Introduction
Protein hydrolysates have gained significant attention in recent years for their ability to deliver high-quality peptides with nutritional and health-promoting benefits in both human and animal nutrition . Among their bioactive properties, their antioxidant potential is particularly noteworthy, along with their potential to modulate microbial growth and anti-inflammatory effects, making them versatile functional ingredients. Beyond their nutritional value and bioactive properties, protein hydrolysates may offer a sustainable solution for waste valorization, particularly within the framework of a circular economy. A deeper understanding of protein hydrolysates and their functional properties is crucial for optimizing their applications in animal nutrition. This study aimed to evaluate the functional and bioactive properties of five protein hydrolysates derived from shark, fish, insect, and swine by-products, using CPSP90 as reference.
Materials and Methods
Four protein hydrolysates (PHs) were produced from different animal by-products: shark cartilage and muscle (SHARK), fish trimmings (FISH), swine meat and bone (SWINE), and whole insect larvae of Hermetia illucens (INSECT). Raw materials were hydrolyzed using either enzymatic or chemical-free thermal processing methods, optimized according to raw material type and target peptide profile. Hydrolysates were subsequently spray-dried and stored under dry conditions before analysis . Each hydrolysate was characterized in terms of d ry matter, crude protein, lipids, ash, phenolic content, chitin, amino acid profile and molecular weight distribution of peptides. Protein solubility was assessed at pH 3.0, 5.0, and 7.0 using the Bicinchoninic Acid (BCA) method. Antioxidant capacity was assessed based on 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) , 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities and Oxygen Radical Absorbance capacity (ORAC). Antibacterial activity was assessed for Eight bacterial pathogens relevant to aquaculture . Bacteria were cultured in 96-well microplates with hydrolysate solutions (40 mg/mL) added to the medium. Optical density (OD600) was measured over time to monitor growth dynamics over a period of 24h. All assays included CPSP90, a commercial soluble fish protein concentrate, with wide application in aquafeeds, as a reference.
Results
All PHs showed high protein content (55.1–89.5% DM), with SHARK and SWINE richest in collagenic amino acids (e.g., hydroxyproline), and FISH and CPSP90 showing highest essential amino acid (EAA) proportions. INSECT, though lowest in protein (55.1% DM), was high in aromatic and branched-chain amino acids (BCAAs), with total phenolics representing 2.7% DM.
Molecular weight analysis revealed that FISH and INSECT had higher proportions of small peptides (<1 kDa), while SHARK was rich in small to medium-sized peptides (1–3 kDa). In contrast, CPSP90 and SWINE exhibited a more uniform distribution of peptides across different molecular weight ranges.
INSECT displayed the highest solubility (61–65%) across all pH levels, significantly higher than CPSP90. On the other hand, SHARK showed poor solubility (26–36%), likely due to its hydrophobic collagenic composition.
In terms of bioactive potential, INSECT and FISH exhibited the strongest antioxidant activity in both ABTS (11.8 and 8.9 Trolox eq %DM, respectively) and ORAC assays. SHARK showed the lowest antioxidant potential, aligning with its low content in aromatic and sulfur-containing amino acids. No DPPH activity was detected in any PH.
Regarding bacterial growth modulation, SWINE hydrolysate consistently inhibited the growth of several pathogenic strains, including Vibrio parahaemolyticus, V. anguillarum, and Aeromonas salmonicida. In contrast, FISH and SHARK hydrolysates often promoted bacterial growth, particularly of V. parahaemolyticus, V. anguillarum, and Photobacterium damselae subsp damselae. INSECT hydrolysate had mixed effects—enhancing the growth of V. anguillarum, Pseudomonas aeruginosa, but inhibiting Tenacibaculum maritimum, A. salmonicida and V. harveyi
Discussion and Conclusion
The functional and nutritional profiles of animal-derived PHs varied widely with source and hydrolysis process . FISH and CPSP90 exhibited the highest essential amino acid content, while SHARK and SWINE hydrolysates were rich in collagenic amino acids . The higher proportion of small and small to medium peptides in INSECT, FISH, and SHARK hydrolysates is likely due to enzymatic hydrolysis using alcalase . In contrast, the more uniform peptide distribution in SWINE may be due to the thermal-pressure process of hydrolysis without added chemicals.
INSECT and FISH hydrolysates demonstrated superior antioxidant activity, while SWINE stood out for its selective antibacterial properties. While INSECT also exhibited the highest protein solubility, its effects on bacterial growth were mixed—it enhanced the proliferation of certain bacterial strains. Similarly, FISH and SHARK hydrolysates promoted the growth of specific bacteria, likely due to their high content of small and small to medium peptides, which may support microbial proliferation. While this could raise concerns in some contexts, it also suggests potential for these hydrolysates to be used as components in microbial culture media, as readily available sources of nitrogen and carbon . The selective growth-promoting properties of some hydrolysates could also be harnessed to cultivate beneficial bacterial strains or modulate microbial communities in aquaculture systems.
Overall, these findings support the targeted application of PHs in sustainable feed and food systems and highlight their potential to replace conventional protein sources, contributing to circular bioeconomy goals. Future research should focus on in vivo validation and detailed peptide characterization to fully unlock their functional potential.
Acknowledgments
This work was supported by the Blue Bioeconomy Pact (C644915664-00000026), for the exercise of activities in the WP6 FEED (Pep4Fish project) through international funds provided by the European Union and by FCT – Foundation for Science and Technology (FCT) , within the scope of UIDB/04423/2020 and UIDP/04423/2020.. L . Rodrigues-dos-Santos acknowledges FCT for the PhD grant (2023.00947.BDANA).