Falkenberg, Susan Skanderup

Der er en stigende interesse i bioaktive peptider fra marine sekundære produkter, idet de er i besiddelse af et stort potentiale for at blive inkorporeret til funktionelle fødevarer og til medicinale formål. Bioaktive peptider fra det marine område er observeret værende i besiddelse af et bredt spekter af fysiologiske funktioner bl.a. antioxidative, blodtrykssænkende, antimikrobielle, anticancer samt effekt mod diabetes 2. Størstedelen af forskningen har dog fokuseret på peptider genereret fra hydrolyse med kommercielle industrielle enzymer og anvendelsen af disse hydrolysater. Det kunne være interessant at finde ud af om nedbrydelse af sekundære produkter fra fisk med fordøjelsesproteaser genererer peptider, som også er i besiddelse af disse sundhedsfremmende egenskaber, enten set i relation til fordøjelse eller som et alternativ til anvendelsen/brugen af industrielle proteaser. Generelt må fisk være udstyret med et særligt bioaktivt forsvar mod den store bakteriemængede i vandet, og må derfor forventes at være i besiddelse af bio-molekyler såsom små peptider. Det kunne derfor være relevant at undersøge om disse naturligt forekommende peptider indeholder andre funktionelle og sundhedsfremmende bioaktive peptider. På denne baggrund har dette PhD projekt haft til formål at finde og karakterisere hidtil ukendte bioaktive peptider fra marine sekundære produkter. Projektet var inddelt i to mere specifikke formål. Del I var at undersøge naturligt forekommende peptider for bioaktiviteter som radikal scavenging aktivitet, Angiotensin I-converting enzym (ACE) og dipeptidyl peptidase (DPP-IV) inbiberende egenskaber og protease inhiberende aktivitet i væv fra sekundære produkter som gæller, buglap muskel og skind fra laks (Salmo salar). Dette blev udført i ekstrakter fra ubehandlet og varmebehandlet væv ved hjælp af in vitro assays. Hvis bioaktivitets molekyler blev påvist, var det desuden et mål at karakterisere de korresponderende bioaktivitets kandidater. I del II var formålet at undersøge peptider i hydrolysater fra buglap muskel og skind genereret ved hjælp af fordøjelsesproteaser for radikal scavenging aktivitet, ACE og DPP-IV inbiberende egenskaber. Yderligere var formålet at studere stabiliteten og mekanismen af muskel hydrolysat overfor ACE og DPP-IV aktivitet. Derudover, hvis bioaktivitets molekyler var påvist, var det ligeledes et mål at karakterisere de korresponderende bioaktivitets kandidater. For de naturligt forekommende peptider undersøgt i del I, blev radikal scavenging aktivitet fundet i &lt;10 kDa ekstrakter fra gæller, buglap muskel og skind med respektive EC50 værdier på 39, 82 og 100 μg/mL. Der kunne ikke detekteres ACE og DPP-IV inhiberende aktivitet. Analyse af dominerende komponenter i aktive fraktioner fra gel kromatografi ved hjælp af massespektrometri viste, at familier fra beslægtede komponenter var i mange fraktioner i forskelligt væv, men mest udtalt i gæller. En familie var defineret i overensstemmelse med en specifik amino syre sekvens (PW). Tre familier var defineret ved hjælp af m/z værdien af den mindste komponent i hver familie (219, 434 og 403). De tre sidstnævnte indeholdt ikke standard umodificerede amino syrer, hvilket indikerer peptider med modificerede amino syrer eller andre typer molekyler. For peptiderne i hydrolysaterne genereret ved hjælp af fordøjelsesproteaser i del II, viste analyse af &lt;10 kDa hydrolysaterne at fordøjelsesproteaser genererede peptider med en tydelig radikal scavenging aktivitet samt DPP-IV og ACE inhiberende aktivitet. Hydrolysater fra pepsin fordøjelse udviste den laveste EC50 værdi for radikal scavenging aktivitet og DPP-IV inhibering, hvorimod EC50 tiltog i hydrolysater efter efterfølgende nedbrydning med proteaser fra pancreatin og mucosa. Interessant, så blev EC50 værdierne for DPP-IV næsten ikke påvirket af sekventiel nedbrydning. Muskel hydrolysatet viste både kompetitive og ikkekompetitiv inhibering, men en forlænget inkubation viste at inhiberingsegenskaberne var ustabile og ville derfor nedbrydes helt som kompetitive substrater af fordøjelsesproteaser. Når hydrolysater blev fraktioneret ved hjælp af gel kromatografi, blev radikal scavenging aktivitet observeret i alle hydrolysaterne, dog udviste hydrolysaterne fra buglap muskel stærkere aktivitet end hydrolysaterne fra skind. DPP-IV og ACE inhiberende aktivitet blev observeret i næsten alle de fraktionerede hydrolysater, kun i det pepsin genererede hydrolysate blev ingen tydelig (eller måske ingen) DPP-IV aktivitet observeret. Dette er bemærkelsesværdigt, idet det ikke var i overensstemmelse med resultaterne fra EC50 værdierne fra tre-folds fortyndingskurverne. Dog er det interessant, idet det kan skyldes en synergi effekt som kun er til stede i hoved hydrolysatet, og som forsvinder når hydrolysatet separeres til fraktioner. Analyse ved hjælp af massespektrometri af de dominerende komponenter fra de aktive fraktioner fra buglap muskel og skind hydrolysaterne genereret af pancreatin/mucosa viste, at mange komponenter var til stede i flere fraktioner. Det har endnu ikke været muligt at identificere mulige kandidat bioaktive komponenter som er ansvarlige for én bestemt bioaktivitet, idet en mere uddybende analyse og karakterisering er påkrævet. Dette PhD projekt viste et tydeligt potentiale for bioaktive peptider med sundhedsfremmende egenskaber fra marine sekundære produkter, både set i relation til fordøjelse og som et alternativ til anvendelse af industrielle proteaser.There is an increasing interest in bioactive peptides from marine secondary products, as they offer a great potential for incorporation into functional food and for medical purposes. Bioactive peptides from marine sources have been found to display a wide range of physiological functions including antioxidative, antihypertensive, antimicrobial, immunomodulatory, anticancer and diabetes 2 effects among others. However, majority of the research has been focusing on the peptides derived from hydrolysis with commercial industrial enzymes and the usefulness of these hydrolysates.It could be interesting whether digestion of fish secondary tissue with gastrointestinal proteases generates peptides, which also have these health promoting properties either in relation to gastrointestinal digestion or as an alternative to the use of industrial proteases. Furthermore, as a bioactive defense system against the bacterial load in the water, fish is expected to possess bio-components as small peptides. It could therefore be relevant whether these naturally occurring peptides exhibit other functional and health promoting bioactive properties.On this background the overall goal of the present PhD research was to discover and characterize novel bioactive peptides from marine secondary products. The research was divided into two more specific objectives in different parts. Part I was to investigate naturally occurring peptides for bioactivities as radical scavenging activity, Angiotensin I-converting enzyme (ACE) and intestinal dipeptidyl peptidase (DPP-IV) inhibiting properties and protease inhibiting activity in tissue of secondary products such as gills, belly flap muscle and skin from salmon (Salmo salar). This was conducted in extracts from untreated and heattreated tissue by using in vitro assays. Furthermore, if any detected, an aim was to characterize the corresponding candidate bioactive molecules. Part II was to investigate peptides in hydrolysates from salmon (Salmo salar) belly flap muscle and skin generated by gastrointestinal proteases for radical scavenging activity, DPP-IV and ACE inhibiting properties. Furthermore it was the aim to study the stability and mechanism of the muscle hydrolysates towards ACE and DPP-IV activity. Also, the corresponding candidate bioactive molecules, - if any, in selected hydrolysates should be characterized.For the naturally occurring peptides investigated in part I, radical scavenging activity was detected in &lt;10 kDa extracts of gills, belly flap muscle and skin with EC50 values of 39, 82 and 100 μg/mL, respectively. No ACE and DPP-IV inhibiting activity could be detected. Mass spectrometry analysis of dominating compounds in active fractions from size exclusion chromatography showed that families of related compounds were found in several fractions from different tissues but most pronounced in gills. One family was defined according to content of a specific amino acid sequence (PW). Three families were defined by the m/z value of the smallest compound reported in each family (219, 434 and 403). The three latter families did not contain standard unmodified amino acids, indicating peptides with modified amino acids or other kinds of molecules.For the peptides in the hydrolysates generated by gastrointestinal proteases investigated in part II, analysis of &lt;10 kDa hydrolysates showed that gastrointestinal proteases generated peptides with clear radical scavenging activity and DPP-IV and ACE inhibiting activity as well. Hydrolysates from pepsin digestion exhibited the lowest EC50 values for radical scavenging activity and ACE inhibition, whereas EC50 increased in hydrolysates after subsequent digestion with pancreatic and mucosal proteases. Interestingly, EC50 values for the DPP-IV inhibition were hardly affected by sequential digestion. Inhibition modes for the muscle hydrolysates were both competitive and non-competitive, but prolonged incubation showed that the inhibitory properties unstable, and therefore properly digested as competitive substrates by gastrointestinal proteases.When fractionated by size exclusion chromatography, radical scavenging activity was found in all obtained hydrolysates, though hydrolysates from belly flap muscle showed a much stronger activity compared to skin hydrolysates. DPP-IV and ACE inhibiting activity was observed in nearly all fractionated hydrolysates, only in the pepsin generated hydrolysates no pronounced (or maybe none) DPP-IV inhibitory effect was observed. This is notable, as it was not in agreement with the obtained results from EC50 values for the three-fold dilution curves. However, it is interesting, as it might be due to a synergy effect only present in the main hydrolysates, which vanishes when the hydrolysates are separated into fractions.Finally, mass spectrometry analysis of dominating compounds in active fractions from size exclusion chromatography from belly flap muscle and skin hydrolysate generated from pancreatin/mucosa digestion, showed that many compounds were present in several fractions. Currently it has not been possible to identify candidate bioactive compounds responsible for a certain bioactivity, as a more thorough analysis and characterization is required as a more thorough analysis and characterization is required.Overall, this PhD research clearly showed a potential for bioactive peptides with health promoting properties from fish secondary tissues, especially when generated with gastrointestinal proteases, both in relation to gastrointestinal digestion and as an alternative to the use of industrial proteases

Online Research Database In Technology

Discovery and characterization of novel bioactive peptides from marine secondary products

http://orbit.dtu.dk/ws/files/103384353/111.pdf

Discovery and characterization of novel bioactive peptides from marine secondary products

Abstract

Similar works

Full text

Available Versions

Online Research Database In Technology