JP2004057206A - Feed - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an inexpensive and efficient feeding method for improving survival rate and vital force of cultured larvae and juveniles. <P>SOLUTION: The feed for larvae and juveniles is obtained by feeding labyrinthula which produces carotenoid together with high unsaturated fatty acid of n-3 series to zooplanktons such as rotifer and Artemia to be bait of the larvae and juveniles. The feed enables stability and efficient transfer of both the effective components. The feed can greatly improve conventional fatty acid, pigment deterioration and feed preservation stability. The culture method using the feed is also provided. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a feed containing a carotenoid containing a carotenoid pigment and / or a xanthophyll pigment and a microorganism capable of producing polyunsaturated fatty acids.
The present invention also relates to a method for producing a feed containing a microorganism capable of producing carotenoids and polyunsaturated fatty acids.
Furthermore, the present invention relates to a method for cultivating fish and larvae using a feed containing a microorganism capable of producing carotenoids and polyunsaturated fatty acids.
[0002]
[Prior art]
Marine fish require n-3 series polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as essential fatty acids, and rotifers (Brachionus sp.) And artemia ( Artemia sp.) And the like are enriched with these fatty acids to improve the growth, survival rate and vitality of larvae and larvae.
Several techniques have been disclosed as methods for obtaining rotifers or artemia that are enriched with such n-3 series polyunsaturated fatty acids. As a typical example, a method of directly adding seafood oil emulsified to a rotifer tank is known. This is a method that expects seaweed oil to be attached to rotifers rather than fed to rotifers and fed to larvae as they are, but there are problems with culture tanks and water pollution and effects. . In addition, yeasts that produce n-3 series highly unsaturated fatty acids called oil-and-fat yeast or yeast microcapsules in the cells, or are added from the outside and incorporated into the cells, have been developed as feed for enriching rotifers. ing. For example, a method of adding n-3 series polyunsaturated fatty acid or ester to a microorganism such as yeast during cultivation and culturing the microorganism to contain n-3 series polyunsaturated fatty acid (Patent Documents 1 to 3) 4), a method using a microorganism having an ability to produce n-3 series polyunsaturated fatty acids (see Patent Documents 5 to 7), and an aqueous component in yeast cells is extracellularly treated by pH treatment or enzyme treatment. Using a yeast residue eluted in a mixture and contacting it with a hydrophobic substance such as an n-3 series polyunsaturated fatty acid, thereby allowing the cells to contain the n-3 series polyunsaturated fatty acid (Patent Document 8-9) have been developed. However, these methods have problems such as deterioration of water quality and lack of lipid accumulation in rotifers. Recently, as a method for solving this problem, a method has been proposed in which labulinchula containing only n-3 series of highly unsaturated fatty acids is fed to zooplankton such as rotifers and artemia and fed to larvae and juveniles (Non-Patent Document 1). reference). In this report, Hayashi et al. Point out that many strains of Labyrinthura have problems in water dispersibility, and die when rotifers and artemia are eaten.
[0003]
On the other hand, carotenoids such as β-carotene, canthaxanthin, and astaxanthin are attracting attention not only as health-enhancing materials but also as feed enhancers in fisheries and fish color tone improvers as an enhancer of immune resistance and a scavenger of active oxygen. . In order to give these pigments to larvae and juveniles as a marine feed, a method of feeding and transferring them to zooplankton such as rotifers and artemia is common as in the case of the above-mentioned n-3 series unsaturated fatty acids. . For example, a method using Phaffia rhodozyma yeast (see Patent Documents 10 to 13), a method using Haematococcus algae (see Patent Document 14), a method using Labyrinthula (see Patent Document 15), and the like are disclosed. ing.
[0004]
If n-series polyunsaturated fatty acids and carotenoids such as astaxanthin are fed simultaneously to rotifers and zooplankton such as artemia and given to larvae and juveniles, both effects derived from each of the above components can be expected, The following three methods are mainly considered as a method for producing zooplankton in which the -3 series of highly unsaturated fatty acids and carotenoids are simultaneously fortified. {Circle around (1)} A method of feeding zooplankton a feed (yeast or the like) containing n-3 series unsaturated fatty acids and a feed (yeast, algae, seafood oil or the like) containing a carotenoid. {Circle over (2)} A method in which carotenoids are adsorbed or encapsulated in a feed (yeast) containing n-3 series of highly unsaturated fatty acids and fed to zooplankton. {Circle over (3)} This is a method in which n-3 series of highly unsaturated fatty acids are adsorbed or included in a feed (yeast or algae) containing carotenoids and fed to zooplankton. However, although the method (1) is the simplest method, it has problems that two kinds of feed must be prepared and that it is difficult to control the degree of reinforcement of both components. The method (2) has problems of carotenoid discoloration deterioration, storage stability, contamination of culture solution, and insufficient carotenoid reinforcement. The method (3) has problems of oxidative deterioration of n-3 series polyunsaturated fatty acids, storage stability, contamination of culture solution, and insufficient nutritional enhancement of n-3 series polyunsaturated fatty acids. Further, the methods (2) and (3) require a process and a preparation technique for additionally adsorbing or encapsulating the other component, and as a result, the feed price becomes high and it is necessary for the method to be put to practical use. Did not reach.
[0005]
It is generally known that polyunsaturated fatty acids such as DHA and EPA are very easily oxidized. When this oxidized oil is given to fish, muscular atrophy may occur in freshwater fish and seawater. Floating fish and edema fish frequently occur in rotifers that are observed in fish or in DHA-rich rotifers, suggesting that one of the causes may be related to the content of antioxidants, etc. The effect of highly unsaturated fatty acids containing lipid peroxides is a problem (see Non-Patent Document 2). However, none of the conventional nutrients that enhance DHA and EPA of rotifers and artemia contain abundant antioxidants that suppress the oxidation of highly unsaturated fatty acids such as DHA and EPA, and inhibit the oxidation of DHA and EPA. Fortified rotifers and artemia fortification have not yet been developed.
[0006]
[Patent Document 1] JP-A-54-105081 [Patent Document 2] JP-A-3-228652 [Patent Document 3] JP-A-4-88954 [Patent Document 4] JP-A-11-98965 [Patent Document 5] JP-A-4-346760 [Patent Document 6] JP-A-8-163990 [Patent Document 7] JP-A-9-191890 [Patent Document 8] JP-A-04-4033 [Patent Document 9] JP-A 08-243378 [Patent Document 10] Japanese Patent Application No. 11-69969 [Patent Document 11] Japanese Patent Application No. 07-61897 [Patent Document 12] Japanese Patent Application No. 06-160713 [Patent Document 13] Japanese Patent Application No. 06-159629 [Patent] Document 14: Japanese Patent Application No. 06-239282 [Patent Document 15] WO 01/62894
[Non-Patent Document 1] Labyrinthura Symposium, published by Masahiro Hayashi et al., 2001 [Non-Patent Document 2] Journal of the Fisheries Society of Japan, 63, 71-84 (1997)
[0007]
[Problems to be solved by the invention]
In view of the current situation, zooplankton, which is a feed for marine larvae, has n-3 series highly unsaturated fatty acids such as DHA, which are essential for larvae and larvae, and has high antioxidant properties, and increases the vitality of larvae and larvae. It is an object of the present invention to provide a method for ingesting and transferring carotenoids which are effective in improving color tone, at the same time efficiently and at a low cost while preventing oxidation of fatty acids. These active ingredients may be separately fed as zooplankton feed ((1) above), or a feed obtained by adding the other active ingredient to a feed containing either one may be fed as zooplankton feed ((2) above). Instead of 3)), the present invention is to provide a feed that simultaneously encapsulates (encapsulates) n-3 series polyunsaturated fatty acids and carotenoids.
[0008]
[Means for Solving the Problems]
The present inventors have intensively studied a method for enriching zooplankton in order to solve the above-mentioned problems, and as a result, it has been found that Labyrinthura, which simultaneously produces and accumulates n-3 series polyunsaturated fatty acids and carotenoids in cells, is obtained in water. For the first time, and found that both active ingredients were simultaneously and stably (preventing fatty acid degradation) and efficiently transferred to zooplankton. Has confirmed that it has a positive effect on the growth of larvae and fry that have been fed as feed, and have completed the present invention.
[0009]
The present invention relates to the provision of a feed for fortification for fisheries, which comprises culturing labirinthura, which simultaneously produces and accumulates n-3 series polyunsaturated fatty acids and carotenoids in cells, or as it is, or After processing with additives, drying, freezing, pulverization, freeze-drying, heating, sterilization, etc., feed on zooplankton such as rotifers and artemia, and cultivate or release fish and fish larvae (Marine, freshwater).
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
The carotenoid of the present invention includes carotene, which is a hydrocarbon carotenoid such as α-carotene, β-carotene, and lycopene, and dyes such as lutein, cryptoxanthin, zeaxanthin, and xanthophylls such as astaxanthin, and one type selected from the dyes. Alternatively, any other dye may be used.
The polyunsaturated fatty acids of the present invention include fatty acid groups such as α-linolenic acid, γ-linolenic acid, octadecatetraenoic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. And a mixture of one or more lipids selected from the fatty acid group may be used.
[0011]
The microorganism used in the present invention is not particularly limited, and may be any microorganism having the ability to produce carotenoids and polyunsaturated fatty acids. Further, any microorganism may be used as long as it is capable of ingesting zooplankton, which is a biological prey such as Rotifer or Artemia, or accumulating zooplankton in a living body. In other words, a microorganism having a high ability to retain carotenoids and polyunsaturated fatty acids after producing and accumulating the same in the microbial cells may be used. Examples of microorganisms that produce carotenoids and n-3 series polyunsaturated fatty acids include Labyrinthura.
Labyrinthula of the present invention refers to marine microorganisms of the Labyrinthulae, Labyrinthulida, Labyrinthulidae, Labyrinthulidae, and Thraustochytriidae, and more specifically to the genus Szochium thyri, and more specifically to the genus Szochium thyri. And a marine microorganism belonging to the genus Thraustochytrium. In particular, it is desirable that Labyrinthura produces and accumulates carotenoids and n-3 series polyunsaturated fatty acids in the cells. The Labyrinthura of the present invention has the ability to produce 0.01% or more of carotenoids on a dry cell basis, and to produce 1% or more of n-3 series of highly unsaturated fatty acids in total lipids. More preferably, it is a rabirin chula which has a capacity to produce carotenoids in an amount of 0.1% or more on a dry cell basis and an n-3 series of highly unsaturated fatty acids in an amount of 10% or more in total lipids. As described above, any rabirinula having an ability to simultaneously produce and accumulate carotenoids and n-3 series polyunsaturated fatty acids in cells can be used. For example, the strain Schizochytrium KH105 strain, which is a microorganism deposited on the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology under the accession number FERM P-18431 on July 25, 2001, contains carotenoids and n- It is preferable in terms of the ability to produce three series of highly unsaturated fatty acids, the amount of accumulation, and the like. Microorganisms having the performance of the present invention have been deposited with IFO and ATCC under accession numbers, and are all available from publicly-known distribution agencies and corporations.
[0012]
The feed of the present invention is a feed containing the above-described carotenoid and a microorganism having an ability to produce n-3 series of highly unsaturated fatty acids, and relates to a feed using the feed for enrichment and a method for enriching a biological feed. The feed of the present invention contains at least a microorganism capable of producing carotenoids and n-3 series polyunsaturated fatty acids, and also has the meaning of a microorganism alone having the ability to produce carotenoids and n-3 series polyunsaturated fatty acids. . That is, the feed of the present invention may be a microorganism capable of producing carotenoids and n-3 series highly unsaturated fatty acids as it is, or may be a mixture with additives, and may be dried, frozen, powdered, freeze-dried, and heated. Processing such as sterilization may be performed, and the form of use, the method of use, the time of use, and the like may be appropriately changed depending on the intended livestock animal, cultured fish, breeding method, growth time, and the like. In addition, the feed of the present invention contains a carotenoid and a microorganism capable of producing n-3 series polyunsaturated fatty acids in an amount of 0.01% or more, preferably 0.1% or more, of carotenoid and total microorganisms based on the dry cell weight. It is preferable that the microorganism contains 5% by weight or more, preferably 50% by weight or more of a microorganism having an ability to produce n-3 series polyunsaturated fatty acids in an amount of 1% or more, preferably 10% or more in the lipid amount.
[0013]
The feed of the present invention may be used as it is, as a zooplankton as a biological feed in which the feed is accumulated, and / or as a feed containing the feed for enrichment, such as chicken, turkey, cow, pig, horse, sheep, and goat. And the like, and can be used as feed and feed for livestock animals such as marine, marine and freshwater farmed fish, and pets. Also, it can be bred as the feed and feed.
The feed containing the feed of the present invention may be a feed obtained by adding and mixing a feed containing a carotenoid and a microorganism capable of producing n-3 series polyunsaturated fatty acids together with a compound feed, and may be a fish meal, soybean meal, wheat gluten, Any compound feed material such as starch can be used and is not particularly limited. The method for producing a feed containing the feed of the present invention may be any feed production step, and the timing of addition of the feed containing carotenoids and microorganisms capable of producing n-3 series polyunsaturated fatty acids is not particularly limited. In the case of moist pellets, for example, the necessary amount may be added at the time of mixing with raw bait, and in the case of dry pellets, the necessary amount may be added in the step of mixing raw materials before molding by an extruder. Alternatively, a method may be used in which an additive such as sodium alginate is used for the pellets to attach the pellet to the surface.
[0014]
Animal planton in which a feed containing the carotenoid of the present invention and a microorganism capable of producing an n-3 series of polyunsaturated fatty acids is stored, for example, rotifers such as rotifers and artemia that store the feed of the present invention. , Tigliopaths, moina, oyster larvae, daphnia and the like. The method for producing an animal planton in which the feed containing the carotenoid and the microorganism having the ability to produce n-3 series polyunsaturated fatty acids of the present invention is accumulated, that is, the method of adding the feed of the present invention, the amount added, and the like are particularly limited. Instead, it may be carried out according to a conventional method, while monitoring the growth status of zooplankton. At the time of culturing the zooplankton, co-cultivation of marine chlorella and feed containing the carotenoid of the present invention and a microorganism capable of producing n-3 series polyunsaturated fatty acids, or carotenoid and n-3 series polyunsaturated fatty acids Production methods by ordinary aquaculture methods, such as microbial culture having the ability to produce Escherichia coli and secondary culture of microorganisms capable of producing carotenoids and n-3 series highly unsaturated fatty acids after high-density cultivation of zooplankton, are applied. . Furthermore, the feeding and breeding method of the cultured fish using the feed and the zooplankton of the present invention is a method of cultivating or releasing the zooplankton of the present invention (marine: red sea bream, black porgy, flounder, flounder, tiger puffer, shrimp, octopus, Yellowtail, freshwater: sweetfish, yamame, char, amago, eel, smelt, etc.). The object of the present invention is to improve the survival rate of larvae and larvae and zooplankton to improve the vitality by accumulating microorganisms belonging to rablinchula and the like having the ability to produce carotenoids and n-3 series polyunsaturated fatty acids. It is related to fortification and does not limit the method of fortification.
[0015]
The culture conditions and production method of a microorganism capable of producing carotenoids and n-3 series polyunsaturated fatty acids according to the present invention include production of carotenoids and n-3 series polyunsaturated fatty acids, and growth conditions of microorganisms having the production. It is preferable to select and use as appropriate according to the above. For example, the culture medium used for cultivation of the Schizochytrium sp. Strain KH105 (Accession number: FERM P-18431) may be any medium as long as this strain can grow well, and may have any composition. Further, two different types of culture media may be used for cell growth and for substance production, respectively. A liquid medium or a solid medium can be used as the medium, and the medium may contain a nitrogen source and inorganic ions in addition to a carbon source. The concentration of seawater in the medium is preferably 50% or more. The carbon source in the culture medium for cell growth is not particularly limited, and a known carbon source can be used.Examples include lower fatty acids such as acetic acid, malic acid, succinic acid, propionic acid, and barmitic acid, starch, and sucrose. preferable. These carbon sources can be added to the medium alone or as a mixture. On the other hand, it is preferable to add one or more of glucose, glycerol and sodium acetate as a carbon source to a medium for producing carotenoids and n-3 series polyunsaturated fatty acids. Further, the concentration of the carbon source is preferably set to 6 to 12%. As the nitrogen source, inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone and yeast extract can be used. It is preferable to use one or more of peptone, sodium nitrate, and potassium nitrate as a medium for producing a carotenoid-based substance and / or a polyunsaturated fatty acid. Furthermore, examples of the inorganic ions include phosphate ions, magnesium ions, calcium ions, potassium ions, iron ions, copper ions, and manganese ions. These inorganic ions may be used in the form of inorganic salts or the like in a medium. .
[0016]
As a method for culturing the microorganism of the present invention, a culture method using a liquid medium such as a shaking culture method, a culture method using a solid medium such as an agar medium, and the like can be used. In the case of producing carotenoids and n-3 series polyunsaturated fatty acids, the culture temperature is, for example, 20 to 35 ° C, preferably 25 to 30 ° C. However, it is preferable to be about 0.5 to 5 days, more preferably about 1 to 3 days.
The culture solution or the like of the microorganism of the present invention may be used as the feed of the present invention as it is. However, when there is a possibility that the components of the remaining medium may stain a farm or the like, a filtration method, a centrifugation method, or a filter filtration method may be used. Collect the cells of microorganisms that have accumulated carotenoids and n-3 series of highly unsaturated fatty acids by a conventional method such as that described above, and use the cells whose surface has been washed with water, an aqueous solution containing a surfactant, ethanol, etc. May be. Further, if necessary, the cells may be dried by a method such as freeze-drying or air-drying, and the dried cells can be used.
[0017]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the following Examples.
[0018]
Embodiment 1
<Culture of Schizochytrium>
Two liters of a medium (pH 7.0) containing 9% of glucose, 1% of yeast extract, 2% of peptone, and 50% of artificial seawater was placed in a 3 liter jar fermenter tank and sterilized at 120 ° C for 20 minutes. Take a platinum loop of the preserved Schizochytrium strain KH105 (Accession number: FERM P-18431), inoculate 50 ml of the above medium in a 200 ml Erlenmeyer flask, and inoculate a reciprocal shaker (160 rpm). ) At 28 ° C for 2 days. This was inoculated into the above-mentioned 2 liter medium, and cultured at 28 ° C. for 7 days at a stirring speed of 100 rpm and an aeration rate of 2 liter / min. During and after the culture, 10 ml of the culture solution was collected, and the cells were collected by centrifugation (3,000 rpm, 10 minutes).
[0019]
Embodiment 2
<Enrichment of zooplankton>
Using the Schizochytrium genus (Schizochytrium) KH105 cells obtained in Example 1, rotifer (Brachionus rotundiformis) as a biological feed was fortified. Using rotifers previously cultured in concentrated freshwater chlorella, the rotifers were adjusted to a density of 8 × 10 6/10 L. In a water tank in which rotifer density was adjusted, 0 ppm (O), 100 ppm (△) or 200 ppm (□) of Schizochytrium genus KH105 cells and 10 parts / L of marine chlorella (Nannochloropsis) as a basic feed were set to 10 10 / L. Then, rotifers were cultured at 25 ° C. over time (0 to 24 hours) to enhance nutrition. The rotifer enriched for each time was collected with a plankton net, washed with running water, and used as an analysis sample.
[0020]
Observation of the collected rotifer with a microscope confirmed that the red pigment of astaxanthin had migrated into the rotifer. This indicates that rotifers can be consumed without aggregation of Schizochytrium KH105 cells, indicating that there is no problem with respect to dispersibility. A portion of the filtrate was directly suspended in chloroform-methanol (2: 1) to extract lipid. An equal amount of a 10% hydrochloric acid-methanol solution was added to the chloroform phase, and the mixture was treated at 60 ° C. for 3 hours to perform methyl esterification. After evaporating to dryness, the residue was dissolved in a small amount of hexane and subjected to gas chromatography analysis for fatty acid analysis. A portion of the filtrate was also suspended in acetone-methanol (7: 3) to extract the dye. After drying under reduced pressure, the residue was dissolved in acetone and the absorbance (478 nm) was measured. The amount of total carotenoid was determined by converting the absorbance of a 1% carotenoid solution to 2,500 (based on astaxanthin).
As shown in FIG. 1, the DHA and carotenoid contents in the rotifer increased with time, indicating that the fortification was enhanced. In addition, the DHA content and the carotenoid content in the rotifer increased with an increase in the amount of added Schizochytrium KH105 cells (○ <△ <□).
[0021]
Embodiment 3
<Cultivation of larvae and fry>
Hatched red sea bream larvae having a total length of about 4 mm were accommodated in a 500-liter panlight aquarium with 10,000 fish each, and a breeding experiment was performed for 25 days. Feeding was performed once a day, and from the second day after the start of the test, 600,000 rotifers per day enriched by the above-described method were gradually increased, and thereafter gradually increased, and 2.4 million individuals per day were given at the end. During the breeding period, marine chlorella (Nannochloropsis) was added to the breeding water every day at a concentration of 1 million cells / ml. The same procedure was used for larvae and larvae of cod and flounder. As a result, ingestion was confirmed as in the case of using normal unenriched zooplankton, and in all test plots, the vitality test (exposed the test fish to the air with a hand net for 60 seconds and re-entered in water again) After returning, the survival rate was examined 24 hours later), and as a result, it was confirmed that the survival rate was improved by 30% or more.
[0022]
【The invention's effect】
A feed capable of simultaneously feeding n-3 series unsaturated fatty acids (eicosapentaenoic acid, docosahexaenoic acid, etc.) essential for the growth of cultured larvae and larvae and pigments of carotenoids effective for improving vitality, and fortified with the feed Can provide zooplankton feed. Resolves water pollution, destabilization of active ingredients, and increased costs associated with conventional mixing and absorption methods of active ingredients, leading to improved vitality of larvae and larvae, increased survival rate, and aquaculture productivity. Could be improved.
[Brief description of the drawings]
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the state of rotifer enrichment using the feed of the present invention, (A) is a diagram showing the accumulated amount of carotenoid, and (B) is a diagram showing the accumulated amount of DHA.

Claims (8)

A feed comprising a microorganism capable of producing carotenoids and polyunsaturated fatty acids. Carotenoid is carotenes, β-carotene, lutein, adonirubin, at least one or more pigments selected from canthaxanthin and astaxanthin, n-3 series of highly unsaturated fatty acids, at least selected from eicosapentaenoic acid and docosahexaenoic acid The feed according to claim 1, which is one or more n-3 series polyunsaturated fatty acids. The feed according to claim 1 or 2, wherein the microorganism capable of producing carotenoids and n-3 series polyunsaturated fatty acids is Labyrinthura. The feed according to any one of claims 1 to 3, wherein the labulintura having the ability to produce carotenoids and n-3 series polyunsaturated fatty acids is a microorganism belonging to the genus Schizochytrium or the genus Thraustochytrium. The method for producing a feed according to claim 1. A zooplankton that has been fed with the feed according to claim 1. A method for enriching zooplankton, comprising feeding the zooplankton with the feed according to claim 1. An aquaculture method using the feed according to claim 1 and / or the zooplankton according to claim 6.

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