Year of grant:
Nano-based, functional food ingredients from marine waste products
Náttúruvísindadeildin, Fróðskaparsetur Føroya
Susan S. Falkenberg, J. Stagsted, H.H. Nielsen, Svein-Ole Mikalsen
Grant from the FRC in DKK:
The project aims to provide an integrated strategy for the exploration and sustainable utilisation of marine waste products. Today, waste products from the commercial fishing industry are used mainly as low-grade animal protein supplements. We wish to combine biomedical and food science competencies to chart novel utilisation of chitosan-based nanoparticles as functional food vehicles for targeted delivery of bioactives, such as sensitive peptides that otherwise will be degraded in the gastro-intestinal tract. As proof-of-concept, we will use purified peptide fractions from commercial fish waste production, e.g. extracts from monkfish skin that is likely to possess antimicrobial activity preventing growth of pathogens. However, peptide-based bioactives in foods will be extensively degraded and will never be active in the gastro-intestinal tract to control growth of pathogens. Thus, we hypothesize that formulation with chitosan nanoparticles will protect bioactive peptides against degradation by digestive enzymes in the proximal gastrointestinal tract, whereas subsequent microbial and cellular digestion of the nanoparticles will release the chitosan-protected cargo and provide novel functionalities locally.
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, the majority of the research has been focusing on the peptides derived from hydrolysis with commercial industrial enzymes and the usefulness of these hydrolysates.
The overall objective of the PhD project was to discover and characterize novel bioactive peptides from marine secondary products and the project has focused on bioactive compounds from salmon tissue that could have a potential effect on oxidative stress, hypertension and diabetes 2. The research was divided into two parts. Part I where the presence of naturally occurring compounds/peptides with radical scavenging activity, Angiotensin I‐converting enzyme (ACE) and intestinal dipeptidyl peptidase (DPP‐IV) inhibiting properties in salmon gills, belly flap muscle and skin was investigated. This was conducted in extracts from untreated and heat treated tissue by using in vitro assays. Part II comprised investigations of low molecular weight peptides generated from salmon (Salmo salar) belly flap muscle and skin by digestion with gastrointestinal proteases for radical scavenging activity, DPP‐IV and ACE inhibiting properties. Furthermore, the study encompassed analysis of stability and mechanism of muscle hydrolysates inhibitory activity towards ACE and DPP‐IV. In both part I and II candidate bioactive molecules were identified and characterised by LC MS/MS
In part I, radical scavenging activity was detected in extracts of gills, belly flap muscle and skin. No ACE and DPP‐IV inhibiting activity could be detected with the in vitro assays used. Mass spectrometry analysis of dominating low molecular weight compounds in active fractions from size exclusion chromatography showed that some compounds were detected in several fractions from all tissues but most pronounced in gills and many compounds were chemically related and could be classified into different families defined by the lowest m/z value found. Only one family was defined according to content of identifiable amino acids and was designated Pro-Trp family (PW). Three families were defined by the m/z values 219, 434 and 403. These three families did not contain standard unmodified amino acids, indicating peptides with modified amino acids or other kinds of molecules.
In part II, digestion of belly flap muscle and skin with different gastrointestinal proteases in combination generated peptides with clear radical scavenging activity and DPP‐IV and ACE inhibiting activity as well. Hydrolysates from pepsin digestion of both muscle and skin exhibited the lowest EC50 values for radical scavenging activity and ACE inhibition, whereas EC50 for the hydrolysates increased after subsequent digestion with pancreatic and mucosal proteases. In contrast EC50 values for the DPP‐IV inhibition remained the same for all hydrolysates from sequential digestion. Results indicated that inhibition modes for muscle hydrolysate after complete in vitro gastrointestinal digestion were both competitive and non‐competitive.
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 presence of many candidate bioactive peptides in several fractions indicating that radical scavenging activity and ACE/DPPIV inhibitory activity in hydrolysate is a result of a synergy effect of many peptides.
Overall, the main result of the PhD project indicate the existence of novel radical scavenging compounds in gills, belly flap muscle and skin from salmon and suggests that digestion of belly flap muscle and skin with gastrointestinal proteases generate novel bioactive peptides with potential effect on oxidative stress, hypertension and diabetes 2. This is both in relation to gastrointestinal digestion of fish muscle and use of gastrointestinal proteases to produce health promoting hydrolysates as an alternative to the use of industrial proteases.
1. Scientific articles, books, thesis etc.
Susan Skanderup Falkenberg: Discovery and characterisation novel bioactive peptides from marine secondary products. PhD thesis, DTU FOOD submitted 28-2-2014, defended 28-5-2014
Falkenberg S.S., Michaelsen S-O., Joensen H., Stagsted J., Nielsen H.H. Extraction and characterization of candidate bioactive compounds in different tissues from salmon (Salmo salar), published in IJARNP Vol 7, No 4 (2014), pp 11-25
Falkenberg, SS., Stagsted J. Nielsen H.H. Hydrolysates from in vitro digestion of salmon (Salmo salar) tissues with gastrointestinal proteases: Radical scavenging activity and inhibition of dipeptidyl peptidase 4 and angiotensin I-converting enzyme. In press JAST
2. Other results, such as unpublished articles, patents, computer systems, original models and new procedures
Falkenberg S.S., Michaelsen S-O., Joensen H., Stagsted J., Nielsen H.H. Digestion of salmon proteins with intestinal proteases: Characterization of radical scavenging, dipeptidyl peptidase 4 and angiotensin I-converting enzyme inhibiting candidate peptides. In preparation
Falkenberg S.S., Michaelsen S-O., Nielsen H.H. Extraction and characterization of naturally occurring bioactive peptides from different tissues from salmon (Salmo salar) Poster presened at the 41st WEFTA meeting, 27-30 September 2011, Gothenburg, Sweden and at the FOOD Denmark PhD congress, november 2011, Copenhagen University
Falkenberg, SS., Stagsted J. Nielsen H.H. Antioxidative, DPP-IV and ACE inhibiting peptides from fish protein hydrolysed with intestinal proteases. Oral presentation at the The 4th Trans Atlantic Fisheries Technology Conference Oct 30-Nov 2 2012, Clearwater Beach, Florida USA