Bioactive polysaccharides

Abstract

Methods for processing microalgae, such as seaweed, in particular brown and green seaweed, are described, to extract polysaccharides with a reduced heavy metal content, reduced iodine content, and increased calcium content. We describe a process including the steps of: providing an algal biomass; extracting the algal biomass with at least one solvent; separating the solvent from algal biomass solids to provide an algal liquid and extracted algal biomass solids; adding a source of calcium ions to the algal liquid; clarifying the algal liquid to remove particulate and / or precipitated alginate from the algal liquid, to provide a clarified algal liquid; providing a protease enzyme to the clarified algal liquid; filtering the protease-treated clarified algal liquid to provide a retentate liquid comprising a bioactive polysaccharide extract and a permeate liquid; and filtering the permeate liquid to provide a liquid comprising a bioactive polysaccharide extract enriched in beta-glucan.

Description

[0001] BIOACTIVE POLYSACCHARIDES

[0002] Field of the Invention

[0003] The present invention relates to bioactive polysaccharides. In particular, the present invention relates to methods for processing seaweed, in particular brown seaweed, to extract polysaccharides which are considered to have bioactivities beneficial to health and well-being. The present invention also provides polysaccharide products with a reduced heavy metal content, reduced iodine content, and increased calcium content.

[0004] Background

[0005] Brown seaweeds contain several beneficial compounds which can be used for health benefits. Examples of such bioactive compounds include fucoidan - a fucose-rich polysaccharide found in brown seaweeds with a number of reported potential uses in health; and laminarin - a polysaccharide present in brown seaweed that has been reported to have potential pharmacological properties such as, for example, use as an antioxidant, having anti-tumour properties, anticancer properties, anti-diabetic properties, and anti-inflammation properties.

[0006] Brown algae seaweeds can contain high concentrations of minerals, heavy metals and other factors such as iodine which are co-extracted alongside desirable polysaccharides.

[0007] Several conventional processes can be used to extract bioactive compounds such as fucoidan from seaweed. US9234051 describes a purification of fucoidan from brown seaweed extracts using a chelating agent, selective precipitation and filtration to remove heavy metal ions.

[0008] Accordingly, there is a need for further processes to provide bioactive polysaccharides from seaweed whilst minimising certain co-products in polysaccharides obtained by the processes.

[0009] Summary of Invention

[0010] According to a first aspect of the present invention there is provided a process for providing a purified bioactive polysaccharide extract from an algal biomass, wherein the process comprises the steps of:

[0011] i) providing an algal biomass;

[0012] ii) extracting the algal biomass with at least one solvent;

[0013] iii) separating the solvent from algal biomass solids to provide an algal liquid and extracted algal biomass solids;

[0014] iv) adding a source of calcium ions to the algal liquid;v) clarifying the algal liquid to remove particulate and / or precipitated alginate from the algal liquid, to provide a clarified algal liquid;

[0015] vi) providing a protease enzyme to the clarified algal liquid;

[0016] vii) filtering the protease-treated clarified algal liquid to provide a retentate liquid comprising a bioactive polysaccharide extract and a permeate liquid; and

[0017] viii) filtering the permeate liquid from step vii) to provide a liquid comprising a bioactive polysaccharide extract enriched in beta-glucan.

[0018] Suitably the solvent extraction step solubilises selected polysaccharides from the algal biomass.

[0019] In certain embodiments, the step of extracting the algal biomass comprises a plurality of solvent extractions.

[0020] In certain embodiments, the solvent extraction step is a liquid extraction step using a mineral acid, optionally a food-safe mineral acid; and / or water.

[0021] In certain examples, the source of calcium ions comprises at least one water-soluble calcium salt.

[0022] In certain examples, the process further comprises a step of diafiltration of the retentate liquid. Suitably, the retentate liquid is diafiltered against several volumes of de-ionised water in order to reach a target purity, such as a target reduction of small molecules. In some examples, a reduction is confirmed by conductivity measurements.

[0023] Optionally further ion-exchange may be performed during a diafiltration using a calcium solution or an acid solution as the diafiltration liquid in one or more cycles of the diafiltration process.

[0024] In certain embodiments the method comprises the steps:

[0025] a) providing algal biomass to at least one water extraction step,

[0026] b) separating liquid from solids after the extraction step of a) to provide an algal liquid and extracted algal biomass solids,

[0027] c) adding a source of calcium ions to algal liquid from b),

[0028] d) clarifying particulate and I or precipitated alginate from the algal liquid of step c) to provide a clarified algal liquid,

[0029] e) providing a protease enzyme to the clarified algal liquid,f) filtering the protease treated clarified algal liquid of step e) to provide a retentate liquid comprising a bioactive polysaccharide extract and a permeate liquid,

[0030] g) filtering the permeate liquid from step f) to provide a second liquid comprising a bioactive polysaccharide extract enriched in beta-glucan.

[0031] In certain embodiments, after the separation step to provide extracted algal biomass solids, the extracted algal biomass solids are treated with at least one acid in at least one acid washing or extraction step, followed by a step of separating liquid from the acid-washed / extracted solids.

[0032] Suitably, the at least one acid washing or extraction step includes one or more acids selected from the group consisting of: hydrochloric acid, acetic acid, phosphoric acid, sulphuric acid, citric acid, malic acid, lactic acid, formic acid, perchloric acid fumaric acid and combinations thereof.

[0033] Suitably at least one of the one or more acids is a strong mineral acid.

[0034] In certain examples, the algal biomass comprises macroalgae. Optionally, the algal biomass comprises seaweed, for example, chopped seaweed. In certain examples, the algal biomass is selected from brown seaweed and / or green seaweed.

[0035] In certain preferred embodiments, the algal biomass is brown seaweed. The bioactive polysaccharide provided by the process may be enriched in fucoidan.

[0036] In other preferred embodiments, the algal biomass is green seaweed. The bioactive polysaccharide provided by the process may be enriched in ulvan.

[0037] Suitably the filtering step is an ultrafiltration step.

[0038] Suitably the filtering step uses a membrane or ceramic filter capable of removing small molecules comprising minerals, mannitol, beta-glucan, small polysaccharides and oligosaccharides and peptides.

[0039] Suitably the membrane or ceramic filter is a >30 kDA molecular weight cut-off filter, preferably a 50 kDa MW cut off filter.Suitably the filtering step provides a retentate wherein a majority of small molecules selected from minerals, mannitol, beta-glucan, small polysaccharides and oligosaccharides, pigments and peptides are removed.

[0040] Suitably diafiltration is used such that at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% by volume of the small molecules present in the clarified liquid stream are removed. In preferred embodiments, the diafiltration step removes between 60 to 95% by volume of small molecules. This allows the degree of purification of polysaccharide to be targeted.

[0041] In certain embodiments, the permeate of the filtering step is subjected to at least a second filtering step. Suitably a second filtering step reduces the level of very small molecules from the permeate.

[0042] Suitably at least a second filtering step uses a < 5 kDA molecular weight cut-off filter, preferably by a 1 kDA molecular weight cut off filter, such that a majority of small molecules selected from minerals, mannitol, beta-glucan, and oligosaccharides, pigments and peptides are removed. In certain examples, the filter is a PES membrane filter (polyethersulfone membrane).

[0043] In certain embodiments, the bioactive polysaccharide extracts provided by a first or second separating step are subjected to a concentrating step to increase a concentration of the bioactive polysaccharide extract. Optionally the concentrating step is a heat-treating step to evaporate solvent.

[0044] In certain embodiments the bioactive polysaccharide extracts are subjected to treatment to reduce a microbial load of the extract. Optionally, the treatment is a heat treating step, such as a pasteurisation step.

[0045] In certain embodiments the bioactive polysaccharide extracts are subjected to a drying step, optionally a spray-drying step or a lyophilisation step.

[0046] In an embodiment, the bioactive polysaccharide extract is enriched in high molecular weight bioactive polysaccharide.

[0047] In certain examples, the high molecular weight bioactive polysaccharide has a molecular weight in the range 3 kDa - 2000 kDa.In certain examples, the high molecular weight bioactive polysaccharide is rich in fucoidan. Fucoidan, is a sulphated polysaccharide which has been shown to have anti-inflammatory activity.

[0048] In certain examples, the high molecular weight bioactive polysaccharide is rich in ulvan. lllvan, is a sulphated polysaccharide which has been shown to have anti-inflammatory activity.

[0049] In certain examples, the bioactive polysaccharide extract is a low molecular weight bioactive polysaccharide. Suitably the low molecular weight bioactive polysaccharide has a molecular weight in the range 1 - 12 kDa.

[0050] Suitably the low molecular weight bioactive polysaccharide is rich in laminarin beta-glucan.

[0051] In certain examples, the algal biomass is brown seaweed and, following the first filtering step, a bioactive polysaccharide extract is provided which is a mixture of high molecular weight bioactive polysaccharide rich in fucoidan. Suitably low molecular weight bioactive polysaccharide rich in laminarin beta-glucan is obtained with further filtering.

[0052] In certain examples, the algal biomass is brown seaweed and, following a second filtering step, a bioactive polysaccharide extract is provided which is rich in laminarin beta-glucan.

[0053] In certain examples, the algal biomass is green seaweed and, following the first filtering step, a bioactive polysaccharide extract is provided which is a mixture of high molecular weight bioactive polysaccharide rich in ulvan. Suitably a low molecular weight bioactive polysaccharide rich in laminarin beta-glucan may be obtained following further filtering.

[0054] In certain embodiments, the process further comprises a hydrolysation step to remove protein impurities from the bioactive polysaccharide extract. Suitably, the hydrolysation step uses one or more proteases to remove one or more proteins. Optionally, the at least one protease selected from: serine proteases, alkaline proteases, commercial enzymes, trypsin, chymotrypsin, aspartic proteases, pepsin, cysteine proteases, papain, bromelain, thermolysin, metalloproteases, neutral proteases, and microbial protease cocktails / protease blends for biomass processing.

[0055] Suitably, an alkaline protease may be subtilisin, for example from Bacillus subtilis, Bacillus licheniformis.Suitably, the at least one protease is an enzyme selected from Savinase (Novozymes), Alcalase (Novozymes), Protex 6L (DuPont / Danisco), or Esperase (Novozymes).

[0056] Suitably, trypsin is obtained from Porcine pancreas, or recombinant microbes and may be commercially provided by TrypZean (Sigma-Aldrich) or NovoTrypsin (Novozymes).

[0057] Suitably, chymotrypsin is obtained from Bovine pancreas. Commercially, chymotrypsin may be Chymotrypsin NF (Sigma-Aldrich) or Biocatalysts SCAT (Biocatalysts Ltd.)

[0058] Suitably, pepsin is obtained from Porcine gastric mucosa. Commercially, Pepsin 1:3000 (Sigma-Aldrich), Biovet Pepsin (Biovet) and Enzeco Pepsin (Enzyme Development Corporation) may be utilised.

[0059] Suitably, a fungal aspartic protease is a fungal aspartic protease (e.g., Rhizopus pepsin) obtained from Rhizopus spp., or Aspergillus spp. Commercially, Acid Protease AP-10 (Amano Enzyme), Promod™ 278P (Biocatalysts Ltd.) or Fungal Acid Protease (Novozymes) may be utilised.

[0060] Suitably, papain is obtained from Carica papaya (papaya latex). Commercially this may be provided by Papain 1000 (Enzyme Development Corporation), Papaya Latex Powder (Sigma-Aldrich), Papain 6000 PU (DuPont).

[0061] Suitably, bromelain is obtained from Ananas comosus (pineapple stem). Commercially this may be provided by Bromelain 2400 GDU (Enzyme Solutions), Enzeco Bromelain (Enzyme Development Corporation), Bromelain 5000 (Sigma-Aldrich).

[0062] Suitably, thermolysin is obtained from Geobacillus stearothermophilus. Commercially thermolysin may be provided by Thermolysin 2000 (Amano Enzyme) or Thermolase (Sigma-Aldrich).

[0063] Suitably neutral proteases (e.g., Bacillus Neutral Protease) may be obtained from Bacillus subtilis, or Bacillus amyloliquefaciens. Commercially this may be provided by Neutrase (Novozymes), Bacillus Neutral Protease (Sigma-Aldrich), or Protex 7L (DuPont / Danisco) Suitably protease blends, for example as obtained from Bacillus spp., Aspergillus spp.

[0064] Commercially, suitable blends include: Protex 6L (DuPont / Danisco) - alkaline protease for biomass processing; Promod™ 439L (Biocatalysts Ltd.) - broad-spectrum protease for plantprotein hydrolysis; and Delvolase (DSM) - protease blend for protein hydrolysis in plant biomass.

[0065] In certain examples, the methods of the present invention are applied to brown seaweeds, for example alginophytes. Suitably algenophytes may be selected from the order Laminariales, Fucales or Dictyotales.

[0066] Suitably the brown seaweed is selected from at least one of: Laminaria abyssalis, Laminaria agardhii, Laminaria appressirhiza, Laminaria brasiliensis, Laminaria brongardiana, Laminaria bulbosa, Laminaria bullata, Laminaria complanata, Laminaria digitata, Laminaria ephemera, Laminaria farlowii, Laminaria groenlandica, Laminaria hyperborea Laminaria inclinatorhiza, Laminaria longipes, Laminaria multiplicata, Laminaria nigripes, Laminaria ochroleuca, Laminaria pallida, Laminaria platymeris, Laminaria rodriguezii, Laminaria ruprechtii, Laminaria sachalinensis, Laminaria setchellii, Laminaria sinclairii, Laminaria solidungula and Laminaria yezoensis

[0067] Suitably the brown seaweed may be from the order Fucales, suitably of the families Fucaceae and Sargassaceae. Suitably the brown seaweed may be from the order Dictyotales suitably of the genera Dictyota, Padina. Suitably the seaweed may be selected from S.latissima, L.digitata, A. escuelnta, L. japonica, U. pinnatifida, M. pyrifera, Sacchoriza polyschides, Lessonia nigrescens and Lessonia trabeculata. Ecklonia cava and Ecklonia stolonifera, Ecklonia maxima, Alaria marginata, Saccharina, Nereocystis luetkeana,Fucus distichus, Eualaria fistulosa, Sargassum sp. A. nodosum and Fucus sp..

[0068] In other examples, the methods of the present invention are applied to green seaweeds, for example, seaweeds selected from the order Ulvales.

[0069] Suitably the green seaweed is selected from at least one of: Ulva Lactuca, Ulva intestinalis, Ulva prolifera, Ulva rigida and Ulva fasciata

[0070] Suitably, the processes of the present invention are used to increase the purity of extraction of polysaccharides. More suitably, the purity of extraction of high molecular weight bioactive polysaccharide extract can be increased to > 80 %. Suitably the process increases the extraction of low molecular weight bioactive polysaccharide extract to >60 %, more suitably to about 66%. Suitably the high molecular weight bioactive polysaccharide extract comprises fucoidan or ulvan (for example depending on the source seaweed used for the process), Suitably the low molecular weight bioactive polysaccharide extract comprises laminarin beta-glucan. Laminarin is a soluble beta-glucan. Herein they may be used interchangeably to describe a polysaccharide extract fraction.

[0071] Suitably, the processes of the present invention are used to decrease unwanted byproducts. More suitably the method reduces iodine levels in the bioactive polysaccharide extract from brown seaweed. More suitably, the method reduces iodine levels down to <10% compared with a method not comprising a hydrolysation step.

[0072] Suitably, the processes of the present invention are used to reduce heavy metal levels in the bioactive polysaccharide extract. Suitably the reduction in heavy metal levels is by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0073] Suitably, the the processes of the present invention are used to increase beneficial compounds within the final filtered product. More suitably, the method is used to increase calcium levels in the bioactive polysaccharide extract.

[0074] The final filtered product is suitable for use in the development of supplements for use in human and veterinary nutrition and medicine. Supplements may comprise powders, capsules or tablets; and / or the supplements may comprise a liquid product.

[0075] The final filtered product may be used for the development of products used in human personal care. Said products may comprise facial skincare including: moisturizers and creams, serums and ampoules, facial mists and essences, cleansing balms, and gels, exfoliators and peels, eye creams and serums, sheet masks and overnight masks, lip balms and treatments; body skincare including, body lotions, creams, and butters, hand and foot creams, firming and anticellulite treatments, body mists and sprays, sun care and after-sun products; specialty skincare including feminine care (intimate washes, gels, and moisturizers), pregnancy and postpartum skincare, baby skincare (lotions, balms, and wipes), scar and wound healing gels, eczema and psoriasis care, tattoo aftercare, and medical and aesthetic skincare (posttreatment soothing gels, barrier creams).

[0076] The products may comprise haircare conditioners, hair masks and treatments, scalp serums and tonics, styling creams and balms, hair elixirs, mousses and foams, hair sprays and mists, pre-wash or overnight treatments, scalp exfoliants and detox treatments.In a second aspect, the present invention provides a composition comprising a high molecular weight bioactive polysaccharide as produced by a process as of the first aspect of the invention.

[0077] Suitably the high molecular weight polysaccharide comprises fucoidan or ulvan; and / or the low molecular weight polysaccharide comprises laminarin.

[0078] Suitably the composition is provided as a nutraceutical, cosmeceutical, pharmaceutical or supplement for humans and animals (e.g. including a veterinary supplement), for local treatment in the form of a liquid or ointment, or as part of an adhesive tape, bandage or wound covering. Suitably the composition may be provided in a form such that it is administered over a sustained period of time, for example as part of a sustained release formulation, as part of a transdermal patch, miniature pump or the like.

[0079] In certain embodiments, the composition comprises an excipient or diluent.

[0080] In a third aspect, the present invention provides a method of treating a subject in need thereof, the method comprising providing a high molecular weight bioactive polysaccharide as produced by a process of the first aspect of the invention.

[0081] In one embodiment, the high molecular weight polysaccharide comprises fucoidan.

[0082] In another embodiment, the low molecular weight polysaccharide comprises laminarin.

[0083] Suitably the method may be prophylactically administered.

[0084] Suitably administration may typically be topical or oral.

[0085] Compositions of the present invention may be administered alone or prior to, concurrent with, or subsequent to another agent, for example an omega oil. For example, the bioactive polysaccharide composition as described herein, may be provided to an individual in combination with another active agent. Suitably the composition may be an injectable composition.

[0086] As used herein, “purified” generally refers to isolation of a substance such that the substance comprising the majority percent of the sample in which it resides. Suitably, in a sample apurified substance comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% of a sample.

[0087] The above and other aspects of the present invention will now be described in further detail, by way of example only, with reference to the following examples and the accompanying figures, in which:

[0088] Figure 1 is a chart showing HMW polysaccharide composition in a first example of an embodiment of a process in accordance with the present invention. The figure illustrates increased presence of fucoidan following use of protease as part of the process to extract a high molecular weight bioactive polysaccharide extract in a process as discussed herein wherein in particular it is shown brown seaweed HMW polysaccharide composition % of dry matter, determined by monosaccharide analysis (HPAE-PAD) and elemental analysis (mean % dm, Invention process N=6, prior art process N=10).

[0089] Figure 2 is a chart showing LMW polysaccharide composition in a second example of an embodiment of a process in accordance with the present invention. The figure illustrates increased presence of laminarin following use of protease as part of the process to extract a low molecular weight bioactive polysaccharide extract in a process as discussed herein wherein in particular it is shown brown seaweed LMW polysaccharide composition % of dry matter, determined by monosaccharide analysis (HPAE-PAD) and elemental analysis (mean % dm, Invention process N=6, prior art process N=13).

[0090] Figure 3 is a chart showing iodine content in example products obtained by an embodiment of a process in accordance with the present invention and illustrates decreased presence of iodine following use of protease as part of the process to extract a high molecular weight bioactive polysaccharide extract and low molecular bioactive polysaccharide extract in a process as discussed herein wherein in particular it is shown that total iodine content of brown seaweed HMW and LMW bioactive polysaccharide products, determined by ICP-MS (mean content mg / kg dry matter ± SEM, invention process N=7, prior art process N=10).

[0091] Figure 4 is a series of four charts showing total arsenic and inorganic arsenic content following use of protease as part of an embodiment of a process of the present invention, to extract a high molecular weight bioactive polysaccharide extract and low molecular bioactive polysaccharide extract in a process as discussed herein wherein in particular it is shown thattotal arsenic and inorganic arsenic content of brown seaweed HMW and LMW bioactive polysaccharide products, determined by ICP-MS (mean content mg / kg dry matter, ± SEM invention process N=6, prior art process N=9).

[0092] Figure 5 is a graph illustrating metal reduction and calcium enrichment in a HMW polysaccharide extract obtained by an embodiment of the present invention, showing mineral content of brown seaweed HMW bioactive polysaccharide product determined by ICP-OES (mean ± SEM, prior art process N=5, invention process N=6).

[0093] Figure 6 is a graph illustrating metal reduction and calcium enrichment in a LMW polysaccharide extract obtained by an embodiment of the present invention, showing mineral content of brown seaweed LMW bioactive polysaccharide product determined by ICP-OES (mean ± SEM, prior art process N=3, invention process N=3).

[0094] Figure 7 is a series of three graphs illustrating a molecular weight (MW) profile of HMW polysaccharide from seaweed wherein in particular MW distribution of brown seaweed HMW (size-exclusion chromatogram - Figure 7A) and LMW polysaccharide products (size-exclusion chromatogram - Figure 7B), and HMW green seaweed polysaccharide product (Figure 7C) from an embodiment of a process in accordance with the present invention (MW distribution was determined by HPLC-SEC analysis with refractive index detection; mean distributions ± SEM).

[0095] Figure 8 is a FTIR spectral comparison of an FTIR spectrum of a HMW polysaccharide from brown seaweed according to a process of the present invention compared with brown seaweed HMW polysaccharide products from a prior art process and a fucoidan reference material. Spectra were collected from dry powders using ATR-FTIR on Bruker Spectrometer. Spectral match was calculated using OPUS software. Product spectra are representative mean spectra, reference material is single representative spectrum.

[0096] Figure 9 is a FTIR spectral comparison of an FTIR spectrum of a LMW polysaccharide from brown seaweed according to a process of the present invention compared with brown seaweed LMW polysaccharide products from a prior art process and a laminarin reference material. Spectra were collected from dry powders using ATR-FTIR on Bruker Spectrometer. Spectral match was calculated using OPUS software. Product spectra are representative mean spectra, reference material is single representative spectrum.Figure 10 is a FTIR spectral comparison of an FTIR spectrum of a HMW polysaccharide from green seaweed according to a process of the present invention compared with green seaweed HMW polysaccharide products from prior art process and a ulvan reference material. Spectra were collected from dry powders using ATR-FTIR on Bruker Spectrometer. Spectral match was calculated using OPUS software. Product spectrum is representative mean spectrum, reference material is single representative spectrum.

[0097] EXAMPLES

[0098] Example 1 Bioactive polysaccharide from brown seaweed

[0099] Seaweed processing to provide a purified bioactive polysaccharide was undertaken using a five stage process:

[0100] - extraction of soluble compounds

[0101] - calcium ion addition

[0102] - clarification

[0103] - filtration to purify and concentrate the extracted bioactive polysaccharide, and - optionally drying of the extracted bioactive polysaccharide into a packaged product.

[0104] Suitably a calcium salt is used as a source of calcium ions.

[0105] Further optionally heat sterilisation of the extracted bioactive polysaccharide into a packed liquid product may be undertaken.

[0106] As part of the first stage, a step of extraction of soluble compounds was undertaken by water washing of chopped seaweed. This stage comprises:

[0107] Water wash 1 and water wash 2

[0108] The purpose of water washes 1 and 2 is to dissolve nutraceuticals, ash, protein, and soluble carbohydrates from chopped seaweed and then separate these from fibre.

[0109] The seaweed may also be subjected to an acid wash step after the water wash step.

[0110] A decanting centrifuge was used between each wash step to separate the liquid and solid streams. The resulting final solid acid cake was further processed to fibre and the liquid.Calcium salts were added to the water wash 1 and water wash 2 liquid to precipitate alginate so the alginate is not carried forward to the filtration step of the second stage of the process.

[0111] Additionally, the addition of calcium salts acts as an ion-exchange step and reduces the heavy metal content of the bioactive polysaccharide comprising bioactive polysaccharide, for example fucoidan.

[0112] A clarification step was undertaken to remove particulates and the precipitated alginate from the liquid.

[0113] The pH of the liquid was adjusted to a pH range 5-6. Protease enzymes were added to hydrolyse protein molecules, which improves separation of proteins and protein-bound components such as organic arsenic compounds.

[0114] Preservatives were added to the liquid to inhibit microbiological growth during hydrolysis and filtration.

[0115] As part of the process, a first ultrafiltration step was carried out using a 50kDA ceramic or membrane filter to purify the bioactive polysaccharide comprising fucoidan from brown seaweed. This first ultrafiltration step removes small molecules including minerals, mannitol, beta-glucan, oligosaccharides, monosaccharides, pigment molecules and peptides from the clarified and protease treated liquid. The retentate from this ultrafiltration step was diafiltered using the 50 kDA ceramic filter to further reduce small molecules and increase the purity of the bioactive polysaccharide extract.

[0116] The permeate water from the first ultrafiltration volume was retained for a second ultrafiltration step. The second ultrafiltration step used a 1kDA PES membrane filter to remove very small molecules from the first ultrafiltration permeate water and subsequently form a beta-glucan concentrate. The further diafiltration using the 1 kDA PES membrane step was used to further reduce small molecules and improve the purity of the bioactive polysaccharide extract.

[0117] As part of the process the filtered bioactive polysaccharide extract was subjected to an evaporation step, then a pasteurisation step. The evaporation step was used to increase the concentration of the liquid comprising the bioactive polysaccharide extract and the pasteurisation step was used to reduce any microbiological activity to a minimum prior to drying.In some examples, the concentrated liquid bioactive polysaccharide extract was dried by spray drying to produce a powder. Suitably this powder can be considered to be a nutraceutical, or cosmeceutical ingredient.

[0118] In other examples, the concentrated liquid bioactive polysaccharide extract was then bottled. Suitably this liquid concentrate can be considered to be a nutraceutical, or cosmeceutical ingredient.

[0119] The process as described herein provides an increase in purity of high molecular weight bioactive polysaccharide, including fucoidan and ulvan from the range 60 -70% to >80 %. It is considered the process as described herein provides an increase in purity of low molecular weight bioactive polysaccharide including laminarin from around17% to > 60% (Figures 1 and 2).

[0120] Seaweed such as kelp is known to have a high natural iodine content. Advantageously the process as described herein reduced the iodine content present in the high molecular weight bioactive polysaccharide, including fucoidan, from around 593 mg / kg to 113 mg / kg and the iodine content in the low molecular weight bioactive polysaccharide including laminarin from around 622 mg / kg to 127 mg / kg (Figure 3).

[0121] Seaweed such as brown seaweed contains high levels of total arsenic (in the range of 21 to 114 mg / kg for common Laminariaceae species. Arsenic is present as both organic species and inorganic species. The inorganic arsenic is considered to be toxic whilst the organic arsenic is considered to be of low or no toxicity. Whilst in seaweeds inorganic arsenic is typically present in seaweed at much lower levels than organic arsenic, total arsenic levels, and in particular inorganic arsenic levels, can limit the consumption of seaweed extracts. Thus, it is advantageous if arsenic levels can be reduced in seaweed extracts. The inventors have determined that the process as described herein can reduce the total arsenic content in high molecular weight bioactive polysaccharide, including fucoidan, and low molecular weight bioactive polysaccharide including laminarin from 79 mg / kg and 68 mg / kg respectively. Analysis of the total arsenic present, has determined that the respective inorganic arsenic is reduced by 63 mg / kg and 57 mg / kg.

[0122] Additionally, seaweed such as brown seaweed contains high levels of minerals (for example of the range 20% to 50% of dry matter). The minerals include a range of salts, typically dominated by K, Na, Ca, Mg and heavy metals, Cd, Hg, Pb. The process as described herein reduces the presence of heavy metals in the bioactive polysaccharide extract. Advantageouslythe presence of heavy metals in the bioactive polysaccharide extract is reduced below regulatory limits.

[0123] Using the process to provide a high molecular weight bioactive polysaccharide, including fucoidan or ulvan, the process can replace non-calcium metal ions present in the seaweed extract with calcium ions, reducing non-calcium metal and particularly heavy metal ion content while at the same time increasing the calcium content of the bioactive extract. As will be appreciated, a fraction of the non-calcium metal ions may be replaced by calcium ions. This is advantageous as it provides a mechanism by which calcium can be provided along with the high molecular weight polysaccharide.

[0124] Example 2 Bioactive polysaccharide from green seaweed

[0125] Seaweed processing to provide a purified bioactive polysaccharide was undertaken using a five stage process:

[0126] - extraction of soluble compounds

[0127] - Calcium ion addition

[0128] - Clarification

[0129] - filtration to purify and concentrate the extracted bioactive polysaccharide, and - optionally drying of the extracted bioactive polysaccharide into a packaged product.

[0130] Suitably a calcium salt is used as a source of calcium ions.

[0131] Optionally the process further comprises heat sterilisation of the extracted bioactive polysaccharide into a packed liquid product.

[0132] As part of the process, a step of extraction of soluble compounds was undertaken by water washing of chopped seaweed. This stage comprises:

[0133] Water wash 1 and water wash 2

[0134] Again, the purpose of water washes 1 and 2 is to dissolve nutraceuticals, ash, protein, and soluble carbohydrates from chopped seaweed and then separate them from fibre.

[0135] A decanting centrifuge was used between each wash step to separate the liquid and solid streams. The resulting solid was further processed to be used as a fibre product.Calcium ions for example as salts were added to the water wash 1 and water wash 2 liquid to precipitate unwanted polysaccharide components and particulates so that they are not carried forward to the filtration step of the second stage of the process. Additionally, this acts as an ion-exchange step undertaken to reduce heavy metal content of the bioactive polysaccharide, comprising ulvan.

[0136] A clarification step was undertaken to remove particulates and the precipitated polysaccharide from the liquid.

[0137] The pH of the liquid was adjusted to a pH range 5-6. Protease enzymes were added to hydrolyse protein molecules, which improves separation of proteins and protein-bound components such as organic arsenic compounds.

[0138] Preservatives were added to the liquid to inhibit microbiological growth during hydrolysis and filtration.

[0139] As part of the process, a first ultrafiltration step was carried out using a 50kDA ceramic or membrane filter to purify the bioactive polysaccharide comprising ulvan from the green seaweed. This first ultrafiltration step removes small molecules including minerals, oligosaccharides, monosaccharides, pigment molecules and peptides from the clarified and protease treated liquid. The retentate from this ultrafiltration step was dia-filtered to further reduce small molecules and increase the purity of the bioactive polysaccharide extract.

[0140] As part of the process the filtered bioactive polysaccharide extract was subjected to an evaporation step, then a pasteurisation step. The evaporation step was used to increase the concentration of the liquid comprising the bioactive polysaccharide extract and the pasteurisation step was used to reduce any microbiological activity to a minimum prior to drying.

[0141] In some examples, the concentrated liquid bioactive polysaccharide extract was dried by spray drying to produce a powder. Suitably this powder can be considered to be a nutraceutical, or cosmeceutical ingredient.

[0142] In other examples, the concentrated liquid bioactive polysaccharide extract was then bottled. Suitably this liquid concentrate can be considered to be a nutraceutical, or cosmeceutical ingredient.It is considered the process as described herein provides an increase in purity of high molecular weight bioactive polysaccharide, including lllvan from the range 60 -70% to >80%.

[0143] The processes of the present invention provide a high molecular weight bioactive polysaccharide, comprising ulvan. The process is able to replace the metal ions present in the seaweed extract with calcium, reducing the metal and heavy metal ion content and increasing the calcium content of the bioactive extract. This is advantageous as it provides a mechanism by which calcium can be provided along with the high molecular weight polysaccharide.

[0144] The present invention provides methods of processing algal biomass, in particular seaweed, which provides a greater purity of extracted polysaccharides, beneficial to health, from algal biomass. In particular, the present invention achieves a higher purity when extracting the polysaccharides fucoidan and laminarin from brown seaweed, and ulvan from green seaweed. Further, the methods of the present invention have been shown to lower the concentration of unwanted side products, such as heavy metals, or iodine, when compared to conventional extraction methods. Suitably, this means a lower heavy metals content in the extracted polysaccharides which is advantageous. Further still, the method of the present invention also leads to a higher concentration of calcium in the extracted product when compared to conventional methods.

[0145] Suitably, the method of the present invention comprises a liquid extraction of whole brown seaweed, decantation of solids, removal of alginates from brown seaweed extract, clarification of brown seaweed extract, hydrolysation, and purification by ultrafiltration.

[0146] Suitably, the method of the present invention comprises a liquid extraction of whole green seaweed, decantation of solids, removal of unwanted polysaccharides from green seaweed extract, clarification of green seaweed extract, hydrolysation, and purification by ultrafiltration.

[0147] Purification of the bioactive polysaccharide extract comprises clarification through use of a clarifying centrifuge and subsequent ultrafiltration.

[0148] Removal of alginates can be achieved by the addition of a calcium salt to the product of the extraction step.

Claims

CLAIMS1. A process for providing a purified bioactive polysaccharide extract from an algal biomass, wherein the process comprises the steps of:i) providing an algal biomass;ii) extracting the algal biomass with at least one solvent;iii) separating the solvent from algal biomass solids to provide an algal liquid and extracted algal biomass solids;iv) adding a source of calcium ions to the algal liquid;v) clarifying the algal liquid to remove particulate and / or precipitated alginate from the algal liquid, to provide a clarified algal liquid;vi) providing a protease enzyme to the clarified algal liquid;vii) filtering the protease-treated clarified algal liquid to provide a retentate liquid comprising a bioactive polysaccharide extract and a permeate liquid; andviii) filtering the permeate liquid from step vii) to provide a liquid comprising a bioactive polysaccharide extract enriched in beta-glucan.

2. A process as claimed in claim 1 wherein the solvent is a solvent which solubilises selected polysaccharides from the algal biomass.

3. A process as claimed in claim 1 or claim 2 wherein the step of extracting the algal biomass comprises a plurality of solvent extractions.

4. A process as claimed in any preceding claim wherein the solvent comprises a mineral acid, optionally a food-safe mineral acid; and / or water.

5. A process as claimed in any preceding claim further comprising a step of diafiltration of the retentate liquid.

6. A process as claimed in claim 5 wherein the step of diafiltration further comprises performing ion-exchange using a calcium solution or an acid solution as the diafiltration liquid in one or more cycles of the diafiltration process.

7. A process as claimed in any preceding claim wherein, after the separation step to provide extracted algal biomass solids, the extracted algal biomass solids are treated with anacid in at least one acid washing or extraction step, followed by a step of separating liquid from the acid-washed / extracted solids.

8. A process as claimed in claim 7 wherein the at least one acid washing or extraction step includes one or more acids selected from the group consisting of: hydrochloric acid, acetic acid, phosphoric acid, sulphuric acid, citric acid, malic acid, lactic acid, formic acid, perchloric acid fumaric acid and combinations thereof; optionally at least one of the one or more acids is a strong mineral acid.

9. A process as claimed in any preceding claim wherein the filtering step:i) is an ultrafiltration step; and / orii) uses a membrane or ceramic filter capable of removing small molecules comprising minerals, mannitol, beta-glucan, small polysaccharides and oligosaccharides and peptides, optionally wherein the membrane or ceramic filter is a >30 kDA molecular weight cut-off filter, or a 50 kDa MW cut off filter.

10. A process as claimed in any preceding claim wherein the permeate of the filtering step is subjected to at least a second filtering step to reduce the level of very small molecules from the permeate; optionally wherein the second filtering step uses a < 5 kDA molecular weight cut-off filter, preferably by a 1 kDA molecular weight cut off filter, such that a majority of small molecules selected from minerals, mannitol, beta-glucan, and oligosaccharides, pigments and peptides are removed, further optionally wherein the filter is a PES membrane filter (polyethersulfone membrane).

11. A process as claimed in any preceding claim wherein the bioactive polysaccharide extract is subjected to a concentrating step to increase a concentration of the bioactive polysaccharide extract.

12. A process as claimed in any preceding claim further comprising a treatment to reduce a microbial load of the extract, optionally a heat treating step, further optionally a pasteurisation step.

13. A process as claimed in any preceding claim further comprising a drying step, optionally a spray-drying step or a lyophilisation step.

14. A process as claimed in any preceding claim further comprising a hydrolysation step to remove protein impurities from the bioactive polysaccharide extract.

15. A process as claimed in claim 14 wherein the hydrolysation step uses one or more proteases to remove one or more proteins; optionally wherein the one or more proteases is selected from: serine proteases, alkaline proteases, commercial enzymes, trypsin, chymotrypsin, aspartic proteases, pepsin, cysteine proteases, papain, bromelain, thermolysin, metalloproteases, neutral proteases, and microbial protease cocktails / protease blends for biomass processing.

16. A process as claimed in claim 15 wherein the protease is:i) an alkaline protease, optionally a subtilisin, further optionally Bacillus subtilis or Bacillus licheniformis', orii) Savinase (Novozymes), Alcalase (Novozymes), Protex 6L (DuPont / Danisco), or Esperase (Novozymes); oriii) a trypsin obtained from Porcine pancreas, or recombinant microbes, optionally TrypZean (Sigma-Aldrich) or NovoTrypsin (Novozymes); oriv) a chymotrypsin obtained from Bovine pancreas, optionally Chymotrypsin NF (Sigma-Aldrich) or Biocatalysts SCAT (Biocatalysts Ltd.); orv) a pepsin obtained from Porcine gastric mucosa, optionally Pepsin 1:3000 (Sigma- Aldrich), Biovet Pepsin (Biovet) or and Enzeco Pepsin (Enzyme Development Corporation); orvi) a fungal aspartic protease, optionally a fungal aspartic protease selected from Rhizopus pepsin, optionally obtained from Rhizopus spp., or Aspergillus spp., further optionally Acid Protease AP-10 (Amano Enzyme), Promod™ 278P (Biocatalysts Ltd.) or Fungal Acid Protease (Novozymes); orvii) a papain obtained from Carica papaya (papaya latex), optionally Papain 1000 (Enzyme Development Corporation), Papaya Latex Powder (Sigma-Aldrich) or Papain 6000 PU (DuPont); orviii) a bromelain obtained from Ananas comosus (pineapple stem), optionally Bromelain 2400 GDU (Enzyme Solutions), Enzeco Bromelain (Enzyme Development Corporation) or Bromelain 5000 (Sigma-Aldrich); orix) a thermolysin obtained from Geobacillus stearothermophilus, optionally Thermolysin 2000 (Amano Enzyme) or Thermolase (Sigma-Aldrich); orx) a neutral protease, optionally Bacillus Neutral Protease, further optionally Bacillus Neutral Protease obtained from Bacillus subtilis, or Bacillus amyloliquefaciens, optionally Neutrase (Novozymes), Bacillus Neutral Protease (Sigma-Aldrich), or Protex 7L (DuPont / Danisco); orxi) a protease blend, optionally a protease blend obtained from Bacillus spp., Aspergillus spp., further optionally Protex 6L (DuPont / Danisco), Promod™ 439L (Biocatalysts Ltd.) and / or Delvolase (DSM).

17. A process as claimed in any preceding claim wherein the algal biomass comprises macroalgae.

18. A process as claimed in claim 17 wherein the macroalgae comprises seaweed.

19. A process as claimed in claim 18 wherein the seaweed comprises brown seaweed.

20. A process as claimed in claim 19 wherein the brown seaweed is at least one alginophyte, optionally at least one algenophyte selected from the order Laminariales, Fucales or Dictyotales.

21. A process as claimed in claim 20 wherein the or each brown seaweed is selected from Laminaria abyssalis, Laminaria agardhii, Laminaria appressirhiza, Laminaria brasiliensis, Laminaria brongardiana, Laminaria bulbosa, Laminaria bullata, Laminaria complanata, Laminaria digitata, Laminaria ephemera, Laminaria farlowii, Laminaria groenlandica, Laminaria hyperborea Laminaria inclinatorhiza, Laminaria longipes, Laminaria multiplicata, Laminaria nigripes, Laminaria ochroleuca, Laminaria pallida, Laminaria platymeris, Laminaria rodriguezii, Laminaria ruprechtii, Laminaria sachalinensis, Laminaria setchellii, Laminaria sinclairii, Laminaria solidungula and Laminaria yezoensis.

22. A process as claimed in claim 20 or claim 21 wherein the or each brown seaweed is selected from:i) the order Fucales, optionally, Fucaceae and Sargassaceae', and / orii) the order Dictyotales, optionally the genera Dictyota, Padina and / oriii) S.latissima, L.digitata, A. escuelnta, L. japonica, U. pinnatifida, M. pyrifera, Sacchoriza polyschides, Lessonia nigrescens and Lessonia trabeculata. Ecklonia cava and Ecklonia stolonifera, Ecklonia maxima, Alaria marginata, Saccharina, Nereocystis luetkeana,Fucus distichus, Eualaria fistulosa, Sargassum sp. A. nodosum and Fucus sp..

23. A process as claimed in any one of claims 18 to 22 wherein the seaweed comprises green seaweed.2124. A process as claimed in claim 23 wherein the at least one green seaweed is selected from the order Ulvales', optionally wherein the green seaweed is at least one green seaweed selected Ulva Lactuca, Ulva intestinalis, Ulva prolifera, Ulva rigida and Ulva fasciata25. A composition comprising a high molecular weight bioactive polysaccharide obtained by a process as claimed in any one of claims 1 to 24.

26. A composition as claimed in claim 25 comprising fucoidan or ulvan.

27. A composition comprising a low molecular weight bioactive polysaccharide obtained by a process as claimed in any one of claims 1 to 24.

28. A composition as claimed in claim 27 comprising laminarin.

29. A composition as claimed in any one of claims 25 to 28 for use as a nutraceutical, cosmeceutical, pharmaceutical or supplement for humans and animals, for topical or local treatment in the form of a liquid or ointment, or as part of an adhesive tape, bandage or wound covering; or as sustained release formulation, as part of a transdermal patch or a miniature pump.

30. A composition as claimed in any one of claims 25 to 29 further comprising an excipient or diluent.

31. A method of treating a subject in need of treatment, the method comprising providing a high molecular weight bioactive polysaccharide or a low molecular weight bioactive polysaccharide obtained by a process as claimed in any one of claims 1 to 24.22

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