JP4778792B2 - Animal plankton feed - Google Patents

Description

The present invention relates to a feed for zooplankton, which is a biological feed for seafood in which seedling production is performed, and a method for producing larvae and juvenile fish using the zooplankton fed with the feed.
The development of artificial seedling production technology has made it possible to produce seedlings for many marine animals such as red sea bream, Japanese flounder, black sea bream, trough pufferfish, sweetfish, flounder, oniokoze, and thistle. Furthermore, due to the dramatic improvement in technology in recent years, it has become possible to produce seedlings such as yellowtail, cucumber, red sea bream, tuna, and octopus, which were previously difficult to produce.
The reason why such seed production of fish species has become possible is the improvement of feed for enrichment of rotifer and artemia fed by larvae.

Eicosapentaenoic acid (hereinafter referred to as “EPA”) and docosahexaenoic acid (hereinafter referred to as “DHA”), which are highly unsaturated fatty acids, are particularly important for the fatty acid requirements of marine seafood during the larval or juvenile stage. It was clarified that it is an essential nutrient for improving production yield and controlling malformations.
However, EPA and DHA are essential nutrients especially in the larval and juvenile stage, and it is common practice today to feed zooplankton such as rotifer, artemia, and daphnia with enriched EPA and DHA. Yes.
The feed for zooplankton for the purpose of fortification includes: (1) a feed in which oils and fats containing fish oil, EPA, and DHA are added to artificially produced microcapsules and cultured microorganisms, and (2 ) There are two types of innately cultured microorganisms that self-produce EPA and DHA.
The type (1) is a classic method that has been conventionally used (for example, see Patent Document 1). On the other hand, in the feed using natural microorganisms of the type (2), cultivated and concentrated microorganisms producing EPA or DHA are individually present as commodities (see, for example, Non-Patent Document 1). ).

In addition, Nannochloropsis, a microorganism containing EPA, has been self-cultured at many seedling production institutions and used for nutrient enhancement. However, because the cell wall is hard, it is possible to enhance nutrition for rotifers that have developed a masticatory organ, but there is a problem that it is not possible to fortify artemia where the masticatory organ is relatively unachievable.
Therefore, by using the cell wall crushing process by forced dispersion treatment (hereinafter referred to as “digestible treatment”) using a high-pressure homogenizer, it is now possible to cultivate Nannochloropsis, a microorganism containing EPA, and enhance the nutrition of artemia. (See, for example, Patent Document 2).
Furthermore, recently, feed for larvae and fry fish focused on n-6 docosapentaenoic acid (hereinafter referred to as “n-6DPA”) has been widely put into practical use as having an effect of preventing malformation in seedling production. (For example, refer to Patent Document 3).
As described above, it has been found that EPA and DHA have an important effect in improving the production yield and controlling malformation in the larval or juvenile stage of seafood, and are currently being used. In order to show these effects, as an evaluation standard for healthy seedlings, that is, fish seedlings, emphasis has been placed on the parts that can be visually judged, such as the shape, color, and organ development.
In recent years, however, the scope of evaluation criteria has been expanded to further improve functional aspects of fish, such as digestive ability, mobility, swimming behavior, and swarming behavior.

Evaluation criteria important in terms of functions include water tank change, physical stress at the transfer stage, for example, resistance to handling stress when captured by a net, adaptability to different water temperatures, etc. (see, for example, Non-Patent Document 2) . Among them, especially for seedlings for release in cultivated fisheries, when released into a low water temperature sea area, if the vitality decreases due to stress after release, it is predicted that it will be immediately preyed by large fish, which is a problem. As an attempt to prevent this, various studies have been conducted on the location of discharge, such as the release to the vicinity of fish reefs.
In the field of early feed, various nutritional approaches have been carried out to improve the morphological abnormalities that have been made in the past, and to use stress as the evaluation criteria, in order to produce healthy seedlings. Yes.
Studies on the effects of lecithin and EPA on stress tolerance of European sea bream show that the increase in EPA in the presence of lecithin improves the resistance to sterilization due to handling and temperature changes (see Non-Patent Document 3).
Studies on the effects of EPA and DHA on vitality tests (handling resistance) of red sea bream larvae have shown that EPA has little effect and DHA has a relatively good effect (see Non-Patent Document 4). ).
However, none of the effects was sufficient, and did not reflect the effects on stress tolerance and adaptability to low water temperature at the actual seedling production site. Moreover, it did not have a sufficient effect in the actual site.
As described above, in recent years, as an index of seed and seedling production, tolerance to handling stress at the time of capture by nets, adaptability to different water temperatures, etc. are important, but the nutritional component approach of initial feed to improve these is The preliminary study is limited and it is extremely insufficient. In particular, there is no comprehensive approach centered on EPA, n-6DPA and DHA.

Patent No. 1992146 Japanese Patent Publication No. 4-8021 Japanese Patent Laid-Open No. 11-276091 Special issue on aquaculture "Additive Goods Best Guide", Midori Shobo Co., Ltd., March 10, 2000, Vol. 37, No. 4, p. 186-191 Takayuki Takahashi, “Environment and Stress in Seedling Production”, Aquanet, Keibunsha, July 2001, Vol. 4, No. 7, p. 62-65 JINGLE LIU, outside six, Necessity of dietary lecithin and eicosapentaenoic acid for growth, survival, stress resistance and lipoprotein formation in gilthead sea bream sparus aurata, "FISHERIES SCIENCE", Japan, Nippon Suisan Journal, 2002, Vol. 68, p. 1165-1172 Toshiro Takeuchi, “Neutral deficiency and required amount in fish”, cultivation and fishery technology training business basic theory course textbook V Development series of juvenile fish, Japan Cultivation Fisheries Association, 1991, Vol. 4, p. 20-23

The objective of this invention is providing the seedlings with which tolerance to the various stress in the seedling production of seafood was provided.
The subject of this invention is providing the feed for zooplankton which can produce a seedling seedling strong to low temperature stress and handling stress.
As a result of diligent efforts to solve these problems (problems), the inventors of the present invention have obtained a cell wall disrupted product of microorganisms containing eicosapentaenoic acid (hereinafter referred to as “EPA”), and n-6 docosapenta. By using a microorganism containing enoic acid (hereinafter referred to as “n-6DPA”) and docosahexaenoic acid (hereinafter referred to as “DHA”) as a feed for zooplankton, The present invention has been completed by finding out that the effect of preventing stress due to exposure and handling is exhibited. Therefore, this invention provides the feed which can provide low water temperature exposure tolerance and handling stress tolerance to fishery products, especially a juvenile fish in seedling production.

Moreover, this invention provides the feed for zooplankton whose eicosapentaenoic acid content in the total fatty acid of the lipid contained in the microorganisms containing an eicosapentaenoic acid is 10-50 mass% in this zooplankton feed.
The present invention also provides an zooplankton feed in which the microorganism containing eicosapentaenoic acid is a true eye-point alga Nannochloropsis.sp.
In the zooplankton feed according to the present invention, the content of n-6 docosapentaenoic acid in the total fatty acids of lipids contained in the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is 10 to 60% by mass. Zooplankton feed is provided.
Further, the present invention provides an zooplankton feed having a docosahexaenoic acid content of 20 to 80% by mass in total fatty acids of lipids contained in a microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid. Provide feed.

The present invention also provides an zooplankton zooplankton in which the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is Schizochytrium.sp or Thraustochytrium.sp. Provide feed.
In the zooplankton feed according to the present invention, the mass ratio of the microorganism-containing cell wall disrupted product containing eicosapentaenoic acid to the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is 1 as a solid matter. A feed for zooplankton that is 9-9: 1 is provided.
Moreover, this invention provides the zooplankton feed in which the zooplankton is 1 type (s) or 2 or more types selected from the group which consists of a rotifer, Artemia, and Daphnia.

In the zooplankton feed according to the present invention, the eicosapentaenoic acid content in the total fatty acids of lipids contained in the zooplankton feed is 5 to 27% by mass, and the n-6 docosapentaenoic acid content is 5 to 5%. There is provided an animal plankton feed having a content of 21% by mass and a docosahexaenoic acid content of 5 to 52% by mass. Thereby, this invention provides the feed for zooplankton containing the lipid which has a characteristic composition regarding EPA, n-6DHA, and DHA.
The present invention also provides a method for producing larvae and larvae, characterized in that the nutritionally enriched zooplankton that has been fortified by feeding the zooplankton feed to the zooplankton is used as a feed for the larvae. This provides a method for producing larvae and fish having resistance to low water temperature exposure and handling stress. In other words, the present invention provides a method for producing larvae and fishes that imparts low water temperature exposure resistance and handling stress resistance to larvae.
The present invention also provides a stress tolerance-imparting agent for fish and shellfish containing a cell wall disrupted product of a microorganism containing eicosapentaenoic acid and a microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid.

The feed for zooplankton characterized by containing a cell wall disrupted product of a microorganism containing eicosapentaenoic acid and a microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid in the present invention, at least ( 1) refers to a feed for zooplankton containing a cell wall disrupted product of a microorganism containing eicosapentaenoic acid, and (2) a microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid.
The “microorganism containing eicosapentaenoic acid (hereinafter referred to as“ EPA ”)” in the present invention is one containing EPA in the microorganism and / or one producing EPA in the microorganism when the microorganism is cultured. Say. EPA may be written as “C20: 5, n-3” or “C20: 5”.
Specific examples include the true eye-point algae Nannochloropsis.sp, the prasinoalga Tetraselmis.sp, and the diatom Keeteros.sp. Of these, the true eye-point alga Nannochloropsis.sp is preferable.

The method for handling these microorganisms is not particularly limited. For example, it is preferably used as a concentrate obtained by separating only microbial cells from a purely cultured culture liquid or a dried product thereof.
The “microorganism” in the present invention broadly includes those derived from microorganisms, and includes a cultured microbial cell, a dry cultured microbial cell, a treated cultured microbial cell, and a culture solution containing the microbial cell and a culture supernatant. Specifically, in addition to the microorganism itself, for example, a microorganism concentrate, a freeze-dried product, a spray-dried product, and the like are included.
The “cell wall disrupted product of microorganism containing EPA” in the present invention refers to a product obtained by forcibly dispersing a microorganism containing EPA using a high-pressure homogenizer or the like and substantially crushing the cell wall of the microorganism ( For example, see Patent Document 3). This process is sometimes referred to as a “digestible process”. The step of the crushing treatment is not limited to any time when preparing microorganisms. For example, digestion treatment may be performed in a state where microorganisms are cultured and cells are collected, After collecting and drying, the dried product may be dissolved or suspended in an arbitrary liquid and subjected to digestion.

The EPA content of the microorganism containing EPA in the present invention is not particularly limited. For example, the EPA content in the total fatty acids of lipids contained in the microorganism containing EPA is 10% by mass or more, preferably 10 to 50% by mass. More preferably, it is 15-48 mass%, More preferably, it is 20-45 mass%, Especially preferably, it is 23-43 mass%, Most preferably, it is 28-38 mass%.
The “lipid” in the present invention is generally sometimes referred to as “oil and fat”, “oil” and “oil”, and refers to, for example, a lipid extracted by the Bligh-Dyer method. The fatty acid content in the total fatty acids of lipids contained in feed and microorganisms is determined by a conventional method, and the method is not particularly limited. For example, after lyophilizing an analytical sample, lipids are extracted from the lyophilized product by the Bligh-Dyer method and methyl esterified, and then the fatty acid composition is calculated by gas chromatography, and the amount of the corresponding fatty acid is determined in the lipid. It can be determined by dividing by the total amount of fatty acids.
The amount of lipid in the microorganism containing EPA in the present invention is not particularly limited. The amount of lipid is greatly affected by the microorganism culture method and is difficult to specify, but is, for example, 1% by mass or more, preferably 3% by mass or more.
The presence state of EPA of microorganisms including EPA in the present invention is not particularly limited. For example, any of complex lipids such as triglyceride, diglyceride, monoglyceride, fatty acid, fatty acid methyl ester, fatty acid ethyl ester, phospholipid and glycolipid may be used.

In the present invention, “microorganism containing n-6 docosapentaenoic acid (hereinafter referred to as“ n-6DPA ”) and docosahexaenoic acid (hereinafter referred to as“ DHA ”)” refers to n-6DPA and DHA in the microorganism. And / or those that produce n-6DPA and DHA in microorganisms when cultured. n-6DPA may be expressed as “C22: 5, n-6” or “C22: 5”, and DHA may be expressed as “C22: 6, n-3” or “C22: 6”.
Specific examples include the genus Schizochytrium. Sp, the genus Thraustochytrium. Sp, the genus Ulkenia, and the genus Altornia. Among them, preferred is Schizochytrium.sp or Thraustochytrium.sp. More specifically, ATCC 20888, 20889, 20891, 24473, 28209, 28221, 34304 and the like can be mentioned. However, the present invention is not limited to these.
The method for handling these microorganisms is not particularly limited. For example, it is preferably used as a concentrate obtained by separating only microbial cells from a purely cultured culture liquid and a dried product thereof.

The n-6DPA content in the microorganism containing n-6DPA and DHA in the present invention is not particularly limited. For example, the n-6DPA content in the total fatty acids of lipids contained in the microorganism containing n-6DPA and DHA is 5 to 5% by mass, preferably 5 to 60% by mass, more preferably 11 to 45% by mass, further preferably 11 to 30% by mass, particularly preferably 11 to 25% by mass, and most particularly preferably 13 to 20%. % By mass.
The DHA content in the microorganism containing n-6DPA and DHA in the present invention is not particularly limited. For example, the DHA content in the lipid of the microorganism containing n-6DPA and DHA is 20% by mass or more, preferably 20 It is -80 mass%, More preferably, it is 30-70 mass%, More preferably, it is 30-60 mass%, Most preferably, it is 30-50 mass%, Most preferably, it is 33-43 mass%.
The ratio of the n-6DPA and DHA content of the microorganism containing n-6DPA and DHA in the present invention is not particularly limited. For example, a microorganism having an arbitrary ratio (arbitrary combination) of the n-6DPA content of the microorganism containing the above n-6DPA and DHA and the DHA content of the microorganism containing the above n-6DPA and DHA, It can be used in the invention. Specifically, for example, the n-6DPA content in the total fatty acids of lipids contained in microorganisms containing n-6DPA and DHA is 10 to 30% by mass, and the DHA content is 30 to 70% by mass. It is done.

The amount of lipid in the microorganism containing n-6DPA and DHA in the present invention is not particularly limited. The amount of lipid is greatly affected by the culturing method of the microorganism and is difficult to specify, but is, for example, 5% by mass or more, and preferably 30% by mass or more.
The presence state of the EPA of the microorganism containing n-6DPA and DHA in the present invention is not particularly limited. For example, any of complex lipids such as triglyceride, diglyceride, monoglyceride, fatty acid, fatty acid methyl ester, fatty acid ethyl ester, phospholipid and glycolipid may be used.
In the zooplankton feed according to the present invention, the mass ratio of the microorganism-containing cell wall disrupted product containing EPA and the microorganism containing n-6DPA and DHA is not particularly limited. 1: 9 to 9: 1 as a product, preferably 1: 1 as a solid product.
The presence of the microorganism used in the present invention can be usually identified by confirming the shape with an optical microscope and the fatty acid composition in the oil. For example, Schizochytrium / Trauskitrium algae having n-6DPA are spherical cells having a diameter of 6 to 7 μm and having no chlorophyll.
In the present invention, the solid material is not particularly limited as long as the appearance is solid, powder or block. Also, it is not liquid, paste or suspension. In the present specification, a powdery material may be referred to as a “dry powder” as being included in the category of a solid material.

When adjusting the mass ratio, the cell wall disrupted product of microorganisms containing EPA may be a solid as described above, and the moisture content is not limited. For example, the moisture content is 10% by mass or less, preferably Is 9 to 0.1 mass%, more preferably 8 to 0.1 mass%.
When adjusting the mass ratio, the microorganism containing n-6DPA and DHA may be a solid substance as described above, and the water content is not limited. For example, the water content is 10% by mass or less, preferably Is 8 to 0.1% by mass, more preferably 7 to 0.1% by mass, still more preferably 6 to 0.1% by mass, particularly preferably 5 to 0.1% by mass, and most particularly preferably 4 to 0. 1% by mass.
The feed for zooplankton according to the present invention can be provided in any form regardless of the form. For example, it can be provided as a solid (solid, powder), paste, liquid (solution, solution) or suspension (suspension).
Moreover, even when it makes liquid (solution, solution form) or suspension (suspension form), the quantity of the feed with respect to the water | moisture content is not limited, A density | concentration does not ask | require. For example, 0.1 to 100 mass of solution can be mixed, dissolved or suspended with respect to 1 mass of feed.

An example of the manufacturing method of the feed for zooplankton concerning this invention is shown.
When adjusting the mass ratio in the present invention, a powdered zooplankton feed can be provided by mixing powdered microorganisms. For example, 100 g of a dry powder having a water content of 8% by mass or less obtained by freeze-drying a cell wall crushed microorganism containing EPA, and a dry powder having a water content of 4% by mass or less spray-dried by a microorganism containing n-6DPA and DHA. What mixed 100g is mentioned. The mixing method of the dry powder derived from each microorganism is not particularly limited.
Moreover, each microorganism can be made into powder form, each can be mixed with arbitrary liquids, can be made into a solution form or a suspension form, and can be mixed after that. Thereby, the feed for solution or suspension zooplankton can be provided. For example, 100 g of dry powder having a water content of 8% by mass obtained by freeze-drying microorganisms containing EPA and adding 1 liter of distilled water to a suspension obtained by crushing the cell wall, and water obtained by spray-drying microorganisms containing n-6DPA and DHA Examples thereof include a mixture of 100 g of dry powder having a content of 4% by mass added to 1 liter of distilled water and agitated suspension. The mixing method of each microorganism-derived solution or suspension is not particularly limited. In addition, the solution or suspension after mixing can be provided again as a dry product in the form of a solid or powder.
When providing a zooplankton feed in the form of a solution or suspension, the amount of microorganisms in the liquid is not particularly limited, but is usually 15% by mass or less, preferably 15 to 5% by mass. 12.5, 10, 7.5, 5 mass%.

The term “zooplankton” as used in the present invention refers to rotifers (such as hornworms), artemia (brine shrimp), daphnia and the like that are generally used as biological feeds in seedling production. These may be used alone or in combination of two or more.
In the present invention, “zooplankton feed” refers to feed for feeding zooplankton. The main purpose of feeding the feed to the zooplankton is to accumulate nutrients (eg, EPA, n-6DPA, DHA, etc.) in the zooplankton (fortification).
In the present invention, the term “enriched zooplankton” refers to an zooplankton in which nutrients in the zooplankton (for example, EPA, n-6DPA, DHA, etc.) are enhanced by feeding the zooplankton feed to the zooplankton. Say. The enriched zooplankton is used by feeding larvae and juveniles during seedling production.
In the present invention, the conditions for primary culture of zooplankton and secondary culture with enrichment of nutrients can be carried out by those skilled in the art by ordinary methods. Specific culture conditions can be corrected with time correction within the knowledge of those skilled in the art.

In the present invention, when such zooplankton feed is used for nutrient enhancement, the amount of the feed added is not particularly limited. This can be done in the usual way by a person skilled in the art. The specific concentration can be corrected with time within the knowledge of those skilled in the art. For example, it can be added at 0.2 g / liter as a solid material of the feed. Specifically, in addition to 100 g of microbial solids containing EPA and 1 liter of distilled water, cell destruction treatment and 100 g of microbial solids containing n-6DPA and DHA are added to 1 liter of distilled water. And agitate the mixture (thus, the feed will be 200 g / 2 liter). The mixture is then used in the zooplankton culture at a concentration of 2 ml / liter (thus, the feed is 200 mg / 2 ml / liter, which translates to 0.2 g / liter of feed).
In the present invention, the contents of EPA, n-6DPA and DHA in the zooplankton feed are not particularly limited.
In the present invention, the EPA content in the zooplankton feed is not particularly limited. For example, the EPA content in the total fatty acids of lipids contained in the feed is 5 to 35% by mass, preferably 5 to 30% by mass. %, More preferably 5 to 27% by mass, still more preferably 5 to 25% by mass, particularly preferably 5 to 20% by mass, and most particularly preferably 10 to 20% by mass.

In the present invention, the n-6DPA content in the zooplankton feed is not particularly limited. For example, the n-6DPA content in the total fatty acids of lipids contained in the feed is 5 to 40% by mass, preferably It is 5 to 30% by mass, more preferably 5 to 21% by mass, further preferably 6 to 20% by mass, particularly preferably 7 to 18% by mass, and most particularly preferably 7 to 15% by mass.
In the present invention, the DHA content in the zooplankton feed is not particularly limited. For example, the DHA content in the total fatty acids of lipids contained in the feed is 5 to 60% by mass, preferably 5 to 55% by mass. %, More preferably 5 to 52% by mass, still more preferably 10 to 40% by mass, particularly preferably 15 to 35% by mass, and most particularly preferably 25 to 35% by mass.
In the present invention, the ratio of the EPA, n-6DPA and DHA content in the total fatty acids of the lipids contained in the zooplankton feed is not particularly limited. For example, lipids having an arbitrary ratio (arbitrary combination) of EPA, n-6DPA and DHA content to the total fatty acids in the lipids of the zooplankton feed can be used in the present invention. Specifically, for example, the fatty acid content in the total fatty acids of lipids contained in such zooplankton feed is 5 to 27% by mass of EPA, 5 to 21% by mass of n-6DPA, and 5 of DHA. A feed for zooplankton that is ˜52 mass% can be provided.

In the present invention, the amount of lipid in the zooplankton feed is not particularly limited. The amount of lipid varies greatly depending on the culture method and mixing ratio of each microorganism, and cannot generally indicate the content.
In the present invention, the presence state of the lipid in the zooplankton feed is not particularly limited. For example, any of complex lipids such as triglyceride, diglyceride, monoglyceride, fatty acid, fatty acid methyl ester, fatty acid ethyl ester, phospholipid and glycolipid may be used.
From another point of view, the present invention adjusts the mixing mass ratio between the microorganism containing EPA or the cell wall crushed material and the microorganism containing n-6DPA and DHA, so that EPA, n-6DPA and DHA Provided is a zooplankton feed for adjusting the content and a method for adjusting or producing a lipid. Moreover, the lipid content obtained by this adjustment method or production method can be adjusted to the content of EPA, n-6DPA, and DHA effective for seedling production of larvae and juvenile fish. Furthermore, the adjustment method or the production method can provide feed and fats and oils with contents of EPA, n-6DPA and DHA that are effective for seedling production of larvae and juveniles.
In another aspect of the present invention, the constituent fatty acid content in the total fatty acids of the lipid is 5 to 27% by mass of EPA, 5 to 21% by mass or more of n-6DPA, and 5 of DHA. The lipid which is -52 mass% can be provided.

The method of using these lipids is not particularly limited, and can be used alone or in combination with others. These can be used for, for example, zooplankton feed, larval and fish production methods, etc., for the production of seeds for all seafood, and can also be used for all pharmaceuticals and foods.
In the present invention, a lipid having a specific ratio of EPA, n-6DPA and DHA content in the total fatty acids of lipids contained in the zooplankton feed, and the feed containing the lipid include microorganisms containing EPA and / or Even without using microorganisms containing n-6DPA and DHA, each component is prepared and mixed, or each component is triglyceride, diglyceride, monoglyceride, fatty acid, fatty acid methyl ester, fatty acid ethyl ester, phospholipid. It can be realized by mixing with any of complex lipids such as glycine and glycolipid. However, the use of a microorganism containing EPA according to the present invention and a microorganism containing n-6DPA and DHA is simple in production, industrially efficient, and inexpensive.
In addition, a zooplankton feed using a microorganism containing EPA according to the present invention and a microorganism containing n-6DPA and DHA, and each component is prepared and included in the zooplankton feed according to the present invention. In the case where the EPA, n-6DPA and DHA contents in the total fatty acid of the lipid are set to the same ratio, the former is preferable in terms of the effect.

In the case of fish, seed production is generally a process of growing from hatched larvae to fry. The fry obtained by these seedling production is then used for release and aquaculture. Therefore, if seedling production is successful, larvae with high survival rate, high vitality, and larvae with various stress tolerances can be produced, it will be very useful for subsequent cultivation.
According to another aspect of the present invention, there is provided a nutrition-enhanced zooplankton that is fortified by feeding the zooplankton feed to the zooplankton.
According to another aspect of the present invention, there is provided a method for producing a nutrition-enhanced zooplankton that is fortified by feeding the zooplankton feed to the zooplankton.

According to another aspect of the present invention, there is provided a zooplankton feeding method for obtaining a nutrition-enhanced zooplankton that is enriched by feeding the zooplankton feed to the zooplankton.
According to another aspect of the present invention, there is provided a larvae and juvenile fish characterized by using the nutrition-enriched zooplankton fortified by feeding the zooplankton feed to the zooplankton as feed for the larvae.
According to another aspect of the present invention, there is provided a feeding method for larvae and larvae, characterized in that the nutritionally enriched zooplankton fortified by feeding the zooplankton feed to the zooplankton is used as bait for the larvae and larvae. provide.
In the present invention, there is provided a method for producing larvae and larvae, characterized in that the nutritionally enriched zooplankton, which has been fortified by feeding the zooplankton feed to the zooplankton, is used as bait for the larvae.

According to another aspect of the present invention, there is provided a larvae and larvae obtained by using the zooplankton feed for seedling production of larvae and juveniles.
According to another aspect of the present invention, there is provided a seedling production method for larvae and larvae characterized in that such zooplankton feed is used for seedling production of larvae and juveniles.
According to another aspect of the present invention, there is provided a production method for larvae and larvae, characterized in that such zooplankton feed is used for seedling production of larvae and larvae.
The zooplankton feed according to the present invention can be used by mixing feed components other than the feed. The feed components other than the feed are not particularly limited, and those used as normal feed for zooplankton can be used. For example, fresh water chlorella, yeast, fish oil, phospholipid and the like can be mentioned. Furthermore, the feed for zooplankton according to the present invention can be used for fish and shellfish in the feed and feed components other than the feed simultaneously or separately.

The fish and shellfish that can perform seed and seedling production using the feed for zooplankton according to the present invention are not particularly limited, and can be used for all those that perform seed and seed production using zooplankton. Examples include fishes such as red sea bream, Japanese flounder, black sea bream, tiger pufferfish, flounder, scorpionfish, sea bream, sweetfish, sea bream, sea bream, red sea bream, tuna, etc., crustaceans such as crab, tiger prawns, cephalopods such as octopus, squid, etc. . Of these, red sea bream, Japanese flounder, black sea bream, trough and flounder are preferred, and red sea bream and Japanese flounder are more preferred.
The feed for zooplankton according to the present invention is excellent in improving the functional aspects of seafood while maintaining the effects that can be recognized with conventional sight such as improving the yield of seafood (improving survival rate) and controlling malformations. It has an effect. Improvement of the functional aspect of seafood means improvement of the internal functional aspect of seafood, although it cannot be recognized visually. For example, it refers to an improvement in resistance such as stress in water tank change, physical stress in the transfer stage, handling stress when captured by a net, and stress when transferred to a different water temperature. Conventionally, when the functional aspects of seafood are low, the survival rate of seafood is reduced by these processes, and seedling production may not be successful.
More specifically, the effect of the zooplankton feed according to the present invention can be obtained by feeding the zooplankton to the zooplankton and using the nutrition-enriched zooplankton as a feed for the larvae and larvae. Because the stress tolerance of larvae and larvae is improved, the survival rate and activity of larvae and larvae will be reduced when the larvae are changed, transferred, captured by the net, or transferred to tanks with different water temperatures. Can be prevented. As a result, seedling production becomes even better than the current situation.

The effects of the zooplankton feed according to the present invention are generally considered to have effects such as production yield improvement (survival rate improvement) and malformation control. (A) It contains a relatively large amount of EPA, and includes DPA and DHA. Compared with (b) a microorganism containing a relatively large amount of DPA and DHA and a microorganism containing a small amount of EPA (5% by mass or less based on the total fatty acid in the lipid). In particular, the feed has a significant effect on the resistance of larvae to air exposure and low water temperature exposure.
In the air exposure test, “handling stress resistance” and “stress prevention effect by handling” referred to in this specification are measured. The test is performed, for example, by placing the larvae after breeding for a certain period of time on a net, exposing them to the air for a certain period of time (for example, 120 seconds), and then transferring them to the same water temperature for a certain period (for example, The measurement can be carried out by measuring the survival rate after 24 hours). As a result, if vitality is maintained and the survival rate of larvae and fish is high (if they do not die), naturally, larvae are less likely to die when handled during seedling production, resulting in successful seedling production. It is very useful for the fishery industry.
In the low water temperature exposure test, “low water temperature exposure resistance (low temperature stress resistance)” and “stress prevention effect by low water temperature exposure” referred to in this specification are measured. In this test, for example, the larvae that have been raised for a certain period of time at a water temperature of 21 ° C. are transferred to another water tank at a water temperature of 12 ° C., 13 ° C., 14 ° C., or 15 ° C. This can be done by measuring the survival rate. As a result, if vitality is maintained and the survival rate of larvae and fish is high (if they do not die), naturally, larvae are less likely to die when handled during seedling production, resulting in successful seedling production. It is very useful for the fishery industry.

According to the present invention, by using a cell wall disrupted product of microorganisms containing EPA and a microorganism containing n-6DPA and DHA as a feed for zooplankton, the low water temperature of larvae and juveniles in seedling production It has the effect of preventing stress from exposure and handling.
That is, according to the present invention, by using the zooplankton fortified by feeding the zooplankton feed to the zooplankton, the nutritionally enriched zooplankton is used as a food for the larvae and larvae. Stress tolerance) and handling stress tolerance are improved. In addition, zooplankton such as Artemia and rotifer fed with the zooplankton feed of the present invention has an advantage that its vitality does not decrease.
These effects are unprecedented and very effective, and are very useful for the production of seafood seedlings, particularly larvae.
Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

[Adjustment of feed for zooplankton and adjustment of nutrient-enriched zooplankton]
(1-1) Preparation of feed for zooplankton As a raw material for feed for zooplankton, about 3 kg of cells were isolated from 100 liters of the culture solution of Schizochytrium ATCC 20891 strain. The cells were then freeze-dried and then pulverized to obtain 1.2 kg of dry powder. Distilled water was added to 100 g of this dry powder to make 1 liter, and test feed A dispersed with a homogenizer was obtained.
Test feed A corresponds to a microorganism containing n-6DPA and DHA.
In addition, 4 kg of cells were obtained from 5000 liters of Nannochloropsis culture and spray-dried. 100 g of the dry powder was added to distilled water to make 1 liter, and the cell wall was crushed with a high-pressure homogenizer to obtain test feed B.
Test feed B corresponds to a microbial cell wall disruption containing EPA.
Separately, 500 ml of A feed and 500 ml of B feed were prepared and mixed to obtain test feed C.
Test feed C corresponds to the feed for zooplankton according to the present invention.
Test feeds A to C were used as fatty acid analysis samples, respectively.
Table 1 shows the fatty acid composition of the test feed. The fatty acid composition was analyzed by gas chromatography after freeze-drying the fatty acid analysis sample, extracting lipids by the Bligh-Dyer method, and methyl esterifying. The ratio (mass%) of the constituent fatty acid was determined from the area percentage.
The water content of the dry powder, which was a freeze-dried microbial cell used in test feed A, was 3.7%, and the oil content was 58.5%. Moreover, the water | moisture content of the dry powder which is the spray dried product used for the test feed B was 7.0%, and the oil content was 7.5%.

(1-2) Adjustment of nutrient-enriched zooplankton The zooplankton culture solution, which is enriched by adding test feed, is prepared by cultivating rotifers in freshwater chlorella to 1000 individuals / ml, and Artemia is adjusted to 100 individuals / ml. In either case, the water temperature was maintained at 25 ° C. Various test feeds were added to each so as to have a concentration of 2 ml / liter, and after 2 hours or more (enriched zooplankton) was used as a fatty acid analysis sample and used as test fish food. Here, the rotifer and artemia enriched with nutrients are referred to as rotifer A to C and artemia A, respectively.
Table 2 shows the fatty acid composition of the fortified rotifer and Table 3 shows the fatty acid composition of the fortified artemia. The fatty acid composition was analyzed by gas chromatography after freeze-drying the fatty acid analysis sample, extracting lipids by the Bligh-Dyer method, and methyl esterifying. The ratio (mass%) of the constituent fatty acid was determined from the area percentage.
From these, it can be seen that the fatty acid composition of the test feed reflects the fatty acid composition of the zooplankton fortified.
Using the rotifer and artemia fortified as described above, breeding tests were carried out using red sea bream in Examples 2 and 3, respectively.

Table 1 shows the fatty acid analysis values of various test feeds. The unit is mass%. “ND” in the table means undetected.
Table 2 shows the fatty acid analysis values of rotifers fortified with various feeds. The unit is mass%. “ND” in the table means undetected.
Table 3 shows fatty acid analysis values of Artemia fortified with various feeds. The unit is mass%.

[Raising test and aerial exposure test in red sea bream using fortified rotifers]
(2-1) Breeding test in red sea bream using nutrient-enriched rotifer Using each of the rotifers (nutrient-enriched rotifers AC) fed with test feed AC obtained in Example 1 for the AC sections, A breeding test was conducted using red sea bream on the test fish.
The self-collected red sea bream fertilized eggs were housed in a 500-liter polycarbonate water tank with 12,500 grains in each group, hatched at the egg-laying water temperature, and then the water temperature was gradually raised to 21 ° C. The hatching rate was 98% or more in each ward, and there was no difference.
The addition of nutrient enriched rotifers A to C was carried out once a day after adjusting the amount of rotifers to be maintained at 5 individuals / ml from the third day after hatching in each group. The amount of rotifer was adjusted to maintain 8 individuals / ml from the 10th day after hatching and 10 individuals / ml from the 15th day and fed twice a day in the morning and evening, and the breeding test was completed on the 20th day after hatching.
The fish body after breeding was used as a fatty acid analysis sample.
After the breeding, the fatty acid composition of the fish was analyzed, and the survival rate and average total length of each section were calculated.

(2-2) Aerial exposure test in red sea bream using nutrient-enriched rotifer Further, as an aerial exposure test, 100 tailed red sea breams were exposed in the air for 60 seconds using a net, and then transferred to another water tank of the same water temperature. The survival rate after 24 hours was observed.
Table 5 shows the fatty acid composition of the fish after the breeding test, and Table 5 shows the results of the average total length, the survival rate after survival, and the survival rate after exposure in the air. Group A refers to the group for administration of fortified rotifer A, Group B refers to the group for administration of fortified rotifer B, and Group C refers to the group for administration of fortified rotifer C. The fatty acid composition was analyzed by gas chromatography after freeze-drying the fatty acid analysis sample, extracting lipids by the Bligh-Dyer method, and methyl esterifying. The ratio (mass%) of the constituent fatty acid was determined from the area percentage.
In Table 4, it can be seen that the A zone has the highest DHA composition compared to the other zones, the B zone has the highest EPA composition, and the C zone contains EPA, n-6DPA and DHA compositions in a well-balanced manner. From these, it can be seen that the fatty acid composition of the test feed and the fortified rotifer is reflected in the fatty acid composition in the fish after the breeding test.

According to Table 5, there was no big difference in the average total length of each ward, the survival rate was inferior to B ward, and A ward and C ward were almost equal. In the aerial exposure test (handling stress test), the survival rate was the highest at 95.3% in the C zone, followed by the A zone and the B zone. That is, the group using the feed containing EPA, n-6DPA and DHA composition in a well-balanced manner was most effective.
Table 4 shows the fatty acid composition of red sea bream fish after the rotifer feeding test. The unit is mass%.
Table 5 shows the test results of red sea bream fed with various nutrient enriched rotifers. The unit is mass%.

[Raising test, air exposure test, and low water temperature exposure test in red sea bream using nutrient-enhanced artemia]
(3-1) Breeding test in red sea bream using nutrient-enhanced Artemia Using Artemia (nutrient-enhanced Artemia A to C) fed with test feeds A to C obtained in Example 1, respectively, for A to C groups, A breeding test was conducted using red sea bream on the test fish.
The red sea bream fertilized eggs collected in-house were used until the 20th day after hatching with rotifers enriched with fish egg extract oil. Each fish tank in each ward was housed in a 500 liter polycarbonate water tank, and the water temperature was 21 ° C. The hatching rate was 98% or more in each ward, and there was no difference.
Up to 30 days after hatching, rotifers enriched with fish egg extract oil and Artemia (nutrient-enhanced Artemia A to C) fortified with each district test feed are used in combination, and then only fortified Artemia A to C until the 36th day Feeding.
Rotifers enriched with fish egg extract oil are fed twice a day in the morning and evening, adjusted to an amount of 10 individuals / ml in the breeding water, and the enriched Artemia A to C are consumed within 2 hours in the morning and evening. The feed was gradually increased.

The fish body after breeding was used as a fatty acid analysis sample.
After the breeding, the fatty acid composition of the fish was analyzed, and the survival rate and average total length of each section were calculated.
(3-2) Aerial exposure test in red sea bream using nutrient-enriched rotifers In addition, as an aerial exposure test, 100 tailed red sea breams were transferred to a separate aquarium after 120 seconds of air exposure using a net, and survived 24 hours later. I saw the rate.
(3-3) Low water temperature exposure test in red sea bream using nutrient-enhanced artemia Further, as a low water temperature exposure test, 100 tailed red sea bream were exposed to low water temperatures of 12 ° C, 13 ° C, 14 ° C, and 15 ° C. The survival rate after 30 minutes was seen.
Table 6 shows the fatty acid composition of the fish after breeding, and Table 7 shows the average length, survival rate, air exposure test, and survival rate in the low-temperature exposure test after breeding. Group A refers to the nutrient enriched Artemia A administration group, Group B refers to the nutrient enriched Artemia B administration group, and Group C refers to the nutrient enriched Artemia C administration group. The fatty acid composition was analyzed by gas chromatography after freeze-drying the fatty acid analysis sample, extracting lipids by the Bligh-Dyer method, and methyl esterifying. The ratio (mass%) of the constituent fatty acid was determined from the area percentage.

In Table 6, it can be seen that the A zone has the highest DHA composition compared to other zones, the B zone has the highest EPA composition, and the C zone contains EPA, n-6DPA and DHA compositions in a well-balanced manner. . From these, it can be seen that the fatty acid composition of the test feed and fortified artemia reflects the fatty acid composition in the fish after the breeding test.
From Table 7, there was no big difference in the average total length of each section, and no difference was seen in the survival rate. On the other hand, in the aerial exposure test, the A and B wards were almost unchanged, and the highest survival rate was seen in the C ward. That is, the group using the feed containing EPA, n-6DPA and DHA composition in a well-balanced manner was most effective.
Moreover, the survival rate by exposure to low water temperature was the highest survival rate in the C zone, followed by the A zone and the B zone when exposed to 12 ° C. The same order was obtained at 13 ° C. At 14 ° C. and 15 ° C., the survival rate was particularly low in the B zone, but no difference was observed between the A and C zones. Group B had a particularly low survival rate compared to Groups A and C in all exposure tests at 12 to 15 ° C. That is, the group using the feed containing EPA, n-6DPA and DHA composition in a well-balanced manner was most effective.

The fact that DHA is effective in improving the resistance of the air exposure test in the air exposure test was a result consistent with previous reports (see Non-Patent Document 4). However, in European sea bream, it has been reported that resistance to low water temperature stress is improved by increasing EPA in the presence of lecithin (see Non-Patent Document 3). However, in this result, the presence of EPA alone in the fish body is low. It was clarified that there was no effect of improving water temperature tolerance, which was slightly improved by the presence of n-6DPA and DHA in the fish body, and that the EPA, n-6DPA and DHA were present in a well-balanced manner in the fish body.
Table 6 shows the fatty acid composition of red sea bream fish after the Artemia feeding test. The unit is mass%.
Table 7 shows the test results of red sea bream fed with various nutrient enriched artemia. The unit is mass%.

Claims (11)

  A cell wall disrupted product of a microorganism having an eicosapentaenoic acid content of 10 to 50% by mass in the total fatty acids of the contained lipid, and a microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid, An agent for imparting resistance to air exposure of larvae and larvae comprising zooplankton fed with a feed containing a microorganism containing 5 to 60% by mass of n-6 docosapentaenoic acid in total fatty acids.   The agent for imparting resistance to air exposure of larvae and juveniles according to claim 1, wherein the microorganism containing eicosapentaenoic acid is a true eye-point alga Nannochloropsis sp.   The agent for imparting resistance to air exposure of larvae and juveniles according to claim 1 or 2, wherein the content of docosahexaenoic acid in the total fatty acid of lipids contained in the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is 20 to 80% by mass. .   The content of n-6 docosapentaenoic acid in the total fatty acids of lipids contained in the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is 5 to 60% by mass, and the content of docosahexaenoic acid is 20 to 80% by mass. The tolerance imparting agent with respect to the air exposure of the larvae and juveniles of any one of Claim 1 to 3.   The n-6 docosapentaenoic acid content in the total fatty acids of lipids contained in the microorganisms containing n-6 docosapentaenoic acid and docosahexaenoic acid is 10-30% by mass, and the docosahexaenoic acid content is 30-70% by mass. The tolerance imparting agent with respect to the air exposure of the larvae and juveniles according to any one of claims 1 to 4.   The larva or juvenile fish according to any one of claims 1 to 5, wherein the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is a genus Schizochytrium.sp or a genus Thraustochytrium.sp. Tolerant to air exposure.   The mass ratio of the cell wall disrupted product of a microorganism containing eicosapentaenoic acid to the microorganism containing n-6 docosapentaenoic acid and docosahexaenoic acid is 1: 9 to 9: 1 as a solid substance. The tolerance imparting agent with respect to the air exposure of the larvae and juveniles of any one of these.   The tolerance imparting agent with respect to the air exposure of the larvae and juvenile fish according to any one of claims 1 to 7, wherein the content of eicosapentaenoic acid in the total fatty acid of the lipid contained in the tolerance imparting agent is 5 to 35% by mass.   The eicosapentaenoic acid content in the total fatty acid of the lipid contained in the tolerance-imparting agent is 5 to 27% by mass, the n-6 docosapentaenoic acid content is 5 to 21% by mass, and the docosahexaenoic acid content is 5 to 5%. It is 52 mass%, The tolerance imparting agent with respect to the air exposure of the larvae and juveniles of any one of Claim 1 to 8. A method for producing larvae and fishes, comprising: providing the larvae with a resistance-imparting agent for exposure to larvae in the air according to any one of claims 1 to 9.   The method for producing larvae and juvenile fish according to claim 10, wherein the zooplankton is one or more selected from the group consisting of rotifer, artemia and daphnia.