Growth promoter for seaweed and method for culturing seaweed

Abstract

To provide an agent which has no adverse influence on biological tissue of seaweed and can promote seaweed growth and a method for culturing seaweed using the same.SOLUTION: A growth promoter for seaweed comprises at least one compound selected from the group consisting of an oxo fatty acid or a salt thereof, and a hydroxy fatty acid or a salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a seaweed growth promoter and a seaweed cultivation method. [Background technology]

[0002] Seaweed is a type of marine algae that grows mostly in coastal areas of the ocean. It serves as food for marine animals and plays an important role as a nursery for young fish. Seaweed is also important as a food source for humans. Nori, kelp, wakame seaweed, and aonori seaweed add color to the dinner table and are important ingredients in creating a rich food culture. Furthermore, the polysaccharides and carotenoids unique to seaweed are beginning to be used in new fields such as medicines and health foods.

[0003] Various fertilizers have been proposed for such seaweed. For example, Patent Document 1 discloses a fertilizer for seaweed cultivation that contains phytic acid as the main component, to which organic fertilizers such as sucrose fatty acid esters are added as needed. Patent Document 2 also discloses a seaweed cultivation system, and discloses ammonium chloride and sodium phosphate as added nutrients. Furthermore, although not related to seaweed, Patent Document 3 discloses a growth promoter for microalgae that contains at least one compound selected from oxo fatty acids, derivatives thereof, or salts thereof, or hydroxylated fatty acids, derivatives thereof, or salts thereof. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 60-21950 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-201480 [Patent Document 3] Japanese Patent Application Publication No. 2023-6709 Summary of the Invention [Problem to be solved by the invention]

[0005] However, according to the inventors' research, it has been found that the seaweed cultivation fertilizer described in Patent Document 1, which contains phytic acid as the main ingredient and to which organic fertilizers such as sucrose fatty acid esters have been added, does not result in sufficient seaweed growth. Furthermore, Patent Document 2 only discloses basic fertilizer components necessary for life support, such as nitrogen and phosphorus, and does not disclose compounds with growth-promoting effects. Furthermore, Patent Document 3 is a growth promoter for microalgae, but does not promote the growth of seaweed, which is a multicellular algae.

[0006] The present invention has been made in consideration of the above problems, and aims to provide an agent for promoting the growth of seaweed without adversely affecting the living tissue of seaweed, and a method for cultivating seaweed using the same. [Means for solving the problem]

[0007] The present invention relates to a seaweed growth promoter comprising at least one compound selected from the group consisting of oxo fatty acids or salts thereof, and hydroxylated fatty acids or salts thereof. The seaweed growth promoter preferably contains an oxo fatty acid or a salt thereof, and a hydroxylated fatty acid or a salt thereof. The at least one compound selected from the group consisting of oxo fatty acids or salts thereof and hydroxylated fatty acids or salts thereof is preferably an unsaturated fatty acid having 18 carbon atoms and not having an α-ketol structure. In the present invention, seaweed refers to a group of marine species of multicellular algae, and does not include microalgae, which are unicellular algae.

[0008] The oxo fatty acid is preferably 13-oxo-9,11-octadecadienoic acid or 9-oxo-10,12-octadecadienoic acid. The hydroxylated fatty acid is preferably 9,10,13-trihydroxy-11-octadecenoic acid or 9,12,13-trihydroxy-10-octadecenoic acid.

[0009] The seaweed growth promoter of the present invention preferably further contains an unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids) or a salt thereof. Moreover, the unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids) is preferably linoleic acid.

[0010] The seaweed is preferably in at least one form selected from sporophytes (thallus, discus, filamentous body) and male and female gametophytes (thallus, discus, filamentous body). The seaweed is preferably one or more of green algae, red algae, and brown algae.

[0011] A preferred method for cultivating seaweed involves cultivating seaweed in a culture solution containing the seaweed growth promoter of the present invention. A preferred method for cultivating seaweed involves cultivating seaweed in a culture solution containing the seaweed growth promoter of the present invention and a fertilizer. Furthermore, the concentration of the growth promoter of the present invention in the culture solution is preferably 0.0001 ppm to 10 ppm. [Effects of the Invention]

[0012] The seaweed growth promoter of the present invention can increase the area, length, and weight of seaweed thallus by simply adding it to seawater in a culture tank compared to when ordinary fertilizer is added. Furthermore, since oxo fatty acids and hydroxylated fatty acids are naturally occurring substances, they have no effect on the human body, and even if they remain in the seaweed, the cultured seaweed and extracts from the seaweed can be used for food, cosmetics, and pharmaceutical applications. In this invention, "promoting the growth of seaweed" refers to promoting an increase in the area, length, and weight of at least one of the forms of seaweed, i.e., sporophytes (thallus, discus, filament), and male and female gametophytes (thallus, discus, filament), as well as promoting an increase in the content of pigments involved in photosynthesis (e.g., chlorophyll, carotenoids, and phycobilins), and amino acids, sugars, fatty acids, lipids (e.g., fatty acid esters, etc.) involved in metabolism, etc. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a graph comparing the daily growth rates (%) of Example 1 and Comparative Example 1 using Enteromorpha japonica. Note that (1) in Example 1 represents the daily growth rate when PES culture medium-containing culture medium (1), (2) in Example 1 represents the daily growth rate when PES culture medium-containing culture medium (2), and (3) in Example 1 represents the daily growth rate when PES culture medium-containing culture medium (3) was used. [Figure 2] 2 is a graph comparing the daily growth rate (%) of Example 2 using P. taoyagisou with that of Comparative Example 2. Note that (1) in Example 2 is the daily growth rate when PES culture medium-containing culture medium (1), (2) in Example 2 is the daily growth rate when PES culture medium-containing culture medium (2), and (3) in Example 2 is the daily growth rate when PES culture medium-containing culture medium (3) were used. [Figure 3] 3 is a graph comparing the fucoxanthin content (mg / g dw) per gram of dry weight between Example 3, which used wakame female gametophytes, and Comparative Example 4. Note that mg / g dw refers to the fucoxanthin content (mg) per gram of dried sample. [Figure 4] 4 is a graph comparing the daily growth rate (%) of Example 4, which used wakame female gametophytes, with that of Comparative Example 5. The daily growth rate was calculated based on the dry weight of the wakame female gametophyte. DETAILED DESCRIPTION OF THE INVENTION

[0014] The seaweed growth promoter of the present invention contains at least one compound selected from the group consisting of oxo fatty acids or salts thereof, and hydroxylated fatty acids or salts thereof.

[0015] By adding oxo fatty acids or their salts or hydroxylated fatty acids or their salts to a seaweed culture tank, the area, length, and weight of the seaweed can be increased compared to when only fertilizer containing nitrogen, phosphorus, and potassium is added.In addition to the area, length, and weight of the seaweed, the content of amino acids, sugars, fatty acids, lipids (e.g., fatty acid esters), etc., which are involved in metabolism, can also be increased.

[0016] It is believed that the oxo fatty acids or salts thereof, and hydroxylated fatty acids or salts thereof of the present invention, when absorbed by seaweed, exert an effect within the seaweed body similar to the stress signal produced when seaweed is subjected to oxidative stress. In other words, it is hypothesized that the oxo fatty acids or salts thereof of the present invention act as a pseudo-stress signal, creating a state of pseudo-oxidative stress in the seaweed. Therefore, seaweed produces energy to respond to oxidative stress, but when not placed in a stressful environment, the produced energy becomes surplus and is stored within the seaweed. It is presumed that the stored energy contributes to the further growth of the seaweed itself. Hydroxylated fatty acids or salts thereof are more hydrophilic than oxo fatty acids or salts thereof, and are thought to improve permeability from the seaweed surface, thereby promoting the absorption of oxo fatty acids or salts thereof. The seaweed growth promoter of the present invention preferably contains both an oxo fatty acid or a salt thereof and a hydroxylated fatty acid or a salt thereof.

[0017] Specific examples of oxo fatty acids of the present invention include ketooctadecadienoic acids, such as, but not limited to, 9-oxo-10,12-octadecadienoic acid (9-oxoODA), 13-oxo-9,11-octadecadienoic acid (13-oxoODA), 5-oxo-6,8-octadecadienoic acid, 6-oxo-9,12-octadecadienoic acid, 8-oxo-9,12-octadecadienoic acid, 10-oxo-8,12-octadecadienoic acid, 11-oxo-9,12-octadecadienoic acid, 12-oxo-9,13-octadecadienoic acid, and 14-oxo-9,12-octadecadienoic acid. In particular, the oxo fatty acids and hydroxylated fatty acids are preferably unsaturated fatty acids having 18 carbon atoms and not having an α-ketol structure. Unsaturated fatty acids having 18 carbon atoms are involved in the metabolism of living organisms, have a high affinity with seaweed, and are also present in terrestrial plants and seaweed, thereby minimizing the environmental impact. Note that an α-ketol structure refers to a structure in which an OH group is bonded to the carbon atom adjacent to the carbonyl group. Fatty acids having an α-ketol structure are susceptible to oxidation and have poor stability, and are therefore not preferred as fatty acids for use in the present invention.

[0018] Oxo fatty acids are so-called rare fatty acids that are known to be produced as intermediates in the metabolism of unsaturated fatty acids. 13-oxo-9,11-octadecadienoic acid or 9-oxo-10,12-octadecadienoic acid or a salt thereof, used as an example of an oxo fatty acid or a salt thereof in the present invention, is a compound having 18 carbon atoms and a structure in which two double bonds form a conjugated system within the molecule. 13-oxo-9,11-octadecadienoic acid and 9-oxo-10,12-octadecadienoic acid are oxo fatty acids produced from the unsaturated fatty acid linoleic acid by enzymatic reaction or other means, and are among the rare fatty acids. 13-oxo-9,11-octadecadienoic acid and 9-oxo-10,12-octadecadienoic acid are known to exist naturally in plants such as tomatoes.

[0019] Hydroxylated fatty acids are fatty acids present in the body that are produced by the metabolism of linoleic acid by intestinal bacteria such as lactic acid bacteria. In the present invention, it is desirable to use 9,10,13-trihydroxy-11-octadecenoic acid or 9,12,13-trihydroxy-10-octadecenoic acid as the hydroxylated fatty acid.

[0020] Until now, it was not known that oxo fatty acids such as 13-oxo-9,11-octadecadienoic acid and 9-oxo-10,12-octadecadienoic acid or their salts, or hydroxylated fatty acids such as 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid, have the effect of promoting the growth of seaweed.

[0021] The seaweed growth promoter of the present invention may contain an oxo fatty acid or its salt, or a hydroxylated fatty acid or its salt, and the origin of the oxo fatty acid or its salt is not particularly limited. Specifically, the oxo fatty acid or its salt, or the hydroxylated fatty acid or its salt may be commercially available, or may be extracted and / or purified from plants such as tomatoes. Alternatively, the oxo fatty acid or its salt, or the hydroxylated fatty acid or its salt may be obtained by the action of an enzyme, such as a plant-derived enzyme, on a substrate such as an unsaturated fatty acid, as described above, or may be obtained by chemical synthesis. For example, the oxo fatty acid or its salt, or the hydroxylated fatty acid or its salt may be produced by enzymatic conversion of linoleic acid as a raw material using lipoxygenase (LOX) and / or a dehydrogenase, such as alcohol dehydrogenase (ADH), or by catalytic reaction using a metal catalyst. The oxo fatty acid or its salt obtained in this manner can be used for seaweed growth promotion at the desired concentration or after appropriate dilution, as needed.

[0022] It is known that oxo fatty acids and hydroxylated fatty acids exist as geometric isomers such as (E,E), (Z,E), (E,Z), and (Z,Z) and optical isomers such as R and S isomers. However, these geometric isomers, optical isomers, and other isomers have similar effects in seaweed growth promoters. Therefore, in the present invention, for example, 13-oxo-9,11-octadecadienoic acid and 9-oxo-10,12-octadecadienoic acid, which can be used as examples of oxo fatty acids or salts thereof, and 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid, which can be used as examples of hydroxylated fatty acids or salts thereof, are considered to include all of their isomers. In other words, regardless of the isomers of the oxo fatty acids and hydroxylated fatty acids contained in the seaweed growth promoter of the present invention, these isomers will have similar effects.

[0023] In the present invention, the oxo fatty acid may be (9Z,11E)-13-oxo-9,11-octadecadienoic acid and / or (10E,12Z)-9-oxo-10,12-octadecadienoic acid, and the hydroxylated fatty acid may be 9(S),10(S),13(S)-trihydroxy-11(E)-octadecenoic acid and / or 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid.

[0024] The seaweed growth promoter of the present invention may contain a desired concentration of oxo fatty acid or its salt, or hydroxylated fatty acid or its salt. For example, a mixture containing oxo fatty acid and hydroxylated fatty acid may be used as the oxo fatty acid or its salt, or hydroxylated fatty acid or its salt.

[0025] In the seaweed growth promoter of the present invention, the oxo fatty acid or hydroxylated fatty acid may be present in the form of a salt. Examples of salts include ammonium salts and metal salts. Metal salts that generate monovalent metal ions are preferred, and for example, sodium salts and potassium salts can be preferably used, although this is not limited thereto.

[0026] The seaweed growth promoter of the present invention is characterized by containing naturally occurring oxo fatty acids or their salts, or hydroxylated fatty acids or their salts, and therefore does not cause problems related to seawater or seabed soil contamination or toxicity. Furthermore, because naturally occurring oxo fatty acids or their salts, and hydroxylated fatty acids or their salts are harmless to the human body, even if these substances remain or accumulate in seaweed, the seaweed can be used for food or pharmaceutical purposes. In other words, by using the seaweed growth promoter of the present invention, seaweed can be cultivated safely, simply, and efficiently.

[0027] In the present invention, a specific example of the seaweed growth promoter of the present invention will be described as containing at least one compound selected from an oxo fatty acid or its salt and a hydroxylated fatty acid or its salt, and an unsaturated fatty acid (excluding oxo fatty acid and hydroxylated fatty acid). The unsaturated fatty acids used in the present invention (excluding oxo fatty acids and hydroxylated fatty acids) are not particularly limited. However, unsaturated fatty acids having approximately 10 to 20 carbon atoms are effective in promoting the absorption of the oxo fatty acids and hydroxylated fatty acids by seaweed. The cell membrane of seaweed is composed of lipids, and the inclusion of unsaturated fatty acids having approximately 10 to 20 carbon atoms facilitates the permeation of the oxo fatty acids and hydroxylated fatty acids through the cell membrane. For example, unsaturated fatty acids having 18 carbon atoms are preferred. Furthermore, the number of unsaturated bonds in the molecular structure of the unsaturated fatty acids used in the present invention is not limited, and they may be monounsaturated or polyunsaturated fatty acids. Examples of unsaturated fatty acids include, but are not limited to, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, and arachidonic acid. Furthermore, the unsaturated fatty acids may be in the form of free fatty acids or salts thereof (commonly usable salts, such as those exemplified above), and one or more of these may be selected. The unsaturated fatty acids used in the present invention may be commercially available products or may be synthesized from commercially available compounds. Furthermore, the unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids) may be one type of unsaturated fatty acid or two or more types.

[0028] Linoleic acid is the most suitable unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids) for use in the present invention. In the present invention, when mixing at least one compound selected from an oxo fatty acid or a salt thereof and a hydroxylated fatty acid or a salt thereof with an unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids), it is desirable to use a stabilizer (emulsifier) ​​to ensure that the at least one compound selected from the oxo fatty acid or a salt thereof and the hydroxylated fatty acid or a salt thereof, and the unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids) exist stably as an emulsion in water, in order to mix them uniformly. As the stabilizer (emulsifier), at least one selected from potassium carbonate and dipotassium hydrogen phosphate can be used.

[0029] When the seaweed growth promoter of the present invention contains unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids), it is preferable that the ratio of at least one compound selected from oxo fatty acids or their salts and hydroxylated fatty acids or their salts relative to the unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids) is 90% or less by weight. If the ratio of at least one compound selected from oxo fatty acids or their salts and hydroxylated fatty acids or their salts relative to the unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids) is greater than 90%, the content of unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids) in the seaweed growth promoter will be low, and the effect of promoting the absorption of oxo fatty acids and hydroxylated fatty acids by seaweed will not be sufficient. It is also preferable that the ratio of at least one compound selected from oxo fatty acids or their salts and hydroxylated fatty acids or their salts relative to the unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids) in the seaweed growth promoter be 10% or more by weight. If the ratio of at least one compound selected from oxo fatty acids or their salts and hydroxylated fatty acids or their salts to the unsaturated fatty acids (excluding oxo fatty acids and hydroxylated fatty acids) is less than 10%, the amount of active ingredient for the growth-promoting effect in the seaweed growth promoter will be small, and the seaweed growth-promoting effect may be insufficient.

[0030] The seaweed growth promoter of the present invention may contain at least one selected from amino acids, nucleic acids, and terpenes, as these substances have the effect of promoting seaweed growth. The amino acid is not particularly limited and may be appropriately selected from isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, histidine, tyrosine, cysteine, aspartic acid, asparagine, serine, glutamic acid, glutamine, proline, glycine, alanine, and arginine, or may be a mixture of two or more of these.

[0031] The nucleic acid may be at least one selected from a nucleic acid base, a nucleoside, a ribonucleoside, a deoxyribonucleoside, a ribonucleotide, and a deoxyribonucleotide. The nucleic acid is not particularly limited, and may be any of five common nucleic acid bases, i.e., adenine, guanine, thymine, cytosine, and uracil; five ribonucleosides in which ribose is bound to a nucleic acid base, i.e., adenosine, guanosine, 5-methyluridine, cytidine, and uridine; five deoxyribonucleosides in which deoxyribose is bound to five nucleic acid bases, i.e., deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine, and deoxyuridine; and 15 ribonucleotides in which one to three phosphates are ester-linked to five ribonucleosides (AMP (adenosine monophosphate), ADP (adenosine diphosphate), ATP (adenosine triphosphate), GMP (guanosine monophosphate), GDP (guanosine diphosphate), and GTP (guanosine triphosphate). phosphate), TMP (thymidylate / thymidine phosphate), TDP (thymidine diphosphate), TTP (thymidine triphosphate), CMP (cytidine monophosphate), CDP (cytidine diphosphate), CTP (cytidine triphosphate), UMP (uridine monophosphate), UDP (uridine diphosphate), UTP (uridine triphosphate)), or 15 types of deoxyribonucleotides in which the hydroxy group at the 2-position of the ribose of these ribonucleotides has been substituted with hydrogen (dAMP, dADP, dATP, dGMP, dGDP, dGTP, dTMP, dTDP, dTTP, dCMP, dCDP, dCTP, dUMP, dUDP, dUTP), or modified bases of these nucleic acid bases, or a mixture of two or more of these may be used.

[0032] Any terpene can be used, but monoterpenes, sesquiterpenes, diterpenes, and combinations thereof are preferred. Particularly preferred examples include monoterpenes such as α-pinene, β-pinene, silvestrene, and limonene, which are core substances of biohormones. Terpineol may also be included. Terpineol includes its isomers α-terpineol, β-terpineol, and γ-terpineol, with α-terpineol being more preferred. However, for example, commercially available terpineol may be a mixture of β-terpineol and γ-terpineol, with α-terpineol being the main component. In other words, as long as it primarily contains α-terpineol, the mixture of isomers can be used as is. Pine oil containing α-pinene as the main component can be used in the present invention.

[0033] Use of the seaweed growth promoter of the present invention may increase the carotenoid and polysaccharide contents in the seaweed. Carotenoids include fucoxanthin and siphonaxanthin, and polysaccharides include alginic acid and fucoidan.

[0034] The seaweed cultured and cultivated in a culture medium to which the seaweed growth promoter of the present invention has been added is in at least one form selected from sporophytes (thallus, discus, filamentous form) and male and female gametophytes (thallus, discus, filamentous form). Regardless of the form, the growth of the seaweed is promoted by culturing it in a culture medium to which the seaweed growth promoter of the present invention has been added.

[0035] The seaweed to which the seaweed growth promoter of the present invention can be applied is not particularly limited, but is preferably one or more of green algae, red algae, or brown algae. Examples of green algae include Enteromorpha spp., Hitotsugusa, Miru, and Caulerpa lentillifera; examples of red algae include Taoyagisou, Asakusa nori, Fukurofunori, Susabinori, and Tanshisai (Japanese name: Haitan Amanori); and examples of brown algae include Undaria pinnatifida, Mozuku seaweed, Hirome seaweed, Aowakame seaweed, Sagarame seaweed, Kayamonori seaweed, Makonbu seaweed, Narrow-skinned kelp, Ecklonia cava, and Kurome seaweed.

[0036] The seaweed growth promoter of the present invention may be added to a culture solution (e.g., filtered seawater, sterilized seawater, etc.) for culturing seaweed, or may be added to seaweed sporophytes (thallus, disc, filament), male and female gametophytes, etc. The seaweed (thallus, disc, filament) may be immersed in the seaweed growth promoter of the present invention and cultured in sterilized seawater. The culture medium may be natural seawater or artificial seawater that has been filtered and sterilized, or may be filtered seawater obtained by filtering natural seawater or sterilized seawater obtained by sterilizing natural seawater. Artificial seawater is prepared to contain mainly cations such as sodium ions, magnesium ions, potassium ions, and calcium ions, and anions such as chloride ions and sulfate ions. The salt concentration of the artificial seawater is preferably 1.0% by mass or more and 3.5% by mass or less. When using natural seawater, deep seawater may be used. Deep seawater is seawater found in the deep sea at depths of 200 meters or more. Because deep seawater has a high salt concentration, bacteria that hinder seaweed cultivation are unlikely to survive. Deep seawater is also free of artificial pollution, has low bacteria levels due to its low temperature, and is not exposed to sunlight, so there is no contamination by living phytoplankton. Deep seawater is also rich in nitrogen (N) from nitrates, phosphorus (P) from phosphates, and silicon (Si) from silicates, making it ideal for growing seaweed.

[0037] The seaweed growth promoter of the present invention may be contained in a porous structure or capsule, or impregnated in a sheet or the like and used as a sustained-release drug. The form of the seaweed growth promoter of the present invention is not particularly limited. For example, it may be in the form of a liquid or gel, or may be in the form of a solid (block, powder, granules, etc.). In the case of a liquid composition, it can be a concentrated type that can be used as is or diluted. The seaweed growth promoter of the present invention may be a powdered freeze-dried product obtained by freeze-drying an aqueous solution containing at least one compound selected from the group consisting of oxo fatty acids or salts thereof and hydroxylated fatty acids or salts thereof, or may be a pulverized or crushed freeze-dried product. The seaweed growth promoter of the present invention may contain additives or carriers such as excipients, binders, disintegrants, etc. that are suitable for application to seaweed, as needed.

[0038] The seaweed growth promoter of the present invention may be prepared by diluting it appropriately to a composition suitable for culturing the type of seaweed. Examples of additives include, but are not limited to, sugars, organic acids, inorganic acids, organic bases, inorganic bases, vitamins, amino acids, peptides, proteins, various ions (contained in natural seawater and artificial seawater), organic fertilizers, inorganic fertilizers, etc. The sugars include, but are not limited to, one or more sugars selected from glucose, galactose, fructose, maltose, sucrose, lactose, oligosaccharides, and sugar alcohols such as glycerol. Examples of organic acids include hydroxycarboxylic acids such as citric acid, gluconic acid, malic acid, heptonic acid, oxalic acid, malonic acid, lactic acid, tartaric acid, succinic acid, fumaric acid, maleic acid, adipic acid, and glutaric acid, polycarboxylic acids, and salts thereof, such as potassium salts, sodium salts, ammonium salts, alkanolamine salts, and aliphatic amine salts. Organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, and the like. Inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide. Examples of inorganic acids include potassium hydroxide, sodium hydroxide, and potassium carbonate. Vitamins include vitamin B12, thiamine (vitamin B1), and biotin (vitamin B7). Examples of organic fertilizers include oil cake, chicken manure, fish meal, rice bran, and wood ash. Inorganic fertilizers are fertilizers whose main components are minerals, such as nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, boron, zinc, molybdenum, copper, and nickel. It is particularly desirable to cultivate seaweed in seawater containing the seaweed growth promoter of the present invention and fertilizer components, such as nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, boron, zinc, molybdenum, copper, nickel, and vitamins.

[0039] In this case, the seaweed growth promoter is preferably used in conjunction with a fertilizer solution, preferably Provasoli's Enriched Seawater (PES). The PES culture medium has the following composition (see "Phycology Experiments and Practices," edited by Ariga Hirokatsu, Inoue Isao, Tanaka Jiro, Yokohama Yasutsugu, and Yoshida Tadao, Kodansha Scientific (2000) and JP 2009-201480 A).

[0040] (PES culture solution) Tris hydroxymethyl aminomethane, 5.0g NaN03, 3.5 g Sodium glucocorophosphate, 500mg Fe stock solution, 250ml P-2 metal mix, 250 ml Vitamine B12 stock solution (0.1mg / ml), 1.0ml Thiamine-HCl stock solution (1.0mg / ml), 5.0ml Biotine stock solution (0.1mg / ml), 0.5ml Distinctive water, 1000ml

[0041] (Fe stock solution) Na2-EDTA·2H2O, 330mg Fe(NH4)2(SO4)2·6H2O, 351 mg Distilled water, 500ml

[0042] (P-2 metal mix) Na2-EDTA·2H2O, 500mg H3BO3, 570mg FeCl3·6H2O, 24.5mg MnSO4·4H2O, 82.0 mg CoSO4·7H2O (4.8 mg / ml), 0.5 ml ZnSO4·7H2O, 11.0 mg Distilled water, 500ml

[0043] PES culture medium contains nitrogen, iron, manganese, cobalt, zinc, phosphorus, etc., making it an excellent culture medium for seaweed.

[0044] It is also possible to use PESI culture medium instead of PES culture medium. PESI culture medium has the following composition:

[0045] (PESI culture solution) Tris hydroxymethyl aminomethane, 5.0g NaN03, 3.5 g Sodium glucocorophosphate, 500mg Fe stock solution, 250ml P-2 metal mix, 250 ml Distinctive water, 300ml KI stock solution(0.1mg / ml) 10ml

[0046] The seaweed growth promoter of the present invention is added to seawater in a culture tank of a culture device. The culture tank may be equipped with one or more devices selected from a stirrer, a vibrator, a temperature controller, a pH regulator, a turbidity meter, a light controller, a device for measuring the concentration of specific gases such as O2 and CO2, and a pressure meter. Seaweed can be cultured by any suitable liquid culture method, such as batch culture, semi-batch culture (fed-batch culture), or continuous culture (perfusion culture). In the cultivation of seaweed in the present invention, artificial seawater or sterilized or disinfected seawater can be used. Sterilization or disinfection of seawater can be carried out by ultraviolet irradiation, blowing in ozone, or autoclaving the seawater.

[0047] In the present invention, seaweed is temporarily cultivated in an artificial environment. The artificial environment can be an environment in which the water temperature, light intensity, and sunlight duration are controlled. Any environment can be used as long as the light intensity, sunlight duration, and water temperature can be strictly controlled. For example, brown algae can be cultivated in a container in an incubator where the light intensity, sunlight duration, and temperature inside the device are regulated, or in an aquarium where the light intensity, sunlight duration, and water temperature can be controlled. In a typical cultivation environment, the light intensity is 5 to 2000 μmol / m 2 / s, but preferably 10 to 100 μmol / m 2 / s. The amount of light irradiation may be changed during cultivation. The water temperature may be between 5 and 30°C, but is preferably between 12 and 22°C. The daylight hours are preferably between 8 and 24 hours. Cultivation is usually carried out under a photoperiod with alternating light and dark periods, but light irradiation may also be continuous. The cultivation period may be between 1 and 40 days, but is preferably between 5 and 10 days. A minimum of 5 days is sufficient.

[0048] The culture tank containing seawater or the like used for the above-mentioned culture can be appropriately agitated or aerated with CO2, O2, or the like, as needed. These conditions can be determined by appropriate experiments to obtain optimal results. The container used for the culture can be selected from those commonly used in the art for seaweed cultivation, or it can be a modified version of a known cultivation device, such as the simple culture container disclosed in JP 2002-101867 A or a large culture container with similar functions.

[0049] In the present invention, the seaweed cultivated by the above-mentioned cultivation method can be used as it is or by extracting carotenoids and polysaccharides from the seaweed, and is considered to be useful as food or as raw materials for cosmetics, pharmaceuticals, supplements, etc. [Example]

[0050] The present invention will be described based on examples, but the present invention is not limited to only the examples.

[0051] (Preparation of PES culture medium) Add the following ingredients to approximately 300ml of distilled water: Tris, NaNO3, disodium β-glycerophosphate (Na2-glycerophosphate), Fe stock solution, P-II metal mix, and Vitamin B 12 The specified amounts of stock solution, Thiamine-HCl stock solution, and Biotin stock solution were added in that order and dissolved thoroughly. The pH of the solution was then adjusted to 7.8, and the volume was adjusted to 1000 mL with distilled water. The resulting PES medium was dispensed into containers and sterilized by autoclaving (121°C, 20 minutes). The sterilized PES medium was then stored refrigerated at 4°C.

[0052] The composition of PES medium is: 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris(hydroxymethyl)aminomethane; Tris), 5.0g; NaNO3, 3.5g; Na2-glycerophosphate, 500mg; Fe stock solution (see composition below), 250 mL; P-II metal mix (see composition below), 250 mL; Vitamin B 12 stock solution (0.1mg / mL), 1.0mL; Thiamine-HCl stock solution (1.0mg / mL), 5.0ml; Add 0.5 mL of biotin stock solution (0.1 mg / mL) and dilute with distilled water to a total volume of 1000 mL (pH 7.8).

[0053] The Fe stock solution consisted of 330 mg of Na2-EDTA·2H2O and 351 mg of Fe(NH4)2(SO4)2·6H2O, which was then diluted with distilled water to a total volume of 500 mL (Fe:EDTA molar ratio = 1:1). The solution was stored refrigerated at 4°C.

[0054] The composition of the P-II metal mix is: Na2-EDTA·2H2O, 500mg; H3BO3, 570mg; FeCl3·6H2O, 24.5 mg; MnSO4·4H2O, 82.0 mg; CoSO4·7H2O stock solution (4.8 mg / mL), 0.5ml; ZnSO4·7H2O, 11.0 mg Add the above ingredients to distilled water in order, and finally add distilled water to make a 500mL solution. Store in a refrigerator at 4℃.

[0055] (Preparation of seaweed growth promoter) A test solution was prepared by adding 580 g of 90% pure linoleic acid (manufactured by NOF Corporation) as a fatty acid-containing raw material, 216 g of potassium carbonate (manufactured by Fujifilm Wako Co., Ltd.), 280 g of dipotassium hydrogen phosphate (manufactured by Fujifilm Wako Co., Ltd.), and 13,000 mL of distilled water, and the pH of the test solution was 9.0.

[0056] 40 mg of lipoxygenase (Nacalai Tesque, soybean-derived) was added to the test solution, and the mixture was reacted at 15°C for 3 hours while being aerated and stirred with oxygen. The reaction mixture was then placed in a water bath at 90°C for 90 minutes. The resulting reaction solution was designated as Solution A. To 6500 mL of Solution A, 35 mL of phosphoric acid (manufactured by Fujifilm Wako Co., Ltd.) was added to adjust the pH to 7.0. This solution was aerated with oxygen and reacted at 50°C for 22 hours while stirring, and then the reaction mixture was placed in a water bath at 90°C for 2 hours. The resulting reaction solution was designated Solution B.

[0057] The entire amount of Solution B obtained above was mixed with the remaining amount of Solution A that was not used to prepare Solution B. The resulting mixture was used as a standard substance for Cayman Chemical's 13-oxoODA (13-oxo-9,11-octadecadienoic acid), 9-oxoODA (9-oxo-10,12-octadecadienoic acid), and Larodan Fine Chemicals' 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. acid) and quantified by liquid chromatography mass spectrometry (LC-MS) using MS2 spectral analysis.

[0058] Ketooctadecadienoic acid (13-oxoODA, 9-oxoODA) was quantified at a UV detection wavelength of 272 nm, and trihydroxyoctadecenoic acid was quantified at a UV detection wavelength of 210 nm using the absolute calibration curve method. The combined yield of 13-oxoODA and its isomers, including the (E,E) and (E,Z) isomers, was 3.70%. The yield of 9-oxoODA was 1.66%. The combined yield of 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid was 1.22% (peaks unseparable by LC-MS), and the recovery of linoleic acid was 84.1%.

[0059] The resulting mixture of 23.7 mL of solution A and B was diluted to a constant volume of 2000 mL with ion-exchanged water to prepare a seaweed growth promoter solution. The seaweed growth promoter solution contained 17.6 ppm of 13-oxoODA, 7.9 ppm of 9-oxoODA, and 5.8 ppm of 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid.

[0060] (Preparation of culture medium for seaweed cultivation) Next, the seaweed growth promoter prepared above was diluted 10 times (culture medium (1)), 100 times (culture medium (2)), and 1000 times (culture medium (3)) with sterilized seawater to prepare three levels of PES culture medium-containing culture medium. 36 ml of PES culture medium was added to 1800 ml of culture medium (1) to prepare PES culture medium-containing culture medium (1) (10-fold diluted solution). 36 ml of PES culture medium was added to 1800 ml of culture medium (2) to prepare PES culture medium-containing culture medium (2) (100-fold diluted solution). 40 ml of PES culture medium was added to 2000 ml of culture medium (3) to prepare PES culture medium-containing culture medium (3) (1000-fold diluted solution).

[0061] The concentration of 13-oxoODA in the PES culture medium-containing culture medium (1) (10-fold diluted solution) was 1.725 ppm, the concentration of 9-oxoODA was 0.775 ppm, and the combined concentration of 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid was 0.569 ppm. The concentration of 13-oxoODA in the PES culture medium-containing culture medium (2) (100-fold diluted solution) was 0.1725 ppm, the concentration of 9-oxoODA was 0.0775 ppm, and the combined concentration of 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid was 0.0569 ppm. The concentration of 13-oxoODA in the PES culture medium-containing culture medium (3) (1000-fold diluted solution) was 0.01725 ppm, the concentration of 9-oxoODA was 0.00775 ppm, and the combined concentration of 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid was 0.00569 ppm.

[0062] Example 1 (Seaweed culture test) (1-1) Natural seawater was heated and pressurized in an autoclave at 121°C, 2 atmospheres, and 20 minutes. 3 liters of sterile seawater was obtained.

[0063] (1-2) Test samples of Green Amorphophallus were prepared according to Patent No. 6861979 and Algal Resources (2019) 12:11-20, “Green Amorphophallus seed production method by adding algal extract to shredded cells” (authors: Ikuya Kita, Akinori Dan, Tatsuo Hamano, Minoru Saito, Naohiro Oka), (https: / / www.jstage.jst.go.jp / article / jsap / 12 / 1 / 12_11 / _pdf / -char / ja).

[0064] (i) Preparation of algal extract The strain used was a Enteromorpha spp. strain, which has an asexual life cycle and produces two-flagellated germ cells, collected near the mouth of the Yoshino River in Tokushima Prefecture and subcultured at the Bioinnovation Laboratory at Tokushima University. We used Enteromorpha spp. (200 wet weight) with 2000 ml of sterilized seawater at 32 psu (practical salinity units, 32 g salinity per kg of seawater). The mixture was ground in a household blender at 22,500 rpm for 1 minute, frozen at -30°C, thawed in a refrigerator at 5°C after 24 hours, and then filtered through a 50 μm filter to separate the filtrate from the residue. The residue was placed in a 9 cm glass mortar and 50 g of sea sand (300–600 μm, 30–50 mesh, Fujifilm Wako Pure Chemical Corporation) was added and ground. The mixture was then suspended in 2000 ml of 32 psu sterilized seawater, placed in a 3 L Erlenmeyer flask, and shaken at 130 rpm for 24 hours using a reciprocating shaker (Multi Shaker MMS, Tokyo Rika Kikai). The shaken solution was then suction-filtered using membrane filters (Cellulose Acetate Filter, Advantec) with pore sizes of 3, 0.45, and 0.2 μm, in that order. The filtrate (algal extract) was stored at −30°C.

[0065] (ii) Preparation of algal fragments The strain used was a Enteromorpha spp. strain, which was collected near the mouth of the Yoshino River in Tokushima Prefecture and subcultured at the Bioinnovation Laboratory at Tokushima University and has an asexual life cycle that produces two-flagellated germ cells. The mother algae was preserved and cultured for one month at 20°C in a culture medium prepared by adding 1.7% v / v of a commercially available algal culture medium (Porphyran Conco, product name, manufactured by Daiichi Seimo Co., Ltd.) to sterilized seawater. After preservation culture, 8 g of algal bodies were weighed out, shredded in a household blender for approximately 60 seconds, and filtered through a 50 μm mesh to prepare algal fragments.

[0066] (iii) Seedling production A culture medium was prepared by adding the above algal extract to sterilized seawater at a ratio of 25 v / v%. 1 g of the above Enteromorpha spp. fragments was added and suspended in 100 ml of this culture medium. 30 ml of the culture medium with the suspended algal fragments was dispensed into 90 mm diameter Petri dishes. Static culture was carried out for 3 days in an incubator at 25°C under conditions of a light intensity of 4500 lux and a photoperiod of 12 L:12 D. This confirmed that the Enteromorpha spp. fragments did not mature, but new shoots formed from the somatic cells.

[0067] (iv) Seedling cultivation A culture solution was prepared by adding 0.05 v / v% of a commercially available algae culture solution (manufactured by Daiichi Seimo Co., Ltd., product name: Porphyran Conco) to sterilized seawater. 50 μl of this culture solution was placed in a 100 ml culture vessel, and the above-mentioned green laver seedlings (10 mg wet weight) were suspended in it. The culture was carried out for 7 days under conditions of a temperature of 20°C, a light intensity of 8000 lux, and a photoperiod of 12 L:12 D. The algae were grown until they reached a length of 2 mm or more, and green laver with radially elongated thallus was produced. This green laver was sliced ​​into leaf lengths of approximately 2 mm to prepare green laver test samples.

[0068] (1-3) A single 2mm specimen of Enteromorpha spp., prepared in (1-2), was placed in all 24 wells of a 24-well multiwell plate together with the PES culture medium (1). Each plate in the 24-well multiwell plate was used as one seawater condition (n=24). The culture conditions were a water temperature of 20°C and a light intensity (photosynthetic photon flux density, PPFD) of 50 μmol m -2 ·s -1 The plates were cultured statically under a 12L:12D (Light:Dark) photoperiod and cool white fluorescent light. The sides of the multiwell plates were covered with vinyl tape to prevent evaporation of the seawater. The culture period for Enteromorpha spp. was set to one week, taking into account the growth rate of the seaweed. Furthermore, the PES culture medium-containing culture medium (2) and the PES culture medium-containing culture medium (3) were also subjected to the culture test in the same manner as the PES culture medium-containing culture medium (1).

[0069] (Comparative Example 1) A culture test for Enteromorpha spp. was carried out in the same manner as in Example 1, except that instead of the PES culture-containing culture solutions (1) to (3), a PES culture-containing sterilized seawater was used as the culture solution for seaweed cultivation, which was a mixture of PES culture solution and sterilized seawater in a weight ratio of PES culture solution:sterilized seawater = 1:50.

[0070] Example 2 (2-1) Natural seawater was heated and pressurized in an autoclave at 121°C, 2 atmospheres, and 20 minutes. 3 liters of sterile seawater was obtained.

[0071] (2-2) Tetraspores were isolated with a pipette from P. thunbergii collected from the Konarutou Strait in Tokushima Prefecture and transferred to a sterile glass slide with a few drops of the sterilized seawater prepared in (1). The slide with the tetraspores was transferred to a sterile petri dish (90 mm diameter, polystyrene) with a lid containing 40 ml of PES culture medium and cultured at a temperature of 20°C, an illumination intensity of 2000 lux, and a photoperiod of 12L:12D (light:dark). After 30 days of culture, the P. thunbergii tetraspores grew from discs to thallus with a diameter of 2 mm. These thallus were used as the P. thunbergii test sample.

[0072] (2-3) A single 2mm specimen of the test sample, prepared in (2-2), was placed in all 24 wells of a 24-well multiwell plate together with the PES culture medium (1). One plate was used for one seawater condition (n=24). The culture conditions were a water temperature of 20°C and a light intensity (photosynthetic photon density, ppdf) of 50 μmol m -2 ·s -1 The plates were cultured statically under a 12L:12D (Light:Dark) photoperiod and cool white fluorescent light. The sides of the multiwell plates were covered with vinyl tape to prevent evaporation of the seawater. The culture period for Taoyagisou was set at one week, taking into account the growth rate of the seaweed. Furthermore, the PES culture medium-containing culture medium (2) and the PES culture medium-containing culture medium (3) were also subjected to the culture test in the same manner as the PES culture medium-containing culture medium (1).

[0073] (Comparative Example 2) A cultivation test of Taoyagisou was carried out in the same manner as in Example 2, except that instead of the PES culture medium-containing culture solutions (1) to (3), PES culture medium and sterilized seawater were mixed in a weight ratio of PES culture medium:sterilized seawater = 1:50 to form the culture solution for seaweed cultivation.

[0074] (Comparative Example 3) (3-1) 30 g of a 50% phytic acid solution and 1 mg of sucrose fatty acid ester were mixed and adjusted to 100 g with ion-exchanged water to prepare a stock solution. (3-2) The stock solution was diluted 100 times with seawater. (3-3) 36 ml of PES culture medium was added to 1800 ml of the diluted solution of (3-2) to prepare a PES culture medium-containing culture medium. (3-4) A culture test of Enteromorpha spp. was carried out in the same manner as in Example 1, except that the PES culture medium-containing culture medium prepared in (3-3) was used as the culture medium for seaweed cultivation. Also, a culture test of Enteromorpha spp. was carried out in the same manner as in Example 2, except that the PES culture medium-containing culture medium prepared in (3-3) was used as the culture medium for seaweed cultivation.

[0075] (Evaluation method) The growth promoting effect of Examples 1 and 2 and Comparative Examples 1 to 3 was confirmed by the following method. The length of the thallus was measured for the cultivated Enteromorpha japonica, and the area of ​​the thallus (disc) for the cultivated Enteromorpha japonica was measured using an inverted microscope (Olympus, product name: IX73) at 40x magnification. The area of ​​the thallus (disc) was measured using a stereo microscope (Olympus, product name: SZX16) at 10x magnification. The length of the thallus and the area of ​​the thallus (disc) were measured using the image processing software "cellSens" that comes with these microscopes.

[0076] The measurement results were evaluated using the daily growth rate DGR (%) expressed by the following formula (1). DGR(%)={(Wt / W0) 1 / t -1}×100 (1) In the above formula (1), Wt: value after t days, W0: value immediately after the start of the experiment, t: number of days of culture For Wt and W0, the area of ​​the thallus or gametophyte of the cultured seaweed, the length of the thallus or gametophyte, or the weight of the thallus or gametophyte can be used. However, the dimensions of Wt and W0 must match.

[0077] (result) As can be seen from Figures 1 and 2, application of a seaweed growth promoter containing 13-oxo-9,11-octadecadienoic acid, 9-oxo-10,12-octadecadienoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid increased the area and length of the seaweed thallus. Furthermore, no growth was observed at all in the Enteromorpha spp. of Comparative Example 3. Furthermore, the Taoyagisou plant of Comparative Example 3 withered and died.

[0078] Example 3 (1) From Naruto wakame seaweed collected near the mouth of the Yoshino River, female gametophytes were cultured and cultivated according to the procedure described below in "(I) Cultivation of seaweed gametophytes."

[0079] (I) Cultivation of seaweed gametophytes The gametophytes that develop from wakame zoospores are separated into one male and one female, grown, mechanically shredded, attached to thread, and fertilized on the thread. The wakame young leaves that develop from these are grown in indoor cultures and in natural seawater. Sporophyll (mekabu) is obtained from the grown wakame thallus (adult).

[0080] 1. Preservation of sporophyll 1) Place the sporophytes in a plastic bag or container and store in a cool, dark place at 15-20°C. If the temperature is lower than 15°C, zoospores will not be released easily when the sporophytes are returned to seawater. If the sporophytes are removed from the seawater and stored in a cool, dark place, zoospores can be released for 2-3 days.

[0081] 2.Collecting zoospores 1) The temperature of the room in which the zoospores are released should be 15-20°C. High temperatures are not recommended as they shorten the swimming time of the zoospores. 2) Cut the sporophyll into pieces about 3-4 cm square. The part closest to the rhizoid is best for releasing spores, but cut off the part of the sporophyll surface that is as clean as possible. Lightly wipe off any dirt from the cut leaf pieces with absorbent paper or similar.

[0082] 3) Prepare three beakers containing 100 mL of sterilized seawater, wash the leaf pieces in turn, and then place them in a petri dish containing 50 mL of sterilized seawater. 4) Place the dish containing the leaf fragment on the stage of a stereomicroscope and shine light from above to release the zoospores. Adjusting the stereomicroscope to a dark field setting makes it easier to observe the release of zoospores. After 10 minutes of illumination with an optical fiber, etc., the zoospores will be sufficiently released.

[0083] 5) Prepare a capillary tube. A capillary tube can be prepared by heating and stretching a hematocrypt tube, or by stretching the tip of a Pasteur pipette until it is sufficiently elongated. 6) Prepare a petri dish filled with 50 mL of PES culture medium.

[0084] 7) Under a stereomicroscope, aspirate an appropriate amount of zoospores and drop them into a petri dish. When aspirating, be careful not to let the capillary tube touch the bottom of the petri dish or the leaf fragment (this is often the case when aspirating diatoms). After dropping, shake the petri dish thoroughly by hand to ensure a uniform zoospore density. 8) Prepare about four different types of petri dishes, each with a different amount of zoospore fluid. If the amount of zoospores is too large, the gametophyte density will be high, and the gametophytes will be too close to each other, making them difficult to isolate.

[0085] 9) After collecting the zoospores, the petri dishes should be cultured at 10-30°C with a 14-12 hour light period (1000-1500 lux). Care must be taken to maintain consistent culture conditions, as gametophytes are subject to large temperature fluctuations and males and females will look similar, making them difficult to distinguish, at high temperatures and high light levels. 10) After two weeks, the gametophytes reach a size where they can be sexed. Because there is a risk of fertilization, sex the gametophytes and isolate them as soon as possible.

[0086] 11) At this stage, there is little contamination with diatoms, but if diatoms do appear, discard the petri dish. If it is not possible to discard the dish, germanium dioxide can be used to suppress diatom growth, allowing you to isolate gametophytes that are not contaminated by diatoms.

[0087] 3. Isolation of Male and Female Gametophytes 1) Place the petri dish containing the gametophytes on the stage of an inverted microscope and search for female gametophytes suitable for isolation. Isolate those that are sufficiently separated from each other and clearly sexed. Attach a tube to a Pasteur pipette, separate the female gametophytes from the petri dish, and aspirate them. Place each aspirated female gametophyte individually into a microplate filled with PES medium. 2) Culture for one month at 10-30°C with a 14-12 hour light period (1500-2000 lux).

[0088] 4. Preservation of female gametophytes 1) After culturing in a microplate, remove the female gametophyte. Usually, the female gametophyte is large enough to be seen with the naked eye, so pick it up from the microplate with ophthalmic tweezers and remove it. If it has not grown to a sufficient size, you can aspirate it using a Pasteur pipette under an inverted microscope. 2) For storage, place in a screw-cap test tube and store at 10-30°C with 14 hours of light (1000-1500 lux). After storage, the medium is changed with PES medium once every two months.

[0089] (2) 1 g (ww) of the above-mentioned wakame female gametophyte (wet weight) was inoculated into 1 L flasks filled with PES-containing culture medium (1) and PES-containing culture medium (2), respectively. Five flasks were used for each culture medium (n = 5). The culture conditions were a water temperature of 20°C and a light intensity of 50 ppfd (μmol m -2 ·s -1 The incubation period was 1 week, with a 12L:12D (Light:Dark) photoperiod and cool white fluorescent light.

[0090] The sample of wakame female gametophyte cultured in PES culture medium-containing culture medium (1) was designated as sample (1), and the sample of wakame female gametophyte cultured in PES culture medium-containing culture medium (2) was designated as sample (2).

[0091] Comparative Example 4 A culture test of wakame female gametophytes was carried out in the same manner as in Example 3, except that the culture medium for seaweed cultivation was a mixture of PES culture medium and sterilized seawater at a weight ratio of PES culture medium:sterilized seawater = 1:50 instead of PES culture medium-containing culture medium (1) and (2). The sample of wakame female gametophytes cultured in the PES culture medium-containing sterilized seawater was designated as sample (3).

[0092] (Evaluation method) The growth-promoting effect was confirmed by measuring the weight of fucoxanthin per gram of dry weight of wakame female gametophyte. [Dry weight] The wakame female gametophytes of Samples (1) and (2) of Example 3 and Sample (3) of Comparative Example 4 were dehydrated from seawater and placed in a zip-top plastic bag in each flask and frozen at -20°C. After freezing, they were dried in a freeze-dryer and their dry weights (dw) were measured.

[0093] [BG extract] 10 mg of each of Sample (1) and Sample (2) of Example 3 and Sample (3) of Comparative Example 4 was suspended in 1 mL of distilled water and stirred at 200 rpm for 1 hour at 25°C using a rotator (RT-5N, manufactured by TAITEC). The solution was removed from the 2 mL tubes containing the stirred female gametophyte solutions of Sample (1), Sample (2), and Sample (3) using a micropipette, and the remaining female gametophytes were removed and thoroughly drained. After that, 10 mg of each was suspended in 1 mL of 100% butylene glycol (1,3-butylene glycol, hereafter referred to as "BG") and stirred at 8000 rpm for 1 hour in a rotator (TAITEC, RT-5N) to prepare BG extracts. The extract from Sample (1) was designated BG extract (1), the extract from Sample (2) was designated BG extract (2), and the extract from Sample (3) was designated BG extract (3).

[0094] [Fucoxanthin concentration measurement] The following liquid chromatography equipment was prepared for detecting fucoxanthin. <Liquid chromatography equipment> Column: diameter 3.0 mm x length 150 mm, packing material used was Kanto Chemical Co., Ltd. 25437-96 RP-18GP 150-3, a packing material for high performance liquid chromatography with a particle size of 5.0 μm. Detector: Absorbance was measured using a spectrophotometer (Shimadzu UV-1800) at a wavelength of 450 nm. Developing solvent A: 10% acetonitrile (a 1:9 mixture of LC / MS grade acetonitrile (Kanto Chemical Co., Ltd. 01033-76) and 0.05 wt% formic acid aqueous solution). Developing solvent B: 80% acetonitrile (a 8:2 mixture of LC / MS grade acetonitrile (Kanto Chemical Co., Ltd. 01033-76) and 0.05 wt% formic acid aqueous solution). Developing solvent C: 100% acetonitrile. Development time gradient: Solvent A (0 min) → Solvent B (1 min) → Solvent C (10 min) → Solvent C. (15 min) → Solvent A (15.1 min) → Solvent A (20 min) Solution flow rate: 0.5 mL / min Injection volume: 5μL Column temperature: 40℃ Sample chamber temperature: 15℃ Liquid pump: Shimadzu Corporation LC10ADVP

[0095] A 0.6589 mg fucoxanthin standard (Fujifilm Wako Pure Chemical Industries, Ltd.) was weighed, added to 1 L of ethanol, and stirred thoroughly to prepare a 1 mM fucoxanthin solution. 1 mL of this solution was further diluted 10-fold with ethanol to prepare a 0.1 mM (100 μM) standard solution. This standard solution was used to confirm the retention time of fucoxanthin in liquid chromatography. The 100 μM fucoxanthin standard solution was also diluted 2-fold (50 μM), 3.33-fold (30 μM), 10-fold (10 μM), and 33.3-fold (3 μM) with ethanol. The standard solution and diluted solution were then used to create a calibration curve for fucoxanthin concentration in liquid chromatography.

[0096] The calibration curve is expressed as y = 110489x + 22725 (x is the fucoxanthin concentration, and y is the peak area). Fucoxanthin at a concentration of 1 M was analyzed using a liquid chromatography device, and it was confirmed that the peak area and fucoxanthin concentration appearing on the chart matched the calibration curve.

[0097] The fucoxanthin concentrations of BG extract (1), BG extract (2), and BG extract (3) were measured. From the results of these concentration measurements, the fucoxanthin content (mg) in BG extract (1), BG extract (2), and BG extract (3) was calculated. Furthermore, the fucoxanthin content in each sample was divided by the dry weight of the wakame female gametophyte in sample (1), sample (2), and sample (3) to calculate the weight of fucoxanthin per gram of dry weight of wakame female gametophyte (FX amount) [mg / g dw]. The results are shown in Figure 3. Note that because n = 5 for each of sample (1), sample (2), and sample (3), the average of the five values ​​for each sample is shown in Figure 3.

[0098] As can be seen from Figure 3, by applying a seaweed growth promoter containing 13-oxo-9,11-octadecadienoic acid, 9-oxo-10,12-octadecadienoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid, the content of the carotenoid fucoxanthin in the seaweed increases, which is thought to have the effect of accelerating photosynthesis, and therefore has the effect of greatly promoting the growth of the seaweed.

[0099] Example 4 (1) 1 g (wet weight) of wakame female gametophytes obtained in the same manner as in Example 3(1) was inoculated into 1 L flasks filled with PES-containing culture solutions (1) to (3), respectively. Five flasks were used for each culture solution (n=5). The culture conditions were a water temperature of 25°C and a light intensity of 50 ppfd (μmol m -2 ·s -1 The incubation period was 1 week, with a 12L:12D (Light:Dark) photoperiod and cool white fluorescent light. (2) The wet weight of the wakame female gametophytes was measured at the start and end of the culture. To measure the wet weight, the wakame female gametophytes were collected from each flask, lightly absorbed with Kimtowel (registered trademark), and then the wet weight was measured. After the culture was completed, the wakame female gametophytes were wiped to remove seawater, placed in a zip-top plastic bag for each flask, and frozen at -50°C. After freezing, they were dehydrated in a freeze-dryer, and the dry weight (Wt) was measured.

[0100] Furthermore, when the weight of the wakame female gametophyte after freeze-drying that was not cultured was compared with the wet weight before freeze-drying, it was found that a wet weight of 1 g ww of wakame female gametophyte corresponds to a dry weight after freeze-drying of 0.3 g dw. Using this proportional equation between wet weight and dry weight, the dry weight (W0) of the wakame female gametophyte at the start of culture was calculated from the wet weight of the wakame female gametophyte at the start of culture.

[0101] For each flask, the daily growth rate (DGR (%)) for one week (t = 7) was calculated using the dry weight (W0) of the wakame female gametophyte at the start of culture (Wt) and the dry weight (Wt) of the wakame female gametophyte at the end of culture (t = 7) using the above formula (1), and the results are shown in Figure 4. The wakame female gametophyte samples cultured in PES culture medium-containing culture solutions (1) to (3) are referred to as samples (1) to (3), respectively.

[0102] (Comparative Example 5) A culture test of wakame female gametophytes was carried out in the same manner as in Example 4, except that instead of the PES culture medium-containing culture solutions (1) to (3), a PES culture medium containing sterilized seawater was used as the culture medium for seaweed cultivation, which was a mixture of PES culture medium and sterilized seawater in a weight ratio of PES culture medium:sterilized seawater = 1:50.The daily growth rate (%) was calculated and is shown in Figure 4. The wakame female gametophyte sample cultured in sterilized seawater containing PES culture medium is designated as sample (4).

[0103] As can be seen from Figure 4, by applying a seaweed growth promoter containing 13-oxo-9,11-octadecadienoic acid, 9-oxo-10,12-octadecadienoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid, the daily growth rate of wakame female gametophytes increased, and the effect of greatly promoting seaweed growth was achieved.

Claims

1. A seaweed growth promoter comprising at least one compound selected from the group consisting of oxo fatty acids or salts thereof and hydroxylated fatty acids or salts thereof.

2. The seaweed growth promoter according to claim 1, comprising an oxo fatty acid or a salt thereof and a hydroxylated fatty acid or a salt thereof.

3. The seaweed growth promoter according to claim 1, wherein the at least one compound selected from the group consisting of oxo fatty acids or salts thereof and hydroxylated fatty acids or salts thereof is an unsaturated fatty acid having 18 carbon atoms and not having an α-ketol structure.

4. 4. The seaweed growth promoter according to claim 3, wherein the oxo fatty acid is at least one selected from the group consisting of 13-oxo-9,11-octadecadienoic acid and 9-oxo-10,12-octadecadienoic acid.

5. 4. The seaweed growth promoter according to claim 3, wherein the hydroxylated fatty acid is at least one selected from the group consisting of 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid.

6. The seaweed growth promoter according to claim 1, further comprising an unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids) or a salt thereof.

7. 2. The seaweed growth promoter according to claim 1, wherein the unsaturated fatty acid (excluding oxo fatty acids and hydroxylated fatty acids) is linoleic acid.

8. The seaweed growth promoter according to claim 1, wherein the seaweed is in at least one form selected from sporophytes (thallus, discus, filamentous body) and male and female gametophytes (thallus, discus, filamentous body).

9. The seaweed growth promoter according to claim 1, wherein the seaweed is one or more of green algae, red algae, and brown algae.

10. A method for cultivating seaweed, comprising cultivating seaweed in a culture solution containing the seaweed growth promoter according to any one of claims 1 to 9.

11. A method for cultivating seaweed, comprising cultivating seaweed in a culture solution containing the seaweed growth promoter according to any one of claims 1 to 9 and a fertilizer.

12. A seaweed cultivation method comprising cultivating seaweed in a culture solution having a concentration of the seaweed growth promoter according to any one of claims 1 to 9 of 0.0001 ppm to 10 ppm.

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