Extraction and characterization of protein from Alaria esculenta, Palmaria palmata and Saccharina latissima
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Global population growth stimulates the search for new, sustainable ingredients in food and feed. The largest potential for production growth is found in the ocean, including cultivation and harvesting of macroalgae. Today, macroalgae are used widely as food in Asia, in addition to non-nutritional related production elsewhere. Macroalgae have the potential to contribute considerably more in the future, e.g. as a protein source in food and feed. The objective of this master's thesis was to find efficient protein extraction methods and to characterize the proteins of three macroalgae found along the Norwegian coastline: the red alga Palmaria palmata (dulse) (harvested in May and December 2015), and the brown algae Alaria esculenta (winged kelp) and Saccharina latissima (sugar kelp) (harvested in June 2015). The study is part of the PROMAC project. Two methods were used to determine the total protein content of the macroalgal raw material: A combined acidic and alkaline treatment with colorimetric determination of the protein content, and acid hydrolysis for determination of the total amino acid content. The latter method is most established, and the results were believed to be more reliable. Accordingto this method, the total protein content of P.palmata (May), P.palmata (Dec.), A.esculenta and S.latissima was 11.1+/-0.3, 11.6+/-0.3, 19.7+/-0.6 and 14.1+/-0.4 % of dry weight, respectively. Extraction procedures were performed with de-ionized water, 0.1 M formic acid and 0.12 M NaOH. The latter gave the best protein yield in all species. Enzyme assisted extractions were performed with polysaccharidases and the protease Alcalase. P.palmata was treated with two xylanases, E-XYLNP and X2629, where E-XYLNP gave the highest protein yield (up to 54 % of total protein content). Sequential treatment of the extracts with Alcalase after xylanase treatment increased the yield further. Similar extractions were performed with alginate lyase, cellulase and Alcalase for A.esculenta, and with alginate lyase and cellulase for S.latissima, but did not give increased protein yield. The polyphenol content of several extracts from the brown species was determined. No relationship was found between protein and polyphenol content. Characterization of the macroalgal proteins was performed by examining the size of the proteins. Gel filtration (FPLC) and analysis of acid soluble peptides showed that all extracts contained considerable amounts of smaller proteins/peptides. Longer incubation times gave higher proportions of these. SDS-PAGE was performed in order to examine larger proteins and protein subunits of P.palmata and A.esculenta extracts. Visual bonds appeared for the red alga at about 20 and 60 kDa, but not for the brown. The composition of free amino acids in several extracts was determined. Glutamate was dominating in P.palmata extracts, followed by aspartate and alanine. For the extracts of the two brown species, alanine was present in highest amount. S.latissima extracts also contained signicant amounts of aspartate and glutamate. The highest protein yield for the two brown species was obtained with 0.12 M NaOH treatment. Enzymatic treatment with alginate lyase, cellulase and Alcalase did not increase protein extractability. NaOH treatment was also effective for P.palmata, but sequential treatment with the xylanase E-XYLNP followed by Alcalase gave the highest protein yield for this species. Characterization procedures showed that the majority of the proteins extracted from A.esculenta and S.latissima had low molecular weights, while P.palmata extracts contained both smaller and larger protein units. More studies are needed on enzymatic treatment of brown species in order to enable extraction of larger protein units in addition to proteins with low molecular weights. Further studies on xylanase/protease treatment on P.palmata can be performed to optimize the extraction process and the protein yield.