Extracting polysaccharides from macroalgae

Abstract

The disclosure pertains to a method for extracting polysaccharides from biomass, in particular from green seaweed. A combination of a washing treatment and an extraction treatment is used.

Description

P136724PC00 Title: EXTRACTING POLYSACCHARIDES FROM MACROALGAE Field

[0001] The invention pertains to the production of compounds that are useful forthe production of e.g. bio-based materials and compositions, and in particular to the extraction of polysaccharides from green seaweed. Introduction

[0002] Seaweed, i.e. marine macroalgae constitute a rich source of biologicalcompounds. The commercial production of seaweed currently only concerns red and brown algae. There is a desire to extract polysaccharides from green seaweed, i.e. from green macroalgae (Chlorophyta). Hence, the invention pertains to the extraction of compounds from green macroalgae, in particular to extract polysaccharides, more in particular ulvan from seaweed of the genus Ulva. Ulvan is a gelling sulfated polysaccharide. It has been described as having biological activities including immunomodulating, antiviral, antioxidant, antihyperlipidemic and anticancer. A background reference about the extraction of ulvan and its applications is provided by: Kidgell et all, Ulvan: A systematic review of extraction, composition and function, Algal Research 39, 2019, article 101422; https: / / doi.org / 10.1016 / j.algal.2019.101422.

[0003] FR2998894A1 describes a method of treating algae. The method comprisesdiffusing the algae to be treated in water to form a pulp, filtering the pulp , ultrafiltration of the pressing juice, demineralizing the ultrafiltration retentate and decanting the demineralized retentate thereby, recovering the vegetable protein present in the algae in one decanted part and sulfated polysaccharide present in the algae in another decanted part (see Fig.1 of that document).

[0004] It is generally advantageous if methods for extracting polysaccharides, e.g.ulvan, from algae have a high yield. It is an aim of the invention to provide a biomass processing method to extract polysaccharides, in particular ulvan, with high yield and desirable composition of the extracted fractions.Summary

[0005] The invention aims to provide, in an aspect, a method of extractingpolysaccharides, in particular ulvan, from biomass, in particular from green seaweed. The method involves two treatments, preferably a washing step and an extraction step. In particular, two product fractions are obtained, one from each treatment, i.e. one from the washing step and another from the extraction step.

[0006] The invention provides a method of extracting a polysaccharide-comprisingfraction from biomass, in particular a green seaweed, the comprising: subjecting the biomass to a treatment, preferably a washing treatment, that preferably involves combining the biomass with a first aqueous liquid to form a first slurry and separating a first solid fraction and a first liquid fraction from the first slurry; purifying the first liquid fraction to obtain a first polysaccharide fraction; subjecting the first solid seaweed fraction to an extraction treatment, wherein in the extraction treatment preferably a second aqueous liquid is used preferably at temperature that is at least 20ºC higher than during the washing treatment and / or preferably at a second pH that is lower than during the washing treatment to obtain a second slurry; separating a second solid fraction and a second liquid fraction from the second slurry; and purifying the second liquid fraction to obtain a second polysaccharide fraction. The washing step and extraction step can also be referred to as first treatment and second treatment.

[0007] A preferred embodiment provides a method of extracting a polysaccharide-comprising fraction from a green seaweed, the comprising: subjecting the green seaweed to a washing treatment that involves combining the green seaweed with a first aqueous liquid to form a first slurry and separating a first solid seaweed fraction and a first liquid fraction from the first slurry; purifying the first liquid fraction to obtain a first polysaccharide fraction; subjecting the first solid seaweed fraction to an extraction treatment with a second aqueous liquid at temperature that is at least 20ºC higher than during the washing treatment and / or at a second pH that is lower than during the washing treatment to obtain a second slurry; separating a second solid fraction and a second liquid fraction from the second slurry; and purifying the second liquid fraction to obtain a second polysaccharide fraction.

[0008] Generally the disclosure pertains to a method for extractingpolysaccharides from biomass, in particular from green seaweed. A combination of a washing treatment and an extraction treatment is used. Brief description of the drawings

[0009] Figure 1 schematically illustrates an example method according to theinvention.

[0010] Figure 2 provides photographs of first ulvan extract (Fig. 2A) and a secondulvan extract (Fig. 2B) obtained in Example 1.

[0011] Figure 3 presents the distribution of main components in Ulva lactucaamong the four main product streams in Example 1.

[0012] Figure 4 shows SEC chromatograms of the first ulvan extract (solid line)and second ulvan extract (dashed line) obtained in Example 1.

[0013] Any embodiments illustrated in the figures are examples only and do notlimit the invention. Detailed description

[0014] Provided is a method of extracting a polysaccharide-comprising fractionfrom green seaweed, and more in particular for obtaining two polysaccharide- comprising fractions from the green seaweed. Hence, the method can alternatively be described as a method of obtaining two polysaccharide-comprising fractions from green seaweed. The method can also be described as a method of recovering polysaccharide fractions from green seaweed.

[0015] As used herein, the term ‘polysaccharide’ includes sulfatedpolysaccharides, such as e.g. ulvan. Hence, the inventive method is preferably a method of obtaining two ulvan-comprising fractions from green seaweed. In the method the polysaccharide-comprising fractions are separately and sequentially obtained from the green seaweed (as opposed to, e.g., separating the ulvan extracted in a single extraction step from green seaweed into two sub-fractions). Preferably, the method gives two ulvan-comprising products, as distinct and different product fractions obtained from the respective method steps. An advantageous high yield can be achieved of polysaccharide products (ulvan products) having desirable product quality and desirable properties.

[0016] The method involves providing a feedstock, in particular a green seaweedfeedstock, in particular comminuted (size-reduced) green seaweed. For instance, milled dried seaweed is used as a feedstock. The drying is for instance performed at a temperature below 60ºC to avoid thermal degradation during the drying, for instance by freeze drying. As another example, ground wet seaweed can be used. The particle size of the seaweed feedstock is preferably less than 4 mm.

[0017] Generally, the feedstock can be macro algae biomass. The green seaweed ise.g. of the genus Ulva and is e.g. Ulva lactuca. The green seaweed feedstock is e.g. cultivated seaweed.

[0018] Figure 1 schematically illustrates an example method according to theinvention. The use of reference numerals in the following discussion, referencing to Fig. 1, is for convenience only and does not limit the invention or the claims.

[0019] The green seaweed feedstock is subjected to a washing treatment (A). Thiswashing step can also be referred to as a first extraction step in the invention.

[0020] Accordingly, the feedstock is combined (mixed) with a first aqueousliquid (AL1) to form a first slurry (SL1) during the washing (A), followed by separation (B) of the solid and liquid fraction, e.g. using a centrifuge. Hence, the first aqueous liquid is used as the washing liquid in the washing treatment (A).

[0021] The first aqueous liquid, before the contacting with seaweed, preferablyhas a total dissolved solids level of less than 10000 ppm by weight, more preferably less than 1000 ppm by weight. These values are lower than typical for seawater; and preferably the first aqueous liquid is less saline than seawater. Accordingly, the washing step provides for demineralization of the green seaweed.

[0022] Preferably, the first aqueous liquid, before the contacting with seaweed,comprises 80 wt.% or more of water by total weight of the first aqueous liquid, or 90 wt.% or more of water, such as 95 wt.% or more of water or 98 wt.% or more water, up to 100% water. Preferably, the first aqueous liquid, before the contacting with seaweed, comprises less than 10 wt.% organic solvents, such as ethanol.

[0023] It was surprisingly found by the present inventors that the spent washingliquid from the washing step contains a useful polysaccharide fraction, further noting that the washing liquid is aqueous. Accordingly, the first liquid fraction is purified in the present invention to obtain a first polysaccharide fraction, in addition to a second polysaccharide fraction that is obtained from the extractionstep. Furthermore, the inventors also observed higher yield than in a comparative process without the washing step.

[0024] As shown in Example 1, the judicious combination of a washing treatmentand an extraction treatment, and recovering the desired polysaccharide product from the liquid phase resulting from each treatment, gives an advantageous high yield of the desired product, especially ulvan.

[0025] The washing step thereby results in the extraction of polysaccharides fromthe seaweed, to as to obtain a first polysaccharide fraction from the washing liquid after purification. In particular, the washing step was found to extract ulvan from the green seaweed. Hence, the ulvan concentration of the washing liquid after the washing step is higher than before the washing step, e.g. at least 2 times higher. As an example, the washing liquid prior to washing is e.g. clean water and after the washing contains the extracted ulvan.

[0026] The washing liquid, i.e. first aqueous liquid, is for instance tap water ordemineralized water.

[0027] The washing step is preferably performed with a liquid to dry solid ratio ofmax. 20, preferably max. 15, more preferably between 8 and 15, on weight basis, e.g. the ratio is in the range 1 – 20, or 5 - 15, or 8 - 15. A too low amount of washing liquid is not effective for washing. A too high amount of washing liquid is less desirable for the recovery of the first aqueous polysaccharide fraction from the washing liquid.

[0028] The washing step is for instance performed for at least 5 minutes or atleast 10 minutes, or at least 1 hour.

[0029] The washing step is suitably performed under gentle mixing. The pH of thefirst aqueous liquid, i.e. before combining with the seaweed, is e.g. in the range of 6.5 to 8.5, e.g. 6.5 to 8.0, such as 7.0 – 8.0.

[0030] The pH of the mixture as obtained at the end of the washing step is e.g. inthe range 5.0 to 6.5.

[0031] The washing step is typically performed at a temperature between 0ºC and50ºC, e.g. 10ºC to 40ºC.

[0032] The washing step also involves separating (B) a first solid seaweedfraction and a first liquid fraction from the first slurry. Hence, the slurry isseparated into at least two fractions, one fraction containing predominantly the solid material and the other material containing predominantly the liquid.

[0033] For example, sieving is used to separate the solid from the liquid.

[0034] For instance, solid / liquid separation based on the density (kg / m³)difference is used. The slurry is e.g. separated using a decanter-centrifuge to recover a liquid supernatant as the liquid fraction, and a solid fraction, i.e. the wet washed seaweed.

[0035] The method involves purifying (C1, C2) the first liquid fraction (LF1) toobtain a first polysaccharide fraction. Hence, a first polysaccharide fraction is recovered from the first liquid from the washing step. The purification can also be referred to as a polysaccharide product recovery step.

[0036] As a generally preference, the purification involves increasing theconcentration of the polysaccharide in the product stream, and involves separating the liquid fraction into (at least) a first fraction and a second fraction, the first fraction having a higher concentration of the polysaccharide than the second fraction, so as to obtain a first polysaccharide product stream.

[0037] This purification involves for instance the removal of species other thanthe desired polysaccharide, e.g. species other than ulvan, from the first liquid fraction. For instance, one or more of the following are removed in the purification step: water, suspended solid particles, molecules with a molecular weight lower than 100 kDa (e.g. proteins), and salts. The purification involves for example one or more purification treatments selected from the group consisting of filtration, dialysis, ultrafiltration, evaporation, drying and precipitation. In Figure 1, the purification step is provided by the filtration (C1) followed by evaporation and drying (C2).

[0038] Filtration can be used to remove suspended solids and particles, e.g.fragments of seaweed. Preferably a filtration aid is used, e.g. diatomaceous silica, such as available under the name Celite®. In an example method, a combination of filtration followed by ultrafiltration is used.

[0039] The purification for example involves water removal, which can beevaporative or non-evaporative or a combination thereof. Depending on the concentration of polysaccharide in the supernatant, (vacuum) evaporation can alsobe performed to increase the concentration and reduce the volume of supernatant before drying.

[0040] Water removal is done, for instance, by drying, e.g. spray draying or freezedrying, or vacuum evaporation. Non-evaporative water removal includes e.g. ultrafiltration and dialysis. Hence, water removal may involve selective water transport through a semi-permeable membrane. Precipitation may also be used to isolate the polysaccharides from the aqueous liquid.

[0041] The method for instance involves the removal of at least 90 wt.% of thewater content of the first liquid fraction to obtain the first polysaccharide fraction. The water removal involves e.g. the removal of at least 10 g water per 1 g of the dry weight of the polysaccharide fraction. This water removal provides advantageously for an increase of the polysaccharide concentration.

[0042] The removed water may be recovered and is optionally recycled as washingliquid or extraction liquid.

[0043] Preferably, the first polysaccharide fraction obtained in step (C)), i.e. thefirst product, contains less than 15 wt.% water or less than 10 wt.% water.

[0044] Preferably, the second polysaccharide fraction obtained in step (F), i.e. thesecond product, contains less than 15 wt.% water or less than 10 wt.% water.

[0045] The purification may also involve, alternatively or in addition, removal ofproteins and salts. For instance, dialysis is used. Dialysis involves removal of molecules with a lower molecular weight (e.g. less than 100 kDa or less than 30 kDa) and salts without (significant) water removal.

[0046] The method furthermore involves subjecting the first solid seaweedfraction (SF1) to an extraction (D) with a second aqueous liquid (AL2) (extraction liquid) to obtain a second slurry (SL2) .

[0047] The extraction step (D) is in particular configured to extractpolysaccharides from the seaweed (the first solid seaweed fraction), so as to obtain a second polysaccharide fraction from the extraction liquid after purification. In particular, the extraction step is preferably to extract ulvan. This provides a clear teaching to the skilled person to perform the extraction with appropriate severity (combination of pH, temperature, extraction duration) to extract ulvan yet without substantial hydrolysis of the ulvan and / or depolymerisation of polysaccharides to monosaccharides.

[0048] The extraction is for example conducted at temperature that is at least20ºC higher than during the washing treatment, or at least 40ºC higher than during the washing treatment (and e.g. max. 140ºC) and / or at a second pH that is lower than during the washing treatment, i.e. at a second pH that is lower than the pH at the end of the washing step.

[0049] The extraction is for example, conducted at a temperature of at least 40ºC,or at least 50ºC, or at least 60ºC. The extraction is, for example, conducted at a pH of max.7, or less than 6, or less than 5, or less than 4.0 or less than 3.0. A pH of minimum 2.0, is preferably used to avoid acid degradation of the polysaccharides. Good results were obtained with pH 2.5. In some embodiments, the pH is minimum 3.0. For instance a mineral acid is used to adjust the pH. The second aqueous liquid preferably has such a temperature and pH. For instance, the second aqueous liquid has a pH in the range from 2.0 – less than 6, or in the range from 2.0 to less than 5, or in the range 2.0 to less than 4.0, or in the range 2.0 to less than 3.0.

[0050] Preferably the temperature is at least 80ºC to obtain good extraction.Preferably the temperature is max. 140ºC to avoid thermal degradation, e.g. preferably up to 120ºC at pH 2.0 – 6.0. For instance, an autoclave is used for temperatures above 100ºC, e.g. a absolute pressure above 1.0 bar, e.g. up to 5 bar). A higher temperature, e.g. above 100ºC, can be used with a less acidic liquid, such as with the second aqueous liquid having a pH in the range 6.5 – 7.5 (for the initial pH of that second aqueous liquid).

[0051] A liquid to dry solid ratio on weight basis of maximum 20, preferablymaximum 15, e.g. between 8 and 15 (i.e. weight basis using dry weight of the solid), is advantageously used for this extraction step (D), for the second aqueous liquid. Hence, the second aqueous liquid is added to the first solid seaweed fraction in a mass of max.20 times the dry weight of the first solid seaweed fraction.

[0052] For example, the liquid to dry solid weight ratio is at least 5 in theextraction step, preferably at least 7. Such a ratio contributes to effective extraction of polysaccharides (especially ulvan). With too low liquid relative to the solid, the extraction step (D) may be not effective or not practical. These ratios apply in particular to batch process steps.

[0053] The extraction is e.g. done under gentle mixing.

[0054] The duration of the extraction treatment is e.g. at least 1 hour andtypically max. 24 hours. A longer duration can be useful when the extraction is conducted at a pH closer to 7 and / or at lower temperatures.

[0055] The extraction step (D) is preferably performed with a combined severityfactor CSF: log(CSF) = log(time*exp[(extraction temperature – 100) / 14.75]) – pH (Formula 1)

[0056] Herein, time is the duration of the extraction in minutes, and temperatureis in ºC.

[0057] Preferably, in the inventive method, log(CSF) is max. 1.0, e.g. in the rangefrom -1.0 to 1.0, more preferably in the range from -1.0 to 0.0, in said step (d). A log(CSF) in this range advantageously minimises polysaccharide hydrolysis (degradation) to monosaccharides while obtaining effective extraction of a second fraction of polysaccharides (ulvan).

[0058] Several combinations of extraction temperature (T), time (duration), andpH can provide such conditions, e.g. for log(CSF) = 1 : (100ºC, 120 min, pH 1) or (120ºC, 30 min, pH 1), for log(CSF) = -1 : (80ºC, 120 min, pH 2.5) or (100C, 50 min, pH 2.8).

[0059] A background reference for the combined severity factor is Wyman andYang, Combined Severity Factor for Predicting Sugar Recovery in Acid-Catalyzed Pretreatment Followed by Enzymatic Hydrolysis, in Hydrothermal Processing in Biorefineries, 2017.

[0060] Some example combinations of duration, temperature, and pH that aresuitable for the extraction step are shown in Table A below. In an embodiment, the extraction treatment of step (D) is conducted with a temperature, duration and pH as specified in Table A, more preferably with pH of at least 2.0. Table A T(ºC) pH Duration (h)0 - 50 1.0 - 2.5 4 to 650 - 80 1.5 - 3.0 1.5 to 280 - 100 2.0 - 3.5 1.5 to 2100 - 120 2.5 - 4.0 1.5 to 2120 - 140 > 3.0 0.5 to 1

[0061] With experimental pilot scale test data using extraction in accordance withthese preferences, no monosaccharides were observed in the liquid from the extraction and in the liquid from the washing, only oligosaccharides and polysaccharides.

[0062] The second slurry, obtained with the extraction, is subjected to separation(E) of solid and liquid, to separate a second solid fraction (SF2) and a second liquid fraction (LF2) from the second slurry. Preferences and details for this step are the same as for the separation of the first slurry, e.g. a decanter-centrifuge is used giving a supernatant as the second liquid fraction and a solid residue.

[0063] The second liquid fraction contains polysaccharides, in particular ulvan.

[0064] Preferably, by virtue of the extraction (D) being extraction and not severehydrolysis, the second liquid fraction from the second slurry has a weight ratio of poly / oligosaccharide to monosaccharide of at least 2.0 : 1 or at least 3.0 : 1 or at least 4.0 : 1. A higher ratio is desired. With log(CSF) = 0.5, a ratio of 4.5:1 was observed.

[0065] The method involves purification (F2) of polysaccharides in the secondliquid fraction , usually with preceding filtration (F1) to remove particles. The purification of the second liquid fraction is configured to increase the concentration of the desired polysaccharides, e.g. ulvan, in the second liquid fraction. The purification typically involves separating the section liquid fraction into at least two fractions, one having a higher concentration than the other, so as to obtain the fraction with a higher concentration of the polysaccharides.

[0066] The purification of the second liquid fraction, step F, is typically directed toisolate the components with a molecular weight higher than 100 kDa (or corresponding large size). The purification is e.g. size selective, to selectively obtain the larger molecular species. Hence, in the preferred purification, the large molecular species are separated from the smaller molecular species, with the large molecules fractions being obtained as the second polysaccharide-containing fraction, giving the second product.

[0067] The second liquid fraction is e.g. subjected to membrane filtration for saidpurification (F2), in particular to obtain the second polysaccharide-containing fraction as a retentate, preferably with a molecular weight higher than 100 kDa.This purification can also be used to remove minerals and proteins comprised in the filtrate (having a smaller molecular size than the ulvan).

[0068] Optionally, the second polysaccharide-containing fraction is after thepurification step (F2) subjected to water removal (G), preferably it is dried, to obtain a solid polysaccharide product; this water removal can also be considered a part of the purification step. For instance, spray drying, vacuum evaporation, or freeze drying is used. Precipitation can also be used to isolate the polysaccharides.

[0069] The first and second polysaccharide-containing fractions are henceobtained as distinct and separate products of the method, from distinct steps. The first and second fraction also have a different composition.

[0070] The method preferably further involves subjecting the first and / or secondpolysaccharide fractions to stabilization, preferably both fractions separately. Stabilization is done e.g. by drying (e.g. freeze drying or spray drying) or by precipitation, e.g. precipitation by adding an antisolvent.

[0071] In the inventive method, two products are obtained, both polysaccharide-containing fractions, which in particular comprise ulvan, namely as the (stabilized) first and second polysaccharide-containing fraction. Hence, also provided is a first poly-saccharide product, in particular first ulvan product, that is obtainable as the first polysaccharide-containing fraction of the inventive method; and a second poly- saccharide product, in particular second ulvan product, that is obtainable as the second polysaccharide-containing fraction of the inventive method.

[0072] Ulvan is a water-soluble polysaccharide and originates from the cell wall ofthe algae. Ulvan has been described as polyanionic heteropolysaccharides with sugar compositions comprising rhamnose, glucuronic acid, iduronic acid and xylose. The composition differs between species and depends also on processing procedures to prepare biomass and ulvan, and eco-physiological factors.

[0073] In an embodiment, the first polysaccharide fraction contains ulvan (firstulvan fraction / first ulvan product), with as components (A) 5 – 10 % rhamnan, up to 1 % galactan, 1 – 2 % glucan, 1 – 2 % xylan, 2 – 3 % glucuronic acid (anhydrous basis), and 2 – 6 % iduronic acid (anhydrous basis), and typically 22 – 28 % SO3, all percentages as wt.% and on the basis of dry weight; i.e. based on dry weight of the product. The first ulvan product, having such constituents, preferably has a weight average molecular weight (Mw) of 350 to 450 kDa, more preferably 400 – 450 kDa,and / or a number average molecular weight (Mn) of 40 – 70 kDa, more preferably 40 – 65 kDa, and preferably has a polydispersity index (PI) Mw / Mn of 5.0 – 8.0; and preferably has this Mw, Mn and PI in combination.

[0074] The first ulvan product can also be defined as such in terms of thiscomposition and / or molecular weight, independent of the preparation method.

[0075] In an embodiment, the second polysaccharide fraction contains ulvan(second ulvan fraction / second ulvan product), with as components (B) 15 – 25 % rhamnan, up to 1 % galactan, up to 2 % glucan, 1 – 3 % xylan, 4 – 9 % glucuronic acid (anhydrous basis), and 10 – 20 % iduronic acid (anhydrous basis), and typically 22 – 28 % SO3, all percentages as wt.% and on the basis of dry weight, i.e. based of dry weight of the product.

[0076] The second ulvan product, having such constituents, preferably has aweight average molecular weight (Mw) of 350 to 450 kDa, more preferably 380 - 430 kDa, and / or a number average molecular weight (Mn) of 70 - 120 kDa, more preferably 90 - 110 kDa, and preferably has a polydispersity index (PI) Mw / Mn of 3 - 6; and preferably has this Mw, Mn and PI in combination.

[0077] The second ulvan product can also be defined as such, in terms ofcomposition and / or molecular weight, independent of the preparation method.

[0078] The invention also provides a polysaccharide-containing productobtainable with the method of the invention. The product is e.g. the first or second polysaccharide fraction, preferably as ulvan fraction. The product is preferably ulvan having such composition (A) or (B).

[0079] The use of ulvans for hydrogel films has been proposed, e.g. for wounddressing. The use of ulvan and ulvan oligosaccharides as a food additive has also been described. The use of ulvan for manufacturing films, e.g. for food packaging, has also been proposed, see e.g. Guidara et al, International Journal of Biological Macromolecules 150 (2020) 714–726.

[0080] Both the first and second polysaccharide fractions were tested and found tohave film-forming properties, on their own and with suitable plasticizers.

[0081] For embodiments wherein the polysaccharide is ulvan, i.e. wherein a firstand second ulvan fraction are obtained from the process and / or are provided as the inventive compositions, the invention also provides a film comprising the first and / or second ulvan fraction. The film is optionally a composite film, e.g.comprising the first and / or second ulvan fraction and a further polysaccharide or plasticiser (other than ulvan); preferably with the first ulvan fraction.

[0082] The invention also provides a process for preparing a coating from the firstand / or second ulvan fraction (provided e.g. as a powder) using methods for preparing ulvan coatings known in the art, i.e. using analogous methods. For example, the process in the above-mentioned publication by Guidara may be used, wherein a film forming solution is prepared by mixing an ulvan extract with water and a plasticizer (e.g. glycerol and / or sorbitol), and subsequently spreading the film forming solution evenly onto a surface to form a film. For example, a suitable method for preparing an ulvan coating from the first and second ulvan product involves the steps of: dissolving the first and / or second ulvan fraction (provided e.g. as a powder) in an aqueous liquid to form an ulvan solution, optionally also containing other film-forming components, and casting the ulvan solution on a substrate, drying of the substrate by solvent evaporation to form a coating or solid ulvan layer, and suitably release of the solid ulvan layer to form a film. Alternatively, a coated article is obtained after the drying.

[0083] The invention also provides a packaging material comprising the inventivefilm comprising the first and / or second ulvan fraction, e.g. as a container comprising the packaging material, and a packaged article comprising the packaging; the packaged material is e.g. a food article.

[0084] A background reference for ulvan-based films and coatings for (food)packaging is Wang et al. Foods 2023, 12(8), 1622; doi: 10.3390 / foods12081622.

[0085] The second polysaccharide fraction may also be very suitable for preparingingredients for e.g. cosmetic and medical compositions because of its higher purity than the first polysaccharide fraction.

[0086] It was surprisingly found that films made of first ulvan fraction (i.e. fromstep (C), hence ulvan extracted during the washing step (A)) had a low water vapor transmission rate, making this type of ulvan fraction advantageous for packaging and coating applications. Furthermore, films made of this first ulvan fraction had a low oxygen transmission rate (OTR), of 1.7 - 1.8 cc / m² / day, which is lower than reported before for ulvan (an OTR of 3 cc / m² / day was reported in said paper of Guidara et al.). This indicates that the first ulvan fraction is advantageous as potential oxygen barrier for coating and packaging applications.

[0087] The method can be conducted as a batch process or as a continuousprocess. Furthermore, one or more batch steps and one or more continuous steps can be combined in the method.

[0088] The term “first” as used herein permits the presence of preceding steps oritems. The terms “first” and “second” are used herein as labels and permit the presence of intermediate steps or items. The term ‘liquid fraction’ as used herein includes suspensions and solutions.

[0089] The term ‘polysaccharide fraction’ as used herein indicates a compositioncomprising one or more polysaccharides; the polysaccharides are e.g. sulfated. Preferably, the composition comprises ulvan. Preferably, the composition (i.e. fraction) comprises at least 30 wt.% of the polysaccharides, and comprises for example at least 30 wt.% ulvan (based on dry weight and relative to total dry weight of the fraction), where ulvan content may be taken to refer to the accumulative contents of rhamnan, iduronic acid, glucuronic acid and sulphate ions. The fraction can also be referred to as polysaccharide-containing product. The fraction is e.g. a liquid solution or a solid material.

[0090] Features that are ‘typical’ or ‘preferable’ are used in some embodiments,not necessarily all embodiments of the invention, and are hence not strictly mandatory. Examples

[0091] The invention will now be further illustrated by the following non-limitingexample(s). These examples do not limit the invention and do not limit the claims. Example 1

[0092] Ulvan was extracted in two stages that included several downstreamseparation processes (DSP). For the first stage (washing step), 6 kg ww of dry grounded green seaweed Ulva lactuca (less than 4 mm size) was mixed with demineralised water in a vessel. The green seaweed was from cultivated seaweed, not wild-harvested. The green seaweed contained 8.3 % rhamnan, 0.5 % galactan, 20.0 % glucan, 2.8 % xylan, 2.2 % glucuronic acid, 4.1 % iduronic acid and 27.1 % ash, all contents in dry weight percentage.

[0093] The slurry had a liquid to solid ratio of 10 kg liquid per kg dw (dry weight)seaweed, adjusted for moisture content of the seaweed as measured with a moisture analyser. The slurry was kept at room temperature (about 20ºC) for 16 h. After this, the slurry was separated in a decanter-centrifuge giving a supernatant and solid residue. The supernatant from the washing step was further filtered under pressure using a glass fibre filter and celite as filtration aid in multiple batches. A filtrate with high viscosity was obtained.

[0094] For the second stage (main extraction), the washed wet solids recoveredfrom the decanter-centrifuge (i.e. the first solid seaweed fraction ) were further mixed with demineralised water to give a liquid to solid ratio of 10 kg liquid per kg dry weight seaweed in an autoclave.

[0095] The autoclave was equipped with a built-in paddle mixer, pH probe and oilheating jacket. The pH was adjusted to 2.5 with the addition of sulphuric acid. The slurry was then mixed in the reactor at 150 rpm and heated to 80°C. After 2 h, the reactor was cooled down and slurry mixture separated in the aforementioned decanter-centrifuge. The separated wet solid residues were dried in an air- ventilated oven at 60°C for storage. The supernatant was filtered under pressure as described before. The filtrate had a lower viscosity (based on visual inspection).

[0096] The filtrate was then subjected to ultra-filtration using a spiral membranemodule with a cut-off of 100 kDa. The ultrafiltration was performed in dialysis mode. This was implemented by manual addition of 5 kg demineralised water to the retentate (feed tank) every time the same amount of permeate was collected. The filtration was performed until reaching 79% permeation. After this time, the retentate was drained from the system and further concentrated through evaporation under vacuum. In this step, approx. half of the water in the retentate was evaporated.

[0097] Small samples of the washing filtrate (first ulvan extract) and theconcentrated retentate (second ulvan extract) were subjected to three different ways of stabilisation: (1) Freeze drying; (2) spray drying; and (3) precipitation with 3 weight parts of concentrated ethanol, centrifugation, washing of separated pellet with 1 weight part of concentrated ethanol, centrifugation and freeze drying. For spray drying, the liquid extracts were heated to 40 °C before feeding and the apparatus was set with an inlet temperature of 150 °C, feed pump at 10 g / min and N2 gas flow of 700 L / h. The observed outlet temperature during spray drying was79 and 90 °C (respectively for the first and second extracts) and observed pressure loss of 60 mbar. The amount of solid recovery through the three methods was compared with no significant differences (coefficient of variation of 4 and 10% for the first ulvan and second ulvan extracts, respectively).

[0098] Filtrate obtained from the washing step was much less coloured than thewashing supernatant and showed gelling behaviour at room temperature without further concentration or demineralization. The filtrate was concentrated under vacuum to reduce the available volume and freeze dried to recover the extract as a stable powder (Fig. 2A). This powder is referred herein as first ulvan extract.

[0099] Filtrate obtained from the main extraction step was subjected toultrafiltration (+100 kDa) in dialysis mode to remove minerals, evaporated under vacuum and further freeze dried to recover a stable powder. However, the material was not obtained in the form of a powder but rather as a fibrous foam (Fig. 2B) This stable product is referred herein as second ulvan extract.

[0100] Analyses of the samples included viscosity and molecular weightdistribution via high performance liquid size exclusion chromatography (HP-SEC) using pullulan polymers for calibration. Analyses also included saccharide and acid-insoluble residue composition determined via sulphuric acid hydrolysis and high performance anion exchange chromatography (HPAEC), ash content (550 °C) and sulphate via determination of total sulphur from Inductive coupled plasma atomic emission spectroscopy (ICP-AES).

[0101] The first ulvan extract had a weight average molecular weight of 300 to450 kDa and the second ulvan extract had a weight average molecular weight of 400-600 kDa.

[0102] Table 1 shows further results of the method. The yields of first and secondulvan extracts corresponded to 27 and 8% based on the initial dry weight of the seaweed, respectively. These corresponded to 48% of the total available rhamnan and 58 % and 55% of the total available glucuronic and iduronic acid (anhydrous basis). Approx. 36% of the dry matter of the seaweed was extracted in the washing stage, where minerals were selectively removed from the seaweed (69% ash in initial seaweed). A lesser fraction of the dry matter was extracted from the seaweed during the main extraction step (~25% of the dry matter of starting seaweed). In this step, the mineral fraction of the seaweed was less preferentially extracted. Asan example, during the washing step, there was a 3:1 mass ratio of ash to organics removed from the seaweed, while for the main extraction step, this ratio was 2:1.

[0103] Ultrafiltration of the main extract filtrate to separate a 100 kDa-richfraction as retentate led to recovery of 40% of the dry weight in the filtrate in the retentate, corresponding to 57% of the organic compounds. Interestingly, 93% of the rhamnan and 100% of the glucuronic and iduronic acid in the filtrate were recovered in the retentate, indicating a selective purification of the ulvan fraction. Table 1 Indicators Units OutcomeWashing extraction DW (dry weight) wt% dw seaweed 36.0extraction efficiency Ash extraction wt% ash in initial seaweed 68.9efficiency Organics extraction wt% organics in initial23.7 efficiency seaweed First ulvan extract wt% dw seaweed 27.3yield Ash yield in first ulvan wt% ash in initial seaweed 53.5extract Organics yield in first wt% organics in initial17.5 ulvan extract seaweed Main extraction DW extraction wt% dw seaweed 25.2efficiency Ash extraction wt% ash in initial seaweed 38.6efficiency Organics extraction wt% organics in initial20.2 efficiency seaweed DW recovery in wt% dw in filtrate 39.8retentate Ash recovery in wt% ash in filtrate 21.9retentate Organics recovery in wt% organics in filtrate 56.8retentate Second ulvan extract wt% dw seaweed 8.4yield Ash yield in second wt% ash in initial seaweed 7.8ulvan extract Organics yield in second wt% organics in initial8.6 ulvan extract seaweed

[0104] Fig. 3 presents the distribution of main components in Ulva lactucaamong the four main product streams of Example 1, namely the final seaweed residue, the first ulvan extract, the second ulvan extract and the permeate fractionfrom the ultrafiltration. Herein Rha indicates rhamnan, Gal: galactan, Glu: glucan; Xyl: xylan; Glucur: glucuronic acid (anhydrous basis); Idurur: iduronic acid (anhydrous basis), Ash550C: ash content after treatment at 550ºC for 12 hours, SO3: sulfate groups content based on total sulphur measured via ICP-AES.

[0105] Taking into account the amount of total free liquid available during thetwo extraction steps, the release yield of rhamnan was 46 and 24% of the available carbohydrate in the starting seaweed. This corresponded to a total of 70% of the available rhamnan, which was higher compared to the released rhamnan yield obtained in a comparative lab-scale single stage extraction at 80 °C, pH 2.5 and 2 h (65% of available rhamnan). In the comparative single-stage process at lab-scale, no washing with water at ambient temperature was used.

[0106] The first ulvan extract powder had a high ash content due to the highsolubilisation of minerals during the washing and to the fact that no mineral removal was applied to the gel-like filtrate of the washing. Further results are shown in Table 2. Table 2 Component First ulvan extract Second ulvan extractAverage SD Average SDBiochemical componentsRhamnan, % 7.88 1.43 21.97 0.26dw Galactan, % dw 0.15 0.21 0.55 0.01Glucan, % dw 1.00 0.16 0.70 0.02Xylan, % dw 1.30 0.41 2.89 0.13Glucuronic acid 2.53 0.01 6.84 0.54(anhydrous basis), % dw Iduronic acid 4.11 0.99 13.65 1.05(anhydrous basis), % dw Acid-insoluble2.15 1.95 0.73 0.07residue, % dw Ash*, % dw 53.18 0.02 25.34 0.34SO3 25.81 n.d. 17.81 n.d.Rha:Xyl,5.5:1 - 6.9:1 -mol:mol Rha:UroAc,1.4:1 - 1.3:1 -mol / mol * Measured at 550 °C; dw = dry weight

[0107] It was found that the first ulvan extract had a higher viscosity and alsoshear-tinning behaviour (pseudo-plastic). The second ulvan extract had a lower viscosity and had rather a Newtonian behaviour (less impact of shear rate on the viscosity). This indicates that first extract is likely more suitable for use as rheology enhancer additive compared to the second extract.

[0108] Figure 4 shows SEC chromatograms of the first ulvan extract (solid line)and second ulvan extract (dashed line) obtained in Example 1. Assignment of MW in this figure was given by pullulan standards. Extract powders were diluted in eluent medium with a dilution factor of 100. Further details are given in Table 3. Table 3 Product WeightNumber Polydispersity average average index molecular molecular (Mw / Mn) weight weight (Mn), (Mw), x 103x 103Da Da First extract 418 59 7.0Second extract 395 96 4.1Example 1A

[0109] The protocol of Example 1 was replicated at 2L scale with a differentsource of Ulva seaweed. This source of Ulva seaweed had lower polysaccharide content (4.5 % dw rhamnan, 4.5 % dw glucan, 1.0 % xylan, 2.6% dw glucuronic acid) and higher ash content (50.2 % dw). Results were consistent with the previous example. In this example, the yields of first and second ulvan extracts corresponded to 4 and 4% based on the initial dry weight of the seaweed, respectively. These corresponded to 78% of the total available rhamnan and 90 % of the total available glucuronic acid (anhydrous basis). Approx.33% of the dry matter of the seaweed was extracted in the washing stage, where minerals were selectively removed from the seaweed (79% ash in initial seaweed). A lesser fraction of the dry matter was extracted from the seaweed during the main extraction step (~7% of the dry matter of starting seaweed). Filtration, dialysis and freeze drying was used to separate and purify the ulvan fractions. The first ulvan fraction contained 10 % rhamnan, 2 % xylan, 6 % glucuronic acid (anhydrous basis), all percentages as wt.% and on the basis of dry weight. The second ulvan fraction contained 20 % rhamnan, 2 % xylan, and 14 % glucuronic acid (anhydrous basis), all percentages as wt.% and on the basis of dry weight.Example 2

[0110] A series of tests was performed of extraction of green seaweed withdifferent (organic) acids at the same concentration that yielded varying pH. The extraction was performed though at 120ºC for 3h on Ulva spp. seaweed. Significant monosaccharide formation was observed at pH < 3 (at the used temperature and duration); such significant monosaccharide is not desired in the context of the present invention. Example 3

[0111] Ulva sp. seaweed was extracted with sulphuric acid at 100ºC for 2h atthree different pH with a total liquid to solid weight ratio of 10 to test the risk of undesirable polysaccharide hydrolysis to monosaccharides. “Eq. acid” indicates equivalent acid mass per dry biomass weight, e.g. 4 % refers to 4 g sulphuric acid per 100 g of dry weight seaweed. Table 4 shows the results. A significant increase in monosaccharides was observed at pH 0.9 compared to pH 2.5 with corresponding lower polysaccharide yield. Table 4 Eq. acid / pH Monomeric(Polymeric) Monomeric (Polymeric) Molarity rhamnose rhamnan glucose glucan yield yield % yield % yield % % 51%0.9 50.1 42.1 25.0 0.00.5 M 8%2.5 3.0 69.2 3.8 0.50.08 M 4%3.5 0.2 39.8 1.0 2.80.04 M

Claims

Claims 1. A method of extracting a polysaccharide-comprising fraction from a green seaweed, comprising: a) subjecting the green seaweed to a washing treatment that involves combining the green seaweed with a first aqueous liquid to form a first slurry; b) separating a first solid seaweed fraction and a first liquid fraction from the first slurry, wherein the first liquid fraction contains polysaccharides, in particular ulvan, from the green seaweed; c) purifying the first liquid fraction to obtain a first polysaccharide fraction; d) subjecting the first solid seaweed fraction to an extraction treatment to extract polysaccharides, preferably ulvan, with a second aqueous liquid at temperature that is at least 20ºC higher than during the washing treatment and / or at a second pH that is lower than during the washing treatment to obtain a second slurry; e) separating a second solid fraction and a second liquid fraction from the second slurry, wherein the second liquid fraction contains extracted polysaccharides, preferably extracted ulvan; and f) purifying the second liquid fraction to obtain a second polysaccharide fraction.

2. The method of claim 1, wherein: step a) involves washing of comminuted seaweed, e.g. milled dried seaweed or ground wet seaweed, at a temperature below 60ºC, and a liquid to dry solid weight ratio of below 20.

3. The method of claim 1 or 2, wherein step c) involves water removal and step f) involves membrane filtration.

4. The method of any of the preceding claims, wherein step d) involves an extraction treatment to extract ulvan at 80 to 120ºC and a pH of 2.0 to 7.0 for at least 1 hour, with a liquid to dry solid weight ratio of below 20, preferably the ratio is 1 – 20, and preferably wherein the pH is in the range 2.0 to 5.0.

5. The method according to any of the preceding claims, wherein the first and second polysaccharide-comprising fractions both comprise ulvan.

6. The method according to any of the preceding claims, wherein the green seaweed is from a species of the Ulva genus.

7. The method according to claim 1, wherein the green seaweed is from a species of the Ulva genus, wherein the polysaccharide-comprising fraction comprises ulvan, and wherein – step a) involves washing of comminuted seaweed, e.g. milled dried seaweed or ground wet seaweed, at a temperature below 60ºC, and a liquid to dry solid weight ratio of below 20, and – step c) involves water removal, and – step d) involves an extraction treatment to extract ulvan at 80 to 120ºC and a pH of 2.0 to 7.0, preferably 2.0 to 5.0, or preferably 3.0 – 5.0, for at least 1 hour, with a liquid to dry solid weight ratio of 1 to 20, and – step f) involves membrane filtration.

8. The method according to any of the preceding claims, wherein in the extraction treatment in step (d), log(CSF) is max. 1.0, preferably between -1.0 and 0.0, wherein log(CSF) = log(time*exp[(extraction temperature – 100) / 14.75]) – pH (Formula 1), wherein time is the duration of the extraction in minutes and temperature is in ºC.

9. Ulvan-containing product obtainable as the first polysaccharide fraction of the method according to any of claims 1-8.

10. Ulvan-containing product with as components 5 – 10 % rhamnan, up to 1 % galactan, 1 – 2 % glucan, 1 – 2 % xylan, 2 – 3 % glucuronic acid (anhydrous basis), and 2 – 6 % iduronic acid (anhydrous basis), and typically 22 – 28 % SO3, all percentages as wt.% and on the basis of dry weight.

11. The ulvan-containing product according to claim 10, with weight average molecular weight (Mw) of 350 to 450 kDa, number average molecular weight (Mn) of 40 – 70 kDa, and a polydispersity index (PI) Mw / Mn of 5.0 – 8.

12. Ulvan-containing product obtainable as the second polysaccharide fraction of the method according to any of claims 1-8.

13. Ulvan-containing product with as components 15 – 25 % rhamnan, up to 1 % galactan, up to 2 % glucan, 1 – 3 % xylan, 4 – 9 % glucuronic acid (anhydrous basis), and 10 – 20 % iduronic acid (anhydrous basis), and typically 22 – 28 % SO3, all percentages as wt.% and on the basis of dry weight.

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