Thermal stability of polyphenols and carotenoids from Laminaria hyperborea

Abstract

The green shift, and the focus on an increased blue bioeconomy, has stimulated the growth of sustainable production lines for marine bioactive products for food-, feed-, cosmetics-, and -pharmaceutical industries. Norway with is long coastline has for decades harvested brown kelp (Laminaria hyperborea) to produce alginate from the kelp stipes. However, the traditional alginate production is not “green”, using large amounts of formalin and the kelp leaves are discarded prior to processing, including loss of valuable compounds such as polyphenols and carotenoids. Alginor ASA, based in Haugesund, is a company committed to the development of a biorefinery that promotes green and sustainable production. A total utilization approach is part of this commitment, including the use of the L. hyperborea leaf fraction.

This master project was done in collaboration with Alginor ASA, and the main goal was to investigate the thermal stability of polyphenols and fucoxanthin in crude methanolic leaf extracts of L. hyperborea, facilitating their utilization from the side-stream of alginate production. This was done using a conventional colorimetric Folin-Ciocâlteu (FC) assay and an inhouse developed selective quantitative NMR method (s-qNMR) (Wekre et al., 2022) for total phenolic quantification, as well as HPLC-DAD, and NMR analysis for fucoxanthin quantification and qualitative observations.

This thesis, divided into five parts, explores the extraction and degradation of polyphenols and fucoxanthin. The initial part focuses on a strategy to minimize degradation during the study, reducing sample preparation and external stressors, and developing the method to track molecular degradation.

The second part develops a gentle maceration extraction method. This method, which compares various solvents, sample-to-solvent ratios, and drying methods, found that macerating freeze-dried leaves in methanol (100 g/L) for 24 hours was the most effective approach.

The third part investigates the relationship between the extraction yields of polyphenols and fucoxanthin against different extraction temperatures. The univariate regression analysis of the extraction yield of fucoxanthin showed a peak yield at an extraction temperature of 48 °C. However, the relationship between the extraction yields of total phenolic content and extraction temperature was less clear, but slightly lower yields and degradation of polyphenols were observed by diminishing 13C-signals in NMR-spectra when extracting at 60 °C.

The fourth part involves producing degradation plots of the filtrate from methanolic extracts that were extracted at room temperature for 24 hours, and for 48 hours at 55 °C. The analysis revealed that fucoxanthin was relatively stable in the matrix of co-extracts in the crude methanolic extract, showing little degradation, at least for the first 24 hours. The total phenolic content remained stable throughout the 48 hours at 55 °C, and no thermal degradation of polyphenols in the crude methanolic extract was observed at a molecular level with the 13C-signals remaining throughout the heating process.

The final part is a qualitative analysis of all the previous parts, attempting to identify or distinguish characteristics of thermally labile polyphenols. The analysis found that polyphenols with ether linkages above 150 ppm are more thermally labile than those with aryl-aryl linkages. An interesting observation was the relationship between two signals at 103 and 103.8 ppm, suggesting that the signal at 103.8 ppm might be a degradation product of the compound at 103 ppm. A table with the strongest crosspeaks from all 2D NMR data was made to aid in the characterization of aromatic compounds in the polyphenolic region.

This comprehensive study provides valuable insights into the extraction and degradation processes of polyphenols and fucoxanthin.