NL2009482C2

NL2009482C2 - Process for mannitol extraction from seaweed.

Info

Publication number: NL2009482C2
Application number: NL2009482A
Authority: NL
Inventors: Jaap Willem Hal; Wouter Johannes Joseph Huijgen
Original assignee: Stichting Energie
Priority date: 2012-09-18
Filing date: 2012-09-18
Publication date: 2014-03-19

Description

Process for mannitol extraction from seaweed

[0001] The present invention relates to an advanced method for isolating mannitol from seaweed.

5

Background

[0002] Mannitol is a prevalent saccharide in life forms, and can be found in a wide variety of natural products, including bacteria, yeasts, fungi, algae, lichens and many plants. Mannitol is particularly abundant in brown algae, such as in Laminaria (kelp).

10 [0003] Mannitol is a valuable compound with many applications, such as in medicine (e g. in osmotherapy) and food products (e g. as sweetener or coating agent). In view of its low glycemic and insulinemic indices, it is used as a low-caloric and low-cariogenic sweetener. Mannitol is not or hardly digestible by human enzymes, but is fermented by the intestinal flora. Its suitability as coating agent stems from its very low 15 hygroscopicity. Mannitol also finds application as excipient in medical formulations, e.g. to mask the unpleasant taste of certain drugs, or as diuretic.

[0004] Mannitol is also valuable as a building block or for further (chemical) modification. First of all, mannitol itself can be used as a starting material for polymer synthesis, such as reaction with isocyanides for polyurethane synthesis (e.g. rigid 20 foams). Secondly, mannitol can be converted into isomannide, which is a valuable chiral diol suitable in the preparation of bioplastics (e.g. polyesters, polyethers, polyamides, polyurethanes). Mannitol behaves similarly as sorbitol, which is convertible to isosorbide. In contrast to isomannide, isosorbide is widely studied (see: Rose and Palkovits, ChemSusChem, 2012, 5, 167 - 176; and Fenouillot el al., Prog. 25 Polym. Sci. 2010, 35, 578 - 622). Because of its different spatial arrangement of the hydroxy-groups, isomannide can lead to the products having different characteristics from isosorbide-derived products. The applicability of isomannide is hampered by the absence of efficient and high yielding production processes of mannitol, while the precursors for isosorbide are readily obtainable from starch. To allow bioplastics to 30 replace fossil fuel based plastics in the future, the price and availability of their precursors needs to be improved.

[0005] Sorbitol is easily obtainable from glucose, which in turn can be prepared by depolymerization of starch or cellulose. Depolymerization of starch is readily 2 performed by enzymatic hydrolysis (by amylases) or acid hydrolysis, but the use of starch for non-food applications competes with the continuously increasing food intake of humans. Depolymerization of cellulose, either for glucose or directly for sorbitol synthesis, is to date not industrially applicable in view of its low efficiency and high 5 costs. Thus, alternatives for isosorbide are desired, and isomannide, readily prepared from mannitol, is a suitable candidate. Mannitol is obtainable via bacterial and yeast-based transformations (see Song and Vieille, Appl. Microbiol. Biotechnol. 2009, 84, 55 - 62). Mannitol may also be prepared from starch, but this requires hydrogenation of fructose over e.g. a nickel catalyst and gives 50/50 molar mixtures of sorbitol and 10 mannitol. In addition, mannitol may be extracted from natural products such as plants or seaweed, for which no efficient and cost-effective process is available for large-scale mannitol production.

[0006] Harvesting and processing seaweed is extensively discussed by McHugh in A Guide to the Seaweed Industry (FAO Fisheries Technical Paper No. 441, Rome, FAO, 15 2003, obtainable from http://www.fao.org/). A method for processing brown seaweed is known from US 2007/0218076, wherein seaweed is extracted with an alcohol having one to six carbon atoms, such as ethanol. This extract comprises low molecular weight compounds from seaweed. The residue is extracted with acidic water (pH below 6), the extract of which contains laminarin and fucoidan. Black etal. (J. Appl. Chem. 1951, 1, 20 414 - 424) describes the laboratory-scale isolation of mannitol from dried and milled seaweed by extraction with boiling methanol or ethanol.

[0007] Adams el al. {Bioresour. Technol. 2011, 102, 226 - 234) describe the determination of mannitol in Laminaria digitata, whereby freeze-dried L. digitata is ground and suspended in water (2% w/w), centrifuged and acidified with 5 mM

25 sulphuric acid to determine the mannitol content in L. digitata on laboratory scale.

[0008] Ross at al. (J. Anal. Appl. Pyrol. 2009, 85, 3 - 10) describe a laboratory scale process for treating seaweed by grinding washed and air-dried seaweed to a powder, and subsequently treating the seaweed powder in water (10 g powder per 200 ml water at reflux for 6 hours) or acid (10 g powder per 50 ml 2.0 M HC1 at 60°C for 6 hours).

30 [0009] In view of the above, there exists a need in the field for a process for the isolation of mannitol from seaweed, which is industrially applicable on large scale, efficient and cost effective.

3

Summary of the invention

[0010] The invention pertains to a process for the treatment of fresh seaweed, in particular seaweed having a high mannitol content, wherein the seaweed is subjected to extraction with water and subsequent filtration. A seaweed having a high mannitol 5 content is a seaweed which during at least part of the seasonal cycle has a mannitol content of at least 5.0 wt% on dry weight basis. Preferred seaweeds are brown algae (Phaeophyceae), such as kelp. Seaweed may contain up to 30 wt% mannitol, based on total dry weight of the seaweed.

[0011] The first step of the process according to the invention is the treatment of fresh 10 seaweed with water, having a pH of 3 to 9 and a salinity of at most 20 g/kg. The inventors surprisingly found that treatment of the fresh seaweed with water, without the need for any pre-treatment, results in an efficient separation of mannitol from the seaweed. Without being bound to a theory, the inventors believe that in view of the high osmotic stress present in seaweed, mannitol will migrate from the cells to the 15 extraction water, without the need of breaking up the cells and/or the cell walls prior to extraction.

[0012] The aqueous extract obtained in the first step of the process comprises large amounts of mannitol, together with laminarin and minerals. In one embodiment, laminarin may be removed from the aqueous extract by ultrafiltration (UF) over a 20 membrane with a molecular weight cut-off of between 3.0 kDa and 200 Da. Optionally, the mannitol solution is demineralized using techniques known in the art, such as reverse osmosis, fractional crystallization (using the high solubility of mannitol in water), ion exchange, simulated moving bed, electrodialysis or nanofiltration over a membrane with small pores (e.g. > 100 kDa).

25

Embodiments 1. A process for isolating carbohydrates from seaweed, comprising: (a) extracting fresh seaweed with water having a salinity of less than 20 g/kg and a pH between 3.0 and 9.0; and 30 (b) isolating carbohydrates from the aqueous extract.

2. The process according to embodiment 1, wherein the pH of the water is between 3.5 and 8.0, preferably between 5.0 and 9.0, more preferably between 6.0 and 8.0.

4 3. The process according to embodiment 1 or 2, wherein step (b) comprises filtering the aqueous extract over an ultrafiltration membrane with a molecular weight cutoff of between 3.0 kDa and 200 Da.

4. The process according to any one of the preceding embodiments, wherein the fresh 5 seaweed is brown algae.

5. The process according to any one of the preceding embodiments, wherein step (a) is performed at a temperature between 10°C and 120°C, preferably between 20°C and 80°C.

6. The process according to any one of the preceding embodiments, further 10 comprising chopping the seaweed prior to step (a).

7. The process according to any one of the preceding embodiments, wherein the carbohydrates comprise mannitol.

8. The process according to embodiment 7, wherein the carbohydrates further comprise glucose and/or glucose oligomers.

15 9. The process according to embodiment 7, wherein the carbohydrates further comprises laminarin.

10. The process according to any one of the preceding embodiments, further comprising: (c) demineralization of the aqueous extract or the ultrafiltration permeate.

20 11. The process according to embodiment 10, wherein the demineralization is performed by reverse osmosis.

Detailed description

[0013] The process for extraction of carbohydrates from seaweed according to the 25 invention is applicable to all types of seaweed, in particular selected from brown algae, green algae and red algae, more preferably from brown algae and green algae, most preferably from brown algae. For reasons of efficiency, it is preferred to use seaweed containing high levels of mannitol, such as at least 5.0 wt%, preferably at least 10 wt% mannitol, more preferably at least 15 wt% mannitol, most preferably at least 20 wt% 30 mannitol, based on total dry weight of the seaweed. Preferred seaweed in this respect are brown algae, but certain green algae, such as Ulva, may also be suitable. In a preferred embodiment, the seaweed is brown algae, in particular belonging to the genera Laminaria, Saccharina, Macrocystis, or Alaria. Preferred species of seaweed 5 are L. digitata (oarweed), S. latissima (sugar kelp), S. japonica (kombu), M. pyrifera (giant kelp) and A. esculenta (winged kelp). Especially preferred seaweeds are kelps. A single species of seaweed can be used, but the process according to the invention is equally suitable to mixtures of two or more species. The seaweed may be naturally 5 grown seaweed or cultured seaweed.

[0014] The mannitol content of seaweed may vary over the year. Hence, it is preferred to use seaweed which is harvested in the right period, i.e. when the mannitol content in the seaweed is the highest. Many studies are devoted on the composition of seaweed throughout the year, which may dependent on many factors, such as location and the 10 particular species involved. For each particular situation, the seasonality may be determined experimentally, by methods known in the art (see e.g. Black, J. Soc. Chem. Ind. 1948, 67, 169 - 172 and 172 - 176; Adams et al, Bioresour. Technol. 2011, 102, 226 - 234; Haug etal., 2ndIntern. SeaweedSymp., Trondheim, 1956, 10 - 15; Haug et al., Seasonal variations in the chemical composition of Alariaesculenta, Laminaria 15 saccharina, Laminaria hyperborea and Laminaria digitata from Northern Norway, Norges Telmisk-Naturvitenskapelige Forskningsrod, 1954, Oslo, Norway).

[0015] The seaweed used for the process according to the invention is preferably fresh seaweed. In the context of the present invention, “fresh seaweed” is meant to include all seaweed having an intact cellular structure or intact cells. Thus, in the process 20 according to the invention, preferably no measures are taken to break up the cells and/or the cell walls of the seaweed. Such measures include grinding dried seaweed to a powder, such as grinding freeze-dried seaweed, grinding heat-dried seaweed (e g. in an oven or furnace) and grinding sun- and/or air-dried seaweed. The inventors have surprisingly found that mannitol is efficiently extracted from whole seaweed cells, e g. 25 from large chunks of seaweed or even from whole seaweed.

[0016] The process of the invention is directed at extracting valuable components from fresh seaweed, in particular carbohydrates. Carbohydrates, as used in the present invention, includes sugars (monosaccharides, oligosaccharides, polysaccharides), as well as their derivatives, such a sugar alcohol, sugar acids, amino sugars, sulphated 30 sugars, etc. The carbohydrates in particular comprise mannitol, glucose and glucose oligomers and polymers.

[0017] In one embodiment, the invention pertains to a process of isolating mannitol from the seaweeds as defined above. In another embodiment, the invention pertains to a 6 process for isolating mannitol and glucose and glucose oligomers and mixtures thereof, e.g. obtained by partial or complete hydrolysis of laminarin. In a further embodiment, the invention pertains to a process of isolating mannitol and (neutral) polysaccharides, in particular laminarins, and their mixtures. A further embodiment pertains to the 5 isolation of mannitol, glucose, uronic acids, fucose and sulphated fucose, and further mono- and oligo-saccharides, and their mixtures, e.g. obtained by extensive hydrolysis of most or all of the polysaccharides contained in the seaweeds.

Pretreatment 10 [0018] Optionally, the seaweed is pretreated prior to the extraction step. For ease of handling, the seaweed may first be chopped to pieces (e.g. 2 to 30 cm). As the process according to the invention is applicable to fresh seaweed, in one embodiment the process does not comprise a step of grinding or milling the seaweed to a powder prior to extraction. Thus, it is preferred that the majority of the seaweed, preferably at least 15 50 wt%, more preferably at least 80 wt%, used in the extraction process is not smaller than 0.5 cm in diameter, preferably not smaller than 1.0 cm in diameter. Washing of the seaweed and removal of large impurities (e.g. shells, sand) may be desired prior to extraction. Also, dried seaweed, e.g. sun-dried and/or air-dried seaweed, may be used in the process according to the invention. The current process may be combined with 20 protein isolation from seaweed, as those proteins are valuable either nutritionally or for carbon fixation (e.g. ribulose-l,5-bisphosphate carboxylase oxygenase (RuBisCo)). Such protein isolation may occur prior to or after mannitol extraction by methods known in the art. Thus, in the context of the invention, the terms “seaweed” and “fresh seaweed” are meant to include chopped seaweed, washed seaweed and dried seaweed.

25 [0019] In one aspect of the invention, the process does not comprise an acidification step prior to extraction. The inventors have found that immersion of the seaweed in e.g. a sulphuric acid bath is not necessary in order to efficiently extract mannitol from the seaweed.

30 Extraction

[0020] The aqueous extraction may be performed in any vessel suitable for performing an extraction. Conveniently, a vessel is used wherein the mixture of seaweed and water can be agitated (e.g. stirred) and heated, such as an autoclave or a fermentor. For the 7 extraction step, the vessel is charged with a mixture of fresh seaweed, optionally pretreated as described above, and water. Water and seaweed may be added simultaneously, or one by one. The resulting mixture preferably has a ratio seaweed : water of between 10:1 and 1:10, more preferably between 5 : 1 and 1 : 5, even more 5 preferably between 2 : 1 and 1:1, based on total (wet) weight of both components. Total wet weight of the seaweed may be determined by weighing the fresh seaweed, which is to be subjected to the extraction step. For the purpose of the invention, the wet weight and the dry weight can be converted by applying a factor 4. The dry weight of seaweed corresponds to the weight of the seaweed after freeze-drying.

10 [0021] For the aqueous extraction, preferably water having a temperature of between 10°C and 120°C, more preferably between 20°C and 100°C, even more preferably between 40°C and 80°C is used. The water that is charged to the vessel may be at any temperature, conveniently at room temperature, and heating towards the desired temperature may take place within in the vessel during extraction. Preferably, the 15 aqueous extraction is not an alkaline extraction. Thus, the pH of the water should not exceed 9.0, preferably not exceed 8.0. Preferably ,the pH of the water is not lower than 3.0, more preferably not lower than 3.5. In one embodiment, the pH of the water used for the extraction is preferably between 5.0 and 9.0, more preferably between 6.0 and 8.0. In another embodiment, some acid may be present in the water that is used for 20 extraction, and the pH of the water is preferably between 3.0 and 5.0, more preferably between 3.5 and 4.5. Laminarin hydrolyses in acid environment, which facilitates the further treatment of laminarin after extraction. Hydrolyzed laminarin is ideally suitable for fermentation, such as fermentation to ethanol, acetone-butanol-ethanol, succinic acid, 1,3-propanediol and/or glyceric acid. Any Bronsted acid may be used. Suitable 25 acids include, but are not limited to, acetic acid, citric acid, formic acid, oxalic acid, hydrochloric acid, hydrofluoric acid and hydrobromic acid. Given the preferred pH ranges given above, the skilled artisan will appreciate how much of each acid is to be added to the extraction water. In a preferred embodiment, acetic acid is used, preferably 0.1 to 2 g per kg water.

30 [0022] It is preferred that the salt content of the extraction water is relatively low, at least lower than sea water. Thus, the salinity is preferably lower than 20 g per kg of water, more preferably lower than 10 g/kg, more preferably lower than 5 g/kg, even more preferably lower than 1 g/kg, most preferably lower than 0.5 g/kg. In terms of 8 sodium the content is preferably less than 5 g/kg, more preferably less than 1 g/kg. In an especially preferred embodiment, demineralized water is used.

[0023] It was found that the duration of the extraction step, i.e. the time during which the extraction vessel is kept at the desired temperature, does not have a marked 5 influence on the amount of mannitol extracted. Preferably, the extraction is continued for at least 0.5 minute, more preferably 1 to 120 minutes, most preferably 5 to 60 minutes. In one embodiment, the mixture is agitated, preferably stirred, during the extraction.

[0024] Alternatively, the extraction is a continuous process, wherein fresh water 10 (substantially absent of mannitol) is added to the extraction vessel and mannitol-rich water is removed from the extraction vessel continuously. In the respect, the vessel may not be a conventional extraction vessel, but any vessel suitable for continuous extraction, e.g. an extruder. Such continuous extraction by reactive extrusion is known for alginate extraction from seaweed (see e.g. Vauchel et al. Food Bioprocess. Technol. 15 2008, 7, 297 - 300) and is also applicable to the process according to the invention.

[0025] The aqueous extraction affords an aqueous extract comprising mannitol, and a viscous solid-like residue comprising alginates. The aqueous extract and the residue may be separated by conventional techniques, including but not limited to filtration and centrifugation. The aqueous extract may further comprise certain amounts of laminarin 20 and minerals, depending on the composition of the treated seaweed, which may optionally be separated from the mannitol in further process steps. Alternatively, when mildly acidic extraction is employed, e.g. at a pH of between 3.5 and 5.0 the aqueous extract comprises mannitol and glucose, and optionally some remaining not fully hydrolyzed laminarin and minerals. Such a mixture of mannitol and glucose is valuable 25 as starting material for alcohol fermentation, such as fermentation to ethanol, acetone-butanol-ethanol, succinic acid, 1,3-propanediol and/or glyceric acid. Optionally, the minerals may be removed from the aqueous extract comprising mannitol and glucose. As further alternative, when more strongly acidic extraction is employed, e.g. at a pH between 2 and 3.5, the aqueous extract comprises mannitol, glucose and further 30 monomeric and oligomeric sugars derived from laminarin, fucoidans and/or alginates. The solid-like residue may be further processed into useful products, such as alginate or furfural. Extraction of alginate from seaweed is known in the art and may be accomplished by e.g. sodium bicarbonate addition to convert alginic acid to water 9 soluble sodium alginate. If no prior protein removal has been performed, the solid-like residue will also contain proteins, which may be separated in a subsequent step.

Filtration andfurther treatment 5 [0026] The aqueous extract obtained by the aqueous extraction of seaweed may be further processed by filtration over an ultrafiltration membrane with a molecular weight cut-off of between 3.0 kDa and 200 Da, preferably between 1.0 kDa and 500 Da. Suitable membranes for the ultrafiltration step include ceramic membranes, polymeric membranes and organic spiral-wound membranes. Ultrafiltration is especially useful to 10 remove laminarin from the aqueous extract. Thus, in a preferred embodiment, the extraction employs water having a pH of 3.0 to 5.0, preferably 3.5 to 4.0 and the resulting aqueous extract is not treated by ultrafiltration. In an especially embodiment, the extraction employs water having a pH of 5.0 to 9.0, preferably 6.0 to 8.0 and the resulting aqueous extract is further treated by ultrafiltration.

15 [0027] Ultrafiltration of the aqueous extract affords a UF retentate comprising laminarin, which can be further processed into useful products. Laminarin, a β(1—>3):β(1—>6) glucan, can be depolymerized to oligosaccharides and to glucose, which has many useful applications, and can be fermented to alcohols, as described above. The UF permeate comprises mannitol and optionally minerals.

20 [0028] In a preferred embodiment, the optionally ultrafiltered aqueous extract is demineralized to remove the majority or all of the minerals. Demineralization may be accomplished by techniques known in the art, such as reverse osmosis, fractional crystallization, ion exchange, simulated moving bed, electrodialysis or nanofiltration over a membrane with small pores (e g. > 100 kDa). Preferably, reverse osmosis is used 25 to demineralize the ultrafiltered solution. In an equally preferred embodiment, the demineralization takes place prior to ultrafiltration and after the aqueous extraction.

Examples

[0029] Example 1: Procedure for extracting mannitol and laminarin from seaweed 30 [0030] Freshly harvested seaweed (Laminaria digitata or Saccharina latissima) was chopped to ~5 cm ribbons and mixed on a 1:1 wet weight basis with demineralized water, optionally containing acetic acid (see table 1). The resulting mixture was charged to an autoclave. The mixture was heated to the desired temperature (T, see 10 table 1) and kept at that temperature for the desired time period (t, see table 1). The mixture was cooled and filtered. The extraction efficiency was determined by measuring the mannitol and glucose concentrations following acidic post-hydrolysis. Acidic post-hydrolysis (2.0 M trifluoroacetic acid, 121 °C, 2 hours) was performed, 5 which converts all oligomeric and polymeric carbohydrates to the corresponding monomers, such as laminarin to glucose. Mannitol and glucose were determined by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD, ICS3000, Dionex, Sunnyvale, CA) equipped with a CarboPac PA1 column. Yields of mannitol and laminarin using various extraction 10 conditions are given in table 1. The composition of the fresh seaweed feedstocks as starting material is given in table 2. In table 1, a yield of 100 % corresponds to extraction of all mannitol or laminarin, which was originally present in the seaweed.

[0031] Table 1. extraction conditions (T, t) and composition of the starting material and 15 the extracts exp. seaweed species T (°C) t (min) yield mannitol yield laminarin (%) (%) 1 Laminaria digitata 20 30 67 *** 2 Laminaria digitata 120 30 66 *** 3* Laminaria digitata 100 120 65 *** 4** Laminaria digitata 100 120 64 *** 5 Saccharina latissima 20 30 47 17 6 Saccharina latissima 80 30 53 72 7 Saccharina latissima 120 30 51 77 8* Saccharina latissima 100 120 55 80 * The extraction water comprises 0.1 M acetic acid (pH = 4.2). ** The extraction water comprises 0.2 M acetic acid (pH = 3.8). *** Not determined.

[0032] Table 2: composition of the seaweeds:

Seaweed__Composition (% w/w dry seaweed)_ __Glucose__Mannitol_

Saccharina Latissima_ 16.0 19.9

Laminaria Digitata 14.6 15.1 20 11

[0033] Example 2: Comparative example

[0034] Seaweed {Laminaria digitata or Saccharina latissima) was treated in the same way as described for example 1 (table 1, exp. 2, 3, 7 and 8), except that no water was added to the seaweed during extraction. The resulting seaweeds turned black 5 (carbonization) without releasing moisture. No aqueous extract could be collected from the autoclave. So, the addition of water is essential for the extraction step, the water as present in the seaweed (which was about 80 wt% in the fresh seaweeds used, based on the total weight of the seaweed) is not sufficient to perform the extraction.

10

Claims

A method for isolating carbohydrates from seaweed, comprising: (a) extracting fresh seaweed with water having a salinity or salinity of less than 20 g / kg and a pH between 3.0 and 9.0; and 5 (b) isolating the carbohydrates from the aqueous extract.

Method according to claim 1, wherein the pH of the water is between 3.5 and 8.0, preferably between 5.0 and 9.0, more preferably between 6.0 and 8.0.

3. A method according to claim 1 or 2, wherein step (b) comprises fusing the aqueous extract over an ultra-filtration membrane with a molecular mass cut-off of between 3.0 kDa and 200 Da.

A method according to any one of the preceding claims, wherein the fresh seaweed is brown seaweed.

5. A method according to any one of the preceding claims, wherein step (a) is carried out at a temperature between 10 ° C and 120 ° C, preferably between 20 ° C and 80 ° C.

The method of any preceding claim, further comprising shredding the seaweed prior to step (a).

The method of any one of the preceding claims, wherein the carbohydrates contain mannitol.

The method of claim 7, wherein the carbohydrates further contain glucose and / or glucose oligomers.

The method of claim 7, wherein the carbohydrates further contain laminarin.

10. A method according to any one of the preceding claims, further comprising: (c) demineralizing the aqueous extract or the ultrafdtration permeate.

The method of claim 10, wherein the demineralization is performed by reverse osmosis.