Introduction
Mushrooms are emerging as an eco-friendly alternative protein source due to their ability to convert organic waste, low production costs, and excellent digestibility (Liu et al., 2025). They are naturally rich in nutrients, particularly carbohydrates (ca. 35–70% dry weight), including complex cell wall polysaccharides such as chitin, β-glucans, and mannans. These compounds are recognized as "biological response modifiers," also referred to as prebiotics, due to their ability to modulate gut microbiota (Zhao et al., 2023). The gut microbiota plays a crucial role in numerous host biological processes, including digestion, absorption, metabolism of nutrients, and immune response, which together may contribute to promoting fish growth performance and health status (Vargas-Albores et al., 2021). For this reason, mushroom meal may be a promising aquafeed ingredient, not only as an alternative protein source but also for its potential functional effects, which may promote fish health status. Therefore, the present study formulated mushroom meal-rich diets derived from Agaricus bisporus, Lentinula edodes and Pleurotus ostreatus by-products and investigated their effects on the growth and gut microbiota of rainbow trout (Oncorhynchus mykiss).
Materials and methods
Three experimental mushroom-based diets derived from A. bisporus (AB), P. ostreatus (PO), and L. edodes (LE) were formulated based on a control diet (CTRL) containing 49% crude protein and 22% crude fat. Mushroom meals (30%) were prepared by mixing with the CTRL diet (70%) and fed to rainbow trout juveniles (initial body weight: 120.1 ± 0.4 g) for 42 days. After 42 days, fish biometry was performed to evaluate growth, and representative fish samples (n = 4 fish/tank, 12 fish/diet) were euthanized, eviscerated, and their gastrointestinal tracts dissected for microbiota analysis. Gut samples were divided into three segments (pyloric caeca, PC; anterior intestine, AI; and posterior intestine, PI), and microbiota analysis was performed in each region. After DNA extraction, quality was checked, and the bacterial V3-V4 region of the 16s rRNA gene was amplified and sequenced on an Illumina MiSeq platform (2 × 300 bp paired-end; FISABIO, Valencia, Spain). Bioinformatic analysis was performed using QIIME2 and DADA2 for cleaning and denoising, and taxonomic assignment with the SILVA database. Diversity and relative abundance were calculated based on the microeco package.
Results
Results showed no significant differences in final fish body weight, body weight gain, and specific growth rate between fish fed the CTRL and mushroom-based diets (p > 0.05). Similarly, somatic indices (condition factor, hepatosomatic index, viscerosomatic index, and perivisceral fat index) and feed efficiency parameters (feed intake and feed conversion ratio) were not affected by the dietary inclusion of mushroom meals compared to fish fed the CTRL diet (p > 0.05).
In terms of microbial diversity across gut regions, alpha and beta diversity showed a stronger similarity between PC and AI than the PI. In addition, phylogenetic diversity, which considers the number of species and the phylogenetic distance, indicates that each region is different. In terms of composition, the three gut regions analyzed in this study were dominated by Firmicutes and Proteobacteria. Regarding the effect of diets, the AB-fed group showed reduced alpha diversity in the PC region compared to the group fed the CTRL diet (p < 0.05), whereas the alpha diversity of fish fed the LE and PO diets remained unchanged (p > 0.05). In terms of beta diversity, fish fed AB and LE diets presented significantly different bacterial community structures compared to the CTRL diet-fed group (p < 0.05). In the AI region, a similar pattern was observed, with fish fed the AB diet showing reduced alpha diversity compared to the CTRL-fed group (p <0.05), whereas PO and LE fed groups were similar to those fed the CTRL diet (p > 0.05). Beta diversity showed significantly different structure among the CTRL, LE, and PO groups (p < 0.05). In the PI region, the alpha diversity of AB and LE was similar to the CTRL fed group, whereas this was reduced in the LE fed group (p < 0.05). Beta diversity showed that all dietary groups had significantly different bacterial community structure (p < 0.05).
In terms of microbial composition, the PC region showed that the CTRL-fed group has a higher relative abundance of Desulfovibrionaceae and Ligilactobacillus than the mushroom-fed groups (p < 0.05). Fish fed with AB showed a higher abundance of Mycoplasma, whereas the LE and PO groups showed dominance of Bacilli (class) and Bacillaceae and Paenobacillus, respectively. In the AI region, microbial composition for the CTRL fed group was dominated by Desulfovibrionaceae, Lactobacillaceae and Weissella. The AB-fed group showed a higher abundance of Mycoplasma, Bacilli (class) and Legionella. The LE-fed group did not reveal any significant change in bacterial abundance, whereas the PO-fed group showed an increase in Ligilactobacillus and Alphaproteobacteria. In the PI region, the CTRL-fed group maintained a higher abundance of Desulfovibrionaceae. The AB-fed group showed a higher abundance of Lactobacillaceae, whereas the PO-fed group did not show any significant change in bacterial abundance.
Conclusions
Overall, the results showed that fish fed with mushroom-based diets presented similar performance to fish fed the control diet, suggesting that mushrooms may be a suitable alternative protein source in aquafeed. Furthermore, dietary inclusion of mushroom meals differentially modulated gut microbiota in terms of diversity and composition depending on the type of mushroom and region of the intestine considered. In particular, mushroom-based diets reduced the abundance of Desulfovibrionaceae across gut regions and shifted to an increase in different bacterial genera, particularly those that are classified as probiotics, such as Lactobacillus, indicating the potential of mushrooms not just as a protein source but also as a prebiotic ingredient.
References
Liu, P., Zhang, Z., Wu, D., Li, W., Chen, W., Yang, Y., 2025. The prospect of mushroom as an alternative protein: From acquisition routes to nutritional quality, biological activity, application and beyond. Food Chem. 469, 142600.
Zhao, J., Hu, Y., Qian, C., Hussain, M., Liu, S., Zhang, A., He, R., Sun, P., 2023. The Interaction between Mushroom Polysaccharides and Gut Microbiota and Their Effect on Human Health: A Review. Biology (Basel). 12, 1–29.
Vargas-Albores, F., Martínez-Córdova, L.R., Hernández-Mendoza, A., Cicala, F., Lago-Lestón, A., Martínez-Porchas, M., 2021. Therapeutic modulation of fish gut microbiota, a feasible strategy for aquaculture? Aquaculture 544, 737050.