JP2017153437A - Feed organisms for fishery organisms and culture methods for fishery organisms - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide feed organisms for fishery organisms and culture methods for fishery organisms.SOLUTION: Feed organisms such as Rotifera and Artemia reinforced in nutrition with sterol-containing algal bodies are fed to fishery organisms such as fish, and fishery organisms are cultured.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a feed organism for aquatic products and a method for culturing aquatic products.

  Zooplanktons such as rotifers and artemia are used as biological feed for seedling production of aquatic products such as fish and crustaceans. As rotifers, for example, those cultured by feeding algae such as Nannochloropsis algae and yeasts such as Saccharomyces cerevisiae are used as biological feed. As Artemia, for example, larvae obtained by hatching dormant eggs are used as biological feed. However, rotifers and artemia obtained in this way are used as biological feed for aquatic products in that they have a low content of highly unsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Has a nutritional deficiency. Therefore, rotifers and artemia are generally fed to aquatic products after enhancing nutrition, specifically, for example, after increasing the content of highly unsaturated fatty acids such as DHA and EPA.

  An object of the present invention is to provide a feed organism for aquatic products and a method for culturing aquatic products.

  As a result of intensive studies, the present inventor has found that when breeding aquatic products such as clownfish, feeding the rotifer and artemia enriched with sterol-containing alga bodies improves the breeding performance and completes the present invention. I let you.

That is, the present invention can be exemplified as follows.
[1]
A fortifying agent for rotifer or artemia containing a sterol-containing algal body.
[2]
The algae bodies are AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM
P-22290), and derivatives thereof, and the above-mentioned nutrient fortifier, which is a cell of an algae selected from a group of Aurantiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5.
[3]
The nutrition enhancer, wherein the sterol is cholesterol.
[4]
A method for producing a feed organism,
A method comprising incorporating a sterol-containing alga into a rotifer or artemia.
[5]
The algae bodies are AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM
P-22290), and derivatives thereof, and an algal cell selected from Aurantiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5.
[6]
The method, wherein the sterol is cholesterol.
[7]
A prey organism,
A feed organism that is a rotifer or artemia containing a sterol-containing algal body.
[8]
The algae bodies are AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM
P-22290), and derivatives thereof, and the bait organism selected from algae cells selected from Aurantiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5.
[9]
The feed organism, wherein the sterol is cholesterol.
[10]
The feed organism comprising cholesterol at a concentration of 0.02% (w / w) or more as a weight relative to the dry weight of the feed organism.
[11]
Furthermore, the said feed organism containing docosahexaenoic acid and / or eicosapentaenoic acid.
[12]
Said feed organism, which is a feed organism for aquatic products.
[13]
The feed organism, wherein the aquatic product is a fish or a crustacean.
[14]
A method for cultivating aquatic products comprising feeding said aquatic organisms to aquatic products.
[15]
The method wherein both the feed organism that is a rotifer and the feed organism that is Artemia are fed.
[16]
The method wherein the bait organism that is a rotifer is fed first and the bait organism that is artemia is fed later.
[17]
The method, wherein the aquatic product is fish or shellfish.
[18]
The method wherein the feed organism is fed to fish during part or all of the larval stage.

  According to the present invention, breeding results in aquaculture of fish and other aquatic products can be improved.

The figure which shows the phylogenetic tree of AJ7866 strain (FERM P-22289) based on the base sequence of 18S rDNA. The figure which shows the result of the fortification test of a rotifer. The figure which shows the result of the breeding test of anemone fish. The figure which shows the result of the fortification test of a rotifer. The figure which shows the result of the breeding test of a flounder.

  Hereinafter, the present invention will be described in detail.

<1> Sterol-containing alga body In the present invention, a sterol-containing alga body is used for enhancing nutrition of feed organisms. By using feed organisms enriched with sterol-containing alga bodies, specifically, by feeding feed organisms enriched with sterol-containing alga bodies to aquatic products and culturing aquatic products, Can improve the breeding performance. That is, by using a feed organism fortified with a sterol-containing alga body, for example, the breeding performance of the aquatic product can be improved as compared with the case of using a feed organism not fortified. Aquaculture is also called “breeding”. The effect of improving the breeding performance of the aquatic product is also referred to as “breeding performance improvement effect”. Examples of the improvement in rearing results include an increase in body weight, an increase in body length, an increase in obesity, an increase in survival rate, and an increase in survival rate of stress test. In the present invention, one kind of rearing result improving effect may be obtained, or two or more kinds of rearing result improving effects may be obtained.

  “Sterol” is a general term for steroid derivatives having a hydroxyl group at the C-3 position. “Steroid” is a general term for compounds having a cyclopentanohydrophenanthrene ring skeleton. That is, “sterol” is a general term for compounds having a cyclopentanohydrophenanthrene ring skeleton having a hydroxyl group at the C-3 position.

  Examples of sterols include animal sterols, plant sterols, and fungal sterols. “Animal sterols”, “plant sterols” and “fungal sterols” refer to sterols found in animals, plants and fungi, respectively. The “plant” mentioned here includes algae.

  Animal sterols include cholesterol, 7-dehydrocholesterol, glycolic acid, taurocholic acid, cholic acid, estradiol, estrone, ethinylestradiol, estriol, dehydroepiandrosterone, methylandrostenediol, 5β-pregnane-3α, 20α-diol , Pregnenolone, chenodeoxycholic acid, dehydrocholic acid, dihydroxycholesterol, digitoxigenin, desmosterol, lasosterol.

  As plant sterols, stigmasterol, β-sitosterol, brassicasterol, campesterol, diosgenin, oleanolic acid, betulinic acid, ursolic acid, hecogenin, salsasapogenin, isofucosterol, abenasterol, Δ7-stigmastenol, Δ7-campestenol, fucosterol, salgasterol may be mentioned.

  Examples of fungal sterols include ergosterol.

  Among them, cholesterol is preferable as the sterol.

  A sterol can exist in forms, such as a free type, a fatty-acid ester type | mold, and a glycoside type | mold, for example. That is, “sterol” may mean a free sterol, a sterol fatty acid ester, a sterol glycoside, or a mixture thereof, unless otherwise specified.

  “Algae” refers to algal cells. That is, the “sterol-containing alga body” refers to an algal cell containing sterol. Examples of sterol-containing algal cells include algal cells having sterol-producing ability, and more specifically, cells of the same algae obtained by culturing algae having sterol-producing ability in a medium.

“Algae having sterol-producing ability” refers to algae that can produce sterol and accumulate in the algae when cultured in a medium. The algae having the ability to produce sterol may have the ability to produce one kind of sterol, or may have the ability to produce two or more kinds of sterols. The algae having the ability to produce sterol preferably have, for example, the ability to produce cholesterol. Algae having sterol-producing ability is, for example, 2.5% (w / w-DCW) or more, 2.7% (w / w-DCW) or more, 3.0% (w / w) when cultured in a medium. Algae capable of producing (accumulating) sterol in an amount of -DCW) or more, 3.3% (w / w-DCW) or more, or 3.5% (w / w-DCW) or more. In addition, algae having sterol-producing ability is, for example, 1.2% (w / w-DCW) or more, 1.5% (w / w-DCW) or more, or 2.0% ( Algae that can produce (accumulate) cholesterol in an amount of (w / w-DCW) or more may be used. “% (W / w-DCW)” indicates the percentage of the weight of the target component (such as sterol) relative to the dry alga body weight (DCW). The culture conditions are not particularly limited as long as algae having sterol-producing ability can sufficiently grow. Examples of such culture conditions include the culture conditions described below. Specific examples of such culture conditions include the culture conditions described in the examples. The amount of sterol can be measured, for example, by a method described later. The “sterol amount” means an amount converted to the weight of free sterol unless otherwise specified.

  The algae having sterol-producing ability may further have the ability to produce highly unsaturated fatty acids. Examples of highly unsaturated fatty acids include docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). The algae having the ability to produce sterols may have the ability to produce one kind of highly unsaturated fatty acid, or may have the ability to produce two or more kinds of highly unsaturated fatty acids. The algae having sterol-producing ability may have, for example, the ability to produce one or both of DHA and EPA.

  The type and amount of a component such as sterol can be determined by a known method used for detection or identification of a compound. Examples of such a method include HPLC, LC / MS, GC / MS, and NMR. These methods can be used alone or in appropriate combination.

  Examples of algae having sterol-producing ability include labyrinthulas. Examples of the labyrinthula having sterol-producing ability include algae of Aurantiochytrium and algae of Thraustochytrium.

  Specific examples of algae having sterol-producing ability include AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM P-22290), and derivatives thereof. . Specific examples of the algae having the ability to produce sterols include algae such as Aulanthiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5.

  AJ7866 shares were entrusted to the Patent Biological Depositary Center (Postal Code 292-0818, Kisarazu Kazusa 2-5-8 Room 120, Chiba, Japan) on August 5, 2015 Deposited under the number FERM P-22289. The AJ7866 strain is considered to be related to the Aurantiochytrium genus by molecular phylogenetic analysis using the 18S rDNA sequence as an index. The base sequence of 18S rDNA of AJ7866 strain is shown in SEQ ID NO: 3.

  AJ7867 shares were commissioned on January 8, 2016, to the Patent Biological Depositary Center (Postal Code 292-0818, Kisarazu Kazusa Kamishi 2-5-8 Room 120, Chiba, Japan) on January 8, 2016 Deposited under the number FERM P-22304. The AJ7867 strain is considered to be related to the Aurantiochytrium genus by molecular phylogenetic analysis using the 18S rDNA sequence as an index. The base sequence of 18S rDNA of AJ7867 strain is shown in SEQ ID NO: 4.

AJ7868 shares were commissioned on August 5, 2015 at the Patent Organism Depositary Center (Postal Code 292-0818, Kisarazu Kazusa Kamashi 2-5-8 120, Chiba, Japan) on August 5, 2015 Deposited under the number FERM P-22290. The AJ7868 strain is considered to be related to the Aurantiochytrium genus by molecular phylogenetic analysis using the 18S rDNA sequence as an index. The base sequence of 18S rDNA of AJ7868 strain is shown in SEQ ID NO: 5.

  The above-mentioned “derived strain” is a strain constructed with the AJ7866 strain, AJ7867 strain, or AJ7868 strain as the parent strain (ancestor strain), and having a sterol production ability equal to or higher than that of the parent strain (ancestral strain). Say. The derived strain preferably has, for example, the same or higher ability to produce cholesterol as the parent strain. The derivative strain may be bred by artificial modification, for example. Artificial alteration includes alteration by genetic engineering techniques and alteration by mutation treatment. Mutation treatments include X-ray irradiation, ultraviolet irradiation, and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). ) And the like. Moreover, the derivative | guide_body strain | stump | stock produced naturally, for example at the time of use of a parent strain (ancestor strain) may be sufficient. Examples of such derived strains include mutants that are naturally generated when culturing the AJ7866 strain, AJ7867 strain, or AJ7868 strain. A derivative strain may be constructed by one type of modification or by a combination of two or more types of modification.

  “The induced strain has a sterol production ability equal to or higher than that of the parent strain” means that when the induced strain is cultured in a medium, the sterol is produced (ie accumulated) when the parent strain is cultured under the same conditions per dry weight of algal bodies. It may mean producing (accumulating) sterols in an amount of 70% or more, 80% or more, 90% or more, or 95% or more of the amount. The derivative strain is, for example, 2.5% (w / w-DCW) or more, 2.7% (w / w-DCW) or more, 3.0% (w / w-DCW) or more, 3.3% ( Sterols may be generated (accumulated) in an amount of w / w-DCW) or more, or 3.5% (w / w-DCW) or more. In addition, the derivative strain is, for example, in an amount of 1.2% (w / w-DCW) or more, 1.5% (w / w-DCW) or more, or 2.0% (w / w-DCW) or more. Cholesterol may be generated (accumulated). The culture conditions are not particularly limited as long as algae having sterol-producing ability can sufficiently grow.

  A sterol-containing algal body is obtained, for example, by culturing an algae having a sterol-producing ability in a medium. The culture conditions are not particularly limited as long as algae having the ability to produce sterol can grow and sterol is produced. The culture can be performed under normal conditions used for culture of heterotrophic algae such as Labyrinthula.

  The culture can be performed using a liquid medium. In culturing, a liquid medium can be directly inoculated with a sterol-producing algae cultured in a solid medium such as an agar medium, or a sterol-producing algae seeded in a liquid medium. You may inoculate the liquid culture medium. That is, the culture may be performed separately for seed culture and main culture. In that case, the culture conditions of the seed culture and the main culture may or may not be the same. The amount of algae having a sterol-producing ability contained in the medium at the start of culture is not particularly limited. The main culture may be performed, for example, by inoculating the seed culture medium with 1 to 50% (v / v) of the main culture medium.

  The culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium at the start of the culture is also referred to as “initial culture medium”. A medium supplied to a culture system (fermentor) in fed-batch culture or continuous culture is also referred to as “fed-batch medium”. In addition, supplying a feeding medium to a culture system in fed-batch culture or continuous culture is also referred to as “fed-batch”. In addition, when culture | cultivation is performed by dividing into seed culture and main culture, for example, both seed culture and main culture may be performed by batch culture. Further, for example, seed culture may be performed by batch culture, and main culture may be performed by fed-batch culture or continuous culture.

The medium to be used is not particularly limited as long as algae having the ability to produce sterol can grow and sterol is produced. As the medium, for example, a normal medium used for culturing heterotrophic algae such as Labyrinthula can be used. The medium may contain a component selected from a carbon source, a nitrogen source, a phosphoric acid source, a sulfur source, and other various organic and inorganic components as necessary. Specific examples of the medium include GPY medium (Example) prepared with 0 to 1 × artificial seawater.

  Specific examples of the carbon source include glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, waste molasses, starch hydrolysate, biomass hydrolyzate and other sugars, and organic acids such as acetic acid and citric acid. Alcohols such as ethanol, glycerol and crude glycerol, and fatty acids. As the carbon source, one type of carbon source may be used, or two or more types of carbon sources may be used in combination.

  Specific examples of the nitrogen source include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen sources such as peptone, yeast extract, meat extract, and soybean protein degradation product, ammonia, and urea. Ammonia gas or ammonia water used for pH adjustment may be used as a nitrogen source. As the nitrogen source, one kind of nitrogen source may be used, or two or more kinds of nitrogen sources may be used in combination.

  Specific examples of the phosphoric acid source include phosphates such as potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphoric acid. As the phosphoric acid source, one type of phosphoric acid source may be used, or two or more types of phosphoric acid sources may be used in combination.

  Specific examples of the sulfur source include inorganic sulfur compounds such as sulfate, thiosulfate, and sulfite, and sulfur-containing amino acids such as cysteine, cystine, and glutathione. As the sulfur source, one kind of sulfur source may be used, or two or more kinds of sulfur sources may be used in combination.

  Other various organic and inorganic components include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium and calcium; vitamin B1, vitamin B2, vitamin B6 and nicotine Examples include vitamins such as acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components such as peptone, casamino acid, yeast extract, and soybean protein degradation products containing these. As other various organic components and inorganic components, one component may be used, or two or more components may be used in combination.

  The culture can be performed, for example, under aerobic conditions. The aerobic condition means that the dissolved oxygen concentration in the liquid medium is 0.33 ppm or more, which is the detection limit of the oxygen membrane electrode, and preferably 1.5 ppm or more. The oxygen concentration may be controlled to, for example, about 5 to 50%, preferably about 10% of the saturated oxygen concentration. Specifically, culture under aerobic conditions can be performed by aeration culture, shaking culture, agitation culture, or a combination thereof. The pH of the medium may be, for example, pH 3 to 10, preferably pH 4.0 to 9.5. During the culture, the pH of the medium can be adjusted as necessary. The pH of the medium is various alkaline or acidic substances such as ammonia gas, aqueous ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, calcium hydroxide, magnesium hydroxide, hydrochloric acid, sulfuric acid, etc. Can be adjusted. The culture temperature may be, for example, 20 to 35 ° C, preferably 25 to 35 ° C. The culture period may be, for example, 10 hours to 120 hours. The culture may be continued, for example, until the carbon source in the medium is consumed or until the activity of the algae having the ability to produce sterols is lost. By culturing algae having the ability to produce sterols under such conditions, algal bodies containing sterols (sterol-containing algal bodies) are generated.

The sterol-containing algal bodies obtained in this way can be used for enhancing the nutrition of feed organisms. “Utilizing algal bodies for the enhancement of nutrients of feed organisms” specifically refers to the use of algal bodies as a component of the nutrient fortifier of the present invention or feeding the algal bodies to the diet organisms. The algal bodies may be used for the fortification of the feed organism while contained in the medium, or may be recovered from the medium and used for the fortification of the feed organism. Moreover, you may utilize an algal body for the nutrient enhancement of a feed organism, after processing suitably. Examples of the treatment include dilution, concentration, freezing, thawing, and drying. These processes may be performed in combination as appropriate.

  The technique for collecting algal cells from the medium is not particularly limited, and for example, a known technique (Grima, E. M. et al. 2003. Biotechnol. Advances 20: 491-515) can be used. Specifically, for example, algal bodies can be recovered from the culture medium by methods such as natural sedimentation, centrifugation, and filtration. At that time, a flocculant may be used. The collected algal bodies can be appropriately washed using an appropriate medium. The collected alga bodies can be appropriately resuspended using an appropriate medium. Examples of the medium that can be used for washing and suspending include an aqueous medium (aqueous solvent) such as water and an aqueous buffer solution.

<2> Nutrition enhancer of the present invention The nutrient enhancer of the present invention is a nutrient enhancer containing a sterol-containing algal body. The nutrient fortifier of the present invention can be used for fortification of feed organisms. That is, the nutrient fortifier of the present invention may specifically be a nutrient fortifier for feed organisms.

  The nutrient fortifier of the present invention may be composed of a sterol-containing algal body, or may contain other components. Examples of other components include water and medium components. In addition, examples of other components include components used for formulation of excipients and the like. That is, the sterol-containing alga bodies (the algal bodies contained in the medium, the alga bodies recovered from the medium, processed products thereof, etc.) as described above are used as they are or in combination with other components. Can be a nutritional enhancer. For example, the sterol-containing alga body of the aspect as described above can be formulated in any form to provide the nutritional enhancer of the present invention.

  The form of the fortifying agent of the present invention is not particularly limited as long as the feed organism to be fortified can feed the sterol-containing alga body. The nutrient fortifier of the present invention may have any shape such as powder, granule, pellet, cube, paste, and liquid. For example, the nutrient fortifying agent of the present invention may be configured to be dispersed in water so that it can be fed by the feed organism to be enriched.

<3> Feed organism of the present invention The feed organism of the present invention is a feed organism fortified with sterol-containing algal bodies. “Nutrified fortification with sterol-containing alga bodies” means that sterol-containing alga bodies are taken in, that is, sterol-containing alga bodies are contained. That is, in other words, the feed organism of the present invention may be a feed organism incorporating a sterol-containing alga body, or may be a feed organism containing a sterol-containing alga body. It should be noted that the organism that is the basis of the feed organism of the present invention (the organism that takes in the sterol-containing alga body) is particularly referred to as a “raw material organism” in distinction from the feed organism of the present invention. The feed organism of the present invention can be used for aquaculture of aquatic products as raw food. That is, the feed organism of the present invention may specifically be a feed organism for aquatic products. Moreover, in one aspect | mode, effects, such as an increase in a stress test survival rate, are acquired by utilizing the feed organism of this invention for aquaculture of aquatic products. Thus, one aspect of the feed organism of the present invention may be a stress relieving agent for aquatic products.

The feed organism of the present invention is selected from rotifers and artemia. That is, the raw material organism is also selected from rotifers and artemia. In particular, the feed organism of the present invention which is a rotifer (the feed organism of the present invention using a rotifer as a raw material organism) is referred to as “the rotifer-based feed organism of the present invention”, and the feed organism of the present invention which is Artemia (a book using the artemia as a raw material organism). The inventive feed organism) is also referred to as “the Artemia feed organism of the invention”.

  "Rotifer" is a general term for organisms belonging to the rotifera. Specific examples of the rotifer include Brachyion plicatilis sp. Complex. The rotifer is generally used as a feed organism in, for example, seed production of marine fish. According to molecular phylogenetic analysis, the rotifer is a complex species consisting of 14 or more species, any of which may be used. Further, the rotifer is roughly classified into three types, L-type, S-type, and SS-type, depending on its size, any of which may be used. The size of the rotifer can be appropriately selected according to various conditions such as the type of aquatic product to be fed and the growth stage. The L-type may be classified as Brachionus plicatilis, the S-type may be classified as Brachionus ibericus, and the SS-type may be classified as Brachionus rotundiformis. In the present invention, these are all treated as Brachyion plicatilis sp. For example, a commercially available product can be used as the rotifer. Specific examples of commercially available hornworms include hornworms commercially available from Chlorella Kogyo Co., Ltd. Moreover, the rotifer can be obtained from the cultivation fisheries center of each place, for example.

  “Artemia” is a general term for organisms belonging to the genus Artemia. Artemia is sometimes called brine shrimp. Specific examples of artemia include Artemia franciscana, Artemia monica, Artemia persimilis, Artemia salina, Artemia sinica, Artemia tibetiana, and Artemia urmiana. As Artemia, for example, a commercially available product can be used. Specific examples of the commercially available artemia include Artemia from Salt Lake, Utah, USA and Artemia from China, which are commercially available from Taiheiyo Trading Co., Ltd.

  The content (uptake amount) of the sterol-containing algal bodies in the feed organism of the present invention is not particularly limited as long as an effect of improving the breeding results is obtained. The content of the sterol-containing algal bodies in the feed organism of the present invention may be an amount such that a desired sterol content can be obtained, for example. The content of sterol in the feed organism of the present invention is, for example, 0.02% (w / w) or more, 0.05% (w / w) or more, 0.1% as the weight relative to the dry weight of the feed organism ( w / w) or more, or 0.15% (w / w) or more. Further, the cholesterol content in the feed organism of the present invention is, for example, 0.02% (w / w) or more, 0.05% (w / w) or more, 0.1% as the weight relative to the dry weight of the feed organism. % (W / w) or more, or 0.15% (w / w) or more.

  The feed organism of the present invention may contain other components. Examples of other components include highly unsaturated fatty acids such as DHA and EPA. The content of DHA in the feed organism of the present invention is, for example, 0.2% (w / w) or more, 0.5% (w / w) or more, or 1% (w / W) or more. The content of EPA in the feed organism of the present invention is, for example, 0.02% (w / w) or more, 0.05% (w / w) or more, 0.1% (weight relative to the dry weight of the feed organism) ( w / w) or more, or 0.15% (w / w) or more.

<4> Production method of feed organism of the present invention The feed organism of the present invention can be produced, for example, by fortifying a source organism with a sterol-containing algal body (that is, allowing the source organism to incorporate a sterol-containing alga body). . That is, the method for producing a feed organism of the present invention may be, for example, a method for producing a feed organism, which includes incorporating a sterol-containing alga into a raw organism. Specifically, the raw material organism can be grown (growth, hatching, etc.) by culture, and then the grown raw material organism can be fortified with sterol-containing alga bodies.

  The culture conditions of the raw material organism are not particularly limited as long as the raw material organism can grow (growth, hatch, etc.). The culture conditions of the raw organism can be appropriately set according to various conditions such as the type of the raw organism. The culture of the raw material organism can be performed, for example, under normal conditions used for culturing the raw material organism.

  In the case of a rotifer, a feed can be fed and cultured in a liquid medium. Examples of the feed for rotifers include algae such as Nannochloropsis, and yeasts such as Saccharomyces cerevisiae. As the liquid medium, a medium having an appropriate salt concentration can be used. The salt concentration of the medium may be, for example, half the salt concentration of seawater to the salt concentration of seawater. Specific examples of the liquid medium include filtered seawater, artificial seawater, and dilutions thereof. The culture temperature may be, for example, 20 ° C to 30 ° C, and preferably about 25 ° C. Culture can be continued until the rotifer grows to the desired extent. The culture can be performed by batch culture, fed-batch culture, continuous culture, or a combination thereof.

  In the case of Artemia, dormant eggs can be hatched by culturing dormant eggs in a liquid medium. As the liquid medium, a medium having an appropriate salt concentration can be used. The salt concentration of the medium may be, for example, half the salt concentration of seawater to the salt concentration of seawater. Specific examples of the liquid medium include filtered seawater, artificial seawater, and dilutions thereof. The hatching temperature may be, for example, 20 ° C. to 30 ° C., and preferably about 28 ° C. In hatching, it is preferable to ventilate the culture tank. After hatching, the eggshell and larvae may be separated. After hatching, the culture can be continued as appropriate. The culture temperature after hatching may be, for example, 20 ° C to 30 ° C, and preferably about 25 ° C. The culture can be continued until the artemia grows to the desired extent, for example, until the artemia is opened.

  Nutrition can be enhanced by allowing the raw organism to coexist with sterol-containing alga bodies in a liquid medium. The conditions for nutrient enrichment are not particularly limited as long as the raw organism can incorporate the sterol-containing alga bodies to a desired degree. The conditions for nutrient enhancement can be appropriately set according to various conditions such as the type of raw material organism. Nutrition fortification can be performed under normal conditions used for nutrient enhancement of raw material organisms, for example, except that sterol-containing alga bodies are used for nutrient enhancement.

  The raw material organisms may be subjected to nutrient enhancement while being contained in the culture medium at the time of culture, or may be recovered from the culture medium at the time of culture and then subjected to nutritional enhancement. For example, the culture solution of the raw organism may be used as it is, or after diluting or concentrating, for nutrient enhancement. As the sterol-containing alga body, for example, the sterol-containing alga body of the aspect as described above (alga bodies still contained in the culture medium, algal bodies recovered from the culture medium, processed products thereof, etc.) can be used. . Moreover, as a sterol containing alga body, the nutrient fortifier of this invention can be used, for example. As the liquid medium, a medium having an appropriate salt concentration can be used. The salt concentration of the medium may be, for example, half the salt concentration of seawater to the salt concentration of seawater. Specific examples of the liquid medium include filtered seawater, artificial seawater, and dilutions thereof. The temperature of nutrient enhancement may be, for example, 20 ° C to 30 ° C, and preferably about 25 ° C.

For example, from the viewpoint of maintaining the activity of the feed organism of the present invention when the feed organism of the present invention is fed to the aquatic product, the environment (salt concentration, temperature, etc.) at the time of culturing or fortifying the source organism is It is preferably close to the environment (salt concentration, temperature, etc.) at the time of aquaculture. If there is a large difference between the environment at the time of cultivation of the raw material organism and the environment at the time of aquaculture of the aquatic product, for example, the environment at the time of fortification of the raw material organism is set to one of the conditions between them, and the feed The organism may be habituated to the environment during aquaculture.

  The density of the raw material organism in the medium at the time of fortification is not particularly limited as long as the raw material organism can incorporate the sterol-containing algal bodies to a desired degree. In the case of a rotifer, the density of the raw material organism at the time of fortification may be, for example, 50,000 / L to 1,000,000 / L, or 100,000 / L to 500,000 / L. In the case of Artemia, the density of the raw material organism at the time of fortification may be, for example, 10,000 / L to 200,000 / L, or 20,000 / L to 100,000 / L.

  The amount of sterol-containing alga bodies to be used is not particularly limited as long as the raw organism can incorporate the sterol-containing alga bodies to a desired degree. The amount of sterol-containing algal bodies used may be, for example, 1 mg or more, 5 mg or more, or 10 mg or more, or 500 mg or less, 200 mg or less, 100 mg or less, or 50 mg or less with respect to 300,000 rotifers. Or a combination thereof. Specifically, the amount of sterol-containing algal bodies may be, for example, 1 mg to 200 mg, 5 mg to 100 mg, or 10 mg to 50 mg with respect to 300,000 rotifers. The amount of sterol-containing alga bodies may be, for example, 2 mg or more, 10 mg or more, or 20 mg or more, and 1000 mg or less, 500 mg or less, 200 mg or less, or 100 mg or less with respect to 50,000 Artemia. Or a combination thereof. Specifically, the amount of sterol-containing alga bodies may be, for example, 2 mg to 500 mg, 10 mg to 200 mg, or 20 mg to 100 mg with respect to 50,000 Artemia.

  The time for fortification is not particularly limited as long as the raw organism can incorporate the sterol-containing alga body to a desired degree. The time for nutrition enhancement may be, for example, 15 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, or 5 hours or more, 48 hours or less, 24 hours or less, 12 hours or less, Alternatively, it may be 9 hours or less, or a combination thereof. Specifically, the time for nutrition enhancement may be, for example, 30 minutes to 24 hours, 1 hour to 12 hours, or 2 hours to 9 hours.

  Nutrition fortification may be carried out using a sterol-containing alga body alone, or may be carried out using a sterol-containing alga body in combination with another nutrient fortification agent.

  By carrying out fortification in this way, the feed organism of the present invention is obtained.

  The feed organism of the present invention may be used for aquaculture of aquatic products while contained in a medium for nutrient enhancement, or may be used for aquaculture of aquatic products after being recovered from the medium for nutrition enhancement. For example, the culture solution after fortification may be used as it is, or after dilution or concentration, for aquaculture. The technique for recovering the feed organism of the present invention from the medium is not particularly limited, and for example, a known technique can be used. Specifically, for example, the feed organism of the present invention can be recovered from the medium by filtration using gauze or the like. The recovered feed organism of the present invention can be appropriately washed using an appropriate medium. The recovered feed organism of the present invention can be appropriately resuspended using an appropriate medium. As a medium that can be used for washing and suspension, for example, a medium used for culturing raw materials or fortifying nutrients can be used.

<5> Method of the Present Invention The method of the present invention is a method for culturing (breding) an aquatic product comprising feeding the aquatic product with the feed organism of the present invention.

  By rearing the aquatic product by the method of the present invention, the breeding performance of the aquatic product can be improved. That is, the method of the present invention may be a method for improving the breeding performance of an aquatic product, including feeding the aquatic product with the feed organism of the present invention.

  Moreover, in one aspect | mode, effects, such as an increase in a stress test survival rate, are acquired by culture | cultivating aquatic products by the method of this invention. Thus, one aspect of the method of the present invention may be a method for alleviating stress in aquatic products, comprising feeding the aquatic product of the present invention to aquatic products.

  The aquatic product is not particularly limited as long as it can feed the feed organism of the present invention. Examples of aquatic products include aquatic products that are generally cultured by feeding rotifers and / or artemia. Examples of aquatic products include fish and crustaceans. Moreover, a marine product is mentioned as a water product. Specific examples of fish include red sea bream, flounder, flounder, amberjack, tiger pufferfish, yellowtail, tuna, eel, tilapia, catfish, sea bass, sweetfish, rainbow trout, Atlantic salmon, coho salmon, horned salmon, and clownfish. Specific examples of the crustacean include vanamae shrimp, tiger prawn, bovine shrimp, white leg shrimp, and crab.

  The feeding mode of the feed organism of the present invention is not particularly limited as long as the effect of improving breeding results is obtained.

  The feed amount of the feed organism of the present invention is not particularly limited as long as an effect of improving the breeding performance is obtained. The feed amount of the feed organism of the present invention can be appropriately selected according to various conditions such as the type of the target aquatic product. The feed amount of the feed organism of the present invention may be, for example, a satiety amount. The feed organism of the present invention may be fed once or multiple times per day. Moreover, the feed organism of the present invention may be fed once every few days. The feed amount of the feed organism of the present invention at the time of each feed may or may not be constant in terms of the amount of sterols such as cholesterol. The concentration of sterols such as cholesterol in the feed organism of the present invention at each feeding may or may not be constant.

  The type of the feed organism of the present invention to be fed can be appropriately selected according to various conditions such as the type of the target aquatic product and the growth stage. In the method of the present invention, only one of the feed organism of the present invention of the rotifer type or the feed organism of the present invention of the artemia type may be fed, or both may be fed. When feeding both the rotifer-based feed organism of the present invention and the artemia-based feed organism of the present invention, the feeding period of the rotifer-based feed organism of the present invention and the artemia-based feed organism of the present invention is one. Some or all of them may overlap, or they may not overlap at all. The feeding order of the rotifer-based feed organism of the present invention and the artemia-based feed of the present invention can be appropriately set. For example, the rotifer-based food of the present invention may be fed first, and the artemia-based food of the present invention may be fed later. "Worm-based feed of the present invention is fed first and Artemia-based feed of the present invention is fed later" means that the feeding period of the feed organism of the present invention of the rotifer system is higher than that of the artemia-based feed organism of the present invention. Is started earlier, the feeding period of the feed organism of the present invention of the rotifer system ends before the feed organism of the present invention of the artemia system, or a combination thereof. In the case of `` feeding the rotifer-based food of the present invention first and feeding the artemia-based food of the present invention later '', the feeding period of the rotifer-based food organism of the present invention and the artemia-based food organism of the present invention is: There may be some overlap or no overlap at all.

The timing and length of feeding the feed organism of the present invention are not particularly limited as long as an effect of improving the breeding results is obtained. The timing and length of feeding the feed organism of the present invention can be appropriately selected according to various conditions such as the type of the target aquatic product. The feed organism of the present invention can be fed, for example, to aquatic products in a specific growth stage. Examples of the growth stage for feeding the feed organism of the present invention include growth stages that are generally cultivated by feeding rotifers and / or artemia. As the growth stage for feeding the feed organism of the present invention, for example, in the case of fish, the larvae and juvenile stages are mentioned. As the growth stage for feeding the feed organism of the present invention, for example, in the case of fish, more specifically, a period after the start of feeding behavior in the larvae and juvenile period is mentioned. Moreover, as a growth stage which feeds the feed organism of this invention, in the case of crustaceans, the period of a larva is mentioned, for example. As the growth stage for feeding the feed organism of the present invention, for example, in the case of crustaceans, more specifically, it is a period of larvae and a period after the start of feeding behavior. The feed organism of the present invention may be fed to the aquatic product during part or all of the growth stage as described above. That is, the feed organism of the present invention is, for example, in the case of fish, a part or all of the period of the larval and juvenile period, more specifically, part or all of the period after the start of feeding behavior in the larval and juvenile period During this period, fish may be fed. When feeding both the rotifer-based feed organism of the present invention and the artemia-based feed organism of the present invention, at least one of them is fed to the aquatic product during part or all of the growth stage as described above. Good. In addition, when feeding both the rotifer-based feed organism of the present invention and the artemia-based feed organism of the present invention, each of them is fed to the aquatic product during part or all of the growth stage as described above. It's okay. The “part of the growth stage” is, for example, a period of 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, or 90% or more of the entire period of the growth stage. Good. Moreover, the feed organism of the present invention may be fed or not fed during a period other than the growth stage as described above. The period of feeding the feed organism of the present invention may be, for example, 3 days or more, 5 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 2 months or more, or 3 months or more. It may be 1 year or less, 6 months or less, 4 months or less, 3 months or less, 2 months or less, 4 weeks or less, 3 weeks or less, or 2 weeks or less, or a non-conflicting combination thereof. When feeding both the rotifer-based feed organism of the present invention and the artemia-based feed organism of the present invention, the period during which at least one of them is fed may be as described above. Moreover, when feeding both the rotifer type feed organism of the present invention and the artemia type feed organism of the present invention, the period during which each of them is fed may be as described above. In addition, the feed organism of the present invention may repeat continuation and interruption of feeding at an arbitrary period.

  Aquaculture (breeding) of the aquatic product can be performed by the same method as a normal method for culturing (breeding) the aquatic product, for example, except for feeding the feed organism of the present invention. That is, aquaculture (breeding) of aquatic products is the same as the usual method of culturing (breeding) aquatic products by feeding forage organisms such as rotifers and artemia, for example, except for feeding the feed organisms of the present invention. It can be done by a method. The aquaculture (breeding) can be performed in, for example, a marine ginger or an onshore water tank. Note that “aquaculture” includes both aquaculture in which only seedling production (such as fry production) is performed and aquaculture in which production is performed up to adults (such as parent fish).

  Hereinafter, the present invention will be described more specifically with reference to examples.

<Example 1> Preparation of sterol-containing algal bodies (1) Acquisition of sterol-accumulating strains of Labyrinthula (1-1) Cultivation of water or soil samples and isolation of strains Rotting of leaves containing river water in Okinawa Prefecture A sample of the object was taken. An appropriate amount of pine pollen (collected in Miyazaki Prefecture) was added to the sample and allowed to stand at room temperature for 8 days. Penicillin G calcium and streptomycin sulfate were added to 0.1 × Raihimani preservation medium (Nissui) prepared with 0.5 × artificial seawater to a final concentration of 50 mg / L, respectively, and final concentrations of 2 mg / L and 0.01 mg / L, respectively. 100 μl of the sample after standing was seeded on a plate of a medium to which vitamin B1 (Nacalai), vitamin B2 (Nacalai), and vitamin B12 (Nacalai) were added so that L and 0.01 mg / L. Each plate was cultured at 28 ° C. for 4 days, and the obtained colonies were streaked with the same medium to purify the colonies. The strain thus obtained was named AJ7866 strain. In addition, the composition of 1 × Raihimani preservation medium (Nissui) and 1 × artificial seawater is as follows.

<Composition of 1x Reichmanni preservation medium (Nissui)>
Yeast extract 8.5 g
Peptone 8.5 g
Glucose 11 g
Potassium dihydrogen phosphate 2.0 g
Tomato juice powder 3.7 g
Polysorbate 80 1.0 g
Agar 15 g
1000 ml of ultrapure water

<1 x artificial seawater composition>
NaCl 30 g
KCl 0.7 g
MgCl2 ・ 6H2O 10.8 g
MgSO4 ・ 7H2O 5.4 g
CaCl2 ・ 2H2O 1 g
1000 ml of ultrapure water

(1-2) Molecular phylogenetic analysis of sterol-accumulating strains About the AJ7866 strain isolated as described above, using the universal primer for amplifying 18S rDNA region of Labyrinthula (SEQ ID NOs: 1 and 2), Molecular phylogenetic analysis was performed using the base sequence as an index. The nucleotide sequence of the 18S rDNA region of the AJ7866 strain determined is shown in SEQ ID NO: 3. Sequence file of 18S rDNA region of known Labyrinthula (Mayumi Ueda, et al., Seasonal dynamics of culturable thraustochytrids (Labyrinthulomycetes, Stramenopile) in estuarine and coastal waters., Aquatic Microbial Ecology. 2015; 74: 187 -204) Non-patent document 2 Alignment files including the above SEQ ID NO: 3 were aligned, and phylogenetic analysis was performed. The phylogenetic analysis was performed by the neighbor join (NJ) method using MEGA 6.06 and applying the Tamura & Nei model. Sites that contain gaps due to alignment are not included in the phylogenetic analysis. The obtained phylogenetic tree is shown in FIG. In the figure, the numerical value of each node indicates the support rate by 1000 bootstrap re-sampling. As a result, it became clear that the AJ7866 strain was related to the Aurantiochytrium genus, so the strain was named Aurantiochytrium sp. AJ7866.

(2) Preparation of sterol-containing algal bodies
Vitamin B1 (Nacalai) and Vitamin B2 so that the final concentrations of AJ7866 strain are 0.2 × Raihimani preservation medium (Nissui) prepared with 0.5 × artificial seawater to 2mg / L, 0.01mg / L, 0.01mg / L respectively. (Nacalai) and a plate of medium supplemented with vitamin B12 (Nacalai), and cultured at 25 ° C. for 60 hours. The cells on the plate medium are scraped into 6 platinum ears, inoculated into an Erlenmeyer flask with 300 ml capacity baffle containing 30 mL of GPY medium prepared with 0.25 x artificial seawater of the following composition, culture temperature 25 ° C, stirring 120 rpm (Rotary), and cultured for 24 hours. 5 ml of the obtained culture solution is inoculated into a 500 ml baffled Erlenmeyer flask containing 45 ml of GTY medium prepared with 0.25 x artificial seawater, and cultured for 51 hours at a culture temperature of 25 ° C and stirring of 120 rpm (rotary). It was.

<Composition of GPY medium>
(A ward)
Glucose 20 g / L
(B ward)
Polypeptone 10 g / L
Yeast extract 5 g / L
(C ward)
Vitamin B1 2 mg / L
Vitamin B2 0.01 mg / L
Vitamin B12 0.01 mg / L
(District D)
MES 8.79 g / L
The B section was adjusted to pH 6.0 using HCl, and then the A section was autoclaved at 120 ° C. for 10 minutes with no pH adjustment. In the D section, pH was adjusted to 6.0 with NaOH, and then in the C section, the pH was not adjusted and the filter filtration was performed. After the A and B sections were cooled to room temperature, the 4 sections were mixed.

<Composition of GTY medium>
(A ward)
Glucose 50 g / L
(B ward)
Tryptone 10 g / L
Yeast extract 5 g / L
(C ward)
Vitamin B1 2 mg / L
Vitamin B2 0.01 mg / L
Vitamin B12 0.01 mg / L
(District D)
MES 8.79 g / L
The B section was adjusted to pH 6.0 using HCl, and then the A section was autoclaved at 120 ° C. for 10 minutes with no pH adjustment. In the D section, pH was adjusted to 6.0 with NaOH, and then in the C section, the pH was not adjusted and the filter filtration was performed. After the A and B sections were cooled to room temperature, the 4 sections were mixed.

  After completion of the culture, the culture solution was centrifuged, concentrated, and lyophilized to obtain a sterol-containing algal body.

<Example 2> Nutrition enhancement test of rotifer
[Experimental procedure]
Using the sterol-containing alga body obtained in Example 1 (hereinafter also referred to as “AJ7866 strain alga body”), the fortification of the rotifer (Brachionus plicatilis sp.complex, hereinafter also simply referred to as “rotifer”) In addition to confirming availability, quantitative nutritional enhancement effects were verified. The rotifer used in this test is an S-type rotifer.

  In this test, the group without additive fortifier (hereinafter referred to as “control group”), the group with comparison alga bodies (Biochromis; manufactured by Chlorella Kogyo Co., Ltd.) (hereinafter referred to as the other company's product group), and the group with added AJ7866 strain alga bodies. Three sections (hereinafter referred to as algal bodies) were set, and a rotifer strengthening test was performed. In addition, the comparative alga body (Biochromis) is sold as an Artemia enhancer rich in DHA.

  The method for preparing the reinforcing agent and the method for reinforcing the rotifer are described below.

  First, the comparative alga bodies and the AJ7866 strain alga bodies were suspended in seawater and stirred for 3 minutes using a biomixer (Nippon Seiki Seisakusho BIO MIXER BM-2) to obtain a strengthening agent. The suspension concentration is 65 g per 1 L of seawater.

Add a fortifying agent to the culture solution of rotifers adjusted to an individual density of 300,000 / L so that the amount of comparison alga and AJ7866 strain algae is 20 mg per 300,000 rotifers. And algal bodies. In addition, the group to which no reinforcing agent was added was defined as a control group. The liquid temperature during the fortification treatment was 25 ± 0.5 ° C. (actual value). 3 hours after the start of strengthening (addition of strengthening agent) and 6
After a time, the rotifer was collected and used as a sample for content analysis. Specifically, the culture solution containing the rotifer strengthened with a siphon was collected from the nutrient enhancement tank, and the rotifer was collected using a 50 μm mueller gauze. Thereafter, the rotifer was washed while passing through filtered seawater, and about 50 g of each sample, which was loosely dehydrated and turned into a paste, was placed in a centrifuge tube and stored frozen. The analytical samples stored frozen were used for analysis of the contents of cholesterol, docosahexaenoic acid (hereinafter DHA), and eicosapentaenoic acid (hereinafter EPA) at the Japan Food Analysis Center.
[result]
The results are shown in FIG. Cholesterol in rotifers was not detected in the control group, and was hardly detected in the competitor's product group. However, 0.12% was detected after 3 hours of algae and 0.17% after 6 hours. DHA was not detected in the control group, but was detected to the same extent in the competitor's product group and the algal body group. EPA was not detected in the control and competitor products, but was detected at 0.18% after 3 hours of the algal body and 0.2% after 6 hours. In addition, the density | concentration of each component is a value calculated per rotifer dry matter weight in consideration of the moisture content analysis result in a rotifer in the figure.

[Consideration conclusion]
In rotifers, cholesterol, DHA, and EPA were not detected in the control group, but were detected in the algal body group, both at 3 hours and 6 hours after the enhancement. The algal body area had the same DHA content but higher cholesterol content than the competitor's product area. In addition, EPA was not detected in the competitor's product area, but only in the algal body area. From this, it was confirmed that AJ7866 strain alga containing cholesterol was taken up by rotifer.

<Example 3> Anemonefish breeding test
[Experimental procedure]
In the larval stage of feeding the rotifer and Artemia franciscana (hereinafter also referred to simply as “Artemia”) fortified with the sterol-containing alga body obtained in Example 1 (AJ7866 strain alga body) A clownfish rearing test was conducted to measure body length and weight at the end of the test, calculate survival rate and obesity, and evaluate stress tolerance.

  In this test, the same rotifer used in Example 2 and Artemia from Utah purchased from Taiheiyo Trading Co., Ltd. were used. Further, the kind of reinforcing agent and the preparation method are the same as those in Example 2. The method for strengthening rotifers and artemia is described below.

  Table 1 shows the conditions for strengthening rotifer and artemia. In the table, “algae” in the column of feed biofortifier indicates AJ7866 strain algae. In the rotifer culture solution adjusted to an individual density of 300,000 / L, fortifying agents prepared from AJ7866 strain algae (hereinafter referred to as algae) and comparative alga (hereinafter referred to as competitors) It was added in the amount described in 1 and was subjected to a nutrient enrichment treatment for 6 hours. Also prepared from AJ7866 strain algae (hereinafter referred to as algae) and a comparison target algae (hereinafter referred to as a product of other companies) in an artemia culture solution in which dormant eggs were hatched and the individual density was adjusted to 50,000 / L. Reinforcing agents were added in the amounts shown in Table 1, respectively, and subjected to a nutrient fortification treatment for 6 hours. The liquid temperature during the fortification treatment was 25 ± 0.5 ° C. (actual value).

  Table 2 shows the conditions of the anemonefish breeding test. The breeding tank used for the anemonefish breeding test is 30L panlite (effective water volume 30L), the breeding water is by continuous pouring of natural filtered seawater adjusted to 25 ± 1 ° C, and the light / dark cycle is 12L: 12D by artificial lighting did. Test fish were produced from 4 parent fish pairs and hatched on the same day and mixed to homogeneity, and 100 fish were used per aquarium. A total of 8 aquariums consisted of 3 lots or 2 lots per aquarium. Set. The aeration rate was set at approximately 100 mL / min, and the feeding amount (feeding biological feeding density) was set so that the feeding was satisfactoried after taking into consideration the water conversion rate.

  During the test period, 1-9 days, the rotifer was fed to the clownfish larvae, 10-12 days after the start of the test, the rotifer and artemia were fed at the same time, and from the 13th day to the 21st day (the end date of the test), only the artemia Was fed. Clownfish larvae were collected at the end of the test on the 21st day, and the body length (0.01 mm) and body weight (individual average) were measured in each test area based on the evaluation method (Table 3). The degree was calculated and stress tolerance was evaluated.

[result]
The results are shown in FIG. The results are shown as relative values when the average value of the control group is taken as 100. The values of the algal body area and the competitor's product area were higher in all items than the control area. Further, the body weight, body length, obesity level, and stress survival rate were higher in the algal body area than in the other company's product area.

[Consideration conclusion]
By feeding the rotifer and artemia enriched with AJ7866 strain alga to the clownfish during the larval stage, the weight of the clownfish compared to the case of feeding the rotifer and artemia enriched with the non-enhanced or comparative alga, Body length, obesity, and survival rate after stress tolerance tests were improved. From this, it was suggested that sterol-containing alga bodies such as cholesterol have an effect of promoting the growth of aquatic products such as clownfish and enhancing stress tolerance.

<Example 4> Nutrition enrichment test for rotifer and flounder breeding test using AJ7867 strain (1) Preparation of sterol-containing alga body AJ7867 strain, a sterol-accumulating strain of Labyrinthula, was obtained by the same procedure as in Example 1. It was. Molecular phylogenetic analysis of the AJ7867 strain was carried out by the same procedure as in Example 1. As a result, it was revealed that the AJ7867 strain was related to the Aurantiochytrium genus, so the strain was named Aurantiochytrium sp. AJ7867. did. According to the same procedure as in Example 1, the AJ7867 strain was cultured to obtain a sterol-containing algal body.

(2) Rotifer fortification test
[Experimental procedure]
The sterol-containing alga body obtained in Example 4 (1) (hereinafter also referred to as “AJ7867 strain alga body”) was used to confirm whether or not rotifers could be fortified and to quantitatively verify the effect of fortification. It was. The rotifer used in this test is an S-type rotifer.

  In this test, the rotifer strengthening test was carried out by setting two sections, the AJ7867 strain alga body addition group (hereinafter referred to as alga body (mh) group) and the comparison target alga body addition group (hereinafter referred to as competitor product area). In addition, the comparison target alga body is a commercially available rotifer / artemia fortifying agent that is concentrated in algae mainly composed of DHA-containing algae and is in a liquid state. The comparative algal bodies contain 0.7% DHA and 0.02% EPA in the liquid.

  The method for preparing the reinforcing agent and the method for reinforcing the rotifer are described below.

  AJ7867 strain alga was suspended in sterilized seawater at a concentration of 0.1 g / ml, and stirred with a mixer for 3 minutes to obtain a strengthening agent. Since the comparative algal bodies were originally provided in liquid form, they were used as they were as reinforcing agents. Add 2 ml of each fortifier (AJ7867 Algae Suspension and Comparative Algae) to 1 L of rotifer culture solution adjusted to an individual density of 2000 / mL. did. The rotifer was collected 6 hours and 16 hours after the start of strengthening (addition of strengthening agent), and used as a sample for analysis of contained components. The rotifer was filtered from the nutrient enrichment tank using a plankton net having a hole size of 60 μm, washed thoroughly with seawater, further washed with tap water, and stored frozen. Samples for analysis that had been stored frozen were used for analysis of cholesterol and DHA content at the Japan Food Analysis Center.

[result]
The results are shown in FIG. Cholesterol in rotifers was hardly detected in the competitor's product group, but 0.11% was detected in the algal (mh) group at 6 hours and 0.17% at 16 hours. DHA was detected at 1.38% for the 6-hour strengthening of the competitor's product line and 0.6% for the 16-hour strengthening, compared with 1.6% for the 6-hour strengthening of the alga body (mh) and 0.68% for the 16-hour strengthening. . In addition, the density | concentration of each component is a value calculated per rotifer dry matter weight in consideration of the moisture content analysis result in a rotifer in the figure.

[Conclusion]
In rotifers, cholesterol and DHA were detected at higher levels in the algal (mh) group than in the competitor's product group, both for 6 hours and 16 hours fortification. From this, it was confirmed that AJ7867 strain alga containing cholesterol was taken up by rotifer.

(3) Flatfish breeding test
[Experimental procedure]
A larvae rearing test was conducted in the larval and juvenile stage fed with rotifer and artemia enriched with the sterol-containing alga obtained in Example 4 (1) (AJ7867 strain alga), and the body length at the end of the test, Body weight and survival rate were evaluated.

  In this test, the same rotifer used in Example 4 (2) and commercially available Artemia from Russia and Salt Lake were used. Moreover, the kind and preparation method of a toughening agent are the same as Example 4 (2). The method for strengthening rotifers and artemia is described below.

  Table 4 shows the conditions for strengthening rotifer and artemia. In the table, “algae (mh)” in the column of feed bioenhancing agent indicates AJ7867 strain algae. To each of the rotifer cultures, each fortifying agent, that is, an AJ7867 strain algae suspension (hereinafter referred to as “algae (mh))” and a comparative algal body (hereinafter referred to as “commercial product”) are added in the amounts shown in Table 4. The nutrition fortification process was performed for 6 hours. In addition, to the culture medium of Artemia obtained by hatching dormant eggs, each strengthening agent, ie, AJ7867 strain alga body suspension (hereinafter referred to as alga body (mh)) and the comparison target nonnochloropsis alga body (hereinafter referred to as “algae body”) And other companies' product areas) were added in the amounts shown in Table 4 and subjected to a nutrient enrichment treatment for 6 hours. In addition, as control, rotifers enriched with freshwater chlorella for 6 hours and artemia enriched with Nannochloropsis for 6 hours were prepared. In addition, the freshwater chlorella used for the fortification of the rotifer in the control group is a raw chlorella V12 (manufactured by Chlorella Kogyo Co., Ltd.), which is a commercial feed for rotifer. In addition, Nannochloropsis used to enhance the nutrition of Artemia in the control group is a commercially available feed for Artemia culture using Nannochloropsis as the main raw material.

  Table 5 shows the conditions of the flounder breeding test. The breeding tank used for the flounder breeding test was a 30L polyethylene tank, and the light / dark cycle was 12L: 12D. The test fish uses 600 flounder 1-day-old after hatching, and each breeding tank has a total of 3 lots (algae (mh) and other competitors) or 2 lots (control) for each test zone. 8 tanks were set. At the age of 14 days, in order to eliminate the influence of the breeding density of each tank, flounder is gathered into one for each test zone, from which 300 fish are randomly selected, 300 fish / zone, 1 lot 30L Housed in a polyethylene aquarium. In addition, the number of times of feeding and the amount of feeding (feeding organism feeding density) were set as shown in Table 5. During the test period 3-13 days, rotifers were fed only to larvae, 14-20 days after the start of the test, rotifers and artemia were fed simultaneously, and only artemia was fed from the 21st day to the 35th day of the end of the test. . The body length (mm), wet body weight (mg), and survival rate (%) of each test group were measured every week of the test period. In addition, about the body length, 30 fish / water tank was taken out at random, each individual was measured, and the average value was calculated. For wet body weight, 30 fish / water tanks were randomly taken on the 7th, 14th and 21st days, and the total body weight was measured.On the 28th and 35th days, 30 fish / water tanks were taken at random and each individual was measured. The average value was also calculated. The survival rate was calculated by counting the number of dead fish per fish tank, and the average value of each test area was calculated on the 14th day, which was 2 or 3 tanks / zone.

[result]
The results are shown in FIG. In the rotifer period (rotifer feeding period), the survival rate at 14 days of age was not significantly different in each test section. Regarding the wet weight and body length during the rotifer period, the algal (mh) group showed higher values than the control group and the competitor's product group. Regarding the survival rate during the Artemia period (Artemia feeding period), the survival rate in the control group had dropped to about 15% at the age of 21 days, so the test in the control group was discontinued. As for the survival rate during the Artemia period, the alga body (AJ7867) group showed a higher value than the other company's product group at all of 21, 28, and 35 days of age. Regarding the wet weight and body length during the Artemia period, the alga body (mh) group showed lower values than the other company's product group.

[Conclusion]
Feeding larvae larvae with rotifers enriched with AJ7867 strain alga body weight, body length compared to feeding larvae larvae with nutrient enrichment using comparison alga bodies There was a tendency for the survival rate to increase. Feeding larvae larvae with Artemia fortified using AJ7867 strain alga body weight and length compared to feeding larvae larvae fortified with larvae larvae There was a tendency for the value of to decrease, but the survival rate tended to increase. It is inferred that the effect of nutrient enhancement by the AJ7867 strain alga during the rotifer period is due to the supply of cholesterol contained in the AJ7867 strain alga to the flounder. On the other hand, since Artemia, which is a crustacean, contains cholesterol at a similar concentration in the alga body (mh) group and the other company's product group, it is presumed that the effect of nutrient enhancement by the AJ7867 strain alga body was not seen during the Artemia period Is done. From the above, it was suggested that feeding rotifers with nutrients enhanced with sterol-containing algal bodies such as cholesterol can promote the growth of aquatic products such as flounder.

<Explanation of Sequence Listing>
SEQ ID NOs: 1, 2: Primer SEQ ID NO: 3: Base sequence of 18S rDNA region of AJ7866 strain (FERM P-22289) SEQ ID NO: 4: Base sequence of 18S rDNA region of AJ7867 strain (FERM P-22304) SEQ ID NO: 5: AJ7868 Of the 18S rDNA region of the strain (FERM P-22290)

Claims (18)

  A fortifying agent for rotifer or artemia containing a sterol-containing algal body. The algae bodies are AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM
P-22290), and a derivative strain thereof, and an algal cell selected from Aurantiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5. Nutritional fortifier.   The nutrient fortifier according to claim 1 or 2, wherein the sterol is cholesterol. A method for producing a feed organism,
A method comprising incorporating a sterol-containing alga into a rotifer or artemia. The algae bodies are AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM
P-22290), and derivatives thereof, and an algal cell selected from a group of Aurantiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5. the method of.   The method according to claim 4 or 5, wherein the sterol is cholesterol. A prey organism,
A feed organism that is a rotifer or artemia containing a sterol-containing algal body. The algae bodies are AJ7866 strain (FERM P-22289), AJ7867 strain (FERM P-22304), AJ7868 strain (FERM
P-22290), and derivatives thereof, and algal cells selected from Aurantiochytrium algae having the base sequence shown in SEQ ID NO: 3, 4, or 5 Feeding organisms.   The bait organism according to claim 7 or 8, wherein the sterol is cholesterol.   The feed organism according to any one of claims 7 to 9, comprising cholesterol at a concentration of 0.02% (w / w) or more as a weight relative to a dry weight of the feed organism.   The bait organism according to any one of claims 7 to 10, further comprising docosahexaenoic acid and / or eicosapentaenoic acid.   The feed organism according to any one of claims 7 to 11, which is a feed organism for aquatic products.   The feed organism according to claim 12, wherein the aquatic product is a fish or a crustacean.   A method for cultivating an aquatic product, comprising feeding the aquatic product with the feed organism according to any one of claims 7 to 13.   15. The method of claim 14, wherein both the feed organism that is a rotifer and the feed organism that is Artemia are fed.   16. The method of claim 15, wherein the bait organism that is a rotifer is fed first and the bait organism that is artemia is fed later.   The method according to any one of claims 14 to 16, wherein the aquatic product is a fish or a crustacean.   18. The method of claim 17, wherein the feed organism is fed to fish during part or all of a larval stage.