Method for producing polysaccharide

Abstract

The present invention addresses the problem of providing: a method for producing a polysaccharide having excellent gelling performance and functionality; and a polysaccharide obtained by the method. The problem is solved by a method for producing a polysaccharide, the method including extracting and recovering a polysaccharide from seaweed cultured in a low nutrient medium in a container.

Description

Polysaccharide manufacturing method

[0001] The present invention relates to a method for producing a polysaccharide.

[0002] Algae that can grow in water are easy to cultivate, and seaweeds in particular can utilize the abundant seawater, which allows for low cultivation costs, so they are attracting attention as a source of biological materials such as polysaccharides such as alginic acid and biofuels.

[0003] It is known that Ulva algae, which mainly live in marine areas, produce a polysaccharide called ulvan (Non-Patent Document 1). It has been confirmed that ulvan extracted from Ulva spp., a species of Ulva spp., gels when dissolved at high concentrations. However, ulvan has poor performance compared to other gelling agents, making it unsuitable for practical use, and has not yet been put to practical use. Furthermore, conventional methods for extracting polysaccharides from Ulva spp. typically involve drying and powdering the algae, followed by hot water extraction and subsequent recovery using ethanol, which requires many steps. Ulvan's functionality is also weaker than that of other polysaccharides, which also prevents its practical use.

[0004] Chem. Pharm. Bull. 69, 432-443 (2021)

[0005] An object of the present invention is to provide a method for producing a polysaccharide having excellent gelling properties and functionality, and a polysaccharide obtained thereby.

[0006]

[0009] In view of the above problems, the present inventors have conducted extensive research and found that the above problems can be solved by a method for producing polysaccharides, which includes extracting and recovering polysaccharides from seaweed cultured in a low-nutrient medium in a container. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0007] Item 1. A method for producing polysaccharides, comprising extracting and recovering polysaccharides from seaweed cultivated in a low-nutrient medium in a container.

[0008] Item 2. The method according to Item 1, wherein the nitrogen concentration of the low-nutrient medium is 0 to 0.042 mg / L.

[0009] Item 3. The method according to Item 2, wherein the phosphorus concentration of the low-nutrient medium is 0 to 0.003 mg / L.

[0010] Item 4. The method according to any one of Items 1 to 3, wherein the seaweed is a seaweed with a high starch granule content, that is, the number of starch granules per cell is 10 or more.

[0011] Item 5. The production method according to any one of Items 1 to 4, wherein the culture period in the low-nutrient medium is 2 days or longer.

[0012] Item 6. The method according to any one of Items 1 to 5, wherein the seaweed is an alga of the genus Ulva.

[0013] Item 7. The method according to any one of Items 1 to 6, wherein the solvent used for extracting and recovering the polysaccharide is water.

[0014] Item 8. A polysaccharide obtained by the production method according to Item 1.

[0015] Item 9. A polysaccharide having a ratio of neutral sugars to acidic sugars of 5.0 or less and / or containing sugars having sulfate groups bound thereto.

[0016] Item 10. The polysaccharide according to Item 9, wherein the ratio of neutral sugars to acidic sugars is 2.2 or less.

[0017] Item 11. The polysaccharide according to Item 9 or 10, wherein the ratio is 1.5 to 2.2.

[0018] Item 12. The polysaccharide according to any one of Items 9 to 11, which is derived from algae of the genus Ulva.

[0019] Item 13. A composition comprising the polysaccharide according to any one of Items 8 to 12.

[0020] Item 14. The composition according to Item 13, which is a gelling agent, a thickener, a disintegrant, a water-absorbing agent, a cosmetic composition, a composition for external use, a food composition, or a pharmaceutical composition.

[0021] Item 15. The composition according to Item 13, which is a gelling agent, a moisturizing agent, a skin barrier function improving agent, a leaky gut inhibitor, an anti-aging agent, or an anti-inflammatory agent.

[0022] The present invention also encompasses the following aspects: The inventions of the following items 1A to 9A can be considered as generic or subordinate concepts of the inventions of the above items 1 to 15.

[0023] Item 1A. A polysaccharide extracted from seaweed, characterized in that a solution of a composition containing the polysaccharide in an aqueous solvent gels at room temperature (20°C) without undergoing a heating step.

[0024] Item 2A: The polysaccharide according to Item 1A, wherein a solution of the polysaccharide-containing composition in an aqueous solvent gels at a concentration of 0.8 w / v % or more.

[0025] Item 3A: The polysaccharide according to Item 1A or 2A, which can form a gel having a gel strength of 300 g cm or more when the polysaccharide solution in an aqueous solvent is 3 w / v % or more.

[0026] Item 4A. The polysaccharide according to any one of Items 1A to 3A, wherein the polysaccharide is extracted from seaweed cultivated in a low-nutrient medium.

[0027] Item 5A. The polysaccharide according to Item 4A, wherein the phosphorus concentration of the low-nutrient medium is 0 to 0.003 mg / L.

[0028] Item 6A: The polysaccharide according to any one of Items 1A to 5A, wherein the polysaccharide is derived from algae of the genus Ulva.

[0029] Item 7A: A composition comprising the polysaccharide according to any one of Items 1A to 6A.

[0030] Item 8A. The composition according to Item 7A, which is a gelling agent, a thickener, a disintegrant, a water-absorbing agent, a cosmetic composition, a composition for external use, a food composition, or a pharmaceutical composition.

[0031] Item 9A. The composition according to Item 7A, which is a gelling agent, moisturizing agent, skin barrier function improving agent, leaky gut inhibitor, anti-aging agent, or anti-inflammatory agent.

[0032] According to the present invention, it is possible to provide a method for producing a polysaccharide having excellent gelling properties and functionality, and a polysaccharide obtained thereby.

[0033] This shows a micrograph of algal cells at the end of culture in Test Example 1. The bar in the lower right corner of the photograph indicates a length of 50 μm. "Normal culture" refers to the case where algal cells were cultured in ES medium and then collected, while "nutrient-depleted culture" refers to the case where algal cells were cultured in sterilized artificial seawater without added nutrients and then collected. The intracellular vesicles are starch granules. This shows an observation image of the gel obtained from a 3 wt.% aqueous solution in Test Example 2 after heating and cooling and returning to room temperature (20°C) for 30 minutes. This shows a photograph of Test Example 2 in which a 2 wt.% aqueous solution of each polysaccharide was prepared in a container, left at room temperature (20°C) for 30 minutes without heating, and then the contents of the container were transferred to a container with a larger diameter. This shows the water retention measurement results for Test Example 3. ** indicates a P value of less than 0.01 between the two groups. This shows the measurement results of mRNA amount (n = 3) in Test Example 4. The measured mRNA is shown on the left side of the vertical axis. On the horizontal axis, "control" indicates a negative control in which only solvent (water) was added, and the other values ​​indicate the substances that were added. The vertical axis indicates the relative amount when the control value is set to 1. ** indicates a P value of less than 0.01 when compared to the control. This shows the measurement results of blood LPS levels and FITC-dextran 4kDa in Test Example 5. * indicates a P value of less than 0.05 between groups, and ** indicates a P value of less than 0.01 between groups. This shows the measurement results of intestinal length in Test Example 5. * indicates a P value of less than 0.05 between groups, and ** indicates a P value of less than 0.01 between groups. This shows the measurement results of ATP production and SA-β-Gal-positive cells in Test Example 6. On the horizontal axis, "control" indicates a negative control in which only solvent (water) was added, and the other values ​​indicate the substances that were added. * indicates a P value of less than 0.05 between groups, and ** indicates a P value of less than 0.01 between groups.

[0034] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0035] 1. Method for Producing Polysaccharides In one aspect, the present invention relates to a method for producing polysaccharides (sometimes referred to as the "production method of the present invention" in this specification), which comprises extracting and recovering polysaccharides from seaweed cultured in a low-nutrient medium in a container. This method is described below.

[0036] The raw material for producing polysaccharides in the production method of the present invention is seaweed. Seaweed is not particularly limited as long as it is marine algae, but typically refers to algae whose algal bodies grow into multicellular or polynuclear bodies. Seaweed may be, for example, unbranched filamentous algae or branched filamentous algae, leaf-like algae, tubular algae, or algae with differentiated tissues. Preferred examples include edible seaweed, which is desirable for cultivation in industrial quantities, and seaweed that can be used as useful substances for livestock feed, pharmaceuticals, etc., or as a raw material for biofuels. Examples of seaweed include red algae, green algae, and brown algae. Examples of red algae include seaweeds belonging to the order Porphyra, family Porphyra, genus Porphyra, order Gracilaria, family Gracilaria, genus Porphyra, order Gelidaceae, order Agaricales, family Porphyra, family Porphyra, order Porphyra, family Porphyra, and order Porphyra, family Porphyra, and the like, of the class Rhodophyceae or class Bangiophyceae. Examples of such seaweeds include Porphyra, Gracilaria, Gracilaria, Asakusa-no-ori, Susabinori, Gelidium, Tosakanori, Igisu, and Mafunori. Examples of green algae include seaweeds belonging to the family Cladophorales, Cladophoraceae, genus Cladophora, family Ulvales, genus Ulva, family Aquilaria, family Aquilaria, etc., of the class Ulvaphyceae, such as Cladophora, lentillifera, laver, Cladophora, cladophora, cladophora perforata, and laver, etc. Examples of brown algae include seaweeds belonging to the family Laminariales, Laminariaceae, Laminariales, Pycnogonaceae, family Pycnogonaceae, family Pycnogonaceae, family Pycnogonaceae, etc., of the class Phaeophyceae, such as Laminaria japonica, wakame seaweed, Cladophora okinawaensis, kayamori, habanori, and Pycnogonaceae.

[0037] Particularly preferred examples of seaweed include algae of the genus Ulva. Ulva algae are algae that belong to the genus Ulva (sometimes called Ulva), and are not particularly limited thereto. Examples of Ulva genus algae include Ulva adhaerens, Ulva aragoensis, Ulva arasakii, Ulva californica, Ulva clathrata, Ulva compressa, Ulva conglobata, Ulva fenestrata, Ulva flexuosa, Ulva intestinalis, Ulva lactuca, Ulva lacinulata, Ulva limnetica, and Ulva limnetica. Examples include linza, Ulva meridionalis, Ulva ohnoi, Ulva australis, Ulva partita, Ulva prolifera, Ulva reticulata, Ulva rigida, Ulva spinulosa, Ulva sublittoralis, Ulva tanneri, and Ulva tepida. Among these, preferred are Ulva meridionalis, Ulva prolifera, and the like.

[0038] The seaweed may be one type alone or a combination of two or more types.

[0039] Cultivation is carried out in a container. The culture is usually aerated culture or static culture, but agitation culture using an agitator is also possible. The container is not particularly limited as long as it can be used for seaweed cultivation, and a suitable capacity can be selected depending on the scale of cultivation. For example, tanks, aquariums, petri dishes, flasks, beakers, etc. made of concrete, glass, plastic, etc. can be used.

[0040] A part of the container (for example, a wall, a bottom, etc.) may be buried underground. In this case, an underground component (for example, soil) may also be a part of the container (for example, a wall, a bottom, etc.).

[0041] The container may be connected to an external environment (eg, the sea), and in this case, it is preferably equipped with a mechanism (eg, a valve, a barrier, etc.) for adjusting the degree of connection with the external environment.

[0042] A low-nutrient medium is a medium that contains fewer nutrients, particularly nitrogen and / or phosphorus, than a normal medium (i.e., a medium intended for growth and cultivation), and is not particularly limited insofar as this is the case. A low-nutrient medium is preferably a medium having a nitrogen concentration of 0 to 0.042 mg / L. In such a medium, the nitrogen concentration is more preferably 0 to 0.03 mg / L, even more preferably 0 to 0.02 mg / L, even more preferably 0 to 0.01 mg / L, particularly preferably 0 to 0.005 mg / L, and particularly preferably 0 to 0.002 mg / L. The phosphorus concentration of the low-nutrient medium is more preferably 0 to 0.003 mg / L, even more preferably 0 to 0.001 mg / L, and even more preferably 0 to 0.0005 mg / L.

[0043] In this specification, the nitrogen concentration refers to dissolved inorganic nitrogen, and the phosphorus concentration refers to dissolved inorganic phosphorus. These concentrations can be measured according to known methods.

[0044] The culture period in a low-nutrient medium is, for example, 1 day or more, preferably 2 days or more, more preferably 3 days or more, and even more preferably 4 days or more. The upper limit of the culture period is not particularly limited, but is, for example, 30 days, 20 days, or 10 days.

[0045] The culture temperature is not particularly limited as long as it is suitable for the vegetative growth of seaweed, and is, for example, 5 to 35°C, preferably 10 to 30°C, more preferably 15 to 27°C, and even more preferably 20 to 27°C.

[0046] The light conditions during cultivation are not particularly limited as long as they are light conditions that allow seaweed to grow, and examples include natural light light-dark cycle conditions, artificial light conditions, and artificial light light-dark cycle conditions.

[0047] In the production method of the present invention, it is preferable to use seaweed with a high content of small starch granules, that is, seaweed having 10 or more small starch granules per cell, as the subject from which polysaccharides are extracted and recovered.

[0048] Starch granules are structures recognized as granules present within chloroplasts, and can be visually recognized as clearly visible vesicular structures within seaweed cells in microscopic images, as shown in Figure 1. From this perspective, one aspect of the present invention relates to a method for producing polysaccharides (also included in the "production method of the present invention"), which comprises extracting and recovering polysaccharides from vesicle-rich seaweed, in which the number of starch granules / vesicles per cell is 10 or more. The major axis of the vesicles / starch granules in microscopic images is, for example, 1 to 10 μm, preferably 2 to 8 μm, and more preferably 3 to 5 μm.

[0049] The number of starch granules / vesicles per cell is preferably 15 or more, more preferably 20 or more, and even more preferably 25 or more. The upper limit of this number is not particularly limited, and is, for example, 40, 60, or 80.

[0050] The number of starch granules / vesicles per cell is measured by the method described in Test Example 1 below.

[0051] A high content of small starch granules can be an indicator of the strain obtained by cultivation in a low nutrient medium.

[0052] In a more preferred embodiment, the seaweed obtained by culturing in a nutrient medium is subjected to culturing in a low-nutrient medium, which allows the number of starch granules / vesicles to be increased more effectively.

[0053] The nutrient medium is a medium with a relatively high concentration of nutrients, particularly nitrogen and / or phosphorus, intended for the purpose of propagation and cultivation. The concentrations vary depending on the type of seaweed and can be set appropriately. For example, in the case of Ulva algae, the nutrient medium is preferably a medium with a nitrogen concentration of 1 to 100 mg / L and / or a phosphorus concentration of 0.1 to 10 mg / L. In this medium, the nitrogen concentration is more preferably 2 to 50 mg / L, even more preferably 3 to 20 mg / L, and even more preferably 5 to 12 mg / L, and the phosphorus concentration is more preferably 0.2 to 5 mg / L, even more preferably 0.3 to 2 mg / L, and even more preferably 0.5 to 1.2 mg / L.

[0054] The cultivation period in the nutrient medium is, for example, 1 to 20 days, or 2 to 10 days.

[0055] The conditions for culturing in a nutrient medium are the same as those for culturing in a low-nutrient medium.

[0056] The medium is not particularly limited as long as it is a liquid suitable for seaweed growth, but examples include ES medium, f / 2 medium, seawater, etc., with the addition of seaweed cultivation nutrients such as nitrates and phosphates as needed. Commercially available nutrients can be used. The seawater is not particularly limited as long as it is a liquid suitable for seaweed tissue growth, but examples include a salt concentration of more than 0% but not more than 5%, more than 1% but not more than 4%, or more than 3% but not more than 3.8%. Either natural seawater or artificial seawater can be used. Artificial seawater refers to freshwater artificially adjusted to mimic the composition of natural seawater by adding salts such as sodium chloride, potassium chloride, calcium chloride, magnesium chloride, and magnesium sulfate, pH buffers, and other trace elements. It also includes seawater from which salt and other components have been removed and / or active ingredients have been added. Seawater may be sterilized or filtered before use.

[0057] The proportion of seaweed with a high content of small starch granules relative to 100% by mass of seaweed used for polysaccharide extraction and recovery is preferably higher, since the higher the proportion, the more polysaccharides with excellent gelling properties and functionality can be obtained. For example, the proportion is 70% by mass or more, preferably 80% by mass or more, more preferably 90% by mass or more, even more preferably 95% by mass or more, even more preferably 99% by mass or more, and particularly preferably 100% by mass.

[0058] From the viewpoint of efficiency of polysaccharide extraction and recovery, the seaweed used for polysaccharide extraction and recovery is preferably formed into small pieces or particles by shearing, crushing, etc. Specifically, cut algae bodies, dried powder of algae bodies, etc. are preferred.

[0059] Extraction and recovery of polysaccharides from seaweed can be carried out according to or in accordance with known methods for extracting and recovering water-soluble polysaccharides. Specifically, water can be used as the solvent for extraction and recovery. While alcohols such as ethanol and other organic solvents can also be used as the solvent, it is preferable not to use them from the standpoints of simplicity, safety, and the like. From this standpoint, the ratio of water to 100% by mass of the solvent used for extraction and recovery is, for example, 70% by mass or more, preferably 80% by mass or more, more preferably 90% by mass or more, even more preferably 95% by mass or more, even more preferably 99% by mass or more, and particularly preferably 100% by mass.

[0060] The amount of extraction solvent used is, for example, 10 mL or more, preferably 15 mL or more, more preferably 20 mL or more per gram of dry weight of seaweed. There is no particular upper limit, and it is, for example, 200 mL, 100 mL, 50 mL, or 30 mL.

[0061] Polysaccharide extraction can be carried out by contacting seaweed with an extraction solvent. This transfers the polysaccharides in the seaweed to the extraction solvent. The contacting method is not particularly limited, and examples include immersing the seaweed in the extraction solvent and spraying the extraction solvent onto the seaweed. From the viewpoint of extraction efficiency, the former is particularly preferred.

[0062] The solvent temperature during extraction is not particularly limited and can be, for example, 0 to 100° C. From the viewpoint of extraction efficiency, the temperature is preferably 50 to 100° C., more preferably 60 to 95° C., and even more preferably 70 to 90° C.

[0063] The extraction time is not particularly limited and is, for example, 1 to 6 hours, preferably 2 to 4 hours.

[0064] During extraction, the mixture may be left to stand or may be stirred.

[0065] The polysaccharide can be recovered by recovering the liquid obtained by extraction (extract). It can also be purified and concentrated as appropriate. Examples include removal of insoluble matter (e.g., removal by solid-liquid separation such as centrifugation or filtration), heat drying, and freeze-drying. In a preferred embodiment, the extract or a concentrate thereof is gelled and then freeze-thawed, allowing for more efficient recovery of the gel-forming polysaccharide. In this case, the gel may be compressed by pressing to remove water, or may be freeze-dried and then washed with water. The resulting polysaccharide may be in a wet state or a dry state (e.g., powder form).

[0066] The polysaccharides obtained by the production method of the present invention tend to have a lower ratio of neutral sugars to acidic sugars compared to polysaccharides derived from conventional seaweed (particularly algae of the genus Ulva). This ratio is, for example, 5.0 or less, 4.0 or less, 3.0 or less, or 2.5 or less. This ratio is preferably 2.2 or less, more preferably 2.1 or less, even more preferably 2.0 or less, and even more preferably 1.95 or less. The lower limit of this ratio is, for example, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.85. This ratio is measured according to the method described in Test Example 1. In one aspect, the present invention relates to polysaccharides obtained by the production method of the present invention, and polysaccharides having a ratio of neutral sugars to acidic sugars of 5.0 or less and / or containing sugars having sulfate groups bound thereto. These may be collectively referred to herein as the "polysaccharides of the present invention."

[0067] 2. Polysaccharide-Containing Composition In one aspect, the present invention relates to a composition containing the polysaccharide of the present invention (sometimes referred to herein as the "composition of the present invention"). This will be described below.

[0068] The polysaccharides of the present invention have excellent gelling properties. For example, they can be gelled without heating. More specifically, they can be dissolved in water or an aqueous solution at, for example, 40°C or below, 30°C or below, or 25°C or below (e.g., 0.8 w / v% or more, 1.2 w / v% or more, 1.5 w / v% or more, 2.0 w / v% or more, 2.5 w / v% or more, 3.0 w / v% or more, and 10 w / v% or less, 8.0 w / v% or less, 7.0 w / v% or less, 6.0 w / v% or less, 5.0 w / v% or less, or 4.0 w / v% or less), and then allowed to stand at the above temperature for a certain period of time (e.g., 10 minutes or more, 20 minutes or more, or 30 minutes or more), thereby forming a gel. Furthermore, the polysaccharides of the present invention can be gelled even after heating. Furthermore, while conventional polysaccharides derived from algae of the genus Ulva tend to dissolve when left at room temperature after gelation, the gel obtained from the polysaccharide of the present invention does not dissolve even when left at room temperature (e.g., 20° C.) As described above, the polysaccharide of the present invention has excellent gelling properties, and therefore the composition of the present invention containing the polysaccharide can be suitably used as a gelling agent.

[0069] The polysaccharide of the present invention has moisturizing effects, skin barrier function improving effects, leaky gut inhibitory effects, anti-aging effects, anti-inflammatory effects, etc. Therefore, the composition of the present invention containing the polysaccharide can be used for these purposes (gelling agent, moisturizing agent, skin barrier function improving agent, leaky gut inhibitor, anti-aging agent, or anti-inflammatory agent). Furthermore, the composition of the present invention can be used as a cosmetic composition, topical composition, food composition, or pharmaceutical composition.

[0070] When the composition of the present invention is applied to animals, the subjects of application are not particularly limited, but include, for example, mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer.

[0071] The composition of the present invention may further contain other components as needed, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, and chelating agents.

[0072] The form of the composition of the present invention is not particularly limited, and may take any form commonly used for each application depending on the application.

[0073] In terms of form, when the application is a pharmaceutical composition, any dosage form can be used, for example, oral preparation forms such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, troches, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies; and parenteral preparation forms such as injectable preparations (for example, drip injections (for example, intravenous drip preparations), intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (for example, ointments, poultices, and lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, and liposomes.

[0074] The route of administration of the composition of the present invention is not particularly limited as long as the desired effect can be obtained, and examples thereof include enteral administration such as oral administration, tube feeding, and enema administration; and parenteral administration such as intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intradermal administration, and intraperitoneal administration.

[0075] In terms of form, when the application is a health promoting agent, a nutritional supplement (such as a supplement), etc., examples include formulation forms suitable for oral ingestion (oral formulation forms), such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, and syrups), and jellies.

[0076] When the application is a food composition, the form may be liquid, gel or solid food, such as juice, soft drinks, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, furikake, infant formula, cake mix, powdered or liquid dairy products, bread, cookies, etc.

[0077] The content of the polysaccharide of the present invention in the composition of the present invention depends on the intended use, mode of use, target of application, and condition of the target of application, and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0078] The dosage of the composition of the present invention (e.g., administration, ingestion, inoculation, etc.) is not particularly limited as long as it is an effective amount that produces the desired effect, and is generally 0.01 to 1000 mg / kg body weight per day in terms of the weight of the active ingredient. The dosage can be administered once a day or multiple times (2 to 3 times a day), and can be increased or decreased as appropriate depending on the patient's age, condition, and symptoms.

[0079] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0080] Test Example 1. Production of polysaccharides from Ulva meridionalis algae. The algae were grown in enriched natural seawater (ES) medium at 25°C under a 12-hour light-dark cycle with a light intensity of 100-200 μmol photons m , until the algae reached a size of 1-3 cm. -2 s -1 0.5 g of the obtained algae (wet weight) was cultured in 5 L of the above ES medium (nitrogen concentration 7.7 mg / L, phosphorus concentration 0.74 mg / L) (normal culture) or in 5 L of sterilized artificial seawater (nitrogen concentration 1.6 μg / L, phosphorus concentration 0 mg / L) without the above nutrients (nutrient-depleted culture) for 5 days at 25°C under a 12-hour light / dark cycle with a light intensity of 100 to 200 μmol photons m -2 s -1 The algae were cultured under the following conditions. Figure 1 shows a micrograph of the algae after the culture was completed.

[0081] For both normal and nutrient-depleted cultures, the number of starch granules per cell was measured based on micrographs of the algal cells after the culture was completed. Specifically, the number of starch granules in 30 randomly selected cells was counted based on the micrographs of the algal cells, and the average was used as the number of starch granules per cell. In the case of nutrient-depleted cultures, the number of starch granules was large and overlapping, making it difficult to accurately count them. The maximum number possible was counted and expressed as "more than." In this case, the average value was also expressed as "more than." The resulting number of starch granules per cell was 5.4 in normal cultures and 21 or more in nutrient-depleted cultures. An increase in the number of starch granules was observed from the second day of culture.

[0082] Polysaccharides were produced from the algae after cultivation as follows. The algae were collected and washed with water. They were then dried and powdered, and 5 g of powder was extracted with 100 mL of 80°C hot water for 2 hours. After filtering insoluble matter from the extract, fine particles were removed by centrifugation, and the supernatant was concentrated. The extract was then stored in a refrigerator to form a gel, which was then frozen and thawed to recover only the gel fibers as polysaccharides. At this time, the gel could be compressed to remove moisture, or it could be freeze-dried and washed with cold water. The resulting polysaccharide was then dried and powdered.

[0083] The ratio of neutral sugars to acidic sugars in the obtained polysaccharides was measured. The specific measurement method is as follows. The ratio of neutral sugars to acidic sugars in the obtained polysaccharides was measured. Acidic sugars and neutral sugars were determined by colorimetric analysis using a spectrophotometer. Specifically, acidic sugars were quantitatively analyzed using the m-hydroxyphenyl method. Neutral sugars were determined by first measuring total sugars using the phenol-sulfuric acid method and then subtracting this from the acidic sugars measured previously. As a result, the ratio of neutral sugars to acidic sugars in the polysaccharides was 2.7 in the case of normal culture and 1.9 in the case of nutrient-depleted culture.

[0084] In the following test examples, the polysaccharide obtained in the case of normal culture is referred to as "general ulvan," and the polysaccharide obtained in the case of nutrient-depleted culture is referred to as "novel polysaccharide."

[0085] We confirmed that novel polysaccharides with similar properties can also be produced by outdoor cultivation in 4-ton tanks. Specifically, after the algae were fully grown, they were cultured in 4-ton tanks using seawater. By limiting the seawater flow rate to less than (tank volume (L) / 50) / hour, the seaweed absorbed nitrogen and phosphorus from the seawater and grew. After a few days of cultivation, cultivation in essentially nutrient-depleted seawater was achieved, with the number of starch granules within the seaweed cells reaching 20 or more. In previous seaweed cultivations, a nutrient-depleted state was considered to exist when the inorganic nitrogen concentration in seawater was 0.042 mg / L or less and the phosphorus concentration was 0.0045 mg / L or less. Therefore, the nitrogen concentration of essentially nutrient-depleted seawater at this time is set at 0.04 mg / L or less. However, by creating essentially nutrient-depleted seawater, outdoor cultivation is also possible, allowing the seaweed to grow gradually while retaining 20 or more starch granules. In this case, the daily growth rate of southern Onori is about 2 to 3.2 times higher in nutrient-rich seawater with sufficient nitrogen and phosphorus added, but about 1.2 to 1.8 times higher in seawater that is essentially depleted of nutrients.In all of the above culture methods, there are more than 20 starch granules, and the polysaccharides have similar properties.

[0086] Test Example 2. Evaluation of the gelling properties of polysaccharides The novel polysaccharide and a common ulvan were each dissolved in water at room temperature (20°C) to prepare a 3 w / v% aqueous solution. The resulting solution was heated to 100°C and then cooled to 4°C to produce a gel. The resulting gel was returned to room temperature (20°C) and observed 30 minutes later. The observed image is shown in Figure 2. While the gel obtained from the common ulvan melted at room temperature, the gel obtained from the novel polysaccharide maintained its gel state even at room temperature.

[0087] The maximum load (g), gel strength (g.cm), and strain rate (%) of the gel obtained above were measured using a rheometer. Specifically, the procedure is as follows: 5 ml of the above solution was added to a 35 mm diameter container to create a gel. Then, measurements were taken using a CR-3000EX (Sun Scientific) with a 12 mm diameter plunger. The results were obtained by analysis using analysis software (REHO DATA ANALYZER PRO). Furthermore, similar measurements were taken on a gel prepared in the same manner as above from a 1.5 w / v% aqueous solution of agarose (Agarose S). The results are shown in Table 1.

[0088]

[0089] Next, the novel polysaccharide (Test Example 1), common ulvan (Test Example 1), and agarose were each dissolved in water at room temperature (20°C) to prepare 2 w / v% aqueous solutions in a container, which were then left to stand at room temperature (20°C) for 30 minutes without heating. Figure 3 shows a photograph of the contents of the container transferred to a container with a larger diameter. Without heating, neither common ulvan nor agarose were able to form a gel, but the novel polysaccharide was able to form a gel even in this case.

[0090] In addition, a gelation test was conducted by heating the polysaccharide solution at different concentrations. The new polysaccharide was able to form a gel at a concentration of 0.8 w / v% or higher, but common ulvan could only form a gel at a concentration of 3 w / v% or higher.

[0091] Furthermore, while agar does not gel after dissolving unless heated to around 100°C, the new polysaccharide gels when left at room temperature for a long time. It was also found that when heated at a concentration of 3 w / v%, it gels at around 60°C.

[0092] Furthermore, it was found that the novel polysaccharide is also capable of gelling dairy products.

[0093] Test Example 3: Evaluation of Moisture-Retaining Capacity of Polysaccharides. The moisture-retaining capacity of 200 mg each of the novel polysaccharide and hyaluronic acid sodium (animal-derived) was measured using the tea bag method. Specifically, the procedure is as follows: Nylon fabric with a mesh size of 57 μm (255 mesh) was cut into a 10 x 40 cm rectangle, folded lengthwise, and heat-sealed at both ends to form a 10 x 20 cm bag (tea bag). Each sample, with a particle size of 60 μm or greater, was weighed and placed in the bag. Then, in a room set at 23°C, 1 L of purified water was filled into a beaker, and the tea bag was immersed for 30 minutes, after which its weight was measured (A mg). As a control, an empty tea bag was immersed in purified water and its weight was measured (B mg). This allows the amount of water absorbed by the sample (moisture-retaining capacity) to be determined by the formula A-200-B mg.

[0094] The results are shown in Figure 4. The novel polysaccharide was found to have a high water retention capacity.

[0095] Similar measurements were also attempted on ordinary ulvan, but ordinary ulvan dissolved and diffused in water, and its water retention capacity was so low that it could not be measured using the above method.

[0096] Test Example 4. Evaluation of the effects of polysaccharides on skin moisturizing power and skin barrier function The novel polysaccharide, common ulvan, and sodium hyaluronate were each added to a culture medium for normal human epidermal keratinocytes (NHEK) to a concentration of 500 μg / mL, and the cells were then harvested after 24 hours of culture. The mRNA levels of the HAS-2 gene (a gene involved in the synthesis of hyaluronic acid), the Filaggrin gene (a gene involved in the skin barrier function), and the SPTLC1 gene and SMDP1 gene (both of which are genes involved in the synthesis of ceramide) were measured using real-time PCR.

[0097] The results are shown in Figure 5. It was suggested that the novel polysaccharide improves skin moisturizing ability and skin barrier function.

[0098] Test Example 5: Evaluation of Effects on Aged Mice. 46-week-old aged mice were orally administered methylcellulose, the novel polysaccharide, or inulin at 50 mg / kg body weight per day for 26 consecutive days. After administration, serum LPS levels were measured. Specifically, as follows: The amount of LPS in the blood is an indicator of leaky gut. Following administration, mice were orally administered FITC-dextran 4kDa, and the amount of FITC-dextran 4kDa in the blood was measured two hours later. Specifically, as follows: 18 46-week-old male C57BL / 6j mice were randomly divided into three groups of six mice each. The novel polysaccharide and inulin were dissolved in purified water to prepare 5 mg / mL aqueous solutions, and administered orally once daily at a dose of 50 mg / kg body weight using an oral administration probe. The control group received the same amount of 0.5 w / v% methylcellulose. Standard feed and water were available ad libitum.

[0099] On the 26th day after the start of administration, after a 4-hour fast, FITC-dextran 4kDa was dissolved in purified water to prepare a 20 mg / mL solution, and a dose of 200 mg / kg body weight was orally administered using an oral administration probe. Two hours later, the animals were euthanized, and blood was collected to obtain serum.

[0100] The FITC fluorescence intensity of the serum samples was measured using a fluorescent plate reader ("infinite 200," Tecan) at emission: 485 nm, excitation: 535 nm. The LPS concentration in the serum samples was also measured using the Pierce Chromogenic Endotoxin Quant Kit (Thermo Scientific). After administration, mice were dissected and their intestinal length was measured. Intestinal length is known to shorten due to inflammation.

[0101] The results of the above tests are shown in Figures 6 and 7. The novel polysaccharide was found to have leaky gut inhibitory, anti-aging, and anti-inflammatory effects.

[0102] Test Example 6: Evaluation of the Effect on Senescent Fibroblasts. The novel polysaccharide, a common ulvan, and other polysaccharides were added to senescent fibroblasts, and ATP production and SA-β-Gal-positive cells were measured. Specifically, the procedure was as follows. Human dermal fibroblasts (NB1RGB cells) were cultured for 80 to 89 days in MEMα medium supplemented with 10% FBS, 100 μg / mL penicillin, and 100 μg / mL streptomycin to produce senescent cells. Each of the substances listed in the table was added to MEMα medium at a concentration of 500 μg / mL, and the human dermal fibroblast senescent cells were further cultured at 37°C for 24 hours. For comparison, a separate culture was performed in the same manner except that no ulvans were added.

[0103] The activity of senescence beta-galactosidase, a marker of senescence, was measured using a Senescence Detection Kit (Abcam) by fixing and staining cultured cells with fixative according to the manufacturer's protocol. Cells with senescence beta-galactosidase activity (senescent cells) stained blue. The number of blue-stained cells was counted among 50 cultured cells. Similarly, ATP production was measured using the CellTiter-Glo® 2.0 Assay (Promega) according to the manufacturer's protocol. Luminescence was measured using a Promega GloMax 20 / 20 Luminometer. ATP production is known to decrease with aging, and SA-β-Gal is a marker of senescence.

[0104] The results are shown in Figure 8. The novel polysaccharide was found to have anti-aging effects.

Claims

1. A method for producing polysaccharides, comprising extracting and recovering polysaccharides from seaweed cultivated in a low-nutrient medium in a container.

2. The method of claim 1, wherein the nitrogen concentration of the low-nutrient medium is 0 to 0.042 mg / L.

3. The method of claim 2, wherein the phosphorus concentration of the low-nutrient medium is 0 to 0.003 mg / L.

4. The method according to claim 1, wherein the seaweed is a seaweed with a high starch granule content, having 10 or more starch granules per cell.

5. The method of claim 1, wherein the culture period in the low-nutrient medium is 2 days or more.

6. The method of claim 1, wherein the seaweed is Ulva algae.

7. The method of claim 1, wherein the solvent used for extracting and recovering the polysaccharide is water.

8. A polysaccharide obtained by the method of claim 1.

9. A polysaccharide having a ratio of neutral sugars to acidic sugars of 5.0 or less and / or containing sugars with sulfate groups attached.

10. The polysaccharide of claim 9, wherein the ratio of neutral sugars to acidic sugars is 2.2 or less.

11. The polysaccharide of claim 9, wherein said ratio is between 1.5 and 2.

2.

12. A polysaccharide extracted from seaweed, characterized in that a solution of a composition containing said polysaccharide in an aqueous solvent gels at room temperature (20°C) without undergoing a heating step.

13. The polysaccharide according to claim 12, wherein a solution of the polysaccharide-containing composition in an aqueous solvent gels at a concentration of 0.8 w / v % or more.

14. The polysaccharide according to claim 12, which can form a gel having a gel strength of 300 g·cm or more when the solution of said polysaccharide in an aqueous solvent is 3 w / v % or more.

15. The polysaccharide according to claim 12, characterized in that the polysaccharide extracted from the seaweed is extracted from seaweed cultivated in a low-nutrient medium.

16. The polysaccharide of claim 15, wherein the phosphorus concentration of the low-nutrient medium is 0 to 0.003 mg / L.

17. The polysaccharide according to claim 9, which is derived from algae of the genus Ulva.

18. A composition comprising the polysaccharide of any one of claims 8 to 17.

19. The composition of claim 18, which is a gelling agent, thickener, disintegrant, water-absorbing agent, cosmetic composition, topical composition, food composition, or pharmaceutical composition.

20. The composition of claim 18, which is a gelling agent, moisturizing agent, skin barrier function improving agent, leaky gut inhibitor, anti-aging agent, or anti-inflammatory agent.

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