Anti-white spot syndrome virus agent

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

Anti-white spot syndrome virus agent

[0001] The present invention relates to an anti-white spot syndrome virus agent.

[0002] As demand for shrimp expands, aquaculture and fish farming are being promoted to increase shrimp numbers. High-density rearing practices aimed at improving productivity can lead to the outbreak of viral infections. In particular, white spot disease (WSD, also known as penaeid acute viremia (PAV)) has caused significant damage to shrimp production.

[0003] Known measures to prevent white spot disease infection include, for example, oral vaccines containing recombinant coat proteins of white spot syndrome virus (WSSV) and DNA vaccines against coat proteins (Patent Document 1, Non-Patent Document 1).

[0004] Japanese Patent Application Laid-Open No. 2008-63302

[0005] Mari Inada et al., Fish Disease Research, Vol. 52 (3), pp. 115-119, 2017 https: / / www.jstage.jst.go.jp / article / jsfp / 52 / 3 / 52_115 / _pdf / -char / ja

[0006] However, vaccines such as those described in Patent Document 1 and Non-Patent Document 1 are expensive and provide protection for a short period of time. For example, the vaccine described in Non-Patent Document 1 provides protection for approximately one month. Therefore, administering a WSSV vaccine to small organisms such as shrimp and other crustaceans is not practical due to the labor and cost involved.

[0007] An object of the present invention is to provide a novel anti-WSSV agent that can prevent WSSV infection.

[0008] 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 sulfite cooking of lignocellulose raw materials. [4] The agent according to any one of [1] to [3], wherein the lignin component contains an extracted component. [5] The agent according to any one of [1] to [4], wherein the agent is for crustaceans. [6] The agent according to any one of [1] to [5], wherein the agent is for crustaceans used in aquaculture or fish farming. [7] The agent according to [5] or [6], wherein the crustaceans include 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.

[0009] The anti-WSSV agent of the present invention can exhibit a good anti-WSSV inhibitory effect. Therefore, when used, for example, as a feed for organisms such as crustaceans or as a treatment agent for breeding water, it can prevent WSSV infection and improve the productivity of these organisms, making it useful in various fields such as the fisheries, aquaculture, and pet supplies.

[0010] Fig. 1 is a graph showing the survival rate of shrimp in Feeding Test 1 (1% lignin) of the Example. Fig. 2 is a graph showing the survival rate of shrimp in Feeding Test 1 (4% lignin) of the Example. Fig. 3 is a graph showing the survival rate of shrimp in Feeding Test 2 (1% lignin) of the Example. Fig. 4 is a graph showing the survival rate of shrimp in Feeding Test 2 (4% lignin) of the Example. Fig. 5 is a graph showing the survival rate of shrimp in Test 2 of the Example.

[0011] The present invention relates to an anti-white spot syndrome virus agent.

[0012] 1. Lignin Component The agent of the present invention contains a lignin component as an active ingredient.

[0013] [1.1. Lignin Derivatives] In this specification, the term "lignin component" refers to 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 powder or liquid form. The method for preparing a liquid lignin component is not particularly limited, but an example of such a method includes dissolving a powdered lignin component in an appropriate solvent (e.g., water or an aqueous sodium hydroxide solution) to obtain a liquid lignin component.

[0014] 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. Among these, lignin sulfonate is preferred. The lignin derivative may be one type or a combination of two or more types, but it is preferable to include at least lignin sulfonate.

[0015] Lignin sulfonate is a lignin derivative having a sulfo group, prepared from lignocellulose raw materials through sulfite treatment. Lignin sulfonate may be in the form of an acid or a salt, but is usually in the form of a salt. Examples of salts include monovalent metal salts, divalent metal salts, ammonium salts, and organic ammonium salts. Of these, calcium salts, magnesium salts, sodium salts, potassium salts, and calcium-sodium mixed salts are preferred.

[0016] 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.

[0017] (Lignocellulose Raw Material) The lignocellulose raw material is not particularly limited as long as it contains lignocellulose in its constituents. Examples include pulp raw materials such as wood and non-wood. Examples of wood include coniferous trees such as radiata pine, spruce, red pine, cedar, and cypress, and broadleaf trees such as white birch and beech. The age and location of the wood are not important. Therefore, wood harvested from trees of different ages or from different locations on a tree may be used in combination. Examples of non-wood include bamboo, kenaf, reed, and rice. The lignocellulose raw material may be used alone or in combination of two or more. Lignin sulfonates may be prepared from raw materials other than lignocellulose raw materials, such as lignin. Examples of lignin include naturally occurring lignin and artificially produced lignin (e.g., dehydrogenation polymerized products of hydroxycinnamic alcohol analogs), and both can be used. Lignin sulfonates can be prepared from lignin, for example, by a method of decomposing lignin and sulfonating it.

[0018] (Sulfite 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 a sulfo group to be introduced into the α-carbon atom of the side chain of lignin contained in the lignocellulosic raw material.

[0019] The sulfite treatment is preferably carried out by sulfite cooking, which allows for more quantitative sulfonation of lignin in lignocellulosic raw materials. The sulfite cooking method is a method in which lignocellulosic raw materials are reacted at high temperatures in a solution of at least one of sulfurous acid and a sulfite salt (e.g., an aqueous solution, cooking liquor). 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.

[0020] When sulfite cooking is carried out, examples of sulfite salts include magnesium salts, calcium salts, sodium salts, and ammonium salts.

[0021] Sulfurous acid (SO ) in a solution of sulfurous acid and / or sulfite salts ２ The concentration of SO 4 is not particularly limited, but is preferably 100 mL of SO 4 per 100 mL of reaction solution. ２ The mass (g) ratio of the SO 4 to the sulphite cooking solution is preferably 1 g / 100 mL or more, and more preferably 2 g / 100 mL or more when sulphite cooking is performed. The upper limit is preferably 20 g / 100 mL or less, and more preferably 15 g / 100 mL or less when sulphite cooking is performed. ２ The 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.

[0022] The pH value of the sulfite treatment is not particularly limited, but is usually 10 or less. When sulfite cooking is performed, it is preferably performed under acidic conditions, more preferably a pH of 5 or less, and even more preferably 3 or less. This allows lignin derivatives (e.g., lignin sulfonates) to be efficiently extracted, resulting in pulp of higher quality. The lower limit of the pH value is preferably 0.1 or more, and when sulfite cooking is performed, more preferably 0.5 or more. The pH value during the sulfite treatment is preferably 0.1 to 10, and when sulfite cooking is performed, more preferably 0.5 to 5, and even more preferably 0.5 to 3.

[0023] The temperature for the sulfite treatment is not particularly limited, but is preferably 170°C or lower, and more preferably 150°C or lower if sulfite cooking is performed. The lower limit is preferably 70°C or higher, and more preferably 100°C or higher if sulfite cooking is performed. The temperature conditions for the sulfite treatment are preferably 70 to 170°C, and more preferably 100 to 150°C if sulfite cooking is performed. The treatment time for the sulfite treatment is not particularly limited, and although it depends on the conditions for the sulfite treatment, 0.5 to 24 hours is preferred, and 1.0 to 12 hours is more preferred.

[0024] 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 and Mg(OH).２ , CaO, Ca(OH) ２ , CaCO ３ , N.H. ３ , N.H. ４ OH, NaOH, NaHCO ３ , Na ２ CO ３ The counter cation is preferably a magnesium ion, a sodium ion, or a calcium ion.

[0025] In the sulfite treatment, when a solution of at least one of sulfurous acid and sulfite salt is used, the solution may contain SO 4 as needed. ２ In addition to the above, counter cations (salts) and digestion penetrants (for example, cyclic ketone compounds such as anthraquinone sulfonates, anthraquinone, and tetrahydroanthraquinone) may be contained.

[0026] There are no limitations on the equipment used for the sulfite treatment, and for example, commonly known dissolving pulp manufacturing equipment can be used.

[0027] The intermediate product can be separated from the solution of at least one of sulfurous acid and sulfite salts by a conventional method, such as a method for separating the sulfite cooking effluent after sulfite cooking (e.g., filtration).

[0028] The lignin sulfonate obtained by the sulfite treatment (e.g., as the filtrate or filtration residue after filtering the insoluble matter in the sulfite solution, preferably as the filtrate) may be used as a lignin derivative either directly or, if necessary, after concentration. On the other hand, if necessary, other treatments may be carried out. This allows the production of lignin derivatives with 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 thereof.

[0029] (Alkali Treatment) The alkali treatment is preferably performed on the filtration residue (insoluble matter) or filtrate after sulfite treatment, or on the treated product after dialysis. 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 with a pH value of 8 or higher, preferably a pH value of 9 or higher. The upper limit of the pH value is usually 14 or lower.

[0030] In the alkali treatment, an alkaline substance is usually brought into contact with the sulfite-treated product. The alkaline substance is not particularly limited, but examples thereof include calcium hydroxide, magnesium hydroxide, sodium hydroxide, potassium hydroxide, sodium carbonate, and ammonia. Among these, sodium hydroxide and calcium hydroxide are preferred. The alkaline substance may be used alone or in combination of two or more.

[0031] Examples of methods for contacting the sulfite-treated product with an alkaline substance include a method in which a dispersion or solution (e.g., an aqueous dispersion or aqueous solution) of the sulfite-treated product is prepared and the alkaline substance is added to the dispersion or solution, and a method in which a solution or dispersion (e.g., an aqueous dispersion or aqueous solution) of the alkaline substance is added to the sulfite-treated product.

[0032] The temperature of the alkali treatment is not particularly limited, but is preferably 40° C. or higher, 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.

[0033] The amount of the alkaline substance in the alkali treatment is preferably 0.5 to 40 mass %, more preferably 1.0 to 30 mass %, relative to the mass of the solids content of the sulfite-treated product, or, when an aqueous solution or dispersion is prepared by dispersing the alkali-treated extract in an aqueous solvent (e.g., water), relative to the mass of the aqueous solution or dispersion.

[0034] The time for the alkali treatment is not particularly limited, but is preferably 0.1 hours or more, more preferably 0.5 hours or more, and is preferably 10 hours or less, more preferably 6 hours or less.

[0035] Prior to the alkali treatment, the sulfite-treated product may be dissolved, dispersed, or its concentration adjusted (prepared as a solution or dispersion in an aqueous solvent such as water) as needed. Dispersion can be carried out, for example, by passing the product through a disc refiner, adding it to a mixer or disperser, or by kneading it. The concentration can be adjusted, for example, by using an aqueous solvent such as water.

[0036] (Oxidation Treatment) The oxidation treatment can be performed on the treated product obtained after the sulfite treatment (e.g., the filtrate after filtration) or the treated product after the alkali treatment. The oxidation treatment can be performed using an appropriate oxidizing agent. When the oxidizing agent is a gas, the oxidation treatment can be performed by bubbling the gas into the filtrate. When the oxidizing agent is a liquid, the oxidation treatment can be performed 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 performed under alkaline conditions (alkaline oxidation treatment). The treatment pH of the alkaline oxidation treatment is usually 8 or higher, preferably 10 or higher, and more preferably 12 or higher. The temperature of the oxidation treatment is usually 20 to 200°C, preferably 50 to 180°C. The oxidation treatment time is usually preferably 0.1 hour or longer, more preferably 0.5 hour or longer. The upper limit is preferably 5 hours or shorter, more preferably 3 hours or shorter.

[0037] (Dialysis or UF Treatment) Dialysis can be performed on the treated product obtained after the sulfite treatment (for example, the filtrate after filtration). Examples of dialysis membranes include cellulose-based membranes such as cellulose acetate, and synthetic polymer 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.

[0038] Instead of dialysis, UF (ultrafiltration) treatment can be used. Known UF membranes can be used. Examples include hollow fiber membranes, spiral membranes, tubular membranes, and flat membranes. Known UF membrane materials can be used. Examples include cellulose acetate, aromatic polyamide, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, and ceramics. The UF membrane may be a commercially available product.

[0039] 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 rate of the treatment liquid from becoming excessively slow. Furthermore, using a UF membrane with a molecular weight cutoff of 30,000 or less can prevent lignin from becoming unable to be separated from the treatment liquid.

[0040] The concentration ratio by UF treatment using a UF membrane can be set as desired. In other words, the UF treatment can be stopped when the outflow volume of the concentrated liquid reaches a desired volume. It is preferable to concentrate the liquid 2 to 6 times. Concentrating 2 to 6 times means that the volume of the raw liquid (black liquor) is reduced to 1 / 2 to 1 / 6.

[0041] The temperature of the treatment liquid during UF treatment is not particularly limited. For example, 20 to 80°C is preferred, and considering the heat resistance of the UF membrane material, 20 to 70°C is more preferred. 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%, more preferably 5 to 20%.

[0042] Another example of a method for producing lignin sulfonates is a method of sulfonating kraft lignin.

[0043] -Kraft Lignin- Kraft lignin is also called thiolignin or sulfate lignin. Examples of kraft lignin include an alkaline solution of kraft lignin, powdered kraft lignin obtained by spray-drying an alkaline solution of kraft lignin to form a powder, and acid-precipitated kraft lignin obtained by precipitating an alkaline solution of kraft lignin with an acid. One of these kraft lignins may be used alone, or two or more may be used in combination.

[0044] As a method for preparing an alkaline solution of kraft lignin, for example, Na ２ An example of such a method is a method in which an alkaline solution containing S is electrolyzed by electrolytic oxidation to produce a NaOH solution on the cathode side (Japanese Patent Laid-Open Publication No. 2000-336589). Examples of methods for preparing acid-precipitated kraft lignin in which an alkaline solution of kraft lignin is precipitated with an acid include methods for preparing powdery acid-precipitated kraft lignin (WO 2006 / 038863, WO 2006 / 031175, and WO 2012 / 005677).

[0045] The method for sulfonating kraft lignin may be sulfonation by a conventional sulfite treatment or sulfite cooking treatment, or may include 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, pp. 669-677 (1997)." However, the method is not limited to the above methods, and other methods may also be used.

[0046] [1.2. Extracted component] The lignin component may further contain an extracted component. By including the extracted component, the anti-WSSV activity can be further enhanced.

[0047] As used herein, extractives (tree extractives) refer to trace components of plants (e.g., plants used as pulp raw materials, preferably wood from plants of the genus Cedar, Chamaecyparis obtusa, Pinus spp., Larch spp., Abies spp., and Eucalyptus spp.) and are typically obtained by extracting wood with organic solvents or other methods. Extractives are typically determinants of wood's physical 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 deposition of cellulose and hemicellulose and lignin, the tree is finally completed as a biological material only after the accumulation of extractives, which are linked to heartwood formation. Examples of organic solvents used to obtain extractives include hexane, benzene, ether, acetone, and alcohol. The extractive content relative to the wood is typically about 5% or less.

[0048] Extracted components typically contain low-molecular-weight compounds (usually low-molecular-weight organic compounds). Many of these are secondary metabolites with molecular weights of several thousand or less, and although they are generally extremely diverse, they can be 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 selected from these.

[0049] Flavonoids include diphenylpropane (C ６ -C ３ -C ６ ) skeleton, examples of which include flavones, flavanones, chalcones, aurones, 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, examples of which include gallotannins and ellagitannins. Hydrolyzable tannins can be decomposed into simple fragments by hydrolysis with acids, alkalis, etc., and hydrolysis of gallotannins and ellagitannins yields gallic acid and ellagic acid, respectively. Condensed tannins include, for example, amorphous polymers whose precursors are catechins or leucoanthocyanidins.

[0050] Lignans include, for example, lignans and related substances. Lignans (resinols) are C phenylpropane units, which are the same structural units as lignin, bonded to each other via carbon-carbon bonds between the β-positions of their side chains. ６ -C ３ -C ３ -C ６ Unlike lignin, it has an asymmetric carbon in the molecule and is optically active. Examples of substances related to lignan include C ６ -C ３ -C ２ -C ６ The stilbenes may be any compounds having an α,β-diphenylethylene skeleton.

[0051] Terpenoids are composed of isoprene units (C ５ H ８ The terpenoid may be a series of compounds in which two or more isoprenoid units are bonded in a chain or ring. 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.

[0052] Extractives have a variety of physiological activities depending on their basic carbon skeleton and substituents. These physiological activities may affect antiviral activity. Examples of physiological activities include biological activity against microorganisms, insects, and plants ("Resistance of wood to biodeterioration by extractives," Wood Preservation 34(2), 48-54, 2008), improved wood durability, and inhibitory effects on chemical and physical processing of wood (e.g., pulping, bleaching, cement hardening).

[0053] [1.3. Amount of Extractable Components, Amount of Methoxy Groups, Sulfo Group S Content, and Solubility in Water] -Amount of Extractable Components- The amount of extractable 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 satisfactorily. Therefore, the amount of extractable 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.

[0054] The amount of extractable components can be measured by the method for measuring hexane extractable substances described in JIS K 0102:2019.

[0055] -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.

[0056] The amount of methoxy groups per solid content of the lignin component is typically 2.0% by mass or more, 2.5% by mass or more, or 3.0% by mass or more, preferably 3.5% by mass or more, and more preferably 4.0% by mass or more. The upper limit is not particularly limited, but is typically 20% by mass or less. Therefore, the amount of methoxy groups per solid content of the lignin component is typically 2.0% by mass or more, 2.5 to 20% by mass or 3.0 to 20% by mass, preferably 3.5 to 20% by mass, and more preferably 4.0 to 20% by mass. This allows the anti-WSSV agent of the present invention to exhibit a favorable WSSV inhibitory effect while ensuring safety.

[0057] The amount of methoxy groups can be measured by the quantitative determination of methoxy groups by the Viebock and Schwappach method (see "Lignin Chemistry Research Methods", pp. 336-340, 1994, published by Uni Publishing Co., Ltd.).

[0058] Extractable component amount / methoxy group amount (mass ratio)—As described above, the extractable component amount can affect safety depending on its physiological activity, while the methoxy group amount is an indicator of the lignin and lignin derivative content. Therefore, the mass ratio of the extractable component amount to the methoxy group amount can be said to represent the balance between safety and WSSV inhibitory effect.

[0059] 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 typically 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, 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 typically 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 within the above range, the WSSV suppression effect can be exhibited well while ensuring safety.

[0060] -Sulfo group- Lignin derivatives are represented by the general formula (1): -SO ３ The lignin derivative may contain two or more groups represented by general formula (1) having different substituents M. In formula (1), M represents 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. The lignin derivative may contain two or more groups represented by general formula (1) having different substituents M.

[0061] -Sulfo Group S Content- The total amount (ratio to the solid content of the lignin derivative) of S content (sulfur atom content: sulfo group S content) of groups represented by general formula (1) in the lignin derivative is typically 0.5% by mass or more, preferably 1.0% by mass or more, and more preferably 1.2% by mass or more. This allows the lignin derivative to be rich in groups represented by general formula (1), exhibit water solubility, and exhibit a good virus inhibitory effect against WSSV. The upper limit is typically 20.0% by mass or less, preferably 15.0% by mass or less, and more preferably 12.0% by mass or less. This allows the lignin derivative to exhibit moderate water solubility and efficiently exhibit a virus inhibitory effect. Therefore, the total amount of sulfo group S content in the lignin derivative is typically 0.5 to 20.0% by mass, preferably 1.0 to 15.0% by mass, and more preferably 1.2 to 12.0% by mass.

[0062] 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%)

[0063] In formula (1), the total S content and inorganic S content of the lignin derivative both represent 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 spectroscopy. The inorganic S content is the SO quantified by ion chromatography. ３ Content and SO ４ It can be calculated as the total amount of content.

[0064] -Solubility in Water- The lignin component has a solubility in water of typically 1.0% by mass or more, preferably 3.0% by mass or more. Since the environment in which the partial chemical structure having a virus-inhibiting effect comes into contact with viruses is an aqueous medium, it is presumed that the above-mentioned range allows the component to have appropriate water solubility, which is advantageous for the virus-inhibiting effect. The upper limit is not particularly limited, and may be 100% by mass or less. Therefore, the lignin component has a solubility in water of typically 1.0 to 100% by mass, preferably 3.0 to 100% by mass.

[0065] The amount of dissolution in water can be calculated as follows: 10 g (dry weight) of a sample is 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) are measured. The mass of the solids in the filtrate is then divided by the mass of the sample (10 g) and multiplied by 100 to calculate the amount.

[0066] 1.4. Optional Components The lignin component may contain components other than the extracted components that are mixed in from the raw materials when preparing the lignin derivative (e.g., during sulfite cooking), such as inorganic salts such as sodium sulfate, sodium sulfite, sodium chloride, magnesium sulfate, magnesium sulfite, magnesium chloride, calcium sulfate, calcium sulfite, calcium chloride, ammonium sulfate, ammonium sulfite, ammonium chloride, and sodium hydroxide.

[0067] The lignin component may also contain sugars as components other than the extracted components. In this specification, sugars refer to at least one type of sugar or a combination of two or more types of sugars.

[0068] The sugars are not limited by the number of carbon atoms constituting them and may be monosaccharides, oligosaccharides, or 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 and amylopectin), cellulose, hemicellulose, dextrin, and glucan. Sugars, particularly polysaccharides, may include polysaccharides contained in plant components, such as pulp, which are lignocellulose raw materials, as well as those produced by decomposition and / or modification during cooking or bleaching. Sugars typically include polysaccharides, reducing sugars, and / or modified sugars. Reducing sugars may be any sugar that exhibits reducing properties. Reducing sugars typically 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. Examples of modified sugars include sugars that have been chemically modified by oxidation or sulfonation, and sugar derivatives substituted with substituents such as hydroxyl groups, aldehyde groups, carbonyl groups, and / or sulfo groups.

[0069] A reducing sugar is a sugar that exhibits reducing properties and generates an aldehyde group or a ketone group 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 degradation products. Examples of degradation products of cellulose and hemicellulose include monosaccharides such as rhamnose, galactose, arabinose, xylose, glucose, mannose, and fructose, and oligosaccharides such as xylooligosaccharides and cellooligosaccharides.

[0070] The sugar-modified product refers to a modified product obtained by chemically modifying a sugar by oxidation, sulfonation, etc. The sugar-modified product may be a sugar derivative in which a functional group such as a hydroxyl group, an aldehyde group, a carbonyl group, and / or a sulfo group has been introduced into the sugar skeleton, or a compound in which two or more sugar derivatives (two types) are bonded.

[0071] The agent of the present invention may further contain other optional components as needed. The other components may be any optional components commonly 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.

[0072] [1.5. Dosage Form] The dosage form of the anti-WSSV agent can be appropriately selected depending on its intended use. Examples of dosage forms include liquid preparations such as solutions, emulsions, suspensions, dispersions, and aerosols; and solid or semi-solid preparations such as granules, tablets, powders, and pastes.

[0073] 1.6. Physiological activity The anti-WSSV agent of the present invention can exert a WSSV inhibitory effect.

[0074] 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.

[0075] The anti-WSSV agent can suppress WSSV infection and its spread in target organisms, which are usually organisms belonging to the order Decapoda (e.g., crustaceans such as shrimp, crabs, and crayfish). The shrimp may be any shrimp that inhabits seawater, brackish water, or freshwater, and examples thereof include Litopenaeus species (Penaeus Fabricius), such as white leg shrimp (Penaeus vannamei Boone), white shrimp (Penaeus setiferus), and western blue shrimp (Penaeus stylirostris Stimpson), as well as kuruma shrimp (Penaeus japonicus Spence Bate), Korean shrimp (Penaeus chinensis), red-tailed shrimp (Penaeus penicillatus Alcock), and Indian shrimp (Penaeus indicus H. Milne). Penaeus Fabricius, such as Penaeus Edwards, Banana prawn (Penaeus merguiensis De Man), black tiger shrimp (Penaeus monodon Fabricius), tiger shrimp (Penaeus semisulcatus De Haan), Japanese shrimp (Penaeus latisulcatus Kishinouye), northern pink shrimp (Penaeus duorarum Burkenroad), and brown shrimp (Penaeus aztecus Ives). the genus Metapenaeus Wood-Mason in Wood-Mason & Alcock, such as Metapenaeus Fabricius, Metapenaeus ensis, Mozambique brown (M. monoceros), Metapenaeus dobsoni, and Metapenaeus elegans De Man; the genus Metapenaeopsis Bouvier, such as Metapenaeopsis acclivis, Metapenaeopsis barbata, and Metapenaeopsis dalei;Examples of shrimp include shrimps that are raised for aquaculture, such as shrimps belonging to the genus Trachysalambria (Trachysalambria Burkenroad), such as monkey shrimp (Trachysalambria curvirostris), and shrimps belonging to the genus Parapenaeopsis (Parapenaeopsis Alcock), such as kiddy shrimp (Parapenaeopsis stylifera).

[0076] [2. Uses] The anti-WSSV agent can be used in various applications where an anti-WSSV effect is expected, such as feed, a treatment agent for breeding water, a disinfectant, and a cleaning agent.

[0077] 2.1. Feed and Medicine By using an anti-WSSV agent as feed or medicine, it is possible to have target organisms ingest the anti-WSSV agent, thereby enabling them to efficiently exert resistance to WSSV.

[0078] The feed and pharmaceuticals may contain an anti-WSSV agent, and the content can be adjusted appropriately depending on the mode of use and the target of application. For example, when calculated based on a daily diet of 5 to 10% of the organism's body weight, the lignin component content is 0.1% by mass or more, preferably 0.5% by mass or more, and more preferably 0.7% by mass or more, relative to the weight of the feed. The upper limit is typically 7.0% by mass or less, preferably 5.0% by mass or less. Therefore, the range is typically 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, for example, once to three times daily for at least three days, at least five days, preferably at least one week, more preferably at least 10 days, and even more preferably at least two weeks. Alternatively, the continuous administration may be repeated by temporarily returning the animal to a normal diet and then repeating the continuous administration after a certain period of time (e.g., one week, two weeks, three weeks, one month, or two months). Furthermore, there is no particular limitation on the age of the crustaceans that can be used.

[0079] Optional components that may be contained in the feed include, for example, nutritional components, such as fish oil, seafood, amino acids, lipids, minerals, vitamins, and other components that are commonly used in feed for target organisms, aquaculture feed, and aquaculture feed, and the content of these components can be selected appropriately.

[0080] The feed may be in any formulation as long as it is similar to that of ordinary feed, and is usually in the form of granules, powder, or powder. The method for producing the feed is not particularly limited, and examples include a method in which the components constituting the sample are mixed, dried, and shaped. The feed may be any feed for the target organism infected with WSSV, and is usually feed for crustaceans, and preferably feed for aquaculture.

[0081] [2.2. Treatment Agent for Rearing Water] By using an anti-WSSV agent as a treatment agent for rearing water, it is possible to inhibit WSSV from entering the body of a target organism via oral ingestion or through the gills, and to efficiently exert WSSV resistance.

[0082] The treatment agent may be any water used to raise target organisms in aquaculture, fish farming, or the like, and may be any of seawater, brackish water, or freshwater (natural or artificially adjusted in composition). The amount of treatment per administration 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, more preferably 0.1 mg / mL or more. The upper limit is preferably 10.0 mg / mL or less, more preferably 7.0 mg / mL or less. Therefore, the amount of treatment per administration is preferably 0.02 to 10.0 mg / mL, 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 (e.g., once every 1 day, 3 days, 5 days, 1 week, 10 days, or 2 weeks).

[0083] 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 treatment method is preferably spraying.

[0084] [2.3. Other Uses] Examples of uses other than the above-mentioned feed and treatment agents include disinfectants and cleaning agents for breeding items. Examples of breeding items include aquariums, filtration devices, aquaculture cages, nets, bottom sand, sanitary products, and other items used in breeding crustacean farming, fish farming, and the like. Examples of materials for the items include, but are not limited to, glass, resin, cloth, and metal. The shape of the item is also not limited.

[0085] 3. Method for preventing white spot disease By administering an anti-WSSV agent to a target organism (a non-human organism such as a crustacean), resistance to WSSV can be increased and a decrease in survival rate can be suppressed. Therefore, the anti-WSSV agent can be used to prevent WSD in crustaceans.

[0086] 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, the method of treating rearing water with a treatment agent containing the anti-WSSV agent, the method of disinfecting or cleaning rearing items with a disinfectant or cleaning agent containing the anti-WSSV agent, and a method combining 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.

[0087] The present invention will be described below with reference to examples, which are not intended to limit the scope of the present invention.

[0088] Example 1 Wood chips (radiata pine) were subjected to sulfite treatment based on the sulfite cooking method. ２ A 4 g / 100 mL solution of sodium sulfite was used, and the treatment temperature was 140°C, pH 2, and treatment time was 3 hours. Next, insoluble matter was filtered off. The resulting filtrate was concentrated using a rotary evaporator until the solid content reached 50%. The pH of the solution was then adjusted to 4.5 with NaOH and powdered using a spray dryer to obtain Lignin Sulfonate A of Example 1 (methoxy group content 6.1% by mass, extractable component content 0.2% by mass, extractable component content / methoxy group content: 0.04, sulfur content of sulfo groups 5.2% by mass, amount dissolved in water 100% by mass).

[0089] Example 2 Ligninsulfonate 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 tubing). The solution in the dialysis tubing was collected and concentrated to a concentration of 25%. The concentrate was then powdered using a spray dryer to obtain Ligninsulfonate 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 in sulfo groups: 3.5% by mass, amount dissolved in water: 100% by mass).

[0090] Example 3 Wood chips (radiata pine) were subjected to sulfite treatment based on the sulfite cooking method. ２ A 2.5 g / 100 mL solution of sodium sulfite was used, and the treatment temperature was 140°C, pH 3, and treatment time was 3 hours. Next, insoluble matter was filtered off, and the resulting filtrate was concentrated using a rotary evaporator until the solids content reached 50%. The pH of the solution was then adjusted to 4.5 with NaOH and powdered using a spray dryer. The resulting powder was dissolved in water to prepare an aqueous solution with a solids content of 25%. The pH was adjusted to 12 with 40% NaOH, and then subjected to alkaline air oxidation at 140°C for 120 minutes. Subsequently, 70% sulfuric acid was added to adjust the pH to 3, and the partially desulfonated lignin sulfonate salt was fractionated and precipitated. The resulting precipitate of partially desulfonated lignin sulfonate salt was washed with water until the filtrate became neutral. 20 parts of the precipitate was suspended in 100 parts of water, heated to 60°C, and then 1 mol / L aqueous sodium hydroxide solution was added with stirring until the pH reached 9, completely dissolving the precipitate. The resulting solution was powdered using a spray dryer to obtain lignin sulfonate C (methoxy group content: 11.2% by mass, extractable component content: 1.6% by mass, extractable component content / methoxy group content: 0.14, S content of sulfo groups: 2.7% by mass, amount soluble in water: 100% by mass).

[0091] [Method for measuring the amount of extracted components]: The amount of extracted components was measured by the method for measuring hexane extractable substances described in JIS K 0102:2019.

[0092] [Methoxy group amount measurement method]: The methoxy group amount was measured by the quantitative determination method of methoxy groups by the Viebock and Schwappach method (see "Lignin Chemistry Research Methods", pp. 336-340, 1994, published by Uni Publishing Co., Ltd.).

[0093] [Method for measuring sulfur content in sulfo groups] The sulfur content in sulfo groups was calculated using the following formula: S content in sulfo groups (mass%) = total sulfur content (mass%) - inorganic sulfur 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 sulfur content was determined by ICP emission spectroscopy. The inorganic sulfur content was determined by SO 2 determined by ion chromatography. ３ Content and SO ４ The total amount of content was used.

[0094] [Method for measuring the amount of lignosulfonate dissolved in water]: The amount of lignosulfonate 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 residue) 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 of lignosulfonate dissolved in water.

[0095] [Shrimp rearing conditions] The shrimp rearing conditions in Tests 1 and 2 described below are as follows: Shrimp used: Approximately 2 g of vannamei shrimp (purchased from a dealer in Kochi Prefecture) Rearing tank: 15 L glass tank Water temperature: 28°C Basic feed: Vannamei shrimp feed (imported from Thailand) Feeding amount: 5% of body weight / day Infection test: Highly virulent WSSV

[0096] [Test 1. Preventive Test for WSSV Infection by Feeding with Lignin] Ligninsulfonates A, B, or C were added to a basal diet at 1% or 4%, respectively, to prepare test diets for vannamei shrimp. Specifically, the basal diet was mixed with 1% or 4% ligninsulfonate and an equal amount of water as the basal diet. The resulting mixture was formed into noodles, dried (at approximately 70°C for approximately 2 hours), and randomly crushed to prepare the test diets. The test diets were fed twice daily by sprinkling them on seawater in an aquarium (ligninsulfonate-added group). The test diets were fed for one week (feeding test 1) or two weeks (feeding test 2). The shrimp were reared according to the above-mentioned [Shrimp Rearing Conditions], except for the diet. The control group was reared in the same manner as the ligninsulfonate-added group, except for the addition of the basal diet instead of the test diet.

[0097] After the end of feeding with the test diet, a WSSV stock suspension (shrimp artificially infected with WSSV were crushed together with artificial seawater in a juicer, and the resulting homogenized sample was filtered to remove any fleshy particles, and stored at -80°C until use) was added to the seawater of each group (15-20 shrimp per group) for virus inoculation. After the addition, the same diet as described above was continued per 10 L of seawater, and rearing was continued according to the above-mentioned "Shrimp growth conditions."

[0098] The number of dead vannamei shrimp was observed two weeks after virus inoculation. The survival rate two weeks after virus inoculation was shown in the following table and figure, with the number of surviving shrimp before virus inoculation set to 100%. The results of Feeding Test 1 are shown in Table 1 and Figure 1 (with 1% lignin sulfonate added), and Table 2 and Figure 2 (with 4% lignin sulfonate added). The results of Feeding Test 2 are shown in Table 3 and Figure 3 (with 1% lignin sulfonate added), and Table 4 and Figure 4 (with 4% lignin sulfonate added).

[0099]

[0100]

[0101]

[0102]

[0103] In Feeding Test 1, the survival rate of the control group began to decline on Day 3, dropping to less than 20% by Day 6. However, all of the lignosulfonate-added groups maintained a survival rate of 95-100% until Day 3-4 and above 45% until Day 6-7 (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).

[0104] [Test 2. WSSV infection control test using lignin seawater] Ten whitenamei shrimp were placed in seawater containing 2 mg / mL of lignin sulfonate A, B, or C, 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].

[0105] Mortality was observed for 10 days after virus inoculation. The survival rate for 10 days after virus inoculation is shown in Table 5 and Figure 5, with the number of surviving shrimp (10 shrimp) before virus inoculation set at 100%.

[0106]

[0107] Compared with the control group, all lignosulfonate-added groups maintained a survival rate of 50% or more up to 5 days after WSSV ingestion, and maintained a survival rate of 20% or more thereafter (Table 5 and FIG. 5).

[0108] These results demonstrate 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 with feed or rearing water.

Claims

1. Lignin is the active ingredient, The lignin component contains at least a lignin sulfonate and an extractive component, and the mass ratio of the amount of the extractive component to the amount of methoxy groups is 1.0 or less.

2. (delete)

3. 10. The agent of claim 1, wherein the lignin component is derived from sulfite cooking of lignocellulosic feedstock.

4. The agent according to claim 1 or 3, wherein the amount of the extracted component per solid content of the lignin component is 0.01 to 1.7% by mass.

5. The agent according to any one of claims 1 and 3 to 4, which is for use on crustaceans.

6. The agent according to any one of claims 1 and 3 to 5, which is for use on crustaceans used in aquaculture or fish farming.

7. The agent according to claim 5 or 6, wherein the crustacean comprises shrimp.

8. A feed comprising the agent according to any one of claims 1 and 3 to 7.

9. A treatment agent for breeding water, comprising the agent according to any one of claims 1 and 3 to 7.

10. A method for preventing white spot disease, comprising administering the agent according to any one of claims 1 and 3 to 7 to crustaceans.