Searching for efficient methods of extraction and characterization of protein from Saccharina latissima and Alaria esculenta.
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AbstractGrowing population and alteration of the environment due to climate change and human im-pact, water scarcity and reduction of the available arable land demand search for alternative food sources. To meet the increasing demand for food, more resources must come from seas and oceans. Exploring possibilities of using seaweed as food and feed is one way of increas-ing the amount of food from the ocean and it is an important path in meeting the increasing demand for nutrient-rich food and feed. Of the three main nutrients: protein, fat and carbo-hydrates, the need for protein is the most difficult to satisfy, therefore there should be an in-creased focus on exploring new protein sources. Successful extraction of macroalgal protein might lead to sustainable production of protein concentrates and other types of food and feed additives rich in protein.The purpose of this thesis was to examine how different conditions influence the extraction of protein from two brown macroalgae: Saccharina latissima and Alaria esculenta. Both contain all the essential amino acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valineA two steps protein extraction was conducted. First, a standard extraction in distilled water was performed. Then, the pellets from this extraction were resuspended in one of the follow-ing: 0.1M NaOH, 0.4M NaOH, 0.1M HCl, 0.4M HCl or distilled water. Further, samples were divided into three groups and were exposed to: no treatment, ultrasound bath and sonicator. then, protein content was measured using Lowry method. Total soluble FAA content and sol-uble FAA profile were determined with HPLC analysis.Protein concentration Saccharina latissima was the highest in samples extracted in alkali and treated with ultrasound. For the first set of samples (originating from sample SL0.1) extracted in 0.1M NaOH, protein content increased from 302.5 µg/mL in SL2.1 (no treatment) to 443.5 µg/mL in SL9.1 (sonicator) and 428.6 µg/mL in SL5.1 (ultrasound bath). For the same extrac-tion from samples originating from SL0.2, a decrease in extracted protein yield was observed. Protein content decreased from 446.6 µg/mL in an untreated sample SL1.2, to 354.5 µg/mL in the sample SL9.2 treated in the sonicator and to 287.8 µg/mL in SL5.2 treated in the ultra-sound bath. For the samples extracted in 0.4M NaOH a similar tendency was observed. While protein yield increased with applied ultrasound treatments in samples originating from SL0.1 to 443.5 µg/mL in SL10.1 (sonciator) and 428.7 µg/mL in SL5.1 (ultrasound bath), it de-creased in samples originating from SL0.2 to 424.7 µg/mL in SL10.2 (sonicator) and SL 5.2 (ultrasound bath). However, these values were still significantly higher than those of corre-sponding samples extracted in 0.1M NaOH and H2O.Using HCl as an extraction solvent did not contribute to the increased yield of the extracted protein.For Alaria esculenta samples where NaOH was used, protein content generally increased after the ultrasound treatment was applied with the exception of sample AE10.1, originating from AE0.1 and extracted in 0.4M NaOH in sonicator. Here the protein content decreased to 69.8 µg/mL. The corresponding sample from the set originating from AE0.1, sample AE9.2 had much higher protein score, 131.7 µg/mL and it was also higher in comparison to the sample that did not undergo any treatment, AE2.2. In samples AE9.1 and AE9.2, where the extrac-tion was performed in 0.1M NaOH with treatment with sonicator the protein content in-creased to 165.3 µg/mL and 176.3 µg/mL, respectively. Protein contents of the samples ex-tracted in the same solvent concentration, where the treatment in the ultrasound bath was ap-plied were higher, 182.3 µg/mL for the sample AE5.1 and 212.4 µg/mL for AE5.2. Protein content in samples extracted in HCl was generally lower than the ones extracted in NaOH and slightly lower than the ones extracted in water (control). Several analyses of content of free amino acids (FAA) with HPLC were performed. However, due to the time limitations of this project only a selection of samples were analyzed.For S. latissima the highest total soluble FAA yield was 74.6 µg/mL and it was measured in sample SL2 that was extracted in 0.4M NaOH and did not undergo ultrasound treatment. Second highest total soluble FAA yield was 57.5 µg/mL and it was measured in sample SL1 that was extracted in 0.1M NaOH and did not undergo ultrasound treatment. The lowest total soluble FAA yield was 16.1 µg/mL measured in sample SL3 that was extracted in 0.1M HCl and did not undergo any ultrasound treatment. Generally, samples SL1 and SL extracted in 0.1M NaOH and 0.4M NaOH that did not undergo any ultrasound treatment had total soluble FAA yield significantly higher than samples extracted in the same solvents and treated with ultrasound bath or with sonicator. Total FAA content of Alaria esculenta supernatants was measured only for extractions in wa-ter. During the first extraction, total soluble FAA content was 174.1 µg/mL in AE0.1 and 221.1 µg/mL in AE0.2. Further the results showed that the second extraction with water did not contribute to a significant increase in the FAA content in the supernatant. Sample AEW3, treated with sonicator was the highest, however only slightly higher than the two other and it was 42.6 µg/mL. The analysis of the soluble FAA profile of Saccharina latissima showed that the three amino acids alanine, glutamic acid and aspartic acid are dominant among FAA found in samples ex-tracted in both solvents. Samples extracted in alkali and in acid had generally the same soluble FAA proportions with the exception of Gly/Arg that had significantly higher peak for the ex-traction in NaOH. FAA were most abundant in sample SL2 (0.4M NaOH, no ultrasound treatment) and in sample SL1 (0.1M NaOH, no ultrasound treatment). Samples SL5 (0.1M NaOH) and SL6 (0.4M NaOH) treated in the ultrasound bath and samples SL9 (0.1M NAOH) and SL10 (0.4M NaOH) treated in sonicator had similar FAA content and it was lower than in untreated samples. FAA after extraction in HCl were most abundant in samples SL4 (no ultrasound treatment), SL7 (ultrasound bath) and SL11 (sonicator) extracted in 0.4M HCl and FAA and it did not increase after ultrasound treatment. The lowest FAA content was found in sample SL3 that was extracted in 0.1M HCl and did not undergo ultrasound treatment.In the analysis of the soluble FAA profile of Alaria esculenta for samples from the first extrac-tion in water (AE0.1 and AE0.2) glutamic acid dominates, followed by a high content of leucine and slightly lower content of alanine. For samples extracted in water for the second time no significant increase of soluble FAA content was measured, however it was shown that sample treated with sonicator (AEW3) had the highest content of FAA.