Ascophyllum Nodosum and Saccharina Latissima) Ingrid Maribu Master’S Thesis in Marine Biotechnology, BIO-3901, May 2021
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Norwegian College of Fishery Science, the Faculty of Bioscience, Fisheries and Economics Chemical and Enzyme-Assisted Extraction of Fucoidan from two Species of Brown Macroalgae (Ascophyllum nodosum and Saccharina latissima) Ingrid Maribu Master’s thesis in Marine Biotechnology, BIO-3901, May 2021 Acknowledgements I would like to start by thanking the Norwegian College of Fishery Science for 5 great years and who led me to discover my interest in macroalgae. I would like to express my gratitude to Nofima, in particular the Marine Bioprospecting group for allowing me to work with such an interesting field and continuous encouragement throughout the year. Especially thanks to my supervisors Tor Haug, Jan Arne Arnesen and Kjetil Elvevold. Enough cannot be said to thank Jan Arne and Kjetil for all the guidance and encouragement they have given me throughout the year, helping me with lab work and analysis, for always answering my many emails and making everyday fun. Thanks to Tor for wanting to go through another round of being my supervisor, for great guidance in writing and expertise in phlorotannins. I would also like to thank Runar Gjerp Solstad for great help with the chromatography methods, Ida Kristine Østnes Hansen for her expertise during the phlorotannin characterization and Kjersti Lian for reading and giving me feedback on my thesis. I have to give a special thanks to my family and friends for their constant support and encouragement (and for pretending to understand what I have been talking about for the last year). Most of all, I would like to thank Elmedina for always putting a smile on my face during stressful times and long hours at the library, for her continuous understanding, ideas and encouragement. Special thanks to Marthe and Christina for taking the time to read my thesis. Tromsø, May 2021 Ingrid Maribu I Abstract Marine environments contain half of the global biodiversity and are a major source of bioactive compounds. Macroalgae are vital species in marine ecosystems and are known to produce several bioactive compounds and polysaccharides that possesses several bioactivities. These molecules are traditionally extracted using methods that heavily rely on hazardous solvents and heat, that more importantly generates waste and emissions. Sustainability has gained worldwide attention and has led scientists to explore more environmentally friendly extraction methods that have a lower environmental impact. The main goal of this thesis was to compare the fucoidan yield of a conventional method against a new, greener extraction method (enzyme- assisted extraction, EAE) from two species of brown macroalgae commonly found in Norway: Ascophyllum nodosum and Saccharina latissima. In addition, the polyphenol/phlorotannin, alginate and monomeric sugars content was analyzed. The chemical extraction (CE) method was conducted at 80 C for 4 hours with 0.1 M HCl. It was tested three different enzymes (Depol692, Depol 793 and Cellulase 13), in addition to a control sample were tested and compared in EAE. The EAE method was conducted at 50 C for 3 hours with pH 5. The polyphenol/phlorotannin content was analyzed after lyophilization, while the carbohydrate-, L- fucose- and alginate content in the macroalgae were analyzed using two different methods of acid hydrolysis. The CE method resulted in a general higher fucose and carbohydrate yield from A. nodosum, indicating higher release of fucoidan and other polysaccharides compared to EAE. However, limited amounts of polyphenols/phlorotannins were detected in the samples after CE, indicating that the process may be harmful for the compounds. In contrast, the extraction yield of polyphenols/phlorotannins after EAE was good. In general, CE resulted in the higher extraction yield of fucoidan, compared to EAE. None of the enzymes used in this study showed a high efficiency compared to the control or CE. However, all enzymes showed potential in degrading the algal cell wall, but further studies are needed to make thorough conclusions on this potential. II Abbreviations A Alginate pellet from EAE ACN Acetonitrile CaCl2 Calcium chloride CE Chemical extraction DAD Diode array detector DM Dry matter DW Dry weight EAE Enzyme-assisted extraction ESI Electrospray ionization EtOH Ethanol FA Formic acid G block 훼-L-guluronic acid HCl Hydrochloric acid HP-SEC High performance – size exclusion chromatography HPLC High performance – liquid chromatography H2SO4 Sulphuric acid IEX Ion exchange chromatography L-FDH L-fucose dehydrogenase M block 훽-D-mannuronic acid Mn Number average molecular weight Mp Peak molecular weight MS Mass spectrometry m/z Mass-to-charge ratio Mw Weight average molecular weight MW Molecular weight III NADP+ / NADPH Nicotinamide adenine dinucleotide phosphate NaOH Sodium hydroxide P Permeate fraction from EAE PAD Pulsed amperometric detector PGE Phloroglucinol equivalents PGU Phloroglucinol units QToF Quadrupole – Time of Flight R Retentate fraction from EAE RID Refractive index detector SEC Size – exclusion chromatography TFA Trifluroacetic acid UPLC Ultra performance – liquid chromatography IV Table of Contents Acknowledgements ..................................................................................................................... I Abstract ..................................................................................................................................... II Abbreviations ........................................................................................................................... III 1 Introduction ........................................................................................................................ 1 1.1 Background ................................................................................................................ 1 1.2 Macroalgae ................................................................................................................. 1 1.3 Brown macroalgae...................................................................................................... 2 1.3.1 Ascophyllum nodosum ........................................................................................ 3 1.3.2 Saccharina latissima .......................................................................................... 3 1.4 Valuable components from brown macroalgae .......................................................... 4 1.4.1 Fucoidan ............................................................................................................. 5 1.4.2 Alginate .............................................................................................................. 7 1.4.3 Polyphenols ........................................................................................................ 8 1.5 Extraction of polysaccharides .................................................................................. 10 1.5.1 Conventional extraction methods ..................................................................... 10 1.5.2 New, greener extraction methods ..................................................................... 11 1.5.2.1 Enzyme assisted extraction .......................................................................... 12 1.6 Methods for polymer analysis .................................................................................. 13 1.6.1 Size exclusion chromatography ....................................................................... 13 1.6.2 Ion exchange chromatography ......................................................................... 13 1.6.3 High performance liquid chromatography – mass spectrometry ..................... 14 1.6.4 Hydrolysis methods .......................................................................................... 14 1.7 Aim of the study ....................................................................................................... 15 2 Materials and Methods ..................................................................................................... 16 2.1 Biological material ................................................................................................... 16 2.2 Enzymes ................................................................................................................... 16 V 2.3 Other chemicals ........................................................................................................ 17 2.4 Methods .................................................................................................................... 17 2.4.1 Proximate analysis............................................................................................ 17 2.4.2 Chemical extraction (CE) ................................................................................. 17 2.4.3 Enzyme assisted extraction (EAE) ................................................................... 19 2.4.4 Molecular weight analysis of polysaccharides ................................................. 20 2.4.5 Hydrochloric acid and sulphuric acid hydrolysis ............................................. 21 2.4.6 Carbohydrate analysis ...................................................................................... 22 2.4.7 L-fucose assay .................................................................................................. 22 2.4.8 Alginate assay .................................................................................................. 23 2.4.9 Polyphenol assay .............................................................................................