## 4 Conclusions  
The findings of this study reveal notable differences in extraction yields, protein, glucan content, and bioactivity across mushroom species and extraction methods, highlighting their potential as functional ingredients for diverse applications. Among the extracts, M1 showed the highest extraction yield and M2B the lowest, though M2B was the most effective for producing protein-rich ingredients, with _A. cylindracea_ identified as the most suitable species for this purpose. Conversely, _A. cylindracea_ was less sustainable for glucan-rich applications, whereas the M2B extract from _P. eryngi_ excelled. All nine extracts were rich in phenolic compounds, but no mushroom species or extract stood out in this regard. Regarding bioactivities, the M2A extract from _A. cylindracea_ demonstrated the strongest antioxidant capacity in both the ABTS and ORAC assays, underscoring its potential as a natural antioxidant. This extract also exhibited the highest antimicrobial activity, inhibiting a broad range of pathogenic bacteria, and high antibiofilm activity, attributed to its phenolic content and robust bioactive properties. In contrast, the M1 and M2B extracts from _A. cylindracea_ showed limited antimicrobial efficacy. The M1 extract from _A. cylindracea_ exhibited the strongest prebiotic effect at a 1% concentration across most of the studied bacterial strains, indicating its potential for enhancing gut health when incorporated into food products. However, additional studies using simulated gut environments would provide a more accurate understanding of how the extract functions as a prebiotic.  
In summary, this study emphasizes the unique functional properties that each mushroom and extraction method offer. _A. cylindracea_ is particularly promising for applications where protein enrichment, antioxidant, antimicrobial, antibiofilm, and prebiotic effects are desired. However, _A. cylindracea_ extracts showed cytotoxicity at the highest concentration tested (40 mg/mL), which may limit their safe use in certain applications. Nevertheless, antimicrobial and prebiotic activities occurred at lower concentrations, suggesting its potential safe use in these applications. To mitigate this, careful optimization of extraction concentrations and doses is recommended, ensuring that the beneficial bioactive effects are retained while minimizing toxicity. Meanwhile, _P. eryngi_ stands out in glucan-rich applications, and _P. ostreatus_ presents a balanced profile in antimicrobial and antibiofilm activities. These findings suggest a wide array of applications for these extracts within the food industry.  
**Author Contributions:** Conceptualization, T.B.A., S.M. and M.P.; methodology, T.B.A., S.M., P.V. and A.S.S.; validation, T.B.A., S.M. and M.P.; formal analysis, T.B.A., S.M., P.V. and A.S.S.; investigation, T.B.A. and S.M.; resources, M.P. and J.N.; data curation, T.B.A. and S.M.; writing--original draft preparation, T.B.A. and S.M.; writing--review and editing, T.B.A., S.M. and M.P.; supervision, M.P.; funding acquisition, J.N. and M.P. All authors have read and agreed to the published version of the manuscript.  
**Funding:** European Agricultural Fund for Rural Development (EAFRD), through the Rural Development Program (PDR2020), within the scope of Project MicoCoating [PDR2020-101-031472]--Valorizacao dos recursos florestais e micologicos para otimizacao de revestimentos. We would also like to thank the scientific collaboration under the FCT project UID/Multi/50016/2019.  
**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. Further inquiries can be directed to the corresponding author.  
**Conflicts of Interest:** The authors declare no conflicts of interest.