JP2002125601A - Feed for animal plankton, method for producing the same, and method for culturing animal plankton - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide feed for animal plankton, enabling Rotatoria, Artemia or the like each used for seedling production of fish and shellfish to efficiently and stably proliferate, to provide a method for producing the feed for animal plankton, and to provide a method for culturing animal plankton. SOLUTION: This animal plankton is produced through culturing Chlorella in the presence of a fatty acid compound having carbon numbers 14-18 to obtain Chlorella in the cell of which the fatty acid compound is effectively stored. The thus obtained Chlorella is used as feed for animal plankton such as Rotatoria, or Artemia.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a feed for zooplankton. More specifically, the present invention relates to a feed for zooplankton capable of efficiently and stably growing (growing) rotifers, artemia, and the like used for the production of seeds of fish and shellfish.

[0002]

2. Description of the Related Art Rotifer is the most important food for fish and shellfish seedlings in the field of marine products. The development of a large-scale cultivation method for rotifers using freshwater concentrated chlorella and the like has led to
A dramatic increase in seed production has been made possible.

[0003] On the other hand, attempts have been made to use chlorella, which is useful as a feed for rotifers, as an important feed for Artemia as an initial feed for marine seedlings. However, unlike rotifers, Artemia cannot digest chlorella in a living cell state due to a disorder of a cell wall.

To improve the above-mentioned problem, Japanese Patent Application Laid-Open No. 62-87060 discloses an artemia feed in which the cell wall of chlorella has been destroyed. Examples of the method for breaking the cell wall include a spray-drying treatment, an ultrasonic treatment, a trituration treatment, and a cell wall lysing enzyme treatment.

[0005]

As described above, large-scale cultivation of rotifers has become possible, but even now, rotifers sometimes fail to proliferate, which may hinder seed production. As for the culture of Artemia, a method of providing chlorella in which the cell wall of chlorella was destroyed as described above has been proposed, but this method alone cannot sufficiently grow Artemia, and until commercialization. Not reached.

Therefore, the present inventors have made intensive research and development to obtain a chlorella capable of efficiently and stably growing (or growing) rotifers and artemia.

[0007] By the way, the present inventors, apart from the research for solving the above-mentioned problems, have proposed an n-3 type such as DHA (docosahexaenoic acid) or EPA (eicosapentaenoic acid) for enriching the feed for larvae. He was developing chlorella with enhanced polyunsaturated fatty acids. In the course of its development, they found that chlorella with enhanced DHA and the like had an effect of promoting rotifer growth. The reason for this is not clear, but it is considered that by enhancing DHA and the like, components such as the fatty acid composition, lipid content, and other fat-soluble substances of chlorella changed, and as a result, the growth of rotifers was promoted. .

Accordingly, the present inventors have further conducted research based on the idea that the fatty acid composition in chlorella cells may have an effect on the promotion of rotifer growth. The present inventors have found a fatty acid having an excellent effect on growth, and have completed the present invention.

(Object of the Invention) It is an object of the present invention to provide a feed for zooplankton capable of efficiently and stably growing (growing) rotifers, artemia and the like used for the production of fish and shellfish seeds, and a method for producing the same. And a method for culturing zooplankton.

[0010]

Means for Solving the Problems In the first invention,
A feed for zooplankton, comprising chlorella that has absorbed a fatty acid compound having 14 to 18 carbon atoms.

[0011] In the second invention, the zooplankton is a rotifer or Artemia.
The feed for zooplankton according to the first invention.

[0012] A third invention is a method for cultivating zooplankton, comprising using the feed for zooplankton according to the first or second invention.

In the fourth invention, the carbon number is 14 to 18;
Culturing chlorella in the presence of a fatty acid compound of the formula (I).

[0014]

DETAILED DESCRIPTION OF THE INVENTION Fatty acid compounds include free fatty acids, salts of fatty acids and alkyl esters of fatty acids.

Free fatty acids refer to saturated or unsaturated, straight-chain or side-chain fatty acids.

The salt of a fatty acid refers to a salt of the above-mentioned free fatty acid, such as an alkali metal such as sodium, potassium or the like.
Salts of alkaline earth metals such as barium, calcium and the like are included.

The alkyl ester of a fatty acid refers to an ester of the above free fatty acid with an alkanol. Examples of the alkanol that can form an ester include monohydric alkanols such as methanol and ethanol.

In the present invention, a fatty acid compound having 14 to 18 carbon atoms is used from the viewpoint that zooplankton can be efficiently propagated (grown) and that raw materials can be easily obtained. There is no particular limitation on the origin of the fatty acid compound, and for example, oils and fats of seafood, beef tallow, olive oil, rice bran oil and the like can be used. Specific examples of the fatty acid compound include myristoleic acid (C14: 1) and palmitoleic acid (C1
6: 1), oleic acid (C18: 1), linoleic acid (C18: 2),
Linolenic acid (C18: 3) and the like, but not particularly limited thereto.

The timing of adding the fatty acid compound to the chlorella culture solution is not particularly limited as long as the chlorella can be cultured in the presence of the fatty acid compound. Chlorella can be added to the culture solution in advance and the chlorella can be cultured. It can be added in the middle, or it can be added to the algal cells after the completion of the culture and re-cultured. However, from the viewpoint of efficiently accumulating fatty acid compounds, reducing the work required for culture, and shortening the culture time, the fatty acid compound should be added during the latter stage of culture, specifically, during the latter half of the logarithmic growth phase to the end of the logarithmic growth phase. Is desirable.

The amount of the fatty acid compound to be added is not particularly limited as long as it is an amount that can be taken up by chlorella. However, if the amount is too small, the effect of taking in the fatty acid compound is reduced. It is not preferable because it lowers. Specifically, it is preferable to adjust the amount of the fatty acid compound added according to the amount of Chlorella alga bodies present in the culture solution,
In a practical range, the concentration of Chlorella algae in the culture solution is 10%.
When g (dry weight) / L, the fatty acid compound is 0.05 to 5.5 mL / L.

The chlorella used is not particularly limited as long as it belongs to the genus Chlorella. As an example of chlorella,
Chlorella vulgaris, Chlorella pyrenoidosa, Chlorella
Chlorella saccharophila, Chlorella regularis, Chlorella sorokiniana and the like can be mentioned. These chlorellas can be easily obtained by those skilled in the art. For example, it can be obtained from the University of Tokyo IAM Culture Collection.

As a culturing method of chlorella, for example, photoautotrophic culturing, heterotrophic culturing, mixed vegetative culturing and the like are known, but the culturing method is not particularly limited.

As the basic medium used for culturing Chlorella, for example, a large number of mediums are known in addition to Bristol medium and Solokin-Klaus medium. It doesn't matter.

The temperature and time for culturing Chlorella, the concentration of Chlorella, the time of light irradiation, the light intensity, the stirring intensity and the like are not particularly limited as long as they can culture Chlorella. It can be set appropriately according to the like.

The chlorella that has been cultured is concentrated and washed with water, for example, by centrifugation. This concentrate can be stored refrigerated and used as it is as a feed for zooplankton. Further, after the concentrated chlorella is freeze-dried or spray-dried, it can be used as a feed for zooplankton as a powder. Furthermore, zooplankton feed can also be prepared by blending dry yeast and other components.

[0026]

The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

Example 1 Effect of Chlorella Enriched with Oleic Acid (C18: 1) on Rotifer Culture (Chlorella cultivation) Chlorella vulgaris C-30, Tokyo IAM using a jar fermenter with a volume of 10 L
Obtained from the company). Medium is glucose 20g /
L, 1.5 g / L urea, 1.2 g / L monopotassium phosphate, 0.6 g / L magnesium sulfate, 0.015 g / L EDTA-Na-Fe, ₅ trace metal A solution
2 mL / L, was used as the addition of vitamin B ₁₂ 30μg / L.
For the culture, 15 × 10 ⁷ cells of Chlorella were added to 5 L of the above culture solution, and aeration and agitation culture was performed at a temperature of 30 ° C. Furthermore, in the late logarithmic growth phase, free oleic acid (manufactured by Wako Pure Chemical Industries)
Was added in an amount of 1 to 5 mL / L of the culture solution. The chlorella that had been cultured was concentrated and washed by centrifugation, and then refrigerated. Chlorella cultured under the same conditions as described above except that oleic acid was not added to the culture solution was used as a control.

Further, as a result of the above culture, it was found that chlorella cultured by adding 5 mL per liter of oleic acid (manufactured by Wako Pure Chemical Industries) was disturbed due to discoloration of cells. From these results, it was found that it was difficult to add oleic acid of 5 mL / L or more. The chlorella algal body concentration of the culture solution when oleic acid was added was about 8 to 9 g (dry weight).
/ L.

(Rotifer cultivation test) Rotifer was cultured using Chlorella cultured by the above method. Rotifer S
A rotifer was used. Aeration cultivation was performed for 4 days in a polycontainer at a culture temperature of 25 ° C. and a volume of 5 L using seawater. The culture was started at a number of rotifers of 50 individuals / mL. Per day, to rotifers 1 × 10 ⁵ individuals were fed separately chlorella concentrate 1mL a (100g dry weight / L) in the morning and evening, investigating changes in the population of rotifers. Table 1 shows the obtained results. In addition, the lipids contained in the fed chlorella cells were analyzed, and the results are shown in Table 1.

[0030]

[Table 1]

As is clear from the results shown in Table 1, it was confirmed that when chlorella was cultured in a culture solution containing oleic acid (C18: 1), oleic acid was effectively accumulated in the chlorella cells. It was found that the oleic acid content of Chlorella increased almost in proportion to the amount of oleic acid added to the culture solution. In addition, it turned out that the total lipid amount of chlorella also increased with the increase in oleic acid content. From the rotifer density, the amount of oleic acid (C18: 1) added 1
It was clarified that those in which oleic acid was further fortified in the range of ４4 mL had an excellent effect as a rotifer feed.

Example 2 As fatty acids, myristoleic acid (C14: 1), palmitoleic acid (C16: 1), oleic acid (C18: 1), linolenic acid (C18: 3), eicosapentaenoic acid (C20: 5) The effect of chlorella on rotifer culture using docosahexaenoic acid (C22: 6) and enriched with each fatty acid.

(Culture of Chlorella) Chlorella (Chlorella vulgaris C-30, obtained from Tokyo University IAM) was cultured using a Sakaguchi flask. The medium is glucose 20 g / L, urea 1.5 g / L, monopotassium phosphate 1.2 g / L, magnesium sulfate
0.6 g / L, EDTA-Na-Fe 0.015 g / L, Trace metal A ₅ solution 2 mL / L,
It was used with the addition of vitamin B ₁₂ 30μg / L. For the culture, 0.3 × 10 ⁷ cells of Chlorella were added to 100 mL of the above culture solution, and aeration and agitation culture was performed at a temperature of 30 ° C.

Furthermore, as fatty acids, free myristoleic acid (manufactured by Sigma), palmitoleic acid (manufactured by Wako Pure Chemical Industries), oleic acid (manufactured by Wako Pure Chemical Industries), linolenic acid (manufactured by Wako Pure Chemical Industries), eicosapentaene Using acid (purity 90%, purified from fish oil) and docosahexaenoic acid (purity 90%, purified from fish oil), in the late logarithmic growth phase of chlorella,
3 mL per 1 L of the culture solution was added to the Sakaguchi flask. The chlorella that had been cultured was concentrated and washed by centrifugation, and then refrigerated. Chlorella cultured under the same conditions as in the above method except that no fatty acid was added to the culture solution was used as a control. The lipid contained in the cultured chlorella cells was analyzed. Table 2 shows the results. In Table 2, the symbols described in the column of fatty acid composition (C14: 1
And the like) indicate the type of fatty acid (for example, C14: 1 is myristoleic acid).

[0035]

[Table 2]

As is clear from the results in Table 2, it was confirmed that when chlorella was cultured in a culture solution to which fatty acids were added, the added fatty acids were effectively accumulated in chlorella.

(Rotifer cultivation test) Rotifer was cultured using Chlorella cultured by the above method. Rotifer S
A rotifer was used. Shaking culture was performed for 6 days in a 100 mL Erlenmeyer flask with a culture temperature of 25 ° C. and a culture solution volume of 20 mL using seawater. The cultivation was started at a rotifer population of 1 individual / mL. One day, 100 rotifers were fed with 1 μL of a chlorella concentrate (100 g / L chlorella dry weight) in the morning and evening, and the change in the number of rotifers was examined. Table 3 shows the obtained results.

[0038]

[Table 3]

As is clear from the results shown in Table 3, it was found that chlorella enriched in fatty acids had a superior effect on rotifer growth. Also, myristoleic acid (C14: 1), palmitoleic acid (C16: 1), oleic acid (C18: 1), and chlorella fortified with eicosapentaenoic acid (C20: 5) and docosahexaenoic acid (C22: 6). It was revealed that chlorella enriched with linolenic acid (C18: 3) had an excellent effect as a rotifer feed.

Example 3 As a fatty acid, two kinds of palmitoleic acid (C16: 1) and oleic acid (C18: 1) were used.
Effect of Chlorella fortified with each fatty acid on Artemia culture

(Culture of Chlorella) Chlorella (Chlorella vulgaris C-30, University of Tokyo)
(Obtained from IAM). Medium is glucose 20g
/ L, urea 1.5g / L, monopotassium phosphate 1.2g / L, magnesium sulfate 0.6g / L, EDTA-Na-Fe 0.015g / L, trace metal A ₅ solution 2m
What added L / L was used. For the culture, 15 × 10 ⁷ cells of Chlorella were added to 5 L of the above culture solution, and aeration and agitation culture was performed at a temperature of 30 ° C.

Further, two kinds of fatty acids, free palmitoleic acid and oleic acid (manufactured by Wako Pure Chemical Industries, Ltd.), were used.
Each L was added to the jar fermenter. Chlorella after the completion of the culture was concentrated and washed by centrifugation, and then freeze-dried. In addition, except that no fatty acid is added to the culture solution,
After culturing under the same conditions as in the above method, freeze-dried chlorella was used as a control. The lipid contained in the cultured chlorella cells was analyzed. Table 4 shows the results. Table 4
Among them, the code (C16: 1, etc.) described in the column of fatty acid composition is
The type of fatty acid (for example, C16: 1 is palmitoleic acid).

[0043]

[Table 4]

As is clear from the results in Table 4, when chlorella was cultured in a culture solution to which palmitoleic acid (C16: 1) or oleic acid (C18: 1) was added, the fatty acids added to chlorella were effectively accumulated. It was confirmed that.

(Culture test of Artemia) Artemia was cultured using Chlorella cultured by the above method. Artemia used hatched larvae. In a polycontainer using seawater, aeration culture was performed at a culture temperature of 25 ° C and a culture volume of 5 L.
Went for days. The cultivation was started at 100 Artemia individuals / mL. To 100 Artemia individuals per day, 0.2 g of chlorella powder was fed separately in the morning and evening, and changes in the average total length of Artemia and the number of individuals were examined. Table 5 shows the obtained results.

[0046]

[Table 5]

As is clear from the results shown in Table 5, in the control group in which the fatty acid was not fortified, the number of Artemia individuals decreased sharply and the growth was poor. On the other hand, the number of survivors decreased slightly in the test group in which palmitoleic acid (C16: 1) or oleic acid (C18: 1) was fortified, but both the growth and the number of survivors were superior to those in the control group. It has been found that this has an excellent effect.

The terms and expressions used in the present specification are for explanation only, are not restrictive, and do not exclude terms and expressions equivalent to the above-mentioned terms and expressions.

[0049]

According to the present invention, by culturing chlorella in the presence of a fatty acid compound having 14 to 18 carbon atoms,
Chlorella in which the above fatty acid compound is effectively accumulated in cells can be obtained. When this chlorella is used as feed for zooplankton such as rotifers and artemia, zooplankton can be efficiently and stably grown (grown).

Continued on the front page F-term (reference) 2B005 GA01 GA02 GA03 GA04 GA07 LA06 LA07 LB06 LB07 MB05 2B104 AA34 CF02 2B150 AA08 AB02 CD26 DA56 DA58 DD49 4B065 AA84X AC20 BB03 BB15 BB20 BC03 BC05 BC08 BC26 BC50 BD15 CA43

Claims (4)

[Claims] 1. A feed for zooplankton comprising chlorella that has absorbed a fatty acid compound having 14 to 18 carbon atoms. 2. The feed for zooplankton according to claim 1, wherein the zooplankton is rotifer or artemia. 3. A method for culturing zooplankton, comprising using the feed for zooplankton according to claim 1 or 2. 4. A method for producing a feed for zooplankton, comprising culturing chlorella in the presence of a fatty acid compound having 14 to 18 carbon atoms.