WO2024080322A1 - Anti-white spot syndrome virus agent - Google Patents

Anti-white spot syndrome virus agent Download PDF

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WO2024080322A1
WO2024080322A1 PCT/JP2023/036956 JP2023036956W WO2024080322A1 WO 2024080322 A1 WO2024080322 A1 WO 2024080322A1 JP 2023036956 W JP2023036956 W JP 2023036956W WO 2024080322 A1 WO2024080322 A1 WO 2024080322A1
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lignin
agent
wssv
mass
treatment
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PCT/JP2023/036956
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French (fr)
Japanese (ja)
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光 相見
大輝 進藤
嘉人 西盛
育生 廣野
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日本製紙株式会社
国立大学法人東京海洋大学
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Publication of WO2024080322A1 publication Critical patent/WO2024080322A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/50—Culture of aquatic animals of shellfish
    • A01K61/59—Culture of aquatic animals of shellfish of crustaceans, e.g. lobsters or shrimps
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/111—Aromatic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs

Definitions

  • the present invention relates to an anti-white spot syndrome virus agent.
  • Measures to prevent white spot disease infection include, for example, oral vaccines containing recombinant coat proteins of the white spot syndrome virus (WSSV) and DNA vaccines against coat proteins (Patent Document 1, Non-Patent Document 1).
  • WSSV white spot syndrome virus
  • Patent Document 1 Non-Patent Document 1
  • vaccines such as those described in Patent Document 1 and Non-Patent Document 1 are expensive and provide protection from infection for a short period of time.
  • the vaccine described in Non-Patent Document 1 provides protection from infection for approximately one month. Therefore, administering the WSSV vaccine to small organisms such as shrimp and other crustaceans is not practical due to the labor and cost involved.
  • the present invention aims to provide a novel anti-WSSV agent that can prevent WSSV infection.
  • the present invention provides the following [1] to [10].
  • [6] The agent according to any one of [1] to [5], which is for use on crustaceans used in aquaculture or fish farming.
  • [7] The agent according to [5] or [6], wherein the crustacean includes shrimp.
  • a feed comprising the agent according to any one of [1] to [7].
  • a treatment agent for breeding water comprising the agent according to any one of [1] to [7].
  • a method for preventing white spot disease comprising administering the agent according to any one of [1] to [7] to crustaceans.
  • the anti-WSSV agent of the present invention can exhibit a good anti-WSSV inhibitory effect. Therefore, for example, by using it as a feed for organisms such as crustaceans, or as a treatment agent for breeding water, it is possible to prevent WSSV infection and improve the productivity of these organisms, making it useful in various fields such as the fisheries, aquaculture, and pet supplies.
  • FIG. 1 is a graph showing the survival rate of shrimp in Feeding Test 1 (1% lignin) of the Examples.
  • FIG. 2 is a graph showing the survival rate of shrimp in Feeding Test 1 (4% lignin) of the Examples.
  • FIG. 3 is a graph showing the survival rate of shrimp in Feeding Test 2 (1% lignin) of the embodiment.
  • FIG. 4 is a graph showing the survival rate of shrimp in Feeding Test 2 (4% lignin) of the embodiment.
  • FIG. 5 is a graph showing the survival rate of shrimp in Test 2 of the Example.
  • the present invention relates to an anti-white spot syndrome virus agent.
  • the agent of the present invention contains a lignin component as an active ingredient.
  • the lignin component means lignin (lignin isolated from a plant), a derivative of lignin, a decomposition product of lignin, or a derivative of a decomposition product of lignin.
  • the lignin derivative may be in a powder or liquid form.
  • the method for preparing the liquid lignin component is not particularly limited, but an example of the method is to dissolve the powdered lignin component in a suitable solvent (e.g., water, an aqueous sodium hydroxide solution) to obtain the liquid lignin component.
  • Lignin components are usually derived from woody biomass, and are classified into several types with different structures and physical properties depending on the processing method.
  • lignin components include lignin sulfonate, kraft lignin, soda lignin, soda-anthraquinone lignin, organosolv lignin, explosive lignin, sulfuric acid lignin, and decomposition products thereof.
  • lignin sulfonate is preferred.
  • the lignin derivative may be one type or a combination of two or more types, but it is preferable that it contains at least lignin sulfonate.
  • Lignosulfonates are lignin derivatives having a sulfo group, which are prepared from lignocellulose raw materials through sulfite treatment. Lignosulfonates may be in the form of an acid or a salt, but are usually in the form of a salt. Examples of salts include monovalent metal salts, divalent metal salts, ammonium salts, and organic ammonium salts, and among these, calcium salts, magnesium salts, sodium salts, potassium salts, and calcium-sodium mixed salts are preferred.
  • Examples of methods for preparing lignin sulfonates include a method in which lignocellulose raw materials or lignin itself is subjected to a sulfite treatment, preferably a method in which lignocellulose raw materials or lignin itself is subjected to a sulfite cooking treatment.
  • the lignocellulose raw material is not particularly limited as long as it contains lignocellulose in the structure.
  • pulp raw materials such as wood and non-wood can be mentioned.
  • wood can be coniferous wood such as radiata pine, Yezo spruce, red pine, cedar, and cypress, and broadleaf wood such as white birch and beech.
  • the age of the wood and the part where it is harvested do not matter. Therefore, wood harvested from trees of different ages or wood harvested from different parts of a tree may be used in combination.
  • non-wood can be bamboo, kenaf, reed, and rice.
  • the lignocellulose raw material may be one type alone or two or more types in combination.
  • Lignosulfonates may be prepared from raw materials other than lignocellulose raw materials, for example, lignin.
  • lignin include naturally occurring lignin and artificially produced lignin (for example, dehydrogenation polymerized products of hydroxycinnamic alcohol analogues), and both can be used.
  • Lignosulfonates can be prepared from lignin, for example, by a method of decomposing lignin and sulfonating it.
  • the sulfite treatment can be carried out by contacting the lignocellulosic raw material with at least one of sulfurous acid and a sulfite salt.
  • the conditions for the sulfite treatment are not particularly limited as long as they allow introduction of a sulfo group to the ⁇ -carbon atom of the side chain of lignin contained in the lignocellulosic raw material.
  • the sulfite treatment is preferably carried out by the sulfite cooking method.
  • the sulfite cooking method is a method in which the lignocellulosic raw materials are reacted at high temperatures in a solution of at least one of sulfurous acid and sulfite salts (e.g., aqueous solution, cooking liquid). This method has been established and is being used industrially as a method for producing sulfite pulp, and is therefore advantageous in terms of economy and ease of implementation.
  • examples of sulfite salts include magnesium salts, calcium salts, sodium salts, and ammonium salts.
  • the sulfurous acid (SO 2 ) concentration in the solution of at least one of sulfurous acid and sulfite is not particularly limited, but the ratio of the mass (g) of SO 2 to 100 mL of reaction solution is preferably 1 g/100 mL or more, and more preferably 2 g/100 mL or more when sulfite cooking is performed.
  • the upper limit is preferably 20 g/100 mL or less, and more preferably 15 g/100 mL or less when sulfite cooking is performed.
  • the SO 2 concentration is preferably 1 g/100 mL to 20 g/100 mL, and more preferably 2 g/100 mL to 15 g/100 mL when sulfite cooking is performed.
  • the pH value of the sulfurous acid treatment is not particularly limited, but is usually 10 or less. When sulfurous acid cooking is performed, it is preferable to perform it under acidic conditions, with a pH of 5 or less being more preferable, and 3 or less being even more preferable. This allows lignin derivatives (e.g., lignin sulfonate salts) to be extracted efficiently, and pulp of higher quality can be obtained.
  • the lower limit of the pH value is preferably 0.1 or more, and when sulfurous acid cooking is performed, 0.5 or more is more preferable.
  • the pH value during sulfurous acid treatment is preferably 0.1 to 10, and when sulfurous acid cooking is performed, 0.5 to 5 is more preferable, and 0.5 to 3 is even more preferable.
  • the temperature of the sulfite treatment is not particularly limited, but is preferably 170° C. or lower, and more preferably 150° C. or lower when sulfite cooking is performed.
  • the lower limit is preferably 70° C. or higher, and more preferably 100° C. or higher when sulfite cooking is performed.
  • the temperature condition of the sulfite treatment is preferably 70 to 170° C., and more preferably 100 to 150° C. when sulfite cooking is performed.
  • the treatment time for the sulfurous acid treatment is not particularly limited, and although it depends on the conditions of the sulfurous acid treatment, it is preferably 0.5 to 24 hours, and more preferably 1.0 to 12 hours.
  • a compound that supplies a counter cation salt: containing the substituent M of the group represented by general formula (1).
  • a compound that supplies a counter cation By adding a compound that supplies a counter cation, the pH value in the sulfite treatment can be kept constant.
  • examples of compounds that supply counter cations include MgO, Mg(OH) 2 , CaO, Ca(OH) 2 , CaCO 3 , NH 3 , NH 4 OH, NaOH, NaHCO 3 , and Na 2 CO 3.
  • the counter cation is preferably a magnesium ion, a sodium ion, or a calcium ion.
  • the solution may contain, in addition to SO2 , the above-mentioned counter cation (salt) and a digestion and penetration agent (for example, a cyclic ketone compound such as anthraquinone sulfonate, anthraquinone, or tetrahydroanthraquinone), as necessary.
  • a digestion and penetration agent for example, a cyclic ketone compound such as anthraquinone sulfonate, anthraquinone, or tetrahydroanthraquinone
  • the intermediate product can be separated from the solution of at least one of sulfurous acid and sulfite salts by a conventional method.
  • a method for separating the sulfurous acid digestion effluent after sulfurous acid digestion e.g., filtration
  • a method for separating the sulfurous acid digestion effluent after sulfurous acid digestion e.g., filtration
  • the lignin sulfonate obtained by the sulfite treatment may be used as a lignin derivative as is, or after concentrating as necessary. On the other hand, if necessary, further treatment may be performed. This makes it possible to obtain a lignin derivative having a high purity and/or an appropriate degree of sulfonation (S content). Examples of other treatments include alkali treatment, oxidation treatment, dialysis treatment, UF treatment, and combinations of these.
  • the alkali treatment is preferably performed on the filtration residue (insoluble matter) or filtrate after the sulfite treatment, or on the treated product after the dialysis treatment.
  • the alkali treatment can be performed by placing the target sample under alkaline conditions. Placing under alkaline conditions usually means placing the sample in an aqueous solution having a pH value of 8 or more, preferably a pH value of 9 or more. The upper limit of the pH value is usually 14 or less.
  • an alkaline substance is usually brought into contact with the sulfite-treated product.
  • the alkaline substance is not particularly limited, but examples include calcium hydroxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, and ammonia. Of these, sodium hydroxide and calcium hydroxide are preferred.
  • the alkaline substance may be used alone or in combination of two or more types.
  • Examples of methods for contacting an alkaline substance with a sulfite-treated product include preparing a dispersion or solution (e.g., an aqueous dispersion or aqueous solution) of the sulfite-treated product and adding an alkaline substance to the dispersion or solution, and adding a solution or dispersion of an alkaline substance (e.g., an aqueous dispersion or aqueous solution) to the sulfite-treated product.
  • a dispersion or solution e.g., an aqueous dispersion or aqueous solution
  • the temperature of the alkali treatment is not particularly limited, but is preferably 40°C or higher, and more preferably 60°C or higher.
  • the upper limit is preferably 200°C or lower, more preferably 180°C or lower, and even more preferably 170°C or lower.
  • the amount of alkaline substance in the alkaline treatment is preferably 0.5 to 40% by mass, more preferably 1.0 to 30% by mass, based on the solids mass of the sulfite-treated product, or, when preparing an aqueous solution or dispersion by dispersing the alkaline-treated extract in an aqueous solvent (e.g., water), based on the mass of the aqueous solution or dispersion.
  • an aqueous solvent e.g., water
  • the duration of the alkali treatment is not particularly limited, but is preferably 0.1 hours or more, and more preferably 0.5 hours or more.
  • the upper limit is preferably 10 hours or less, and more preferably 6 hours or less.
  • the sulfite-treated product Prior to the alkali treatment, the sulfite-treated product may be dissolved, dispersed, or its concentration adjusted (preparation of a solution or dispersion in an aqueous solvent such as water) as necessary.
  • Dispersion can be performed, for example, by passing the product through a disc refiner, adding it to a mixer or disperser, or by kneading. Concentration can be adjusted, for example, by using an aqueous solvent such as water.
  • the oxidation treatment can be carried out on the treated product obtained after the sulfurous acid treatment (for example, the filtrate after filtration) or the treated product after the alkali treatment.
  • the oxidation treatment can be carried out by using an appropriate oxidizing agent.
  • the oxidizing agent is a gas
  • the oxidation can be carried out by bubbling the gas into the filtrate.
  • the oxidizing agent is a liquid
  • the oxidation can be carried out by adding the liquid to the filtration residue or the filtrate.
  • the oxidizing agent is preferably air, oxygen, hydrogen peroxide, ozone, or a combination thereof.
  • the oxidation treatment is preferably carried out under alkaline conditions (alkaline oxidation treatment).
  • the treatment pH of the alkaline oxidation treatment is usually 8 or more, preferably 10 or more, and more preferably 12 or more.
  • the temperature of the oxidation treatment is usually 20 to 200° C., and preferably 50 to 180° C.
  • the time of the oxidation treatment is usually preferably 0.1 hours or more, and more preferably 0.5 hours or more.
  • the upper limit is preferably 5 hours or less, and more preferably 3 hours or less.
  • the dialysis treatment can be performed on the treated product obtained after the sulfite treatment (for example, the filtrate after filtration).
  • the dialysis membrane include cellulose-based membranes such as cellulose acetate, and synthetic polymer-based membranes such as ethylene vinyl alcohol, polyacrylonitrile, polymethyl methacrylate, polysulfone, and polyethersulfone.
  • the molecular weight fraction is usually 5,000 to 100,000, preferably 7,000 to 80,000, and more preferably 10,000 to 50,000.
  • UF ultrafiltration
  • Any known UF membrane can be used. Examples include hollow fiber membranes, spiral membranes, tubular membranes, and flat membranes. Any known material can be used for the UF membrane. Examples include cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, and ceramics.
  • the UF membrane may be a commercially available product.
  • the molecular weight cutoff of the UF membrane is preferably 5,000 to 30,000, more preferably 10,000 to 25,000, and even more preferably 15,000 to 23,000.
  • Using a UF membrane with a molecular weight cutoff of 5,000 or more can prevent the separation speed of the treatment liquid from becoming excessively slow.
  • using a UF membrane with a molecular weight cutoff of 30,000 or less can prevent lignin from not being separated from the treatment liquid.
  • the concentration rate by UF treatment using a UF membrane can be set as desired.
  • the UF treatment can be stopped when the amount of concentrated liquid flowing out reaches a desired amount. It is preferable to concentrate it 2 to 6 times. Concentrating 2 to 6 times means that the amount of raw liquid (black liquor) becomes 1/2 to 1/6 of its original amount.
  • the temperature of the treatment liquid during UF treatment is not particularly limited. For example, 20 to 80°C is preferable, and considering the heat resistance of the UF membrane material, 20 to 70°C is more preferable.
  • the pH value of the treatment liquid during UF treatment is preferably 2 to 11.
  • the solids concentration (w/w) of the black liquor during UF treatment is preferably 2 to 30%, and more preferably 5 to 20%.
  • Another example of a method for producing lignin sulfonates is the sulfonation of kraft lignin.
  • - Kraft lignin - Kraft lignin is also called thiolignin or sulfate lignin.
  • the kraft lignin include an alkaline solution of kraft lignin, powdered kraft lignin obtained by spray-drying an alkaline solution of kraft lignin to obtain a powder, and acid-precipitated kraft lignin obtained by precipitating an alkaline solution of kraft lignin with an acid.
  • the kraft lignin may be one of these, or a combination of two or more of them.
  • An example of a method for preparing an alkaline solution of kraft lignin is a method in which an alkaline solution containing Na 2 S flowing in a kraft pulp production process is electrolyzed by electrolytic oxidation to produce a NaOH solution on the cathode side (JP 2000-336589 A).
  • An example of a method for preparing acid-precipitated kraft lignin by precipitating an alkaline solution of kraft lignin with an acid is a method for preparing powdery acid-precipitated kraft lignin (WO 2006/038863, WO 2006/031175, WO 2012/005677).
  • the method of sulfonating kraft lignin may be sulfonation by ordinary sulfite treatment or sulfite cooking treatment, or may be the method described in "Development of New Lignin Derivatives as Soil Conditioning Agents by Radical Sulfonation and Alkali-Oxygen Treatment: Mokuzai Gakkaishi, Vol. 43, No. 8, 669-677 (1997)", but is not limited to the above methods and other methods may be used.
  • the lignin component may further include an extractive component.
  • the anti-WSSV activity can be further enhanced.
  • extractive components refers to trace components of plants (e.g., plants used as pulp raw materials, preferably wood from plants of the genus Cryptomeria, Chamaecyparis obtusa, Pinus serrata, Larch, Abies monad, and Eucalyptus), and can usually be obtained by extraction treatment of wood with organic solvents or the like. Extractive components are usually determining factors for wood properties such as color, odor, durability, adhesiveness, and biological activity, and are said to be the components that chemically characterize wood.
  • extractive components which are linked to the formation of heartwood, occurs.
  • organic solvents used to obtain extractive components include hexane, benzene, ether, acetone, and alcohol.
  • the content of extractive components in wood is usually about 5% or less.
  • Extracted components usually contain low molecular weight compounds (usually low molecular weight organic compounds). Many of these are secondary metabolic products with molecular weights of several thousand or less, and although they are generally extremely diverse, they are broadly classified into aromatic extracted components and terpenoids. Examples of aromatic extracted components include flavonoids, tannins, lignans, and stilbenes. The extracted components contain at least one type selected from these.
  • Flavonoids are a general term for compounds having a diphenylpropane (C 6 -C 3 -C 6 ) skeleton, and examples thereof include flavones, flavanones, chalcones, aurone, isoflavones, catechins, and leucoanthocyanidins. Tannins may be either hydrolyzable tannins or condensed tannins. Hydrolyzable tannins are tannins having a structure in which a phenolic carboxylic acid such as gallic acid is ester-bonded to a nucleus such as glucose, and examples thereof include gallotannins and ellagitannins.
  • Hydrolyzable tannins can be decomposed into simple fragments by hydrolysis with an acid, an alkali, or the like, and when gallotannins and ellagitannins are hydrolyzed, gallic acid and ellagic acid are obtained, respectively.
  • Condensed tannins include, for example, amorphous polymers having catechins or leucoanthocyanidins as precursors.
  • lignans examples include lignans and substances closely related thereto.
  • Lignans (resinols) have a structure having a C 6 -C 3 -C 3 -C 6 skeleton in which phenylpropane units, which are the same structural units of lignin, are carbon-carbon bonded between the ⁇ -positions of the side chains. Unlike lignin, they have an asymmetric carbon in the molecule and are optically active.
  • substances closely related to lignans include norlignans having a C 6 -C 3 -C 2 -C 6 skeleton, which has one less carbon atom than the lignin skeleton.
  • Stilbenes may be compounds having an ⁇ , ⁇ -diphenylethylene skeleton.
  • the terpenoid may be a series of compounds in which two or more isoprene units ( C5H8 ) are bonded in a chain or ring shape.
  • Terpenoids consisting of 2, 3, 4, and 6 isoprenoid units are called monoterpenes (10 carbon atoms), sesquiterpenes (15 carbon atoms), diterpenes (20 carbon atoms: for example, abietic acid), and triterpenes (30 carbon atoms), respectively, and any of these may be used.
  • Extractives have a variety of physiological activities depending on the basic carbon skeleton and substituents of the extractives. Some of these physiological activities may affect the antiviral action. Examples of physiological activities include biological activity against microorganisms, insects, and plants ("Resistance of wood to biological deterioration by extractives", Wood Conservation 34 (2), 48-54, 2008), improvement of wood durability, and inhibitory action against chemical and physical processing of wood (e.g. pulping, bleaching, cement hardening).
  • physiological activities include biological activity against microorganisms, insects, and plants ("Resistance of wood to biological deterioration by extractives", Wood Conservation 34 (2), 48-54, 2008), improvement of wood durability, and inhibitory action against chemical and physical processing of wood (e.g. pulping, bleaching, cement hardening).
  • the amount of the extracted components per solid content of the lignin component is preferably 1.7% by mass or less, more preferably 1.65% by mass or less, and even more preferably 1.63% by mass or less. This ensures the safety of the agent.
  • the lower limit is usually 0.01% by mass or more, preferably 0.03% by mass or more, and more preferably 0.05% by mass or more. This allows the WSSV suppression effect to be exhibited well.
  • the amount of the extracted components per solid content of the lignin component is preferably 1.7% by mass or less, more preferably 0.01 to 1.65% by mass, even more preferably 0.03 to 1.63% by mass, and even more preferably 0.05 to 1.63% by mass.
  • the amount of extractable components can be measured using the method for measuring hexane extractable substances described in JIS K 0102:2019.
  • lignin has a structure containing methoxy groups bonded to aromatic nuclei, and therefore the amount of methoxy groups is an index of the content of lignin and lignin derivatives.
  • the amount of methoxy groups per solid content of the lignin component is usually 2.0 mass% or more, 2.5 mass% or more, or 3.0 mass% or more, preferably 3.5 mass% or more, and more preferably 4.0 mass% or more. There is no particular upper limit, but it is usually 20 mass% or less. Therefore, the amount of methoxy groups per solid content of the lignin component is usually 2.0 mass% or more, 2.5 to 20 mass% or 3.0 to 20 mass%, preferably 3.5 to 20 mass%, and more preferably 4.0 to 20 mass%. This allows the anti-WSSV agent of the present invention to exhibit a good WSSV inhibitory effect while ensuring the safety of the agent.
  • the amount of methoxy groups can be measured by the quantitative determination of methoxy groups using the Viebock and Schwappach method (see “Lignin Chemical Research Methods,” pp. 336-340, 1994, published by Uni Publishing Co., Ltd.).
  • the extracted components may affect safety depending on their physiological activity, while the amount of methoxy groups is an index of the content of lignin and lignin derivatives. Therefore, the mass ratio of the amount of extracted components to the amount of methoxy groups can be said to represent the balance between safety and WSSV inhibitory effect.
  • the mass ratio of the amount of extractable components to the amount of methoxy groups per solid content of the lignin component is usually 1.0 or less, preferably 0.8 or less, more preferably 0.6 or less, even more preferably 0.4 or less, and even more preferably 0.3 or less.
  • the lower limit is preferably 0.001 or more, and more preferably 0.005 or more. Therefore, the mass ratio of the amount of extractable components to the amount of methoxy groups per solid content of the lignin component is usually 1.0 or less, preferably 0.001 to 0.8, more preferably 0.001 to 0.6, even more preferably 0.005 to 0.4, and even more preferably 0.005 to 0.3.
  • the lignin derivative contains a sulfo group represented by the general formula (1): -SO 3 M.
  • M is a hydrogen atom, a monovalent metal salt, a divalent metal salt, an ammonium salt, or an organic ammonium salt, and examples thereof include a sodium ion, a calcium ion, a potassium ion, a magnesium ion, or an ammonium ion, with the sodium ion, magnesium ion, and calcium ion being preferred.
  • the lignin derivative may contain two or more types of groups represented by the general formula (1) having different substituents M.
  • the total amount (ratio to the solid content of the lignin derivative) of the S content (sulfur atom content: sulfo group S content) of the group represented by the general formula (1) of the lignin derivative is usually 0.5% by mass or more, preferably 1.0% by mass or more, more preferably 1.2% by mass or more.
  • the lignin derivative is rich in the group represented by the general formula (1), and can exhibit water solubility and can exhibit a good virus inhibitory effect against WSSV.
  • the upper limit is usually 20.0% by mass or less, preferably 15.0% by mass or less, more preferably 12.0% by mass or less.
  • the total amount of the sulfo group S content of the lignin derivative is usually 0.5 to 20.0% by mass, preferably 1.0 to 15.0% by mass, more preferably 1.2 to 12.0% by mass.
  • the sulfo group S content can be calculated by the following formula (1).
  • Sulfo group S content (mass%) total S content (mass%) of lignin derivative - inorganic S content (mass%)
  • the total S content and inorganic S content of the lignin derivative both indicate the S content relative to the solid content of the lignin derivative.
  • the total S content is the total S content contained in the lignin component and can be quantified by ICP atomic emission spectrometry.
  • the inorganic S content can be calculated as the sum of the SO3 content and SO4 content quantified by ion chromatography.
  • the lignin component has a water solubility of usually 1.0% by mass or more, preferably 3.0% by mass or more.
  • the environment in which the partial chemical structure having the virus inhibitory effect comes into contact with the virus is an aqueous medium, and it is presumed that the above range allows the lignin component to have a moderate water solubility, which is advantageous for the virus inhibitory effect.
  • the upper limit is not particularly limited, and may be 100% by mass or less. Therefore, the lignin component has a water solubility of usually 1.0 to 100% by mass, preferably 3.0 to 100% by mass.
  • the amount of solubility in water can be calculated as follows: Disperse 10 g (dry weight) of the sample in 300 g of water, stir for 60 minutes, and then filter. Measure the mass of the filtrate and the mass of the solids (dried filtrate). Then, divide the mass of the solids in the filtrate by the mass of the sample (10 g) and multiply by 100 to calculate the amount.
  • the lignin component is a component other than the extracted component that is mixed from the raw material when preparing the lignin derivative (for example, during sulfite cooking), such as sodium sulfate, sodium sulfite, sodium chloride, magnesium sulfate, magnesium sulfite, and magnesium chloride.
  • the inorganic salts may include calcium sulfate, calcium sulfite, calcium chloride, ammonium sulfate, ammonium sulfite, ammonium chloride, sodium hydroxide, and the like.
  • the lignin component may also contain sugars as components other than the extractable components.
  • sugars refers to at least one type of sugar or a combination of two or more types of sugars.
  • the number of carbon atoms constituting the sugar is not limited, and the sugar may be any of monosaccharides, oligosaccharides, and polysaccharides.
  • monosaccharides include trioses such as aldotriose and ketotriose; tetraoses such as erythrose, threose, and erythrulose; pentoses such as xylose, ribose, arabinose, lyxose, ribulose, and xylulose; hexoses such as glucose, mannose, allose, altrose, glucose, gulose, idose, galactose, talose, psicose, fructose, sorbose, tagatose, fucose, fructose, and rhamnose; and heptoses such as sedoheptulose.
  • oligosaccharides include disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose; trisaccharides such as raffinose, melezitose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and oligosaccharides such as xylooligosaccharides, cellooligosaccharides, fructooligosaccharides, galactooligosaccharides, and mannanoligosaccharides.
  • disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose
  • trisaccharides such as raffinose, melezitose, and maltotriose
  • tetrasaccharides such as acarbose and stachyose
  • oligosaccharides such
  • polysaccharides examples include glycogen, starch (amylose, amylopectin), cellulose, hemicellulose, dextrin, and glucan.
  • Sugars, particularly polysaccharides may include polysaccharides contained in plant components that are raw materials for lignocellulose such as pulp, and those that are produced by decomposition and/or modification of these during cooking or bleaching treatment.
  • the sugars generally include polysaccharides, reducing sugars, and/or modified sugars.
  • the reducing sugar may be any sugar that exhibits reducing properties.
  • the reducing sugar generally produces an aldehyde group or a ketone group in a basic solution.
  • Examples of the reducing sugar include all monosaccharides, disaccharides such as maltose, lactose, arabinose, and invert sugar of sucrose, and polysaccharides.
  • Examples of the modified sugar include modified sugars that are chemically modified by oxidation, sulfonation, and the like, and sugar derivatives that are substituted with substituents such as hydroxyl groups, aldehyde groups, carbonyl groups, and/or sulfo groups.
  • Reducing sugars are sugars that exhibit reducing properties and generate aldehyde or ketone groups in a basic solution.
  • reducing sugars include all monosaccharides, disaccharides such as maltose, lactose, arabinose, and invert sugars of sucrose, and polysaccharides.
  • Reducing sugars typically include cellulose, hemicellulose, and their decomposition products. Examples of decomposition products of cellulose and hemicellulose include monosaccharides such as rhamnose, galactose, arabinose, xylose, glucose, mannose, and fructose, and oligosaccharides such as xylooligosaccharides and cellooligosaccharides.
  • sugar modification product refers to a product in which sugar has been chemically modified, such as by oxidation or sulfonation.
  • sugar modification products include sugar derivatives in which functional groups such as hydroxyl groups, aldehyde groups, carbonyl groups, and/or sulfo groups have been introduced into the sugar skeleton, and compounds in which two or more sugar derivatives (two types) are bonded together.
  • the agent of the present invention may further contain other optional components as necessary.
  • the other components may be any optional components normally used in the intended use of the agent, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, various nutritional components, and anti-WSSV agents other than the above-mentioned lignin components.
  • the dosage form of the anti-WSSV agent can be appropriately selected depending on the application.
  • dosage forms include liquids such as liquids, emulsions, suspensions, dispersions, and aerosols; granules, tablets, and powders.
  • the solid or semi-solid agent include a solid or semi-solid agent such as a paste agent.
  • the anti-WSSV agent of the present invention can exert a WSSV suppression effect.
  • WSSV White Spot Syndrome Virus
  • WSSV is a rod-shaped, double-stranded DNA virus belonging to the Whispovirus genus. WSSV is transmitted by ingesting infected organisms and through water entering through the gills.
  • Anti-WSSV agents can suppress WSSV infection and its spread in target organisms.
  • the target organisms are usually organisms belonging to the order Decapoda (e.g., crustaceans such as shrimp, crabs, and crayfish).
  • Shrimp may be any shrimp that lives in seawater, brackish water, or fresh water, and examples of such shrimp include Litopenaeus genus (Penaeus Fabricius), such as white leg shrimp (Penaeus vannamei Boone), white shrimp (Penaeus setiferus), western blue shrimp (Penaeus stylirostris Stimpson), kuruma shrimp (Penaeus japonicus Spence Bate), Korean shrimp (Penaeus chinensis), red-tailed shrimp (Penaeus s penicillatus Alcock), Indian shrimp (Penaeus indicus H.
  • Metapenaeopsis Bouvier including red shrimp (Metapenaeopsis barbata) and Japanese prawn (Metapenaeopsis dalei)
  • Trachysalambria Burkenroad including monkey shrimp (Trachysalambria curvirostris)
  • Parapenaeopsis Alcock including kiddy shrimp (Parapenaeopsis stylifera).
  • anti-WSSV agent can be used in various applications where an anti-WSSV effect is expected, such as feed, breeding water treatment agents, disinfectants, and cleaning agents.
  • the target organism can be made to ingest the anti-WSSV agent, thereby efficiently exerting resistance to WSSV.
  • the feed and medicine may contain an anti-WSSV agent, and the content can be adjusted appropriately depending on the mode of use and the subject of application.
  • the lignin component when calculated as a daily feed of 5 to 10% of the body weight of the organism, is contained in an amount of 0.1% by mass or more, preferably 0.5% by mass or more, and more preferably 0.7% by mass or more, based on the weight of the feed.
  • the upper limit is usually 7.0% by mass or less, preferably 5.0% by mass or less. Therefore, it is usually 0.1 to 7.0% by mass, preferably 0.5 to 7.0% by mass, and more preferably 0.7 to 5.0% by mass.
  • the feed is preferably administered continuously, and may be administered once to three times a day for, for example, 3 days or more, 5 days or more, preferably 1 week or more, more preferably 10 days or more, and even more preferably 2 weeks or more.
  • the feed may be temporarily returned to normal feed, and then continuous administration may be repeated after a certain period of time (for example, 1 week, 2 weeks, 3 weeks, 1 month, or 2 months). Additionally, there are no particular limitations on the age of the crustaceans that can be used.
  • Optional components that may be contained in the feed include, for example, nutritional components.
  • nutritional components include fish oil, seafood meat, amino acids, lipids, minerals, vitamins, and other components that are typically used in feed for target organisms, aquaculture feed, and aquaculture feed, and the content of these components can be selected as appropriate.
  • the feed may be in the same form as normal feed, and is usually in the form of granules, pellets, or powder. There are no particular limitations on the method for producing the feed, and examples include a method in which the components constituting the sample are mixed, dried, and shaped.
  • the feed may be feed for the target organism infected with WSSV, and is usually feed for crustaceans, and preferably feed for aquaculture.
  • Treatment agents for breeding water By using an anti-WSSV agent as a treatment agent for breeding water, it is possible to suppress the entry of WSSV into the body of the target organism via oral ingestion or gills, etc., thereby efficiently exerting resistance to WSSV.
  • the treatment agent may be any water used to raise the target organism in aquaculture, fish farming, or the like, and may be any of seawater, brackish water, and freshwater (either natural or artificially adjusted).
  • the amount of treatment per time is not particularly limited and can be adjusted appropriately depending on the mode of use and the target of application.
  • the weight of the lignin component relative to the amount of water is preferably 0.02 mg/mL or more, and more preferably 0.1 mg/mL or more.
  • the upper limit is preferably 10.0 mg/mL or less, and more preferably 7.0 mg/mL or less. Therefore, the amount of treatment per time is preferably 0.02 to 10.0 mg/mL, and more preferably 0.1 to 7.0 mg/mL, in terms of the lignin component.
  • Treatment may be performed once to three times a day continuously (for example, once every 1 day, 3 days, 5 days, 1 week, 10 days, or 2 weeks).
  • the dosage form of the treatment agent may be, for example, granules, powders, or tablets, and may be selected appropriately based on the ease of treatment.
  • the preferred treatment method is spraying.
  • Examples of the agent other than the above-mentioned feed and treatment agent include disinfectants and cleaning agents for breeding articles.
  • breeding articles include aquariums, filtration devices, culture cages, nets, bottom sand, sanitary products, and other articles used in breeding crustacean farming, fish farming, and the like.
  • materials for the articles include, but are not limited to, glass, resin, cloth, and metal. The shape of the article is also not limited.
  • the method of administering the anti-WSSV agent is not particularly limited. Examples include the above-mentioned method of feeding the animals with feed or medicine containing the anti-WSSV agent, treating rearing water with a treatment agent containing the anti-WSSV agent, disinfecting or cleaning rearing items with a disinfectant or cleaning agent containing the anti-WSSV agent, or a combination of two or more of these.
  • the timing of administration can be selected depending on the administration method, for example, a method of administering for a certain period of time or a method of administering periodically.
  • Example 1 Wood chips (radiata pine) were subjected to sulfite treatment based on the sulfite cooking method.
  • sulfite treatment a solution of sodium sulfite with a SO2 concentration of 4 g/100 mL was used, and the temperature was 140 ° C., pH 2, and treatment time was 3 hours.
  • insoluble matter was filtered off.
  • the obtained filtrate was concentrated with a rotary evaporator until the solid content was 50%.
  • the pH of the solution was then adjusted to pH 4.5 with NaOH, and the solution was powdered with a spray dryer to obtain lignin sulfonate A of Example 1 (methoxy group amount 6.1 mass%, extractable component amount 0.2 mass%, extractable component amount/methoxy group amount: 0.04, sulfo group S content 5.2 mass%, solubility in water 100 mass%).
  • Example 2 The lignin sulfonate A obtained in Example 1 was dissolved in water to prepare an aqueous solution with a solid content of 25%, and the solution was dialyzed for 3 days using a dialysis membrane (molecular weight cutoff: 20,000, Spectra/Por (registered trademark) cellulose ester dialysis tube). The solution in the dialysis tube was collected and concentrated to a stock solution volume of 25%, and powdered with a spray dryer to obtain lignin sulfonate B (methoxy group content 11.1% by mass, extractable component content 0.09% by mass, extractable component content/methoxy group content: 0.008, sulfur content of sulfo group 3.5% by mass, amount dissolved in water 100% by mass).
  • a dialysis membrane moleukin sulfonate B
  • Example 3 Wood chips (radiata pine) were subjected to sulfite treatment based on the sulfite cooking method.
  • sulfite treatment a solution of sodium sulfite with a SO2 concentration of 2.5 g/100 mL was used, and the temperature was 140 ° C., pH 3, and treatment time was 3 hours.
  • insoluble matter was filtered off, and the obtained filtrate was concentrated with a rotary evaporator until the solid content was 50%.
  • the pH of the solution was then adjusted to 4.5 with NaOH, and powdered with a spray dryer.
  • the obtained powder was dissolved in water to prepare an aqueous solution with a solid content of 25%, and the pH was adjusted to 12 with 40% NaOH, and then the solution was oxidized with alkaline air at 140 ° C. for 120 minutes. Then, 70% sulfuric acid was added to adjust the pH to 3, and the partially desulfonated lignin sulfonate was fractionated and precipitated. The resulting precipitate of partially desulfonated lignin sulfonate was washed with water until the filtrate became neutral, 20 parts of the precipitate was suspended in 100 parts of water, and the precipitate was completely dissolved by adding 1 mol/L aqueous sodium hydroxide solution with stirring after heating to 60° C. until the pH reached 9.
  • the resulting solution was powdered with a spray dryer to obtain Ligninsulfonate C (methoxy group amount 11.2% by mass, extractable component amount 1.6% by mass, extractable component amount/methoxy group amount: 0.14, S content of sulfo group 2.7% by mass, amount dissolved in water 100% by mass).
  • S content of the sulfo group was calculated according to the following formula.
  • Sulfo group S content (mass%) total S content (mass%) - inorganic S content (mass%) (The S content in the formula indicates the S content relative to the solid content of the lignin sulfonate.)
  • the total S content was quantified by ICP emission spectrometry.
  • the inorganic S content was the sum of the SO3 content and the SO4 content quantified by ion chromatography.
  • the amount of lignin sulfonate dissolved in water was calculated as follows. 10 g (dry weight) of a sample was dispersed in 300 g of water, stirred for 60 minutes, and then filtered. The mass of the filtrate and the mass of the solids (dried filtrate) were measured. The mass of the solids in the filtrate was then divided by the mass of the sample (10 g) and multiplied by 100 to calculate the amount.
  • Test 1 Prevention of WSSV infection by feeding with lignin
  • Lignosulfonate A, B, or C was added to the basic diet at 1% or 4%, respectively, to prepare a test diet for vannamei shrimp.
  • the basic diet was mixed with 1% or 4% lignosulfonate and the same amount of water as the basic diet, and the resulting mixture was formed into noodles, dried (at about 70°C for about 2 hours), and randomly crushed to prepare the test diet.
  • the shrimp were fed twice a day by dispersing the mixture in seawater in an aquarium (lignosulfonate-added group).
  • the test diet was fed for one week (feeding test 1) or two weeks (feeding test 2).
  • the shrimp were reared according to the above-mentioned [Rearing conditions for shrimp], except for the feed.
  • the shrimp were reared in the same manner as the lignosulfonate-added group, except for the addition of the basic diet instead of the test diet, and used as the control group.
  • a WSSV stock suspension (shrimp artificially infected with WSSV were crushed in a juicer together with artificial seawater, and the resulting homogenized sample was filtered to remove any fleshy parts, and stored at -80°C until use) was added to the seawater of each group (15-20 shrimp per group) and the virus was inoculated. After addition, the same feed as described above was continued per 10 L of seawater, and rearing was continued according to the shrimp growth conditions described above.
  • the number of dead shrimp was observed two weeks after the virus inoculation.
  • the survival rate of shrimp before the virus inoculation was set as 100%, and the survival rate two weeks after the virus inoculation is shown in the following table and figure.
  • the results of feeding test 1 are shown in Table 1 and Figure 1 (1% lignin sulfonate added), and Table 2 and Figure 2 (4% lignin sulfonate added).
  • the results of feeding test 2 are shown in Table 3 and Figure 3 (1% lignin sulfonate added), and Table 4 and Figure 4 (4% lignin sulfonate added).
  • Test 2 WSSV infection prevention test using lignin seawater
  • Ten whitenamei shrimp were placed in seawater containing lignin sulfonate A, B, or C dissolved at 2 mg/mL, and the same WSSV stock suspension as used in Test 1 was added for virus inoculation.
  • the WSSV used for virus inoculation was prepared in the same manner as in Test 1.
  • the other growth conditions were the same as those in the above-mentioned [Shrimp growth conditions].
  • the anti-WSSV agent of the present invention is useful for preventing infection with white spot disease and can be used by mixing it into feed or rearing water.

Abstract

The purpose of the present invention is to provide a novel anti-WSSV agent which can prevent infection by a white spot syndrome virus (WSSV) before the infection occurs. The present invention provides: an anti-WSSV agent containing a lignin component such as lignin sulfonate as an active ingredient; and a method for preventing infection of white spot disease, the method comprising administering the agent to a crustacean. The lignin component is a component derived from a sulfite digestion method of a lignocellulose raw material, and preferably comprises an extracted component. The anti-WSSV agent can be used preferably for a crustacean that is subjected to marine culture or fish cultivating, and can be used more preferably for shrimp. The anti-WSSV agent can be used as a feed for crustaceans such as shrimp or a water treatment agent for marine culture or fish cultivating.

Description

抗ホワイトスポットシンドロームウイルス剤Anti-white spot syndrome virus agent
　本発明は、抗ホワイトスポットシンドロームウイルス剤に関する。 The present invention relates to an anti-white spot syndrome virus agent.
　エビ類の需要拡大に伴い、増殖のための養殖、栽培漁業が進められている。生産性向上のため高密度で飼育を行うことにより、ウイルスによる感染症が発生することがある。特に、ホワイトスポット病（ＷＳＤ、ホワイトスポット病、急性ウイルス血症（ｐｅｎａｅｉｄ　ａｃｕｔｅ　ｖｉｒｅｍｉａ：ＰＡＶ））は、エビの生産に大きな被害を与えてきた。 As demand for shrimp increases, shrimp farming and aquaculture are being promoted to increase their numbers. High-density rearing to improve productivity can lead to the outbreak of viral infections. In particular, white spot disease (WSD, penaeid acute viremia: PAV) has caused great damage to shrimp production.
　ホワイトスポット病感染を未然に防ぐための対策として、例えば、ホワイトスポットシンドロームウイルス（ＷＳＳＶ：ホワイトスポット病ウイルス）の組換え外被タンパク質を含有する経口ワクチン、外被タンパク質に対するＤＮＡワクチンが知られている（特許文献１、非特許文献１）。 Measures to prevent white spot disease infection include, for example, oral vaccines containing recombinant coat proteins of the white spot syndrome virus (WSSV) and DNA vaccines against coat proteins (Patent Document 1, Non-Patent Document 1).
特開２００８－６３３０２号公報JP 2008-63302 A
　しかしながら、特許文献１及び非特許文献１のようなワクチンは高価であり、感染防御期間も短い。例えば、非特許文献１のようなワクチンは感染防御期間が約１カ月である。したがって、エビ等の甲殻類のような小さな個体にＷＳＳＶワクチンを投与することは、作業の手間、コストから実用的とは言えない。 However, vaccines such as those described in Patent Document 1 and Non-Patent Document 1 are expensive and provide protection from infection for a short period of time. For example, the vaccine described in Non-Patent Document 1 provides protection from infection for approximately one month. Therefore, administering the WSSV vaccine to small organisms such as shrimp and other crustaceans is not practical due to the labor and cost involved.
　本発明は、ＷＳＳＶ感染を未然に防ぐことのできる新規な抗ＷＳＳＶ剤の提供を目的とする。 The present invention aims to provide a novel anti-WSSV agent that can prevent WSSV infection.
　本発明は、以下の〔１〕～〔１０〕を提供する。
〔１〕リグニン成分を有効成分とする、抗ホワイトスポットシンドロームウイルス剤。
〔２〕リグニン成分が、リグニンスルホン酸塩を少なくとも含む、〔１〕に記載の剤。
〔３〕リグニン成分は、リグノセルロース原料の亜硫酸蒸解法に由来する、〔１〕又は〔２〕に記載の剤。　
〔４〕リグニン成分は、抽出成分を含む、〔１〕～〔３〕のいずれか１項に記載の剤。
〔５〕甲殻類用である、〔１〕～〔４〕のいずれか１項に記載の剤。
〔６〕養殖又は栽培漁業に供される甲殻類用である、〔１〕～〔５〕のいずれか１項に記載の剤。
〔７〕甲殻類がエビを含む、〔５〕又は〔６〕に記載の剤。
〔８〕〔１〕～〔７〕のいずれか１項に記載の剤を含む、飼料。
〔９〕〔１〕～〔７〕のいずれか１項に記載の剤を含む、飼育用水用処理剤。
〔１０〕〔１〕～〔７〕のいずれか１項に記載の剤を甲殻類に投与することを含む、ホワイトスポット病の予防方法。 The present invention provides the following [1] to [10].
[1] An anti-white spot syndrome virus agent containing a lignin component as an active ingredient.
[2] The agent according to [1], wherein the lignin component contains at least a lignin sulfonate.
[3] The agent according to [1] or [2], wherein the lignin component is derived from a sulfite cooking method of a lignocellulosic raw material.
[4] The agent according to any one of [1] to [3], wherein the lignin component includes an extract component.
[5] The agent according to any one of [1] to [4], which is for use on crustaceans.
[6] The agent according to any one of [1] to [5], which is for use on crustaceans used in aquaculture or fish farming.
[7] The agent according to [5] or [6], wherein the crustacean includes shrimp.
[8] A feed comprising the agent according to any one of [1] to [7].
[9] A treatment agent for breeding water, comprising the agent according to any one of [1] to [7].
[10] A method for preventing white spot disease, comprising administering the agent according to any one of [1] to [7] to crustaceans.
　本発明の抗ＷＳＳＶ剤は、良好な抗ＷＳＳＶ抑制効果を発揮することができる。そのため、例えば、甲殻類等の生物の飼料、飼育用水用の処理剤等に利用することにより、ＷＳＳＶ感染を予防でき、これらの生物の生産性を向上させることができ、水産業、養殖業、ペット用品等の各分野において有用である。 The anti-WSSV agent of the present invention can exhibit a good anti-WSSV inhibitory effect. Therefore, for example, by using it as a feed for organisms such as crustaceans, or as a treatment agent for breeding water, it is possible to prevent WSSV infection and improve the productivity of these organisms, making it useful in various fields such as the fisheries, aquaculture, and pet supplies.
図１は、実施例の給餌試験１（１％リグニン）におけるエビの生存率を示す図である。FIG. 1 is a graph showing the survival rate of shrimp in Feeding Test 1 (1% lignin) of the Examples. 図２は、実施例の給餌試験１（４％リグニン）におけるエビの生存率を示す図である。FIG. 2 is a graph showing the survival rate of shrimp in Feeding Test 1 (4% lignin) of the Examples. 図３は、実施例の給餌試験２（１％リグニン）におけるエビの生存率を示す図である。FIG. 3 is a graph showing the survival rate of shrimp in Feeding Test 2 (1% lignin) of the embodiment. 図４は、実施例の給餌試験２（４％リグニン）におけるエビの生存率を示す図である。FIG. 4 is a graph showing the survival rate of shrimp in Feeding Test 2 (4% lignin) of the embodiment. 図５は、実施例の試験２におけるエビの生存率を示す図である。FIG. 5 is a graph showing the survival rate of shrimp in Test 2 of the Example.
　本発明は抗ホワイトスポットシンドロームウイルス剤に関する。 The present invention relates to an anti-white spot syndrome virus agent.
〔１．リグニン成分〕
　本発明の剤は、リグニン成分を有効成分として含有する。 1. Lignin Components
The agent of the present invention contains a lignin component as an active ingredient.
〔１．１．リグニン誘導体〕
　本明細書において、リグニン成分は、リグニン（植物体から単離されたリグニン）、リグニンの誘導体、リグニンの分解物、又はリグニンの分解物の誘導体を意味する。リグニン誘導体は、粉体状でも、液体状でもよい。液体状のリグニン成分の調製方法は特に限定されないが、例えば、粉末状のリグニン成分を適当な溶媒（例えば水、水酸化ナトリウム水溶液）に溶解して液体状のリグニン成分を得る方法が挙げられる。 [1.1. Lignin derivatives]
In this specification, the lignin component means lignin (lignin isolated from a plant), a derivative of lignin, a decomposition product of lignin, or a derivative of a decomposition product of lignin. The lignin derivative may be in a powder or liquid form. The method for preparing the liquid lignin component is not particularly limited, but an example of the method is to dissolve the powdered lignin component in a suitable solvent (e.g., water, an aqueous sodium hydroxide solution) to obtain the liquid lignin component.
　リグニン成分は、通常は木質バイオマス由来であり、その処理方法によって、構造及び物性が異なるいくつかの種類に分類される。リグニン成分としては、例えば、リグニンスルホン酸塩、クラフトリグニン、ソーダリグニン、ソーダ－アントラキノンリグニン、オルガノソルブリグニン、爆砕リグニン、硫酸リグニン及びそれらの分解物が挙げられる。これらのうち、リグニンスルホン酸塩が好ましい。リグニン誘導体は１種でも２種以上の組み合わせでもよいが、リグニンスルホン酸塩を少なくとも含むことが好ましい。 Lignin components are usually derived from woody biomass, and are classified into several types with different structures and physical properties depending on the processing method. Examples of lignin components include lignin sulfonate, kraft lignin, soda lignin, soda-anthraquinone lignin, organosolv lignin, explosive lignin, sulfuric acid lignin, and decomposition products thereof. Of these, lignin sulfonate is preferred. The lignin derivative may be one type or a combination of two or more types, but it is preferable that it contains at least lignin sulfonate.
－リグニンスルホン酸塩－
　リグニンスルホン酸塩は、リグノセルロース原料から亜硫酸処理を経て調製される、スルホ基を有するリグニン誘導体である。リグニンスルホン酸塩は、酸の形態でも塩の形態でもよいが、通常は塩の形態である。塩としては、例えば、一価金属塩、二価金属塩、アンモニウム塩ならびに有機アンモニウム塩が挙げられ、このうち、カルシウム塩、マグネシウム塩、ナトリウム塩、カリウム塩、カルシウム・ナトリウム混合塩が好ましい。 -Lignosulfonate-
Lignosulfonates are lignin derivatives having a sulfo group, which are prepared from lignocellulose raw materials through sulfite treatment. Lignosulfonates may be in the form of an acid or a salt, but are usually in the form of a salt. Examples of salts include monovalent metal salts, divalent metal salts, ammonium salts, and organic ammonium salts, and among these, calcium salts, magnesium salts, sodium salts, potassium salts, and calcium-sodium mixed salts are preferred.
　リグニンスルホン酸塩の調製方法としては、例えば、リグノセルロース原料又はリグニンそのものを亜硫酸処理して調製する方法、好ましくは、リグノセルロース原料又はリグニンそのものを亜硫酸蒸解処理して調製する方法が挙げられる。 Examples of methods for preparing lignin sulfonates include a method in which lignocellulose raw materials or lignin itself is subjected to a sulfite treatment, preferably a method in which lignocellulose raw materials or lignin itself is subjected to a sulfite cooking treatment.
（リグノセルロース原料）
　リグノセルロース原料は、構成体中にリグノセルロースを含むものであれば特に限定されない。例えば、木材、非木材等のパルプ原料が挙げられる。木材としては、たとえば、ラジアータパイン、エゾマツ、アカマツ、スギ、ヒノキ等の針葉樹木材、シラカバ、ブナ等の広葉樹木材が挙げられる。木材の樹齢、採取部位は問わない。そのため、互いに樹齢の異なる樹木から採取された木材や、互いに樹木の異なる部位から採取された木材を組み合わせて用いてもよい。非木材としては、例えば、竹、ケナフ、葦、稲が挙げられる。リグノセルロース原料は、１種単独でもよいし、２種以上の組み合わせでもよい。
　リグニンスルホン酸塩は、リグノセルロース原料以外の原料、例えば、リグニンから調製されてもよい。リグニンとしては、天然由来のもの、人工的に製造されたもの（例えば、ヒドロキシケイ皮アルコール類縁体の脱水素重合物）が例示され、いずれも利用できる。リグニンからのリグニンスルホン酸塩の調製は、例えば、リグニンを分解し、スルホン化する方法によることができる。　 (Lignocellulosic raw material)
The lignocellulose raw material is not particularly limited as long as it contains lignocellulose in the structure. For example, pulp raw materials such as wood and non-wood can be mentioned. For example, wood can be coniferous wood such as radiata pine, Yezo spruce, red pine, cedar, and cypress, and broadleaf wood such as white birch and beech. The age of the wood and the part where it is harvested do not matter. Therefore, wood harvested from trees of different ages or wood harvested from different parts of a tree may be used in combination. For example, non-wood can be bamboo, kenaf, reed, and rice. The lignocellulose raw material may be one type alone or two or more types in combination.
Lignosulfonates may be prepared from raw materials other than lignocellulose raw materials, for example, lignin. Examples of lignin include naturally occurring lignin and artificially produced lignin (for example, dehydrogenation polymerized products of hydroxycinnamic alcohol analogues), and both can be used. Lignosulfonates can be prepared from lignin, for example, by a method of decomposing lignin and sulfonating it.
（亜硫酸処理）
　亜硫酸処理は、亜硫酸及び亜硫酸塩の少なくともいずれかをリグノセルロース原料に接触させて行うことができる。亜硫酸処理の条件は、特に限定されず、リグノセルロース原料に含まれるリグニンの側鎖のα炭素原子にスルホ基が導入され得る条件であればよい。 (Sulfurous acid treatment)
The sulfite treatment can be carried out by contacting the lignocellulosic raw material with at least one of sulfurous acid and a sulfite salt. The conditions for the sulfite treatment are not particularly limited as long as they allow introduction of a sulfo group to the α-carbon atom of the side chain of lignin contained in the lignocellulosic raw material.
　亜硫酸処理は、亜硫酸蒸解法により行うことが好ましい。これにより、リグノセルロース原料中のリグニンをより定量的にスルホ化することができる。亜硫酸蒸解法は、亜硫酸及び亜硫酸塩の少なくともいずれかの溶液（例えば、水溶液、蒸解液）中で、リグノセルロース原料を高温下で反応させる方法である。当該方法は、サルファイトパルプの製造方法として工業的に確立されており、実施されているため、経済性及び実施容易性の面で有利である。　　 The sulfite treatment is preferably carried out by the sulfite cooking method. This allows the lignin in the lignocellulosic raw materials to be sulfonated more quantitatively. The sulfite cooking method is a method in which the lignocellulosic raw materials are reacted at high temperatures in a solution of at least one of sulfurous acid and sulfite salts (e.g., aqueous solution, cooking liquid). This method has been established and is being used industrially as a method for producing sulfite pulp, and is therefore advantageous in terms of economy and ease of implementation.
　亜硫酸塩の塩としては、亜硫酸蒸解を行う場合、例えば、マグネシウム塩、カルシウム塩、ナトリウム塩、アンモニウム塩が挙げられる。　　 When sulfite cooking is performed, examples of sulfite salts include magnesium salts, calcium salts, sodium salts, and ammonium salts.
　亜硫酸及び亜硫酸塩の少なくともいずれかの溶液における亜硫酸（ＳＯ２）濃度は、特に限定されないが、反応薬液１００ｍＬに対するＳＯ２の質量（ｇ）の比率が、１ｇ／１００ｍＬ以上が好ましく、亜硫酸蒸解を行う場合には２ｇ／１００ｍＬ以上がより好ましい。上限は、２０ｇ／１００ｍＬ以下が好ましく、亜硫酸蒸解を行う場合には１５ｇ／１００ｍＬ以下がより好ましい。ＳＯ２濃度は、１ｇ／１００ｍＬ～２０ｇ／１００ｍＬが好ましく、亜硫酸蒸解を行う場合には２ｇ／１００ｍＬ～１５ｇ／１００ｍＬがより好ましい。　　 The sulfurous acid (SO 2 ) concentration in the solution of at least one of sulfurous acid and sulfite is not particularly limited, but the ratio of the mass (g) of SO 2 to 100 mL of reaction solution is preferably 1 g/100 mL or more, and more preferably 2 g/100 mL or more when sulfite cooking is performed. The upper limit is preferably 20 g/100 mL or less, and more preferably 15 g/100 mL or less when sulfite cooking is performed. The SO 2 concentration is preferably 1 g/100 mL to 20 g/100 mL, and more preferably 2 g/100 mL to 15 g/100 mL when sulfite cooking is performed.
　亜硫酸処理のｐＨ値は特に限定されないが、通常は１０以下である。亜硫酸蒸解を行う場合、酸性下で行うことが好ましく、ｐＨ５以下がより好ましく、３以下が更に好ましい。これにより、リグニン誘導体（例えば、リグニンスルホン酸塩）を効率よく取り出すことができ、より高品質のパルプを得ることができる。ｐＨ値の下限は、０．１以上が好ましく、亜硫酸蒸解を行う場合には０．５以上がより好ましい。亜硫酸処理の際のｐＨ値は、０．１～１０が好ましく、亜硫酸蒸解を行う場合には０．５～５がより好ましく、０．５～３が更に好ましい。　　 The pH value of the sulfurous acid treatment is not particularly limited, but is usually 10 or less. When sulfurous acid cooking is performed, it is preferable to perform it under acidic conditions, with a pH of 5 or less being more preferable, and 3 or less being even more preferable. This allows lignin derivatives (e.g., lignin sulfonate salts) to be extracted efficiently, and pulp of higher quality can be obtained. The lower limit of the pH value is preferably 0.1 or more, and when sulfurous acid cooking is performed, 0.5 or more is more preferable. The pH value during sulfurous acid treatment is preferably 0.1 to 10, and when sulfurous acid cooking is performed, 0.5 to 5 is more preferable, and 0.5 to 3 is even more preferable.
　亜硫酸処理の温度は特に限定されないが、１７０℃以下が好ましく、亜硫酸蒸解を行う場合には１５０℃以下がより好ましい。下限は、７０℃以上が好ましく、亜硫酸蒸解を行う場合には１００℃以上がより好ましい。亜硫酸処理の温度条件は、７０～１７０℃が好ましく、亜硫酸蒸解を行う場合には１００～１５０℃がより好ましい。
　亜硫酸処理の処理時間は特に限定されなく、亜硫酸処理の諸条件にもよるが、０．５～２４時間が好ましく、１．０～１２時間がより好ましい。　　 The temperature of the sulfite treatment is not particularly limited, but is preferably 170° C. or lower, and more preferably 150° C. or lower when sulfite cooking is performed. The lower limit is preferably 70° C. or higher, and more preferably 100° C. or higher when sulfite cooking is performed. The temperature condition of the sulfite treatment is preferably 70 to 170° C., and more preferably 100 to 150° C. when sulfite cooking is performed.
The treatment time for the sulfurous acid treatment is not particularly limited, and although it depends on the conditions of the sulfurous acid treatment, it is preferably 0.5 to 24 hours, and more preferably 1.0 to 12 hours.
　亜硫酸処理においては、カウンターカチオン（塩：一般式（１）で表される基の置換基Ｍを含む）を供給する化合物を添加することが好ましい。カウンターカチオンを供給する化合物を添加することにより、亜硫酸処理におけるｐＨ値を一定に保つことができる。カウンターカチオンを供給する化合物としては、例えば、ＭｇＯ、Ｍｇ（ＯＨ）２、ＣａＯ、Ｃａ（ＯＨ）２、ＣａＣＯ３、ＮＨ３、ＮＨ４ＯＨ、ＮａＯＨ、ＮａＨＣＯ３、Ｎａ２ＣＯ３が挙げられる。カウンターカチオンは、マグネシウムイオン、ナトリウムイオン、およびカルシウムイオンが好ましい。　　 In the sulfite treatment, it is preferable to add a compound that supplies a counter cation (salt: containing the substituent M of the group represented by general formula (1)). By adding a compound that supplies a counter cation, the pH value in the sulfite treatment can be kept constant. Examples of compounds that supply counter cations include MgO, Mg(OH) 2 , CaO, Ca(OH) 2 , CaCO 3 , NH 3 , NH 4 OH, NaOH, NaHCO 3 , and Na 2 CO 3. The counter cation is preferably a magnesium ion, a sodium ion, or a calcium ion.
　亜硫酸処理において、亜硫酸及び亜硫酸塩の少なくともいずれかの溶液を用いる場合、溶液には必要に応じて、ＳＯ２のほかに、上記カウンターカチオン（塩）、蒸解浸透剤（例えば、アントラキノンスルホン酸塩、アントラキノン、テトラヒドロアントラキノン等の環状ケトン化合物）を含ませてもよい。　　 In the sulfurous acid treatment, when a solution of at least one of sulfurous acid and sulfite is used, the solution may contain, in addition to SO2 , the above-mentioned counter cation (salt) and a digestion and penetration agent (for example, a cyclic ketone compound such as anthraquinone sulfonate, anthraquinone, or tetrahydroanthraquinone), as necessary.
　亜硫酸処理を行う際に用いる設備に限定はなく、例えば、一般に知られている溶解パルプの製造設備を用いることができる。　　 There are no limitations on the equipment used for sulfite treatment; for example, commonly known dissolving pulp manufacturing equipment can be used.
　亜硫酸及び亜硫酸塩の少なくともいずれかの溶液から中間生成物を分離するには、常法に従って行えばよい。分離方法としては、例えば、亜硫酸蒸解後の亜硫酸蒸解排液の分離方法（例えば、ろ過）が挙げられる。　　 The intermediate product can be separated from the solution of at least one of sulfurous acid and sulfite salts by a conventional method. For example, a method for separating the sulfurous acid digestion effluent after sulfurous acid digestion (e.g., filtration) can be used.
　亜硫酸処理により得られる（例えば、亜硫酸溶液の不溶解物をろ過後のろ液又はろ過残渣として、好ましくはろ液として得られる）リグニンスルホン酸塩は、そのまま、又は必要に応じて濃縮してリグニン誘導体として用いてもよい。一方、必要に応じてさらに他の処理を行ってもよい。これにより、純度の高い、及び／又は、適度なスルホン化度（Ｓ含量）を有するリグニン誘導体を得ることができる。他の処理としては、例えば、アルカリ処理、酸化処理、透析処理、ＵＦ処理、及びこれらの組み合わせが挙げられる。　 The lignin sulfonate obtained by the sulfite treatment (e.g., as the filtrate or filtration residue after filtering the insoluble matter of the sulfite solution, preferably as the filtrate) may be used as a lignin derivative as is, or after concentrating as necessary. On the other hand, if necessary, further treatment may be performed. This makes it possible to obtain a lignin derivative having a high purity and/or an appropriate degree of sulfonation (S content). Examples of other treatments include alkali treatment, oxidation treatment, dialysis treatment, UF treatment, and combinations of these.
（アルカリ処理）
　アルカリ処理は、亜硫酸処理後のろ過残渣（不溶解物）やろ液、透析処理後の処理物に対して行うことが好ましい。アルカリ処理は、対象サンプルをアルカリ性条件下におけばよい。アルカリ性条件下におくとは、通常、ｐＨ値が８以上、好ましくはｐＨ値が９以上の水溶液下におくことをいう。ｐＨ値の上限は、通常、１４以下である。　　 (Alkaline treatment)
The alkali treatment is preferably performed on the filtration residue (insoluble matter) or filtrate after the sulfite treatment, or on the treated product after the dialysis treatment. The alkali treatment can be performed by placing the target sample under alkaline conditions. Placing under alkaline conditions usually means placing the sample in an aqueous solution having a pH value of 8 or more, preferably a pH value of 9 or more. The upper limit of the pH value is usually 14 or less.
　アルカリ処理においては、通常、アルカリ性物質を亜硫酸処理物に接触させる。アルカリ性物質は、特に限定されないが、例えば、水酸化カルシウム、水酸化マグネシウム、水酸化ナトリウム、水酸化カリウム、炭酸ナトリウム、アンモニアが挙げられる。中でも、水酸化ナトリウム、水酸化カルシウムが好ましい。アルカリ性物質は、１種単独で用いてもよく、２種以上を組み合わせて用いてもよい。　　 In the alkaline treatment, an alkaline substance is usually brought into contact with the sulfite-treated product. The alkaline substance is not particularly limited, but examples include calcium hydroxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, and ammonia. Of these, sodium hydroxide and calcium hydroxide are preferred. The alkaline substance may be used alone or in combination of two or more types.
　亜硫酸処理物にアルカリ性物質を接触させる方法としては、亜硫酸処理物の分散液又は溶液（例えば、水分散液、水溶液）を調製し、該分散液又は溶液中にアルカリ性物質を添加する方法や、亜硫酸処理物にアルカリ性物質の溶液又は分散液（例えば、水分散液、水溶液）を添加する方法が例示される。　　 Examples of methods for contacting an alkaline substance with a sulfite-treated product include preparing a dispersion or solution (e.g., an aqueous dispersion or aqueous solution) of the sulfite-treated product and adding an alkaline substance to the dispersion or solution, and adding a solution or dispersion of an alkaline substance (e.g., an aqueous dispersion or aqueous solution) to the sulfite-treated product.
　アルカリ処理の温度は特に限定されないが、４０℃以上が好ましく、６０℃以上がより好ましい。上限は、２００℃以下が好ましく、１８０℃以下がより好ましく、１７０℃以下がさらに好ましい。　　 The temperature of the alkali treatment is not particularly limited, but is preferably 40°C or higher, and more preferably 60°C or higher. The upper limit is preferably 200°C or lower, more preferably 180°C or lower, and even more preferably 170°C or lower.
　アルカリ処理におけるアルカリ性物質の量は、亜硫酸処理物の固形分質量に対して、或いは、アルカリ処理抽出物を水性溶媒（例えば、水）に分散した水溶液又は分散液を調製する場合、水溶液又は分散液の質量に対して、０．５～４０質量％が好ましく、１．０～３０質量％がより好ましい。　　 The amount of alkaline substance in the alkaline treatment is preferably 0.5 to 40% by mass, more preferably 1.0 to 30% by mass, based on the solids mass of the sulfite-treated product, or, when preparing an aqueous solution or dispersion by dispersing the alkaline-treated extract in an aqueous solvent (e.g., water), based on the mass of the aqueous solution or dispersion.
　アルカリ処理の時間は特に限定されないが、０．１時間以上が好ましく、０．５時間以上がより好ましい。上限は、１０時間以下が好ましく、６時間以下がより好ましい。　　 The duration of the alkali treatment is not particularly limited, but is preferably 0.1 hours or more, and more preferably 0.5 hours or more. The upper limit is preferably 10 hours or less, and more preferably 6 hours or less.
　アルカリ処理に先立ち、必要に応じて、亜硫酸処理物の溶解、分散処理、濃度の調整（水等の水性溶媒の溶液又は分散液の調製）を行ってもよい。分散処理は、例えばディスクリファイナーの通過、ミキサー、ディスパーザーへの添加、ニーダー処理により行うことができる。濃度の調整は、例えば、水等の水性溶媒を用いて行うことができる。　 Prior to the alkali treatment, the sulfite-treated product may be dissolved, dispersed, or its concentration adjusted (preparation of a solution or dispersion in an aqueous solvent such as water) as necessary. Dispersion can be performed, for example, by passing the product through a disc refiner, adding it to a mixer or disperser, or by kneading. Concentration can be adjusted, for example, by using an aqueous solvent such as water.
（酸化処理）
　酸化処理は、亜硫酸処理後に得られる処理物（例えば、ろ過後のろ液）、又はアルカリ処理後の処理物に対して行うことができる。酸化処理は、適宜酸化剤を用いて行えばよく、酸化剤が気体の場合、気体をろ液中に通気することにより行うことができる。酸化剤が液体の場合、液体をろ過残渣やろ液に添加することにより行うことができる。酸化剤は、空気、酸素、過酸化水素、オゾン、又はこれらの組み合わせが好ましい。酸化処理は、アルカリ条件で行うこと（アルカリ酸化処理）が好ましい。アルカリ酸化処理の処理ｐＨは、通常８以上であり、１０以上が好ましく、１２以上がより好ましい。酸化処理の温度は、通常、２０～２００℃であり、好ましくは５０～１８０℃である。酸化処理の時間は、通常、０．１時間以上が好ましく、０．５時間以上がより好ましい。上限は、５時間以下が好ましく、３時間以下がより好ましい。　 (Oxidation Treatment)
The oxidation treatment can be carried out on the treated product obtained after the sulfurous acid treatment (for example, the filtrate after filtration) or the treated product after the alkali treatment. The oxidation treatment can be carried out by using an appropriate oxidizing agent. When the oxidizing agent is a gas, the oxidation can be carried out by bubbling the gas into the filtrate. When the oxidizing agent is a liquid, the oxidation can be carried out by adding the liquid to the filtration residue or the filtrate. The oxidizing agent is preferably air, oxygen, hydrogen peroxide, ozone, or a combination thereof. The oxidation treatment is preferably carried out under alkaline conditions (alkaline oxidation treatment). The treatment pH of the alkaline oxidation treatment is usually 8 or more, preferably 10 or more, and more preferably 12 or more. The temperature of the oxidation treatment is usually 20 to 200° C., and preferably 50 to 180° C. The time of the oxidation treatment is usually preferably 0.1 hours or more, and more preferably 0.5 hours or more. The upper limit is preferably 5 hours or less, and more preferably 3 hours or less.
（透析処理又はＵＦ処理）
　透析処理は、亜硫酸処理後に得られる処理物（例えば、ろ過後のろ液）に対して行うことができる。透析膜としては、例えば、セルロースアセテート等のセルロース系膜、エチレンビニルアルコール、ポリアクリロニトリル、ポリメチルメタクリレート、ポリスルホン、ポリエーテルスルホン等の合成高分子系膜が挙げられる。分子量分画は通常５，０００～１００，０００、好ましくは７，０００～８０，０００、より好ましくは１０，０００～５０，０００である。　 (Dialysis or UF treatment)
The dialysis treatment can be performed on the treated product obtained after the sulfite treatment (for example, the filtrate after filtration). Examples of the dialysis membrane include cellulose-based membranes such as cellulose acetate, and synthetic polymer-based membranes such as ethylene vinyl alcohol, polyacrylonitrile, polymethyl methacrylate, polysulfone, and polyethersulfone. The molecular weight fraction is usually 5,000 to 100,000, preferably 7,000 to 80,000, and more preferably 10,000 to 50,000.
　透析処理の代わりに、ＵＦ（限外ろ過）処理を用いることができる。ＵＦ膜としては、公知のＵＦ膜を用いることができる。例えば、中空糸膜、スパイラル膜、チューブラー膜、平膜が挙げられる。ＵＦ膜の素材は、公知のものを用いることができる。例えば、酢酸セルロース、芳香族ポリアミド、ポリビニルアルコール、ポリスルホン、ポリフッ化ビニリデン、ポリエチレン、ポリアクリロニトリル、セラミックが挙げられる。なお、ＵＦ膜は市販品であってもよい。　 Instead of dialysis, UF (ultrafiltration) treatment can be used. Any known UF membrane can be used. Examples include hollow fiber membranes, spiral membranes, tubular membranes, and flat membranes. Any known material can be used for the UF membrane. Examples include cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, and ceramics. The UF membrane may be a commercially available product.
　ＵＦ膜の分画分子量は、５，０００～３０，０００が好ましく、１０，０００～２５，０００がより好ましく、１５，０００～２３，０００がさらに好ましい。分画分子量が５，０００以上のＵＦ膜を用いると、処理液の分離速度が過度に遅くなることを防止し得る。また、分画分子量が３０，０００以下のＵＦ膜を用いると、処理液からリグニンが分離されなくなることを防止し得る。　 The molecular weight cutoff of the UF membrane is preferably 5,000 to 30,000, more preferably 10,000 to 25,000, and even more preferably 15,000 to 23,000. Using a UF membrane with a molecular weight cutoff of 5,000 or more can prevent the separation speed of the treatment liquid from becoming excessively slow. Also, using a UF membrane with a molecular weight cutoff of 30,000 or less can prevent lignin from not being separated from the treatment liquid.
　ＵＦ膜を用いたＵＦ処理による濃縮倍率は、任意に設定できる。すなわち、濃縮液の流出量が任意の量になった時に、ＵＦ処理を停止すればよい。好ましくは２～６倍に濃縮することが好ましい。２～６倍に濃縮とは、原液（黒液）量が１／２～１／６量になることを意味する。　 The concentration rate by UF treatment using a UF membrane can be set as desired. In other words, the UF treatment can be stopped when the amount of concentrated liquid flowing out reaches a desired amount. It is preferable to concentrate it 2 to 6 times. Concentrating 2 to 6 times means that the amount of raw liquid (black liquor) becomes 1/2 to 1/6 of its original amount.
　ＵＦ処理時の処理液の温度は特に限定されない。例えば、２０～８０℃が好ましく、ＵＦ膜材質の耐熱面を考慮すると、２０～７０℃がより好ましい。ＵＦ処理時の処理液のｐＨ値は、２～１１が好ましい。ＵＦ処理時の黒液の固形分濃度（ｗ／ｗ）は、２～３０％が好ましく、５～２０％がより好ましい。　 The temperature of the treatment liquid during UF treatment is not particularly limited. For example, 20 to 80°C is preferable, and considering the heat resistance of the UF membrane material, 20 to 70°C is more preferable. The pH value of the treatment liquid during UF treatment is preferably 2 to 11. The solids concentration (w/w) of the black liquor during UF treatment is preferably 2 to 30%, and more preferably 5 to 20%.
　リグニンスルホン酸塩の製造方法の別の例としては、クラフトリグニンをスルホン化する方法が挙げられる。　 Another example of a method for producing lignin sulfonates is the sulfonation of kraft lignin.
－クラフトリグニン－
　クラフトリグニン（Ｋｒａｆｔ　Ｌｉｇｎｉｎ）は、チオリグニン（Ｔｈｉｏｌｉｇｎｉｎ）、サルフェートリグニン（Ｓｕｌｐｈａｔｅ　Ｌｉｇｎｉｎ）とも呼ばれる。クラフトリグニンとしては、例えば、クラフトリグニンのアルカリ溶液、クラフトリグニンのアルカリ溶液をスプレードライして粉末化した粉末化クラフトリグニン、クラフトリグニンのアルカリ溶液を酸で沈殿させた酸沈殿クラフトリグニンが挙げられる。クラフトリグニンは、これらのうち１種を用いてもよく、２種以上の組み合わせでもよい。 - Kraft lignin -
Kraft lignin is also called thiolignin or sulfate lignin. Examples of the kraft lignin include an alkaline solution of kraft lignin, powdered kraft lignin obtained by spray-drying an alkaline solution of kraft lignin to obtain a powder, and acid-precipitated kraft lignin obtained by precipitating an alkaline solution of kraft lignin with an acid. The kraft lignin may be one of these, or a combination of two or more of them.
　クラフトリグニンのアルカリ溶液の調製方法としては、例えば、クラフト法パルプ製造プロセス内を流れるＮａ２Ｓを含むアルカリ性溶液を電解酸化法により電解し、陰極側でＮａＯＨ溶液を生じさせる方法（特開２０００－３３６５８９号公報）が挙げられる。クラフトリグニンのアルカリ溶液を酸で沈殿させた酸沈殿クラフトリグニンの調製方法としては、例えば、粉末状の酸沈殿クラフトリグニンの調製方法（国際公開第２００６／０３８８６３号、国際公開第２００６／０３１１７５号、国際公開第２０１２／００５６７７号）が挙げられる。　 An example of a method for preparing an alkaline solution of kraft lignin is a method in which an alkaline solution containing Na 2 S flowing in a kraft pulp production process is electrolyzed by electrolytic oxidation to produce a NaOH solution on the cathode side (JP 2000-336589 A). An example of a method for preparing acid-precipitated kraft lignin by precipitating an alkaline solution of kraft lignin with an acid is a method for preparing powdery acid-precipitated kraft lignin (WO 2006/038863, WO 2006/031175, WO 2012/005677).
　クラフトリグニンのスルホン化の方法は、通常の亜硫酸処理や亜硫酸蒸解処理によるスルホン化によればよく、また、「Ｄｅｖｅｌｏｐｍｅｎｔ　ｏｆ　Ｎｅｗ　Ｌｉｇｎｉｎ　Ｄｅｒｉｖａｔｉｖｅｓ　ａｓ　Ｓｏｉｌ　Ｃｏｎｄｉｔｉｏｎｉｎｇ　Ａｇｅｎｔｓ　ｂｙ　Ｒａｄｉｃａｌ　Ｓｕｌｆｏｎａｔｉｏｎ　ａｎｄ　Ａｌｋａｌｉ－Ｏｘｙｇｅｎ　Ｔｒｅａｔｍｅｎｔ：木材学会誌，第４３巻，第８号，６６９－６７７（１９９７）」に記載されている方法なども挙げられるが、上記方法に限定されず、他の方法によることができる。　 The method of sulfonating kraft lignin may be sulfonation by ordinary sulfite treatment or sulfite cooking treatment, or may be the method described in "Development of New Lignin Derivatives as Soil Conditioning Agents by Radical Sulfonation and Alkali-Oxygen Treatment: Mokuzai Gakkaishi, Vol. 43, No. 8, 669-677 (1997)", but is not limited to the above methods and other methods may be used.
〔１．２．抽出成分〕
　リグニン成分は、抽出成分をさらに含んでもよい。抽出成分を含むことにより、抗ＷＳＳＶ活性をより促進させることができる。 1.2. Extracted Components
The lignin component may further include an extractive component. By including the extractive component, the anti-WSSV activity can be further enhanced.
　本明細書において抽出成分（樹木抽出成分）は、植物（例えばパルプ原料として用いられる植物、好ましくはスギ属植物、ヒノキ属植物、マツ属植物、カラマツ属植物、モミ属植物、ユーカリ属植物の木材）の微量成分であり、通常、木材の有機溶媒等の抽出処理により得ることができる。抽出成分は、通常、色調、におい、耐久性、接着性、生物活性等の木材物性の決定因子であり、木材を化学的に特徴付ける成分であると言われている。樹木は、セルロース及びヘミセルロースの堆積、並びにリグニンの沈着により骨格が形成された後、心材形成と連動する抽出成分の蓄積を待って、初めて生物材料として完成する。抽出成分を得る際使用される有機溶媒としては、例えば、ヘキサン、ベンゼン、エーテル、アセトン、アルコールが挙げられる。木材に対する抽出成分の含有量は、通常、約５％以下である。 In this specification, the term "extractive components (tree extractive components)" refers to trace components of plants (e.g., plants used as pulp raw materials, preferably wood from plants of the genus Cryptomeria, Chamaecyparis obtusa, Pinus serrata, Larch, Abies monad, and Eucalyptus), and can usually be obtained by extraction treatment of wood with organic solvents or the like. Extractive components are usually determining factors for wood properties such as color, odor, durability, adhesiveness, and biological activity, and are said to be the components that chemically characterize wood. After the skeleton of a tree is formed by the accumulation of cellulose and hemicellulose and the deposition of lignin, the tree is not completed as a biological material until the accumulation of extractive components, which are linked to the formation of heartwood, occurs. Examples of organic solvents used to obtain extractive components include hexane, benzene, ether, acetone, and alcohol. The content of extractive components in wood is usually about 5% or less.
　抽出成分は、通常、低分子化合物（通常、低分子有機化合物）を含む。その多くは分子量数千以下の二次代謝産物であり、一般には極めて多様であるが、芳香族抽出成分とテルペノイドに大別される。芳香族抽出成分としては、例えば、フラボノイド類、タンニン類、リグナン類、スチルベン類が挙げられる。抽出成分は、これらから選ばれる少なくとも１種を含む。 　Extracted components usually contain low molecular weight compounds (usually low molecular weight organic compounds). Many of these are secondary metabolic products with molecular weights of several thousand or less, and although they are generally extremely diverse, they are broadly classified into aromatic extracted components and terpenoids. Examples of aromatic extracted components include flavonoids, tannins, lignans, and stilbenes. The extracted components contain at least one type selected from these.
　フラボノイド類は、ジフェニルプロパン（Ｃ６－Ｃ３－Ｃ６）骨格を持つ化合物の総称であり、例えば、フラボン、フラバノン、カルコン、オーロン、イソフラボン、カテキン、ロイコアントシアニジンが挙げられる。タンニン類は、加水分解型タンニンと縮合型タンニンのいずれでもよい。加水分解型タンニンは、グルコースなどを核として、これに没食子酸などのフェノールカルボン酸がエステル結合した構造を有するタンニンであり、例えば、ガロタンニン、エラグタンニンが挙げられる。加水分解型タンニンは、酸、アルカリなどによる加水分解により単純なフラグメントに分解され得るところ、ガロタンニン及びエラグタンニンを加水分解すると、それぞれ没食子酸、エラグ酸が得られる。縮合型タンニンとしては、例えば、カテキン類又はロイコアントシアニジン類を前駆物質とする無定形高分子が挙げられる。　 Flavonoids are a general term for compounds having a diphenylpropane (C 6 -C 3 -C 6 ) skeleton, and examples thereof include flavones, flavanones, chalcones, aurone, isoflavones, catechins, and leucoanthocyanidins. Tannins may be either hydrolyzable tannins or condensed tannins. Hydrolyzable tannins are tannins having a structure in which a phenolic carboxylic acid such as gallic acid is ester-bonded to a nucleus such as glucose, and examples thereof include gallotannins and ellagitannins. Hydrolyzable tannins can be decomposed into simple fragments by hydrolysis with an acid, an alkali, or the like, and when gallotannins and ellagitannins are hydrolyzed, gallic acid and ellagic acid are obtained, respectively. Condensed tannins include, for example, amorphous polymers having catechins or leucoanthocyanidins as precursors.
　リグナン類としては、例えば、リグナン及びその近縁物質が挙げられる。リグナン（レジノール）は、リグニンの構成単位と同じフェニルプロパン単位が、その側鎖β位間で炭素－炭素結合したＣ６－Ｃ３－Ｃ３－Ｃ６骨格を有する構造を有する。リグニンと異なり、分子内に不斉炭素をもち、光学活性を有する。リグナン近縁物質としては、例えば、リグニン骨格より炭素数が１個少ないＣ６－Ｃ３－Ｃ２－Ｃ６骨格を有するノルリグナン類が挙げられる。スチルベン類は、α、β－ジフェニルエチレン骨格を有する化合物であればよい。　 Examples of lignans include lignans and substances closely related thereto. Lignans (resinols) have a structure having a C 6 -C 3 -C 3 -C 6 skeleton in which phenylpropane units, which are the same structural units of lignin, are carbon-carbon bonded between the β-positions of the side chains. Unlike lignin, they have an asymmetric carbon in the molecule and are optically active. Examples of substances closely related to lignans include norlignans having a C 6 -C 3 -C 2 -C 6 skeleton, which has one less carbon atom than the lignin skeleton. Stilbenes may be compounds having an α,β-diphenylethylene skeleton.
　テルペノイドは、イソプレン単位（Ｃ５Ｈ８）が２個以上、鎖状又は環状に結合した一連の化合物であればよい。イソプレノイド単位２、３、４、６個からなるテルペノイドをそれぞれモノテルペン（炭素原子数１０）、セスキテルペン（炭素原子数１５）、ジテルペン（炭素原子数２０：例えば、アビエチン酸）、トリテルペン（炭素原子数３０）というが、これらのいずれでもよい。　 The terpenoid may be a series of compounds in which two or more isoprene units ( C5H8 ) are bonded in a chain or ring shape. Terpenoids consisting of 2, 3, 4, and 6 isoprenoid units are called monoterpenes (10 carbon atoms), sesquiterpenes (15 carbon atoms), diterpenes (20 carbon atoms: for example, abietic acid), and triterpenes (30 carbon atoms), respectively, and any of these may be used.
　抽出成分は、抽出成分の基本的な炭素骨格、置換基により多様な生理活性を有する。これらの生理活性によっては抗ウイルス作用に影響することがある。生理活性としては、例えば、微生物、昆虫、植物に対する生物活性（「抽出成分による木材の生物劣化抵抗性」、木材保存　３４（２）、４８－５４、２００８）、材の耐久性の向上、材の化学的、物理的加工（例えばパルプ化、漂白、セメント硬化）への阻害作用が挙げられる。 　Extractives have a variety of physiological activities depending on the basic carbon skeleton and substituents of the extractives. Some of these physiological activities may affect the antiviral action. Examples of physiological activities include biological activity against microorganisms, insects, and plants ("Resistance of wood to biological deterioration by extractives", Wood Conservation 34 (2), 48-54, 2008), improvement of wood durability, and inhibitory action against chemical and physical processing of wood (e.g. pulping, bleaching, cement hardening).
〔１．３．抽出成分量、メトキシ基量、スルホ基Ｓ含量、水への溶解性〕
－抽出成分量－
　リグニン成分の固形分あたりの抽出成分量は、１．７質量％以下が好ましく、１．６５質量％以下がより好ましく、１．６３質量％以下が更に好ましい。これにより、剤の安全性を確保することができる。下限は、通常、０．０１質量％以上、好ましくは０．０３質量％以上、より好ましくは０．０５質量％以上である。これにより、ＷＳＳＶ抑制効果を良好に発揮できる。したがって、リグニン成分の固形分あたりの抽出成分量は、１．７質量％以下が好ましく、０．０１～１．６５質量％がより好ましく、０．０３～１．６３質量％が更に好ましく、０．０５～１．６３質量％が更により好ましい。 [1.3. Amount of extractable components, amount of methoxy groups, sulfo group S content, and solubility in water]
- Amount of extracted components -
The amount of the extracted components per solid content of the lignin component is preferably 1.7% by mass or less, more preferably 1.65% by mass or less, and even more preferably 1.63% by mass or less. This ensures the safety of the agent. The lower limit is usually 0.01% by mass or more, preferably 0.03% by mass or more, and more preferably 0.05% by mass or more. This allows the WSSV suppression effect to be exhibited well. Therefore, the amount of the extracted components per solid content of the lignin component is preferably 1.7% by mass or less, more preferably 0.01 to 1.65% by mass, even more preferably 0.03 to 1.63% by mass, and even more preferably 0.05 to 1.63% by mass.
　抽出成分量は、ＪＩＳ　Ｋ　０１０２：２０１９記載のヘキサン抽出物質の測定法によって測定できる。 The amount of extractable components can be measured using the method for measuring hexane extractable substances described in JIS K 0102:2019.
－メトキシ基－
　一般にリグニンの構造中には芳香核に結合したメトキシ基が存在するため、メトキシ基量は、リグニンおよびリグニン誘導体含量の指標である。 -Methoxy group-
Generally, lignin has a structure containing methoxy groups bonded to aromatic nuclei, and therefore the amount of methoxy groups is an index of the content of lignin and lignin derivatives.
　リグニン成分の固形分あたりのメトキシ基量は、通常、２．０質量％以上、２．５質量％以上又は３．０質量％以上、好ましくは３．５質量％以上、より好ましくは４．０質量％以上である。上限は特に制限されないが、通常２０質量％以下である。したがって、リグニン成分の固形分あたりのメトキシ基量は、通常、２．０質量％以上、２．５～２０質量％又は３．０～２０質量％、好ましくは３．５～２０質量％、より好ましくは４．０～２０質量％である。これにより、本発明の抗ＷＳＳＶ剤の安全性を確保しつつＷＳＳＶ抑制効果を良好に発揮できる。 The amount of methoxy groups per solid content of the lignin component is usually 2.0 mass% or more, 2.5 mass% or more, or 3.0 mass% or more, preferably 3.5 mass% or more, and more preferably 4.0 mass% or more. There is no particular upper limit, but it is usually 20 mass% or less. Therefore, the amount of methoxy groups per solid content of the lignin component is usually 2.0 mass% or more, 2.5 to 20 mass% or 3.0 to 20 mass%, preferably 3.5 to 20 mass%, and more preferably 4.0 to 20 mass%. This allows the anti-WSSV agent of the present invention to exhibit a good WSSV inhibitory effect while ensuring the safety of the agent.
　メトキシ基量は、Ｖｉｅｂｏｃｋ及びＳｃｈｗａｐｐａｃｈ法によるメトキシ基の定量法（「リグニン化学研究法」、Ｐ．３３６～３４０、平成６年、ユニ出版（株）発行、参照）により測定できる。 The amount of methoxy groups can be measured by the quantitative determination of methoxy groups using the Viebock and Schwappach method (see "Lignin Chemical Research Methods," pp. 336-340, 1994, published by Uni Publishing Co., Ltd.).
－抽出成分量／メトキシ基量（質量比）－
　上述のとおり、抽出成分は、その生理活性によっては安全性に影響することがあり、一方、メトキシ基量はリグニンおよびリグニン誘導体含量の指標である。そのため、抽出成分量のメトキシ基量に対する質量比は、安全性及びＷＳＳＶ抑制効果のバランスを表すと言える。 --Amount of extracted components/Amount of methoxy groups (mass ratio)--
As described above, the extracted components may affect safety depending on their physiological activity, while the amount of methoxy groups is an index of the content of lignin and lignin derivatives. Therefore, the mass ratio of the amount of extracted components to the amount of methoxy groups can be said to represent the balance between safety and WSSV inhibitory effect.
　リグニン成分においては、リグニン成分の固形分当たりの、抽出成分量のメトキシ基量に対する質量比が、通常、１．０以下、好ましくは０．８以下、より好ましくは０．６以下、更に好ましくは０．４以下、更により好ましくは０．３以下である。下限は、０．００１以上が好ましく、より好ましくは０．００５以上である。したがって、リグニン成分の固形分当たりの、抽出成分量のメトキシ基量に対する質量比は、通常、１．０以下であり、好ましくは０．００１～０．８、より好ましくは０．００１～０．６、更に好ましくは０．００５～０．４、更により好ましくは０．００５～０．３である。質量比が上記範囲であることにより、安全性を確保しつつＷＳＳＶ抑制効果を良好に発揮できる。 In the lignin component, the mass ratio of the amount of extractable components to the amount of methoxy groups per solid content of the lignin component is usually 1.0 or less, preferably 0.8 or less, more preferably 0.6 or less, even more preferably 0.4 or less, and even more preferably 0.3 or less. The lower limit is preferably 0.001 or more, and more preferably 0.005 or more. Therefore, the mass ratio of the amount of extractable components to the amount of methoxy groups per solid content of the lignin component is usually 1.0 or less, preferably 0.001 to 0.8, more preferably 0.001 to 0.6, even more preferably 0.005 to 0.4, and even more preferably 0.005 to 0.3. By having the mass ratio in the above range, it is possible to achieve a good WSSV suppression effect while ensuring safety.
－スルホ基－
　リグニン誘導体は、一般式（１）：－ＳＯ３Ｍで表されるスルホ基を含む。式（１）中、Ｍは、水素原子、一価金属塩、二価金属塩、アンモニウム塩ならびに有機アンモニウム塩であり、例えば、ナトリウムイオン、カルシウム、カリウムイオン、マグネシウムイオン、又はアンモニウムイオンが挙げられ、ナトリウムイオン、マグネシウムイオン、カルシウムイオンが好ましい。リグニン誘導体は、置換基Ｍが異なる２種以上の一般式（１）で表される基を含んでもよい。 -Sulfo group-
The lignin derivative contains a sulfo group represented by the general formula (1): -SO 3 M. In formula (1), M is a hydrogen atom, a monovalent metal salt, a divalent metal salt, an ammonium salt, or an organic ammonium salt, and examples thereof include a sodium ion, a calcium ion, a potassium ion, a magnesium ion, or an ammonium ion, with the sodium ion, magnesium ion, and calcium ion being preferred. The lignin derivative may contain two or more types of groups represented by the general formula (1) having different substituents M.
－スルホ基Ｓ含量－
　リグニン誘導体の、一般式（１）で表される基のＳ含量（硫黄原子の含有量：スルホ基Ｓ含量）の合計量（リグニン誘導体の固形分量に対する割合）は、通常は０．５質量％以上であり、好ましくは１．０質量％以上、より好ましくは１．２質量％以上である。これにより、一般式（１）で表される基を豊富に含み、リグニン誘導体が水溶性を示すことができ、ＷＳＳＶに対し良好なウイルスの抑制効果を示すことができる。上限は、通常２０．０質量％以下、好ましくは１５．０質量％以下、より好ましくは１２．０質量％以下である。これにより、適度な水溶性を示すことができ、効率よくウイルスの抑制効果を発揮できる。したがって、リグニン誘導体の、スルホ基Ｓ含量の合計量は、通常は０．５～２０．０質量％であり、好ましくは１．０～１５．０質量％、より好ましくは１．２～１２．０質量％である。 --Sulfo group S content--
The total amount (ratio to the solid content of the lignin derivative) of the S content (sulfur atom content: sulfo group S content) of the group represented by the general formula (1) of the lignin derivative is usually 0.5% by mass or more, preferably 1.0% by mass or more, more preferably 1.2% by mass or more. As a result, the lignin derivative is rich in the group represented by the general formula (1), and can exhibit water solubility and can exhibit a good virus inhibitory effect against WSSV. The upper limit is usually 20.0% by mass or less, preferably 15.0% by mass or less, more preferably 12.0% by mass or less. As a result, it can exhibit moderate water solubility and can efficiently exhibit a virus inhibitory effect. Therefore, the total amount of the sulfo group S content of the lignin derivative is usually 0.5 to 20.0% by mass, preferably 1.0 to 15.0% by mass, more preferably 1.2 to 12.0% by mass.
　スルホ基Ｓ含量は、下記式（１）より算出できる。
式（１）：
　スルホ基Ｓ含量（質量％）＝リグニン誘導体の全Ｓ含量（質量％）－無機態Ｓ含量（質量％）　 The sulfo group S content can be calculated by the following formula (1).
Formula (1):
Sulfo group S content (mass%) = total S content (mass%) of lignin derivative - inorganic S content (mass%)
　式（１）中、リグニン誘導体の全Ｓ含量及び無機態Ｓ含量は、いずれもリグニン誘導体の固形分量に対するＳ含量を示す。式（１）中、全Ｓ含量は、リグニン成分に含まれるすべてのＳ含量であり、ＩＣＰ発光分光分析法により定量することができる。また、無機態Ｓ含量は、イオンクロマト法により定量したＳＯ３含量及びＳＯ４含量の合計量として算出できる。　 In formula (1), the total S content and inorganic S content of the lignin derivative both indicate the S content relative to the solid content of the lignin derivative. In formula (1), the total S content is the total S content contained in the lignin component and can be quantified by ICP atomic emission spectrometry. The inorganic S content can be calculated as the sum of the SO3 content and SO4 content quantified by ion chromatography.
－水への溶解性－
　リグニン成分は、水への溶解性が、通常１．０質量％以上、好ましくは３．０質量％以上である。ウイルスの抑制効果を有する部分化学構造とウイルスが接触する環境は水媒体中であるところ、上記範囲であることにより、適度な水溶性を有することができ、ウイルスの抑制効果に有利に働くものと推測される。上限は、特に限定されず、１００質量％以下であればよい。したがって、リグニン成分は、水への溶解性が、通常１．０～１００質量％、好ましくは３．０～１００質量％である。 -Solubility in water-
The lignin component has a water solubility of usually 1.0% by mass or more, preferably 3.0% by mass or more. The environment in which the partial chemical structure having the virus inhibitory effect comes into contact with the virus is an aqueous medium, and it is presumed that the above range allows the lignin component to have a moderate water solubility, which is advantageous for the virus inhibitory effect. The upper limit is not particularly limited, and may be 100% by mass or less. Therefore, the lignin component has a water solubility of usually 1.0 to 100% by mass, preferably 3.0 to 100% by mass.
　水への溶解量は、次のようにして算出できる。１０ｇ（乾燥重量）のサンプルを水３００ｇに分散し、６０分攪拌した後に濾過する。ろ液の質量及び固形分（ろ物を乾燥したもの）の質量を測定する。そして、ろ液の固形分の質量をサンプルの質量（１０ｇ）で割り、１００倍することにより算出する。 The amount of solubility in water can be calculated as follows: Disperse 10 g (dry weight) of the sample in 300 g of water, stir for 60 minutes, and then filter. Measure the mass of the filtrate and the mass of the solids (dried filtrate). Then, divide the mass of the solids in the filtrate by the mass of the sample (10 g) and multiply by 100 to calculate the amount.
〔１．４．任意成分〕
　リグニン成分は、リグニン誘導体を調製する際（例えば、亜硫酸蒸解の際）に原料より混入してくる抽出成分以外の成分、例えば、硫酸ナトリウム、亜硫酸ナトリウム、塩化ナトリウム、硫酸マグネシウム、亜硫酸マグネシウム、塩化マグネシウム、硫酸カルシウム、亜硫酸カルシウム、塩化カルシウム、硫酸アンモニウム、亜硫酸アンモニウム、塩化アンモニウム、水酸化ナトリウム等の無機塩を含んでもよい。 [1.4. Optional components
The lignin component is a component other than the extracted component that is mixed from the raw material when preparing the lignin derivative (for example, during sulfite cooking), such as sodium sulfate, sodium sulfite, sodium chloride, magnesium sulfate, magnesium sulfite, and magnesium chloride. The inorganic salts may include calcium sulfate, calcium sulfite, calcium chloride, ammonium sulfate, ammonium sulfite, ammonium chloride, sodium hydroxide, and the like.
　またリグニン成分は、抽出成分以外の成分として、糖類を含んでいてもよい。本明細書において、糖類は、少なくとも１種類の糖又は２種類以上の糖の組み合わせである。 The lignin component may also contain sugars as components other than the extractable components. In this specification, sugars refers to at least one type of sugar or a combination of two or more types of sugars.
　糖は、構成する炭素数に制限はなく、単糖、少糖、多糖のいずれでもよい。単糖としては以下が例示される：アルドトリオース、ケトトリオースなどの三炭糖；エリトロース、トレオース、エリトルロースなどの四炭糖；キシロース、リボース、アラビノース、リキソース、リブロース、キシルロースなどの五炭糖；グルコース、マンノース、アロース、アルトロース、ルコース、グロース、イドース、ガラクトース、タロース、プシコース、フルクトース、ソルボース、タガトース、フコース、フルクトース、ラムノースなどの六炭糖；セドヘプツロースなどの七炭糖。少糖としては以下が例示される：スクロース、ラクトース、マルトース、トレハロース、ツラノース、セロビオースなどの二糖；ラフィノース、メレジトース、マルトトリオースなどの三糖；アカルボース、スタキオースなどの四糖；キシロオリゴ糖、セロオリゴ糖、フラクトオリゴ糖、ガラクトオリゴ糖、マンナンオリゴ糖などのオリゴ糖。多糖としては、グリコーゲン、でんぷん（アミロース、アミロペクチン）、セルロース、ヘミセルロース、デキストリン、グルカンが例示される。糖類、中でも多糖類は、パルプ等のリグノセルロース原料である植物成分に含有される多糖類、及びそれらが蒸解又は漂白処理の際に分解及び／又は変性して生成するものを含んでもよい。
　糖類は、通常、多糖、還元性糖、及び／又は糖変性物を含む。還元性糖は、還元性を示す糖であればよい。還元性糖は通常、塩基性溶液中でアルデヒド基又はケトン基を生じる。還元性糖としては、すべての単糖、マルトース、ラクトース、アラビノース、スクロースの転化糖などの二糖、及び多糖などが例示される。糖変性物としては例えば、糖が酸化、スルホン化などの化学変性を受けてなる変性物、ヒドロキシ基、アルデヒド基、カルボニル基、又は／及びスルホ基などの置換基で置換されている糖誘導体が挙げられる。 The number of carbon atoms constituting the sugar is not limited, and the sugar may be any of monosaccharides, oligosaccharides, and polysaccharides. Examples of monosaccharides include trioses such as aldotriose and ketotriose; tetraoses such as erythrose, threose, and erythrulose; pentoses such as xylose, ribose, arabinose, lyxose, ribulose, and xylulose; hexoses such as glucose, mannose, allose, altrose, glucose, gulose, idose, galactose, talose, psicose, fructose, sorbose, tagatose, fucose, fructose, and rhamnose; and heptoses such as sedoheptulose. Examples of oligosaccharides include disaccharides such as sucrose, lactose, maltose, trehalose, turanose, and cellobiose; trisaccharides such as raffinose, melezitose, and maltotriose; tetrasaccharides such as acarbose and stachyose; and oligosaccharides such as xylooligosaccharides, cellooligosaccharides, fructooligosaccharides, galactooligosaccharides, and mannanoligosaccharides. Examples of polysaccharides include glycogen, starch (amylose, amylopectin), cellulose, hemicellulose, dextrin, and glucan. Sugars, particularly polysaccharides, may include polysaccharides contained in plant components that are raw materials for lignocellulose such as pulp, and those that are produced by decomposition and/or modification of these during cooking or bleaching treatment.
The sugars generally include polysaccharides, reducing sugars, and/or modified sugars. The reducing sugar may be any sugar that exhibits reducing properties. The reducing sugar generally produces an aldehyde group or a ketone group in a basic solution. Examples of the reducing sugar include all monosaccharides, disaccharides such as maltose, lactose, arabinose, and invert sugar of sucrose, and polysaccharides. Examples of the modified sugar include modified sugars that are chemically modified by oxidation, sulfonation, and the like, and sugar derivatives that are substituted with substituents such as hydroxyl groups, aldehyde groups, carbonyl groups, and/or sulfo groups.
　還元性糖とは、還元性を示す糖であり、塩基性溶液中でアルデヒド基又はケトン基を生じる糖を意味する。還元性糖としては、例えば、すべての単糖、マルトース、ラクトース、アラビノース、スクロースの転化糖などの二糖、及び多糖が例示される。還元性糖は、通常、セルロース、ヘミセルロース、及びそれらの分解物を含む。セルロース及びヘミセルロースの分解物としては、例えば、ラムノース、ガラクトース、アラビノース、キシロース、グルコース、マンノース、フルクトースなどの単糖、キシロオリゴ糖、セロオリゴ糖などのオリゴ糖が挙げられる。 Reducing sugars are sugars that exhibit reducing properties and generate aldehyde or ketone groups in a basic solution. Examples of reducing sugars include all monosaccharides, disaccharides such as maltose, lactose, arabinose, and invert sugars of sucrose, and polysaccharides. Reducing sugars typically include cellulose, hemicellulose, and their decomposition products. Examples of decomposition products of cellulose and hemicellulose include monosaccharides such as rhamnose, galactose, arabinose, xylose, glucose, mannose, and fructose, and oligosaccharides such as xylooligosaccharides and cellooligosaccharides.
　糖変性物とは、糖が酸化、スルホン化などの化学変性を受けてなる変性物を意味する。糖変性物は、ヒドロキシ基、アルデヒド基、カルボニル基、又は／及びスルホ基などの官能基が糖の骨格中に導入された糖誘導体であってもよいし、糖誘導体２つ（２種）以上が結合した化合物などが例示される。 The term "sugar modification product" refers to a product in which sugar has been chemically modified, such as by oxidation or sulfonation. Examples of sugar modification products include sugar derivatives in which functional groups such as hydroxyl groups, aldehyde groups, carbonyl groups, and/or sulfo groups have been introduced into the sugar skeleton, and compounds in which two or more sugar derivatives (two types) are bonded together.
　本発明の剤は、必要に応じてさらに他の任意成分を含んでいてもよい。他の成分としては、剤の用途に応じて、その用途において通常用いられる任意成分であればよく、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤、各種栄養成分、上述のリグニン成分以外の抗ＷＳＳＶ剤が挙げられる。 The agent of the present invention may further contain other optional components as necessary. The other components may be any optional components normally used in the intended use of the agent, such as bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, various nutritional components, and anti-WSSV agents other than the above-mentioned lignin components.
〔１．５．剤形〕
　抗ＷＳＳＶ剤の剤型は、その用途に応じて適宜選択できる。剤形としては、例えば、液剤、乳剤、懸濁剤、分散剤、エアゾール剤等の液剤；粒剤、顆粒剤、錠剤、粉剤、ペースト剤等の固形又は半固形剤が挙げられる。 [1.5. Dosage form
The dosage form of the anti-WSSV agent can be appropriately selected depending on the application. Examples of dosage forms include liquids such as liquids, emulsions, suspensions, dispersions, and aerosols; granules, tablets, and powders. Examples of the solid or semi-solid agent include a solid or semi-solid agent such as a paste agent.
〔１．６．生理活性〕
　本発明の抗ＷＳＳＶ剤は、ＷＳＳＶ抑制効果を発揮できる。 1.6. Biological activity
The anti-WSSV agent of the present invention can exert a WSSV suppression effect.
　ＷＳＳＶ（ホワイトスポットシンドロームウイルス）は、ウイスポウイルス属（Ｗｈｉｓｐｏｖｉｒｕｓ）に属する桿状の二本鎖ＤＮＡウイルスである。ＷＳＳＶは、感染生物の捕食、及び鰓から入る水を介して感染する。 WSSV (White Spot Syndrome Virus) is a rod-shaped, double-stranded DNA virus belonging to the Whispovirus genus. WSSV is transmitted by ingesting infected organisms and through water entering through the gills.
　抗ＷＳＳＶ剤は、ＷＳＳＶの対象生物への感染、及びその拡大を抑制できる。対象生物は、通常、十脚目（Ｄｅｃａｐｏｄａ）に属する生物（例えば、エビ、カニ、ザリガニ等の甲殻類）である。エビとしては、海水、汽水、淡水のいずれに生息するものでもよく、例えば、バナメイエビ（ホワイトレッグシュリンプ、Ｐｅｎａｅｕｓ　ｖａｎｎａｍｅｉ　Ｂｏｏｎｅ）、ホワイトシュリンプ（Ｐｅｎａｅｕｓ　ｓｅｔｉｆｅｒｕｓ）、ウェスタンブルーシュリンプ（Ｐｅｎａｅｕｓ　ｓｔｙｌｉｒｏｓｔｒｉｓ　Ｓｔｉｍｐｓｏｎ）等のＬｉｔｏｐｅｎａｅｕｓ属（Ｐｅｎａｅｕｓ　Ｆａｂｒｉｃｉｕｓ）、クルマエビ（Ｐｅｎａｅｕｓ　ｊａｐｏｎｉｃｕｓ　Ｓｐｅｎｃｅ　Ｂａｔｅ）、コウライエビ（Ｐｅｎａｅｕｓ　ｃｈｉｎｅｎｓｉｓ）、アカオエビ（Ｐｅｎａｅｕｓ　ｐｅｎｉｃｉｌｌａｔｕｓ　Ａｌｃｏｃｋ）、インドエビ（Ｐｅｎａｅｕｓ　ｉｎｄｉｃｕｓ　Ｈ．　Ｍｉｌｎｅ　Ｅｄｗａｒｄｓ）、Ｂａｎａｎａ　ｐｒａｗｎ（Ｐｅｎａｅｕｓ　ｍｅｒｇｕｉｅｎｓｉｓ　Ｄｅ　Ｍａｎ）等のコウライエビ属（Ｐｅｎａｅｕｓ　Ｆａｂｒｉｃｉｕｓ）、ウシエビ（ブラックタイガー、Ｐｅｎａｅｕｓ　ｍｏｎｏｄｏｎ　Ｆａｂｒｉｃｉｕｓ）、クマエビ（Ｐｅｎａｅｕｓ　ｓｅｍｉｓｕｌｃａｔｕｓ　Ｄｅ　Ｈａａｎ）、フトミゾエビ（Ｐｅｎａｅｕｓ　ｌａｔｉｓｕｌｃａｔｕｓ　Ｋｉｓｈｉｎｏｕｙｅ）、ノーザンピンクシュリンプ（Ｐｅｎａｅｕｓ　ｄｕｏｒａｒｕｍ　Ｂｕｒｋｅｎｒｏａｄ）、ブラウンシュリンプ（Ｐｅｎａｅｕｓ　ａｚｔｅｃｕｓ　Ｉｖｅｓ）等のクルマエビ属（Ｐｅｎａｅｕｓ　Ｆａｂｒｉｃｉｕｓ）、ヨシエビ（Ｍｅｔａｐｅｎａｅｕｓ　ｅｎｓｉｓ）、モザンビークブラウン（Ｍ．ｍｏｎｏｃｅｒｏｓ）、プーバラン（Ｍ．ｄｏｂｓｏｎｉ）、Ｍｅｔａｐｅｎａｅｕｓ　ｅｌｅｇａｎｓ　Ｄｅ　Ｍａｎ等のヨシエビ属（Ｍｅｔａｐｅｎａｅｕｓ　Ｗｏｏｄ－Ｍａｓｏｎ　ｉｎ　Ｗｏｏｄ－Ｍａｓｏｎ　＆　Ａｌｃｏｃｋ）、トラエビ（Ｍｅｔａｐｅｎａｅｏｐｓｉｓ　ａｃｃｌｉｖｉｓ）、アカエビ（Ｍｅｔａｐｅｎａｅｏｐｓｉｓ　ｂａｒｂａｔａ）、キシエビ（Ｍｅｔａｐｅｎａｅｏｐｓｉｓ　ｄａｌｅｉ）等のアカエビ属（Ｍｅｔａｐｅｎａｅｏｐｓｉｓ　Ｂｏｕｖｉｅｒ）、サルエビ（Ｔｒａｃｈｙｓａｌａｍｂｒｉａ　ｃｕｒｖｉｒｏｓｔｒｉｓ）等のサルエビ属（Ｔｒａｃｈｙｓａｌａｍｂｒｉａ　Ｂｕｒｋｅｎｒｏａｄ）、キディシュリンプ（Ｐａｒａｐｅｎａｅｏｐｓｉｓ　ｓｔｙｌｉｆｅｒａ）等のＰａｒａｐｅｎａｅｏｐｓｉｓ属（Ｐａｒａｐｅｎａｅｏｐｓｉｓ　Ａｌｃｏｃｋ）に属するエビ等の、養殖用に飼育されているエビが挙げられる。 Anti-WSSV agents can suppress WSSV infection and its spread in target organisms. The target organisms are usually organisms belonging to the order Decapoda (e.g., crustaceans such as shrimp, crabs, and crayfish). Shrimp may be any shrimp that lives in seawater, brackish water, or fresh water, and examples of such shrimp include Litopenaeus genus (Penaeus Fabricius), such as white leg shrimp (Penaeus vannamei Boone), white shrimp (Penaeus setiferus), western blue shrimp (Penaeus stylirostris Stimpson), kuruma shrimp (Penaeus japonicus Spence Bate), Korean shrimp (Penaeus chinensis), red-tailed shrimp (Penaeus s penicillatus Alcock), Indian shrimp (Penaeus indicus H. Milne Edwards), Banana prawn (Penaeus merguiensis De Man), etc. of the Korean shrimp genus (Penaeus Fabricius), black tiger shrimp (Penaeus monodon Fabricius), Japanese tiger shrimp (Penaeus semisulcatus De Haan), Japanese giant shrimp (Penaeus latisulcatus Kishinouye), northern pink shrimp (P Penaeus duorarum Burkenroad), brown shrimp (Penaeus aztecus Ives) and other species of the genus Penaeus Fabricius, Metapenaeus ensis, Mozambique brown (M. monoceros), Poobaran (M. dobsoni), Metapenaeus elegans De Man and other species of the genus Metapenaeus Wood-Mason in Wood-Mason & Alcock, and tiger shrimp (Metapenaeopsis acclivis) These include shrimps that are raised for aquaculture, such as Metapenaeopsis Bouvier, including red shrimp (Metapenaeopsis barbata) and Japanese prawn (Metapenaeopsis dalei), Trachysalambria Burkenroad, including monkey shrimp (Trachysalambria curvirostris), and Parapenaeopsis Alcock, including kiddy shrimp (Parapenaeopsis stylifera).
〔２．用途〕
　抗ＷＳＳＶ剤は、抗ＷＳＳＶ効果を期待する各種用途に利用できる。用途としては、例えば、飼料、飼育用水用処理剤、消毒剤、洗浄剤が挙げられる。 [2. Applications
The anti-WSSV agent can be used in various applications where an anti-WSSV effect is expected, such as feed, breeding water treatment agents, disinfectants, and cleaning agents.
〔２．１．飼料、医薬〕
　抗ＷＳＳＶ剤を飼料、医薬として利用することにより、対象生物に抗ＷＳＳＶ剤を捕食させることができ、効率的にＷＳＳＶ抵抗性を発揮させることができる。 2.1. Feed and medicine
By using the anti-WSSV agent as feed or medicine, the target organism can be made to ingest the anti-WSSV agent, thereby efficiently exerting resistance to WSSV.
　飼料、医薬は抗ＷＳＳＶ剤を含有すればよく、その含有量は、使用態様、適用対象により適宜調整可能である。例えば、１日あたり生物の体重の５～１０％量の飼料を投与する場合に換算して、リグニン成分を飼料の重量に対して、０．１質量％以上含有し、０．５質量％以上が好ましく、０．７質量％以上がより好ましい。上限は、通常、７．０質量％以下、好ましくは５．０質量％以下である。したがって通常、０．１～７．０質量％、好ましくは０．５～７．０質量％、より好ましくは０．７～５．０質量％である。飼料は、継続的に投与することが好ましく、例えば、３日以上、５日以上、好ましくは１週間以上、より好ましくは１０日以上、さらに好ましくは２週間以上継続して１日１回から３回投与してもよい。また、継続的な投与を行った後いったん通常の飼料に戻して一定期間（たとえば、１週間、２週間、３週間、１カ月、２か月）経過後に再び継続的な投与を行うことを繰り返すことでもよい。また、甲殻類の週齢は特に限定なく使用できる。 The feed and medicine may contain an anti-WSSV agent, and the content can be adjusted appropriately depending on the mode of use and the subject of application. For example, when calculated as a daily feed of 5 to 10% of the body weight of the organism, the lignin component is contained in an amount of 0.1% by mass or more, preferably 0.5% by mass or more, and more preferably 0.7% by mass or more, based on the weight of the feed. The upper limit is usually 7.0% by mass or less, preferably 5.0% by mass or less. Therefore, it is usually 0.1 to 7.0% by mass, preferably 0.5 to 7.0% by mass, and more preferably 0.7 to 5.0% by mass. The feed is preferably administered continuously, and may be administered once to three times a day for, for example, 3 days or more, 5 days or more, preferably 1 week or more, more preferably 10 days or more, and even more preferably 2 weeks or more. After continuous administration, the feed may be temporarily returned to normal feed, and then continuous administration may be repeated after a certain period of time (for example, 1 week, 2 weeks, 3 weeks, 1 month, or 2 months). Additionally, there are no particular limitations on the age of the crustaceans that can be used.
　飼料が含み得る任意成分としては、例えば、栄養成分が挙げられる。栄養成分としては、例えば、魚油、魚介肉、アミノ酸、脂質、ミネラル、ビタミン等の、対象生物の飼料、水産用、養殖用飼料に通常用いられる成分が挙げられ、その含有量は適宜選定できる。 Optional components that may be contained in the feed include, for example, nutritional components. Examples of nutritional components include fish oil, seafood meat, amino acids, lipids, minerals, vitamins, and other components that are typically used in feed for target organisms, aquaculture feed, and aquaculture feed, and the content of these components can be selected as appropriate.
　飼料の剤型は、通常の飼料の剤型と同様であればよく、通常は、粒剤、顆粒剤、粉剤である。飼料の製造方法は、特に限定されず、例えば、試料を構成する成分を混合、乾燥、成形を行う方法が挙げられる。飼料は、ＷＳＳＶが感染する対象生物用の飼料であればよく、通常は甲殻類用飼料であり、養殖用飼料であることが好ましい。 The feed may be in the same form as normal feed, and is usually in the form of granules, pellets, or powder. There are no particular limitations on the method for producing the feed, and examples include a method in which the components constituting the sample are mixed, dried, and shaped. The feed may be feed for the target organism infected with WSSV, and is usually feed for crustaceans, and preferably feed for aquaculture.
〔２．２．飼育用水用処理剤〕
　抗ＷＳＳＶ剤を飼育用水用処理剤として利用することにより、対象生物の体内に経口または鰓を介する等によるＷＳＳＶの侵入を抑制でき、効率的にＷＳＳＶ抵抗性を発揮させることができる。 2.2. Treatment agents for breeding water
By using an anti-WSSV agent as a treatment agent for breeding water, it is possible to suppress the entry of WSSV into the body of the target organism via oral ingestion or gills, etc., thereby efficiently exerting resistance to WSSV.
　処理剤は、養殖、栽培漁業等の飼育において対象生物を飼育するために用いる水であればよく、（天然の、又は人工的に成分調整した）海水、汽水、淡水のいずれでもよい。１回あたりの処理量は特に限定されず、使用態様、適用対象に応じて適宜調整可能である。例えば、水の量に対するリグニン成分の重量として、０．０２ｍｇ／ｍＬ以上が好ましく、０．１ｍｇ／ｍＬ以上であることがより好ましい。上限は、１０．０ｍｇ／ｍＬ以下であることが好ましく、７．０ｍｇ／ｍＬ以下であることがより好ましい。したがって、１回あたりの処理量は、リグニン成分として、０．０２～１０．０ｍｇ／ｍＬが好ましく、０．１～７．０ｍｇ／ｍＬがより好ましい。処理の回数は、１日に１回～３回、継続的に（例えば、１日、３日、５日、１週間、１０日又は２週間に一度）行ってもよい。 The treatment agent may be any water used to raise the target organism in aquaculture, fish farming, or the like, and may be any of seawater, brackish water, and freshwater (either natural or artificially adjusted). The amount of treatment per time is not particularly limited and can be adjusted appropriately depending on the mode of use and the target of application. For example, the weight of the lignin component relative to the amount of water is preferably 0.02 mg/mL or more, and more preferably 0.1 mg/mL or more. The upper limit is preferably 10.0 mg/mL or less, and more preferably 7.0 mg/mL or less. Therefore, the amount of treatment per time is preferably 0.02 to 10.0 mg/mL, and more preferably 0.1 to 7.0 mg/mL, in terms of the lignin component. Treatment may be performed once to three times a day continuously (for example, once every 1 day, 3 days, 5 days, 1 week, 10 days, or 2 weeks).
　処理剤の剤型としては、例えば、粒剤、顆粒剤、粉剤、錠剤が挙げられ、処理の作業性から適宜選択すればよい。処理方法としては、散布が好ましい。 The dosage form of the treatment agent may be, for example, granules, powders, or tablets, and may be selected appropriately based on the ease of treatment. The preferred treatment method is spraying.
〔２．３．他の用途〕
　上述の飼料、処理剤以外の例としては、飼育用物品の消毒剤、洗浄剤が挙げられる。飼育用物品としては、水槽、ろ過装置、養殖篭、網、底砂、衛生用品等の、甲殻類の養殖、栽培漁業等の飼育に用いられる物品が挙げられる。物品の素材としては、例えば、ガラス、樹脂、布、金属が挙げられ、特に限定されない。また、物品の形状も限定されない。 2.3. Other Uses
Examples of the agent other than the above-mentioned feed and treatment agent include disinfectants and cleaning agents for breeding articles. Examples of breeding articles include aquariums, filtration devices, culture cages, nets, bottom sand, sanitary products, and other articles used in breeding crustacean farming, fish farming, and the like. Examples of materials for the articles include, but are not limited to, glass, resin, cloth, and metal. The shape of the article is also not limited.
〔３．ホワイトスポット病の予防方法〕
　抗ＷＳＳＶ剤を対象生物（甲殻類等のヒト以外の生物）に投与することにより、ＷＳＳＶへの抵抗性を高め、生存率の低下を抑制できる。そのため、抗ＷＳＳＶ剤は、甲殻類のＷＳＤの予防に利用できる。 3. How to prevent white spot disease
By administering an anti-WSSV agent to a target organism (an organism other than a human, such as crustaceans), it is possible to increase resistance to WSSV and suppress a decrease in survival rate. Therefore, the anti-WSSV agent can be used to prevent WSD in crustaceans.
　抗ＷＳＳＶ剤の投与方法は特に限定されない。例えば、上述の、抗ＷＳＳＶ剤を含む飼料又は医薬を捕食させる方法、抗ＷＳＳＶ剤を含む処理剤で飼育用水を処理する方法、抗ＷＳＳＶ剤を含む消毒剤又は洗浄剤で飼育用物品を消毒又は洗浄する方法、これらの２つ以上を組み合わせる方法が挙げられる。投与時期としては、例えば、一定期間投与する方法、定期的に投与する方法が挙げられ、投与方法によって選択できる。 The method of administering the anti-WSSV agent is not particularly limited. Examples include the above-mentioned method of feeding the animals with feed or medicine containing the anti-WSSV agent, treating rearing water with a treatment agent containing the anti-WSSV agent, disinfecting or cleaning rearing items with a disinfectant or cleaning agent containing the anti-WSSV agent, or a combination of two or more of these. The timing of administration can be selected depending on the administration method, for example, a method of administering for a certain period of time or a method of administering periodically.
　以下、本発明を実施例により説明する。以下の実施例は、本発明を限定するものではない。 The present invention will be described below with reference to examples. The following examples are not intended to limit the present invention.
＜実施例１＞
　木材チップ（ラジアータパイン）を亜硫酸蒸解法に基づき亜硫酸処理した。亜硫酸処理においては、ＳＯ２濃度４ｇ／１００ｍＬの亜硫酸ナトリウムの溶液を用いて、温度１４０℃、ｐＨ２、処理時間３時間とした。次に不溶解物をろ別した。得られたろ液を固形分が５０％となるまでロータリーエバポレーターで濃縮した。その後溶液のｐＨをＮａＯＨでｐＨ４．５に調整し、スプレードライヤーで粉末化して、実施例１のリグニンスルホン酸塩Ａを得た（メトキシ基量６．１質量％、抽出成分量０．２質量％、抽出成分量／メトキシ基量：０．０４、スルホ基のＳ含量５．２質量％、水への溶解量１００質量％）。 Example 1
Wood chips (radiata pine) were subjected to sulfite treatment based on the sulfite cooking method. In the sulfite treatment, a solution of sodium sulfite with a SO2 concentration of 4 g/100 mL was used, and the temperature was 140 ° C., pH 2, and treatment time was 3 hours. Next, insoluble matter was filtered off. The obtained filtrate was concentrated with a rotary evaporator until the solid content was 50%. The pH of the solution was then adjusted to pH 4.5 with NaOH, and the solution was powdered with a spray dryer to obtain lignin sulfonate A of Example 1 (methoxy group amount 6.1 mass%, extractable component amount 0.2 mass%, extractable component amount/methoxy group amount: 0.04, sulfo group S content 5.2 mass%, solubility in water 100 mass%).
＜実施例２＞
　実施例１にて得られたリグニンスルホン酸塩Ａを水に溶解して固形分が２５％の水溶液となるように調製し、透析膜（分子量分画：２０，０００、Ｓｐｅｃｔｒａ／Ｐｏｒ（登録商標）　セルロースエステル透析用チューブ）を用い、３日間透析処理した。透析チューブ内の溶液を集め、原液量が２５％となるまで濃縮し、スプレードライヤーにより粉末化してリグニンスルホン酸塩Ｂを得た（メトキシ基量１１．１質量％、抽出成分量０．０９質量％、抽出成分量／メトキシ基量：０．００８、スルホ基のＳ含量３．５質量％、水への溶解量１００質量％）。 Example 2
The lignin sulfonate A obtained in Example 1 was dissolved in water to prepare an aqueous solution with a solid content of 25%, and the solution was dialyzed for 3 days using a dialysis membrane (molecular weight cutoff: 20,000, Spectra/Por (registered trademark) cellulose ester dialysis tube). The solution in the dialysis tube was collected and concentrated to a stock solution volume of 25%, and powdered with a spray dryer to obtain lignin sulfonate B (methoxy group content 11.1% by mass, extractable component content 0.09% by mass, extractable component content/methoxy group content: 0.008, sulfur content of sulfo group 3.5% by mass, amount dissolved in water 100% by mass).
＜実施例３＞
　木材チップ（ラジアータパイン）を亜硫酸蒸解法に基づき亜硫酸処理した。亜硫酸処理においては、ＳＯ２濃度２．５ｇ／１００ｍＬの亜硫酸ナトリウムの溶液を用いて、温度１４０℃、ｐＨ３、処理時間３時間とした。次に不溶解物をろ別し、得られたろ液を固形分が５０％となるまでロータリーエバポレーターで濃縮した。その後ＮａＯＨで溶液のｐＨを４．５に調整し、スプレードライヤーで粉末化した。得られた粉末を水に溶解して固形分が２５％の水溶液となるように調製し、４０％ＮａＯＨでｐＨ１２とした後、１４０℃で１２０分間アルカリ空気酸化した。その後、７０％硫酸を加えてｐＨ３とし、部分脱スルホン化したリグニンスルホン酸塩を分別沈殿させた。得られた部分脱スルホン化したリグニンスルホン酸塩の沈殿物を、ろ液が中性となるまで水で洗浄し、沈殿物の２０部を１００部の水に懸濁し、６０℃に加温後に攪拌ながらｐＨが９となるまで１ｍｏｌ／Ｌの水酸化ナトリウム水溶液を加えて沈殿物を完全に溶解した。得られた溶液をスプレードライヤーで粉末化して、リグニンスルホン酸塩Ｃを得た（メトキシ基量１１．２質量％、抽出成分量１．６質量％、抽出成分量／メトキシ基量：０．１４、スルホ基のＳ含量２．７質量％、水への溶解量１００質量％）。 Example 3
Wood chips (radiata pine) were subjected to sulfite treatment based on the sulfite cooking method. In the sulfite treatment, a solution of sodium sulfite with a SO2 concentration of 2.5 g/100 mL was used, and the temperature was 140 ° C., pH 3, and treatment time was 3 hours. Next, insoluble matter was filtered off, and the obtained filtrate was concentrated with a rotary evaporator until the solid content was 50%. The pH of the solution was then adjusted to 4.5 with NaOH, and powdered with a spray dryer. The obtained powder was dissolved in water to prepare an aqueous solution with a solid content of 25%, and the pH was adjusted to 12 with 40% NaOH, and then the solution was oxidized with alkaline air at 140 ° C. for 120 minutes. Then, 70% sulfuric acid was added to adjust the pH to 3, and the partially desulfonated lignin sulfonate was fractionated and precipitated. The resulting precipitate of partially desulfonated lignin sulfonate was washed with water until the filtrate became neutral, 20 parts of the precipitate was suspended in 100 parts of water, and the precipitate was completely dissolved by adding 1 mol/L aqueous sodium hydroxide solution with stirring after heating to 60° C. until the pH reached 9. The resulting solution was powdered with a spray dryer to obtain Ligninsulfonate C (methoxy group amount 11.2% by mass, extractable component amount 1.6% by mass, extractable component amount/methoxy group amount: 0.14, S content of sulfo group 2.7% by mass, amount dissolved in water 100% by mass).
［抽出成分量の測定法］：
　抽出成分量は、ＪＩＳ　Ｋ　０１０２：２０１９記載のヘキサン抽出物質の測定法によって測定した。 [Method for measuring the amount of extracted components]:
The amount of extractable components was measured by the method for measuring hexane extractable substances described in JIS K 0102:2019.
［メトキシ基量の測定法］：
　メトキシ基量は、Ｖｉｅｂｏｃｋ及びＳｃｈｗａｐｐａｃｈ法によるメトキシ基の定量法（「リグニン化学研究法」、Ｐ．３３６～３４０、平成６年、ユニ出版（株）発行、参照）によって測定した。 [Methoxy group amount measurement method]:
The amount of methoxy groups was measured by the quantitative determination of methoxy groups by the Viebock and Schwappach method (see "Lignin Chemical Research Methods", pp. 336-340, 1994, published by Uni Publishing Co., Ltd.).
［スルホ基のＳ含量の測定方法］：
　スルホ基のＳ含量は、以下の式により求めた。
　スルホ基のＳ含量（質量％）＝全Ｓ含量（質量％）－無機態Ｓ含量（質量％）
　（数式中のＳ含量は、いずれもリグニンスルホン酸塩の固形物量に対するＳ含量を示す。）
　数式中、全Ｓ含量は、ＩＣＰ発光分光分析法により定量した。また、無機態Ｓ含量は、イオンクロマト法により定量したＳＯ３含量及びＳＯ４含量の合計量を用いた。 [Method for measuring S content in sulfo groups]:
The S content of the sulfo group was calculated according to the following formula.
Sulfo group S content (mass%) = total S content (mass%) - inorganic S content (mass%)
(The S content in the formula indicates the S content relative to the solid content of the lignin sulfonate.)
In the formula, the total S content was quantified by ICP emission spectrometry. The inorganic S content was the sum of the SO3 content and the SO4 content quantified by ion chromatography.
［リグニンスルホン酸塩の水への溶解量の測定法］：
　リグニンスルホン酸塩の水への溶解量は、次のようにして算出した。１０ｇ（乾燥重量）のサンプルを水３００ｇに分散し、６０分攪拌した後に濾過した。ろ液の質量及び固形分（ろ物を乾燥したもの）の質量を測定した。そして、ろ液の固形分の質量をサンプルの質量（１０ｇ）で割り、１００倍することにより算出した。 [Method for measuring the amount of lignosulfonate dissolved in water]
The amount of lignin sulfonate dissolved in water was calculated as follows. 10 g (dry weight) of a sample was dispersed in 300 g of water, stirred for 60 minutes, and then filtered. The mass of the filtrate and the mass of the solids (dried filtrate) were measured. The mass of the solids in the filtrate was then divided by the mass of the sample (10 g) and multiplied by 100 to calculate the amount.
［エビの育成条件］
　後述する試験１及び２におけるエビの育成条件は以下のとおりである。
　使用したエビ：２ｇ前後のバナメイエビ（高知県業者より購入）
　飼育水槽：１５Ｌガラス水槽
　水温：２８℃
　基本飼料：バナメイエビ用飼料（タイからの輸入品）
　給餌量：体重の５％／日
　感染試験：強毒性ＷＳＳＶ [Shrimp cultivation conditions]
The shrimp were grown under the following conditions in Tests 1 and 2.
Shrimp used: Vannamei shrimp of about 2g (purchased from a Kochi Prefecture dealer)
Breeding tank: 15L glass tank Water temperature: 28℃
Basic feed: Vannamei shrimp feed (imported from Thailand)
Feeding amount: 5% of body weight/day Infection test: Highly virulent WSSV
〔試験１．リグニン添加給餌によるＷＳＳＶ感染予防試験〕
　リグニンスルホン酸塩Ａ、Ｂ、又はＣをそれぞれ１％あるいは４％となるように基本飼料に添加し、試験用のバナメイ用飼料を調製した。具体的には、基本飼料に対し、１％あるいは４％相当のリグニンスルホン酸塩、及び基本飼料と同量の水を混合し、得られた混合物を麺状に成形、乾燥（約７０℃で約２時間）させ、ランダムに破砕して試験用飼料とした。給餌は１日に２回、水槽中の海水に散布して行った（リグニンスルホン酸塩添加群）。試験用飼料の給餌期間は１週間（給餌試験１）又は２週間（給餌試験２）とした。飼育は、飼料以外は上述の［エビの育成条件］に従って行った。また、試験用飼料の代わりに基本飼料を添加した他はリグニンスルホン酸塩添加群と同様に飼育し、コントロール群とした。 [Test 1. Prevention of WSSV infection by feeding with lignin]
Lignosulfonate A, B, or C was added to the basic diet at 1% or 4%, respectively, to prepare a test diet for vannamei shrimp. Specifically, the basic diet was mixed with 1% or 4% lignosulfonate and the same amount of water as the basic diet, and the resulting mixture was formed into noodles, dried (at about 70°C for about 2 hours), and randomly crushed to prepare the test diet. The shrimp were fed twice a day by dispersing the mixture in seawater in an aquarium (lignosulfonate-added group). The test diet was fed for one week (feeding test 1) or two weeks (feeding test 2). The shrimp were reared according to the above-mentioned [Rearing conditions for shrimp], except for the feed. The shrimp were reared in the same manner as the lignosulfonate-added group, except for the addition of the basic diet instead of the test diet, and used as the control group.
　試験用飼料の給餌終了後、各群（１５～２０匹／群）の海水にＷＳＳＶストック懸濁液（人為的にＷＳＳＶに感染させたエビを人工海水とともにジューサで破砕し、得られるホモジナイズ試料からろ過等により肉片等を除き、使用時まで－８０℃で保存していたもの）を添加しウイルス接種を行った。添加後は、海水１０Ｌに対し引き続き、上記記載のものと同じ飼料を用い、上述の［エビの生育条件］に従って飼育を継続した。 After feeding of the test feed was completed, a WSSV stock suspension (shrimp artificially infected with WSSV were crushed in a juicer together with artificial seawater, and the resulting homogenized sample was filtered to remove any fleshy parts, and stored at -80°C until use) was added to the seawater of each group (15-20 shrimp per group) and the virus was inoculated. After addition, the same feed as described above was continued per 10 L of seawater, and rearing was continued according to the shrimp growth conditions described above.
　ウイルス接種後２週間におけるバナメイエビの斃死個体数を観察した。ウイルス接種前のエビの生存数を１００％とし、ウイルス接種後２週間の生存率を以下の表及び図に示す。
　給餌試験１の結果をそれぞれ、表１及び図１（１％リグニンスルホン酸塩添加）、表２及び図２（４％リグニンスルホン酸塩添加）に示す。給餌試験２の結果をそれぞれ、表３及び図３（１％リグニンスルホン酸塩添加）、表４及び図４（４％リグニンスルホン酸塩添加）に示す。 The number of dead shrimp was observed two weeks after the virus inoculation. The survival rate of shrimp before the virus inoculation was set as 100%, and the survival rate two weeks after the virus inoculation is shown in the following table and figure.
The results of feeding test 1 are shown in Table 1 and Figure 1 (1% lignin sulfonate added), and Table 2 and Figure 2 (4% lignin sulfonate added). The results of feeding test 2 are shown in Table 3 and Figure 3 (1% lignin sulfonate added), and Table 4 and Figure 4 (4% lignin sulfonate added).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　給餌試験１において、コントロール群は３日目から生存率が低下し６日目には生存率２０％未満まで低下したが、リグニンスルホン酸塩添加群はいずれも３～４日目まで生存率９５～１００％を維持できており、６～７日目まで４５％以上の生存率を維持できていた（表１～２、図１～２）。給餌試験２（４％リグニン）において、リグニンスルホン塩酸添加群において生存率が４０％以下に低下することがなく、給餌試験１と比較すると、生存率をより長期間維持できることがわかった（表３～４、図３～４）。 In feeding test 1, the survival rate of the control group began to decline from the third day, dropping to less than 20% by the sixth day, whereas all of the lignosulfonate-added groups maintained a survival rate of 95-100% until the third or fourth day, and above 45% until the sixth or seventh day (Tables 1-2, Figures 1-2). In feeding test 2 (4% lignin), the survival rate of the lignosulfonate-added group never dropped below 40%, demonstrating that survival rates could be maintained for a longer period of time compared to feeding test 1 (Tables 3-4, Figures 3-4).
〔試験２．リグニン海水を用いたＷＳＳＶ感染防除試験〕
　リグニンスルホン酸塩Ａ、Ｂ、又はＣをそれぞれ２ｍｇ／ｍＬで溶かした海水にバナメイエビを１０匹入れ、試験１で用いたのと同じＷＳＳＶストック懸濁液を添加し、ウイルス接種を行った。ウイルス接種に用いたＷＳＳＶの調整方法は試験１と同様に行った。その他の育成条件は上述の［エビの育成条件］と同様である。 [Test 2. WSSV infection prevention test using lignin seawater]
Ten whitenamei shrimp were placed in seawater containing lignin sulfonate A, B, or C dissolved at 2 mg/mL, and the same WSSV stock suspension as used in Test 1 was added for virus inoculation. The WSSV used for virus inoculation was prepared in the same manner as in Test 1. The other growth conditions were the same as those in the above-mentioned [Shrimp growth conditions].
　ウイルス接種後１０日間の斃死を観察した。ウイルス接種前のエビの生存数（１０尾）を１００％とし、ウイルス接種後１０日間の生存率を表５及び図５に示す。 Mortality was observed for 10 days after virus inoculation. The number of surviving shrimp before virus inoculation (10 shrimp) was set as 100%, and the survival rate for 10 days after virus inoculation is shown in Table 5 and Figure 5.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　コントロール群と比較して、いずれのリグニンスルホン酸塩添加群も、ＷＳＳＶ摂取後５日目まで生存率５０％以上を維持しており、その後も２０％以上を維持していた（表５及び図５）。 Compared to the control group, all lignosulfonate-added groups maintained a survival rate of 50% or higher up until the fifth day after WSSV ingestion, and maintained a survival rate of 20% or higher thereafter (Table 5 and Figure 5).
　これらの結果は、本発明の抗ＷＳＳＶ剤がホワイトスポット病の感染予防に有用であり、飼料又は飼育用水へ混合して用いることができることを示している。 These results indicate that the anti-WSSV agent of the present invention is useful for preventing infection with white spot disease and can be used by mixing it into feed or rearing water.

Claims (10)

  1. 　リグニン成分を有効成分とする、抗ホワイトスポットシンドロームウイルス剤。 An anti-white spot syndrome virus agent whose active ingredient is lignin.
  2. 　リグニン成分が、リグニンスルホン酸塩を少なくとも含む、請求項１に記載の剤。 The agent according to claim 1, wherein the lignin component contains at least lignin sulfonate.
  3. 　リグニン成分は、リグノセルロース原料の亜硫酸蒸解法に由来する、請求項１又は２に記載の剤。 The agent according to claim 1 or 2, wherein the lignin component is derived from sulfite cooking of lignocellulosic raw materials.
  4. 　リグニン成分は、抽出成分を含む、請求項１～３のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 3, wherein the lignin component includes an extract component.
  5. 　甲殻類用である、請求項１～４のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 4, which is for use on crustaceans.
  6. 　養殖又は栽培漁業に供される甲殻類用である、請求項１～５のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 5, which is for use on crustaceans used in aquaculture or fish farming.
  7. 　甲殻類がエビを含む、請求項５又は６に記載の剤。 The agent according to claim 5 or 6, wherein the crustacean includes shrimp.
  8. 　請求項１～７のいずれか１項に記載の剤を含む、飼料。 A feed containing the agent according to any one of claims 1 to 7.
  9. 　請求項１～７のいずれか１項に記載の剤を含む、飼育用水用処理剤。 A treatment agent for breeding water, comprising the agent according to any one of claims 1 to 7.
  10. 　請求項１～７のいずれか１項に記載の剤を甲殻類に投与することを含む、ホワイトスポット病の予防方法。 A method for preventing white spot disease, comprising administering the agent according to any one of claims 1 to 7 to crustaceans.
PCT/JP2023/036956 2022-10-13 2023-10-12 Anti-white spot syndrome virus agent WO2024080322A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03206043A (en) * 1990-01-06 1991-09-09 Hiroshi Sakagami Preventive for viral infection
JP2006304761A (en) * 2005-05-02 2006-11-09 Kazuo Sakuma Lonicera caerulea l. product containing component inhibiting vancomycin-resisting enterococcus (vre), methicillin resistant staphylococcus aureus (mrsa)
JP2011074047A (en) * 2009-10-02 2011-04-14 Enzyme Kk Process of humus extract
JP2020048468A (en) * 2018-09-26 2020-04-02 エンザイム株式会社 Cultivation method of aquatic animal

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03206043A (en) * 1990-01-06 1991-09-09 Hiroshi Sakagami Preventive for viral infection
JP2006304761A (en) * 2005-05-02 2006-11-09 Kazuo Sakuma Lonicera caerulea l. product containing component inhibiting vancomycin-resisting enterococcus (vre), methicillin resistant staphylococcus aureus (mrsa)
JP2011074047A (en) * 2009-10-02 2011-04-14 Enzyme Kk Process of humus extract
JP2020048468A (en) * 2018-09-26 2020-04-02 エンザイム株式会社 Cultivation method of aquatic animal

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