! ! ! ! ! ! ! Extraction, characterization and application of antioxidants from the Nordic brown alga Fucus vesiculosus PhD thesis by Ditte Baun Hermund National Food Institute Technical University of Denmark February 2016 ! ! DATA SHEET Title: Extraction, characterization and application of antioxidants from the Nordic brown alga Fucus vesiculosus Author: Ditte Baun Hermund Affiliation: Division of Food Technology National Food Institute Technical University of Denmark Søltofts Plads, DK-2800 Kgs. Lyngby, Denmark e-mail: [email protected], [email protected] Supervisors: Charlotte Munch Jacobsen, Professor MSO Division of Food technology National Food Institute Technical University of Denmark Kristian Fog Nielsen, Associate Professor Department of Systems Biology DTU Systems Biology Technical University of Denmark Funding: This project is financially supported by Nordic Innovation and the Technical University of Denmark ! ! ! PREFACE ! PREFACE This PhD project entitled “Extraction, characterization and application of antioxidants from the Nordic brown alga Fucus vesiculosus” was carried out at the National Food Institute (DTU Food), Technical University of Denmark (DTU). The work was supervised by Professor Charlotte Jacobsen (main supervisor) from DTU Food and Associate Professor Kristian Fog Nielsen (co- supervisor) from DTU Systems Biology. The project started in May 2012 and continued until February 2016, interrupted for 8 months due to maternity leave. The aim of this project was to extract and characterise highly antioxidative polyphenolic secondary metabolites, phlorotannins, derived from Nordic brown alga Fucus vesiculosus. Furthermore, it was to study possible applications for F. vesiculosus extracts rich in phlorotannins to inhibit lipid oxidation in food and skin care products. The PhD work was part of the project "Novel bioactive seaweed based ingredients and products" which was part of the Nordic marine 3 innovation programme financed by Nordic Innovation. Our part in this project was to study the application possibilities of extracts from Icelandic F. vesiculosus plants. This was done in collaboration with Matís in Iceland. An additional part of the PhD project, was to extract, identify and characterise phlorotannins in Nordic F. vesiculosus plants. Integrated during this part was a research stay at Kemicentrum at Lund University in Sweden. I was awarded a travel grant from the European Section of AOCS to participate in the 105th AOCS Annual Meeting & Expo in San Antionio, Texas, USA, May 4-7, 2014. Furthermore, I was granted the Young Scientist Award from Nordic Lipid Forum which supported my participation in the 28th Nordic Lipid Symposium in Reykjavik, Iceland, June 3-6, 2015. The stay in Iceland also gave me a possibility to visit Matís and their research facilities. February 21, 2016 Kgs. Lyngby, Denmark Ditte Baun Hermund i ! ACKNOWLEDGEMENT ! ACKNOWLEDGEMENTS I would like to thank my supervisors, Professor Charlotte Jacobsen and Ass. Professor Kristian Fog Nielsen for their great supervision and knowledge sharing. Warm thanks go to my main supervisor, Charlotte Jacobsen, for her guidance and encouragement, and for keeping me on the right track. Moreover, I sincerely thank all the people who contributed to the achievements of this project; (cid:120) Rósa Jónsdóttir at Matís for her knowledge sharing, collaboration and for providing the seaweed extracts (cid:120) Postdoc Merichel Plaza from Kemicentrum, for her help, expertizes and being such a loving and joyful person. It was a pleasure working with her and I hope there will be more collaboration in the future (cid:120) Postdoc Ayse (cid:46)(cid:68)(cid:85)(cid:68)(cid:71)(cid:68)(cid:247) from TUBITAK in Turkey, for her collaboration and for teaching me new emulsion techniques (cid:120) PhD Candela Poyato for her great work on the joint project with Birgitte Raagaard Thomsen (cid:120) Ulf Andersen and Betina Mikkelsen at Arla food Ingredients in Brabrand for having us visiting their facilities to do CLSM (cid:120) Primex and Arla Food for providing ingredients for some of my experiments (cid:120) Louise Helene Søgaard Jensen who took time making microscopic images of granola bars on her super advanced microscope (cid:120) Lab technicians at DTU Food Lyngby, Inge, Trang, Lis and Victoria, for their expertise and assistance in the lab (cid:120) Lab technicians Birgitte Koch Herbst and Vera Lykkerask Berg at DTU Food in Mørkhøj for their great help and expertise in metal measurements and for operating the ASE300 (cid:120) My great and talented master students, Betül and Philipp, it has been fun and challenging (in a good way) to teach you about lipid oxidation and seaweed I would also like to thank all my Food-colleagues for your great help, support and scientific as well as non-scientific discussions. Especially thanks to my PhD fellow students for their support and for all the fun (and beers) that we shared. Lastly, thanks to my friends and family for your help and support. A special thanks to my awesome husband for his eternal love and devotion. Ditte Baun Hermund iii ! SUMMARY ! SUMMARY Marine algae are a huge underutilized resource in the Nordic countries with a potential to be used in the development of new natural ingredients for the food, cosmetics and pharmaceutical industry. Such ingredients can act as natural preservatives and prevent product deterioration during storage, in particular in the form of rancidity due to oxidation of unsaturated fatty acids in the products. A characteristic feature of Fucus vesiculosus, also known as bladder wrack, is a high content of phlorotannins – a particular type of polyphenol group. Previous studies have shown positive correlations between the phlorotannin content and radical scavenging capacity of extracts derived from Nordic F. vesiculosus. Radical scavenging capacity is an important antioxidant property in terms of preventing the oxidation of unsaturated fatty acids. The high content of antioxidative phlorotannins in F. vesiculosus therefore makes this alga particularly attractive for the development of new natural antioxidants. While the in vitro antioxidant properties of F. vesiculosus extracts are widely studied, studies evaluating the antioxidant efficacy of such extracts in food and skin care products are scarce. This PhD study investigated the possibilities of using extracts from Nordic F. vesiculosus as natural antioxidants in food and skin care products. All tested food products were fortified with fish oil rich in polyunsaturated omega-3 fatty acids. The fish oil was added specifically in order to examine the effectiveness of the antioxidants in systems which are more likely to oxidize compared with conventional products. The products tested were all oil-in-water emulsions except for granola bars, which were instead added 70% fish oil-in-water emulsions. Tests were made on a selections of extracts made from water, acetone, and ethanol, as well as a fraction of purified phlorotannins. Investigations also highlighted the influence of the extraction medium on the antioxidant properties, the phlorotannin content as well as other co-extracted substances. Moreover, it was examined which phlorotannins were present in each of the extracts, and how each specific phlorotannin contributed to the overall antioxidant activity. All extracts examined and also the phlorotannin-rich fraction were somewhat able to improve the oxidative stability of the food and skin care products. The effectiveness of these extracts was to a large degree dependant on their antioxidant properties and composition, which in turn depended on the extraction medium used. In general, water was efficient in extracting iron chelating compounds. However, it was also found that water was not effective in extracting phlorotannins, and that the iron chelating ability, according to our results, to a greater extent was due to the presence of the pigment 19-hex-fucoxanthin. It has also been discussed whether algal sugars with iron chelating ability may be extracted with water and hence affect the antioxidant properties of the v ! SUMMARY ! water extract. However, this aspect was not investigated. The high iron chelating ability of the water extract proved particularly effective in FO-enriched mayonnaise. Previous studies have also shown that iron chelating ability is an important property of antioxidants to work efficiently in this particular food. Acetone and ethanol were highly effective in extracting phlorotannins, which were found to have good radical scavenging capacity as well as reducing power. In addition, these phlorotannins exhibited a high affinity to the interface between the hydrophilic and the hydrophobic phase, compared to phlorotannins extracted with water. The more amphiphilic phlorotannins were also found to be effective antioxidants in FO-enriched granola bars. It was examined from microscopy how the emulsified fish oil added seaweed extracts localized when added to the granola bars. Emulsions added extracts with more amphiphilic phlorotannins clearly improved incorporation of the fish oil emulsions into the granola bars, which in turn had a major impact on the oxidative stability of these products. It was concluded that the surface active phlorotannins were important radical scavengers in granola bars. These phlorotannins are chain-breaking antioxidants that deactivate lipid radicals formed in the first part of lipid oxidation. In addition, it was discussed whether some of these phlorotannins also regenerated antioxidative tocopherols from the oil phase. A structural characterization and on-line detection of phlorotannins in the purified fraction was carried out in support of a further characterisation of phlorotannins and how they each contribute to the overall antioxidant activity. By mass spectrometry 13 phlorotannin isomers were identified with molecular weights between 374 and 870 Da (3 to 7 phloroglucinol units). It was found that the antioxidant activity is decreasing with increased molecular weight and hence with increased polymerization of the phlorotannins. This PhD work has contributed basic knowledge of relevance to future large scale development of natural antioxidants from seaweeds to the benefit of the food, cosmetic and pharmaceutical industrial sectors. It is clearly demonstrated that it is possible to produce antioxidants from seaweed thallus, and also that it is possible to use alternative environment-friendly extraction methods. In addition, the studies highlight examples of application possibilities of seaweed extracts as natural antioxidants, e.g. in the formulation of functional foods enriched with fish oil. vi ! RESUMÈ ! RESUMÉ Tang er en stor uudnyttet ressource i Norden, som har potentiale til at kunne anvendes til udvikling af nye naturlige ingredienser til fødevare-, kosmetik- of lægemiddelindustrien. Sådanne ingredienser kan virke som naturlige konserveringsmidler for at undgå produktforringelse under lagring, bl.a. i form af harskning grundet oxidation af umættede fedtsyrer i produkterne. Fucus vesiculosus, også kaldet blæretang, er særligt kendetegnet ved at have et højt indhold af en særlig slags polyphenoler, kaldet phlorotanniner. Tidligere studier har påvist en sammenhæng mellem et højt phlorotanninindhold og høj in vitro antioxidant aktivitet, særligt radikal indfangningsevne, af ekstrakter fra bl.a. nordisk F. vesiculosus. Radikalindfangningsevnen er en vigtig egenskab i forhold til at forhindre oxidation af umættede fedtsyrer. Det høje indhold af de antioxidative phlorotanniner i F. vesiculosus gør derfor denne art særlig attraktiv til udvikling af nye naturlige antioxidanter udvundet fra tang. Mens studier, der undersøger in vitro antioxidant egenskaber i antioxidant assays af F. vesiculosus ekstrakter, er udbredte, er der noget længere mellem studier, som behandler antioxidant- aktiviteten af lignende ekstrakter i bl.a. fødevare- og hudplejeprodukter. Dette ph.d. studie undersøgte mulighederne for at anvende ekstrakter fra nordisk F. vesiculosus som naturlige antioxidanter i fødevare- og hudplejeprodukter. Alle testede fødevareprodukter var berigede med fiskeolie rig på flerumættede omega-3 fedtsyrer. Fiskeolien blev tilsat netop for at undersøge, hvor effektive antioxidanterne var i systemer, som er mere tilbøjelige til at oxidere sammenlignet med konventionelle produkter. De testede produkter var alle olie-i-vand emulsioner på nær müslibarerne, som i stedet var tilsat 70% fiskeolie-i-vand emulsioner. Et udvalg af ekstrakter lavet på vand, acetone og ethanol, samt en fraktion af oprensede phlorotanniner blev testet. Indflydelsen af ekstraktions-mediet på de antioxidative egenskaber, indholdet af phlorotanniner og de øvrige ekstraherede stoffer blev kortlagt. Det blev ydermere undersøgt, hvilke phlorotanniner der er til stede i disse ekstrakter, og hvordan hver enkel phlorotannin kan bidrage til den overordnede antioxidant-aktivitet. Alle undersøgte ekstrakter og den phlorotannin-rige fraktion var i nogen grad i stand til at forbedre den oxidative stabilitet af fødevare- og hudplejeprodukter. Effektiviteten af disse ekstrakter afhang dog i høj grad af deres antioxidative egenskaber og komposition, som igen afhang af, hvilket ekstraktionsmedie der var blevet brugt under ekstraktionen. Generelt var vand effektivt til at ekstrahere stoffer med jernkelerende evne. Dog viste det sig, at vand ikke var effektivt til at ekstrahere phlorotanniner, og at den jernkelerende evne ifølge vores resultater i højere grad skyldes tilstedeværelsen af 19-hex-fucoxanthin. Dog blev det også diskuteret, hvorvidt sukre med vii ! RESUMÈ ! jernkelerende evne muligvis i højere grad ekstraheres med vand og dermed kan have indflydelse på vandekstraktets antioxidative egenskaber. Dette blev dog ikke undersøgt nærmere. Den høje jernkelerende evne for vandekstraktet viste sig særligt effektiv i fiskeolieberiget mayonnaise. Tidligere studier har også vist, at jernkelerende aktivitet er en vigtig egenskab hos antioxidanter, som er effektive i netop denne fødevare. Acetone og ethanol var i høj grad effektive til at ekstrahere phlorotanniner, som viste sig at have gode radikalindfangnings- samt reducerende evner. Ydermere udviste disse phlorotanniner en høj affinitet til grænsefladen mellem den hydrofile og hydrofobe fase sammenlignet med de phlorotanniner, som var ekstraheret med vand. De mere amphiphile phlorotanniner viste sig også at være effektive antioxidanter i fiskeolieberigede müslibarer. Med mikroskopi blev det undersøgt, hvordan for-emulgerede fiskeolieemulsioner tilsat tangekstrakter lokaliseredes, når de kom i müslibardejen. Det viste sig, at ved tilsætningen af emulsioner, hvor de mere amphiphile phlorotanniner var til stede, forblev oliedråberne i højere grad intakte, hvilket havde stor betydning for den oxidative stabilitet af müslibarerne. Det blev konkluderet at de overfladeaktive phlorotanniner var vigtige radikalindfangere i müslibarer. Disse phlorotanniner virkede i høj grad som kædebrydende antioxidanter ved at deaktivere lipidradikaler dannet i første del af lipidoxidationen. Derudover blev det diskuteret, hvorvidt nogle af disse phlorotanniner også regenererede antioxidative tocoferoler i oliefasen. Ved nærmere undersøgelse af phlortanninernes sammensætning, og hvordan disse hver især bidrog til den overordnede antioxidant aktivitet, blev der foretaget strukturkarakterisering samt on- line detektion af phlorotanninerne i den oprensede fraktion. Ved massespektrometri blev der identificeret 13 phlorotanninisomerer med molekylevægte mellem 374 og 870 Da (3 til 7 phloroglucinolenheder). Det viste sig, at antioxidant aktiviteten var faldende med øget molekylevægt og dermed også med øget polymerisering af phlorotanninerne. Dette ph.d. arbejde har bidraget med central basisviden, som er nødvendig for en fremtidig udvikling af naturlige antioxidanter udvundet fra tang i industriel skala til gavn for fødevare-, kosmetik- og lægemiddelindustrien. Studierne har således demonstrereret, at det er muligt at producere antioxidative tangekstrakter og har også åbnet mulighederne for at bruge alternative miljøvenlige ekstraktionsmetoder til ekstraktion af antioxidanter fra tang. Derudover har studierne anvist eksempler på anvendelsesmuligheder der er for sådanne tangekstrakter som naturlige antioxidanter bl.a. i formuleringen af funktionelle fødevarer beriget med fiskeolie. viii !