JP3302123B2 - Animal Plankton Culture Feed - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a feed for culturing zooplankton such as Rotifer, Artemia and the like, which are indispensable for the production of aquatic seedlings in cultivation, fishery and aquaculture. More specifically, the present invention relates to a feed for culturing zooplankton that can enhance docosahexaenoic acid in nutritional aspects.

[0002]

2. Description of the Related Art In the production of fish and crustaceans in the field of fisheries, i.e., the production of juveniles and larvae (hereinafter referred to simply as juveniles), zooplankton such as rotifers and artemia are useful as biological feeds. Is essential. For larvae, it is known that ω-3 highly unsaturated fatty acids represented by eicosapentaenoic acid (hereinafter abbreviated as EPA) are essential fatty acids. However, zooplankton, which is a feed for larvae, contains almost no ω-3 highly unsaturated fatty acids.

[0003] For this reason, attempts have been made to enhance ω-3 polyunsaturated fatty acids in zooplankton and accumulate them in the body. For example, Nannochloropsis (Nannoc chloropsis), commonly known as marine chlorella
algae of the genus hloropsis) contain EPA in their algal bodies,
It is used as a suitable feed when cultivating rotifers and artemia. The zooplankton cultured using this has enhanced EPA and is effective in improving the growth rate and decreasing the mortality in the production of larvae and larvae. ("Oil", Vol. 42, No. 12, pp. 242-246, 1989, etc.)

[0004] In recent years, it has been reported that docosahexaenoic acid (hereinafter abbreviated as DHA) is more effective as an essential fatty acid for marine juveniles such as red sea bream and red horse mackerel than EPA. (Proceedings of the Spring Meeting of the Fisheries Society of Japan, pp. 39, and Abstracts of the Spring Meeting of the Fisheries Society of Japan, pp. 44), namely, seedlings reared by changing the EPA and DHA content in artificial feed In the larvae, the DHA-added group has a growth promoting effect.

[0005] Therefore, zooplankton for breeding larvae and larvae preferably contains DHA, which not only allows the larvae to easily feed DHA as an essential fatty acid, Oil and other feed components disperse in the water, deposit on the bottom of the water and become a source of contamination, resulting in a more efficient biological feed compared to artificial formula feed, which may inhibit the growth of larvae and larvae. there is a possibility.

[0006] When looking at algae that produce DHA, for example, J. Protozoal, Vol. 117 (2), Vol.
On pages 13 to 219, 1970, DHA-containing algae such as genus Amphidinium are reported. JP-A-4-346760 discloses that alga bodies of the genus Isochrysis as DHA-containing algae are used as feed for zooplankton. However, these algae are all grown under autotrophic conditions that require light irradiation in their culture.If such algae are used as feed for zooplankton, the production cost becomes extremely expensive,
Impractical.

On the other hand, PHATOCHEMISTRY, Vol. 27 (6), No. 1
679-1683, 1988, describe algae of the genus Crypthecodinium.
According to this prior art document, Crypthecodinium cohnii (Crypthecodinium cohnii) is a neutral lipid composed mainly of triacylglyceride in about 72% of lipids in the cultured algal cells, and about 28% is phosphatidylcholine or the like. Is a polar lipid mainly composed of phospholipids. And 8.8% of the constituent fatty acids of the total lipids is DHA.

[0008] However, there has been little research on industrial use development of DHA-producing algae growing on such heterotrophs. Although it is known that an algae containing a large amount of DHA is effective for culturing zooplankton and fortifying DHA as described above, it is known that an alga containing DHA is effective for culturing zooplankton efficiently. Was not known.

[0009]

In view of the above situation, the present inventors have conducted intensive studies on a feed for culturing zooplankton, which is useful as a biological feed for larvae and larvae in the production of marine seeds and seedlings. Was completed.
That is, an object of the present invention is to provide an inexpensive feed that can fortify DHA, which is one of the essential fatty acids of larvae, to early zooplankton, and that has high production efficiency of the zooplankton.

[0010]

SUMMARY OF THE INVENTION To this end, the present invention is directed to a heterotrophic growing, comprising DHA-containing polar lipid,
The gist of the present invention is a feed for cultivating animal plankton, which is made from algae having a DHA content of 10% by weight or more in total fatty acids. First, the zooplankton to be used in the present invention may be any substance that can be used as a biological feed for fry and larvae, and specific examples include rotifers, artemia and daphnia.

Next, algae related to the feed of the present invention will be described. The alga used in the present invention is not particularly limited as long as it is an algae capable of culturing with heterotrophic nutrition and capable of producing DHA. Examples of the algae that meet this condition include, according to “Microbiological Dictionary 1992 Edition” (published by Gihodo), Colored Algae, Dinoflagellate,
Crypthecodinium cohn, belonging to the genus Crypsecodinium
ii) Also, brown plant phylum, dichomycete, Thraustochytrium aureu (Thraustochytrium aureu)
m) and the like. These are American Type Culture
Collection (US, abbreviated as ATCC) and other algal research institutions.
1, 30334, 30336, 50052, and the like, and examples of the latter are ATCC 34304, 28211, and the like.

Algae suitable as a raw material for the feed of the present invention include:
For example, a culture of the algae, which contains polar lipids such as phospholipids and glycolipids containing DHA as lipids accumulated in the algae, and DHA is 10% by weight of the total fatty acids constituting the lipids It is mandatory to occupy more than%. It is important that algae used as feed materials for culturing zooplankton accumulate DHA in algal bodies in the form of the polar lipids rather than neutral lipids such as triacylglycerides. Is preferably 30% by weight or more in the total lipids. The DHA content is 10% by weight of the total fatty acids of the lipids in the alga body.
Or more, and preferably 30% by weight or more. If the amount is less than 10% by weight, it becomes difficult to enhance DHA in zooplankton.

The cultivation of the algae according to the present invention may be carried out as follows. That is, a natural or artificial seawater medium having a salt concentration of 12 to 25% by weight, or a carbon source such as glucose or sucrose, a nitrogen source such as ammonium salt, yeast extract, amino acid or its salt, various vitamins and minerals Using a medium containing pH
The culture was aerated at 20 to 40 ° C for 2 to 10 days.
The cultivation end time may be any time at which the required amount of algal cells is secured, but the algal cell diameter is 5 to 1 around mid-logarithmic growth.
5 μm and 10 to 25 μm in the stationary phase, and the culture may be stopped according to the size of the zooplankton to which this is applied.

In the culturing, the salt concentration in the medium is adjusted at any time after the middle exponential growth period in the growth curve of the algae, preferably during the period from the logarithmic growth period to the stationary phase, by changing the salt concentration suitable for the growth of the algae. Concentration, ie 12-25
0.1 to 10% by weight, preferably 0.1 to 3% by weight
Weight% higher, and further culture at that salt concentration for 3 more days.
By carrying out for about 50 hours, the DHA-containing polar lipid is 30% by weight or more, more practically about 50 to 65% by weight, and the DHA content in the total fatty acids is 30% by weight or more, more practically 45 to 45%. Algae of about 50% by weight can be obtained.

After completion of the cultivation, the alga thus obtained is concentrated by a suitable means such as centrifugation, precision or ultrafiltration, and if necessary, washed, washed under reduced pressure or spray dried, and crushed. This is used alone or mixed with other known feed components to obtain the above-mentioned zooplankton feed. The zooplankton cultivated and cultivated with such a feed for 12 to 100 hours or the zooplankton cultivated in the secondary culture is highly active, and the DHA content in the total fatty acids is enhanced to 10 to 30% by weight.

[0016]

【Example】

Reference Example 1 Crypsecodinium coney (ATCC3033
6) was inoculated into 3 liters of a medium having the composition shown in Table 1, and aerated with a jar fermenter at 30 ° C. while adjusting the pH to 6.8. During the culture, samples were taken periodically. D. The turbidity at 660 nm was measured. A growth curve was drawn from the turbidity, and the cultured algal cells were collected by centrifugation at the middle stage of logarithmic growth (85 hours), and freeze-dried to obtain 32 g of dried algal cells.

[0017]

[Table 1]

* Vitamin mix aqueous solution Biotin: 0.003 g / liter-solution Thiamine: 1.000〃 ** Metal mix aqueous solution Na ₂ EDTA: 1.00 g / liter-solution FeCl ₂ .6H ₂ O: 0.05０．０５ H ₃ BO _3: 1.00 〃 MnCl ₂ · 4H ₂ O: 0.15 〃 ZnCl _2: 0.01 〃 CoCl · 6H ₂ O: 0.005 〃

3.2 g of the dried algal cells were crushed with Hiscotron in a mixed solvent of chloroform / methanol = 1: 1 (by weight) and then extracted to obtain 0.3 g of oil. The extracted oil was developed with a mixed solvent of chloroform / methanol / acetic acid by thin layer chromatography (TLC) using a silica gel plate to separate a polar lipid fraction and a neutral lipid fraction. Each spot was scraped off from this silica gel plate, and chloroform / methanol = 1: 1.
(Weight) When reextracted with a mixed solvent, the polar lipid content was 60% by weight and the neutral lipid content was 40% by weight.

About 200 ml of the extracted oil was added with about 5 ml of a mixed solution of sulfuric acid / methanol and refluxed in a hot water bath for 1.5 hours. After methyl esterification, hexane and water (5 ml each) were added, and the mixture was extracted. Was analyzed by gas chromatography (GLC) to find that the DHA content in the total fatty acids was 42% by weight. Further, the polar lipid fraction was treated in the same manner and the constituent fatty acid composition was analyzed by GLC. As a result, the DHA content in the polar lipid was 50% by weight.

Reference Example 2 Crypsecodinium coney (ATCC3033)
6) was cultured under the same conditions as in Reference Example 1 except that the culture time was set to the stationary phase (124 hours). The yield of dried alga bodies is 50
g. As a result of oil extraction, TLC and GLC analysis using 5.0 g of the dried algal cells in the same manner as in Reference Example 1, oil yield: 0.7 g, polar lipid content: 25% by weight, neutral lipid content: 75% by weight, DHA content in total fatty acids of oil:
18% by weight, DHA content in polar lipid: 50% by weight.

Reference Example 3 Traustochytrium aureum (ATCC3430)
4) was cultured in the same manner as in Reference Example 1. However, during the culture, in the growth curve obtained from the turbidity of the culture solution, at 120 hours after the start of the culture corresponding to the mid-logarithmic growth period, 20 g / liter of sodium chloride was added to increase the salt concentration of the culture solution by 2% by weight. And further cultured for 24 hours. 65 g of dried alga body
Met. As a result of oil extraction, TLC and GLC analysis using 6.5 g of the dried algal cells in the same manner as in Reference Example 1, oil yield = 0.7 g, polar lipid content: 58% by weight, neutral lipid content: 42% by weight, DHA content in total fatty acids of oil: 5
0% by weight, DHA content in polar lipid: 65% by weight.

Example 1 A rotifer was produced using the alga bodies obtained by the methods of Reference Examples 1 and 2. Two 20-liter water tanks containing 70% seawater were adjusted to 20 ° C., and the ventilation rate was 0.3 V / V / M.
After setting the air blowing rate (liter per minute per liter of culture solution (liter)), the rotifer (mixture of S-type and L-type) was set at 100 individuals / ml at the start of the culture. Then, 0.1 g of the cultured alga bodies (referred to as alga bodies) obtained in Reference Examples 1 and 2 were added to each aquarium.
(Wet algae body weight) / liter, and the number of rotifers per ml was measured over time. The result is shown in FIG.

The rotifer, cultured for 72 hours, was collected with a nylon net, washed with water to remove the algal cells, and the fatty acid composition of lipids in the rotifer was analyzed in the same manner as in Reference Example 1. As a result, DHA in total fatty acids
The content was 28% by weight when using algal cells, and 18% by weight when using algal cells. The difference between these two values indicated the effectiveness of the polar lipid. The egg-bearing rate of the rotifer was 50% when the algal body was used as a feed, and 45% when the algal body was used as a feed, all of which had high vitality.

Example 2 US-made durable Artemia eggs were cultivated at 75-1 ° C at a water temperature of 25-27 ° C.
After putting in 00% seawater and blowing air so strongly that the eggs did not settle, 24 hours later, the hatched Artemia nauplius and the eggshell were separated. While maintaining a 5-liter water tank filled with seawater at 27 ° C. and ventilating air at 0.3 V / V / M, the above-mentioned Artemia nauplius was set to an initial concentration of 100 individuals / ml. The fresh algal cells obtained by the method of Reference Example 1 were administered thereto at 0.2 g (wet algal cell weight) / liter, cultured for 24 hours and fed. When the fatty acid composition of the lipid in Artemia was analyzed in accordance with the method of Reference Example 1, the DHA content in the total fatty acids was 1% by weight or less immediately after hatching, that is, before the start of cultivation in algae. After culturing for an hour, the content increased to 29% by weight.

Example 3 A 5-liter aquarium filled with 50% seawater was maintained at 25 ° C., and air was supplied at a flow rate of 0.3 V / V / M so that the initial concentration of brackish water daphnia was 50 individuals / ml. I set it. 0.1 g of the dried algal cells obtained by the method of Reference Example 3
(Dry algae body weight) / liter
They were cultured for a period of time and fed. When the fatty acid composition of the lipids in the daphnia was analyzed in accordance with the method of Reference Example 1, the initial DHA content in the total fatty acids was 0.8% by weight, and the DHA content was very low. 25
It was fortified to weight percent.

Example 4 Crypsecodinium coney (ATCC 3033)
6) was cultured under the same conditions as in Reference Example 1 to obtain a wet algal body. A part of this wet algal body was frozen at -20 ° C and freeze-dried by a conventional method. By using the method of cultivating the rotifer of the rotifer described in Example 1, the concentration of the rotifer at the start of the cultivation was adjusted to 100 individuals / ml, and the wet alga body, the frozen alga body, or the lyophilized alga body was obtained. 0.1 g (in terms of dry alga body weight) / liter was added, and the cells were cultured in the same manner.
As a result, the growth rates of the rotifer were almost the same, and the DHA content in the total fatty acids of the lipid of the rotifer was 25 to 28% by weight after culturing for 72 hours.
(28% by weight for wet algal bodies, 26% by weight for frozen algal bodies, 25% by weight for freeze-dried algal bodies), and the egg carrying rate was 45 to 50%.

Comparative Example 1 In the method for cultivating the rotifer, Rotifer, 0.1 g / l of the alga body according to the present invention was prepared by using a fat oil yeast (manufactured by Kyowa Hakko Co., Ltd., product name "oil fat yeast": fish oil). (Assimilated yeast) was replaced with 0.5 g / liter and cultured under the same other conditions. DHA content in total fatty acids in the lipid of Rotifer, Rotifer was 72% by weight after culturing for 72 hours.
The egg-carrying rate was only 35%.

Comparative Example 2 In the method for cultivating the rotifer, Rotifer, 0.1 g / l according to the present invention, freshwater chlorella (manufactured by Nippon Chlorella, product name "Fresh Green 600"): The fresh algae of freshwater chlorella cultured by nutrient) were replaced with 0.3 g / liter, and cultured under the same other conditions. The growth rate and egg carrying rate of the rotifer were almost the same as the results of Example 1, but the DHA content in the total fatty acids in the lipid of the rotifer was 0.5 weight even after culturing for 120 hours. % Or less.

Comparative Example 3 In the method for cultivating the rotifer, Rotifer, 0.1 g / l of the algae according to the present invention, powdered fish oil (product name "Sea Oil Powder", manufactured by KUP Co., Ltd .: DHA) A 15% by weight fish oil adsorbed on an egg white peptide) was replaced with 0.5 g / liter and cultured under the same other conditions. The DHA content in the total fatty acids in the lipid of the rotifer, Rotifer rotifer after 72 hours of cultivation was only 5% by weight, and the egg-carrying rate was 30%.

[0031]

According to the present invention, there is provided a useful feed for nurturing zooplankton, which is a biological feed for juvenile larvae in the production of marine seedlings. The feed of the present invention can be produced at low cost by using algae that can be cultured by heterotrophic nutrition, can efficiently fortify DHA to zooplankton, and its content is about 20 to 20% of the total fatty acids of lipids of zooplankton. Reaches 30% by weight. In addition, the zooplankton, which has a high egg carrying rate and high vitality, can be stably cultivated.

[Brief description of the drawings]

FIG. 1 is a diagram showing the relationship between the number of rotifers per unit culture and the cultivation time. Algae refers to the alga body obtained by the method of Reference Example 1, and alga body refers to the alga body obtained by the method of Reference Example 2.

Claims (6)

(57) [Claims] 1. A feed for cultivating zooplankton, which is grown under heterotrophic conditions, contains docosahexaenoic acid-containing polar lipids, and has a docosahexaenoic acid content in total fatty acids of at least 10% by weight as an ingredient. 2. The feed according to claim 1, wherein the content of the polar lipid in the algal lipid is 30% by weight or more. 3. The feed according to claim 1, wherein the content of docosahexaenoic acid among the fatty acids constituting the polar lipid in the algal lipid is 30% by weight or more. 4. The method according to claim 1, wherein the algae is Crypthecdinium.
The feed according to any one of claims 1 to 3, which is a unicellular algae belonging to the genus odinium). 5. The method according to claim 5, wherein the algae is 0.1 to 10% by weight of a salt concentration suitable for growth after the middle of exponential growth in the culture.
The feed according to any one of claims 1 to 4, wherein the feed is cultured at a high salt concentration. 6. The feed according to claim 1, wherein the zooplankton is a rotifer, Artemia or Daphnia.