 <2 <2 <2 <2 Cu ** <10 <10 14.9 ±0.4 15.25 ±0.65 16.1 ±0.5 17.65 ±1.75 15.75 ±0.05 Fe ** <50 <50 70.8 ±2.4 49.85 ±2.45 <50 <50 71.5 Hg ** <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 K * 20,800 ±600 24,300 ±0 25,550 ±50 21,300 ±300 20,800 ±200 20,400 ±100 23,400 ±400 Mg * 1240 ±60 1305 ±15 1160 ±10 1020 ±10 965.5 ±10.5 871.5 ±9.5 1055 ±25 Mn * 7.25 ±0.32 9.83 ±0.46 9.89 ±0.41 7.59 ±0.09 7.8 ±0 6.92 ±0.17 9.41 ±0.35 Mo ** <1 <1 <1 <1 <1 <1 <1 Ni ** <1 <1 <1 <1 <1 <1 <1 P * 1715 ±5 4215 ±5 5860 ±40 5610 ±10 5715 ±125 5475 ±115 7815 ±35 Pb ** <10 <10 <10 <10 <10 <10 <10 S * 1510 ±30 1655 ±30 2040 ±70 1530 ±10 1480 ±20 1430 ±50 2200 ±30 Se ** <10 <10 <10 <10 <10 <10 <10 Si * 145 ±19 157.5 ±4.5 190 ±9 155.5 ±2.5 126 ±5 123 ±2 88.75 ±2.95 Sn ** <5 <5 <5 <5 <5 <5 <5 V ** <2 <2 <2 <2 <2 <2 <2 Zn <50 <50 51 ±0 <50 <50 <50 62.8 *p-value ≥0.05; ** p-value < 0.05. 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