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Alternative proteins have emerged as promising sources for the development of more sustainable, nutritious, and technologically viable foods. This study aimed to compare the functional properties and digestibility of alternative and conventional proteins. The alternative sources evaluated were mushroom powder (CP; 24.42% protein) and freeze-dried fungal mycelium (MP; 30.07% protein). The conventional sources were soy protein isolate (PS; 69.77% protein), whey protein isolate (PL; 73.53% protein), freeze-dried pork loin (LL; 79.78% protein), and freeze-dried egg white (CL; 76.92% protein). Water-holding capacity, oil-holding capacity, protein solubility, emulsifying properties, foam formation and stability, gel formation, and in vitro gastrointestinal digestion using the standardized INFOGEST protocol were evaluated. After digestion, the bioaccessible <3 kDa fraction was analyzed for protein digestibility, based on solubilized nitrogen, and for its free amino acid profile. Water-holding capacity was highest for PS and CP, with values of 3.86 and 3.12 g/g, respectively, whereas oil-holding capacity was highest for LL (2.75 g/g) and CP (2.52 g/g). Foam stability was high, particularly for MP (99.24%) and CP (98.46%). PL and CL showed the highest emulsifying capacity, with values of 93.32% and 91.29%, respectively, whereas CP exhibited the highest emulsifying activity (23.80 m²/g). Protein solubility increased under alkaline conditions, with the highest values observed for CL, PL, and PS at pH 12. Regarding the digestibility index of the soluble fraction in the <3 kDa filtrate, PS showed the highest mean value (90.85%), followed by LL (63.09%), PL (57.92%), CL (42.27%), MP (40.15%), and CP (33.28%). In the bioaccessible fraction, the free amino acid profile indicated a predominance of leucine, lysine, arginine, and alanine, compounds associated with muscle metabolism and protein synthesis. PS showed the highest leucine (51.52 mg/g) and lysine (47.72 mg/g) contents, whereas CP had a higher alanine concentration (39.87 mg/g) and a relevant arginine content (20.63 mg/g). Overall, the samples exhibited distinct functional behaviors and digestibility, indicating that alternative and conventional proteins may be directed toward specific applications in innovative food formulations, particularly protein-based systems and hybrid products, depending on the desired technological and nutritional function.
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