WO2015072539A1 - Culture feed for aquatic organisms - Google Patents

Culture feed for aquatic organisms Download PDF

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Publication number
WO2015072539A1
WO2015072539A1 PCT/JP2014/080167 JP2014080167W WO2015072539A1 WO 2015072539 A1 WO2015072539 A1 WO 2015072539A1 JP 2014080167 W JP2014080167 W JP 2014080167W WO 2015072539 A1 WO2015072539 A1 WO 2015072539A1
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parachlorella
algae
feed
base sequence
aquatic
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PCT/JP2014/080167
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French (fr)
Japanese (ja)
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憲司 山下
達也 小田
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株式会社カネカ
国立大学法人長崎大学
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Publication of WO2015072539A1 publication Critical patent/WO2015072539A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms

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  • the present invention relates to an aquaculture feed and an aquaculture method that can effectively promote the growth of aquatic organisms.
  • Aquaculture refers to increasing the number and quantity of raw aquatic life by managing and breeding useful aquatic life, and has the advantage that stable supply is possible rather than capturing natural aquatic life.
  • live feed such as Brachionus plicatiris is used at the stage of fry.
  • algal organisms such as Nannochloropsis oculata are used as feed for young shellfish before being cultured in the sea.
  • living cells of the genus Chlorella are mainly used as the feed.
  • Patent Document 1 discloses a feed for rotifer culture that is prepared by adding phospholipids to a living cell of a genus Chlorella algae and then freezing or drying it. According to this technique, it is said that aggregation of chlorella algae cells during freezing and drying can be suppressed by dispersing and adsorbing phospholipids between cells of the genus Chlorella algae.
  • Patent Document 2 discloses a fry feed prepared by drying and granulating marine chlorella algae. According to the feed, fry can be grown without using a rotifer, and there is no problem of off-flavor due to oxidation of fish oil.
  • livestock feed is useful for aquatic culture, especially for larval shellfish and juvenile fish, but there is a problem that stable supply is difficult for livestock feed, and that production is expensive or expensive. There is.
  • a dry feed using chlorella algae as a raw material has been studied, there is a problem that the growth effect of aquatic organisms is not sufficient for the chlorella feed.
  • an object of the present invention is to provide an aquatic aquaculture feed that exhibits an effective growth effect on aquatic organisms, even though it is not a biological feed, and an aquatic organism culture method using the feed.
  • the inventors of the present invention have made extensive studies to solve the above problems. As a result, the dried body of Parachlorella algae, which is closely related to Chlorella algae but different from Chlorella algae, exhibits excellent growth effects on aquatic organisms even though it is not a living feed And the present invention was completed.
  • An aquatic aquaculture feed comprising a dried body of Parachlorella algae as an active ingredient.
  • a method for cultivating aquatic organisms comprising a step of feeding aquatic organisms with a feed containing a dried body of Parachlorella algae as an active ingredient.
  • the active ingredient of the aquatic aquaculture feed according to the present invention is not a living cell but a dry body, a stable supply is possible. Moreover, since the excellent growth effect with respect to aquatic organisms is shown, normal and further growth of aquatic organisms can be promoted with a smaller amount. Furthermore, self-contamination of cultured water due to residual feed is a particular problem in aquatic terrestrial culture, but because the feed of the present invention is efficiently digested and absorbed by aquatic organisms, the residual amount can be reduced. Yes, self-contamination can be suppressed. Therefore, the aquatic aquaculture feed according to the present invention is capable of effectively cultivating aquatic organisms, and is very useful industrially as an excellent substitute for conventional aquaculture feed.
  • FIG. 1 is a graph showing the degree of growth in each case where juveniles are cultured for a certain period of time using living cells of microalgae which are conventional feeds, aquaculture feed for aquatic organisms according to the present invention, or chlorella dry powder which is conventional feeds. It is.
  • FIG. 2 is an enlarged photograph of juveniles cultured for a certain period using the living cells of microalgae as a conventional feed, the cultured feed for aquatic organisms according to the present invention, or the chlorella dry powder as a conventional feed.
  • FIG. 3 is a fluorescence micrograph of juvenile shellfish and its excreta cultured using the aquatic aquaculture feed according to the present invention or the conventional feed chlorella dry powder.
  • FIG. 4 is a graph showing changes in the number of rotifers cultured for a certain period using the living cells of microalgae as a conventional feed, the aquatic aquaculture feed according to the present invention, or the chlorella dry powder as a conventional feed.
  • FIG. 5 is a graph showing changes in body weight of red sea bream cultivated using the aquatic aquaculture feed according to the present invention.
  • FIG. 6 is a graph showing changes in body weight of prawns cultured using the aquatic aquaculture feed according to the present invention or a normal mixed feed.
  • the aquatic aquaculture feed according to the present invention is characterized by containing, as an active ingredient, a dried body of the genus Parachlorella.
  • the genus Parachlorella belongs to the Treboxya algae among the genus Chlorella that straddles the Treboxyphyceae and Chlorophyceae, but it is determined by molecular phylogenetic analysis using 18S rDNA and 16S rDNA. For example, it forms a different group from other genus Chlorella.
  • Parachlorella algae do not have a strong cell wall structure like Chlorella algae, and instead are covered with a thick film mainly composed of polysaccharides. This is considered to be the reason why Parachlorella algae are easily digested and absorbed by aquatic organisms compared to Chlorella algae, and their growth effect and feed efficiency are high.
  • Parachlorella algae can be obtained by isolating colonies from freshwater samples collected in the field by subculture using a general medium, and finally identifying the genus species by molecular phylogenetic analysis. Obtainable. Moreover, what is necessary is just to acquire and use if there exists a commercially available thing.
  • Parachlorella algae can grow in aerobic and anaerobic conditions in common media such as freshwater and LB media, but proliferate particularly well at room temperature to 30 ° C. under bright and aerobic conditions. .
  • the para Chlorella algae for example, Parachlorella kessleri, Parachlorella beyerinckii, Parachlorella marinichlorella, Parachlorella dictyoshaerium, Parachlorella mucidosphaerium, Parachlorella closteriopsis, Parachlorella dicloster, include Parachlorella beijerinc. Of these, Parachlorella kessleri and Parachlorella beyerinckii are particularly suitable.
  • KNK-A001 strain (Accession No .: FERM BP-22256) is deposited at the depository as follows.
  • the morphological characteristics and the like of the KNK-A001 strain according to the present invention are as follows.
  • SEQ ID NO: 1 The partial base sequence of 18S rRNA of KNK-A001 strain is shown in SEQ ID NO: 1 (SEQ ID NO: 1).
  • the 18S rRNA has the following base sequence (2) or (3) with respect to the base sequence (1) corresponding to SEQ ID NO: 1, the same as the KNK-A001 strain, Parachlorella algae It is considered that it has an excellent growth effect on aquatic organisms as in the case of the KNK-A001 strain.
  • the number of mutations such as deletion is preferably 30 or less, 20 or less, or 10 or less, more preferably 9 or less, 8 or less, 6 or less, or 5 or less, and further preferably 4 or less, 3 or less. 2 or less or 1 or less is more preferable.
  • the percentage of base sequence identity is preferably 99.0% or more, 99.2% or more, or 99.4% or more, more preferably 99.5% or more, 99.6. % Or more or 99.7% or more is more preferable, and 99.8% or more or 99.9% or more is more preferable.
  • parachlorella algae used in the present invention are preferably those heterotrophic cultured at least in the final culture stage.
  • Parachlorella algae may differ in the composition of cells and components contained in cells when autotrophic culture and heterotrophic culture are performed, but at least the superiority of parachlorella algae cultured under heterotrophic culture The growth effect has been confirmed.
  • the Parachlorella algae according to the present invention include mutants whose dried bodies exhibit an excellent growth effect on aquatic organisms.
  • the “mutant” means a parachlorella algae improved by artificial selection, hybridization, mutation, genetic recombination and the like.
  • the dried body of Parachlorella algae itself which is an active ingredient of the aquatic aquaculture feed according to the present invention, can be obtained by drying live cells of Parachlorella algae and a culture solution containing the living cells. That is, the living cells may be separated from the culture solution of Parachlorella algae by filtration or centrifugation, and dried, or the culture solution may be dried as it is. Parachlorella algae release secretions into the culture solution, and this secretion may be one of the effective components for the growth of aquatic organisms. When the culture solution is dried as it is, the dried product contains such secretions.
  • Drying of the living cells of Parachlorella algae and the culture solution containing the living cells can be performed according to a conventional method.
  • the living cells or the culture solution may be dried at 50 ° C. or higher and 200 ° C. or lower for 10 seconds or longer and 10 hours or shorter.
  • the pressure may be reduced during drying.
  • drying time can be shortened by drying the culture solution while flowing it into a thin film.
  • Specific drying conditions can be appropriately adjusted according to the amount of Parachlorella algae to be dried, the amount of the culture solution, the equipment to be used, and the like.
  • the obtained dried body is generally aggregated, it may be further pulverized. What is necessary is just to adjust the grinding
  • the “dry body” in the present invention is not particularly limited, and the moisture content is not limited unless the parachlorella algae is not a living cell and is not in a clear wet state.
  • the moisture content of the dried product can be 30% by mass or less.
  • the water content the lower the value, the higher the storage stability of the feed and the easier the transportation, so 20% by mass or less is preferable, 15% by mass or less is more preferable, and 10% by mass or less is more preferable. 8% by mass or less is even more preferable.
  • the water content is preferably 1% by mass or more, more preferably 2% by mass or more, and further preferably 5% by mass or more.
  • the drying is performed in order to kill a cell or to improve the storage stability, the water content in an actual feed product or feed during feeding does not matter.
  • the dried body of Parachlorella algae may be fed after being dispersed in aquaculture water or the like. Further, the moisture content in the dried product can be easily measured by the Karl Fischer method.
  • the “active ingredient” refers to a substance that does not exhibit at least toxicity to aquatic organisms to be cultured and promotes the increase in the number of aquatic organisms and the body weight.
  • the feed amount of the aquatic aquaculture feed according to the present invention may be adjusted as appropriate according to the type of aquatic organisms to be fed, the degree of growth, etc., for example, larvae cultured on land, fry, and ring animals.
  • the number of Parachlorella algae cells in the dried body relative to the aquaculture water should be about 1 ⁇ 10 4 cells / mL or more and 1 ⁇ 10 6 cells / mL or less per feeding Can do. What is necessary is just to increase the said feed amount according to the growth degree of the aquatic organism which is a culture target.
  • the feed amount of the aquatic aquaculture feed according to the present invention for general fish may be appropriately adjusted in consideration of the type, the degree of growth, the blending ratio with other feeds, etc.
  • the intake of Parachlorella algae per feed can be about 0.2 g / kg body weight or more.
  • the number of feeds per day of the aquatic aquaculture feed according to the present invention may be adjusted as appropriate, but can be adjusted to, for example, 1 to 3 times per day.
  • the content of the present feed is preferably 1.0% by mass or more.
  • the upper limit is not particularly limited, but is preferably 10% by mass or less in order to reduce nutritional bias.
  • Other feeds to be blended include, for example, fish meal, soybean meal, rice bran, wheat flour, starch, yeast, fats and oils, vitamins, minerals, etc., if selected appropriately according to the type and growth degree of the target fish Good.
  • the form of the aquatic aquaculture feed according to the present invention is not particularly limited, and may be appropriately determined depending on the type of aquatic organism to be fed, the degree of growth, and the like.
  • the dried body of Parachlorella algae can be crushed so that it can be easily eaten by juvenile shellfish and microorganisms.
  • the crushed material as described above may diffuse into the aquaculture water and the larvae may not be eaten, so the pulverized product of Parachlorella algae can be used as needed for other feeds.
  • it is preferably molded into a suitable size to form a solid.
  • the target organism to be fed with the aquatic aquaculture feed according to the present invention is not particularly limited as long as it can grow effectively with the feed according to the present invention.
  • shellfish such as oysters, abalone, scallops, scallops, scallops, cypresses
  • saltwater fish such as Thailand, flounder, tuna, yellowtail, amberjack, striped horse mackerel, isaki
  • freshwater fish such as eel, char, trout, trout
  • feed zooplankton such as tigriops.
  • Parachlorella Algae Culture Process In this process, Parachlorella algae are cultured. Although implementation of this process is arbitrary, it is preferable to implement this process and to increase the total amount of Parachlorella algae from the viewpoint of production of aquatic aquaculture feed according to the present invention.
  • the culture conditions for the Parachlorella algae may be appropriately determined according to the optimal conditions for the Parachlorella algae to be cultured.
  • the culture may be performed at room temperature to 30 ° C. under aerobic conditions or anaerobic conditions in a general medium such as a fresh water medium or an LB medium.
  • Parachlorella algae generally grow particularly well under light conditions. Therefore, from the viewpoint of increasing the total amount of Parachlorella algae, it is preferable to culture under bright conditions, particularly under autotrophic conditions. However, since an excellent growth effect of Parachlorella algae cultured at least in heterotrophic culture has been confirmed, it is also preferable to cultivate under heterotrophic conditions while shielding light. In addition, after the Parachlorella algae are sufficiently grown under autotrophic conditions, they may be cultured under heterotrophic conditions.
  • Feeding step In this step, feed containing a dry body of Parachlorella algae as an active ingredient is fed to aquatic organisms. Specific feeding conditions may be appropriately determined according to the type of aquatic organisms to be cultured and the degree of growth as described above.
  • the present invention it is possible to promote normal or higher growth of aquatic organisms in a smaller amount than when feeding a general feed, and it is possible to suppress the amount of feeding.
  • the number of aquatic organisms and the total weight can be significantly increased as compared with the case where the same amount of general feed is fed.
  • Example 1 Culture experiment of oyster larvae (1) Preparation of aquaculture feed for aquatic organisms according to the present invention Parachlorella kessleri KNK-A001 strain (accession number: FERM BP-22256) on a sterilized liquid medium And light-shielded with aluminum foil, and then precultured at 30 ° C. for 72 hours. Next, a pre-culture medium was added to a larger volume of a sterilized liquid medium, light-shielded with aluminum foil, and cultured for 143 hours at an internal temperature of 30 ° C., an aeration rate of 2 L / min, an agitation rate of 450 rpm, and a pH of 6-7.
  • Parachlorella kessleri KNK-A001 strain accession number: FERM BP-22256
  • the culture solution was dried with a double drum dryer, and the obtained dried aggregate was pulverized with a feather mill to obtain a dried cell product of KNK-A001 strain.
  • Parachlorella beyerinckii seed algae product name “Bayerink”, purchased from Mitsui & Co., Ltd. was subjected to the same experiment as the Parachlorella algae.
  • the KNK-A001 dry powder, Parachlorella Bayelinceae algae dry powder and Chlorella dry powder are suspended in filtered seawater and then subjected to sonication to break up the partial cell aggregation and uniform cell suspension. It was made into a turbid liquid and fed at a rate of 20 ⁇ 10 4 cells / mL / day, which was adjusted to a suitable concentration in a preliminary experiment. Keat cellos was fed at a rate of 3.75 ⁇ 10 4 cells / mL / day or 20 ⁇ 10 4 cells / mL / day with reference to a technical report (Vasquez et al., 2009).
  • the oyster larvae were bred for 1 week in an incubator at 25 ° C. while feeding each feed once a day at the above rate and aeration with a Pasteur pipette.
  • FIG. 1 A graph showing the increase in shell length of each oyster larva after one week of breeding is shown in FIG. 1, and an enlarged photograph of each oyster larva is shown in FIG.
  • “Kaneka Chlorella” in FIG. 1 indicates the KNK-A001 strain.
  • a and B are fed with live Keatoceros cells at 3.75 ⁇ 10 4 cells / mL / day and 20 ⁇ 10 4 cells / mL / day, and C is fed with dry powder of KNK-A001 strain.
  • D is a section fed with chlorella dry powder.
  • the average growth amount which is the difference between the shell length immediately after the start of the experiment and the shell length after one week of breeding, was measured. The results are shown in Table 2.
  • the growth rate of the oyster larvae fed with the dry powder of chlorella was significantly higher, whereas the growth rate of the oyster larvae fed with the dry powder of the genus Parachlorella according to the present invention was significantly higher.
  • the dry powder of the genus Parachlorella showed a growth effect comparable to that of Keatoceros, which is a living cell, even though it was a dry body.
  • Example 2 Rotation experiment of rotifer Two rotifers with 500 ⁇ L of EMS medium were added to each well of a 24-well plate, and Parachlorella querseri KNK-A001 strain dry powder, Parachlorella similar to that used in Example 1 above.
  • -Bayerink seed algae dry powder or chlorella dry powder was fed at 20 ⁇ 10 4 cells / mL / day, respectively.
  • the culture was similarly performed using only the EMS medium without adding the feed. The results are shown in FIG.
  • the aquatic aquaculture feed according to the present invention is superior to chlorella dry powder as a feed for rotifers.
  • Example 3 Red sea bream culture experiment Five cylindrical pools having a diameter of 3 m and a height of 1 m were prepared, and 30 red sea breams weighing about 57.5 g were placed in each pool. 0% by mass and 0.5% by mass of dry powder of Parachlorella kesleri KNK-A001 similar to that used in Example 1 above for powdered red sea bream mixed feed (manufactured by Higashimaru, product name “Himezakura”) %, 1% by mass, 2.5% by mass, and 5% by mass, and formed into pellets having a diameter of 2 mm and a length of 3 mm. The sample feed was bred once a day for 1 month while being fed with satiation to the extent that no excess feed was produced.
  • the water temperature during the breeding period was 26.4 to 28.4 ° C., and the average was 27.4 ° C.
  • the body weight was measured every two weeks, and the average value was calculated. The results are shown in FIG.
  • Example 4 Aquatic Shrimp Culture Experiment A total of 4 200 L water tanks were prepared, each with the same amount of sand, and warm seawater at about 24 ° C. was constantly injected in a flowing water system. Each fish tank contained 15 prawns with a body weight of 3 to 5 g. A mixed feed for shrimp (manufactured by Higashimaru, product name “Himezakura”) is mixed with 0.02% by mass of the dry powder of Parachlorella kesleri KNK-A001 obtained in Example 1 above, once a day for 2 tanks. After sunset, the fish tank was fed with food, and the next morning, the remaining food was collected and reared for 6 weeks.
  • Higashimaru, product name “Himezakura” is mixed with 0.02% by mass of the dry powder of Parachlorella kesleri KNK-A001 obtained in Example 1 above, once a day for 2 tanks. After sunset, the fish tank was fed with food, and the next morning, the remaining food was collected and reared for

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Abstract

　The objective of the present invention is to provide a culture feed for aquatic organisms that exhibits an effective growth effect in aquatic organisms despite not being a live feed, and to provide a method for culturing aquatic organisms using said feed. The culture feed for aquatic organisms according to the present invention is characterized by including dried Parachlorella algae as an active ingredient.

Description

水生生物用養殖飼料Aquatic aquaculture feed
　本発明は、水生生物の成長を有効に促進することができる養殖飼料と養殖方法に関するものである。 The present invention relates to an aquaculture feed and an aquaculture method that can effectively promote the growth of aquatic organisms.
　養殖とは、主に有用な水生生物を人が管理して飼育することにより数や量の増収を図ることをいい、天然水生生物を捕獲するよりも安定供給が可能になるという利点がある。 Aquaculture refers to increasing the number and quantity of raw aquatic life by managing and breeding useful aquatic life, and has the advantage that stable supply is possible rather than capturing natural aquatic life.
　マダイやヒラメなどの高級魚を養殖するに当たっては、稚魚の段階ではシオミズツボワムシ（Ｂｒａｃｈｉｏｎｕｓ　ｐｌｉｃａｔｉｌｉｓ）などの生体飼料が用いられている。また、海中で養殖する前の稚貝には、Ｎａｎｎｏｃｈｌｏｒｏｐｓｉｓ　ｏｃｕｌａｔａなどの藻類の生体が飼料として用いられている。また、生体飼料であるシオミズツボワムシ自体の養殖においては、クロレラ（Ｃｈｌｏｒｅｌｌａ）属藻類の生体細胞が飼料として主に用いられる。 In order to cultivate luxury fish such as red sea bream and flounder, live feed such as Brachionus plicatiris is used at the stage of fry. In addition, algal organisms such as Nannochloropsis oculata are used as feed for young shellfish before being cultured in the sea. Further, in the cultivation of the rotifer itself, which is a biological feed, living cells of the genus Chlorella are mainly used as the feed.
　これら生体飼料生物は、一般的に、養殖場またはその近辺で養殖し、養殖生物に給餌される。ところが、これら生体飼料生物では予期できない増殖不良がしばしば起こり、餌不足が原因となって養殖生物が身痩せしたり大量斃死したりすることが大きな問題となっている。このような生体飼料生物の安定的な飼育にはコストがかかり、購入するにしても高価であるため、養殖業者の悩みの種になっている。 These living organisms are generally cultivated at or near a farm and fed to the aquaculture organism. However, unpredictable growth failure often occurs in these live-feed organisms, and it is a big problem that the aquaculture organisms are leaned or drowned in large quantities due to lack of food. Such stable feeding of live feed organisms is costly and expensive even if purchased, which has been a problem for farmers.
　そこで、生体飼料ではない乾燥飼料が求められている。乾燥飼料であれば、生体飼料とは異なり大量生産した後に保管しておくことができ、安定供給が可能である。また、運搬コストが生体飼料に比べてかなり低いため、より安価であるという利点がある。 Therefore, there is a demand for dry feed that is not biological feed. If it is dry feed, it can be stored after mass production, unlike living feed, and can be stably supplied. Moreover, since the transportation cost is considerably lower than that of living feed, there is an advantage that it is cheaper.
　生体飼料ではない養殖飼料としては、例えば特許文献１には、クロレラ属藻類の生体細胞にリン脂質を添加して凍結または乾燥したものであるワムシ培養用飼料が開示されている。当該技術では、クロレラ属藻類の細胞間にリン脂質を分散・吸着させることにより、クロレラ属藻類細胞の凍結や乾燥の際における凝集を抑制できるとされている。 As a cultured feed that is not a biological feed, for example, Patent Document 1 discloses a feed for rotifer culture that is prepared by adding phospholipids to a living cell of a genus Chlorella algae and then freezing or drying it. According to this technique, it is said that aggregation of chlorella algae cells during freezing and drying can be suppressed by dispersing and adsorbing phospholipids between cells of the genus Chlorella algae.
　また、特許文献２には、海産クロレラ属藻類を乾燥して造粒した稚魚用飼料が開示されている。当該飼料によれば、ワムシを使わなくても稚魚を成長させることができ、また、魚油の酸化による異臭の問題が無いとされている。 Further, Patent Document 2 discloses a fry feed prepared by drying and granulating marine chlorella algae. According to the feed, fry can be grown without using a rotifer, and there is no problem of off-flavor due to oxidation of fish oil.
特開昭６３－１６４８２４号公報JP 63-164824 A 特開昭６１－７４５５０号公報JP-A-61-74550
　上述したように、水生生物の養殖、特に稚貝や稚魚の養殖では生体飼料が有用であるが、生体飼料には安定供給が難しいといった問題や、その生産にコストがかかったり高価であるという問題がある。一方、クロレラ属藻類を原料とする乾燥飼料も検討されてはいるが、クロレラ飼料には水生生物の成長効果が十分でないという問題がある。 As mentioned above, livestock feed is useful for aquatic culture, especially for larval shellfish and juvenile fish, but there is a problem that stable supply is difficult for livestock feed, and that production is expensive or expensive. There is. On the other hand, although a dry feed using chlorella algae as a raw material has been studied, there is a problem that the growth effect of aquatic organisms is not sufficient for the chlorella feed.
　そこで本発明は、生体飼料ではないにもかかわらず、水生生物に対して有効な成長効果を示す水生生物用養殖飼料と、当該飼料を用いる水生生物の養殖方法を提供することを目的とする。 Therefore, an object of the present invention is to provide an aquatic aquaculture feed that exhibits an effective growth effect on aquatic organisms, even though it is not a biological feed, and an aquatic organism culture method using the feed.
　本発明者らは、上記課題を解決するために鋭意研究を重ねた。その結果、クロレラ属藻類に近縁なものではあるがクロレラ属藻類とは異なるパラクロレラ属藻類の乾燥体が、生体飼料ではないにもかかわらず、水生生物に対して優れた成長効果を示すことを見出して本発明を完成した。 The inventors of the present invention have made extensive studies to solve the above problems. As a result, the dried body of Parachlorella algae, which is closely related to Chlorella algae but different from Chlorella algae, exhibits excellent growth effects on aquatic organisms even though it is not a living feed And the present invention was completed.
　以下、本発明を示す。 Hereinafter, the present invention will be described.
　［１］　パラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体を有効成分として含むことを特徴とする水生生物用養殖飼料。 [1] An aquatic aquaculture feed comprising a dried body of Parachlorella algae as an active ingredient.
　［２］　パラクロレラ属藻類がパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類である上記［１］に記載の水生生物用養殖飼料。 [2] The aquatic aquaculture feed according to the above [1], wherein the Parachlorella algae is a Parachlorella kessleri algae.
　［３］　パラクロレラ・ケスレリ種藻類がＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）である上記［２］に記載の水生生物用養殖飼料。 [3] The aquatic aquaculture feed according to the above [2], wherein the Parachlorella quercerii seed algae is KNK-A001 strain (Accession number: FERM BP-22256).
　［４］　パラクロレラ属藻類の１８Ｓ　ｒＲＮＡが、下記（１）～（３）のいずれかの塩基配列を有する上記［１］～［３］のいずれかに記載の水生生物用養殖飼料。
　（１）　配列番号１の塩基配列
　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列 [4] The aquatic aquaculture feed according to any one of [1] to [3] above, wherein the 18S rRNA of Parachlorella algae has any one of the following base sequences (1) to (3):
(1) Base sequence of SEQ ID NO: 1 (2) In the base sequence defined in (1) above, a base sequence in which 1 to 31 bases have been deleted, substituted and / or added (3) (1) ) Base sequence having a sequence identity of 98.5% or more with respect to the base sequence defined in
　［５］　パラクロレラ属藻類がパラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類である上記［１］に記載の水生生物用養殖飼料。 [5] The aquatic aquaculture feed according to [1] above, wherein the Parachlorella algae is a Parachlorella beyerinckii species algae.
　［６］　従属栄養培養したパラクロレラ属藻類の乾燥体を含む上記［１］～［５］のいずれかに記載の水生生物用養殖飼料。 [6] The aquatic aquaculture feed according to any one of [1] to [5] above, comprising a dried body of Parachlorella algae cultured in heterotrophic culture.
　［７］　水生生物が、貝類、魚類、飼料用動物プランクトンから選択されるものである上記［１］～［６］のいずれかに記載の水生生物用養殖飼料。 [7] The aquatic aquaculture feed according to any one of [1] to [6] above, wherein the aquatic organism is selected from shellfish, fish, and zooplankton for feed.
　［８］　水生生物用養殖飼料の有効成分としてのパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 [8] Use of a dried body of Parachlorella algae as an active ingredient of aquatic aquaculture feed.
　［９］　パラクロレラ属藻類がパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類である上記［８］に記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 [9] Use of a dried body of Parachlorella algae according to [8] above, wherein the Parachlorella algae is a Parachlorella kessleri seed algae.
　［１０］　パラクロレラ・ケスレリ種藻類がＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）である上記［９］に記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 [10] Use of a dried body of Parachlorella algae as described in [9] above, wherein the Parachlorella quercerae algae is KNK-A001 strain (Accession number: FERM BP-22256).
　［１１］　パラクロレラ属藻類の１８Ｓ　ｒＲＮＡが、下記（１）～（３）のいずれかの塩基配列を有する上記［８］～［１０］のいずれかに記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。
　（１）　配列番号１の塩基配列
　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列 [11] The Parachlorella algae according to any one of [8] to [10] above, wherein the 18S rRNA of Parachlorella algae has any one of the following base sequences (1) to (3): Use of dry body.
(1) Base sequence of SEQ ID NO: 1 (2) In the base sequence defined in (1) above, a base sequence in which 1 to 31 bases have been deleted, substituted and / or added (3) (1) ) Base sequence having a sequence identity of 98.5% or more with respect to the base sequence defined in
　［１２］　パラクロレラ属藻類がパラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類である上記［８］に記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 [12] Use of the dried body of Parachlorella algae according to [8] above, wherein the Parachlorella algae is a Parachlorella beyerinckii species algae.
　［１３］　パラクロレラ属藻類が従属栄養培養されたものである上記［８］～［１２］のいずれかに記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 [13] Use of the dried body of Parachlorella algae according to any one of [8] to [12] above, wherein the parachlorella algae are heterotrophically cultured.
　［１４］　水生生物が、貝類、魚類、飼料用動物プランクトンから選択されるものである上記［８］～［１３］のいずれかに記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 [14] Use of a dried body of Parachlorella algae according to any one of [8] to [13] above, wherein the aquatic organism is selected from shellfish, fish and zooplankton for feed.
　［１５］　パラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体を有効成分として含む飼料を水生生物に給餌する工程を含むことを特徴とする水生生物の養殖方法。 [15] A method for cultivating aquatic organisms, comprising a step of feeding aquatic organisms with a feed containing a dried body of Parachlorella algae as an active ingredient.
　［１６］　パラクロレラ属藻類がパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類である上記［１５］に記載の方法。 [16] The method according to [15] above, wherein the Parachlorella algae is a Parachlorella kessleri seed algae.
　［１７］　パラクロレラ・ケスレリ種藻類がＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）である上記［１６］に記載の方法。 [17] The method according to [16] above, wherein the Parachlorella kucereri seed algae is KNK-A001 strain (accession number: FERM BP-22256).
　［１８］　パラクロレラ属藻類の１８Ｓ　ｒＲＮＡが、下記（１）～（３）のいずれかの塩基配列を有する上記［１５］～［１７］のいずれかに記載の方法。
　（１）　配列番号１の塩基配列
　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列 [18] The method according to any one of [15] to [17] above, wherein the 18S rRNA of Parachlorella algae has any one of the following base sequences (1) to (3):
(1) Base sequence of SEQ ID NO: 1 (2) In the base sequence defined in (1) above, a base sequence in which 1 to 31 bases have been deleted, substituted and / or added (3) (1) ) Base sequence having a sequence identity of 98.5% or more with respect to the base sequence defined in
　［１９］　パラクロレラ属藻類がパラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類である上記［１５］に記載の方法。 [19] The method according to [15] above, wherein the Parachlorella algae is a Parachlorella beyerinckii algae.
　［２０］　さらに、パラクロレラ属藻類を従属栄養培養する工程と、従属栄養培養したパラクロレラ属藻類を乾燥してパラクロレラ属藻類の乾燥体を得る工程を含む上記［１５］～［１９］のいずれかに記載の方法。 [20] The above [15] to [19], further comprising a heterotrophic culture of Parachlorella algae and a step of drying the heterotrophic cultured Parachlorella algae to obtain a dried body of Parachlorella algae The method according to any one.
　［２１］　水生生物が、貝類、魚類、飼料用動物プランクトンから選択されるものである上記［１５］～［２０］のいずれかに記載の方法。 [21] The method according to any one of [15] to [20] above, wherein the aquatic organism is selected from shellfish, fish, and feed zooplankton.
　本発明に係る水生生物用養殖飼料の有効成分は、生体細胞などではなく乾燥体であるので、安定的な供給が可能である。また、水生生物に対する優れた成長効果を示すことから、より少ない量で水生生物の正常な、また、それ以上の成長を促すことができる。さらに、残留飼料による養殖水の自家汚染は水生生物の陸上養殖において特に問題となるが、本発明飼料は水生生物にとり効率的に消化吸収されるものであることから、残留量の低減が可能であり、自家汚染を抑制することができる。よって本発明に係る水生生物用養殖飼料は、水生生物を効果的に養殖できるものであり、従来の養殖飼料にとって代わり得る優れたものとして産業上非常に有用である。 Since the active ingredient of the aquatic aquaculture feed according to the present invention is not a living cell but a dry body, a stable supply is possible. Moreover, since the excellent growth effect with respect to aquatic organisms is shown, normal and further growth of aquatic organisms can be promoted with a smaller amount. Furthermore, self-contamination of cultured water due to residual feed is a particular problem in aquatic terrestrial culture, but because the feed of the present invention is efficiently digested and absorbed by aquatic organisms, the residual amount can be reduced. Yes, self-contamination can be suppressed. Therefore, the aquatic aquaculture feed according to the present invention is capable of effectively cultivating aquatic organisms, and is very useful industrially as an excellent substitute for conventional aquaculture feed.
図１は、従来飼料である微細藻類の生細胞、本発明に係る水生生物用養殖飼料、または従来飼料であるクロレラ乾燥粉末を用いて稚貝を一定期間養殖した各場合の成長度合いを示すグラフである。FIG. 1 is a graph showing the degree of growth in each case where juveniles are cultured for a certain period of time using living cells of microalgae which are conventional feeds, aquaculture feed for aquatic organisms according to the present invention, or chlorella dry powder which is conventional feeds. It is. 図２は、従来飼料である微細藻類の生細胞、本発明に係る水生生物用養殖飼料、または従来飼料であるクロレラ乾燥粉末を用いて一定期間養殖した稚貝の拡大写真である。FIG. 2 is an enlarged photograph of juveniles cultured for a certain period using the living cells of microalgae as a conventional feed, the cultured feed for aquatic organisms according to the present invention, or the chlorella dry powder as a conventional feed. 図３は、本発明に係る水生生物用養殖飼料、または従来飼料であるクロレラ乾燥粉末を用いて養殖した稚貝とその排泄物の蛍光顕微鏡写真である。FIG. 3 is a fluorescence micrograph of juvenile shellfish and its excreta cultured using the aquatic aquaculture feed according to the present invention or the conventional feed chlorella dry powder. 図４は、従来飼料である微細藻類の生細胞、本発明に係る水生生物用養殖飼料、または従来飼料であるクロレラ乾燥粉末を用いて一定期間養殖したワムシの個体数変化を示すグラフである。FIG. 4 is a graph showing changes in the number of rotifers cultured for a certain period using the living cells of microalgae as a conventional feed, the aquatic aquaculture feed according to the present invention, or the chlorella dry powder as a conventional feed. 図５は、本発明に係る水生生物用養殖飼料を用いて養殖したマダイ稚魚の体重変化を示すグラフである。FIG. 5 is a graph showing changes in body weight of red sea bream cultivated using the aquatic aquaculture feed according to the present invention. 図６は、本発明に係る水生生物用養殖飼料または通常の配合飼料を用いて養殖したクルマエビの体重変化を示すグラフである。FIG. 6 is a graph showing changes in body weight of prawns cultured using the aquatic aquaculture feed according to the present invention or a normal mixed feed.
　本発明に係る水生生物用養殖飼料は、パラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体を有効成分として含むことを特徴とする。 The aquatic aquaculture feed according to the present invention is characterized by containing, as an active ingredient, a dried body of the genus Parachlorella.
　パラクロレラ属は、トレボキシア藻綱（Ｔｒｅｂｏｕｘｉｏｐｈｙｃｅａｅ）と緑藻綱（Ｃｈｌｏｒｏｐｈｙｃｅａｅ）とにまたがるクロレラ属のうち、トレボキシア藻綱に属するものであるが、１８Ｓ　ｒＤＮＡおよび１６Ｓ　ｒＤＮＡを用いた分子系統学的解析によれば他のクロレラ属とは別のグループを形成するものである。 The genus Parachlorella belongs to the Treboxya algae among the genus Chlorella that straddles the Treboxyphyceae and Chlorophyceae, but it is determined by molecular phylogenetic analysis using 18S rDNA and 16S rDNA. For example, it forms a different group from other genus Chlorella.
　パラクロレラ属藻類にはクロレラ属藻類のような強固な細胞壁構造が認められず、代わりに多糖類を主体とした厚い膜で覆われている。このことが、クロレラ属藻類に比べてパラクロレラ属藻類が水生生物に消化吸収され易く、その成長効果や飼料効率が高い理由であると考えられる。 Parachlorella algae do not have a strong cell wall structure like Chlorella algae, and instead are covered with a thick film mainly composed of polysaccharides. This is considered to be the reason why Parachlorella algae are easily digested and absorbed by aquatic organisms compared to Chlorella algae, and their growth effect and feed efficiency are high.
　一般的に、パラクロレラ属藻類は、野外で採取した淡水サンプルから、一般的な培地を使った継代培養によりコロニーを分離し、最終的に分子系統学的解析により属種を特定することにより得ることができる。また、市販のものなどがあれば、入手して使用すればよい。 In general, Parachlorella algae can be obtained by isolating colonies from freshwater samples collected in the field by subculture using a general medium, and finally identifying the genus species by molecular phylogenetic analysis. Obtainable. Moreover, what is necessary is just to acquire and use if there exists a commercially available thing.
　パラクロレラ属藻類は、淡水培地やＬＢ培地などの一般的な培地中、好気条件、嫌気条件のいずれでも生育可能であるが、室温～３０℃、明条件、好気条件で特によく増殖する。 Parachlorella algae can grow in aerobic and anaerobic conditions in common media such as freshwater and LB media, but proliferate particularly well at room temperature to 30 ° C. under bright and aerobic conditions. .
　パラクロレラ属藻類としては、例えば、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｍａｒｉｎｉｃｈｌｏｒｅｌｌａ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｄｉｃｔｙｏｓｈａｅｒｉｕｍ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｍｕｃｉｄｏｓｐｈａｅｒｉｕｍ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｃｌｏｓｔｅｒｉｏｐｓｉｓ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｄｉｃｌｏｓｔｅｒ、Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｉｊｅｒｉｎｃが挙げられる。これらの中でも特にＰａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉとＰａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉが好適である。 The para Chlorella algae, for example, Parachlorella kessleri, Parachlorella beyerinckii, Parachlorella marinichlorella, Parachlorella dictyoshaerium, Parachlorella mucidosphaerium, Parachlorella closteriopsis, Parachlorella dicloster, include Parachlorella beijerinc. Of these, Parachlorella kessleri and Parachlorella beyerinckii are particularly suitable.
　パラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類のうち、特に好適なＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）は、下記の通り寄託機関に寄託されている。
（ｉ）　寄託機関の名称およびあて名
　名称：　独立行政法人製品評価技術基盤機構　特許生物寄託センター
　あて名：　日本国　千葉県木更津市かずさ鎌足２－５－８－１　１２０号室
（ｉｉ）　受託日：　２０１３年９月３日
（ｉｉｉ）　受託番号：　ＦＥＲＭ　ＢＰ－２２２５６
　本発明に係るＫＮＫ－Ａ００１株の形態的特徴などは、以下のとおりである。 Among Parachlorella kessleri seed algae, particularly preferred KNK-A001 strain (Accession No .: FERM BP-22256) is deposited at the depository as follows.
(I) Name and address of depositary institution Name: National Institute of Technology and Evaluation Patent Biological Depositary Center Address: 2-5-8-1 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan Room 120 (ii) Date of accession: 2013 September 3 (iii) Accession Number: FERM BP-22256
The morphological characteristics and the like of the KNK-A001 strain according to the present invention are as follows.
Figure JPOXMLDOC01-appb-T000001
 　また、ＫＮＫ－Ａ００１株の１８Ｓ　ｒＲＮＡの部分塩基配列を、配列番号１（ＳＥＱ　ＩＤ　ＮＯ：１）に示す。
Figure JPOXMLDOC01-appb-T000001
 The partial base sequence of 18S rRNA of KNK-A001 strain is shown in SEQ ID NO: 1 (SEQ ID NO: 1).
　また、その１８Ｓ　ｒＲＮＡが、配列番号１に相当する（１）の塩基配列に対して、下記（２）または（３）の塩基配列を有する場合には、ＫＮＫ－Ａ００１株と同じくパラクロレラ属藻類に属し、且つＫＮＫ－Ａ００１株と同様に水生生物に対して優れた成長効果を示すと考えられる。 Further, when the 18S rRNA has the following base sequence (2) or (3) with respect to the base sequence (1) corresponding to SEQ ID NO: 1, the same as the KNK-A001 strain, Parachlorella algae It is considered that it has an excellent growth effect on aquatic organisms as in the case of the KNK-A001 strain.
　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列
　なお、欠失などの変異の導入により１８Ｓ　ｒＲＮＡの塩基配列の塩基数が変化する場合においても、変異数が上記のとおり１以上、３１以下の範囲内にあるか、配列同一性のパーセンテージが上記のとおり９８．５％以上の範囲内にあれば、変異導入後の配列において、変異導入前の特定位置に相当する位置を特定することは当業者にとり容易である。具体的には、塩基配列の多重アラインメント用プログラムで比較すべき配列をアライメントし、位置を決定することが可能である。また、塩基配列の同一性も、多重アラインメント用プログラムで容易に求めることができる。 (2) A base sequence in which one or more and 31 or less bases are deleted, substituted and / or added in the base sequence defined in (1) above (3) For the base sequence defined in (1) above In addition, even when the number of bases of the 18S rRNA base sequence changes due to the introduction of mutation such as deletion, the number of mutations is 1 or more and 31 or less as described above. If the percentage of sequence identity is within the range of 98.5% or more as described above, it is possible to specify a position corresponding to a specific position before mutagenesis in the sequence after mutagenesis. Easy for those skilled in the art. Specifically, the sequences to be compared can be aligned and the position determined by a program for multiple alignment of base sequences. In addition, the identity of the base sequence can be easily determined by a program for multiple alignment.
　上記塩基配列（２）において、欠失などの変異の数としては、３０以下、２０以下または１０以下がより好ましく、９以下、８以下、６以下または５以下がさらに好ましく、４以下、３以下、２以下または１以下がさらに好ましい。 In the base sequence (2), the number of mutations such as deletion is preferably 30 or less, 20 or less, or 10 or less, more preferably 9 or less, 8 or less, 6 or less, or 5 or less, and further preferably 4 or less, 3 or less. 2 or less or 1 or less is more preferable.
　また、上記塩基配列（３）において、塩基配列の同一性のパーセンテージとしては、９９．０％以上、９９．２％以上または９９．４％以上がより好ましく、９９．５％以上、９９．６％以上または９９．７％以上がさらに好ましく、９９．８％以上または９９．９％以上がさらに好ましい。 In the base sequence (3), the percentage of base sequence identity is preferably 99.0% or more, 99.2% or more, or 99.4% or more, more preferably 99.5% or more, 99.6. % Or more or 99.7% or more is more preferable, and 99.8% or more or 99.9% or more is more preferable.
　本発明で用いるパラクロレラ属藻類としては、少なくとも最終の培養段階で従属栄養培養したものが好ましい。パラクロレラ属藻類は、独立栄養培養した場合と従属栄養培養した場合とでは細胞の構成や細胞に含まれる成分に相違が生じる場合があり得るが、少なくとも従属栄養培養したパラクロレラ属藻類の優れた成長効果は確認されている。 The parachlorella algae used in the present invention are preferably those heterotrophic cultured at least in the final culture stage. Parachlorella algae may differ in the composition of cells and components contained in cells when autotrophic culture and heterotrophic culture are performed, but at least the superiority of parachlorella algae cultured under heterotrophic culture The growth effect has been confirmed.
　本発明に係るパラクロレラ属藻類には、その変異体であって、その乾燥体が水生生物に対して優れた成長効果を示すものも含むものとする。ここで「変異体」とは、人為的な選択、交雑、突然変異、遺伝子組み換えなどにより改良したパラクロレラ属藻類をいうものとする。 The Parachlorella algae according to the present invention include mutants whose dried bodies exhibit an excellent growth effect on aquatic organisms. Here, the “mutant” means a parachlorella algae improved by artificial selection, hybridization, mutation, genetic recombination and the like.
　本発明に係る水生生物用養殖飼料の有効成分であるパラクロレラ属藻類自体の乾燥体は、パラクロレラ属藻類の生細胞や、当該生細胞を含む培養液を乾燥することにより得ることができる。即ち、パラクロレラ属藻類の培養液から濾過や遠心分離などにより生細胞を分離して乾燥したり、或いは培養液をそのまま乾燥すればよい。なお、パラクロレラ属藻類は、培養液中に分泌物を放出し、この分泌物が水生生物の成長に有効な成分の一つである可能性がある。培養液をそのまま乾燥した場合には、乾燥体にはかかる分泌物が含まれることになる。 The dried body of Parachlorella algae itself, which is an active ingredient of the aquatic aquaculture feed according to the present invention, can be obtained by drying live cells of Parachlorella algae and a culture solution containing the living cells. That is, the living cells may be separated from the culture solution of Parachlorella algae by filtration or centrifugation, and dried, or the culture solution may be dried as it is. Parachlorella algae release secretions into the culture solution, and this secretion may be one of the effective components for the growth of aquatic organisms. When the culture solution is dried as it is, the dried product contains such secretions.
　パラクロレラ属藻類の生細胞や当該生細胞を含む培養液の乾燥は、常法に従って行うことができる。例えば、当該生細胞または培養液を５０℃以上、２００℃以下で１０秒間以上、１０時間以下程度乾燥すればよい。乾燥時には減圧してもよい。なお、培養液を薄膜状に流下させつつ乾燥すれば、乾燥時間を短縮することができる。また、凍結乾燥してもよい。具体的な乾燥条件は、乾燥すべきパラクロレラ属藻類や培養液の量、使用する機器などに応じて適宜調整することができる。また、得られた乾燥体は、一般的には凝集しているので、さらに粉砕してもよい。粉砕程度は、給餌すべき水生生物が摂取し易い程度に調整すればよい。 Drying of the living cells of Parachlorella algae and the culture solution containing the living cells can be performed according to a conventional method. For example, the living cells or the culture solution may be dried at 50 ° C. or higher and 200 ° C. or lower for 10 seconds or longer and 10 hours or shorter. The pressure may be reduced during drying. It should be noted that drying time can be shortened by drying the culture solution while flowing it into a thin film. Moreover, you may freeze-dry. Specific drying conditions can be appropriately adjusted according to the amount of Parachlorella algae to be dried, the amount of the culture solution, the equipment to be used, and the like. Moreover, since the obtained dried body is generally aggregated, it may be further pulverized. What is necessary is just to adjust the grinding | pulverization grade to the grade which the aquatic organism which should be fed can ingest easily.
　なお、本発明における「乾燥体」は特に制限されず、パラクロレラ属藻類が生細胞ではなく、また、明らかな湿潤状態になければその水分含量は問わないものとする。例えば、乾燥体の水分含量としては３０質量％以下とすることができる。当該水分含量としては、その値が低いほど飼料の保存安定性が高く、また、運搬が容易であるので、２０質量％以下が好ましく、１５質量％以下がより好ましく、１０質量％以下がさらに好ましく、８質量％以下がよりさらに好ましい。一方、過剰に乾燥させる必要はないので、当該水分含量としては１質量％以上が好ましく、２質量％以上がより好ましく、５質量％以上がさらに好ましい。なお、上記の乾燥は細胞を殺すためや保存安定性を向上させるために行うものであるので、実際の飼料製品や給餌時の飼料における水分含量は問題にならない。例えば、パラクロレラ属藻類の乾燥体を養殖水などに分散した上で給餌してもよい。また、乾燥体中の水分含量は、カールフィッシャー法により容易に測定することができる。 In addition, the “dry body” in the present invention is not particularly limited, and the moisture content is not limited unless the parachlorella algae is not a living cell and is not in a clear wet state. For example, the moisture content of the dried product can be 30% by mass or less. As the water content, the lower the value, the higher the storage stability of the feed and the easier the transportation, so 20% by mass or less is preferable, 15% by mass or less is more preferable, and 10% by mass or less is more preferable. 8% by mass or less is even more preferable. On the other hand, since it is not necessary to dry excessively, the water content is preferably 1% by mass or more, more preferably 2% by mass or more, and further preferably 5% by mass or more. In addition, since said drying is performed in order to kill a cell or to improve the storage stability, the water content in an actual feed product or feed during feeding does not matter. For example, the dried body of Parachlorella algae may be fed after being dispersed in aquaculture water or the like. Further, the moisture content in the dried product can be easily measured by the Karl Fischer method.
　また、本発明において「有効成分」とは、養殖対象である水生生物に対して少なくとも毒性を示すことはなく、水生生物の個体数や体重の増加を促進するものをいう。 Further, in the present invention, the “active ingredient” refers to a substance that does not exhibit at least toxicity to aquatic organisms to be cultured and promotes the increase in the number of aquatic organisms and the body weight.
　本発明に係る水生生物用養殖飼料の給餌量は、給餌すべき水生生物の種類や成長度合いなどに応じて適宜調整すればよいが、例えば、陸上養殖される稚貝や稚魚、輪形動物のような飼料用動物プランクトンの場合、養殖水に対する乾燥体中のパラクロレラ属藻類細胞の数で、一回の給餌当たり１×１０4ｃｅｌｌ／ｍＬ以上、１×１０6ｃｅｌｌ／ｍＬ以下程度とすることができる。当該給餌量は、養殖対象である水生生物の成長度合いに応じて増やしていけばよい。 The feed amount of the aquatic aquaculture feed according to the present invention may be adjusted as appropriate according to the type of aquatic organisms to be fed, the degree of growth, etc., for example, larvae cultured on land, fry, and ring animals. In the case of zooplankton for feed, the number of Parachlorella algae cells in the dried body relative to the aquaculture water should be about 1 × 10 4 cells / mL or more and 1 × 10 6 cells / mL or less per feeding Can do. What is necessary is just to increase the said feed amount according to the growth degree of the aquatic organism which is a culture target.
　また、一般的な魚類に対する本発明に係る水生生物用養殖飼料の給餌量も、その種類や成長度合い、他の飼料との配合割合などを考慮して適宜調整すればよいが、例えば、一日の施餌当たりのパラクロレラ属藻類の摂取量を０．２ｇ／ｋｇ体重以上程度とすることができる。 Further, the feed amount of the aquatic aquaculture feed according to the present invention for general fish may be appropriately adjusted in consideration of the type, the degree of growth, the blending ratio with other feeds, etc. The intake of Parachlorella algae per feed can be about 0.2 g / kg body weight or more.
　本発明に係る水生生物用養殖飼料の一日当たりの給餌回数も適宜調整すればよいが、例えば、一日当たり１回以上、３回以下とすることができる。 The number of feeds per day of the aquatic aquaculture feed according to the present invention may be adjusted as appropriate, but can be adjusted to, for example, 1 to 3 times per day.
　本発明に係る水生生物用養殖飼料を、魚類用に他の飼料等と混合する場合には、本発明飼料の含有割合を１．０質量％以上とすることが好ましい。上限は特に制限されないが、栄養の偏りを低減するために、１０質量％以下とすることが好ましい。配合すべき他の飼料としては、例えば、魚粉、大豆粕、米糠、小麦粉、でんぷん、酵母、油脂、ビタミン類、ミネラル類などが挙げられ、対象魚類の種類や成長度合いに応じて適宜選択すればよい。 When the aquatic aquaculture feed according to the present invention is mixed with other feeds for fish, the content of the present feed is preferably 1.0% by mass or more. The upper limit is not particularly limited, but is preferably 10% by mass or less in order to reduce nutritional bias. Other feeds to be blended include, for example, fish meal, soybean meal, rice bran, wheat flour, starch, yeast, fats and oils, vitamins, minerals, etc., if selected appropriately according to the type and growth degree of the target fish Good.
　本発明に係る水生生物用養殖飼料の形態は特に制限されず、給餌すべき水生生物の種類や成長度合いなどに応じて適宜決定すればよい。例えば、稚貝や微生物には、摂食し易いようパラクロレラ属藻類の乾燥体を粉砕したものとすることができる。一方、稚魚の場合、上記のような粉砕物では、養殖水中に拡散してしまい稚魚が摂食できないおそれがあるので、パラクロレラ属藻類の乾燥体の粉砕物を、必要に応じて他の飼料と共に適度な大きさに成形して固形状とすることが好ましい。 The form of the aquatic aquaculture feed according to the present invention is not particularly limited, and may be appropriately determined depending on the type of aquatic organism to be fed, the degree of growth, and the like. For example, the dried body of Parachlorella algae can be crushed so that it can be easily eaten by juvenile shellfish and microorganisms. On the other hand, in the case of larvae, the crushed material as described above may diffuse into the aquaculture water and the larvae may not be eaten, so the pulverized product of Parachlorella algae can be used as needed for other feeds. At the same time, it is preferably molded into a suitable size to form a solid.
　本発明に係る水生生物用養殖飼料を給餌すべき対象生物は、本発明飼料により有効に成長できるものであれば特に制限されない。例えば、カキ、アワビ、ホタテ、シンジュガイ、イタヤガイ、ヒオウギガイなどの貝類；タイ、ヒラメ、マグロ、ブリ、カンパチ、シマアジ、イサキなどの海水魚；ウナギ、イワナ、アマゴ、マスなどの淡水魚；シオミズツボワムシやチグリオプスなどの飼料用動物プランクトンを挙げることができる。 The target organism to be fed with the aquatic aquaculture feed according to the present invention is not particularly limited as long as it can grow effectively with the feed according to the present invention. For example, shellfish such as oysters, abalone, scallops, scallops, scallops, cypresses; saltwater fish such as Thailand, flounder, tuna, yellowtail, amberjack, striped horse mackerel, isaki; freshwater fish such as eel, char, trout, trout; And feed zooplankton such as tigriops.
　以下、本発明に係る水生生物の養殖方法につき、簡単に説明する。 Hereinafter, the aquatic organism cultivation method according to the present invention will be briefly described.
　１．　パラクロレラ属藻類の培養工程
　本工程では、パラクロレラ属藻類を培養する。本工程の実施は任意であるが、本発明に係る水生生物用養殖飼料の生産の観点からは、本工程を実施してパラクロレラ属藻類の総量を増やすことが好ましい。 1. Parachlorella Algae Culture Process In this process, Parachlorella algae are cultured. Although implementation of this process is arbitrary, it is preferable to implement this process and to increase the total amount of Parachlorella algae from the viewpoint of production of aquatic aquaculture feed according to the present invention.
　パラクロレラ属藻類の培養条件は、培養すべきパラクロレラ属藻類の至適条件に応じて適宜決定すればよい。例えば、上記のとおり、淡水培地やＬＢ培地などの一般的な培地中、好気条件または嫌気条件下、室温～３０℃で培養すればよい。 The culture conditions for the Parachlorella algae may be appropriately determined according to the optimal conditions for the Parachlorella algae to be cultured. For example, as described above, the culture may be performed at room temperature to 30 ° C. under aerobic conditions or anaerobic conditions in a general medium such as a fresh water medium or an LB medium.
　パラクロレラ属藻類は、一般的には明条件で特によく増殖する。よって、パラクロレラ属藻類の総量を増やすという観点では、明条件で、特に独立栄養条件で培養することが好ましい。しかし、少なくとも従属栄養培養したパラクロレラ属藻類の優れた成長効果は確認されていることから、遮光し、従属栄養条件下で培養することも好ましい。その他、独立栄養条件でパラクロレラ属藻類を十分に増殖せしめた後に、従属栄養条件下で培養してもよい。 Parachlorella algae generally grow particularly well under light conditions. Therefore, from the viewpoint of increasing the total amount of Parachlorella algae, it is preferable to culture under bright conditions, particularly under autotrophic conditions. However, since an excellent growth effect of Parachlorella algae cultured at least in heterotrophic culture has been confirmed, it is also preferable to cultivate under heterotrophic conditions while shielding light. In addition, after the Parachlorella algae are sufficiently grown under autotrophic conditions, they may be cultured under heterotrophic conditions.
　２．　乾燥工程
　本工程では、パラクロレラ属藻類を乾燥してパラクロレラ藻類の乾燥体を得る。乾燥方法や乾燥の程度などは、上記に従えばよい。 2. Drying step In this step, Parachlorella algae are dried to obtain a dried body of Parachlorella algae. What is necessary is just to follow the drying method, the degree of drying, etc. above.
　３．　給餌工程
　本工程では、パラクロレラ属藻類の乾燥体を有効成分として含む飼料を水生生物に給餌する。具体的な給餌条件は、上記のとおり、養殖対象である水生生物の種類や成長度合いなどに応じて適宜決定すればよい。 3. Feeding step In this step, feed containing a dry body of Parachlorella algae as an active ingredient is fed to aquatic organisms. Specific feeding conditions may be appropriately determined according to the type of aquatic organisms to be cultured and the degree of growth as described above.
　本発明によれば、一般的な飼料を給餌した場合に比べ、より少ない量で水生生物を正常な、またはそれ以上の成長を促すことができ、給餌量を抑制することが可能である。また、一般的な飼料を同量給餌した場合に比べ、水生生物の個体数や総重量を顕著に増加させることができる。 According to the present invention, it is possible to promote normal or higher growth of aquatic organisms in a smaller amount than when feeding a general feed, and it is possible to suppress the amount of feeding. In addition, the number of aquatic organisms and the total weight can be significantly increased as compared with the case where the same amount of general feed is fed.
　本願は、２０１３年１１月１８日に出願された日本国特許出願第２０１３－２３７８３３号に基づく優先権の利益を主張するものである。２０１３年１１月１８日に出願された日本国特許出願第２０１３－２３７８３３号の明細書の全内容が、本願に参考のため援用される。 This application claims the benefit of priority based on Japanese Patent Application No. 2013-237833 filed on Nov. 18, 2013. The entire contents of Japanese Patent Application No. 2013-237833 filed on November 18, 2013 are incorporated herein by reference.
　以下、実施例を挙げて本発明をより具体的に説明するが、本発明はもとより下記実施例によって制限を受けるものではなく、前・後記の趣旨に適合し得る範囲で適当に変更を加えて実施することも勿論可能であり、それらはいずれも本発明の技術的範囲に包含される。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.
　実施例１：カキ稚貝の養殖実験
　（１）　本発明に係る水生生物用養殖飼料の調製
　殺菌した液体培地にパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）ＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）を接種し、アルミホイルで遮光した後、３０℃で７２時間前培養した。次に、より大容量の殺菌液体培地に前培養培地を加え、アルミホイルで遮光した後、内温３０℃、通気量２Ｌ／分、撹拌数４５０ｒｐｍ、ｐＨ６～７で１４３時間培養した。 Example 1: Culture experiment of oyster larvae (1) Preparation of aquaculture feed for aquatic organisms according to the present invention Parachlorella kessleri KNK-A001 strain (accession number: FERM BP-22256) on a sterilized liquid medium And light-shielded with aluminum foil, and then precultured at 30 ° C. for 72 hours. Next, a pre-culture medium was added to a larger volume of a sterilized liquid medium, light-shielded with aluminum foil, and cultured for 143 hours at an internal temperature of 30 ° C., an aeration rate of 2 L / min, an agitation rate of 450 rpm, and a pH of 6-7.
　次いで、ダブルドラム型乾燥機により培養液を乾燥し、得られた乾燥凝集体をフェザーミルで粉砕することによりＫＮＫ－Ａ００１株の細胞乾燥物を得た。 Next, the culture solution was dried with a double drum dryer, and the obtained dried aggregate was pulverized with a feather mill to obtain a dried cell product of KNK-A001 strain.
　また、パラクロレラ属藻類として、パラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類（製品名「バイエリンク」，三井物産株式会社より購入）を同様の実験に供した。 Also, Parachlorella beyerinckii seed algae (product name “Bayerink”, purchased from Mitsui & Co., Ltd.) was subjected to the same experiment as the Parachlorella algae.
　また、比較のために、マガキ養殖の初期飼料として用いられる微細藻類であるキートセロス（Ｃｈａｅｔｏｃｅｒｏｓ　ｇｒａｃｉｌｉｓ）を継代培養し、生きたまま実験に用いた。さらに、市販のクロレラ（Ｃｈｌｏｒｅｌｌａ　ｖｕｌｇａｒｉｓ）乾燥粉末も実験に供した。 For comparison, the microalgae, Chaetoceros gracilis, used as an initial feed for oyster culture was subcultured and used alive for experiments. Furthermore, a commercially available chlorella (Chlorella vulgaris) dry powder was also used for the experiment.
　（２）　養殖実験
　ガラスプレートに付着してから１週間が経過したマガキ（Ｃｒａｓｓｏｓｔｅｒａ　ｇｉｇａｓ）の２３～２７日目のＤ型幼生１０個体を、濾過海水（５００ｍＬ）を入れた１０００ｍＬビーカー内に入れた。 (2) Aquaculture Experiment Ten D-type larvae of oysters (Crassostera gigas) 23 to 27 days after one week had adhered to the glass plate were placed in a 1000 mL beaker containing filtered seawater (500 mL). .
　ＫＮＫ－Ａ００１株乾燥粉末、パラクロレラ・バイエリンク種藻類乾燥粉末およびクロレラ乾燥粉末は、濾過海水に懸濁後、部分的な細胞凝集を解砕するために超音波処理を行い、均一な細胞懸濁液とし、予備実験で好適濃度とされた２０×１０4ｃｅｌｌ／ｍＬ／ｄａｙの割合で給餌した。キートセロスは、技術報告書（Vasquezら，2009）を参照して、３．７５×１０4ｃｅｌｌ／ｍＬ／ｄａｙまたは２０×１０4ｃｅｌｌ／ｍＬ／ｄａｙの割合で給餌した。 The KNK-A001 dry powder, Parachlorella Bayelinceae algae dry powder and Chlorella dry powder are suspended in filtered seawater and then subjected to sonication to break up the partial cell aggregation and uniform cell suspension. It was made into a turbid liquid and fed at a rate of 20 × 10 4 cells / mL / day, which was adjusted to a suitable concentration in a preliminary experiment. Keat cellos was fed at a rate of 3.75 × 10 4 cells / mL / day or 20 × 10 4 cells / mL / day with reference to a technical report (Vasquez et al., 2009).
　各飼料を上記割合で１日１回給餌しつつ、パスツールピペットを使って通気しつつ、２５℃のインキュベーター内でマガキ幼生を１週間飼育した。 The oyster larvae were bred for 1 week in an incubator at 25 ° C. while feeding each feed once a day at the above rate and aeration with a Pasteur pipette.
　１週間飼育後の各マガキ幼生の殻長の増加分のグラフを図１に、各マガキ幼生の拡大写真を図２に示す。なお、図１中の「カネカクロレラ」はＫＮＫ－Ａ００１株を示す。また、図２において、ＡとＢはそれぞれキートセロス生細胞を３．７５×１０4ｃｅｌｌ／ｍＬ／ｄａｙ、２０×１０4ｃｅｌｌ／ｍＬ／ｄａｙ給餌した区、ＣはＫＮＫ－Ａ００１株乾燥粉末を給餌した区、Ｄはクロレラ乾燥粉末を給餌した区である。さらに、実験開始直後の殻長と１週間飼育後の殻長の差である平均成長量を測定した。結果を表２に示す。 A graph showing the increase in shell length of each oyster larva after one week of breeding is shown in FIG. 1, and an enlarged photograph of each oyster larva is shown in FIG. In addition, “Kaneka Chlorella” in FIG. 1 indicates the KNK-A001 strain. In FIG. 2, A and B are fed with live Keatoceros cells at 3.75 × 10 4 cells / mL / day and 20 × 10 4 cells / mL / day, and C is fed with dry powder of KNK-A001 strain. D is a section fed with chlorella dry powder. Furthermore, the average growth amount, which is the difference between the shell length immediately after the start of the experiment and the shell length after one week of breeding, was measured. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
 　以上の結果より、クロレラ乾燥粉末を給餌したマガキ幼生では成長度が劣っていたのに対して、本発明に係るパラクロレラ属藻類乾燥粉末を給餌したマガキ幼生の成長度は有意に高かった。また、パラクロレラ属藻類乾燥粉末は、乾燥体であるにもかかわらず、生細胞であるキートセロスと同程度の成長効果を示した。
Figure JPOXMLDOC01-appb-T000002
 From the above results, the growth rate of the oyster larvae fed with the dry powder of chlorella was significantly higher, whereas the growth rate of the oyster larvae fed with the dry powder of the genus Parachlorella according to the present invention was significantly higher. In addition, the dry powder of the genus Parachlorella showed a growth effect comparable to that of Keatoceros, which is a living cell, even though it was a dry body.
　（３）　成長効果の違いの原因検討実験
　パラクロレラ属藻類乾燥粉末とクロレラ乾燥粉末のマガキ幼生に対する成長効果の相違の原因を検討するために、マガキ幼生の排泄物を調べた。パラクロレラ・ケスレリ　ＫＮＫ－Ａ００１株乾燥粉末を給餌した際のマガキ幼生の周辺に存在する排泄物の、クロロフィル由来の傾向を観察する波長での蛍光顕微鏡写真（図３Ａ）と、クロレラ乾燥粉末を給餌した場合の顕微鏡写真（図３Ｂ）を示す。 (3) Cause examination experiment of difference in growth effect In order to examine the cause of the difference in growth effect of Parachlorella algae dry powder and Chlorella dry powder on oyster larvae, the excrement of oyster larvae was examined. Fluorescence micrograph at the wavelength to observe the tendency of chlorophyll-derived excreta present around oyster larvae when fed with Parachlorella quercerium KNK-A001 strain dry powder and feeding with Chlorella dry powder A micrograph (FIG. 3B) is shown.
　図３Ｂのとおり、クロレラ乾燥粉末を給餌した場合には、排泄物の中に粒状の強い蛍光が多数観察された。これは、クロレラ細胞が消化されずにそのまま排出されていることを示している。一方、図３Ａのとおり、ＫＮＫ－Ａ００１株乾燥粉末を給餌した場合には不定形の弱い蛍光が排泄物全体に観察され、粒状物は見られない。かかる結果のとおり、同量を給餌しているにもかかわらずクロレラ乾燥粉末よりもパラクロレラ属藻類乾燥粉末で有効な成長効果が得られたのは、クロレラ乾燥粉末は十分に消化されず栄養源として有効に利用されないのに対して、パラクロレラ属藻類乾燥粉末は栄養源として効率的に利用されていることによると考えられる。 As shown in FIG. 3B, when the chlorella dry powder was fed, many granular strong fluorescence was observed in the excreta. This indicates that the chlorella cells are excreted without being digested. On the other hand, as shown in FIG. 3A, when the dry powder of KNK-A001 strain is fed, amorphous weak fluorescence is observed in the whole excrement, and no particulate matter is seen. As can be seen from these results, the more effective growth effect was obtained with Parachlorella algae dry powder than with Chlorella dry powder despite being fed the same amount. However, it is considered that Parachlorella algae dry powder is efficiently used as a nutrient source.
　実施例２：ワムシの養殖実験
　２４ウェルプレートの各ウェルに５００μＬのＥＭＳ培地と共にワムシを２匹加え、上記実施例１で用いたものと同様のパラクロレラ・ケスレリ　ＫＮＫ－Ａ００１株乾燥粉末、パラクロレラ・バイエリンク種藻類乾燥粉末またはクロレラ乾燥粉末を，それぞれ２０×１０4ｃｅｌｌ／ｍＬ／ｄａｙ給餌した。２４時間毎に立体顕微鏡（Ｎｉｋｏｎ社製，製品名「ＳＭＺ１０００」）で観察して泳いでいる個体数を計数しながら、明暗１２時間サイクルの白色蛍光（２００±５μｍｏｌ／ｍ2ｓ）、２６℃の条件で１週間培養した。また、比較のため、飼料を添加せずＥＭＳ培地のみでも同様に培養を行った。結果を図４に示す。 Example 2: Rotation experiment of rotifer Two rotifers with 500 μL of EMS medium were added to each well of a 24-well plate, and Parachlorella querseri KNK-A001 strain dry powder, Parachlorella similar to that used in Example 1 above. -Bayerink seed algae dry powder or chlorella dry powder was fed at 20 × 10 4 cells / mL / day, respectively. White fluorescence (200 ± 5 μmol / m 2 s) at a light / dark 12 hour cycle, 26 ° C. while counting the number of individuals swimming by observation with a stereomicroscope (product name “SMZ1000”, manufactured by Nikon) every 24 hours For 1 week. In addition, for comparison, the culture was similarly performed using only the EMS medium without adding the feed. The results are shown in FIG.
　図４のとおり、ＥＳＭ培地のみで無給餌の場合ではワムシ個体数は増加せず、７日目ですべて死滅した。一方、クロレラ乾燥粉末給餌区ではワムシ個体数は増加し、７日目に４６±９匹となった。また、ＫＮＫ－Ａ００１株乾燥粉末給餌区およびパラクロレラ・バイエリンク種藻類乾燥粉末では、同じくワムシ固体数は増加し、且つその成長効果はクロレラ乾燥給餌区の場合に比べて常に高いものであり、７日目では５３±２匹となった。以上の結果から、本発明に係る水生生物用養殖飼料は、クロレラ乾燥粉末よりも、ワムシに対する飼料として優れたものであるといえる。 As shown in FIG. 4, in the case of ESM medium alone and no feeding, the number of rotifers did not increase and all died on the 7th day. On the other hand, in the chlorella dry powder feeding section, the number of rotifers increased to 46 ± 9 on the seventh day. In addition, in the KNK-A001 dry powder feeding area and Parachlorella Bayelinceae algae dry powder, the number of rotifer solids is also increased, and the growth effect is always higher than in the Chlorella dry feeding area, On day 7, the number was 53 ± 2. From the above results, it can be said that the aquatic aquaculture feed according to the present invention is superior to chlorella dry powder as a feed for rotifers.
　実施例３：マダイの養殖実験
　直径３ｍ、高さ１ｍの円柱形プールを５個用意し、各プールに体重が約５７．５ｇのマダイ稚魚を３０尾ずつ入れた。粉末状のマダイ用配合飼料（ヒガシマル社製，製品名「ひめ桜」）に上記実施例１で用いたものと同様のパラクロレラ・ケスレリ　ＫＮＫ－Ａ００１株乾燥粉末を０質量％、０．５質量％、１質量％、２．５質量％、５質量％混合し、直径２ｍｍ、長さ３ｍｍのペレットに成形した。当該サンプル飼料を１日１回、余剰飼料が生じない程度に飽食給餌しつつ１ヶ月間飼育した。飼育期間中の水温は２６．４～２８．４℃であり、平均２７．４℃であった。残留飼料が生じた場合にはその都度除去した。２週間毎に体重を測定し、平均値を算出した。結果を図５に示す。 Example 3: Red sea bream culture experiment Five cylindrical pools having a diameter of 3 m and a height of 1 m were prepared, and 30 red sea breams weighing about 57.5 g were placed in each pool. 0% by mass and 0.5% by mass of dry powder of Parachlorella kesleri KNK-A001 similar to that used in Example 1 above for powdered red sea bream mixed feed (manufactured by Higashimaru, product name “Himezakura”) %, 1% by mass, 2.5% by mass, and 5% by mass, and formed into pellets having a diameter of 2 mm and a length of 3 mm. The sample feed was bred once a day for 1 month while being fed with satiation to the extent that no excess feed was produced. The water temperature during the breeding period was 26.4 to 28.4 ° C., and the average was 27.4 ° C. When residual feed was generated, it was removed each time. The body weight was measured every two weeks, and the average value was calculated. The results are shown in FIG.
　図５のとおり、本発明に係るパラクロレラ属藻類乾燥体を飼料に０．５質量％配合した場合にはそれほどの効果は見られないが、１．０質量％以上配合した場合には明確な成長促進効果が認められた。 As shown in FIG. 5, when 0.5% by mass of the dried parachlorella algae according to the present invention is blended in the feed, not much effect is seen, but when 1.0% by mass or more is blended, it is clear. A growth promoting effect was observed.
　実施例４：クルマエビの養殖実験
　２００Ｌの水槽を合計４槽用意し、それぞれに同一量の砂を入れ、約２４℃の加温海水を流水方式で常時注入した。各水槽へ体重３～５ｇのクルマエビを１５尾ずつ収容した。エビ用配合飼料（ヒガシマル社製，製品名「ひめ桜」）に上記実施例１で得たパラクロレラ・ケスレリ　ＫＮＫ－Ａ００１株乾燥粉末を０．０２質量％配合し、１日１回、２槽の水槽へ日没後に飽食給餌し、翌朝、残餌を回収しつつ６週間飼育した。また、比較の為に、ＫＮＫ－Ａ００１株乾燥粉末を配合しないエビ用配合飼料のみを２槽の水槽へ給餌して、同様に飼育した。飼育開始時と、飼育開始から２週間後、４週間後および６週間後に各エビの体重を測定し、平均値を算出した。結果を図６に示す。 Example 4: Aquatic Shrimp Culture Experiment A total of 4 200 L water tanks were prepared, each with the same amount of sand, and warm seawater at about 24 ° C. was constantly injected in a flowing water system. Each fish tank contained 15 prawns with a body weight of 3 to 5 g. A mixed feed for shrimp (manufactured by Higashimaru, product name “Himezakura”) is mixed with 0.02% by mass of the dry powder of Parachlorella kesleri KNK-A001 obtained in Example 1 above, once a day for 2 tanks. After sunset, the fish tank was fed with food, and the next morning, the remaining food was collected and reared for 6 weeks. For comparison, only shrimp formula feed containing no KNK-A001 strain dry powder was fed to two tanks and reared in the same manner. The body weight of each shrimp was measured at the start of breeding and after 2 weeks, 4 weeks and 6 weeks from the start of breeding, and the average value was calculated. The results are shown in FIG.
　図６に示された結果のとおり、本発明に係るバイノス乾燥粉末を含む飼料を給餌した場合には、通常の飼料のみを給餌した場合に比べて常にクルマエビの成長度合いが高かった。よって、本発明に係るパラクロレラ属藻類給餌区では、クルマエビの成長効果は対照飼料の場合に比べて常に高いものであった。以上の結果から、本発明に係る水生生物用養殖飼料は、エビ類の養殖飼料としても優れたものであることが明らかとなった。 As shown in the results shown in FIG. 6, when the feed containing the binos dry powder according to the present invention was fed, the degree of growth of the prawns was always higher than when only the normal feed was fed. Therefore, in the Parachlorella algae feeding area according to the present invention, the growth effect of prawns was always higher than that of the control feed. From the above results, it was revealed that the aquatic aquaculture feed according to the present invention is also excellent as a shrimp aquaculture feed.

Claims (21)

  1. 　パラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体を有効成分として含むことを特徴とする水生生物用養殖飼料。 Aquaculture feed for aquatic organisms containing a dried body of Parachlorella as an active ingredient.
  2. 　パラクロレラ属藻類がパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類である請求項１に記載の水生生物用養殖飼料。 The aquaculture feed for aquatic organisms according to claim 1, wherein the Parachlorella algae is Parachlorella kessleri seed algae.
  3. 　パラクロレラ・ケスレリ種藻類がＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）である請求項２に記載の水生生物用養殖飼料。 The aquatic aquaculture feed according to claim 2, wherein the Parachlorella quercerae seed algae is KNK-A001 strain (Accession No .: FERM BP-22256).
  4. 　パラクロレラ属藻類の１８Ｓ　ｒＲＮＡが、下記（１）～（３）のいずれかの塩基配列を有する請求項１～３のいずれかに記載の水生生物用養殖飼料。
    　（１）　配列番号１の塩基配列
    　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
    　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列     The aquatic aquaculture feed according to any one of claims 1 to 3, wherein the 18S rRNA of the Parachlorella algae has any one of the following base sequences (1) to (3):
    (1) Base sequence of SEQ ID NO: 1 (2) In the base sequence defined in (1) above, a base sequence in which 1 to 31 bases have been deleted, substituted and / or added (3) (1) ) Base sequence having a sequence identity of 98.5% or more with respect to the base sequence defined in
  5. 　パラクロレラ属藻類がパラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類である請求項１に記載の水生生物用養殖飼料。 The aquatic aquaculture feed according to claim 1, wherein the Parachlorella algae is a Parachlorella beyerinckii seed algae.
  6. 　従属栄養培養したパラクロレラ属藻類の乾燥体を含む請求項１～５のいずれかに記載の水生生物用養殖飼料。 The aquatic aquaculture feed according to any one of claims 1 to 5, comprising a dried body of Parachlorella algae cultured in heterotrophic culture.
  7. 　水生生物が、貝類、魚類、飼料用動物プランクトンから選択されるものである請求項１～６のいずれかに記載の水生生物用養殖飼料。 The aquatic aquaculture feed according to any one of claims 1 to 6, wherein the aquatic organism is selected from shellfish, fish and zooplankton for feed.
  8. 　水生生物用養殖飼料の有効成分としてのパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 Use of a dried body of Parachlorella algae as an active ingredient of aquatic aquaculture feed.
  9. 　パラクロレラ属藻類がパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類である請求項８に記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 Use of the dried body of Parachlorella algae according to claim 8, wherein the Parachlorella algae is Parachlorella kessleri seed algae.
  10. 　パラクロレラ・ケスレリ種藻類がＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）である請求項９に記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 10. Use of a dried body of Parachlorella algae according to claim 9, wherein the Parachlorella quercerae seed algae is KNK-A001 strain (Accession number: FERM BP-22256).
  11. 　パラクロレラ属藻類の１８Ｓ　ｒＲＮＡが、下記（１）～（３）のいずれかの塩基配列を有する請求項８～１０のいずれかに記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。
    　（１）　配列番号１の塩基配列
    　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
    　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列     Use of the dried body of Parachlorella algae according to any one of claims 8 to 10, wherein the 18S rRNA of the genus Parachlorella has a base sequence of any one of the following (1) to (3).
    (1) Base sequence of SEQ ID NO: 1 (2) In the base sequence defined in (1) above, a base sequence in which 1 to 31 bases have been deleted, substituted and / or added (3) (1) ) Base sequence having a sequence identity of 98.5% or more with respect to the base sequence defined in
  12. 　パラクロレラ属藻類がパラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類である請求項８に記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 Use of the dried body of Parachlorella algae according to claim 8, wherein the Parachlorella algae is a Parachlorella beyerinckii seed algae.
  13. 　パラクロレラ属藻類が従属栄養培養されたものである請求項８～１２のいずれかに記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 Use of a dried body of Parachlorella algae according to any one of claims 8 to 12, wherein Parachlorella algae are heterotrophically cultured.
  14. 　水生生物が、貝類、魚類、飼料用動物プランクトンから選択されるものである請求項８～１３のいずれかに記載のパラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体の使用。 The use of a dried body of Parachlorella algae according to any one of claims 8 to 13, wherein the aquatic organism is selected from shellfish, fish and zooplankton for feed.
  15. 　パラクロレラ（Ｐａｒａｃｈｌｏｒｅｌｌａ）属藻類の乾燥体を有効成分として含む飼料を水生生物に給餌する工程を含むことを特徴とする水生生物の養殖方法。 A method for cultivating aquatic organisms, comprising a step of feeding aquatic organisms with a feed containing a dried body of Parachlorella as an active ingredient.
  16. 　パラクロレラ属藻類がパラクロレラ・ケスレリ（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｋｅｓｓｌｅｒｉ）種藻類である請求項１５に記載の方法。 The method according to claim 15, wherein the Parachlorella algae is a Parachlorella kessleri seed algae.
  17. 　パラクロレラ・ケスレリ種藻類がＫＮＫ－Ａ００１株（受託番号：ＦＥＲＭ　ＢＰ－２２２５６）である請求項１６に記載の方法。 The method according to claim 16, wherein the Parachlorella quercerii species algae is KNK-A001 strain (Accession No .: FERM BP-22256).
  18. 　パラクロレラ属藻類の１８Ｓ　ｒＲＮＡが、下記（１）～（３）のいずれかの塩基配列を有する請求項１５～１７のいずれかに記載の方法。
    　（１）　配列番号１の塩基配列
    　（２）　上記（１）に規定される塩基配列において、１以上、３１以下の塩基が欠失、置換および／または付加された塩基配列
    　（３）　上記（１）に規定される塩基配列に対して９８．５％以上の配列同一性を有する塩基配列     The method according to any one of claims 15 to 17, wherein the 18S rRNA of the genus Parachlorella has any one of the following base sequences (1) to (3).
    (1) Base sequence of SEQ ID NO: 1 (2) In the base sequence defined in (1) above, a base sequence in which 1 to 31 bases have been deleted, substituted and / or added (3) (1) ) Base sequence having a sequence identity of 98.5% or more with respect to the base sequence defined in
  19. 　パラクロレラ属藻類がパラクロレラ・バイエリンク（Ｐａｒａｃｈｌｏｒｅｌｌａ　ｂｅｙｅｒｉｎｃｋｉｉ）種藻類である請求項１５に記載の方法。 The method according to claim 15, wherein the Parachlorella algae is a Parachlorella beyerinckii seed algae.
  20. 　さらに、パラクロレラ属藻類を従属栄養培養する工程と、従属栄養培養したパラクロレラ属藻類を乾燥してパラクロレラ属藻類の乾燥体を得る工程を含む請求項１５～１９のいずれかに記載の方法。 The method according to any one of claims 15 to 19, further comprising heterotrophic culture of Parachlorella algae, and a step of drying the Parachlorella algae subjected to heterotrophic culture to obtain a dried body of Parachlorella algae. .
  21. 　水生生物が、貝類、魚類、飼料用動物プランクトンから選択されるものである請求項１５～２０のいずれかに記載の方法。 The method according to any one of claims 15 to 20, wherein the aquatic organism is selected from shellfish, fish and zooplankton for feed.
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Citations (2)

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WO2013035797A1 (en) * 2011-09-09 2013-03-14 株式会社カネカ Method for cultivating seaweed and method for producing alginic acid-containing composition
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WO2013035797A1 (en) * 2011-09-09 2013-03-14 株式会社カネカ Method for cultivating seaweed and method for producing alginic acid-containing composition
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