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Understanding carrageenan extraction principles to build insight in red seaweed cell wall structural organization

Book - Dissertation

Carrageenan is a type of sulfated galactan found in the cell wall of red seaweeds called the carrageenophytes. In 2015, more than 60 thousand tonnes of dry carrageenophytes were processed globally to meet the demand of industrial carrageenan. These industries use carrageenan as a gelling or thickening agent in many of their product applications. Carrageenan gelling properties depend strongly on the carrageenan types and cations used to make the gels. In general, there are three major carrageenan types. The first two, κ- (kappa) and ι- (iota) carrageenan are used to form a strong and weak gel, respectively. These two carrageenan types are found at varying ratios in the gametophyte life phase of the carrageenophyte. The third type, λ-(lambda) carrageenan, is a non-gelling (precursor type) and acts as a thickening agent. λ-carrageenan exists by itself in the tetrasporophyte life phase of the carrageenophyte. The conventional carrageenan extraction process relies on high temperatures (80-90oC) and relatively long extraction times (4-8 h) to optimally solubilize carrageenan from the carrageenophyte cell wall, before the carrageenan is recovered through isopropanol (IPA) precipitation. For seaweed species that have a high ratio of gelling carrageenan (κ-,ι-carrageenan), alkali is usually added during the extraction process to transform the precursor carrageenan into gelling carrageenan, maximizing the gelling carrageenan concentration, thus improving its gelling properties. The combination of these high extraction temperatures, long extraction time, as well as the use of alkali effectively degrades the seaweed biomass, allowing the recovery of optimal carrageenan yields. However, as a consequence, many other temperature-sensitive seaweed components that are still valuable such as phycobiliproteins (light harvesting protein complex unique to red seaweeds and cyanobacteria) will be denatured under these intense conditions and will have lost their functionality (e.g. color). Furthermore, the use of the isopropanol filtration aid to separate the residual biomass from the water-soluble carrageenan makes it difficult to recover the biomass, as the biomass adheres to the filtration aid. This is unfortunate, since the biomass is still relatively rich in other compounds (e.g. proteins, pigments, fibres etc.). Due to the large volume of carrageenophytes being processed, valorization of even a fraction of the side stream components will add value to the carrageenan industry. To address this challenge, an understanding of the carrageenan extraction mechanism is needed to build a strategy for a more sustainable carrageenan extraction. Knowledge on the carrageenan native structure, its solubility from the cell wall matrix, and whether there are physical barriers restricting their extractions are some of the questions that need to be answered to build a better understanding of their structural organization. This doctoral project aims to provide better understanding on the principles of carrageenan extraction by studying the effect of varying extraction parameters on the carrageenan yield (how much carrageenan can be obtained at the extraction parameters tested) and quality (what types of carrageenan are obtained under the extraction parameters tested). Rheological properties of the carrageenan gels made from the experimental carrageenan extractions were also characterized. The extraction parameters studied includes time, temperature and effect of salt addition. Additionally, the effect of varying starting seaweed particle size was also investigated to determine whether carrageenan extraction is surface area dependent or not. The resulting yield and quality of the carrageenan-rich precipitates (CRP) extracted from the different extraction conditions could give insight on the carrageenan structural organization within the seaweed cell wall. The carrageenan from Chondrus crispus was chosen as the research object, as it contains hybrid carrageenan (κ-, ι-carrageenan, and a small portion of their precursors (μ-, and ν-carrageenan, respectively) in their gametophyte life phase, whereas λ-carrageenan can be found in its tetrasporophyte life phase. In other words, the extraction behaviour of κ-, ι-, and λ-carrageenan can be determined. Our results showed that the two seaweed life phases exhibited different behaviour when extraction was performed at room temperature. On the one hand, the λ-carrageenan from tetrasporophyte seaweed was easier to extract, as almost all of the carrageenan could already be extracted at room temperature over several sequential extractions. On the other hand, the hybrid carrageenan from the gametophyte could not be well extracted at room temperature, as only a very small yield was obtained. When the temperature was increased, hybrid-carrageenan yield increased as well. It was also found that only a short time was needed (<2 h) for efficient carrageenan extraction, as increasing the extraction time >2 h did not result in a substantial increase in yield. With regard to the effect of salt addition, it was observed that the type of salts in the extraction medium played a role in carrageenan extraction. When the endogenous seaweed salts were removed by a wash step, a small but significant increase in carrageenan extraction at room temperature was observed. Meanwhile, addition of divalent cations in the extraction medium strengthened the seaweed resistance to heat extraction at 60°C. Since carrageenan structure is also highly affected by cations, it could be hypothesized that the difference in yield between the different types of salt added was due to the carrageenan structure being affected through the manipulation of salts in the extraction medium, ultimately affecting its carrageenan extractability. Compositional and rheological analysis of the carrageenan extracted from the different T-t (temperature-time) conditions seemed to suggest that precursor units (non-gelling carrageenan) have higher extractability at lower temperatures and can leach out faster than transformed units (gelling carrageenan). This was concluded from the lower levels of 3,6-anhydrogalactose detected in the CRP extracted at room temperature (22oC) compared to extractions from 45oC and 90oC. 3,6-anhydrogalactose is only found in gelling carrageenan as it is the result of transformation from the precursor units and is the component responsible for the gelling functionality. Furthermore, carrageenan from 15 min (0.25 h) extraction also showed higher amount of sulfate compared to extractions for 2 up to 8 h. Higher sulfate content can also indicate precursor units, since precursor units originally have a sulfate group in the C6 of the galactose, before it is cleaved to form the anhydro ring in the transformed 3,6-anhydrogalactose. Rheological properties of the gels made from the experimental carrageenan extractions also supported the observed compositional trend. Gels made from CRP extracted at 22oC had the lowest G' value compared to gels made from CRPs extracted at 45oC/90oC extractions, which can be explained by the lower amount of 3,6-anhydrogalactose detected in the former. The viscosity of the gels made from CRPs extracted for 0.25 h extractions were also higher compared to gels made from CRPs extracted for 2 up to8 h. This could be explained by the higher amount of sulfate detected in the CRPs in the former. It is known that precursor units cannot gel due to their bulky structure (having one additional sulfate group compared to the gelling carrageenan), giving a more viscous property in solution. No compositional nor rheological differences were found in the CRP or the gels made from these CRPs extracted at 45 and 90oC and between 2 and 8 h. Mechanical disintegration of the starting dried seaweed chips caused an increase in specific surface area and sieving them into distinct size fractions allowed us to study the relationship between carrageenan extraction and surface area. Carrageenan extraction from these decreasing size fractions (which have an increasing specific surface area) at room temperature and 45oC only resulted in a slight increase of CRP yield with decreasing size fractions. At 90oC, there was no differences in the CRP yield for the extraction time that was tested. This showed that at milder temperature, hybrid carrageenan extraction is only slightly surface area dependent. The combination of smaller particle size and mild extraction temperature could not replace the effectivity of high temperature. This result showed that carrageenan extraction is not surface area dependent, and therefore its native structure plays a big role in its extractability. On another note, at the same time, the red pigment phycoerythrin was observed to diffuse out of the seaweed strongly with decreasing seaweed size fractions. This difference in extraction mechanism could be exploited to first valorize the pigments prior to the carrageenan extraction. Based on these experimental results and postulation of the carrageenan structural organization, an alternative extraction method was tested as proof of concept. This novel method was successful in extracting carrageenan with optimal yields at room temperature.
Publication year:2021
Accessibility:Closed