Almeida, André

Borges, Sandra

Martins, Rui

Odila, Joana

Pintado, Manuela

Rosa, Ana

Vanderpoorten, Wouter

English

Repositório Institucional da Universidade Católica Portuguesa

ResultsFish by-products as a source of proteolytic enzymes and bioactive peptidesSandra Borges1, Joana Odila1, Rui Martins2, Ana Rosa2, Wouter Vanderpoorten2, André Almeida2, Manuela Pintado11 CBQF - Centro de Biotecnologia e Química Fina - Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Porto, Portugal 2 ETSA, Empresa Transformadora de Subprodutos, Loures, PortugalIntroductionMethodsConclusionsAcknowledgementsThis work was supported by National Funds from project MOREPEP (POCI-01-0247-FEDER-017638), funded by Fundo Europeu de Desenvolvimento Regional (FEDER), under Programa OperacionalCompetitividade e Internacionalização (POCI). We would also to thank the scientific collaboration of CBQF under the FCT project UID/Multi/50016/2013.The increasing of fish production, at an average annual rate of 3.2%, and the growth in per capita fish consumption from 9.9 kg in 1960 to 19.7 in 2013 have generated large amounts of organic wastes. These wastes includeviscera, carcass, head, skin and bones, that are discarded without recovery efforts and which represent for more than 60% of biomass [1]. This situation creates a burdensome disposal problem and environmental concerns asthey are highly perishable. So, marine bioprocess industry has a prominent potential to reuse and apply more by-products as valuable products.Proteases are one of the most important enzymes used worldwide, accounting for approximately 50% of the industrial enzymes market. These enzymes have several industrial applications in food, feed, agriculture, cosmetics andpharmaceutics [2]. Fish internal organs constitute approximately 20% of the marine biomass, being fish viscera a rich source of proteases, i.e., different digestive enzymes (pepsin, trypsin, chymotrypsin, elastase).Enzymatic hydrolysis has gained more attention to obtain hydrolysates with improved and more defined nutritional, functional and bioactive properties. Enzymatic processes have been implemented in a broad range of industriesin recent decades because they are specific, fast in action and often save raw materials, energy, chemicals and/or water [3].In this context, enzymes from fish viscera can be employed to produce protein hydrolysates, enhancing the value of processing by-products. For this reason, the use of fish viscera become the use of the enzymatic hydrolysiseconomically more appealing and more sustainable.Currently, some research studies have been carried out with commercial enzymes in order to obtain bioactive peptides from different animal matrices. Bioactive peptides are special proteins segments that have numerouspotential physiological functions within the body, namely antioxidant and antihypertensive or angiotensin converting enzyme (ACE) inhibition.Therefore, the present study aims to use a commercial enzyme (alcalase) and an enzymatic extract from fish viscera to generate bioactive peptides from fish by-products and evaluate their potential antioxidant andantihypertensive activity.Fish viscera are usually discarded as waste or low-value by-products. This research work describes a process to extract the enzymes of fish viscera and apply them to hydrolyze fish by-products. The selected conditions for theenzymatic hydrolysis was 10% (E/S) during 6 h using the viscera enzymatic extract and 3% (E/S) during 2 h using alcalase. Fish protein hydrolysates, in addition to their high protein content, demonstrated to contain smallpeptides. All protein hydrolysates proved to have a notable antioxidant capacity, similar results were achieved for hydrolysates obtained with viscera enzymatic extract and alcalase. Fish protein hydrolysates were also able toinhibit the ACE, however, hydrolysates obtained with alcalase revealed a higher antihypertensive activity. Therefore, the viscera enzymatic extract showed a great potential to hydrolyze fish by-products demonstrating an addedvalue because, in this way, the enzymes and the hydrolysates are obtained using the same by-products, being aligned with the concept of a circular economy. In this way, it will be possible to use the animal by-products based ona sustainable approach and to promote added value compounds for animal and human nutrition.References1 - Villamil, O., Váquiro H. and Solanilla J.F. (2017) “Fish viscera protein hydrolysates: Production, potential applications and functional and bioactive properties”. Food Chemistry. 224: p. 160-1712 - Bougatef, A. (2013) “Trypsins from fish processing waste: characteristics and biotechnological applications - comprehensive review”. Journal of Cleaner Production. 57: p. 257-2653 - Jegannathan, K.R. and Nielsen, P.H. (2013) “Environmental assessment of enzyme use in industrial production - a literature review”. Journal of Cleaner Production. 42: p. 228-240Homogenization and addition of an extraction buffer to the viscera CentrifugationCollection of supernatant that comprises the viscera enzymesHydrolysis of fish with Viscera enzymatic extract (70 U/mL)orAlcalase (2600 U/mL)Fish Protein Hydrolysates(freeze-dried)- Degree of hydrolysis- FPLC- Antioxidant activity (ABTS, DPPH and ORAC)- Antihypertensive activity (ACE inhibition)AlcalaseViscera enzymatic extractFigure 1 - Degree of hydrolysis obtained for fish during hydrolysis with viscera enzymatic extract and alcalaseViscera enzymatic extract: 10% E/S, 6hAlcalase: 3% E/S, 2hDegree of hydrolysis(%)Protein content(%)Viscera enzymaticextract32.5 ± 2.8 78.8 ± 3.5Alcalase 52.1 ± 2.7 83.6 ± 2.8Bioactivity Antioxidant activity Antihypertensive activityABTS (mg ascorbic acid equivalent/g hydrolysate)DPPH(mg Trolox equivalent/g hydrolysate)ORAC(mg Trolox equivalent/g hydrolysate)IC50(μg of protein/mL)Visceraenzymatic extract10.4 ± 0.9 4.0 ± 0.1 142.0 ± 4.2 554.4Alcalase 11.0 ± 0.5 4.0 ± 0.4 153.2 ± 29.4 101.1Table 2 – Bioactivity of fish protein hydrolysatesFigure 2 – Peptide profile of fish protein hydrolysatesTable 1 – Characterization of fish protein hydrolysatesAbsorbance 280 nm (mAU)Elution volume (mL)

Fish by-products as a source of proteolytic enzymes and bioactive peptides

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Fish by-products as a source of proteolytic enzymes and bioactive peptides

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