method

Abstract

The present invention provides a method for obtaining alginate from macroalgae, in particular brown algae such as Laminaria hyperborea. The present invention further relates to alginate obtained by such a method. More specifically, the present invention is a method for extracting alginate from macroalgae or a part thereof, comprising: (i) contacting the macroalgae or a part thereof with an aqueous solution of a weak organic acid, such as lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, or glycolic acid; (ii) subsequently contacting the macroalgae or a part thereof with an aqueous solution of a mineral acid, thereby forming a pretreated macroalgal material; and (iii) extracting alginate from the pretreated macroalgal material. Such a method may produce light-colored alginate without the need to use toxic chemicals such as formaldehyde. The method may be controlled to adjust the final composition of the extracted alginate, for example, its molecular weight, polydispersity, its viscosity when dissolved in water, or its M / G ratio. This allows the production of alginates with properties tailored according to their intended use.

Description

[Technical field]

[0001] The present invention relates to a method for processing macroalgae and to products produced by such a method. More specifically, the present invention relates to a method for obtaining alginate from macroalgae, in particular brown macroalgae such as Laminaria hyperborea. The present invention further relates to alginate obtainable, obtained or directly obtained by such a method.

[0002] In certain aspects, the present invention relates to improvements in and relating to the extraction of alginate compared to methods currently used in industrial processing of macroalgae. Improvements include, but are not limited to, improved alginate quality and / or yield and improved sustainability of the method. Advantageously, the method produces light-colored alginate without the need to use highly toxic chemicals such as formaldehyde. The reduced CO2 emissions and ability to carry out the method at ambient temperature additionally provide a more environmentally acceptable process than those currently used in industry.

[0003] In certain aspects, the present invention further relates to methods of processing macroalgae that can be controlled to tailor the final composition of the extracted alginate, e.g., its molecular weight and / or its M / G ratio can be adjusted such that the functional properties of the extracted alginate material, such as its viscosity when dissolved in water, its gelling ability, etc., can be tailored depending on its intended use. [Background technology]

[0004] Macroalgae, also known as "seaweed", are a source of commercially useful products for a variety of applications, such as use in the food, cosmetic, and pharmaceutical industries, as well as in agriculture and animal feed. To obtain such products, the macroalgae generally need to be processed and often the products extracted. This is the case for alginate, a polysaccharide that can be extracted from brown macroalgae. Alginate is the main structural component of the cell wall of kelp (Laminaria hyperborea) and is present in high concentrations in the main stem ("stalk") and leaf ("thallus") parts.

[0005] "Alginate" is a term commonly used in the industry to refer to alginic acid and any derivative of alginic acid, such as salts of alginic acid. Alginates are composed of linear chains formed from two monomers, β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. The M and G monomers are covalently linked to form a linear copolymer. There are three types of segments in the linear structure: M blocks consisting of consecutive M units, G blocks consisting of consecutive G units, and MG blocks containing heterogeneous or alternating M and G units. Alginates are present in the cell walls of brown algae in the form of insoluble salts of alginic acid and multivalent cations such as calcium or aluminum. They exist primarily as the calcium salt of alginic acid. Potassium and sodium salts may also exist. Sodium alginate is a water-soluble polymer that gives a highly viscous solution. In the presence of multivalent cations such as calcium, sodium alginate has the ability to form a gel. Divalent cations, such as calcium ions, bind to the G-blocks of aligned alginate chains, creating cross-links between separate alginate chains or within the same alginate chain, a process that creates a gel network.

[0006] Alginates have beneficial applications in many industries, such as pharmaceutical, medical, nutraceutical and health, agriculture, cosmetics, food, paper and textile industries.For example, alginates are used in wound dressings due to their hypoallergenic properties.Further uses include as thickeners, emulsifiers or stabilizers in food and as weight loss supplements.Alginates are also useful products in paper and printing.

[0007] Alginates are traditionally extracted from brown macroalgae as soluble sodium salts. Conversion of insoluble calcium alginate to soluble sodium alginate makes alginate "extractable". The extraction method influences the chemical and mechanical properties of the alginate and determines its applications. The properties of alginate, such as its viscosity when dissolved in water or the strength of the gel obtained upon addition of calcium salts, are determined by its molecular weight, the arrangement of M and G residues in the polymer chain, and the overall M / G ratio of the alginate chain. For example, Ca 2+ In the presence of ions, the G blocks form ionic complexes to produce cross-linked structures known as the "egg box model" responsible for strong gel formation. The proportions of M, G, and MG blocks determine the physical properties of the alginate. Alginates with high G have higher gelling properties, but alginates with high M do not form strong gels in the presence of multivalent cations and are therefore preferred for use as viscosity modifiers. Alginates with high M / G ratios result in elastic gels, whereas those with low M / G ratios produce brittle gels. The M / G ratio can be altered by chemical or enzymatic modification of the alginate. The arrangement of M and G residues as well as the overall M / G ratio can be altered by extraction processes. Different uses of alginates often require that they have certain predictable chemical and physical properties, such as molecular weight range and distribution, purity, viscosity, M and G content, M / G ratio, etc. G-rich alginates are particularly useful, for example, for pharmaceutical applications.

[0008] Natural alginates present in macroalgae have a high molecular weight and contain multivalent cations, both of which render them insoluble. The objective of the extraction process is to obtain a dry, powdered alginate, typically sodium alginate, ideally in high yield, with a high molecular weight and minimal color. Extraction of alginates generally requires a multi-step process that includes treatment in an acid solution, typically hydrochloric acid or sulfuric acid, to convert the natural alginate to alginic acid, followed by treatment with sodium carbonate to convert the insoluble alginic acid to soluble (i.e., extractable) sodium alginate. Treatment with sodium hydroxide at high pH (typically pH 11 or higher) and heating may also be required to promote hydrolysis of the alginate chains to reduce their molecular weight to the point where they become soluble. The result of this process is a viscous fluid that requires "thickening" (e.g., by dilution with water) to allow filtration to separate the soluble alginate from the remaining seaweed residue. The dissolved alginate is then recovered from the aqueous solution, for example by adding an acid to precipitate alginic acid, by adding a calcium salt to precipitate calcium alginate (from any alginate fragments containing G-blocks), or by adding a poor solvent such as ethanol.

[0009] The current "industry standard" process for producing sodium alginate from brown macroalgae relies on the use of highly caustic solutions containing approximately 4% by weight sodium carbonate in addition to sodium hydroxide to reduce the molecular weight of the native alginate chains and convert the insoluble alginate (e.g., calcium alginate) to its corresponding soluble sodium form. These chemicals are used in excess. Thus, the process is wasteful in terms of the amount of chemicals used. Typical yields of alginate are also low, e.g., in the range of 15-20% by weight (dry weight basis). As part of the alginate recovery process, excess amounts of CO2 are also released when the sodium carbonate is neutralized.

[0010] For use in many of its industrial applications, colorless or light-colored alginates are required to prevent the product to which they are added from being stained by the color of the alginate. A particular problem when extracting alginate from macroalgae, especially brown macroalgae, is the undesirable color in the extracted product. Most of the color of the macroalgae stalks is caused by the presence of colored compounds (i.e., pigments), such as polyphenols (e.g., phlorotannins), carotenoids, and chlorophyll, that are mainly present in the outermost surface layer of the stalk (i.e., the "bark"). When the macroalgae are processed as a whole or when raw stalks are used, these color-producing compounds are incorporated into the alginate extraction process and form an irreversible colored alginate solution after the extraction.

[0011] Traditionally, the problem of undesirable color has been addressed by preventing its extraction using formaldehyde / formalin (which acts as a color fixative by complexing with the pigments and making them insoluble) or by the use of chemical bleaching agents, such as hypochlorite bleaching agents. Formaldehyde also acts as a preservative and is often used post-harvest and before processing to prevent microbial degradation of the macroalgae. However, the use of formaldehyde is regulated due to its toxicity to humans and animals, and there is a general need to reduce its use, and the use of chemical bleaching agents, to provide a more sustainable and environmentally acceptable process. The use of these agents in the production of alginate materials to be ingested by humans or animals or used on the human or animal body is particularly undesirable. The presence of formaldehyde in the macroalgae residue remaining after alginate extraction also reduces its commercial value, and it is treated as waste, disposed of without attempting to recover other potentially useful materials, such as cellulose.

[0012] An alternative to the use of formaldehyde and chemical bleaching agents to address the problem of undesirable color in extracting alginate from macroalgae has been suggested in International Patent Application Publication No. WO 2015 / 067971 (Marine Biopolymers Ltd.). This prior application proposes removing the outer surface layer of the stalk by mechanical peeling or grinding prior to extraction of the alginate. While this partially addresses the color problem described above, it requires peeling or grinding the stalk, which not only adds an additional processing step that increases the cost of the process, but is also wasteful in that the entire stalk is not utilized. Mechanical peeling is also difficult to control when performed on stalks that are not uniform in diameter along their length, and therefore the process inevitably has to remove a thicker portion of the stalk than is strictly required to address the color problem. This is also not ideal, as the portion of the stalk just below the bark contains the "high G" alginates that are particularly commercially valuable. Pigments such as polyphenols are also present to some degree throughout the stalk structure. Removal of the outer surface layer of the stems does not address the color problems arising from these. Summary of the Invention

[0013] The present invention provides an alternative method for obtaining alginate or alginate-containing materials from macroalgae that addresses or mitigates at least some of these problems. In at least certain aspects, the present invention provides an improvement over methods previously known and previously used in the art, particularly methods used to process macroalgae on an industrial scale.

[0014] Proposed herein is a method for producing alginate, which includes a pretreatment of the macroalgae, before extraction, i.e. before converting the alginate present in the macroalgae to a soluble (i.e. extractable) form that can be extracted and recovered. The pretreatment includes exposing the macroalgae, or a portion thereof, to a weak organic acid, followed by cation exchange with a mineral acid. These pretreatment steps, also referred to herein as the "pre-extraction" phase of the method, are very effective in converting the native alginate (e.g. calcium alginate) to alginic acid. In the "extraction" phase, subsequent treatment of the macroalgae material, for example with an alkaline solution, typically a sodium-containing alkaline solution such as sodium carbonate, forms a water-soluble salt of the alginate (e.g. sodium alginate), which can then be recovered using conventional methods. Importantly, it is possible to use a much lower concentration of sodium carbonate for the extraction, compared to that used in current industrial methods. This results in a reduction in CO2 emissions. The methods described herein also allow the use of sodium hydroxide instead of sodium carbonate, which results in a zero CO2 emission profile for the extraction part of the process.

[0015] Importantly, and unexpectedly, the yield of alginate produced using the "pre-extraction" treatment described herein is higher than that obtained using conventional industrial methods, without compromising the quality of the alginate, such as its viscosity when dissolved in water. The light-colored alginate also does not require the use of formaldehyde, formalin or any chemical bleaching agents in its production, and most surprisingly, does not require the removal of the pigment-containing bark from the stems. Thus, the entire stems can be used without generating large amounts of waste, allowing direct access to other materials from the remaining residue. The "pre-extraction" stage of the method can be further controlled to recover alginate materials with predictable and desired functional properties. Advantageously, the method described herein therefore allows the properties of the extracted alginate to be tailored according to its intended use.

[0016] In one aspect, the present invention provides a method for extracting alginate from macroalgae or a part thereof, the method comprising: (i) contacting the macroalgae, or a portion thereof, with an aqueous solution of a weak organic acid; (ii) subsequently contacting the macroalgae, or a portion thereof, with an aqueous solution of a mineral acid, thereby forming a pretreated macroalgae material; (iii) extracting alginate from the pretreated macroalgae material.

[0017] In another aspect, the present invention provides an alginate or alginate derivative obtained, obtainable or directly obtained from the method described herein, in particular sodium alginate obtained, obtainable or directly obtained from the method.

[0018] In a further aspect, the present invention provides products comprising an alginate or alginate derivative as described herein, such as products comprising sodium alginate, including, but not limited to, foods, pharmaceuticals, medical products, dietary supplements and health products, products for use in agriculture, cosmetics, and products for use in the paper and textile industries. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] As used herein, unless otherwise specified, the term "alginate" is used broadly to refer to alginate salts (sometimes referred to in the art as "alginates") as well as any other derivatives of alginic acid and alginic acid itself. As used herein, alginic acid is a polysaccharide consisting of blocks of (1-4)-linked β-D-mannuronate (M), blocks of α-L-guluronate (G), and blocks with alternating structures (MG). Any reference herein to "natural insoluble alginate" is intended to refer to alginate in its naturally occurring form, particularly calcium alginate. When referring to "soluble alginate", it will be understood that this refers to a soluble form, for example, a soluble sodium form. However, it may also refer to any other monoionic form that is soluble, such as potassium alginate or ammonium alginate. As will be understood, any reference herein to "soluble alginate" refers to an alginate that is soluble in water. "Insoluble alginate" will be understood to refer to alginates that are insoluble in water, such as insoluble salts of alginic acid with multivalent cations, such as calcium or aluminum. Typically, naturally insoluble alginates include calcium alginate. Examples of soluble alginates include sodium alginate, potassium alginate, and ammonium alginate. Typically, the soluble alginate is sodium alginate. Any "soluble" form of alginate may also be referred to herein as "extractable alginate," meaning that it can be extracted from macroalgae by direct solubilization.

[0020] The terms "macroalgae" and "seaweed" are used interchangeably herein and are intended to refer to any species of macroscopic multicellular seaweed. Any macroalgae containing alginate may be used in the methods of the present invention. Brown macroalgae, such as kelp, are known to contain high concentrations of alginate and are particularly suitable. The term "kelp" refers to large brown macroalgae that form part of the marine order Laminariales. Suitable macroalgae for use in the present invention include, but are not limited to, those selected from the group consisting of Laminaria, Ascophyllum, Durvillaea, Ecklonia, Lessonia, Macrocitis species, and Sargasum. Examples of specific species include Laminaria hyperborea, Laminaria digitata, Lessonia trabuculata, Lessonia flavicans, and Lessonia braziliensis. Laminaria species, such as Laminaria hyperborea, are particularly suitable.

[0021] Macroalgae typically include three distinct morphological parts or sections: thallus (also known as "leaf" or "blade"), stalk ("stem-like" structure), and stolon ("root-like" structure that anchors the macroalgae to the seabed, sometimes called "appressorium"). These parts differ in terms of their physical properties and chemical composition. Harvesting methods include cutting the stalk near the appressorium. After harvesting, the thallus and stalk are typically separated from each other to form different "parts". The methods described herein may be performed on the whole macroalgae (i.e., stalk and thallus), but are typically performed on one or more separated parts. When separated parts are used together in the methods of the invention, they may be combined in any desired ratio depending on the desired properties of the extracted alginate. For example, a combination of separated leaves and stalks may be used, for example, in a 50:50 weight ratio. The size of the macroalgae or parts thereof is typically reduced to increase its surface area before being processed according to the methods of the invention. Suitable methods are described herein.

[0022] The alginate is concentrated in the stalks of the macroalgae. In one embodiment, the method is carried out on the stalks of the macroalgae that contain the highest alginate content. Thus, the macroalgae parts used in the method of the present invention may substantially only comprise the stalks. The use of the stalks of Laminaria hyperborea is particularly preferred. Alternatively, the method of the present invention may be carried out on the thallus of the macroalgae, or on a part of the thallus. If a part of the thallus is used, this is generally the thickest part taken from the base of the thallus. The use of the thallus or any part of the thallus of Laminaria hyperborea is preferred. Alternatively, the method of the present invention may be carried out on the entire macroalgae, for example a combination of both the stalks and the thallus. The selection of the appropriate part (or parts) of the macroalgae for use in the method will affect the physicochemical properties of the resulting material and may be selected accordingly.

[0023] Epiphytes are organisms that grow on the surface of macroalgae in a marine environment. These include other species of algae, bacteria, fungi, sponges, bryozoans, ascidians, protozoans, crustaceans, mollusks, and other sessile organisms. These may be beneficially removed (or substantially removed) before use of the macroalgae or any part of the macroalgae in the methods described herein. If it is desirable to remove epiphytes from the surface of the macroalgae or part thereof, any conventional method may be used. These may be removed by washing with water, for example using a high pressure water jet. However, in some embodiments, the epiphytes do not need to be removed. Thus, the macroalgae or part thereof used in the methods of the present invention may have epiphytes on its surface.

[0024] Macroalgal stalks may be selected for use in the methods of the invention because they have a higher alginate content and a higher proportion of G-blocks (i.e., a higher G / M ratio) than leaves. The stalk may be substantially cylindrical and includes three characteristic regions defined based on radial distance from the central axis of the stalk. The radially inner portion includes a core region of the stalk, referred to as the "inner core." The radially intermediate portion surrounding the core includes a tissue region, referred to as the "outer core." The radially outermost portion includes a protective surface layer, which may be referred to as the "outer layer." This outer layer may also be referred to as the "bark," "skin," or "skin" of the stalk.

[0025] The stalks may be treated to remove some or all of their outer surface layer prior to treatment according to the methods described herein. However, in a preferred embodiment of the present invention, it need not be removed. This is particularly advantageous. Stalks that have not been subjected to any chemical or physical process to remove the outermost surface layer (i.e., the outer layer remains substantially "untreated") are particularly preferred for use in the methods of the present invention. Such stalks may be referred to as "unpeeled" stalks. Thus, in one set of embodiments, the macroalgae used in the present method may be whole macroalgae (i.e., stalks and thallus), in which the stalks retain their outer surface layer, or stalks that have been separated from the leaves but still retain their outer surface layer. The use of unpeeled stalks of Laminaria hyperborea is particularly preferred for use in the present invention.

[0026] Although generally less desirable, it is possible to carry out the methods described herein on stalks from which the outermost layer has been substantially removed, i.e., "peeled" stalks. Thus, in one embodiment, the method may include removing an outwardly facing surface layer, containing undesirable pigments such as polyphenols, from the stalk or portion of the stalk. The outwardly facing surface layer to be removed includes at least the epidermis layer and may also include the split cortex layer. Typically, the outwardly facing surface layer to be removed includes at least the epidermis and the split cortex layer. Removal of the surface layer may be carried out using any method known in the art. For example, it may be removed by a chemical peeling process or a mechanical method. Mechanical methods include peeling, grinding, scraping, or treatment with a high pressure water jet. Peeling, grinding or scraping may be carried out manually (i.e. by hand), but is more typically carried out using an automated machine, such as a peeling and / or grinding machine known in the art for peeling and / or grinding vegetables. A suitable peeling method is described in International Patent Application Publication No. 2015 / 067971, the entire contents of which are incorporated herein by reference. The thickness of the outer-facing surface layer of the stalk to be removed depends on the type, age, and thickness (i.e., diameter) of the macroalgae, but can be easily determined by a person skilled in the art. The outer-facing surface layer of the stalk to be removed may have a thickness of at least 0.5 mm, preferably at least 1.5 mm. For example, it may have a thickness in the range of 0.5 mm to 2.5 mm.

[0027] The methods described herein may be performed on the whole macroalgae (i.e. unprocessed) or on a portion thereof. For example, it may be performed on the stalks. Before performing the pre-extraction steps of the method, it is generally preferred that the macroalgae or a portion thereof is reduced in size to increase its surface area and thus improve the efficiency of the processing method. The method used to reduce its size is not particularly important, and any known method may be used to reduce the size of the material, i.e. to divide the material into multiple portions, e.g., multiple stalk portions. For example, the macroalgae or a portion thereof (e.g., stalks or thallus) may be divided by any combination of cutting, chopping, flaking, blending, and milling. If appropriate, it may be cut into smaller pieces before flaking, blending, or grinding. This may be useful to aid in the handling of the material during the flaking, blending, or grinding steps.

[0028] In one embodiment, the macroalgae or parts thereof may be divided into portions by cutting and subsequent grinding. Cutting may be suitable to reduce the size of the macroalgae into smaller portions. For example, the stalks may be cut to lengths of 5-100 mm, such as 5-10 mm.

[0029] The grinding may be carried out using any conventional grinding machine known in the art. If desired, the grinding may include two or more grinding stages, including the use of progressively finer screens, to provide the desired particle size. The ground portion may have a particle size in the range of 0.1 mm to 10 mm, preferably 1 mm to 5 mm, such as 1 mm to 2 mm. In one embodiment, the ground portion may have a particle size in the range of 2 mm to 10 mm, such as 4 mm to 8 mm.

[0030] The method described herein may include the further step of washing the macroalgae or parts thereof with water prior to carrying out the pretreatment step. For example, it may include washing a plurality of macroalgae parts (e.g., stem parts and / or thallus parts) with water. Although deionized water may be used, it is generally preferred to use potable water (which contains calcium ions) to reduce loss of low molecular weight "G"-containing alginates from the material.

[0031] Advantageously, washing with water removes salts and, in part, other undesirable water-soluble components such as polyphenols. One or more washing steps may be performed if necessary. The temperature of the water and the washing time may be easily determined by one skilled in the art. Lower temperatures and / or shorter treatment times are generally preferred to reduce the energy requirements of the process and to avoid any harsh treatment of the material that may adversely affect the extracted alginate material. When washing the macroalgae parts, water may be added to the parts, then agitated in the water, and then drained through a filter. If desired, any water-soluble material may be recovered from the washing water. In one embodiment, no additional washing steps are required at this point in the process.

[0032] The method described herein may be performed on living or dead macroalgae. For example, it may be performed on fresh, frozen or dried macroalgae or any part(s) thereof. "Living" macroalgae retain some biological activity, such as respiration. In one embodiment, the method is performed on fresh macroalgae or any part(s) thereof. By "fresh" it is meant that the macroalgae or any part(s) thereof has not been dehydrated to any appreciable extent after harvesting. Fresh macroalgae includes material that has been harvested alive, i.e., material that is a living, breathing plant. Alternatively, after harvesting, the macroalgae or any part(s) thereof may be processed such that it no longer has biological activity, such as respiration. For example, the macroalgae may be squeezed to remove seawater, thus reducing the volume of the plant material to aid in its transportation. Squeezing may result in "dead" plant material in some cases. Alternatively, the macroalgae or any part(s) thereof may be frozen or dried. For example, it may be air dried at ambient or elevated temperature, or it may be dried in a fluidized bed dryer. Prior to drying, the macroalgae or portions thereof are typically chopped or flaked to reduce the energy requirements of the drying process. Following drying, the macroalgae may be further chopped, flaked, or ground (e.g., by grinding or milling) to produce a material that can be stored prior to treatment by the methods described herein. Any dried macroalgae material is typically rehydrated before being subjected to a pre-extraction process as described herein. The addition of water to the dried material may also be beneficial to extract any water-soluble pigments (e.g., polyphenols) that are not attached to the alginate chains, and to remove undesirable salts and other low molecular weight components.

[0033] Rehydration of any dried macroalgae material is typically carried out by contacting the material with water. As with the washing step, deionized water may be used for rehydration purposes, but it is generally preferred to use potable water (which contains calcium ions) to reduce loss of low molecular weight "G"-containing alginates from the material. The use of potable water also reduces the cost of the process when carried out on an industrial scale. A suitable hydration ratio (wet mass:dry mass) can be easily determined, but may be, for example, greater than about 8:1, preferably greater than 10:1. It may be, for example, in the range of about 8:1 to about 12:1. Hydration may be carried out by adding the dried macroalgae material, e.g., dried flakes, to water, stirring, and allowing to settle. It may be carried out in a continuous or batch process. Multiple hydration steps may be carried out, in which case water is removed from the hydrated mass at the end of each step and the solid mass is collected and transferred to a subsequent hydration stage. This aids in the removal of undesirable salts, water-soluble pigments, and other components from the material. The hydration step may be carried out until the conductivity of the water removed from the material is sufficiently reduced to indicate that a sufficient amount of undesirable salts have been removed from the material. For example, a conductivity of less than about 200 μS for deionized water may be appropriate. For potable water, an acceptable conductivity may be its native conductivity + 200 μS. The required hydration time depends on the particle size of the dry material, but can be easily determined by one of ordinary skill in the art. Hydration may take several hours. The final material is typically treated to remove excess water before further processing. The hydrated mass may then be treated as described herein.

[0034] The pre-extraction stage of the method involves an initial step of contacting the macroalgae, or a portion thereof, with a weak organic acid as described herein. This step is effective to reduce the molecular weight of the native alginate and decolorize the material. Without wishing to be bound by theory, it is believed that the organic acid is effective to break down any chlorophyll and pigment residues. The decolorized material is then treated with a mineral acid, thereby exchanging metal ions (e.g., calcium) present in the alginate structure for hydrogen ions to facilitate subsequent extraction.

[0035] The pre-extraction stage of the method may be limited to treatment of the macroalgae or parts thereof with organic acids and treatment with mineral acids, i.e., no additional pre-treatment steps are performed (apart from any size reduction and / or washing steps of the macroalgal material as described herein). However, in some embodiments, the pre-extraction stage may include additional pre-treatment steps, such as those commonly known and used in the art. If performed, these are typically performed before contacting the macroalgal material with organic acids. The additional pre-treatment steps may include, for example, methods known to adjust the M / G ratio of the alginate. For example, the macroalgae or parts thereof may be subjected to additional pre-treatments that can enrich the G content of the material (i.e., increase the G / M ratio). Acid treatment at high temperatures (e.g., treatment using an acid with a low pH, such as a mineral acid) may be performed, for example, to hydrolyze the "M" blocks and thereby enrich the G content of the alginate. Such treatments are particularly suitable in the case of leaf alginate, which has a lower G content than alginate present in the stems, for example. Other known methods that can be used to hydrolyze the "M" blocks include enzymatic treatment, for example using lyase enzymes. Suitable methods for increasing the G content of alginates include those described in European Patent No. 0980391, the entire contents of which are incorporated herein by reference.

[0036] In some embodiments, the method may include an additional pretreatment step involving treatment with an alcohol, such as propan-2-ol. This may be beneficial when treating macroalgae leaves (i.e., thallus) to aid in the removal of colored pigments. However, this step is not required. As described herein, light-colored alginate material can still be obtained from leaves without the need to perform this additional pretreatment step. Any pigments removed in this step may be recovered and purified as a separate product, if desired.

[0037] In some embodiments of the invention, the method may include an additional pretreatment step in which the macroalgae or a portion thereof is contacted with calcium ions. 2+ The addition of ions serves to bind the G-blocks in the alginate and protect them from degradation during subsequent processing. If performed, this step is generally performed prior to the organic acid treatment. The calcium ions may be provided, for example, in the form of a calcium chloride solution. A typical concentration of calcium chloride can be readily determined by one skilled in the art, but may be in the range of 0.5-10% w / v, preferably 1.0-7.5% w / v, for example 5.0% w / v.

[0038] In certain embodiments, any additional pretreatment of the macroalgae, which would degrade the target alginate material to a significant extent, should be avoided or at least minimized. Microwave treatment of macroalgae is conventionally used to degrade complex polysaccharides into their corresponding monomers, for example in the production of biofuels. Such treatment should be avoided in the method of the present invention. Thus, in one embodiment, the method described herein excludes any step involving exposing the macroalgae or parts thereof, any of the intermediate products produced during the method, and any of the recovered alginate to microwaves. Any harsh acid or alkali treatment, which would hydrolyze the alginate chains to any significant extent, should also be minimized (preferably avoided) to reduce the degree of degradation of the alginate chains and avoid any reduction in their molecular weight.

[0039] As used herein, the term "organic acid" refers to an organic compound having acidic properties. Organic acids for use in the present invention may have one or more acid groups.

[0040] As used herein, the term "weak organic acid" refers to a substance that partially dissociates when dissolved in a solvent, such as water. The strength of an acid is determined by its acid dissociation constant, K, which can be determined experimentally by known methods such as titration. a Weak acids have a lower K than strong acids.a and higher pK a It has pK a is the dissociation constant (K) of the acid measured in aqueous medium at a temperature of 25°C a ) is the negative logarithm (to the base 10). Weak acids have very high K a value and slightly negative pK a Very low values ​​of K compared to strong acids with a (hence the higher value of pK a An acid may have more than one dissociation constant depending on how many protons it can give up, and thus the pK a1 , pK a2 Two or more pKs are indicated as a The pK of an acid may have a value. a Values ​​can be readily found in the literature, for example, CRC Handbook of Chemistry and Physics, 97th Edition, June 2016, Ed. William M. Haynes.

[0041] Organic acids for use in the present invention should not induce acid hydrolysis of alginate to a significant extent, so that degradation of the alginate chains is minimized. Advantageously, the organic acid has a pK value less than that of alginic acid. a Alginic acid has a pK in the range of 1.5 to 3.5. a In a preferred embodiment, the organic acid has the lowest pK of alginic acid. a pK greater than a or the lowest pK, if appropriate a (i.e., "pK a1 "). Thus, preferably, the organic acid has a pK greater than 1.5. a It has a pK in the range of 2 to 6, preferably 2.5 to 5.5, more preferably 3 to 5, for example 3 to 4.5. a (or, where appropriate, the lowest pK a ) are preferred for use in the present invention.

[0042] In one embodiment, the organic acid for use in the present invention has the highest pK of alginic acid. a pK a (or lowest pK, if appropriate) a ) and therefore has a pK of 3.5 or less. a (or, where appropriate, the lowest pK a ) are particularly preferred.

[0043] Organic acids suitable for use in the present invention are those having a pK a The organic acid can be easily selected by those skilled in the art based on the E number. If the alginate is intended for use in any pharmaceutical or food application, the organic acid should be selected accordingly. Therefore, food-grade acids, i.e., those that are acceptable for use in foods intended for human consumption, may be suitable. Typically, it is an organic acid that is approved for use as a food additive by a food-related government agency (e.g., the European Food Safety Authority or the United States Food and Drug Administration). Organic acids that have an E number and are therefore approved for use as a food additive in the European Union are particularly suitable.

[0044] Organic acids that can be used in the present invention include, for example, carboxylic acids. They may contain one or more carboxylic acid groups, i.e., they may be mono- or polycarboxylic acids. As used herein, the term "polycarboxylic acid" refers to a carboxylic acid that contains at least two carboxylic acid functional groups (i.e., -COOH). The acid may be, for example, mono-, di-, or tri-carboxylic acid.

[0045] In one embodiment, the organic acid may be a polycarboxylic acid, such as a di- or tricarboxylic acid. 2+ Carboxylic acids that also have the ability to chelate multivalent cations such as ions may be particularly suitable. This includes, in particular, tricarboxylic acids such as citric acid.

[0046] The carboxylic acid is typically an aliphatic acid. The aliphatic carboxylic acid may be linear, branched, or cyclic, and may be saturated or unsaturated. Typically, the carboxylic acid is saturated. The carboxylic acid may contain, for example, 2 to 20 carbon atoms. Optionally, it may contain one or more additional hydroxy groups. The aliphatic carboxylic acid may contain 2 to 16 carbons, preferably 2 to 14 carbon atoms, for example 2 to 12 carbon atoms. The carboxylic acid may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Advantageously, the carboxylic acid may contain 4, 5, 6, 7, 8, 9, or 10 carbon atoms, for example 4, 5, 6, 7, or 8 carbon atoms. For example, the carboxylic acid may contain 4, 5, or 6 carbon atoms.

[0047] Carboxylic acids that also contain a hydroxy group are particularly suitable for use in the present invention. Thus, carboxylic acids suitable for use in the present invention include alpha-hydroxy acids. As used herein, the term "alpha-hydroxy acid" (or "AHA") refers to a carboxylic acid substituted with a hydroxy group at the alpha-carbon atom. It includes lactones that have a hydroxy group at the alpha position and may be saturated or unsaturated. Examples of AHAs provided in the form of lactones include, but are not limited to, ascorbic acid. In addition to the hydroxy group at the alpha-carbon atom, alpha-hydroxy acids or "AHAs" as defined herein may contain one or more additional hydroxy groups.

[0048] In one embodiment, the carboxylic acid for use in the present invention is food grade AHA.Examples of AHA suitable for use in the present invention include lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, and glycolic acid.Of these, lactic acid (E270), malic acid (E296), tartaric acid (E334), citric acid (E330), and ascorbic acid (E300) have E numbers and are generally preferred.Particularly preferred for use in the present invention are malic acid, citric acid, and ascorbic acid.The use of citric acid is particularly preferred.

[0049] Examples of other carboxylic acids that may be used in the present invention include, but are not limited to, acetic acid (E260) and formic acid (E326).

[0050] pK of Carboxylic Acids Suitable for Use in the Present Invention a value and pK if necessary a1 Values ​​are: Lactic acid: pK a =3.86 Malic acid: pK a1 =3.40 Tartaric acid:pK a1 =2.98 Citric acid: pK a1 =3.13 Ascorbic acid: pK a1 =4.17 Glycolic acid: pK a =3.83 Acetic acid: pKa = 4.76 Formic acid: pKa = 3.75

[0051] Any pH and pK mentioned herein a Values ​​are measured at ambient temperature, typically and preferably at 25°C.

[0052] The step of contacting the macroalgae or a portion thereof with the organic acid may be carried out in any known manner. For example, it may include adding an aqueous solution of the acid to the macroalgae or a portion thereof and stirring to ensure good contact. The stirring may include simple mixing, or other techniques such as blending, high shear mixing, etc. The appropriate mixing ratio (macroalgae:organic acid) may be easily determined by one skilled in the art. Typically, the organic acid solution is used in excess to ensure good contact with the macroalgae and to aid in the diffusion of the organic acid into the macroalgae. For example, a volume ratio of macroalgae:organic acid in the range of about 1:1.5 to about 1:5 or about 1:2 to about 1:3 may be used. A volume ratio of about 1:2 may be appropriate.

[0053] The exact conditions for the organic acid treatment, such as the concentration of the acid, the temperature duration of the treatment, etc., can be readily selected by those skilled in the art, taking into account factors such as the intended use of the extracted alginate material and its desired properties. As described herein, the organic acid treatment conditions can be varied to appropriately adjust the properties of the resulting alginate. As demonstrated in the examples, the exposure time of the macroalgal material to the organic acid, its concentration, and the temperature of the organic acid treatment affect the molecular weight of the extracted alginate. This in turn essentially affects the viscosity of the alginate when dissolved in solution. Longer treatment times and / or higher temperatures are, for example, effective in reducing the molecular weight and viscosity of the alginate. The use of higher concentrations of organic acid also reduces the molecular weight (and therefore the viscosity) of the extracted alginate. Advantageously, the conditions of the pretreatment with organic acid can be adjusted to recover alginate with desired functional properties.

[0054] Typically, the organic acid may be used in the form of an aqueous solution having a concentration of 0.1-10.0% w / v, 0.25-5.0% w / v, 0.75-2.5% w / v, 1.0-2.0% w / v, or 1.0-1.5% w / v, preferably about 1% w / v. If it is desired to provide an alginate with a higher molecular weight (and therefore a higher viscosity), the use of a lower concentration of the organic acid may be preferred. If a lower molecular weight (and therefore a lower viscosity) of the extracted alginate is desired, a higher concentration may be appropriate and selected accordingly. For example, an aqueous solution of the organic acid having a concentration in the range of 5.0-10.0% w / v, 6.0-10.0% w / v, or 8.0-10.0% w / v may be used.

[0055] The temperature of the organic acid treatment may be selected depending on the desired molecular weight (and therefore viscosity) of the extracted alginate. Generally, temperatures up to about 100°C may be used. However, lower temperatures are generally preferred to reduce the overall energy requirements of the process. The use of lower temperatures may also provide a greater degree of control over the organic acid pretreatment step (and therefore its effect on the properties of the extracted alginate). Temperatures in the range of 10-100°C, preferably 10-50°C, more preferably 15-30°C, for example 20-25°C, may be used. Advantageously, however, this step of the process is carried out at ambient temperature, for example in the range of 18-25°C. As will be appreciated, at ambient temperatures no additional heating is required. Thus, in one set of embodiments, the invention provides a method for extracting alginate from macroalgae, or a portion thereof, the method comprising: (i) contacting the macroalgae, or a portion thereof, with an aqueous solution of a weak organic acid at ambient temperature, for example a temperature of 18-25° C.; (ii) subsequently contacting the macroalgae, or a portion thereof, with an aqueous solution of a mineral acid, thereby forming a pretreated macroalgae material; and (iii) extracting alginate from the pretreated macroalgae material.

[0056] If a lower molecular weight (and therefore lower viscosity) of the extracted alginate is desired, higher temperatures for the organic acid treatment may be appropriate and selected accordingly. If higher temperatures are used, these may be in the range of 60-100°C, such as 65-100°C, 70-100°C, 80-100°C, 90-100°C, or 95-99°C. Thus, in another set of embodiments, the invention provides a method for extracting alginate from macroalgae or a portion thereof, the method comprising: (i) contacting the macroalgae or a portion thereof with an aqueous solution of a weak organic acid at a temperature of 60-100°C; (ii) subsequently contacting the macroalgae or a portion thereof with an aqueous solution of a mineral acid, thereby forming a pretreated macroalgal material; and (iii) extracting alginate from the pretreated macroalgal material.

[0057] The duration of the organic acid treatment may be appropriately selected by one skilled in the art. For example, the duration of the treatment may range from a few minutes to a few hours. As will be appreciated, the duration of the treatment is influenced by the selected concentration of the organic acid and the temperature used in this step of the method. When a low concentration of organic acid is used, the duration of the treatment may be extended, for example, to several days or even weeks. Typically, however, the organic acid treatment may be performed for up to 2 hours, for example up to 1.5 hours, for example up to 1 hour. The treatment may be performed for a shorter time, for example less than 1 hour, especially when a high temperature and / or high concentration of organic acid is used. For example, the treatment time may be at most about 2 minutes, or about 5 minutes. The treatment time may be, for example, in the range of 2 to 60 minutes, or 5 to 50 minutes, or 10 to 40 minutes, or 20 to 30 minutes.

[0058] The appropriate combination of temperature and duration of the organic acid treatment can be selected by one skilled in the art. For example, treatment at ambient temperature for about 1 hour may be particularly suitable for producing alginates with high viscosity, e.g., greater than 800 cp, greater than 900 cp, greater than 1000 cp, greater than 1500 cp, greater than 1600 cp, greater than 1700 cp, greater than 1800 cp, or greater than 1900 cp. When a higher temperature, e.g., about 60°C, is used, a treatment time in the range of about 5-10 minutes may be selected to produce an alginate with a medium viscosity, e.g., a viscosity in the range of 400-800 cp, and a treatment time in the range of about 30-40 minutes may be selected to produce an alginate with a low viscosity, e.g., a viscosity in the range of 50-400 cp. When an ultra-low viscosity alginate is desired, a higher treatment temperature up to about 100°C, e.g., about 95°C to about 99°C, e.g., about 95°C for about 20 minutes, may be suitable. Higher processing temperatures for about 20-45 minutes, e.g., 35-40 minutes, may be appropriate to provide an ultra-low viscosity alginate. The ultra-low viscosity may be in the range of 5-50 cp. All viscosities referred to herein refer to the viscosity of a 1 wt.% solution of the alginate in water at 20° C., measured using a Brookfield viscometer.

[0059] The selection of temperature and duration of the organic acid pretreatment step of the present method should take into account the concentration of the organic acid solution used. For example, if a higher concentration of organic acid is used, a shorter treatment time and / or lower temperature may be appropriate to provide the desired degree of control in producing an extracted alginate having the required functional properties.

[0060] Following organic acid pretreatment, the liquid is typically separated from the macroalgae or parts thereof, i.e., undissolved solids, for example by filtration or centrifugation. To improve process efficiency, the filtrate or liquid phase from the centrifuge may be collected and reused in another pretreatment process. At this stage, an additional washing step may be performed, for example using deionized water.

[0061] The treatment with organic acid is followed by a metal cation exchange step, the purpose of which is to convert the insoluble alginate to alginic acid by exchanging metal cations with protons. In the method of the present invention, this metal cation exchange step is carried out "subsequently" to the organic acid treatment step. In this context, the term "subsequently" is not intended to exclude the option of one or more intermediate treatment steps between steps (i) and (ii) of the method. Step (ii) may, but need not, immediately follow step (i). As detailed above, after the organic acid pretreatment step, for example, the treated macroalgae or a part thereof may be separated from any liquid and, optionally, the treated macroalgae or a part thereof may be subjected to one or more washing steps.

[0062] Step (ii) involves contacting the macroalgae or a portion thereof with an aqueous solution of a mineral acid to form a pretreated macroalgae material. This is done by the addition of a mineral acid, which is added to the reaction mixture to lower the overall pH, for example to a pH in the range of about 1.5 to about 2, for example 1.7 to 1.9. Suitable mineral acids include hydrochloric acid and / or sulphuric acid. Conveniently, the mineral acid will be hydrochloric acid. More preferably, the mineral acid will be sulphuric acid. The material may be allowed to stand for up to 60 minutes, for example up to 30 minutes, for example up to about 15 minutes. As will be appreciated, the contact time will depend on the particle size of the material and may be readily selected by the skilled artisan. The mixture may be agitated (e.g. stirred) during contact with the mineral acid. Generally, the mineral acid treatment is carried out at ambient temperature, i.e. at a temperature in the range of 18-25°C. If desired, the step of treatment with mineral acid may be repeated.

[0063] Following the mineral acid treatment, the method typically includes a step of separating the resulting mixture into a solution phase and residual solids. For example, the material can then be drained through a filter or transferred to a centrifuge. The resulting gel or precipitate may be rinsed with water in one or more rinse steps to remove excess mineral acid. The water used in this part of the process typically contains Ca 2+ The water is deionized to avoid reintroduction of ions. The water rinse is effective to raise the pH of the material, for example to a pH in the range of 4 to 5, and may be repeated as necessary. The filtrate or liquid phase from the centrifuge, which is the mineral acid solution, may be collected and can be used in a subsequent metal cation exchange step, if desired, thereby improving process efficiency.

[0064] After the pretreatment process described herein, the native alginate is present in an insoluble form, i.e. mainly in the form of alginic acid. Calcium alginate residues may still be present. The next step in the process is the "extraction step", which involves converting the insoluble alginate and / or alginic acid present in the macroalgae into a soluble form (e.g., a soluble sodium form) and, optionally, recovering it.

[0065] Extraction of alginate involves the conversion of insoluble alginate and / or alginic acid present in the macroalgae to a soluble sodium (or potassium) form, which is extracted into solution for recovery. Methods for extracting alginate from macroalgae are well known in the art, and any known method can be used to obtain the desired alginate after the pretreatment process described herein.

[0066] After extraction, the solubilized alginate may be separated from the remaining solid components of the macroalgae (e.g., by filtration or centrifugation) and further processed to recover the alginate, e.g., sodium alginate, potassium alginate, or ammonium alginate, e.g., in dry powder form.

[0067] The step of extracting alginate from macroalgae or a part thereof may include contacting the macroalgae or a part thereof with an alkaline solution, i.e., this step is an alkaline extraction process. As used herein, the term "extraction" is intended to refer to a process involving solubilization of alginate present in the macroalgae or a part thereof in an insoluble form, typically as calcium alginate. After extraction, some or all of the resulting solution containing solubilized alginate may be separated (e.g., filtered) from the remaining solid components of the macroalgae.

[0068] Typically, the alkaline solution used for extraction may be selected from sodium hydroxide, potassium hydroxide, ammonium hydroxide, and sodium carbonate. Conveniently, it may contain carbonate ions, for example a sodium carbonate solution. For example, the step of extracting alginate from macroalgae or parts thereof includes the use of sodium carbonate and / or sodium hydroxide, preferably sodium carbonate (for example a saturated sodium carbonate solution). The alkaline solution, for example sodium carbonate, may be used at any suitable concentration. It is preferably used at a low concentration. For example, it may be used at a concentration of 0.05-4%, preferably 0.1-1%, or 0.1-0.5%, for example about 0.25%. Conventional industrial processes for the production of alginate use high concentrations of highly caustic substances to achieve the required molecular weight reduction of native alginate and the desired pH level (for example in the region of pH 11 or 12) for its extraction. The use of 4% sodium carbonate in combination with sodium hydroxide is typical, resulting in high CO2 emissions when neutralizing sodium carbonate in the alginate recovery process. The ability to use much lower concentrations of sodium carbonate in the method of the present invention results in a significant reduction in CO2 emissions. For example, it is estimated that using sodium carbonate at a concentration of 0.25% or less results in at least 11 times less CO2 per ton of alginate produced. As also described herein, further reductions in CO2 emissions of up to 40 times can be achieved by effectively titrating alginic acid with sodium carbonate solution.

[0069] In one embodiment, sodium hydroxide may be used instead of sodium carbonate in the alkaline extraction step. Advantageously, the use of sodium hydroxide results in zero CO2 emissions for this step of the process.

[0070] The contact with the alkaline solution may include soaking the macroalgae or a part thereof in the alkaline solution or may include mixing, e.g., high shear mixing, with the alkaline solution. The soaking or mixing may be carried out for a period of about 2 minutes to about 24 hours, e.g., 30 minutes to 24 hours, e.g., 30 to 45 minutes. During the contact, the pH should be maintained within the range of about 7 to 9, preferably about 7 to about 8.5, more preferably about 7 to about 8, e.g., about 7 to about 7.5. The pH may be maintained, for example, at 7, 7.1, 7.2, 7.3, 7.4, or 7.5. If necessary, additional alkali may be added as needed. The reaction temperature and reaction time may be easily varied. For example, the reaction temperature may be in the range of 10 to 80° C., preferably 20 to 60° C., e.g., 20 to 30° C., or 40 to 60° C. Preferably, the alkaline extraction will be carried out at ambient temperature, i.e., without additional heating.

[0071] In one embodiment, the alkaline solution is gradually added to the acidified macroalgae or part thereof until the pH increases and stabilizes within the desired pH range, for example, about 7 to about 9.5, preferably about 7 to about 9, more preferably about 7 to about 8.5, even more preferably about 7 to about 8, for example about 7 to about 7.5. For example, the alkaline solution may be added until the pH increases to 7, 7.1, 7.2, 7.3, 7.4, or 7.5. Effective control of the pH during this step may be achieved by gradually adding the alkaline solution while simultaneously monitoring the pH, for example, with a pH meter. In this way, the amount of alkaline solution added is effectively balanced with the amount of alginic acid present, and ideally does not exceed the amount strictly necessary to achieve conversion of the insoluble alginate to a soluble form (e.g., sodium form). For example, the final concentration of the alkali (e.g., sodium carbonate) may be about 0.1%. Stabilization of the pH of the material is indicative of the conversion of the alginate to a soluble form (e.g., sodium alginate). Minimizing the pH reduces the extent of hydrolysis of the alginate chains.

[0072] The step of extracting alginate from macroalgae or parts thereof may further comprise a step of separating the solubilized alginate from the residual solids. The separation of the solubilized alginate from the residual solids can be carried out by known methods, for example by dilution with water (if necessary) and filtration, for example by centrifugation. These separation steps can be repeated as necessary. Any solids removed can be used, for example, in cellulose production.

[0073] If desired, alginate may be recovered from any separated sodium (or potassium or ammonium) alginate solution using conventional methods, such as the well-known "alginic acid" or "calcium alginate" methods described herein. Such methods are well-known and described in the prior art, for example, McHugh (Dennis J. McHugh - Chapter 5 (Alginate) in "A guide to the seaweed industry", FAO Fisheries Technical Paper 441, Food and Agriculture Organisation of the United Nations, 2003), the entire contents of which are incorporated herein by reference.

[0074] In the alginic acid process, the pH of the solution is adjusted by contacting it with a mineral acid, such as hydrochloric acid and / or sulfuric acid, to form an alginic acid precipitate. The acid may be used in an amount and concentration sufficient to lower the pH of the solution to about 2 or less, preferably 1.7 to 1.9, thereby forming an alginic acid precipitate. Preferably, hydrochloric acid is used. The alginic acid precipitate is recovered in the form of a gel, for example by centrifugation. The resulting gel may optionally be rinsed with water to remove excess acid and the pH may be raised to provide a solution having a pH of about 3.5 to about 4.0. If desired, the alginic acid gel may then be converted to sodium alginate by the addition of an alkali containing sodium ions, for example by the addition of a sodium carbonate solution. The addition should be done under continuous stirring. The amount and concentration of the sodium carbonate solution may be readily adjusted, but is typically sufficient to adjust the pH of the solution to 7.0 to 7.3. Alternatively, other soluble alginates may be prepared using appropriate counterions. For example, potassium alginate may be prepared using an alkali containing potassium ions.

[0075] To recover the desired alginate product, the resulting solution may be contacted with an anti-solvent, such as an alcohol or mixture of alcohols, or acetone. Suitable alcohols include, for example, propan-2-ol and ethanol. This displaces the sodium alginate from the solution as a thick gel or precipitate. The precipitate may then be removed from the solvent mixture, for example by centrifugation. The anti-solvent may be recovered and reused, improving the efficiency of the process. The resulting alginate may then be dried, for example in a vacuum oven, at a temperature of, for example, up to 100°C, for example up to 95°C, for example up to 85°C, for example up to 50°C, for example up to 30°C, preferably 30°C.

[0076] In the "calcium alginate" method, calcium chloride is added to precipitate calcium alginate or form a gel, which may then be recovered. The pH of the precipitate or gel is then reduced to less than about 2.3 using a mineral acid, such as hydrochloric acid and / or sulfuric acid. The resulting alginate precipitate or gel is recovered, for example, by centrifugation. It may optionally be rinsed with water to remove excess acid and the pH increased to provide a solution having a pH of about 3 to about 4. If desired, the alginate material may then be converted to sodium alginate by the addition of an alkali containing sodium ions, for example, by the addition of a sodium carbonate solution. The addition may be performed with stirring. The amount and concentration of the sodium carbonate solution may be readily adjusted, but is typically sufficient to adjust the pH of the solution to 7.0 to 7.3. Alternatively, other soluble alginates may be prepared using appropriate counterions. For example, potassium alginate may be prepared using an alkali containing potassium ions. To recover the desired alginate product, the resulting solution may be contacted with an anti-solvent, such as an alcohol or mixture of alcohols, or acetone, as described above for the "alginic acid" process.

[0077] A particular embodiment of the method of the present invention is described with reference to FIG. 1, which uses citric acid as the selected organic acid. In FIG. 1, the process involves obtaining macroalgae (Laminaria hyperborea) having stalks and leaves, and removing the non-stalk portions to provide macroalgae portions consisting only of stalks. The non-stalk portions are removed by manual or automatic cutting, for example using a cutting machine commonly used in the art. The stalks are then chopped and dried, for example by air drying or fluidized bed drying. Prior to the pre-treatment stage of the process, the dried stalks are rehydrated in potable water for 2 hours. Salts and other undesirable water-soluble components such as polyphenols are extracted during the rehydration process. The rehydrated stalks are then separated from the remaining water. At this point, the rehydrated stalks are ready for the pre-treatment step with organic acids.

[0078] As described herein, the method of the present invention can be easily adapted to tailor the final properties of the alginate product. This is illustrated in FIG. 1 with various modifications intended to produce alginates with the desired "high", "medium", "low" or "ultra-low" viscosity. For example, a "high" viscosity may be greater than 800 cp, a "medium" viscosity may be in the range of 400-800 cp, a low viscosity may be in the range of 50-400 cp, and an ultra-low viscosity may be in the range of 5-50 cp. If a "high" viscosity alginate is desired, the citric acid pretreatment may be carried out for 60 minutes under ambient conditions. If an alginate with a "medium" viscosity is desired, the citric acid pretreatment may be carried out at a higher temperature, i.e., 60° C., for 5-10 minutes, whereas if an alginate with a "low" viscosity is desired, the period of citric acid pretreatment at 60° C. may be extended to 30-40 minutes. If a very low viscosity alginate is desired, i.e., one in which the alginate has been degraded to form oligosaccharides, an additional pretreatment step may be performed. Prior to the citric acid pretreatment, the alginate is treated with calcium ions to bind the G-blocks and protect them from degradation. This is followed by a citric acid treatment at 95°C for at least 20 minutes.

[0079] The remainder of the process shown in Figure 1 is common to each of the different target viscosities. In each case, the solids are rinsed with deionized water to remove excess citric acid, and the undissolved solids are separated by filtration. Mineral acid (e.g., hydrochloric acid or sulfuric acid) is then added to the undissolved solids to adjust the pH to 1.7-1.9, thereby mobilizing calcium ions bound to alginates in the macroalgae matrix. The mineral acid treatment is carried out for approximately 15 minutes. The resulting mixture is then drained, and the solid residue is rinsed with deionized water to remove excess mineral acid.

[0080] The mineral acid treated sample is then extracted using a saturated sodium carbonate solution. This solution is slowly added to the solid material and the pH is maintained at 7-7.5 for 45-60 minutes with stirring. During this process, the alginic acid is neutralized by the sodium carbonate. This produces soluble sodium alginate, which can be extracted into solution and recovered. The solid and liquid components of the mixture are then separated. Optionally, the solid component can be used in cellulose production.

[0081] The liquid component is treated to recover sodium alginate in powder form. Specifically, it is contacted with a mineral acid (e.g., hydrochloric acid or sulfuric acid) to lower the pH to 1.7-1.9. This converts the sodium alginate back to alginic acid, which is insoluble and precipitates as a thick alginic acid gel, which is recovered from the solution by filtration. The gel is rinsed to remove excess acid and then converted to sodium alginate by adding saturated sodium carbonate solution until the pH reaches 7.0. Addition of propan-2-ol precipitates sodium alginate, which is recovered as a solid by filtration. Drying at 30°C under vacuum gives the desired sodium alginate product in the form of a white powder.

[0082] Thus, the method of the present invention provides a process in which the viscosity of the alginate can be easily adjusted by varying the exact conditions of the citric acid pretreatment step. All downstream processing steps remain the same regardless of the initial citric acid treatment stage. This allows the same downstream processing equipment to be used, which is advantageous in an industrial environment. Although the particular embodiment of the method shown in Figure 1 is described in relation to the use of Laminaria hyperborea and citric acid, it will be understood that other macroalgae and other weak organic acids, such as any of those described herein, can be used in the method according to the present invention.

[0083] In one or more embodiments, the method of the present invention provides an improvement over conventional industrial methods used in the production of alginate from macroalgae. Such improvements include, but are not limited to, the yield of alginate, the quality, purity, and properties of the alginate produced by the method, and the sustainability of the process. Advantageously, the method provides the ability to fine-tune the properties of the extracted alginate material, such as its molecular weight, its M / G ratio, etc., as needed.

[0084] As shown herein, the use of organic acids is effective in breaking down undesirable pigments (e.g., polyphenols) in macroalgae, including those present in the outer layer or "bark" of the stalk. Thus, it aids in their removal, thus providing a lighter colored alginate product. Thus, at least in certain embodiments, the present invention provides an alternative method for addressing the problem of undesirable color in extracted alginate. Importantly, it avoids the need to chemically or mechanically remove the bark from the stalk prior to processing. This reduces the amount of wasted material and simplifies the manufacturing process when implemented on an industrial scale. Furthermore, it avoids the need to use known color fixing agents (e.g., formaldehyde and formaldehyde derivatives) and / or chemical bleaching agents such as hypochlorite to address color issues. This produces an alginate material that does not require additional bleaching after production, and a residual cellulose-containing residue that is free of toxic chemicals such as formaldehyde.

[0085] Thus, in one set of embodiments, the methods of the invention do not include a step of treating the macroalgae, or a portion thereof, with formaldehyde or a formaldehyde derivative.

[0086] In another set of embodiments, the method of the invention does not include treating the macroalgae or a portion thereof with a bleaching agent. In a further set of embodiments, the method does not include bleaching the recovered alginate material. In one set of embodiments, the method of the invention does not include a bleaching step, i.e., the method does not include the use of a bleaching agent. For example, the method does not include contacting any of the macroalgae or a portion thereof, any of the intermediate products produced during the method, and the recovered alginate with a bleaching agent. As used herein, the term "bleaching agent" refers to a chemical agent that can lighten or whiten a substrate through a chemical reaction. Typically, a bleaching agent is one that participates in a bleaching reaction that involves an oxidation or reduction process that breaks down colored pigments. Examples of bleaching agents include compounds that contain or act as a source of peroxides or peroxyacids, such as hydrogen peroxide, peroxide salts, peroxyacids, hydroperoxides, carbonates, percarbonates, 6-(phthalimido)peroxyhexanoic acid (PAP), peracetic acid; oxidation catalysts, such as mononuclear or binuclear transition metal catalysts (e.g., manganese) (e.g., the oxidation catalyst is [(Mn IV )2(uO)3(Me3-TACN)2] 2+ , [(Mn III )2(uO)(u-CH3COO)2(Me3-TACN2] 2+ , and [Mn III Mn IV (uO)2(u-CH3COO)(Me4-DTNE)] 2+ and their suitable salts; peroxide activators (i.e., compounds that react with a source of peroxide groups to provide peroxide groups), such as tetraacetylethylenediamine (TAED); peroxyacid activators (i.e., compounds that react with a source of peroxyacid to provide peroxyacid groups), such as tetraacetylethylenediamine (TAED); hypochlorite; compounds that contain chlorite or act as a source of chlorite; chlorine dioxide; chlorite; and chlorine. Exemplary bleaching agents include hydrogen peroxide, peroxyacids, persulfates, organic peroxides, and hypochlorites.

[0087] Although the methods described herein advantageously provide an alginate material that is light in appearance, it will be understood that the desired color of the final alginate material will ultimately be determined by its end use. In certain applications, it may be desirable to bleach the final alginate material. However, whatever bleaching agent is used, it may be used in low concentrations.

[0088] As demonstrated herein, the method of the present invention unexpectedly provides an increased yield of alginate. As used herein, the term "increased yield" refers to an increased production of alginate from the treatment of macroalgae. It may be assumed that the increased yield may be due to hydrolysis of alginate chains by organic acids (resulting in more alginate chains being extracted), but the evidence presented herein does not support this assumption. Contrary to expectations, the viscosity of the alginate (indicative of its molecular weight) does not decrease at the expense of the increased yield. Thus, treatment with organic acids not only allows for the recovery of a higher alginate yield, but also, unexpectedly, preserves the molecular weight (i.e., chain length) of the alginate, resulting in a higher viscosity when dissolved in water. Typical yields when carrying out the method of the present invention may be greater than 25%, preferably greater than 30% (on a dry weight basis). In some cases, the yield may be increased by more than 40% (on a dry weight basis).

[0089] As a direct result of the methods used in their preparation, the alginate materials described herein differ from those produced using conventional industrial processes. Thus, in another aspect, the present invention provides novel alginate materials, i.e., alginates or alginate derivatives, obtainable, obtained or directly obtained by the methods described herein.

[0090] The color of the resulting alginate depends on the nature of the starting material, i.e., the part or parts of the macroalgae used in its production. For example, leaves are known to contain a higher percentage of pigment than stalks and may produce a white to off-white product, compared to those from stalks, which may be "bone white". However, alginate products from leaves are sufficiently colorless that they do not require additional bleaching to be useful as a product.

[0091] As demonstrated herein, regardless of the input material selected, the extracted alginate material produced according to the method of the present invention is therefore "light" in color. The method of the present invention allows for the extraction of high quality, clean (i.e., decolorized) alginate material from any starting material, even from stems, including bark and epiphytes. In at least certain embodiments, the color of the produced alginate material can be described as "off-white," "white," or "bone white." Due to its reduced color (i.e., reduced content of pigments), the alginate material is particularly suitable for use in applications requiring low concentrations of color. Also, the absence of toxic chemicals used in its production makes it particularly suitable for pharmaceutical and food applications that cannot tolerate even trace amounts of chemicals traditionally used to address color issues.

[0092] In certain embodiments, the alginate material is substantially free of any pigments, such as polyphenols. For example, it may contain less than about 2% by weight, preferably less than about 1% by weight, such as less than about 0.5% by weight, or less than about 0.3% by weight of any pigments. In particular, it may contain less than about 2% by weight, preferably less than about 1% by weight, such as less than about 0.5% by weight, or less than about 0.3% by weight of any polyphenols. As will be appreciated, the traditional use of chemical bleaching agents to address color issues does not necessarily remove contaminants, but may reduce their color by converting them into other components with different light absorption and / or reflection properties. Although bleached alginate materials are not colored, they may still contain contaminants resulting from the original pigments.

[0093] In certain aspects, the extracted alginate material has a reduced content of any residual formaldehyde or any derivative of formaldehyde, such as glutaraldehyde, compared to that produced using conventional industrial processes.Thus, in one set of embodiments, the alginate material is substantially free of any derivative of formaldehyde, such as formaldehyde or glutaraldehyde.For example, it may have a residual content of formaldehyde or any derivative of formaldehyde that is less than about 2% by weight, preferably less than about 1% by weight, such as less than about 0.5% by weight, or less than about 0.3% by weight.Most preferably, the content of formaldehyde or any derivative thereof is below the detection limit, i.e., undetectable.

[0094] In certain aspects, the alginate material has a reduced content of any residual chemical bleaching agent, as described herein. In one set of embodiments, the alginate material is substantially free of any chemical bleaching agent, as defined herein, such as hypochlorite bleaching agent. For example, it may have a residual content of chemical bleaching agent that is less than about 2% by weight, preferably less than about 1% by weight, such as less than about 0.5% by weight, or less than about 0.3% by weight. Most preferably, the content of any chemical bleaching agent is below the detection limit, i.e., undetectable.

[0095] The alginate produced according to the method of the present invention is characterized by its molecular weight, polydispersity index, viscosity (i.e. the resulting viscosity of the solution in which it is dissolved), its M and G content, its M / G ratio, and its gelling properties (i.e. Ca 2+ The polymer may be further characterized in terms of its ability to form a gel upon contact with ions.

[0096] Unless otherwise specified, as used herein, "molecular weight" refers to weight average molecular weight (Mw). Weight average molecular weight is the sum of the products of the molecular weight of any polymer fraction multiplied by its weight fraction. Molecular weight may be measured, for example, by size exclusion chromatography with multi-angle static light scattering (SEC-MALS) using a mobile phase of Na3PO4+EDTA for the sample. A calibration curve for determining molecular weight may be made using pullulan molecular weight standards. SEC-MALS analysis may provide the weight average molecular weight (Mw) and polydispersity index (PDI). Molecular weight (Mw) may be determined, for example, according to the procedure in the Examples shown herein. The molecular weight of the alginate may be adjusted by varying the parameters of the pretreatment with organic acids described herein. In this way, the molecular weight may be adjusted according to the desired use of the material. The molecular weight of the alginate may range from about 30 to about 650 kDa, for example from about 40 to about 500 kDa, or from about 50 to about 400 kDa, for example from about 60 to about 350 kDa. Due to the mild conditions used in certain embodiments of the method of the present invention, the molecular weight of the alginate can be higher than that obtained by the current industry standard method.The molecular weight of the alginate can be, for example, at least 300 kDa.For example, it can be at least 310 kDa, preferably at least 320 kDa, more preferably at least 330 kDa, at least 340 kDa, at least 400 kDa, at least 450 kDa, at least 500 kDa, at least 550 kDa, or at least 600 kDa.By adjusting the exact conditions used in the organic acid pretreatment step as described herein, for example, when using a higher treatment temperature and / or a longer treatment time, an alginate with a lower molecular weight can be obtained as desired.

[0097] As referred to herein, the polydispersity index (PDI) of a polymer is calculated by dividing the weight average molecular weight of the polymer by its number average molecular weight. The number average molecular weight may be measured, for example, using SEC-MALS, as described herein. The closer the polydispersity index is to 1.0, the more uniform the molecular weight range of the polymer is. The polydispersity index of the alginate may be in the range of 1.2 to 3.5, such as 1.2 to 2.7, 1.2 to 2.6, 1.2 to 2.3, or 1.2 to 2.0, such as 1.3 to 1.9. In a preferred embodiment, the polydispersity index is low, such as in the range of 1.2 to 2.0, such as 1.2 to 1.8, such as 1.2 to 1.5, for example about 1.4. The ability to produce alginates with low PDI is advantageous, as its uniformity allows for greater flexibility in any downstream processes that may be used to further adjust its Mw according to the desired end use.

[0098] The α-L-guluronate (G) content of the alginate may be determined using methods known in the art, such as 1H-NMR. For example, it may be measured using the method of Grasdalen et al., as described in the Examples. The α-L-guluronate (G) content of the alginate obtained by the methods described herein may range from about 55-80%, such as about 60-80%, or 65-75%. In certain embodiments, the α-L-guluronate (G) content is greater than 70%, such as greater than 75%.

[0099] The present invention also relates to products containing the alginates and alginate materials described herein, including food products, pharmaceutical products, agricultural chemical products, health care products, biomedical products, cosmetics, textile products, paper products, and cardboard products. [Brief description of the drawings]

[0100] The invention will now be described in more detail in the following non-limiting examples and with reference to the accompanying figures. [Figure 1] FIG. 2 is a schematic diagram illustrating a method according to an embodiment of the present invention. [Diagram 2] FIG. 13 shows images of whole stalks treated in 1% (w / v) citric acid. [Diagram 3] FIG. 13 is an image showing peeled stalks treated in 1% (w / v) citric acid. [Figure 4] 13A-C are images comparing whole stalks treated in 1% (w / v) citric acid and 2% formaldehyde solutions. [Diagram 5] 1 is an image showing an alginate sample produced after pretreatment with citric acid and after treatment with formaldehyde according to the present invention. [Figure 6] 1 is an image showing an alginate sample produced after pretreatment with citric acid according to the present invention. [Figure 7] FIG. 1 shows the effect of duration of exposure to citric acid on alginate solution viscosity using citric acid solutions having concentrations of 0.10%, 0.25%, 0.50%, and 1.00% (w / v). [Figure 8] FIG. 1 shows the effect of increasing exposure time to citric acid at 60° C. on alginate solution viscosity. [Figure 9] FIG. 1 shows the effect of duration of exposure to citric acid on alginate solution viscosity using a citric acid solution having a concentration of 10% (w / v). EXAMPLES

[0101] General procedure: Preparation of starting materials: Preparation of dried, flaky Laminaria hyperborea: leaves and epiphytes were removed but the skins were left on before being chopped and dried to produce a stable intermediate (dried stalks), which were rehydrated by addition of water before carrying out the pretreatment process. Rehydration and removal of residual water by water washing helped to extract salts and other undesirable water-soluble components, including polyphenols, from the stalk matrix.

[0102] Preparation of freshly chopped Laminaria hyperborea stems: Leaves and epiphytes were removed and the skinned stems were immersed in demineralized water to remove salts such that the conductivity of the solution was less than 200 μS. The soaked stems were then blended using a Tefal Blendforce II, type BL42 blender with a 600 W motor at the highest power setting.

[0103] For both dry and fresh material, the particle size of the stalks was approximately in the range of 500-1000 μm.

[0104] Pretreatment with organic acids: The rehydrated stems prepared as above were added to a blender. A 1% w / v solution of the selected organic acid in demineralized water was prepared and enough organic acid solution was added to the reaction vessel to cover the rehydrated stems in the blender. The resulting reaction mixture was then blended using two 5 second blending pulses or one 10 second blending pulse. After blending, the resulting mixture was transferred to a container. An additional aliquot of the 1% w / v organic acid solution was used to rinse the blender and the rinse solution was also transferred to the container. The contents of the container were then allowed to soak for 60 minutes with agitation.

[0105] Metal cation exchange: The liquid was separated from the undissolved solids by filtrate or centrifugation. The liquid phase from the filtrate or centrifugation, containing the organic acid solution, was collected. The sample in the filter or centrifuge was then rinsed with demineralized water to remove excess organic acid, the sample was returned to the container, and more water was added to rinse. The liquid was again separated from the undissolved solids by filtration. The sample was then transferred to a blender and hydrochloric acid was added to the reaction mixture to lower the overall pH to 1.7-1.9. The acidified sample was then blended using two 5 second blending pulses or one 10 second blending pulse, and the sample was allowed to stand with or without agitation. The sample was then drained through a filter or transferred to a centrifuge, and the resulting solid fraction was transferred to a container and rinsed with demineralized water to remove excess hydrochloric acid. The filtrate or liquid phase from the centrifugation, which is the hydrochloric acid solution, was collected.

[0106] Extraction of alginate; The mineral acid treated sample was then drained through a filter or separated using a centrifuge and the solids transferred to a blender. Saturated sodium carbonate solution was added to the blender and the resulting mixture was blended using two 5 second blending pulses or one 10 second blending pulse. The pH of the blended mixture was approximately 9, but rapidly decreased as the carbonate reacted with the alginic acid. The blended mixture was then transferred to a reaction vessel and more saturated sodium carbonate solution was added with stirring over a period of 30-45 minutes to maintain the pH of the solution at approximately 7.0-7.2. During this process, the alginic acid is neutralized by the sodium carbonate. This produces soluble sodium alginate, which can be extracted into solution and recovered.

[0107] After stirring for 30-45 minutes, the solid particles were removed from the solution by filtration or centrifugation to obtain the primary extract. Once all the liquid had been collected, the solids on the filter were transferred to a beaker and mixed with demineralized water. This mixture was allowed to stand or mix for 10 minutes, during which time most of the remaining alginate was extracted, and then filtered to obtain the secondary extract. The primary and secondary extracts were then combined to form the alginate solution.

[0108] Alginate Recovery ("Alginate Pathway"): Hydrochloric acid was added to the alginate solution with mixing to lower the pH to 1.7-1.9. This converts the sodium alginate to alginic acid, which is insoluble and precipitates as a thick, clear gel. The solution was then filtered and the gel was either retained on the filter or the solution was transferred to a centrifuge and the gel was collected. To convert the gel to sodium alginate, it was transferred to a reaction vessel and saturated sodium carbonate solution was added slowly with stirring until the pH reached 7.0-7.3.

[0109] An equal volume (1:1 ratio) of propan-2-ol was added to the solution of sodium alginate. The solution was then mixed, which displaced the sodium alginate from the solution as a thick gel. The resulting mixture was then transferred to a blender and pulsed for 5 seconds to disperse the gel and complete the precipitation process. The mixture was then filtered or transferred to a centrifuge to recover the product, and a rotary evaporator was used to recover the propan-2-ol.

[0110] To remove the remaining water from the resulting gel-like product, the sample was returned to the blender and an additional portion of propan-2-ol was added. The resulting mixture was blended for 5 seconds and then filtered or transferred to a centrifuge. The sodium alginate was obtained as a pellet on the filter or in the centrifuge and the propan-2-ol was recovered. The alginate pellets were then dried in a vacuum oven at 30°C. The evaporated propan-2-ol was directly condensed and recovered.

[0111] Example 1: Pretreatment with organic acids To produce alginate, dried stems of Laminaria hyperborea were rehydrated and subjected to pretreatment and subsequent extraction with organic acids as described in the general procedure above. Readily available food-grade acids (ascorbic acid, lactic acid, citric acid, malic acid, and acetic acid) were tested in the pretreatment step.

[0112] Alginate yields were determined. The viscosity of a 1 wt% solution of the resulting alginate was measured using a falling ball viscometer method. The results are shown in Table 1 below, along with the molecular weights and pKa values ​​of the organic acids tested. For comparison, the pKa of alginic acid ("alginate") is in the range of 1.5 to 3.5, and the pKa of hydrochloric acid is -5.9.

[0113] [Table 1]

[0114] Pretreatment with each organic acid resulted in an increase in the yield and viscosity of the solution of alginate produced. The high viscosity indicates that the alginate was not appreciably degraded in the alginate extraction process. Citric and malic acids were the most effective with respect to the highest yield and viscosity of the alginate produced. Without being bound by theory, the reason why citric and malic acids are more effective than other organic acids may be due to their primary pKa values, both of which are close to the upper limit of alginic acid (pKa=3.5). This may allow selective alginate chain scission without significant degradation as seen with treatment with high strength acids or alkalis.

[0115] The alginate obtained without pretreatment with organic acids was light brown in color, whereas all alginate samples produced after pretreatment with organic acids were white in color.

[0116] Example 2: Treatment with Citric Acid vs. Formaldehyde Whole (i.e., unpeeled) and peeled stalks were treated in 1% (w / v) citric acid solution for 7 days and the colour was observed. For comparison, whole stalk samples were also treated with 2% formaldehyde, in accordance with current industry standards.

[0117] Citric acid removed the brown pigmentation from the entire stalk (including the bark) leaving behind traces of chlorophyll which are green in color. The images in Figure 2 show the color reduction for whole stalks treated with citric acid solution. As can be seen in the images, over time the original brown pigmentation of the stalks deteriorates and the green color of remaining chlorophyll residues is visible. Similar results were observed when treating stalk samples blended with 1% (w / v) citric acid versus simply immersed in water.

[0118] When the bark was removed, the stalks still had a light brown color, but when treated with 1% (w / v) citric acid, the color quickly disappeared and the stalks turned white. The images in Figure 3 show the loss of color for peeled stalks when treated with citric acid solution.

[0119] When the entire stalk is examined, the color change is slow due to the density of the stalk structure, but when the size of the stalk is reduced (e.g., by chopping, grinding, etc.), the color change is rapid.

[0120] In comparison, samples treated with 2% formaldehyde had a very different response. They darkened and became more brown over time. This is believed to be the result of polymerization of phenolic species present, which have no color in the unpolymerized form. The images in Figure 4 show stalks after 7 days of treatment with 1% (w / v) citric acid and 2% formaldehyde solution.

[0121] Alginate was extracted from the citric acid and formaldehyde treated stem samples using the extraction method described in the general procedure above. Images of the resulting alginate are shown in Figure 5. The formaldehyde treated sample produced a brown alginate material that required bleaching to achieve the same color as that produced from the citric acid pretreated stems. In contrast to current industrial processes, the citric acid pretreatment method of the present invention produces clean, substantially colorless alginate without the use of formaldehyde.

[0122] Example 3: Pretreatment with Citric Acid To produce alginate material, dried stems of Laminaria hyperborea were rehydrated and subjected to pretreatment with citric acid and subsequent extraction as described in the general procedure above.

[0123] The stalk powder was rehydrated using demineralized water. This also helped to remove undesirable soluble components such as polyphenols that were extracted into the water. The deep orange color of the wash water at this stage indicates the presence of oxidized polyphenols. The molecular weight of the polyphenols is not significantly affected, so they remain water soluble and a high percentage of the polyphenols are removed. Subsequent treatment with citric acid solution at room temperature (1% w / v for 60 minutes) extracts the remainder of the free polyphenols and breaks down the carotenoid and chlorophyll residues, which are then washed away when the acid solution is drained. This further reduces the concentration of color-contributing materials present in the starting matrix while reducing the molecular weight of the native alginates improving solubilization and extraction efficiency. The citric acid treated material is further rinsed to remove remaining contaminants before being sent to the rest of the extraction process.

[0124] Standard industrial processes treat macroalgae with formaldehyde after harvesting to prevent microbial degradation of the macroalgae and sequester the polyphenols by polymerization. Formaldehyde complexes the polyphenols, thereby increasing their molecular weight and rendering them insoluble and unable to impart color to the alginate. In contrast to the method according to the invention, in which the polyphenols are removed prior to alginate extraction, the polymerized polyphenols produced as a result of the formaldehyde treatment are carried forward to the further extraction stage.

[0125] method: The rehydrated stalk powder was added to a blender with 250 mL of 1% w / v citric acid (anhydrous) and blended for two 5-second cycles. The blended sample was transferred to a container and held at room temperature with agitation for 60 minutes. The particles became lighter in color as colored compounds were destroyed or removed. After 60 minutes, the mixture was drained through a 160 mesh nylon bowl filter (approximately 100 μm) to retain the solids. The solids were then transferred to a blender with 300 mL of demineralized water and 10 mL of 10% HCl and blended for two 5-second cycles to obtain a pH of 1.7-1.9.

[0126] The mixture was then transferred to another container and held for 15 minutes with agitation. After 15 minutes, the mixture was drained through a 160 mesh filter and pressured to remove as much liquid as possible. The solids were then transferred to a jug with 300 mL of water to remove excess residual acid. After 15 minutes, the mixture was drained through a 160 mesh filter and pressured to remove as much liquid as possible, then the solids were transferred to a blender with 500 mL of 0.25% w / v sodium carbonate solution and blended two times for 5 seconds. The resulting mixture was transferred to a container and rinsed with an additional 0.25% w / v sodium carbonate solution. The volume was brought to 700 mL. The pH was checked and adjusted to 8-8.5 with saturated sodium carbonate solution as needed, then the solution was held for 30 minutes with agitation. The solution was transferred to a blender and blended two times for 5 seconds and then held for an additional 30 minutes. After a total extraction time of 60 minutes, the highly viscous solution was filtered through a 160 mesh nylon filter, retaining the solids. The solids were then re-extracted with an additional 400 mL of water, the pH was adjusted to 8-8.5 by the addition of saturated sodium carbonate solution and held for 15 minutes, and the solids were then filtered through a 160 mesh nylon filter and all the liquid was collected.

[0127] The resulting extract was then acidified to pH 1.7-1.9 with hydrochloric acid while gently stirring to avoid dissociating the alginate gel. After 10 minutes of gel formation, the gel was filtered through a 200 mesh nylon filter to recover the alginate gel. Excess water was allowed to drain from the gel before transferring it to a beaker. The pH of the gel was then adjusted to 7.1-7.4 using saturated sodium carbonate solution to ensure complete deprotonation by replacing the protons in the alginate with sodium ions. The buffered gel was then added to a blender along with an equal volume of methanol, ethanol, or propan-2-ol (depending on availability) and blended. This dissociated and dehydrated the gel, which was then filtered and squeezed to dry. The filtered and squeezed sodium alginate was then transferred to a coffee grinder and gently pulsed to break up the fibrous clumps before being transferred to a drying dish and dried in an oven at 95°C for 30-60 minutes (depending on sample size).

[0128] The experiments were performed in triplicate (Tests A, B, and C) and the results are shown in Table 2 below.

[0129] [Table 2]

[0130] The resulting alginate was white in color, despite the absence of any bleaching agents in the process. This was also achieved using a completely ambient process with no heat input. Furthermore, the process time was less than 4 hours from hydration of the stalk powder, and the high viscosity of the extraction solution indicates that a high molecular weight alginate was obtained.

[0131] Example 4: Analysis of molecular weight and α-L-guluronate (G) content of alginates produced using citric acid pretreatment Using the general procedure described above, samples of alginate were obtained from eight separate laboratory extractions. The recovered alginate was a "bone white" fibrous solid (see Figure 4). The average yield over the eight extractions was 36.8% (based on input dry matter content) and the alginate (1% solution) had an average viscosity of 6200 mPa·s (measured using the falling ball method).

[0132] The molecular weight of the alginate samples was determined by size exclusion chromatography (SEC-MALS), i.e., high performance liquid chromatography (HPLC) with online multi-angle static light scattering (MALS). Separation was performed using 1+3 columns by Shodex, in the following order: OHpak LB-G, LB-806, LB-805 and LB-804. The temperature of the columns was adjusted to 40°C using an Agilent 1260 Infinity II Multicolumn Thermostat, but measurements were performed at 25°C. Measurements were performed with a Dawn HELEOS-8+ multi-angle laser light scattering photometer (Wyatt, Santa Barbara, CA, USA) (λ0=660 nm) followed by an Optilab T-rEX differential refractometer. A mobile phase of 0.10 mol / L Na3PO4 (pH=7) + 0.01 mol / L EDTA was used. The flow rate was 0.5 mL / min. The injection volume was 50 μL and the alginate concentration was 3 g / L. Pullulan standard (GPC 50,000) from Sigma Aldrich was used for instrument standardization. Data were acquired and processed using Astra (v.7) software (Wyatt, Santa Barbara, CA, USA). Polydispersity (Mw / Mn) of the samples was calculated from the obtained Mw (mass weighted molecular weight) and Mn (number weighted molecular weight) measurements.

[0133] The α-L-guluronate (G) content was determined by 1H-NMR. 1H-NMR analysis was performed on a Bruker BioSpin 500 WB at 500 MHz and 368 K (95.035 °C). The solvent was DO and the alginate concentration was 30 g / L. The M / G ratio, and therefore the G content, was determined using the method described by Grasdalen et al., 13C NMR Studies of Monomeric Composition and Sequence in Alginate, Carbohydr. Res., 1981, 89, 179-191 and Grasdalen, High-field, 1H-NMR spectroscopy of alginate: sequential structure and linkage conformations, Carbohydr. Res., 1983, 118, 255-260.

[0134] Alginate produced by the citric acid method was found to have a molecular weight of 348.6 kDa and an α-L-guluronate (G) content of 67%. Typical parameters for alginate from Laminaria hyperborea are approximately 300 kDa and a "G" content of 65-70%. The molecular weight is higher than expected. The alginate has a polydispersity of 1.42, indicating that a narrow molecular weight fraction was extracted. This is in contrast to standard alginate production methods, which provide material with a broader molecular weight distribution.

[0135] Example 5: Effects of chronic exposure to citric acid Rehydrated Laminaria hyperborea stem material was exposed to 0.10, 0.25, 0.50, and 1.00 (% w / v) solutions of citric acid under ambient conditions for 7, 14, 21, and 28 days. Alginate was then extracted using the method described in the general procedure above, and its viscosity was measured using the falling ball method.

[0136] As can be seen from Figure 7, the viscosity of the extracted alginate decreases as a function of time when the stems are exposed to citric acid. The higher the concentration of citric acid used for pretreatment, the lower the viscosity of the alginate. Even though the resulting solutions were of low viscosity, they all showed a decrease in the viscosity of the Ca 2+ It was possible to produce a stable cohesive gel when exposed to calcium ions (calcium chloride solution). 2+ It has been demonstrated that there is a predominance of α-L-guluronate (G block), which can crosslink in the presence of ions.

[0137] The results demonstrate that prolonged exposure to concentrations as low as 0.1% (w / v) can reduce the viscosity of the subsequently extracted alginate, which is intrinsically related to its molecular weight. Thus, the molecular weight of the alginate can be adjusted as needed by varying the citric acid concentration and treatment time.

[0138] Example 6: Use of Citric Acid at High Temperatures Rehydrated Laminaria hyperborea stem material was exposed to a 1% (w / v) citric acid solution for 5-20 minutes at 60° C. Alginate was extracted and recovered as described in the general procedure above, and the viscosity of the resulting 1% alginate solution was measured.

[0139] The results in Figure 8 show a predictable rate of decrease in viscosity with increasing exposure time. From the measured viscosity, the rate of decrease in viscosity over time can be calculated according to the following formula:

[0140]

number

[0141] This relationship can be used to adjust the exposure time to the organic acid in order to adjust the viscosity (and therefore molecular weight) of the alginate as desired.

[0142] Increasing the temperature of the citric acid solution to 95 °C resulted in a much faster rate of viscosity decrease, Δviscosity = 598 mPa s min -1 It was calculated to give

[0143] Example 7: Treatment with calcium ions Since calcium ions are known to cross-link G-blocks in alginate complexes, it was speculated that pre-saturating the alginate in the stalk matrix might inhibit G-block degradation. To test this hypothesis, two samples were obtained from a single rehydrated batch of stalk material. One sample was pretreated with 5% calcium chloride solution prior to citric acid pretreatment and extraction of alginate, and the other was pretreated with citric acid only prior to alginate extraction. Citric acid pretreatment was performed with 1% (w / v) citric acid at 95°C for 10 min.

[0144] The alginate yield in the calcium ion-treated sample was found to be approximately 10% higher than that of the untreated one (45.7% vs. 36.0%), indicating that the alginate was protected from degradation by calcium ions. Both samples were also found to produce alginate that had the same resulting viscosity of 19 mPa sec and formed a gel when exposed to calcium ions.

[0145] Samples of alginate from each experiment were subjected to analysis as described in Example 4 using size exclusion chromatography to find the molecular weight and 1H-NMR to confirm the "G" content. The results are shown in Table 3.

[0146] [Table 3]

[0147] The addition of calcium ions as a pretreatment protects the alginate from degradation, resulting in a higher yield; however, in so doing, there are more "M blocks", which results in a lower overall "G" content when compared to the untreated sample (72% vs. 76% "G").

[0148] The use of citric acid at elevated temperatures has been shown to reduce alginate molecular weight and increase the α-L-guluronate (G) content in the resulting oligomers via the degradation of β-D-mannuronate (M). These conditions (longer duration or higher temperature) can be further applied, which can enhance the reduction in molecular weight of the oligomers and result in a further increase in the α-L-guluronate (G) content. They can also be used to increase the α-L-guluronate (G) content of the extracted leaf alginate (usually about 50 / 50 or about 45 / 55 (G / M)) to a content closer to that of the stem alginate (typically about 70 / 30 (G / M)).

[0149] Example 8: Comparison of organic acid pretreatments To evaluate the effectiveness of organic acid pretreatment, experiments were conducted using the general procedure outlined above, but with different pretreatments and different parts of seaweed. 1) Desalinated water 2) 1% Citric Acid 3) propan-2-ol, and 4) 2% formaldehyde.

[0150] The seaweed samples were as follows: (a) stalk + bark + epiphyte (i.e., "unpeeled stalk" with epiphyte); (b) stalk + bark (i.e., "unpeeled stalk" without epiphyte); (c) peeled stalk. "Unpeeled stalk" refers to stalks with bark and "peeled stalk" refers to stalks from which the bark has been removed. The seaweed samples were vacuum packed and chilled for transport.

[0151] (a) Extraction of alginate from stems + bark + epiphytes The results of the different pretreatments are shown in Table 4 below.

[0152] [Table 4]

[0153] As can be seen from Table 4, the alginate obtained from the samples subjected to citric acid pretreatment was obtained in substantially higher yield and with higher viscosity than the samples obtained using other industry standard pretreatments. It was also observed that citric acid pretreatment provided a lighter alginate than pretreatment using formaldehyde, which is typically used to reduce the color of extracted alginate.

[0154] (b) Extraction of alginate from stems + bark

[0155] [Table 5]

[0156] A similar trend was observed for samples from which epiphytes had been removed, but the yield and viscosity of alginate extracted using citric acid was higher. Samples treated with 2% formaldehyde provided relatively dark colored alginate material (this was especially evident when the material was dissolved for viscosity testing), while the remaining samples provided colorless or light colored alginate material.

[0157] (c) Extraction of alginate from peeled stalks.

[0158] [Table 6]

[0159] All samples produced colorless or light colored alginate materials.

[0160] Conclusion: Alginate recovered using citric acid pretreatment had consistently higher yields and viscosities compared to other pretreatment methods. The viscosity of citric acid treated samples of peeled stems was significantly lower than that from samples with the bark still present. This suggests that the oldest alginate (and therefore containing the highest proportion of "G" blocks) is closest to the bark layer and is likely lost when the stems are peeled.

[0161] These experiments demonstrate that pretreatment with citric acid allows the extraction of excellent quality alginate from both peeled and unpeeled stem parts, giving a clean product in all cases. It was also observed that the alginate with the strongest color in each case came from the material pretreated with 2% formaldehyde.

[0162] Example 9: Extraction of alginate from leaf powder Leaf powder was pretreated with 50% propan-2-ol. It was not hydrated with water to avoid the release of fucoidan, which would make the material difficult to handle due to the resulting viscosity. Alginate was extracted from the leaf powder samples as follows. A: Pretreatment with 50% propan-2-ol; extraction with mineral acids only. B: Pretreatment with 50% propan-2-ol containing 1% citric acid; extraction with mineral acids C: Pretreatment with 50% propan-2-ol containing 1% malic acid; extraction with mineral acids

[0163] Each sample was washed with 50% propan-2-ol (3 x 200 mL) and acid as required, then filtered and treated with 100% propan-2-ol as a final wash. This final wash removed all green coloration and the remaining solids turned light brown in color. Each sample was then extracted by treatment with hydrochloric acid at pH 1.8 for 15 minutes, followed by extraction with 0.25% sodium carbonate (700 mL, pH 8-8.5). The resulting products were dried overnight at 30°C and the yields were recorded. The results are provided in Table 7.

[0164] [Table 7]

[0165] Pretreatment with citric and malic acids provided a much higher yield of alginate, and the resulting alginate had a much higher viscosity than when propan-2-ol was used alone.

[0166] The resulting leaf alginate is white to off-white and does not require further bleaching to be useful as a product. In contrast, leaf alginate produced using formaldehyde is typically dark brown and requires significant bleaching before it can be used.

[0167] Example 10: Sodium Citrate Pretreatment Two samples of stalk powder were rehydrated as described above. Each sample was then treated with a solution containing 1 wt% sodium citrate and 1 wt% citric acid for 60 minutes. After pretreatment, the samples were drained, rinsed, and then treated with hydrochloric acid at pH 1.8 for 5 minutes. The samples were then drained and rinsed to remove excess acid before being extracted with water (700 mL) buffered to pH 7.5 with saturated sodium carbonate. The samples were then dried overnight and the alginate yield and viscosity were recorded. The results are provided in Table 8.

[0168] [Table 8]

[0169] Example 11: Calculating CO2 emissions compared to current industry standard processes The majority of CO2 emissions in the alginate process are directly attributable to the use of sodium carbonate (Na2CO3), and CO2 is emitted whenever acid is encountered. For example, when using either sulfuric or hydrochloric acid to neutralize the sodium carbonate, direct emissions result from the following reaction:

[0170] [ka]

[0171] The stoichiometric balance is the same in both reactions, and therefore the amount of CO2 produced by either acid is the same. For every kg of unreacted sodium carbonate, neutralization produces 0.415 kg of CO2, regardless of the acid used in the reaction.

[0172] When the stem material is reacted with mineral acids, the metal cations present in the alginate are exchanged for protons to form alginic acid ("alginate-H"). + In the next extraction step, this reacts with sodium carbonate to produce soluble sodium alginate, which proceeds according to the following reaction:

[0173] [ka]

[0174] The molecular weights cannot be used directly to calculate the stoichiometric balance, but the reaction is + and CO3 2- This can be simplified to CO3 reacting directly with alginic acid. 2- For every 1 kg, 0.365 kg of CO2 is produced. When the process according to the invention is operated at neutral pH, there are no additional dissolved solids since the "salt" is sodium alginate and there is no excess of sodium carbonate. However, this is not the case for the current industrial methods used to produce alginate, where there is a large excess of sodium carbonate in the extraction solution.

[0175] On a process basis (assuming no excess process chemicals), the CO2 emissions for the process according to the invention are calculated per 1000 kg of starting stem material with salt removed and are shown in Table 9 below.

[0176] [Table 9]

[0177] This indicates that processing 1000 kg of salt-depleted stalks will result in 118.6 kg of CO2 emissions, which translates to 296.6 kg of CO2 per tonne of alginate produced.

[0178] In certain embodiments of the present invention, a sodium carbonate concentration of 0.2-0.25% (depending on the residual concentration of mineral acid present after rinsing) in the extraction solution may be used. Comparing a concentration of 0.25% to the industry standard of using a minimum of 4% sodium carbonate, per 1000 L of extraction solution, the following can be calculated:

[0179] [Table 10]

[0180] This indicates that the industry standard method produces 16.6 times more CO2 than the method of the present invention.

[0181] Additionally, using sodium hydroxide instead of sodium carbonate results in a zero CO2 emissions profile for this part of the process.

[0182] Example 12: Effects of chronic exposure to citric acid - 10% w / v solution The experiment described in Example 5 was repeated using a 10% w / v solution of citric acid. The results are presented in Figure 9. As observed for the lower citric acid concentrations used in Example 5, the viscosity of the extracted alginate decreases as a function of the exposure time of the stems to citric acid. However, the decrease in viscosity is more rapid when the stems are treated with the higher 10.00% w / v citric acid solution than with the lower concentrations. Although the resulting alginate solutions were low in viscosity, they all contained Ca. 2+It was possible to produce stable cohesive gels when exposed to ions (calcium chloride solution), confirming the predominance of G-blocks in the extracted alginate.

[0183] These results demonstrate that by increasing the concentration of citric acid used in the pretreatment step, a more rapid, yet still predictable reduction in the viscosity (and therefore molecular weight) of the alginate can be obtained.

[0184] Example 13: Comparison of pretreatment with citric acid + mineral acid according to the present invention versus citric acid or mineral acid alone. Experiments were carried out to compare the effect of pretreatment according to the invention with pretreatment using either organic or mineral acids alone. Tests A to C were each carried out according to the general procedure described above. Test A was carried out in the absence of citric acid. Test B was carried out in the absence of hydrochloric acid. In test C, the citric acid treatment was followed by a hydrochloric acid treatment. When citric acid was used, it was used in all cases in the form of a 2.5% w / v solution. In test A, the stalks were provided in the form of dry, flaky Laminaria hyperborea, whereas in tests B and C, the dry, flaky material was further milled to obtain a fraction with a particle size between 200 and 700 μm. The results regarding the alginate yield are shown in Table 11.

[0185] [Table 11]

[0186] As can be seen from the results in Table 11, the combination of both the organic acid and metal cation exchange treatments results in a significantly increased yield of alginate compared to the use of either treatment alone.

[0187] Example 14: Use of Citric Acid at Different Temperatures A series of comparative experiments was carried out to investigate the effect of varying citric acid treatment conditions on the extracted alginate.

[0188] In tests A1 and A2, a stalk powder having a particle size of about 250 μm was prepared from dried, flaked Laminaria hyperborea from which leaves and epiphytes had been removed.

[0189] In trials B1 and B2, the stalk powder used in trials A1 and A2 was mixed in a 50:50 weight ratio with leaf powder prepared by grinding dry leaf flakes to a particle size of approximately 250 μm.

[0190] In trials C1 and C2, the stalk powder used in trials A1 and A2 was mixed in a 50:50 weight ratio with leaf powder prepared by grinding dried leaf flakes obtained from whole dried leaves to a particle size of approximately 250 μm.

[0191] All tests were performed according to the general procedure described above. For tests A1, B1, and C1, the citric acid treatment was performed for 60 minutes at ambient temperature. For tests A2, B2, and C2, the citric acid treatment was performed for 35-40 minutes at 95-99°C.

[0192] The viscosity of the resulting alginate was measured using a Brookfield viscometer at 20° C. The molecular weight, polydispersity index, and G and M contents were measured according to the methods described in Example 4. The results are shown in Table 12 below.

[0193] [Table 12] 1 Measured using a 1% w / v solution 2 Measured using a 10% w / v solution

[0194] From the data in Table 12, a similar trend is observed across all three sets of experiments. The alginate extracted after pretreatment with citric acid at higher temperatures has a lower molecular weight, lower polydispersity index, higher G content and lower M content than the alginate extracted from the same starting material but after pretreatment with citric acid at lower temperatures. Therefore, pretreatment with citric acid at higher temperatures can be used to obtain G-enriched low molecular weight alginate with a relatively narrow molecular weight distribution.

[0195] Leaf alginates typically have a higher M content than stem alginates. This is consistent with the G and M contents found for the alginates obtained in tests A1, B1, and C1, which included a lower temperature citric acid pretreatment intended to substantially preserve the native alginate structure. However, it was observed that a higher temperature citric acid pretreatment of a mixture of stem powder + leaf powder provides an alginate with a similar G and M content to that obtained from stem alginate, in other words the G content in the leaf alginate is enriched.

[0196] The results demonstrate that the conditions of the organic acid pretreatment can be tailored to provide an alginate product with desired properties depending on its intended use.

Claims

**Claim 1** A method for extracting alginate from macroalgae or a part thereof, comprising: (i) contacting the macroalgae or a part thereof with an aqueous solution of a weak organic acid; (ii) subsequently contacting the macroalgae or a part thereof with an aqueous solution of a mineral acid to form a pretreated macroalgae material; (iii) extracting alginate from the pretreated macroalgae material. **Claim 2** The organic acid has a pK greater than 1.5 ａ , preferably a pK in the range of 2 to 6 ａ The method according to claim 1, which has such a property. **Claim 3** wherein the organic acid has a pKa of 3.5 or less ａ The method according to claim 1 or 2 **Claim 4** The method according to claim 1, wherein the organic acid is an α-hydroxy acid, preferably a food-grade α-hydroxy acid. **Claim 5** The method according to claim 1, wherein the organic acid is selected from the group consisting of lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glycolic acid, acetic acid, and formic acid. **Claim 6** The method according to claim 1, wherein the organic acid is selected from the group consisting of lactic acid, malic acid, tartaric acid, citric acid, and ascorbic acid. **Claim 7** The method according to claim 1, wherein the organic acid is citric acid or malic acid. **Claim 8** The method according to claim 1, wherein the organic acid is citric acid. **Claim 9** The method according to claim 1, wherein the concentration of the organic acid is 0.1 to 10.0% w / v. **Claim 10** The method according to claim 1, wherein step (i) is carried out at a temperature in the range of 10 to 100°C, preferably at ambient temperature. **Claim 11** The method according to claim 1, wherein the mineral acid is hydrochloric acid or sulfuric acid. **Claim 12** The method according to claim 1, wherein step (ii) is carried out over a time of up to 60 minutes. **Claim 13** The method according to claim 1, wherein step (iii) includes contacting the pretreated macroalgae material with an alkaline solution. **Claim 14** The method according to claim 13, wherein the alkaline solution is sodium carbonate and / or sodium hydroxide. **Claim 15** The method according to claim 14, wherein the alkaline solution is sodium carbonate and is used at a concentration in the range of 0.05 to 4%, for example 0.1 to 0.5%. **Claim 16** The method according to claim 1, further comprising, prior to step (i), contacting the macroalgae or a part thereof with a calcium chloride solution, preferably wherein the concentration of the calcium chloride solution is in the range of 0.5 to 10% w / v. **Claim 17** The method according to claim 1, wherein the macroalgae is selected from the group consisting of the genus Laminaria, Ascophyllum, Durvillaea, Ecklonia, Lessonia, Macrocystis species, and Sargassum.

18. The method according to claim 17, wherein the macroalgae is Laminaria hyperborea.

19. The method according to claim 1, wherein the macroalgae portion is a stem-like part, a leaf, or a combination thereof.

20. The method according to claim 19, wherein the stem-like part is not skinned.

21. The method according to claim 1, which does not include the step of treating the macroalgae or a part thereof with formaldehyde or any derivative of formaldehyde.

22. The method according to claim 1, which does not include any step of treating the macroalgae or a part thereof with a bleaching agent.

23. (i) contacting the macroalgae or a part thereof with an aqueous solution of a weak organic acid, which is an α-hydroxy acid; (ii) subsequently contacting the macroalgae or a part thereof with an aqueous solution of a mineral acid at ambient temperature to form a pretreated macroalgae material, wherein the mineral acid is hydrochloric acid or sulfuric acid; (iii) extracting alginate from the pretreated macroalgae material by contacting the pretreated macroalgae material with an alkaline solution, which is sodium carbonate and / or sodium hydroxide; The method according to claim 1, comprising:

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