In vivo assessment of marine vs bovine origin collagen-based composite scaffolds promoting bone regeneration in a New Zealand rabbit model.

The ability of human tissues to self-repair is limited, which motivates the scientific community to explore new and better therapeutic approaches to tissue regeneration. The present manuscript provides a comparative study between a marine-based composite biomaterial, and another composed of well-established counterparts for bone tissue regeneration. Blue shark skin collagen was combined with bioapatite obtained from blue shark's teeth (mColl:BAp), while bovine collagen was combined with synthetic hydroxyapatite (bColl:Ap) to produce 3D composite scaffolds by freeze-drying. Collagens showed similar profiles, while apatite particles differed in their composition, being the marine bioapatite a fluoride-enriched ceramic. The marine-sourced biomaterials presented higher porosities, improved mechanical properties, and slower degradation rates when compared to synthetic apatite-reinforced bovine collagen. The in vivo performance regarding bone tissue regeneration was evaluated in defects created in femoral condyles in New Zealand rabbits twelve weeks post-surgery. Micro-CT results showed that mColl:BAp implanted condyles had a slower degradation and an higher tissue formation (17.9 ± 6.9 %) when compared with bColl:Ap implanted ones (12.9 ± 7.6 %). The histomorphometry analysis provided supporting evidence, confirming the observed trend by quantifying 13.1 ± 7.9 % of new tissue formation for mColl:BAp composites and 10.4 ± 3.2 % for bColl:Ap composites, suggesting the potential use of marine biomaterials for bone regeneration.

Production and Partial Characterization of Bioactive Compounds from Underutilized Marine Bioresources for a Cosmetic Formulation: Cytotoxicity and Bioactivity Evaluation.

Hydrolyzed collagen, glycogen, and hyaluronic acid, obtained through the biotechnological valorization of underutilized marine bioresources, fulfill cosmetic industry requirements for sustainable products produced under circular economy principles. Hydrolyzed collagen was obtained by hydrolyzing blue shark collagen with papain and ultrafiltration. Glycogen was isolated from industrial mussel cooking wastewaters through ultrafiltration, precipitation, and selective polysaccharide separation. Hyaluronic acid was produced by fermentation, purification, and depolymerization. The main objective was to test the feasibility of including these three biomolecules in a cosmetic formulation as bioactive compounds. For this, the in vitro irritant potential of the three ingredients and also that of the cosmetic formulation was assayed according to the Reconstituted Human Epithelium Test method OECD 439. Moreover, an in vitro assessment of the effect of hydrolyzed collagen and hyaluronic acid combinations on mRNA expression and collagen type I synthesis was evaluated in adult human fibroblasts. This study establishes, for the first time, the potential use of particular hydrolyzed collagen and hyaluronic acid combinations as stimulators of collagen I synthesis in fibroblast cultures. Besides, it provide safety information regarding potential use of those biomolecules in the formulation of a cosmetic preparation positively concluding that both, ingredients and cosmetic preparation, resulted not irritant for skin following an international validated reference method.

Assessing non-synthetic crosslinkers in biomaterial inks based on polymers of marine origin to increase the shape fidelity in 3D extrusion printing.

In the past decade, there has been significant progress in 3D printing research for tissue engineering (TE) using biomaterial inks made from natural and synthetic compounds. These constructs can aid in the regeneration process after tissue loss or injury, but achieving high shape fidelity is a challenge as it affects the construct's physical and biological performance with cells. In parallel with the growth of 3D bioprinting approaches, some marine-origin polymers have been studied due to their biocompatibility, biodegradability, low immunogenicity, and similarities to human extracellular matrix components, making them an excellent alternative to land mammal-origin polymers with reduced disease transmission risk and ethical concerns. In this research, collagen from shark skin, chitosan from squid pens, and fucoidan from brown algae were effectively blended for the manufacturing of an adequate biomaterial ink to achieve a printable, reproducible material with a high shape fidelity and reticulated using four different approaches (phosphate-buffered saline, cell culture medium, 6% CaCl2, and 5 mM Genipin). Materials characterization was composed by filament collapse, fusion behavior, swelling behavior, and rheological and compressive tests, which demonstrated favorable shape fidelity resulting in a stable structure without deformations, and interesting shear recovery properties around the 80% mark. Additionally, live/dead assays were conducted in order to assess the cell viability of an immortalized human mesenchymal stem cell line, seeded directly on the 3D printed constructs, which showed over 90% viable cells. Overall, the Roswell Park Memorial Institute cell culture medium promoted the adequate crosslinking of this biopolymer blend to serve the TE approach, taking advantage of its capacity to hamper pH decrease coming from the acidic biomaterial ink. While the crosslinking occurs, the pH can be easily monitored by the presence of the indicator phenol red in the cell culture medium, which reduces costs and time.

Deepening in the understanding of the role of collagen subunits on the differential molecular arrangement of P. glauca and M. merluccius marine collagens.

Decades of extensive efforts on marine collagen extraction and characterization allowed to recognize the unique and excellent characteristics of marine collagen offering advantages over that obtained from terrestrial sources. However, not all marine collagens have the same biochemical characteristics; understanding those at molecular and supramolecular level, is crucial for optimal design of applications. One relevant aspect of collagen characterization is the analysis of its different subunits (α-chains) and their intermolecular cross-links (ß- and γ-components), which ultimately determine the specific functions of a particular collagen. Collagens from a teleost and an elasmobranch species were analyzed to understand the influence of their subunit composition and intermolecular crosslinking pattern on their different physicochemical behaviour. For comparative purposes a commercial mammal collagen was included in the study. Although electrophoretic profiles showed the typical composition of type I collagen for hake, blue shark and calf collagen, molar ratios of their α-chains were different indicating a different degree of dimerization of their α2-chains with implications in the presence of a different crosslinking degree pattern. Electrophoresis, amino acid composition, hydrophobicity (RP-HPLC) and molecular weight analysis (GPC-HPLC) results, besides a peptide mapping and an antioxidant activity study of the resultant peptides, would help to understand the role of different subunit collagen composition and different crosslinking pattern in the conformation of a differential quaternary supramolecular structure within different species and its biofunctional implications. The experiments developed would allow to progress in the valorization potential of fish discards and byproducts to explore commercial uses of collagens from marine origin.

Development of a Ready-to-Eat Fish Product Enriched with Fish Oil Entrapped in a κ-Carrageenan Egg White Fish Protein Hydrolysate Dry Powder.

This work describes the development of a ready-to-eat (RTE) product based on an equal mixture of fish mince from three undervalued fish species with different fat contents and protein gelling capacity, which was enriched with fish oil entrapped in a κ-carrageenan egg white fish protein hydrolysate powder, obtained by either spray drying (SD) or heat drying (HD) at 80 °C (HD80). Previously, the spray-dried (SD) powder and heat-dried powders obtained at 45 °C, 60 °C and 80 °C (HD45, HD60 and HD80) were characterised in terms of water solubility, lipid oxidation (TBARS), hygroscopicity and ζ potential. All HD powders showed higher hygroscopicity and lower TBARS than the SD powder. The dry powder was incorporated into a blend composed of salt-ground batter and raw mince to improve binding and textural properties. Changes in water-holding capacity, colour, shear strength and microorganisms were monitored during the processing steps. The RTE product presented a high protein content and a noticeable amount of long-chain ω-3 fatty acids. The use of undervalued fish species together with fish oil and a protein hydrolysate from fish waste contribute to improving the sustainability of fishery resources, being conducive to obtaining a potentially functional RTE product.

Advanced Polymeric Membranes as Biomaterials Based on Marine Sources Envisaging the Regeneration of Human Tissues.

The self-repair capacity of human tissue is limited, motivating the arising of tissue engineering (TE) in building temporary scaffolds that envisage the regeneration of human tissues, including articular cartilage. However, despite the large number of preclinical data available, current therapies are not yet capable of fully restoring the entire healthy structure and function on this tissue when significantly damaged. For this reason, new biomaterial approaches are needed, and the present work proposes the development and characterization of innovative polymeric membranes formed by blending marine origin polymers, in a chemical free cross-linking approach, as biomaterials for tissue regeneration. The results confirmed the production of polyelectrolyte complexes molded as membranes, with structural stability resulting from natural intermolecular interactions between the marine biopolymers collagen, chitosan and fucoidan. Furthermore, the polymeric membranes presented adequate swelling ability without compromising cohesiveness (between 300 and 600%), appropriate surface properties, revealing mechanical properties similar to native articular cartilage. From the different formulations studied, the ones performing better were the ones produced with 3 % shark collagen, 3% chitosan and 10% fucoidan, as well as with 5% jellyfish collagen, 3% shark collagen, 3% chitosan and 10% fucoidan. Overall, the novel marine polymeric membranes demonstrated to have promising chemical, and physical properties for tissue engineering approaches, namely as thin biomaterial that can be applied over the damaged articular cartilage aiming its regeneration.

A next-generation sequencing approach for the detection of mixed species in canned tuna.

Tuna cans are relevant seafood products for which mixtures of different tuna species are not allowed according to European regulations. In order to support the prevention of food fraud and mislabelling, a next-generation sequencing methodology based on mitochondrial cytochrome b and control region markers has been tested. Analyses of defined mixtures of DNA, fresh tissue and canned tissue revealed a qualitative and, to some extent, semiquantitative identification of tuna species. While the choice of the bioinformatic pipeline had no influence in the results (p = 0.71), quantitative differences occurred depending on the treatment of the sample, marker, species, and mixture (p < 0.01). The results revealed that matrix-specific calibrators or normalization models should also be used in NGS. The method represents an important step towards a semiquantitative method for routine control of this analytically challenging food matrix. Tests of commercial samples uncovered mixed species in some cans, being not in compliance with EU regulations.

Marine origin biomaterials using a compressive and absorption methodology as cell-laden hydrogel envisaging cartilage tissue engineering.

In the recent decade, marine origin products have been growingly studied as building blocks complying with the constant demand of the biomedical sector regarding the development of new devices for Tissue Engineering and Regenerative Medicine (TERM). In this work, several combinations of marine collagen-chitosan-fucoidan hydrogel were formed using a newly developed eco-friendly compressive and absorption methodology to produce hydrogels (CAMPH), which consists of compacting the biopolymers solution while removing the excess of water. The hydrogel formulations were prepared by blending solutions of 5% collagen from jellyfish and/or 3% collagen from blue shark skin, with solutions of 3% chitosan from squid pens and solutions of 10% fucoidan from brown algae, at different ratios. The biopolymer physico-chemical characterization comprised Amino Acid analysis, ATR-FTIR, CD, SDS-PAGE, ICP, XRD, and the results suggested the shark/jellyfish collagen(s) conserved the triple helical structure and had similarities with type I and type II collagen, respectively. The studied collagens also contain a denaturation temperature of around 30-32 °C and a molecular weight between 120 and 125 kDa. Additionally, the hydrogel properties were determined by rheology, water uptake ability, degradation rate, and SEM, and the results showed that all formulations had interesting mechanical (strong viscoelastic character) and structural stability properties, with a significant positive highlight in the formulation of H3 (blending all biopolymers, i.e., 5% collagen from jellyfish, 3% collagen from skin shark, 3% chitosan and 10% of fucoidan) in the degradation test, that shows a mass loss around 18% over the 30 days, while the H1 and H2, present a mass loss of around 35% and 44%, respectively. Additionally, the in vitro cellular assessments using chondrocyte cells (ATDC5) in encapsulated state revealed, for all hydrogel formulations, a non-cytotoxic behavior. Furthermore, Live/Dead assay and Phalloidin/DAPI staining, to assess the cytoskeletal organization, proved that the hydrogels can provide a suitable microenvironment for cell adhesion, viability, and proliferation, after being encapsulated. Overall, the results show that all marine collagen (jellyfish/shark)-chitosan-fucoidan hydrogel formulations provide a good structural architecture and microenvironment, highlighting the H3 biomaterial due to containing more polymers in their composition, making it suitable for biomedical articular cartilage therapies.

The Influence of DNA Extraction Methods on Species Identification Results of Seafood Products.

In terms of species identification, the ultimate aim of extracting DNA is the subsequent amplification of the selected marker; therefore, the quality and quantity of the extracted DNA must be sufficient for PCR-based methods. The purpose of this study is to compare five DNA extraction methods according to the parameters of quantity, quality and simplicity, among others, in order to determine the most suitable method for identification for Cephalopoda, Gadiformes and Pleuronectiformes. The Wizard DNA clean-up system kit (Promega), MPure-12TM automated nucleic acid purification system (MP Biomedicals), Chelex 100 resin (Biorad), DNeasy blood and tissue kit (Qiagen) and a swab method were examined. The obtained DNA quantity was determined by fluorescence, and quality was evaluated with ratios of absorbance of A260/A280 and A260/A230 by agarose gel visualization of the extracts and by analyzing the success of PCR amplifications of 720 bp fragments of cytochrome c oxidase I (COI) for Cephalopods and 465 bp fragments of cytochrome b for Gadiformes and Pleuronectiformes. Statistical results confirmed significant differences between the tested methods according to yield, efficiency and purity and no significant differences with respect to the species employed. The best yields were obtained with the Wizard kit, whereas other methods stand out in terms of their affordability (Chelex) and automation (Mpure).

Mineralized collagen as a bioactive ink to support encapsulation of human adipose stem cells: A step towards the future of bone regeneration.

Bioprinting - printing with incorporated living cells - has earned special attention on tissue engineering approaches, aiming to closer reproduce the 3D microenvironment of the target tissue. However, it raises extra complexity related to the need to use cell-friendly printing conditions that still comply with material printing fidelity. Inspired by the composite nano structural organization of mineralized tissues, this work reports the efficiency of the chemical approach followed to in situ mineralize blue shark skin collagen, at a nano scale level, to ultimately produce stable inks. The influence of initial cellular density was evaluated by assessing three different concentrations (2.5, 5 and 7.5 × 106 cells·ml-1) of human adipose stem cells (hASC), with the higher density of encapsulated cells presenting improved viability in a long culture term. Immunodetection of osteogenic-related markers, like RUNX2 and osteopontin, 21 days after cell culture in basal conditions confirmed the potential of the ink to be applied for osteogenic purposes, which may be associated with the success of the cell-to-ink interaction and the Ca2+ ions released from the co-precipitated hydroxyapatite. A combination of mineralized shark collagen, alginate and hASC is thus proposed as a bioactive bioink with potential properties for regeneration of bone tissue.

Characterization of Gelatin and Hydrolysates from Valorization of Farmed Salmon Skin By-Products.

Salmon processing commonly involves the skinning of fish, generating by-products that need to be handled. Such skin residues may represent valuable raw materials from a valorization perspective, mainly due to their collagen content. With this approach, we propose in the present work the extraction of gelatin from farmed salmon and further valorization of the remaining residue through hydrolysis. Use of different chemical treatments prior to thermal extraction of gelatin results in a consistent yield of around 5%, but considerable differences in rheological properties. As expected from a cold-water species, salmon gelatin produces rather weak gels, ranging from 0 to 98 g Bloom. Nevertheless, the best performing gelatins show considerable structural integrity, assessed by gel permeation chromatography with light scattering detection for the first time on salmon gelatin. Finally, proteolysis of skin residues with Alcalase for 4 h maximizes digestibility and antihypertensive activity of the resulting hydrolysates, accompanied by the sharpest reduction in molecular weight and higher content of essential amino acids. These results indicate the possibility of tuning salmon gelatin properties through changes in chemical treatment conditions, and completing the valorization cycle through production of bioactive and nutritious hydrolysates.

A New Rapid Method for the Authentication of Common Octopus (Octopus vulgaris) in Seafood Products Using Recombinase Polymerase Amplification (RPA) and Lateral Flow Assay (LFA).

The common octopus (Octopus vulgaris) is a highly valued cephalopod species which is marketed with different grades of processing, such as frozen, cooked or even canned, and is likely to be mislabeled. Some molecular methods have been developed for the authentication of these products, but they are either labor-intensive and/or require specialized equipment and personnel. This work describes a newly designed rapid, sensitive and easy-to-use method for the detection of Octopus vulgaris in food products, based on Recombinase Polymerase Amplification (RPA) and a detection using a Lateral Flow assay (LFA). After studying several gene markers, a system of primers and nfo-probe was designed in the COI (Cytochrome Oxidase I) region and was successfully tested in 32 reference samples (covering 14 species) and 32 commercial products, after optimization. The method was also validated in a ring trial with eight European laboratories and represents a useful tool for food authenticity control at all levels of the value chain.

Prionace glauca skin collagen bioengineered constructs as a promising approach to trigger cartilage regeneration.

Representing a strategy of marine by-products valorization, based on isolation of biocompounds and assessment of biomedical applicability, the potential of blue shark (Prionace glauca (PG)) skin collagen to induce chondrogenic differentiation of human adipose stem cells (hASC) was investigated, with and without exogenous stimulation. For that, a cryogelation method was applied to produce highly interconnected porous 3-dimensional (3D) constructs made of collagen and collagen:hyaluronic acid (20:1). In vitro studies reveal that hASC adhere abundantly to the constructs which then suggests the early chondrogenic differentiation of those cells. These findings are supported by the mRNA expression encoding chondrogenic-related markers like Coll II and Sox-9 that are markedly upregulated at an early stage for both conditions, with and without exogenous stimulation. The introduction of hyaluronic acid (Hya) seems to play a crucial role at later time points, as shown by the evident immunodetection of aggrecan (ACAN), even without exogenous stimulation. It is hypothesized that the PG collagen itself can support chondrogenic differentiation at early time points, but exogenous stimulation is required to ensure phenotype maintenance. The present work highlights the relevance of using blue shark collagen biopolymer as a building block to produce highly effective temporary matrices for cartilage applications.

Molecular Weight Analysis of Blue Shark (Prionace glauca) Collagen Hydrolysates by GPC-LS; Effect of High Molecular Weight Hydrolysates on Fibroblast Cultures: mRNA Collagen Type I Expression and Synthesis.

High molecular weight (Mw) collagen hydrolysates have been demonstrated to produce a higher synthesis of collagen type I mRNA. Mw determination is a key factor maximizing the effect of collagen hydrolysates on collagen type I synthesis by fibroblasts. This work aimed to achieve a high average Mw in Blue Shark Collagen Hydrolysate, studying different hydrolysis parameters by GPC-LS analysis and testing its effect on mRNA Type I collagen expression. Analysis revealed differences in blue shark collagen hydrolysates Mw depending on hydrolysis conditions. Papain leads to obtaining a significantly higher Mw hydrolysate than Alcalase at different times of hydrolysis and at different enzyme/substrate ratios. Besides, the time of the hydrolysis factor is more determinant than the enzyme/substrate ratio factor for obtaining a higher or lower hydrolysate Mw when using Papain as the enzyme. Contrary, Alcalase hydrolysates resulted in similar Mw with no significant differences between different conditions of hydrolysis assayed. Blue shark collagen hydrolysate showing the highest Mw showed neither cytotoxic nor proliferation effect on fibroblast cell culture. Besides, it exhibited an increasing effect on both mRNA expression and pro-collagen I production.

Does Subunit Composition Influence the Intermolecular Crosslinking of Fish Collagen? A Study with Hake and Blue Shark Skin Collagens.

Acid-soluble collagens from European hake and Blue shark skin were isolated, characterized, and compared. As the structure of collagen determines its function, the final objective of this study was to investigate biochemical differences between both collagens to identify future potential applications. Chromatographic behavior revealed differences in collagen from both species. Increases of temperature and stirring time produced no effect on European hake collagen solubility in the mobile phase, resulting in the same chromatographic profiles. Conversely, the application of temperature and stirring-time increments showed a positive effect on Blue shark collagen solubility, resulting in different chromatographic profiles and observing higher molecular weight components when sample is incubated at 50 °C (15 min) after 48 h stirring. To test if the different chromatographic behavior exhibited by both collagens could be influenced by differences in subunit composition (alpha-chains), cation exchange chromatography was employed to separate collagen subunits. The electrophoretic patterns and gel permeation chromatography with light-scattering detection (GPC-LS) results of the obtained cation exchange peak fractions revealed differences regarding subunit composition between both species, influencing the crosslinking pattern. This is the first comparative study using GPC-LS to provide information of European hake and Blue shark collagen subunit composition.

Characterization and potential strategies for the valorisation of the Southwest Atlantic butterfish (Stromateus brasiliensis).

The waste of fish resources constitutes a serious environmental problem that must be avoided. The valorisation of by-catch species and decreasing the discard rate constitute a more efficient and sustainable use of these marine biomasses. In this work, we characterize and propose different potential uses for Stromateus brasiliensis, another frequently discarded (≥ 90%) and poorly studied by-catch species captured in the South Atlantic Ocean (FAO 41) by trawler fishing fleets. Furthermore, in the case of this species, freezing and frozen storage of the whole fish is the only strategy currently employed for its exploitation. The results revealed that muscle from S. brasiliensis presented a high content of polyunsaturated fatty acids (20.34%) and that the concentrations of both total diacyl glyceryl ethers (2.41%) and heavy metals (Hg 0.038, Pb 0.006 and Cd 0.018 mg/kg) were below the established limits for safe human consumption. Likewise, the protein hydrolysates proved to be a good source of amino acids for human consumption or animal feeding. Minced muscle blocks could be made by a mechanical separation process of the flesh, and the composition of minced muscle did not differ much from that of the whole fish. Furthermore, this process allows the incorporation of cryoprotectants and antioxidants to extend the frozen shelf life of this fatty fish. An extraction process from mechanically mixed skin and bones yielded a good source of collagen that should not be neglected.

A Real-Time PCR Method for the Authentication of Common Cuttlefish (Sepia officinalis) in Food Products.

Cephalopods are very relevant food resources. The common cuttlefish (Sepia officinalis) is highly appreciated by consumers and there is a lack of rapid methods for its authentication in food products. We introduce a new minor groove binding (MGB) TaqMan real-time PCR (Polymerase Chain Reaction) method for the authentication of S. officinalis in food products to amplify a 122 base pairs (bp) fragment of the mitochondrial COI (Cytochrome Oxidase I) region. Reference and commercial samples of S. officinalis showed a threshold cycle (Ct) mean of 14.40, while the rest of the species examined did not amplify, or showed a significantly different Ct (p < 0.001). The calculated efficiency of the system was 101%, and the minimum DNA quantity detected was 10-4 ng. No cross-reactivity was detected with any other species, thus, the designed method differentiates S. officinalis from other species of the genus Sepia and other cephalopod species and works for fresh, frozen, grilled, cooked and canned samples of Sepia spp. The method has proved to be reliable and rapid, and it may prove to be a useful tool for the control of fraud in cuttlefish products.

Production, Characterization, and Bioactivity of Fish Protein Hydrolysates from Aquaculture Turbot (Scophthalmus maximus) Wastes.

The valorization of wastes generated in the processing of farmed fish is currently an issue of extreme relevance for the industry, aiming to accomplish the objectives of circular bioeconomy. In the present report, turbot (Scophthalmus maximus) by-products were subjected to Alcalase hydrolysis under the optimal conditions initially defined by response surface methodology. All the fish protein hydrolysates (FPHs) showed a high yield of digestion (>83%), very remarkable degrees of hydrolysis (30-37%), high content of soluble protein (>62 g/L), an excellent profile of amino acids, and almost total in vitro digestibility (higher than 92%). Antioxidant and antihypertensive activities were analyzed in all cases, viscera hydrolysates being the most active. The range of average molecular weights (Mw) of turbot hydrolysates varied from 1200 to 1669 Da, and peptide size distribution showed that the hydrolysate of viscera had the highest content of peptides above 1000 Da and below 200 Da.

Cell-Laden Biomimetically Mineralized Shark-Skin-Collagen-Based 3D Printed Hydrogels for the Engineering of Hard Tissues.

Mineralization processes based on coprecipitation methods have been applied as a promising alternative to the most commonly used methods of polymer-ceramic combination, direct mixing, and incubation in simulated body fluid (SBF) or modified SBF. In the present study, for the first time, the in situ mineralization (ideally hydroxyapatite formation) of blue shark (Prionace glauca (PG)) collagen to fabricate 3D printable cell-laden hydrogels is proposed. In the first part, several parameters for collagen mineralization were tested until optimization. The hydroxyapatite formation was confirmed by FT-IR, XRD, and TEM techniques. In the second part, stable bioinks combining the biomimetically mineralized collagen with alginate (AG) (1:1, 1:2, 1:3, and AG) solution were used for 3D printing of hydrogels. The addition of Ca2+ ions into the system did present a synergistic effect: by one side, the in situ mineralization of the collagen occurred, and at same time, they were also useful to ionically cross-link the blends with alginate, avoiding the addition of any cytotoxic chemical cross-linking agent. Mouse fibroblast cell line survival during and after printing was favored by the presence of PG collagen as exhibited by the biological performance of the hydrogels. Inspired in a concept of marine byproduct valorization, 3D bioprinting of in situ mineralized blue shark collagen is thus proposed as a promising approach, envisioning the engineering of mineralized tissues.

Valorization of Aquaculture By-Products of Salmonids to Produce Enzymatic Hydrolysates: Process Optimization, Chemical Characterization and Evaluation of Bioactives.

In the present manuscript, various by-products (heads, trimmings, and frames) generated from salmonids (rainbow trout and salmon) processing were evaluated as substrates for the production of fish protein hydrolysates (FPHs), potentially adequate as protein ingredients of aquaculture feeds. Initially, enzymatic conditions of hydrolysis were optimized using second order rotatable designs and multivariable statistical analysis. The optimal conditions for the Alcalase hydrolysis of heads were 0.1% (v/w) of enzyme concentration, pH 8.27, 56.2°C, ratio (Solid:Liquid = 1:1), 3 h of hydrolysis, and agitation of 200 rpm for rainbow trout and 0.2% (v/w) of enzyme, pH 8.98, 64.2 °C, 200 rpm, 3 h of hydrolysis, and S:L = 1:1 for salmon. These conditions obtained at 100 mL-reactor scale were then validated at 5L-reactor scale. The hydrolytic capacity of Alcalase and the protein quality of FPHs were excellent in terms of digestion of wastes (Vdig > 84%), high degrees of hydrolysis (Hm > 30%), high concentration of soluble protein (Prs > 48 g/L), good balance of amino acids, and almost full in vitro digestibility (Dig > 93%). Fish oils were recovered from wastes jointly with FPHs and bioactive properties of hydrolysates (antioxidant and antihypertensive) were also determined. The salmon FPHs from trimmings + frames (TF) showed the higher protein content in comparison to the rest of FPHs from salmonids. Average molecular weights of salmonid-FPHs ranged from 1.4 to 2.0 kDa and the peptide sizes distribution indicated that hydrolysates of rainbow trout heads and salmon TF led to the highest percentages of small peptides (0-500 Da).