WO2019211442A1 - Fucoidans from diatoms - Google Patents

Abstract

The present invention relates to the use of microalgae as a source of fucoidan.

Description

Fucoidans from diatoms

The present invention relates to the use of microalgae as a source of fucoidan.

In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Fucoidans are ubiquitous, sulfated heteropolysaccharides from marine brown seaweeds. Generally, fucoidans are a group of fucose-containing sulfated polysaccharides with high structural diversity. Fucose can be the backbone of a branched polysaccharide (called homofucans). The fucose monosaccharide that forms the polymer backbone can be linked by a-1-2, a-1-3 and a-1-4 glycosidic bonds with sulfate groups at C2, C3 or C4 and additional acetylations. But fucose can also be the branch on a backbone composed of another monosaccharide (heterofucans: xylofucoglycuronans, glycuronofucoglycan, pseudo- fucoidans). Common other monosaccharides than fucose in homofucans and heterofucans are xylose, galactose, mannose, glucosamine, uronic acid and mannuronic acid. The fucoidans in brown seaweeds in the order of Fucales, e.g. Fucus evanescens and Fucus serratus, represent the canonical chemical structures that define this class of molecules. These structures contain a large proportion of both a(1 ,3)- and a(1 ,4)-linked L-fucopyranose residues, which may be substituted with sulfate (S0₃ ^~) on C-2 and C-4. Other typical fucoidan structures include the ones found in the alga Ascophyllum nodosum, which also belongs to the order Fucales. These fucoidan molecules host a backbone consisting of a repeating structure of a(1 ,3)-linked L-fucopyranose residues, with sulfate at the C-2 position and with the a(1 ,3)-linked L-fucopyranose residues linked to a(1 ,4)-L-fucopyranose residues with additional disulfate at C-2 and C-3. These structural elements composed of mainly linear backbone structures of alpha-linked, highly sulfated L-fucose are also found in fucoidans extracted from F. vesiculosus the brown seaweed commonly known as bladderwrack. Fucoidans in other macroalgae such as the order laminarinales can have related structures, while so called pseudo-fucoidans have different backbone structures frequently composed of a(1 ,6)-linked galactose residues and only side chains are composed of alpha-configured, sulfated L-fucose residues. There are many potential therapeutic applications described for these different types of fucoidan molecules (for review: (Fitton (2011 ). Therapies from fucoidan; multifunctional marine polymers. Mar Drugs 9: 1731-1760; Ale et al. (2011 ). Important determinants for fucoidan bioactivity: A critical review of structure-function relations and extraction methods for fucose-containing sulfated polysaccharides from brown seaweeds. Mar Drugs 9: 2106-2130).

Owing to the possible therapeutic potential of fucoidan, the nutraceutical and cosmetic industries increased their efforts in recent years to develop and sell fucoidan products. A quick search with a search engine, with fucoidan as search term, reveals a multitude of food supplements in form of beverages or tablets. Most of these products are already commercially available and mostly marketed over internet in the US, UK but also Germany. Brown seaweeds including Cladosiphon okamuranus (Mozuku), Undaria pinnatifida (Mekabu) and Laminaria japonica (Kombu) are the relevant sources of fucoidans in Japan. Several companies sell fucoidan products. The company Kanefuku Co, Ltd, Japan produces and supplies fucoidan polysaccharides (Umi No Shizuku) as dietary supplements in capsules and drinks that are thought to enhance the immune system and increase longevity. Fucoidan products from Takara Bio Inc, Japan are marketed as an anti-inflammatory and immune stimulating formulation. These formulations are used in nutritional drinks and in functional foods. Production of pure fucoidan products is challenging because of the presence of other polysaccharides, such as alginate. In Korea, Seaherb Co. Ltd. has patented a technology for the manufacture of low molecular weight fucoidan saccharides aimed at increasing absorption rate.

Applicability of fucoidans as “cosmeceuticals” in the cosmetic industry has also been studied. Most of these studies focus on the inhibitory effects of topically applied fucoidans on aging and photo-damaged skin. One possible mechanism for this beneficial effect may be the inhibition of metalloproteinases that digest tissue fiber in the skin (Fitton (2011 ). Therapies from fucoidan; multifunctional marine polymers. Mar Drugs 9: 1731-1760). Fucoidans may enhance dermal fibroblast proliferation and deposition of collagen; protect the elastic fiber network in human skin culture and to have potential for inclusion in lotion as a natural whitening agent. Developing fucoidans as protective agents in topical cosmetic formulations thus appear to be a promising strategy. Since fucoidans are highly water soluble, they can easily be incorporated into lotions, creams and other beauty products. An unexplored aspect of fucoidan lies in its use as a nutrient for beneficial gut microbes (prebiotics). As a pharmaceutical or nutraceutical ingredient, fucoidans clearly offer distinctive possibilities for immuno-modulation and anti-cancer effects, results that are based on many scientific publications. However, new sources of fucoidan are required to improve the supply and quality of this promising resource.

Fucoidans are produced by macroalgae, which are the major source of fucoidans on the market; however, different reasons motivate the search for new fucoidan sources. Despite notable bioactivities and a lack of oral toxicity, fucoidans remain relatively unexploited as a source of therapeutics due to their macroalgae source and heterogeneity. The limited reproducibility of the quality of fucoidan from macroalgae due to varying harvesting times and seasonality hinders the use of fucoidan in pharmacological applications (Fitton (2011 ). Therapies from fucoidan; multifunctional marine polymers. Mar Drugs 9: 1731-1760). The chemical heterogeneity of fucoidan complicates scientific studies aiming at constraining structure-function relationships of these molecules in pharmacological evaluations. Moreover, in Europe macroalgae are currently not cultivated on a large scale. Most of the macroalgae are harvested from natural stocks that are prone to changes in productivity and may suffer from overharvesting, climate change and other environmental threats such as pollution or eutrophication (Morand and Merceron (2005). Macroalgal population and sustainability. J Coast Res 21 : 1009-1020).

In view of the shortcomings of the prior art, the technical problem underlying the present invention can be seen in the provision of improved means and methods for obtaining fucoidan. This technical problem has been solved by the subject-matter of the attached claims.

In a first aspect, the present invention relates to the use of microalgae as a source of fucoidan.

The term “microalgae” is art-established and refers to unicellular, microscopic algae. Microalgae include, among others, diatoms and coccoliths. Diatoms constitute a significant fraction of the marine phytoplankton. In the field of taxonomy, diatoms are also referred to as Bacillariophyceae .

The term“fucoidan” has its art-established meaning. It designates a sulfated polysaccharide, which contains fucose as building blocks. The art-established sources of fucoidans are brown algae. The definition of fucoidan and the discussion of various subgroups thereof as provided in the introductory section herein above is part of the invention. A description of fucoidans can also be found in Deniaud-Bouet et al, A review about brown algal cell walls and fucose-containing polysaccharides, Carbohydr. Polym. 175, 395-408 (2017). Obtaining fucoidan from microalgae does not require transduction (or a related process) of microalgae, e.g. with genes encoding enzymes involved in fucoidan biosynthesis. Fucose content of fucoidans may vary widely, e.g. between 5% and 60% by mass.

Having said that, microalgae can be genetically modified, e.g. in order to increase fucoidan production. As such, in a preferred embodiment, microalgae as recited in the uses and methods of the invention can be genetically modified to optimize the fucoidan pathway. This can be done to modify the product fucoidan and/or to generate larger amounts thereof. Modifications of the fucoidan pathway include the increased expression of specific glycosyl transferases involved in fucoidan biosynthesis (several genes proposed to be involved in the synthesis of fucoidan in brown macroalgae can be found in Michel et al. (2010) The cell wall polysaccharide metabolism of the brown alga Ectocarpus siliculosus. Insights into the evolution of extracellular matrix polysaccharides in Eukaryotes. New Phytol 188: 82-97).

The present inventors discovered the occurrence of fucoidan in microalgae. Microalgae are taxonomically quite distinct from the art-established source of fucoidan which are brown macroalgae. As such, this discovery is unexpected.

The availability of microalgae including diatoms as a source of fucoidan is advantageous in that diatoms are amenable to cultivation. Different from harvesting from natural sources, cultivation is, at least in principle, not limited in scale. Furthermore, cultivation allows establishing and precise control of defined culture conditions (see Aden et al. ((2012). Production cost of a real microalgae production plant and strategies to reduce it. Biotechnol Adv 30: 1344-1353) for suitable culture conditions), which improve reproducibility of the quality of fucoidan. Not being dependent on harvesting microalgae from natural stocks avoids suffering from changes in productivity as well as dealing with overharvesting, climate change and other environmental threats such as pollution or eutrophication (Morand and Merceron (2005) Macroalgal population and sustainability. J Coast Res 21: 1009-1020). Microalgae grow fast and produce high levels of biomass. Furthermore cultivation allows optimization of yield.

A further advantage of microalgae, in particular of diatoms, is that, according to a surprising discovery of the present inventors, these organisms not only are a source of fucoidan, but also secrete or exudate fucoidan into the surrounding water or medium. This simplifies the process of obtaining fucoidans from this new source because, deviant from the prior art procedures for the sources of fucoidan known so far, breaking up cells is dispensable. In view of the multiple and beneficial uses of fucoidans as reviewed herein above, the easier availability of fucoidans in accordance with the present invention is an important step forward.

In a second aspect, the present invention provides a method of producing fucoidan, said method comprising: (a) cultivating microalgae in a culture medium; and (b) recovering fucoidan produced by said microalgae from the culture.

Suitable media include ESAW media as described, for example, in Harrison et al ((1980). A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J Phycol 16: 28-35). Other enriched seawater media designed for growing microalgae, such as the widely used f/2 media (Guillard and Ryther (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervaceae (Cleve) Gran. Can. J. Microbiol. 8: 229- 239). Generally speaking, a suitable culture medium requires trace elements and vitamins that are always included in the list of components of media for marine algae. In the case of diatoms silica is also required. The two media mentioned (ESAW and f/2) include silica among their components.

Exemplary culture conditions are disclosed in the examples. In particular, culturing is preferably effected at temperatures in the range from about 10°C to about 20°C, such as at 15°C. Preferably, a light/dark cycle is applied, such as about 12 hours illumination followed by 12 hours darkness. Illumination is preferably in a range between about 100 and 200 pmol photons m ²s ¹, such as between 140 and 150 pmol photons mV. Also, it is common to air bubble the cultures. Alternatively, a magnetic stirrer may be used for agitaton.

Preferred methods of recovering fucoidan are subject of preferred embodiments disclosed further below.

Related to the second aspect, the present invention provides, in a third aspect, a method of enriching or purifying fucoidan, said method comprising: (a) cultivating microalgae in a culture medium; and (b) recovering fucoidan produced by said microalgae from the culture.

In a preferred embodiment of the method of the second and third aspect, said recovering comprises: (ba) subjecting polysaccharides obtained from said microalgae to a preparative analytical method; and (bb) separating those fractions which (i) yield fucose upon hydrolysis, and/or (ii) comprise fucoidan; thereby obtaining fucoidan. As can be seen from the above, fucoidan-containing fractions can be identified by determining presence of fucoidan (option (ii)) or based on the fact that fucoidan is particularly rich in fucose (option (i)). Preferred preparative analytical methods for step (ba) are subject of a preferred embodiment disclosed further below.

Furthermore, the separation of fucoidan containing fractions may be based on prior knowledge. To explain further, once a method in accordance with the present invention is established (for example by choice of chromatographic devices and matrices, such as a particular column, ion-exchange resin and a particular buffer system), the elution properties of the process may be determined, thereby obtaining knowledge of the point in time when fucoidan elutes (or equivalently, of the fractions comprising fucoidan). Based on such information, there is no necessity in subsequent implementations using the same experimental setup to determine once again which particular fractions contain fucoidan. Such implementation of the methods of the invention based on prior knowledge is comprised by the term“fractions which comprise fucoidan”.

It is understood that between steps (a) and (b), preferably a step of harvesting said microalgae is preferred. Harvesting of microalgae is preferably done by centrifugation.

Harvesting is preferably followed by homogenization, which preferably is effected prior to step (b). Homogenising can be done with art-established methods such as pestle and mortar.

Prior to homogenization, microalgae are preferably freeze-dried.

Alternatively, and noting that in accordance with the invention microalgae, in particular diatoms, secrete fucoidan, harvesting is dispensable. In other words, the medium or seawater in which said microalgae have been present or are present may be subjected to said preparative analytical method. When doing so, the step corresponding to harvesting may still optionally be performed, but it would not serve to obtain said microalgae, but instead said medium or seawater. If harvesting is by centrifugation, the supernatant would be processed further.

In a particularly preferred embodiment, said polysaccharides of (ba) are obtained (a) from said culture medium, wherein said culture medium is optionally desalted and/or concentrated; or (b) by extraction, preferably using water as a solvent. Other solvents, such as saline buffer, can be used as well for polysaccharide extraction. It is preferred to desalt the culture medium. Desalting may be effected by dialysis, preferably with a 1 kDa membrane. Alternatively, preferably in addition, the medium is concentrated. Concentrating may be effected by freeze-drying or ultrafiltration. Ultrafiltration can be done with a 1 kDa membrane. Further details of preferred or exemplary procedures for obtaining fucoidans from medium or sea water can be found in the Examples.

Extraction in accordance with this preferred embodiment is preferably implemented by extracting (a) an alcohol insoluble residue (AIR) obtained from homogenized microalgae; or (b) homogenized microalgae. The preparation of the AIR may be effected by adding solvent to the biomass followed by mixing. Thereafter, samples are subject to centrifugation, the supernatant is discarded, and the pellet is resuspended. Resuspension may be either in a second solvent to be used for the purpose of preparing the AIR, or the solvent to be used for polysaccharide extraction (such as water). Preferred solvents for the AIR procedure include ethanol, and furthermore a 1 :1 chloroform: methanol mixture and acetone. The three solvents may be in the indicated order.

As can be seen from the examples enclosed herewith, preparation of the AIR is a preferred option, but not a requirement. If AIR is not used, the homogenized microalgae may be directly extracted.

In a further preferred embodiment, said preparative analytical method is selected from (a) chromatography, preferably anion exchange chromatography; (b) a graded precipitation method; (c) a quaternary ammonium salt precipitation method; and (d) size exclusion chromatography.

Particularly preferred is anion exchange chromatography. Anion exchange chromatography exploits the presence of negative charges on fucoidan. As noted above, fucoidan carries sulfate groups which have a negative charge.

Purification of fucoidan with methods of the invention may yield at least 90%, at least 95%, at least 98%, at least 99% or completely pure (100%) fucoidan.

The term“graded precipitation method” is known in the art and is based on different solubility of different polysaccharides in alcohol; see, for example, Peng et al. (2009) Comparative study of hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. J. Agric. Food Chem. 57, 6305-6317. The result obtained by the preparative analytical method may be subjected to desalting and/or lyophilisation. Lyophilisation will yield a powder of purified fucoidan. Preferred degrees of purity are disclosed above.

In a further preferred embodiment, the presence or absence of fucose in step (bb)(i) is determined by drawing a sample from a given fraction, subjecting it to hydrolysis and analysing the hydrolysate for fucose. Preferably, hydrolysis is acid hydrolysis, for example effected by adding HCI with a final concentration of 1 M HCI and incubating at 100°C for 24 hours.

This preferred embodiment is a specific implementation of step (bb)(i) disclosed further above.

Fucoses (as well as other monosaccharides) are preferably analyzed with HPLC, e.g. HPLC with pulsed amperometric detection. To identify fucose, a standard, e.g. a known amount of L-fucose may be used.

In a preferred embodiment, fucose is L-fucose. Generally speaking, L-fucose is the prevalent form of fucose in nature.

In an alternative or additional preferred embodiment, the presence or absence of fucoidan in step (bb)(ii) is determined by drawing a sample from a given fraction and subjecting it to analysis using a fucoidan-specific antibody, preferably using enzyme-linked immunosorbent assay (ELISA).

Fucoidan-specific antibodies are available and mentioned in the examples.

In a further preferred embodiment of the use of the first aspect and of the methods of the second and third aspect, said microalgae are (a) diatoms, preferably selected from the families Thalassiosiraceae, Chaetocerotaceae, Phaeodactylaceae and Bacillariaceae, more preferably from the genera Thalassiosira, Chaetoceros, Phaeodactylum and Nitzschia ; or (b) Coccolithophorida, preferably Noelaerhabdaceae, more preferably of the genus Emiliania.

Particularly preferred species are Thalassiosira weissflogii, T. pseudonana, Chaetoceros affinis, C. socialis, Phaeodactylum tricornutum, Nitzschia frustulum and Emiliania huxleyi.

In a further preferred embodiment, said fucoidan is a homofucoidan or a heterofucoidan. As noted above, while fucose is a compulsory constituent of fucoidans, the fucose contents may vary widely, such as between about 5% and about 60% by mass, preferably between about 10% and about 50% by mass.

In a fourth aspect, the present invention provides a method of producing a pharmaceutical composition, said method comprising the method of the second or third aspect.

As noted herein above, fucoidan has been described for uses in medicine. As a consequence, the method of the second and the third aspect either amounts to a method of producing a pharmaceutical composition or may comprise, as one or more additional steps, which are to be effected after the steps recited further above, the addition of carriers, diluents and/or excipients.

Examples of suitable pharmaceutical carriers, diluents and/or excipients are well-known in the art and include phosphate buffered saline, various types of excipients, binders, wetting agents, sterile solutions and coatings. Examples include gelatine, magnesium stearate and microcrystalline cellulose.

Optionally, the method of producing a pharmaceutical composition in accordance with the fourth aspect may furthermore comprise the addition of further pharmaceutically active substances, preferably pharmaceutically active substances having effects related to the beneficial effects of fucoidan.

An alternative to the fourth aspect is provided by the fifth aspect of the present invention, which relates to a method of producing a nutraceutical, a food supplement or a cosmetic composition, said method comprising the method of the second or third aspect.

The terms“nutraceutical” and“food supplement” have their art-established meaning. Such composition, while generally being beneficial, do not or do not necessarily serve the purpose of treating, alleviating or preventing a disease or disorder. In the same vein, also a method of producing a cosmetic composition is provided.

In a sixth aspect, the present invention provides a fucoidan obtained by the method of the second or third aspect. Table 1 as comprised in the examples provides detailed information about preferred fucoidans in accordance with this aspect of the invention.

As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The figures show:

Figure 1 : Purification of fucoidan from T. weissflogii cells that were cultivated in the laboratory. A) The fucoidan present in the water extract obtained from concentrated T. weissflogii cells was purified by anion exchange chromatography. The black line shows the percentage of 5 M NaCI used for the gradient. B) The chromatographic fractions corresponding to the gradient were analyzed. To determine the presence of fucoidan in these fractions we performed monosaccharide analysis by high performance liquid chromatography with pulsed amperometric detection (HPLC-PAD). This experiment showed the fraction that contained the highest concentration of L-fucose, the building block of fucoidan. Figure 2: Microarray analysis and ELISA of polysaccharides extracted from the microalgae Thalassiosira weissflogii analysed with the specific antibody BAM2 reveal the presence of fucoidan in this diatom species. A) T. weissflogii water extracts were printed as microarrays and probed with a panel of polysaccharide-specific monoclonal antibodies including BAM2, which specifically recognizes fucoidan. In addition we tested a beta-(1-3)-glucan recognizing antibody (positive control) and xyloglucan and pectin recognizing antibodies (negative controls). Binding results are presented as a heat map in which colour intensity correlates to the mean antibody signal values. The rows represent triplicates. B) The presence of fucoidan was confirmed in an ELISA experiment where decreasing concentrations of the 7. weissflogii water extract were probed with the fucoidan specific antibody BAM2 revealing a dose dependent response.

Figure 3: Immunolabelling of T. weissflogii and other diatoms species with the mAb BAM2 indicates the microalgae synthesize and secrete fucoidan. A) A representative image showing the binding of the mAb BAM2 (green) to 7. weissflogii cell surfaces. B) and C) Images of single 7. weissflogii cells showing the binding of BAM2 (green) to the fucoidan located on the cell surface and of DAPI, a DNA stain (blue), to the nucleus of the cells. D) The diatom species 7. pseudonana shows green fluorescence due to bound BAM2 antibody indicating fucoidan is produced by this microalgae. E) The diatom Chaetoceros affinis shows green fluorescence due to bound BAM2 antibody indicating fucoidan is produced by this microalgae. DAPI stain (blue) is observed at the diatom nucleus. The negative controls with only the FITC-conjugated secondary antibody but not BAM2 did not show the green fluorescence (data not shown).

Figure 4: Chart showing the monosaccharide composition as % in the purified fucoidan-rich fractions (highest fucoidan signal) from different microalgae species.

The sample“microalgae from a bloom” represents values obtained from the water extract of material retained in a 3 pm filter during a spring microalgae bloom at the North Sea (Helgoland, Germany - on the 12^th of May 2016).

Figure 5: Analyses of polysaccharides secreted to the media by the microalgae Thalassiosira weissflogii, with both microarray analysis and epifluorescence microscopy, reveal the presence of fucoidan in the diatoms exudates. A) 7. weissflogii exudates were harvested and re-dissolved in water. These exudates were printed as microarrays and probed with a panel of polysaccharide-specific monoclonal antibodies including BAM2, which specifically recognizes fucoidan. In addition we tested a pectin and a xyloglucan recognizing antibodies (negative controls). Binding results are presented as a heat map in which colour intensity correlates to the mean antibody signal values. The rows represent triplicates. B) The presence of fucoidan in T. weissflogii exudates was confirmed by immunolabeling. T. weissflogii cultures were fixed and filtered through a 0.2 pm filter, which was then probed with the mAb BAM3 specific for fucoidan. Two representative images show the binding of the fucoidan mAb, indicating that the diatom produced and secreted fucoidan, which aggregated forming particles. The negative controls with only the FITC- conjugated secondary antibody did not show the green fluorescence (data not shown). Scale bar = 10 pm.

Figure 6: Epitope detection chromatography analysis shows negatively charged fucoidan is present in different diatom species. Purification of fucoidan from different water extracts of bloom samples and pure diatom lab cultures was done by performing an anion exchange chromatography run with a gradient (dashed line) of salt from 0 to 100% 5 M NaCI. All 48 chromatographic fractions were collected and aliquots were analyzed by ELISA using the fucoidan-specific probe BAM1 (see detected signal as continuous line). Highest antibody signal shows the chromatographic fraction containing the purified fucoidan. Panel on the top left shows fucoidan purification in microalgae cells from a bloom, which was dominated by the diatom Chaetoceros socialis, at the North Sea. Panel on the top right illustrates the purification of fucoidan in microalgae exudates from the bloom - i.e. the fucoidan was present in the dissolved organic fraction, therefore was not present in the diatom cells but secreted into the sea water. Samples from the bloom were harvested near Helgoland on May 12, 2016. The further four panels show purification of fucoidan from several diatom lab cultures.

The examples illustrate the invention.

Example 1

Materials and methods

Microalgae growth

The microalgae species Thalassiosira weissflogii, T. pseudonana, Chaetoceros affinis, C. socialis and Emiliania huxleyi were grown in ESAW media (for details of media see Harrison et al. ((1980). A broad spectrum artificial seawater medium for coastal and open ocean phytoplankton. J Phycol 16: 28-35). Microalgae cultures were grown in an incubation chamber at 15°C, with a constant 12-h/12-h light/dark cycle and without shaking. Illumination was applied with five Sylvania F36W/GRO tubes, resulting in an irradiance of about 140 pmol photons rrf² s ¹. Once per week 5 ml of the growing culture were inoculated into 20 ml of new ESAW media and the old culture was discarded. For the experiments described here we prepared either 1 L or 20 L cultures, which were inoculated with 7 ml or 1 L, respectively, of a culture at 7 days after inoculation. All cultures grew under the above described conditions.

The microalgae cultures were harvested at 10 days after inoculation (at stationary phase) by centrifugation at 6800 g for 20 minutes. The pellet, containing the microalgal biomass, was collected and frozen. Next, the microalgae biomass was freeze dried - in order to get rid of the water. For a 1 L culture we obtained around 50 mg of diatom dry biomass while with the 20L we obtained around 2000 mg. (Note: when bubbled, T. weissflogii cultures have been reported to produce 146 mg dry biomass/L (d’lppolito et al. (2015). Potential of lipid metabolism in marine diatoms for biofuel production. Biotechnol Biofuels 8.)). Afterwards, the biomass was homogenized.

Homogenized freeze-dried material may be directly subjected to fucoidan extraction; see further below.

Alternatively, the alcohol insoluble residue (AIR) may be obtained prior to fucoidan extraction. Essentially, the AIR procedure consists of adding 6:1 volumes of solventvolume of biomass, vortexing and then rotating the samples at room temperature for 10 minutes. Next samples are spun down at 4500 rpm for 15 minutes. The supernatant is discarded and the pellet is re-suspended in the subsequent solvent. The solvents used are (in this order): 99.9% ethanol, chlorofornrmethanol (1 :1 ) and pure acetone. After the last centrifugation, the supernatant is discarded and the pellet is left to air dry overnight at room temperature in the fume hood.

Fucoidan extraction

Polysaccharide extraction from the AIR material was performed by adding 1 :1 ml milliQ watenmg sample (or also at higher concentration of biomass such as 1 :30 milliQ water: mg sample), vortexing and stirring for 1 h at 60°C at 650 rpm. Extraction by rotating at room temperature for 1 h was also tested and was proven successful as well. After the extraction, samples were spun down at 4500 rpm for 15 min and the supernatants, which contain the water-extracted polysaccharides, were collected and stored at 4°C. The water-extracted polysaccharides accounted for approximately 15% of the total dry AIR microalgae weight (based on our analysis by the phenol sulfuric acid method (Dubois et al. (1956). Colorimetric method for determination of sugars and related substances. Anal Chem 28: 350-356).

Fucoidan purification

The fucoidan present in the microalgae water-extract was purified by using anion-exchange chromatography. The microalgae water extract was run through an anion-exchange chromatography column using 50 mM sodium acetate buffer pH 5.0 as buffer A and 50 mM sodium acetate buffer 5M NaCI pH 5.0 as buffer B. Alternatively, Tris buffer pH 7.5 may be used as buffer A and Tris buffer 5 M NaCI pH 7.5 as buffer B. The chromatographic fractions containing the purified fucoidan were identified by monosaccharide analysis. The purification is highly reproducible, i.e. the particular chromatographic fraction that shows highest fucoidan content is the same in independent chromatography runs. Therefore it is not necessary to perform monosaccharide analysis to detect the fucoidan-rich fraction when using the same protocol run (buffer B gradient). The fucoidan-rich fraction may be dialyzed using 1 kDa dialysis tubing to remove the NaCI. Then it can be frozen and freeze dried.

Based on the quantitative method monosaccharide analysis we determined that the yield of fucoidan rich-polysaccharide fraction in T. weissflogii AIR is around 1.8 %. Biomass production (culture harvested at stationary phase) of T. weissflogii has been reported as 146 mg dry weight/L (d’Ippolito et al., 2015). Therefore, about 260 mg of purified fucose- containing polysaccharide would be obtained per each 100 L of T. weissflogii culture.

Collecting microalgae exudates

The microalgae cultures were harvested by centrifugation and the pellet was collected and frozen, as described above. The supernatant, which contained the polysaccharides secreted to the media, was kept and concentrated. Alternatively, the microalgae cultures can be filtered through 0.2 pm filter and the filtrate will contain the polysaccharides secreted to the media. The supernatant (or filtrate) was concentrated by ultrafiltration (using an Amicon® stirred cell) with a 1 kDa membrane. Membranes of 5 kDa were tested and proven suitable for concentration of fucoidan as well. For supernatant volumes greater than 1 L, concentration was done by tangential flow filtration (TFF) using membranes with a cut-off of 1 kDa. The microalgae culture supernatant was concentrated to approximately 10 to 40 ml. At this step ethanol precipitation by addition of three volumes of pure ethanol was tested and proven effective to get a precipitate with the polysaccharides (including fucoidan). Alternatively, the concentrated sample was collected and placed inside a 1 kDa dialysis tubing and then dialysed overnight against 5 L milliQ water in a beaker with stirring. After overnight, water was changed and left stirring for further 4 h. The dialysed sample (containing the polysaccharides that diatoms secreted to the media) was frozen and freeze dried. The freeze-dried material was directly subjected to fucoidan extraction and fucoidan purification in identical manner as for the microalgal biomass; see above. Note that after dialysis, instead of freezing and freeze drying, the sample can be further concentrated with ultrafiltration (1 kDa membrane) and this can be directly injected into an anion-exchange chromatography for fucoidan purification.

Example 2

Results

Fucoidan from Thalassiosira weissflogii

We first harvested the microalgal biomass, then extracted polysaccharides with water and finally we separated the different polysaccharides in the extract by anion exchange chromatography using a column with weak anion exchange resin. The fucoidan was bound to the column under law salt conditions and eluted with an increasing concentration of sodium chloride (Figure 1 ). The chromatographic fractions corresponding to the gradient were investigated by hydrolyzing the polysaccharides with HCI. The liberated monosaccharides were analyzed with HPLC with pulsed amperometric detection. Therefore, the detection of the purified fucoidan was achieved with monosaccharide analysis revealing a defined fucoidan peak (highest concentration of L-fucose) at approximately 1.75 M NaCI concentration (Figure 1). We reproduced this experiment multiple times (testing both weak and strong anion-exchange columns) obtaining nearly equal results with a clearly defined peak eluting at the same concentration of NaCI. Before performing acid hydrolysis, the chromatographic fractions were dialyzed with 1 kDa dialysis tubing to remove NaCI. Presence of fucoidan seemed to be independent of the presence or absence of bacteria as fucoidan was detected in both axenic and non-axenic T. weissflogii cultures (data not shown). Altering the growth conditions of the diatom did not significantly change the chromatography profile. Therefore, the result is highly reproducible and the fractions containing the highest amount of fucose-containing polysaccharide were consistent. In combination these two methods, chromatography and monosaccharide analysis, enabled the identification of fucoidan in diatoms. The microalgae fucoidan purification by anion exchange chromatography was reliable and consistent. However, other commonly used methods to isolate and purify polysaccharides from their natural sources (for some examples see review (Shi (2016). Bioactivities, isolation and purification methods of polysaccharides from natural products: A review. Int J Biol Macromol 92: 37-48) can be used instead.

Composition of the purified fucoidan-enriched fraction

Fucoidan purification by anion exchange chromatography was performed in different microalgae species. Specifically, T. weissflogii, T. pseudonana, Chaetoceros socialis and Emiliania huxleyi. The building blocks present in the chromatographic fractions that showed the highest fucoidan peak are shown in Table 1. Furthermore, the fucoidan-rich fraction from microalgae species present during a phytoplankton bloom were analyzed as well. Microalgal biomass retained on a 3 pm filter during a spring microalgae bloom at the North Sea (at Helgoland - 54^° 11.3’ N, 7^° 54.0’ E) was collected. The material was extracted with water and the polysaccharide extract was purified by anion exchange chromatography. Figure 4 displays the monosaccharide analysis results in a chart.

Table 1: Monosaccharide composition of the purified fucoidan-rich fractions (highest fucoidan peak) from different microalgae species. Values correspond to the % of total monosaccharides detected in the microalgae purified chromatographic fraction that gave the highest fucoidan peak. Two different strains of C. socialis (T18 and T20) were included in the study. The sample“microalgae from a bloom” corresponds to values obtained from the water extract of material retained in a 3 pm filter during a spring microalgae bloom at the North Sea (Helgoland, Germany - on the 12^th of May 2016). Ara, arabinose; Gleam, glucosamine; -, not detected.

Additional confirmation of the presence of fucoidan in Thalassiosira weissflogii

To further proof the presence of fucoidan in diatoms we used a new, alternative technology that has only recently been developed and which has to the best of our knowledge never been applied to diatoms. Comprehensive microarray polymer profiling (CoMPP) is an established high-throughput method for screening polysaccharides in land plants (Moller et al. (2007). High-throughput mapping of cell-wall polymers within and between plants using novel microarrays. Plant J 50: 1118-28). CoMPP has recently been used to investigate the presence of glycosylated proteins in macroalgae (Herve et al. (2016). Arabinogalactan proteins have deep roots in eukaryotes: identification of genes and epitopes in brown algae and their role in Fucus serratus embryo development. New Phytol 209: 1428-1441). The principle is that the polysaccharides from a given plant or in this case microalga are extracted with defined solvents (in our case under mild conditions with water - limiting acid based hydrolysis common in conventional extraction protocols), and then spotted onto microarray slides. These slides are incubated with antibodies each of which is specific for a certain type of polysaccharide. A positive reaction with one of the antibodies, akin to immunological ELISA experiments, indicates the presence of the particular polysaccharide in the sample. We combined the CoMPP technology with another recent innovation to further confirm the presence of fucoidan in diatoms. This second innovation is a recently developed new set of fucoidan specific antibodies that specifically recognize the fucoidan from brown seaweeds with very high specificity (Torode et al. (2015). Monoclonal antibodies directed to fucoidan preparations from brown algae. PLoS One 10: e01 18366).

Our microarray experiments revealed that the BAM2 antibody, which specifically recognizes fucoidan in macroalgae (Torode et al. (2015). Monoclonal antibodies directed to fucoidan preparations from brown algae. PLoS One 10: e01 18366) shows strong binding signal to extracts of diatoms such as Thalassiosira weissflogii (Figure 2A). The negative controls consisting of antibodies that recognize polysaccharides that are not present in diatoms, such as pectins and xyloglucans, did not show positive binding. The positive control experiment included an antibody that specifically recognizes b-1 ,3-glucan, an abundant polysaccharide in diatoms. This antibody showed strong binding to the diatom extract owing to the presence of this polysaccharide in the extract. These controls confirmed the fidelity of the detection method. Moreover, the microarray contained additional known, commercially available polysaccharides, including the fucoidan from brown algae to which the BAM2 antibody effectively bound (data not shown). We repeated the extraction and performed an experiment with a different method in an enzyme linked immunosorbent assay, which is a classical way to identify macromolecules with antibodies. Results confirmed that fucoidan was present in the water extracts of T. weissfiogii (Figure 2B).

Visualization of fucoidan in different diatom species with immunolabelling and fluorescence microscopy

To expand on the results that were obtained with monosaccharide analysis, microarrays and with the ELISA assay we used immunolabelling and fluorescence microscopy to visualize and analyze the localization of the fucoidan molecule on the diatom cell walls. T. weissfiogii and two additional diatom species, T. pseudonana and C. affinis, were grown in liquid culture with a constant light regime, at 15 degrees Celsius and without shaking (details on growing conditions are depicted at the“experimental procedures” section). The microalgae cells were fixed with paraformaldehyde, collected on filters and those were incubated with the mAb BAM2 followed by a secondary antibody that was specific for the primary antibody (BAM2) and that was coupled to a fluorescent fluorophore (FITC: green). We identified fucoidan signal on the surface of T. weissfiogii (Figure 3A-C), on T. pseudonana (Figure 3D) and on C. affinis (Figure 3E). The visible green fluorescence signal associated with all three species indicated that fucoidan production is a common trait in diatoms. Control experiments (not shown) did not show green fluorescence.

Fucoidan in exudates of Thalassiosira weissfiogii

Samples containing T. weissfiogii exudates were collected and concentrated. Polysaccharides present in the samples were extracted (re-dissolved) in water and analyzed by CoMPP (method described above). Results of our microarray analysis showed strong binding signal of BAM2 antibody, which is specific for fucoidan, to the diatom secreted polysaccharides. (Figure 5A). These data indicate that fucoidan was produced and secreted to the media by the diatoms. Control antibodies specific for pectins and xyloglucans, structures not produced by diatoms, did not show binding. Secretion of fucoidan to the media by T. weissfiogii was further confirmed by fluorescent microscopy. T. weissfiogii was grown in liquid culture following the growing conditions described at the“experimental procedures” section. Diatom cultures were fixed with paraformaldehyde and filtered through a 0.2 pm filter. Filters were incubated with the antibody BAM3, one of the new set of fucoidan specific monoclonal antibodies (Torode et al. (2015). Monoclonal antibodies directed to fucoidan preparations from brown algae. PLoS One 10: e0118366). After that, filters were incubated with a secondary antibody specific for the primary antibody (BAM3) that was conjugated to a fluorescent fluorophore (FITC: green). We detected binding signal on aggregates (Figure 5B). This indicates that T. weissflogii secreted fucoidan into the media and that at least a fraction of this secreted fucoidan aggregated into particles. The negative controls with only FITC-conjugated secondary antibody did not show green fluorescence (data not shown). Fucoidan in diatom exudates during a bloom

Additionally, we detected fucoidan in the dissolved organic matter from a diatom bloom at the North Sea. During a spring bloom we harvested samples corresponding to diatom cells in 3 pm filters. We also harvested samples corresponding to the dissolved molecules in the sea water, which were obtained by filtering the sea water through a 0.2 pm filters and concentrating the filtrate by tangential flow filtration (TFF). Polysaccharides present in the diatom-containing and the dissolved molecule-containing samples were extracted with water. Each extract was purified by anion exchange chromatography. The chromatographic fractions were collected and analyzed by ELISA using the recently developed epitope detection chromatography (Cornuault et al. (2014), Plant J. 78, 715-722). Figure 6 (top panels) shows the mAb BAM1 (Torode et al. (2015). Monoclonal antibodies directed to fucoidan preparations from brown algae. PLoS One 10: e0118366), signal intensity detected in the different fractions (continuous lines). The dashed line shows the percentage of 5 M NaCI used for the gradient. Results revealed that there was presence of fucoidan in the diatom cell-fraction as well as in the exudate cell free-fraction. The extra four panels show examples of epitope detection chromatography in different diatom species Figure 6.

Claims

1. Use of microalgae as a source of fucoidan.

2. A method of producing fucoidan, said method comprising:

(a) cultivating microalgae in a culture medium; and

(b) recovering fucoidan produced by said microalgae from the culture.

3. A method of enriching or purifying fucoidan, said method comprising:

(a) cultivating microalgae in a culture medium; and

(b) recovering fucoidan produced by said microalgae from the culture.

4. The method of claim 2 or 3, said recovering comprising:

(ba) subjecting polysaccharides obtained from said microalgae to a preparative analytical method; and

(bb) separating those fractions which

(i) yield fucose upon hydrolysis, and/or

(ii) comprise fucoidan;

thereby obtaining fucoidan.

5. The method of claim 4, wherein said polysaccharides are obtained

(a) from said culture medium, wherein said culture medium is optionally desalted and/or concentrated; or

(b) by extraction, preferably using water as a solvent.

6. The method of claim 5(b), wherein the material to be extracted is

(a) an alcohol insoluble residue obtained from homogenized microalgae; or

(b) homogenized microalgae.

7. The method of any one of claims 4 to 6, wherein said preparative analytical method is selected from

(a) chromatography, preferably anion exchange chromatography;

(b) a graded precipitation method;

(c) a quaternary ammonium salt precipitation method; and

(d) size exclusion chromatography.

8. The method of any one of the preceding claims, wherein the presence or absence of fucose in step (bb)(i) is determined by drawing a sample from a given fraction, subjecting it to acid hydrolysis, and analysing the hydrolysate for fucose.

9. The method of any one of claim 2 to 8, wherein fucose is L-fucose.

10. The method of any one of claims 4 to 9, wherein the presence or absence of fucoidan in step (bb)(ii) is determined by drawing a sample from a given fraction and subjecting it to analysis using a fucoidan-specific antibody, preferably using enzyme-linked immunosorbent assay (ELISA).

11. The use or method of any one of the preceding claims, wherein said microalgae are

(a) diatoms, preferably selected from the families Thalassiosiraceae, Chaetocerotaceae, Phaeodactylaceae and Bacillariaceae, more preferably from the genera Thalassiosira, Chaetoceros, Phaeodactylum and Nitzschia; or

(b) Coccolithophorida, preferably Noelaerhabdaceae, more preferably of the genus Emiliania.

12. The use or method of any one of claims 1 to 11 , wherein said fucoidan is a homofucoidan or a heterofucoidan.

13. A method of producing a pharmaceutical composition, said method comprising the method of any one of claims 2 to 12.

14. A method of producing a nutraceutical, a food supplement or a cosmetic composition, said method comprising the method of any one of claims 2 to 12.

15. A fucoidan obtained by the method of any one of claims 2 to 12.