Method for cascading treatment of fresh algae

Abstract

The present invention describes a method for processing fresh algae at ambient temperature by osmotic shocking the algae and treating the disrupted algae with an enzyme composition containing cell wall-degrading enzymes. This gentle process at ambient temperature allows for the isolation of algal proteins that have good solubility in the presence of salts and good foaming, emulsifying, and water-binding properties. Another advantage is that this protein isolation method enables cascade biorefineries, since after protein isolation, the remaining biomass can be treated with carbohydrate-degrading enzymes to produce high yields of clean biogas and a mineral-rich water stream for anaerobic digestion.

Description

Technical Field

[0001] The present invention relates to a method for treating algae to recover algal proteins and biogas. The present invention also relates to the proteins recovered by this method.

Background Art

[0002] Algae are a valuable source of polysaccharides, proteins, minerals, and oils. In order to separate these valuable components from algae, it is necessary to treat the algae. Freshwater algae are harvested and treated in situ. In the case of saltwater algae, so-called seaweeds, harvesting and processing are carried out at different locations. Seaweeds are harvested offshore and transported to the mainland for onshore treatment. By the time treatment begins, decomposition and deterioration of the seaweeds have already started. As a result, the algae cannot be used for food, pharmaceutical, or cosmetic applications after intensive treatment or without using a significant amount of antibacterial preservatives. Furthermore, a large amount of water is transported while carrying the algae onshore, which is very heavy and costly.

[0003] Alternatively, algae may be harvested offshore, stored offshore, and then, before processing, their storage or shelf life extended. Stored seaweed is transported overland for further processing. This avoids the transport of large amounts of water, but does not avoid damage to algal components such as proteins. Storage is often carried out in the form of drying or freezing. Non-patent document 1 describes the extraction of proteins from frozen or freeze-dried seaweed. Non-patent document 2 is a study of various protein extraction methods from dried seaweed. Non-patent document 3 describes the extraction of optimized alkaline proteins from dried seaweed. Non-patent document 4 describes the extraction of other optimized alkaline proteins from oven-dried dulse (Palmaria palmate). The heat used for storage, particularly drying seaweed, can cause deformation or denaturation of proteins, leading to a decrease in functionality and associated value. Furthermore, heating to dry large amounts of moist algal biomass requires a considerable amount of (costly) energy. Another drawback is that preservatives are usually added to the algal biomass. These preservatives can remain in the final food product, and in the case of acid (storage) treatment, they also destroy algal components, which is undesirable. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Vilg & Undeland (2017) J. Appl. Phycol. 29:585 [Non-Patent Document 2] Postma et al J Appl Phycol (2018) 30:1281 [Non-Patent Document 3] Maehre et al. 2016 Mar. drugs 14:196 [Non-Patent Document 4] Harnedy & FitzGerald 2013 Food Sci technol 51:375 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] It would be desirable to have a more efficient, more economical, and gentler method for seaweed processing that yields high-quality, functional, and typically intact protein products that can be used in food applications and allow for the extraction of further value from the remaining biomass after protein separation. [Brief explanation of the drawing]

[0006] [Figure 1] One embodiment of the apparatus according to the present invention includes a harvesting and washing unit 2; a processing unit 3 for enzymatic digestion; a recovery unit 4 for protein separation and concentration; an anaerobic digestion processing unit 6; a recovery unit 7 for recovering the product from 6; and a blending unit 5 for blending the product 8 from the recovery unit 4 or 7. [Modes for carrying out the invention]

[0007] Detailed description of the invention In one embodiment, the present invention relates to a method for treating algae. This method comprises (i) subjecting algae to osmotic shock; (ii) treating the shocked algae with an enzyme composition containing a cell wall-degrading enzyme; and (iii) separating the enzyme-treated algae into a solid phase and a liquid phase, wherein the enzyme treatment of the algae is started within 3 hours after harvest, and the temperature of steps (i) to (iii) is in the range of 4 to 30°C.

[0008] In conventional industries, when harvesting and processing take place in different locations, biomass is typically transported from the harvesting site to the processing site. In the method according to the present invention, instead of transporting harvested biomass to the processing site, the processing plant is transported to the location where the biomass is harvested so that the biomass is processed as fresh as possible to obtain the highest functional value of its components. In the method according to the present invention, algae are processed at the harvesting site or very close to the harvesting site. This is particularly advantageous when the algae are harvested in a location separate from the main processing site, for example, offshore, which is common in the case of marine algae, also known as seaweed or macro-algae. Using the method of the present invention, there is little opportunity for the algae to decompose or deteriorate before processing begins. For example, the high cost of water transport for transporting seaweed (seaweed is composed of 90% or more water) to processing plants is also avoided. Therefore, the protein obtained from the algae is of good quality, has functionality, such as good solubility, can be produced more economically, and can be used for food applications.

[0009] Another advantage of the method according to the present invention is that, after protein extraction, the remaining biomass can be used for further valuable extraction. For example, the biomass can be used for biogas production or mineral recovery. In one embodiment of the present invention, the biomass remaining after protein extraction is exposed to a carbohydrase enzyme mixture to release sugars. The carbohydrate-rich hydrolyzed biomass is then fed into an anaerobic digester to produce a stream of biogas and mineral-rich water. The latter can be used as or in a fertilizer composition, which is also part of the present invention. Other food components such as sugars, alginates, carrageenan, and fucoidan can also be recovered from this carbohydrate-rich fraction.

[0010] This method is highly advantageous for processing seaweed, but it can be used for processing any type of algae, either on land or offshore. In one embodiment, this method is used for offshore processing of algae, particularly seaweed or macroalgae, to produce proteins, and optionally, biogas or fertilizer compositions from the algal biomass remaining after protein recovery. The term "biogas" refers to the products of anaerobic digestion or anaerobic fermentation of biomass. Biogas consists mainly of methane and carbon dioxide, and may contain small amounts of hydrogen sulfide, water, and siloxanes.

[0011] The method of the present invention is particularly advantageous when harvesting biomass such as algae in situations where the processing site and the harvesting site are far apart, as it enables immediate processing of biomass. Processing is usually started quickly to minimize the time the algae are exposed to air or oxygen, for example, within 30 minutes, 60 minutes, 90 minutes, 2 hours, 2.5 hours, or 3 hours after harvesting.

[0012] In this context, the term "processing" refers to a process that includes the destruction of algal cells or the treatment of algal biomass with cell wall-degrading enzymes. Simply drying the algal biomass without further cell destruction or enzymatic treatment is not considered the start of processing.

[0013] In the context of this invention, “offshore” means a marine or freshwater location such as a sea, ocean, estuary, river or lake, coast or riverbank. In one embodiment, the marine or freshwater location is at least 1 km from the coast, shore, or riverbank, for example, 1 to 200 km, 1 to 50 km, or 50 to 200 km from the coast, shore, or riverbank. Offshore does not mean a pond or waterway on land.

[0014] The temperature during processing does not exceed 30°C, preferably 4-30°C, 5-25°C, or 15-25°C, so that the proteins isolated from algae do not undergo deformation or denaturation, and their functional properties such as solubility are preserved. Using the method according to the present invention, dried or concentrated liquid algal proteins can be obtained.

[0015] The method according to the present invention uses fresh algae. In the context of the present invention, “fresh algae” means algae as harvested, without further processing such as drying and freezing. Therefore, the term “fresh algae” does not include dried, powdered, rehydrated, frozen, siloed, or thawed algae. Fresh algae typically have a water content in the range of 80–95% w / w. Freshly harvested algae are those harvested more than four hours ago, such as three hours ago, two hours ago, or one hour ago.

[0016] Apart from algae, the harvest may contain other solids such as small marine organisms and plastics, which are preferably removed before processing the algae. Algae may constitute 86%–100% w / w of marine organisms at harvest. In preferred embodiments, algae constitute 90%–100% w / w or 99%–100% w / w of marine organisms in the harvest. Harvesting means separating algae from the surrounding water, such as recovering algae from a saltwater or freshwater location such as a lake, river, sea, estuary, or ocean. Algae can be harvested by any suitable means, for example, by collecting freely floating algae, or by cutting or stripping algal biomass from nets and ropes on which they have been seeded and grown. In one embodiment, algae or seaweed are seeded on cultivation ropes. Harvesting then involves stripping the algae or seaweed from the ropes.

[0017] Within 30 minutes to 2 or 3 hours after harvesting algae from a marine or freshwater location, subject the algae to an osmotic shock to disrupt the algae and release the cell contents. The cell contents can be partially or completely released by the osmotic shock. The osmotic shock can disrupt all or part of the algal cells, for example, at least 50%, at least 60% or at least 75% of the algal cells. The osmotic shock can be carried out by any suitable means, such as using water, as long as the temperature does not exceed 30°C. The temperature during the osmotic shock is preferably between 4 and 30°C, 5 and 25°C, or 15 and 25°C. In one embodiment, deionized water is used to subject the seaweed to an osmotic shock. The deionized water is preferably used at a ratio of algae:water = 1:1 to 1:10 based on the weight of the wet algae. The osmotic shock treatment is usually carried out for 5 to 60 minutes, preferably 5 to 20 minutes.

[0018] Before subjecting them to an osmotic shock, the algae may be sized into small pieces, for example, by cutting or slicing them, or by placing them in a blender or cutter. In one embodiment, the algae are sized into small pieces in deionized water, which means that they are sized into small pieces while being subjected to an osmotic shock.

[0019] Immediately after or almost immediately after harvesting, before subjecting the algae to an osmotic shock, the algae may be washed to remove small marine organisms such as fish, crustaceans, crabs, etc., and debris such as plastics, driftwood, buoys, etc. For washing, for example, excess seawater at 4 to 30°C can be used.

[0020] Immediately after or almost immediately after harvesting, before subjecting the algae to an osmotic shock, the adhering seawater may be removed. The adhering seawater can be removed by low-speed centrifugation so as not to damage the algae. In one embodiment, the adhering seawater is removed by centrifugation at 100 to 500 rpm, preferably 100 to 200 rpm or 200 to 300 rpm.

[0021] In the context of the present invention, the term “algae” means a group of unicellular or multicellular photosynthetic eukaryotic non-vascular aquatic organisms that inhabit or submerge in seawater, brackish water, or freshwater and contain a variety of bioactive compounds used in agriculture, the cosmetics industry, the food or feed industry, or the pharmaceutical industry. Seaweed, in particular, is rich in polysaccharides that exhibit antimicrobial, antioxidant, and antiviral activity. Seaweed contains valuable molecules such as proteins, vitamins, spore elements, polyphenols, iodine, alginic acid and its derivatives, carrageenan, chlorophyll, carotenoids, and agar. The methods according to the present invention can be used to treat algae, in particular also called seaweed or macroalgae, including red algae (Rhodophyta), green algae (Chlorophyta), and brown algae (Ochrophyta, Phaeophyceae). Algae that can be used in the method according to the present invention include, but are not limited to, Alaria, Ascophyllum, Caulerpa, Chondrus, Durvillaea, Enteromorpha, Fucus, Gracilaria, Laminara, Pelvetia, Pyropia, Porphyra, Sargassum, Saccharina, Ulva, and Undaria species.Of particular interest are Ascophyllum nodosum, Chondrus crispus, Enteromorpha intestinalis, Fucus spiralis, Fucus vesiculosus, Gracilaria bursa-pastoris, Gracilaria crassa, Gracilaria dura, Gracilaria longa, Gracilaria verrucosa, Laminaria digitata, Laminaria ochroleuca, Laminaria pallida, and Lessonia nigressence. nigrescens), Macrocystis integrifolia, Macrocystis pyrifera, Nemacystus decipiens, Nereocystis luetkeana, Palmaria palmata, Porphyra purpurea, Porphyra umbilicalis, Saccharina japonica, Saccharina latissima, Saccharina longicruris, Saccharina sessilis, Sargassum filipendula, Sargassum Examples include fusiforme, Sargassum muticum, Ulva intestinalis, Ulva compressa, Ulva lactuca, and Undaria pinantifida.In one embodiment, one or more of the above species, or a combination of two or more of the mentioned species, are processed by the method according to the invention. In a preferred embodiment, green algae, particularly of the genus Ulva, more particularly Ulva lactuca, are processed.

[0022] Within 2 to 3 hours after harvesting, the disrupted algal biomass is treated with a composition containing cell wall degrading enzymes. This composition is used to release proteins that are linked or associated with other cell masses such as cell walls and cell organelles. In one embodiment, the enzyme composition comprises at least 0.10% w / w of cell wall degrading enzymes, for example 0.10% w / w to 100% w / w, 0.20% w / w to 95% w / w, 0.50% w / w to 90% w / w, 1.0% w / w to 90% w / w, 1.0% w / w to 10% w / w, 1.0% w / w to 20% w / w, 10% w / w to 20% w / w, 20% w / w to 65% w / w, 70% w / w to 95% w / w or 80% w / w to 95% w / w, based on the total weight of the enzyme composition. In other embodiments, the enzyme composition consists of cell wall degrading enzymes.

[0023] Suitable cell wall-degrading enzymes that may be present in the enzyme composition include, but are not limited to, cellulase (EC3.2.1.4), xylanase (EC3.2.1.8 and EC3.2.1.32), beta-glucanase (EC3.2.1.6), amylase (EC3.2.1.1 and EC3.2.1.2), phytase (EC3.1.3.8 and EC3.1.3.26), phospholipase (PLA1, PLA2, PLB, PLC, PLD, EC3.1.1.4, EC3.1.4.11 and EC3.1.4.4) and polygalacturonase (EC3.2.1.15). In one embodiment, the cell wall-degrading enzyme is the primary activity in the enzyme composition, while in other embodiments, the cell wall-degrading enzyme is a secondary or minor activity. The enzyme composition comprises one or more of these cell wall-degrading enzymes. Those skilled in the art will understand that the optimal mixture of enzymes may vary between algal species and seasons. Therefore, any mixture of cell wall-degrading enzymes can be used, as long as it is used on fresh or newly harvested algae. In one embodiment, the destroyed algae are treated with an enzyme composition comprising a combination of cellulase, endoxylanase, beta-glucanase, alpha-amylase, beta-amylase, phytase, polygalacturonase, and phospholipase PLA2. In another embodiment, the destroyed algal biomass is treated with an enzyme composition comprising phytase (EC3.1.3.8) and phospholipases (PLA1, PLA2, PLB, PLC, PLD, EC3.1.1.4, EC3.1.4.11, and EC3.1.4.4). In other embodiments, the destroyed algal biomass is treated with an enzyme composition comprising endoxylanase (EC3.2.1.8) and phospholipases (PLA1, PLA2, PLB, PLC, PLD, EC3.1.1.4, EC3.1.4.11, and EC3.1.4.4). In one embodiment, an enzyme composition is used that comprises enzymes active against cellulose, xylan, beta-glucan, amylase, phytate, pectin, galacturonic acid, or phospholipids. In other embodiments, an enzyme composition is used that comprises beta-glucanase, phytase, poly-galacturonase, and phospholipase.In other embodiments, an enzyme composition is used comprising polygalacturonase having at least 20,000 AVJP / g activity; endo-1,3β-glucanase having at least 80 BGLU / g activity; fungal beta-glucanase having at least 100,000 BGF / g activity; bacterial amylase having at least 7,500 U / g activity; phytase having at least 5,000 FTU / g activity; phospholipase A2; and xylanase. Before use, these enzymes are mixed in a ratio of 1:1:1:1:1:1:1, and the mixture is used at a dose of 500 ml per 1000 kg of 100% dry biomass. These enzymes are commercially available, for example, from DSM (Delft, Netherlands).

[0024] Cell wall-degrading enzymes in enzyme compositions can be obtained by isolation from plants, bacteria, fungi, etc., by novel synthesis, or by mutagenesis of known enzymes.

[0025] Cell wall-degrading enzymes are used to release some or all of the intracellular proteins, including those that bind to or are associated with other cell clumps. In one embodiment, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, for example, 30% w / w to 60% w / w, 45% w / w to 70% w / w, or 50% to 90% w / w of intracellular proteins are released.

[0026] Any suitable dose of the enzyme preparation can be used. In one embodiment, a cell wall-degrading enzyme composition is used at a concentration of 0.001% w / w to 5% w / w, 0.005% w / w to 2% w / w, or 0.01% w / w to 1% w / w per 1000 kg of 100% dry matter biomass. The dry matter content of algae can be measured by any method known in the art, typically including, for example, drying a representative algal sample in an oven to remove all moisture from the algal sample or algae by evaporation, and measuring the weight of the sample before and after drying.

[0027] Depending on the enzyme dose, it may take approximately 5 minutes to 50 hours, 30 minutes to 48 hours, 6 hours to 24 hours, 18 hours to 30 hours, or 30 hours to 50 hours to make some or all of the intracellular proteins available. In one embodiment, the disrupted algal biomass is enzymatically treated for 24 to 48 hours. During the enzymatic treatment, the temperature does not exceed 30°C, preferably 4 to 30°C, 5 to 25°C, or 15 to 25°C.

[0028] It is not necessary to adjust the pH at any stage of the process. The pH used is typically in the range of pH 5.5 to pH 7.5, for example, pH 6.0 to 7.5.

[0029] After enzymatic treatment, the solid and liquid phases are separated, preferably by centrifugation or filtration (step (iii)). Centrifugation is used to obtain a pellet and a protein-containing supernatant. Filtration is used to obtain a retaining solution and a protein-containing filtrate.

[0030] Proteins can typically be recovered from the supernatant or filtrate by concentration. Concentration can be carried out by any suitable means such as ultrafiltration, centrifugation, precipitation, or nanofiltration, as long as the temperature does not exceed 30°C, preferably between 4–30°C, 5–25°C, or 15–25°C. The concentrated protein can be stored until use. The concentrated protein can be easily transported to another location, for example, on land if harvesting and processing are carried out offshore. Proteins isolated from fresh algae are more functional than commercially available algal proteins mainly prepared from dried algae and can be used in known applications of the protein or algal protein in the food, feed, cosmetics, or pharmaceutical industries, such as as foaming agents, gelling agents, thickeners, emulsifiers, colorants, pigments, antioxidants, or antimicrobial agents.

[0031] Optionally, the protein is dried onshore or offshore, preferably by an immediate drying method such as spray drying. In one embodiment, the protein is dried immediately by spray drying using a box dryer (Sanovo technology A / S, Odense, Denmark). Suitable conditions for box drying are, for example, an inlet temperature in the range of 175–185°C and an outlet temperature in the range of 90–97°C. The protein may be concentrated before or during drying.

[0032] Using the method according to the present invention, proteins can be obtained in high yields, such as at least 65% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 85% w / w, or at least 90% w / w, based on the total protein of fresh algae.

[0033] The solid phase obtained in step (iii) after protein separation, such as pellets or holding liquid, contains carbohydrates including sugars, fats, oils, and minerals. The carbohydrates, which are the main components of the solid phase, can be used in the production of renewable energy such as biogas, bioethanol, and bioplastics. The minerals can be used as fertilizer. The oils and fats can be used in animal feed, food, cosmetics, and pharmaceuticals. Therefore, after protein extraction, the remaining biomass can be used to extract further value.

[0034] Many suitable methods for producing biogas from biomass by anaerobic digestion have been published, such as a one-step process using one reactor and a two-step process using two reactors, both of which include (a) hydrolysis and acidification, and (b) acetic acid production or methane production. In the two-step process, steps (a) and (b) are carried out in separate reactors or compartments. Both of these steps can be carried out microbiologically. Alternatively, as described in WO2013 / 000928, for example, step (a) is carried out enzymatically using an enzyme preparation and step (b) is carried out microbiologically. In a preferred embodiment of biogas production, the solid phase remaining after protein extraction is subjected to a two-step process, first to enzymatic treatment before microbial digestion by methane-producing organisms. Suitable carbohydrate-degrading enzymes that may be present in the enzyme composition include, but are not limited to, amylase (EC3.2.1.1 and EC3.2.1.2), glucose oxidase (EC1.1.3.4), cellulase (EC3.2.1.4), xylanase (EC3.2.1.8 and EC3.2.1.32), beta-glucanase (EC3.2.1.6), phytase (EC3.1.3.8 and EC3.1.3.26), phospholipase (PLA1, PLA2, PLB, PLC, PLD, EC3.1.1.4, EC3.1.4.11 and EC3.1.4.4) and polygalacturonase (EC3.2.1.15). The enzyme composition may contain one or more carbohydrate-degrading enzymes. Those skilled in the art will understand that the optimal mixture of enzymes may vary between algal species and seasons. Therefore, any mixture of carbohydrate-digesting enzymes can be used, as long as it is used on fresh or newly harvested algae. In one embodiment, the carbohydrate-digesting enzyme is the primary activity in the enzyme composition, while in other embodiments, the carbohydrate-digesting enzyme is a secondary or minor activity in the enzyme composition. All enzymes are commercially available, for example, from DSM (Delft, Netherlands). Enzyme preparations can be used in any appropriate dose.In one embodiment, an enzyme mixture is used that comprises cellulose having an activity of at least 3500 CMC U / g, glucanase having an activity of at least 100,000 BGF / g, xylanase having an activity of at least 120,000 AVJP / g, and phytase having an activity of at least 5000 FTU / g, preferably containing the enzymes in a weight ratio of 1:2:2:1, and the mixture is used at a dose of 0.05 ml per kg of biomass after protein extraction.

[0035] Preferably, the enzymatic hydrolysis and microbial digestion of carbohydrates are carried out in separate reactors or separate compartments. Any suitable reactor configuration, such as a continuous stirred tank reactor (CSTR), a sequential batch reactor (SBR), or an anaerobic membrane bioreactor (AnMBR), can be used for biogas production according to the present invention. Preferably, more sophisticated systems such as an upward flow anaerobic sludge blanket (UASB) or an expanded granular sludge bed (EGSB) are used.

[0036] Biogas production from fresh algae from which the protein fraction has been removed first is highly advantageous compared to conventional biogas processes in which proteins are not removed before enzymatic treatment, as it can result in higher biogas yields and cleaner biogas with fewer nitrogen impurities. Biogas yields can be 250-600 Nm3, e.g., 300-500 Nm3 or 400-500 Nm3, per 1000 kg of 100% dry weight algae with a residence time of 12 hours, at 60%-80%, e.g., 70% methane. In one embodiment, 460 Nm3 of biogas was produced per 1000 kg of 100% dry weight seaweed with a residence time of 12 hours, at 70% methane.

[0037] Therefore, clean biogas produced from fresh seaweed after removing almost all or all algal proteins is another embodiment of the present invention. Those skilled in the art will understand that after separating the solid phase for biogas production, proteins may or may not be extracted from the protein-containing liquid phase. In either case, clean biogas is obtained. Therefore, in one embodiment, the present invention is (i) Subject algae to osmotic shock; (ii) Treat the shock-treated algae with an enzyme composition containing a cell wall-degrading enzyme; (iii) Separate the enzyme-treated algae into solid and liquid phases, where the enzyme treatment of the algae is started within 3 hours after harvest, and the temperature from step (i) to step (iii) is in the range of 4 to 30°C; (iv) The solid phase obtained in step (iii) is used to produce biogas, preferably by enzymatic hydrolysis using a carbohydrate-degrading enzyme followed by methane production by microorganisms; (v) Optionally, recover the protein from the liquid phase obtained in step (iii). Regarding the method.

[0038] Biogas is preferably produced by a two-step process involving enzymatic hydrolysis of a solid phase using a carbohydrase enzyme preparation, followed by methane production by microorganisms. Alternatively, the solid phase can be converted into synthesis gas using gasification processes known in the art.

[0039] In other embodiments, the present invention relates to algal protein preparations obtained by the method according to the present invention, i.e., by processing fresh algae. Algal protein preparations obtained from fresh algae have excellent functional protein properties, such as solubility. Their solubility is typically, for example, two or three times better than that of proteins prepared from preserved seaweed, such as dried seaweed. In one embodiment, protein preparations obtained from fresh algae have a solubility of at least 55% w / w, at least 60% w / w, at least 65% w / w, or at least 70% w / w, based on total protein dispersed at a pH in the range of pH 3 to 10. The solubility is also good in the presence of NaCl, and is typically, for example, three or four times better than the solubility of proteins prepared from preserved seaweed, such as dried seaweed. In one embodiment, a protein preparation obtained from fresh algae has a solubility of at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, or at least 80% w / w in the presence of NaCl, based on the dispersed total protein. In one embodiment, the protein according to the present invention has a solubility of 1 mg / ml at any NaCl concentration in the range of 0.05 to 3% w / v NaCl. In one embodiment, the protein according to the present invention has a solubility of at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, or at least 80% w / w at any pH in the range of pH 3 to 10, and a solubility of at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, or at least 80% w / w in the presence of 0.05 to 3% w / v NaCl. Solubility at different pH values ​​or NaCl concentrations can be measured, for example, by dispersing a certain amount of the dried product in desalted water, then setting the pH with phosphoric acid or sodium hydroxide, or setting the salt concentration with NaCl, and then centrifuging the dispersion / solution. The solubilized protein can be measured, for example, by Pierce TMThe protein can be measured in the supernatant by measuring the absorption at 562 nm using a commercially available kit such as the BCA protein assay kit (Thermo Scientific, Bleiswijk, Netherlands) with a spectrometer. Proteins may have an isoelectric point in the pH range of 7.5 to 8.5.

[0040] Those skilled in the art will understand that the exact amino acid composition of the resulting protein may vary depending on the type of seaweed, but the protein according to the present invention may have high levels of asparagine or glutamine, for example, at least 10% w / w or at least 15% w / w based on the weight of total amino acids. It may also have substantial amounts of glycine, proline or alanine, for example, at least 5% w / w, at least 7% w / w or at least 10% w / w based on the weight of total amino acids.

[0041] Proteins can also possess good nutritional properties as well as functional properties such as foaming, emulsifying, or water-binding capabilities, and can be used in a variety of applications, particularly in food, pharmaceutical, cosmetic, and animal feed.

[0042] In one embodiment, the present invention has the following features: a) Containing at least 10% w / w or at least 15% w / w of asparagine or glutamine based on total amino acid weight; b) Based on total amino acid weight, containing at least 5% w / w, at least 7% w / w, or at least 10% w / w of glycine, proline, or alanine; c) containing solubility of at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, or at least 80% w / w at any pH in the range of pH 3 to 10; d) Solubility of at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, at least 75% w / w, or at least 80% w / w at any NaCl concentration in the range of 0.05 to 3% w / v NaCl; e) Contains a solubility of at least 1 mg / ml at any NaCl concentration in the range of 0.05 to 3% w / v NaCl; f) The isoelectric point is located within the pH range of pH 7 to pH 9; g) Contains emulsifying properties comparable to egg protein, The present invention relates to algal proteins or protein preparations containing two or more of the following features. In one embodiment, the algal protein or protein preparation according to the present invention has all of these features.

[0043] In other embodiments, the present invention relates to an apparatus 1 (Figure 1) for processing algae according to the processing method of the present invention. Apparatus 1 is mobile and can therefore be moved to or transported to a harvesting site. The apparatus includes a harvesting unit 2 for harvesting, cleaning and washing; a processing unit 3 for enzymatic digestion of the harvested material; and a recovery unit 4 for separation and concentration of proteins. The apparatus may optionally further include an anaerobic digestion processing unit 6, a recovery unit 7 for recovery of products from 6, optionally concentration, and a blending unit 5 for blending products from recovery units 4 or 7. The apparatus may include a mobile unit that can be navigated or towed to a place where algae are proliferating, growing or being cultivated, such as a ship, barge, or vessel, or may be installed in, on, or on the mobile unit. In this way, algae can be harvested at a specific location where algae are present at a specific time of year.

[0044] In one embodiment, the present invention relates to a method for processing algae, and this method is (i) Subject algae to osmotic shock; (ii) Treat the shock-treated algae with an enzyme composition containing a cell wall-degrading enzyme; (iii) Separate the enzyme-treated algae into solid and liquid phases, where the enzyme treatment of the algae is started within 3 hours after harvest, and the temperature from step (i) to step (iii) is in the range of 4 to 30°C; (iv) Dry the liquid phase of step (iii), preferably by spray drying, to obtain the protein; (v) Optionally, use the solid phase obtained in step (iii) to produce biogas, bioplastics, or bioethanol. Here, steps (i) to (v) are performed offshore on a mobile device according to the present invention. Includes.

[0045] Those skilled in the art will understand that new embodiments can be formed by combining the embodiments described above. The embodiments and preferred embodiments of the processing methods described can also be applied to products of the processing methods according to the present invention, such as proteins, biogas, mineral streams, and mobile devices, and vice versa. [Examples]

[0046] material and method Enzyme preparation A The enzyme preparations used to release the cell contents contained the following enzymes (all from DSM, Delft, and the Netherlands unless otherwise specified): -2ml Cellulase (Filtrase BRX) -2ml xylanase (Filtrase NLC), -2ml Amylase (MATS classic), -2ml Phytase (Phytase 5000L), -2 ml phospholipase A2 (Purifinae PLA2), and -90ml desalinated water -2g of beta-glucanase / endoxylanase (Battonage, Oenobrands, Montferrer-sur-lez, France) was dissolved in liquid form with gentle stirring. When diluting the enzyme preparation, it was diluted with desalted water.

[0047] Example 1 Treatment of green algae using the method according to the present invention Fresh green algae (Ulva Lactuca, 5 kg) were harvested and washed with excess fresh seawater at a maximum temperature of 20°C. The attached water was removed by slow centrifugation. Next, 5 liters of desalinated cold water were added, and the mixture was shredded in a blender until fragments of approximately 1 square mm were obtained. The shredded biomass was divided into two parts. One part was given 25 ml of desalinated water as a control. The other part was incubated with 25 ml of enzyme preparation A diluted 10-fold to release cellular contents within 1 hour after harvesting.

[0048] Both portions were stirred at room temperature for 24 hours (actually below 20°C), then centrifuged at 4°C and 4500 rpm for 10 minutes, and the supernatant was collected. The pellet was resuspended in desalinated water, centrifuged again, and the supernatant was collected and added to the first supernatant. The collected supernatant was frozen until further analysis. Further analysis showed that the supernatant contained protein. This demonstrates that protein can be separated from algae using the gentle process according to the present invention. The protein was dried by spray drying the supernatant and stored for further analysis.

[0049] Example 2 Treatment of red algae using the method according to the present invention Fresh red algae (Gracillaria, 5 kg) were harvested and washed with excess fresh seawater at a maximum temperature of 20°C. The attached water was removed by slow centrifugation, the algae were cut into approximately 1 square mm fragments, and further processed as described in Example 1, including enzymatic treatment within 2 hours of harvesting. The recovered supernatant was frozen for further analysis. Further analysis showed that the Gracillaria supernatant contained protein. Another type of red algae, fresh Chondrus, was also harvested and processed using the protocol described in Example 1, including enzymatic treatment within 2 hours of harvesting. The recovered supernatant was frozen for further analysis. Further analysis showed that the Chondrus supernatant contained protein. This demonstrates that proteins can be isolated from red algae using the mild process according to the present invention.

[0050] Example 3 Treatment of brown algae using the method according to the present invention Fresh brown algae (Fucus) were harvested and washed with excess fresh seawater at a maximum temperature of 20°C. The attached water was removed by slow centrifugation, and the algae were cut into approximately 1 square mm fragments and further processed as described in Example 1, including enzymatic treatment within 2 hours of harvesting. The recovered supernatant was frozen until further analysis. Further analysis showed that the supernatant contained protein. This example demonstrates that the method according to the present invention can also be used for protein isolation from brown algae.

[0051] Example 4: Large-scale processing of fresh Ulva for protein separation and biogas production in a cascade process. Green algae (Ulva lactuca, 76 kg) were harvested and washed with excess fresh seawater at a maximum temperature of 20°C. The attached water was removed by slow centrifugation. One kg portion of seaweed was mixed with one liter of desalinated water and shredded into approximately 1 square mm fragments using a robotic coupe cutter R10. An additional liter of desalinated water and the shredded seaweed were placed in a new 1000 liter IBC. The total of 76 kg of Ulva was incubated for 24 hours in 600 liters of desalinated water with gentle agitation within 3 hours of harvesting. At the beginning of the 24-hour incubation period, enzyme preparation A (500 ml of enzyme mixture / 1000 kg of 100% dry biomass) was added. After the incubation period, the remaining seaweed solids were removed by filtering the entire slurry through a four-fold cheesecloth. The liquid, protein-containing fraction, and filtrate passing through cheesecloth were collected, and the protein was dried by spray-drying the supernatant using a box dryer (Sanovo technology A / S, Odense, Denmark) with an inlet temperature of 180°C and an outlet temperature of 94°C. The spray-dried protein was then characterized as described in the following examples. The protein extraction efficiency of the method according to the present invention was approximately 81% w / w, based on dry weight and as a percentage of the total protein present in the starting material, as shown in Table 1. The protein was measured using a BCA assay (Thermo Scientific, Bleiswijk, Netherlands).

[0052] [Table 1]

[0053] After protein extraction, to hydrolyze the carbohydrates as much as possible into a solution, the solids held in cheesecloth were then re-incubated for 48 hours with a carbohydrase enzyme preparation containing cellulase Methaplus L100 (>3500 CMC U / g) / glucanase Axiase 100 (>120000 AVJP / g) / xylanase Filtrase NLC (>100000 BGF / g) and phytase Maxamyl P (>5000 FTu / g) in a weight ratio of 1:2:2:1 (all enzymes from DSM, Delft, Netherlands). The mixture was administered at a rate of 0.05 ml (mix) per 1 kg of wet biomass. These hydrolyzed solids were transferred to an anaerobic digester (Hydothane's EGSB-skid) to produce biogas and a mineral-rich water stream. The mineral stream can be used as fertilizer. Biogas conversion demonstrated that no pH stabilizers or additional nutrients were needed, and with a residence time of 12 hours, it was possible to achieve a result of 460 Nm3 biogas / 1000 kg of seaweed at 70% methane and 100% dry weight. This is a much higher yield than would have been expected if all microbial processes were used, i.e., without enzymatic treatment, or if the whole seaweed was used without first extracting the protein.

[0054] Example 5: SDS-Page analysis of GOA protein isolated according to the method of the present invention. The spray-dried proteins obtained in Example 4 were analyzed using SDS-PAGE. Electrophoresis was performed according to Laemmli (1970) Nature 227:5259. Seaweed was dried with hot air, ground into flakes, stored for 8 weeks, and then the proteins were separated. A protein sample obtained from Ulva (Hello Seaweed, Fuzhou Beautiful Agricultural Development Co, China) was used as a reference. For SDS-PAGE analysis, both samples were exposed to fresh water. Both were rehydrated for several hours with gentle agitation, while the dried seaweed sample and the GOA sample were rehydrated for the same amount of time. After centrifugation, the pellet was discarded and the supernatant containing the proteins was collected. Aliquots of each sample were pipetted from the supernatant and diluted with extraction buffer (1.5% SDS, 20% glycerol, 0.01% bromophenol blue) to similar protein concentrations. Gel protein bands were created using a fast-stain ready-to-use gel (SERVA electrophoresis GmbH, Germany). Gel analysis was performed using the freeware GelAnalyzer2010. A marker set of 6kDa–67kDa was used. SDS-PAGE analysis showed that GOA protein samples obtained from fresh Ulva seaweed contained more proteins with molecular sizes greater than 40kDa than the Ulva reference sample obtained from dried material. The GOA samples had almost twice the amount of 27–30kDa proteins and fewer smaller proteins in the 6kDa range (Table 2). These results indicate that protein samples obtained from fresh seaweed contain more intact proteins and less degraded proteins than samples from dried seaweed. More complete proteins also mean more functional proteins. Protein degradation is detrimental to functional properties such as emulsification, viscosity, and heat-set gelation.

[0055] [Table 2]

[0056] Example 6 Amino acid composition of the protein sample according to the present invention The amino acid composition of the spray-dried protein sample from Example 4 was measured by acid hydrolysis and HPLC and is shown in Table 3. The protein contained high levels of asparagine (15% of total amino acids) and glutamine (20% of total amino acids), as well as substantial amounts of glycine, proline, and alanine.

[0057] [Table 3]

[0058] Example 7: Protein solubility at different pH values To analyze the protein solubility of the spray-dried protein samples (GOA samples) obtained in Example 4, 1 g of the dried GOA protein sample was dispersed in 100 ml of desalted water. The same experiment was performed on samples obtained from dried and pulverized Ulva. For the Ulva samples, an equivalent amount of protein was loaded into the test using 2 g of dried Ulva / 100 ml of desalted water. The dispersions were set to pH 3, 4, 4.5, 5, 6, 7, 8, and 9 using phosphoric acid (low pH) and sodium hydroxide (high pH). The dispersions / solutions were then centrifuged. 100 microliter aliquots were pipetted from the supernatant of each test tube and pierced using a Shimadzu UV / VIS spectrometer at 562 nm. TMThe samples were diluted to a concentration range suitable for soluble protein analysis using the BCA protein assay kit (Thermo Scientific, Bleiswijk, Netherlands). The measured protein mg / ml was then corrected for the sample weight and dilution factor used in the BCA assay. The solubility of seaweed protein remained relatively constant with little variation. Table 4 compares the solubility of Ulva and GOA proteins, clearly showing that the solubility of the GOA protein sample is 3–12 times higher than that of the Ulva reference sample. The average protein solubility of the GOA protein sample is 5 times higher. The results show that the variability of Ulva is 38%, compared to only 5% for GOA. This indicates that Ulva is much more pH-sensitive than GOA, and that Ulva exhibits minimal solubility at pH 6. Seaweed offers much higher solubility and allows for the preparation of protein samples that are much more consistent across the pH range than protein samples from dried seaweed.

[0059] [Table 4]

[0060] The presented results also show the different isoelectric points of the three protein samples. The decrease in solubility indicates the isoelectric point. Thus, the GOA sample has an isoelectric point around pH 8, while the Ulva reference sample has an isoelectric point around pH 6. These results indicate that proteins isolated from fresh seaweed are proteins with different isoelectric points, and consequently exhibit different pH behaviors in their applications.

[0061] [Table 5]

[0062] Example 8 Salt sensitivity of protein samples from fresh seaweed To analyze the solubility of proteins in the presence of different concentrations of NaCl, 1 g of the spray-dried protein sample (GOA sample) obtained in Example 4 was dispersed in 100 ml of desalinated water. The same experiment was performed as a reference for egg albumin (EA) samples and samples obtained from dried and ground Ulva. 2 g of the Ulva sample was used to load an equivalent amount of protein in the test. Solubilized proteins from GOA, Ulva, and EA were diluted with desalinated water to reduce the salinity effect of the samples. The diluted samples were then set to a NaCl concentration of 0-3%. The protein solutions were centrifuged and pierced using a Shimadzu UV / VIS spectrometer at 562 nm. TM For soluble protein analysis using the BCA protein assay kit (Thermo Scientific, Bleijswijk, Netherlands), 100 μl of the supernatant from each sample was pipetteed. The measured protein mg / mL was then corrected for the sample weight and dilution factor used in the BCA assay. Measurements were performed twice. At all salt concentrations, GOA protein samples exhibited significantly higher solubility than Ulva samples. For Ulva, on average, 44 ± 12 mg of protein was soluble with increasing salt concentration. For GOA, 192 ± 11 mg of protein was soluble with increasing salt percentage. The percentage of solubility in the presence of NaCl is shown in Table 6. The average protein solubility of GOA was four times that of Ulva, and the variability of Ulva was 27%, while the variability of GOA was only 6%. This indicates that Ulva is more sensitive to changes in salt concentration. This indicates that protein samples obtained from fresh seaweed according to the present invention have higher solubility than protein samples obtained from dried seaweed, and their stability is not affected by high salt concentrations.

[0063] [Table 6]

[0064] Example 9 Preparation of sponge cake Standard recipe (Italian cake version): 9 grams whole egg flour, 221 grams water, 210 grams sugar, 150 grams wheat flour, 75 grams potato starch. Whole egg flour was replaced with protein from soy, whey, sunflower, or GOA protein according to the present invention. The mixture was kneaded into the dough using Hobart N50. The dough was cut into 10 cm circles to prepare the cakes. The dough was baked in an oven at 180°C for 22 minutes, until the cakes were golden / yellow. The cakes were removed from the oven and allowed to cool. The cakes prepared from seaweed (GOA) protein had a very nice appearance, comparable to the cakes made from whole egg protein. Unlike cakes made from other proteins such as soy, whey, and sunflower, which crumbled to the touch or had a very rough / uneven surface, the (GOA) seaweed cakes maintained their round shape and did not crumble. This indicates that GOA (seaweed) protein can be used as a substitute for whole egg flour due to the consistency and baking properties of the dough.

[0065] Example 10: Preparation of 75% Mayonnaise A standard recipe (Sanovo egg group) was used: 15% egg yolk powder, 107 grams of water, 2 grams of salt, 375 grams of (canola) oil, and 10 grams of vinegar. All ingredients were added and mixed in a gentle, continuous order using a constant-speed mixer (Bosch 300W type 4179 or Hobart N50). The emulsion was stored at 2-5°C for 24 hours to stabilize. After 24 hours, viscosity was measured. For comparison, egg yolk powder was replaced with GOA (seaweed) protein, soy, pea, and sunflower protein. A whole egg powder / GOA (seaweed) protein mix (50 / 50) was also tested.

[0066] The mayonnaise derived from seaweed (GOA) protein obtained in Example 4 showed good consistency. The soy protein-mayonnaise emulsion or pea protein-mayonnaise emulsion was broken down before formation. This indicates that GOA protein has excellent emulsifying properties comparable to egg yolk. The GOA protein emulsion also exhibited excellent scoopability and spreadability. The whole egg powder / GOA protein mixture did not perform inferiorly to the individual pure proteins.

[0067] The main difference observed with GOA protein was the extremely low syneresis of the emulsion. The latter indicates that GOA protein forms a more stable emulsion compared to whole egg powder, and significantly more stable than the other tested emulsions of soy, pea, or sunflower protein. After refrigeration at 2-5°C for 4 weeks, the GOA (seaweed) protein emulsion still showed no syneresis, while all others became almost completely liquid. The experiment was stopped due to mold growth in all samples. In conclusion, a) the protein isolated by the method according to the present invention enables superior emulsion formation. b) the mixture of protein and egg according to the present invention did not impair the emulsion properties. c) the protein according to the present invention exhibits significantly lower syneresis than tested proteins such as soy protein, whey protein, sunflower protein, or pea protein, offering new opportunities for the use of such emulsions in food applications.

Claims

1. (i) Subject seaweed to osmotic shock, (ii) The seaweed subjected to shock is treated with an enzyme composition containing a cell wall-degrading enzyme. (iii) Separating the enzyme-treated seaweed into solid and liquid phases. A method for processing seaweed, comprising the following characteristics: the enzymatic treatment of the seaweed is started within three hours of harvesting the algae, and the temperature from step (i) to step (iii) is in the range of 4 to 30°C.

2. The method according to claim 1, characterized in that the seaweed is processed at a temperature in the range of 5 to 25°C.

3. The method according to claim 2, characterized in that the seaweed is processed at a temperature in the range of 15 to 25°C.

4. The method according to any one of claims 1 to 3, characterized in that the osmotic shock lasts for 5 to 20 minutes.

5. The method according to any one of claims 1 to 4, characterized in that the pH is not adjusted during processing.

6. The method according to claim 5, characterized in that the pH during processing is pH 5.5 to 7.

5.

7. The method according to any one of claims 1 to 6, characterized in that the cell wall-degrading enzyme composition comprises one or more of cellulase, xylanase, alpha-amylase, beta-amylase, phytase, polygalacturonase, and phospholipase, and beta-glucanase.

8. The method according to any one of claims 1 to 7, further comprising drying the liquid phase of step (iii) to obtain seaweed protein.

9. The method according to claim 8, wherein the drying is spray drying.

10. The method according to claim 8 or 9, characterized in that the yield of seaweed protein is at least 65% w / w based on the total protein in the seaweed.

11. The method according to any one of claims 1 to 10, further comprising using the solid phase obtained in step (iii) to produce biogas, bioplastics, bioethanol, minerals, fats, or oils.

12. The method according to any one of claims 1 to 11, characterized in that biogas is produced from a solid phase obtained in step (iii) by a two-step anaerobic digestion process including hydrolysis by a carbohydrate-degrading enzyme preparation and subsequent methane production by microorganisms.

13. The method according to claim 12, characterized in that anaerobic digestion generates a mineral stream to be recovered.

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