JPH0740890B2 - Feed for zooplankton, method for culturing flagella algae used in the feed, and method for producing DHA-rich oil - 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 zooplankton having a high content of docosahexaenoic acid, a method for culturing a microorganism having a high content of docosahexaenoic acid which can be suitably used for this feed, and a method for producing an oil having a high content of docosahexaenoic acid. .

[0002]

2. Description of the Related Art Docosahexaenoic acid (hereinafter referred to as "DHA") has attracted attention because of its ability to suppress the growth of infants' brains and the deterioration of their learning function associated with aging. It is a fatty acid. This DH
Although A is currently extracted from fish such as tuna, the ratio of total fatty acid contained in fish is as low as 15%, and it is difficult to extract A efficiently because it contains many impurities. There is. In addition, since the source of supply depends on natural resources, stable supply is difficult, and the reduction of resources and the effects of marine pollution are also problems.

By the way, for the cultivation of marine fish, polyunsaturated fatty acids such as DHA and icosapentaenoic acid (EPA) are necessary, and growth and vitality are enhanced and survival rate is improved by feeding a feed enriched with these. It has become clear that the number of malformed fish is decreasing. In particular, DHA
When strengthened, the effect is remarkable. EFA
-free, EPA strengthening, DHA strengthening and EPA + DHA
Fig. 4 shows changes in fish weight of juvenile striped horse mackerel and Fig. 5 shows changes in survival rate when using (1: 1) fortified feeds.
Are shown respectively.

[0004]

However, the amount of DHA contained in zooplankton such as rotifer, artemia, shellfish, shrimp, and crab larvae which are currently used as feed for these fish is very small. This is because microalgae such as Nannochloropsis, diatoms, tetracelmis, chlorella, and baker's yeast, which are feeds for zooplankton, do not contain DHA in their lipid components. As described above, it has been difficult to extract the DHA itself in the related art. Therefore, at present, a method of administering emulsified fish oil or the like together with the feed is used to supplement this. However, this method has a drawback that the breeding water is dirty and inefficient, and in addition, a large amount of fish oil is administered, which results in the production of lipid-rich fish bodies. Therefore, an object of the present invention is to provide a feed for zooplankton in which DHA is enhanced. Another object of the present invention is to provide a method for culturing a microorganism having a high DHA content, which can be suitably used as a feed for DHA-enriched zooplankton. Further, the present invention aims to provide a method for producing an oil having a high DHA content.

[0005]

[Means for Solving the Problems] In view of the above circumstances, the present inventors have made earnest studies on a biological feed having a high DHA content and a high feed value with respect to zooplankton. The inventors have found that the above-mentioned problems can be solved by using chrysecodinium, and have completed the present invention. That is, the present invention is characterized by using the flagellated chryseocodinium alga as a feed for zooplankton.

Chrysecodinium is a unicellular alga with a cell size of 8 to 20 μm, which is classified into dinoflagellates and is a plankton that lives in the ocean and can be easily collected.

[0007] The pure culture of chrysecodinium is preferably carried out at 25-30 ° C or lower using a flask, a jar fermenter or the like while aeration or stirring is performed. As the medium used at this time, for example, seawater having a salt content of 3 to 30% or artificial seawater to which a carbon source and a nitrogen source are added can be used. Examples of the carbon source added to the medium include glucose, glycerol, ethanol and sodium acetate, and examples of the nitrogen source include histidine, betaine, ammonium sulfate, glutamate, and yeast extract. Each can be listed. Furthermore, as components other than the carbon source and the nitrogen source, for example, phosphates, trace metals, and vitamins can be added to the medium.

[0008] The cultured alga can be used as a feed for plankton after being made into powder by means such as freeze-drying or spray-drying. Alternatively, the alga can be used as it is by washing it with water, concentrating it, suspending it in seawater, etc., and refrigerating it. Furthermore, the alga thus obtained can be used by adding substances conventionally used as a feed for zooplankton such as Nannochloropsis, Chlorella, and baker's yeast.

[0009] Chrysecodinium has excellent intracellular components and has a high feed value for zooplankton. Also, because of the high DHA content, D of zooplankton such as rotifer and artemia, which are important as feed for marine fish,
It can also be suitably used as a nutrition-enhancing feed for increasing the HA content.

As described above, chrysecodinium is useful because it has a high DHA content even in the usual culture method. However, the present inventors have also found that it is possible to further increase the DHA content of chrysecodinium by changing the culture conditions.

First, by changing the components of the medium, DH
The production amount of A can be increased. In a commonly used culture medium, histidine, betaine, etc. are used as the nitrogen source as described above. Glutamic acid, glutamate such as sodium glutamate, etc. Doubled. As described above, glucose or the like is usually used as the carbon source, but by changing it to an alcohol such as glycerol or ethanol, DHA can be obtained.
Production will be further increased about twice. Furthermore, chrysecodinium usually grows at 10 to 30 ° C., but when cultured at 10 to 20 ° C., the DHA content is particularly high, which is preferable.

The concentration of the medium conventionally used for culturing chrysecodinium was as low as 6 g / l of carbon source. Therefore, the cell density after culture is 2.5 g.
It was as low as dry density / l, and it was difficult to increase it. The present inventors have examined the influence of the medium concentration on the growth of chrysocodinium, and have found that even if the carbon source concentration is about 50 g / l, there is no effect on the growth. As a result of culturing with a carbon source concentration of 50 g / l, the cell density after culturing was 25 g / l, and the DHA production amount was increased to 850 mg / l. This is 10 times higher in cell density and 20 times higher in DHA production amount than the conventional method.

When the chrysecodinium is cultured under the optimum conditions shown above, the proportion of DHA in the total fatty acids of the chrysecodinium can be increased to about 55%. Therefore, by drying after culturing to obtain a DHA-rich algal body, it can be used not only as a sample for zooplankton but also as a food or a feed for other animals. Further, an oil containing a high amount of DHA can be obtained by extracting lipids from this alga by a conventional method using hexane or the like. The DHA-rich oil thus prepared can be suitably used as a raw material for medicines and a raw material for health foods.

[0014]

EXAMPLES The present invention will now be described in more detail based on examples. Example 1 Production of an important rotifer, Rotifer (hereinafter referred to as rotifer), which is important as an initial feed for marine fish, was carried out using chrysecodinium.

Chrysecodinium as a feed for rotifers
The space was cultured in a medium having the composition shown in Table 1 below for 7 days, washed with water and concentrated by centrifugation, and stored in a refrigerator. Table _{1 ------------------------- NaCl 10 g MgCl 2 ·} 7H 2 O 1.67 g KCl 0.27 g CaCl 2 · 2H 2 O 0.27 g glucoside -Su 20 g Glutamic acid 10 g (NH ₃ ) ₂ SO ₄ 0.33 g NH ₂ PO ₄ 0.66 g Biotin 6.6 μg Vitamin B ₁ 333 μg Vitamin B ₁₂ 3.3 μg EDTA-Na 1-Fe 19.5 mg A ₅ solution ⁽¹⁾ 3.3 ml −−−−−−−−−−−−−−−−−−−−−−−−−− (1) H ₃ BO ₃ 2.86 g, MgCl ₂ .4H ₂ O 1.81
_{g, ZnSO 4 · 7H 2 O} 0.22g, CuSO 4 · 5H
Prepared by dissolving 0.08 g of ₂ O and 0.021 g of Na ₂ MoO ₄ in 1 liter of pure water, and adding 1 drop of H ₂ SO ₄ . The chrysecodinium produced in this way contained DHA, which accounted for 45% of its total fatty acids.

Cultivation of rotifers was carried out in a 500-liter Panlite water tank once daily for 8 days while feeding the above-mentioned chrysecodinium. As a result, the rotifer grew as shown in FIG. In FIG. 1, the horizontal axis shows the number of culture days and the vertical axis shows the rotifer density in the culture solution expressed in the number of individuals per ml. The increased rotifer is 4
Harvested on days 1 and 8. 8 days of culture, 2.8 ×
10 ⁷ It was possible to produce individual rotifers. Example 2 Artemia, which is important as a biofeed for marine fish, was cultivated using chrysecodinium. Crycecodinium spices, which is a feed for Artemia, was cultured in the same manner as in Example 1.

The training of Artemia was carried out by hatching Artemia reared larvae in a Panlite water tank of 100 liters at 5 individuals / ml.
It was carried out by culturing for 10 days while feeding the above chrysecodinium once a day. The result is shown in FIG. In Fig. 2, the horizontal axis is the number of training days,
The vertical axis represents the survival rate (%) of Artemia and the average value (mm) of the entire length.

As is clear from FIG. 2, 10 times from the start of training.
On day one, the average total length of Artemia reached 3.0 mm.
In addition, the survival rate during the training period was as high as 70%.
Therefore, it is clear that chrysecodinium is excellent as a feed for artemia training. Example 3 Rotifer and Artemia were cultured for a short period of time using chrysecodinium, and the effect of increasing DHA content was examined. The chrysecodinium used for culturing rotifer and artemia was cultured in the same manner as in Example 1.

The rotifer and artemia to be cultured were suspended in seawater so as to have a density of 300 individuals / ml and 100 individuals / ml, respectively, and cultured for 30 hours while feeding chrycecodinium together. The results of fatty acid analysis at the start and end of culture of rotifer and artemia are shown in Table 2 below.
Shown in. Table 2 Fatty acid analysis results of rotifer and artemia --------------------------- Rotifer artemia ----- −−−−−−−−−− At start At end At start At end −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Myristin Acid 1.3 6.2-- 12.2 Palmitic acid 19.0 12.7 13.2 3.8 Palmitooleic acid 4.0 2.5 ---- Stearic acid 4.4 4.0 5.8 3.0 Oleic acid 2.9 12.7 33.4 43.5 Linoleic acid 23.0 5.3 5.5 4.6 Linolenic acid 28.6 3.8 21.3 16.1 Icosapentaenoic acid --5.7 2.6 5.7 Docosahexaenoic acid (DHA) --40.3 --10.6 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− %

As is clear from Table 2, in the case of rotifer, the content of DHA was 40% after 30 hours of culturing, although it did not contain DHA at the start of culturing.
In addition, the content of DHA could be dramatically increased in a short time. Also, in the case of Artemia, DH was not contained at the start of the culture, but DH
It was possible to increase the content of A to 10%. Therefore, it is clear that chrysecodinium has a very excellent effect as a diet for enrichment of zooplankton. Comparative example 1

The rotifer was cultivated in the same manner as in Example 1 except that various feeds shown in Table 3 below were used instead of chrysecodinium as the rotifer feed, and the rotifer was contained in the rotifer after culturing. The amounts of icosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were measured. The results are also shown in Table 3. Each numerical value in Table 3 indicates a ratio (%) to the total fatty acid, and tr indicates a trace amount. Table 3 EPA and DHA content of rotifers --------------------------- Feed EPA DHA ------------- −−−−−−−−−−−−−−−− Nannochloropsis 22.8 3.4 Tetrasermis 5.2 0.3 Chlorella tr 0 Baker's yeast 0 0 Oil and fat yeast 7.5 3.1 === ======================== Chrysecodinium 5.7 40.3 ------------------ −−−−−−−−−−−

As is clear from Table 3, in the conventional feeds, only the nannochloropsis was found to be EPA.
There is no diet that enhances DHA.
In contrast, chrysecodinium is found to significantly enhance DHA. Example 4 Chrysecodinium spesces was cultured in a medium containing various nitrogen compounds, and the DHA production amount in each case was compared.

First, a basic medium having the composition shown in Table 4 below.
On the ground, various nitrogen compounds shown in Table 5 below were used as nitrogen sources.
Prepare the medium by adding it to a nitrogen concentration of 0.3 g / l
did. Put each prepared medium into a Sakaguchi flask and keep at 25 ℃.
Crycecodinium spesces culture for 7 days at
No, DHA production was measured. The results are also shown in Table 5.
Write down. Table 4 Foundation composition₂・ 7H₂O 1.67 g KCl 0.27 g CaCl₂・ 2H₂O 0.27 g Glucose 6 g KH₂PO_Four 0.2 g biotin 2 μg vitamin B₁ 100 μg vitamin B₁₂ 1 μg EDTA-Na-Fe 5.8 mg A_Fivesolution⁽¹⁾  1 ml −−−−−−−−−−−−−−−−−−−−−−−−−− (1) H₃BO₃2.86g, MgCl₂・ 4H₂O 1.81
g, ZnSO_Four・ 7H₂O 0.22g, CuSO_Four・ 5H
₂O 0.08g and Na₂MoO_Four 0.021 g of pure water 1 liter
Dissolve in tor, H₂SO_FourPrepared by adding 1 drop. Table 5 Amount of DHA produced when Crycecodinium spices was cultured in a medium supplemented with various nitrogen compounds ----------------------- -------- Nitrate source DHA production (mg / l) ------------------------------------------- ammonium sulfate 1.0 histidine 3.6 betaine hydrochloride 1.8 Casamino acid 27.3 Glutamic acid 42.5 Sodium glutamate 45.3 Yeast extract 40.1 −−−−−−−−−−−−−−−−−−−−−−−−−−−

As is clear from Table 5, when sodium glutamate, glutamic acid and east extract were added to the medium, the production amount of DHA exceeded 40 mg / l. Compared with these nitrogen compounds, they were slightly inferior when casamino acids were used, and remarkably inferior when other compounds were used. Example 5 Chrysecodinium spesces was cultured in a medium supplemented with various carbon sources, and the production amount of DHA in each case was compared.

First, 3.7 g / l of sodium glutamate was added to the basal medium having the composition shown in Table 4 above, and various carbon sources shown in Table 6 below were added instead of glucose.
The medium was prepared by adding them so as to be g / l. On this occasion,
Ethanol and organic acid were added little by little depending on the amount consumed during the culture. Each of the prepared culture media was placed in a Sakaguchi flask, and the chrysecodinium spices were cultured at 25 ° C. for 7 days, and the DHA production amount was measured after the culture. The results are also shown in Table 6. Table 6 Amount of DHA produced when Crycecodinium spices was cultured in a medium to which various carbon sources were added ----------------------- ----- Carbon source DHA production (mg / l) ---------------------------------------- Glucose 45.3 Acetic acid 37.0 Galactose 44.1 Sucrose 1.5 Glycerol 100.6 Ethanol 93.6 Succinic acid 1.5 Waste molasses 5.8 −−−−−−−−−−−−−−−−−−−−−−−−−−−−

As is clear from Table 6, when glycerol or ethanol is used as the carbon source, the amount of DHA is about twice as much as that when glucose or the like used in the conventional method is used. Was obtained. Further, glycerol and ethanol are remarkably excellent as compared with other carbon sources. Also, molasses, which is known as an inexpensive fermentation raw material, is a carbon source that enables DHA production although its production is low. Example 6 Chrysecodinium spices were cultured at different temperatures, and the amount of DHA produced in each case was compared. Crycecodinium spesces was cultured for 7 days at 25 ° C. using a basal medium having the composition shown in Table 4 above. The results are shown in Table 7 below. Table 7 Production of DHA when culturing Chrysecodinium spesces at different temperatures ----------------- Culture temperature (° C.) DHA production (mg / l) −−−−−−−−−−−−−−−−−−−−−−−− 30 43.2 25 45.3 20 47.0 15 72.3 12 126.0 10 75.0 −−−−−−−−−−−−−−−−−−−−−−−−−

As is clear from Table 7, in the temperature range of 10 to 30 ° C., chrysecodinium spices grow and DHA is also produced. Particularly, at 10 to 20 ° C, the production amount exceeds 70 mg / l, which is preferable from the viewpoint of DHA production. Example 7 Chrysecodinium spices were cultivated at high density with increasing medium concentration to produce DHA.

As the medium, a medium having a concentration of 8.3 times that of the basal medium having the composition shown in Table 4 above was prepared, to which 60 g / l of glycerol as a carbon source and 25 g / l of sodium glutamate as a nitrogen source were added. What was done was used. The culture is
It was carried out at 25 ° C. using a jar fermenter. Changes in cell density and DHA concentration during culture are shown in FIG. In FIG. 3, − ◯ − indicates the cell concentration in g / l, −−− indicates D
HA concentration is shown in mg / l. The horizontal axis represents the culture time. As is clear from FIG. 1, the DHA concentration increased almost in proportion to the cell concentration, and 8 to 70 hours after the initiation of the culture.
Reached 50 mg / l. In addition, the lipid contained in the cultured cells was analyzed. The results are shown in Table 8 below. Table 8 Lipid content and fatty acid composition of the produced algal cells ----------------------- Total lipid amount (% algal dry weight) 1) Fatty acid composition (% total fatty acid) Lauric acid 1.8 Myristic acid 9.6 Palmitic acid 16.8 Palmitooleic acid 1.4 Stearic acid 1.4 Oleic acid 12.8 Docosahexaenoic acid (DHA) 51. 9 −−−−−−−−−−−−−−−−−−−−−−−−−−− As shown in Table 8, the produced algal cells contained 12% lipid. 51.9% of the fatty acid composition was DHA. Example 8 Oil was extracted from the DHA-rich algal cells obtained by culturing.

Algal bodies used for extraction (cresecodinium
Was cultured in the same manner as in Example 7, and extraction was carried out by a conventional method using n-hexane as a solvent.
As a result, about 80 g of oil could be produced from 1 kg of algal cells. The fatty acid composition of the obtained oil is shown in Table 9 below.
Shown in. In Table 9, tr represents a trace amount. Table 9 Fatty acid composition of produced oil (% total fatty acid) ---------- Lauric acid tr Myristic acid 7.7 Palmitic acid 13.1 Palmitooleic acid tr Stearic acid tr Oleic acid 21.0 Docosahexaenoic acid (DHA) 58.2 -------------------- As shown in Table 9. In the fatty acid composition of the obtained oil, 50% or more thereof was DHA.

[0030]

INDUSTRIAL APPLICABILITY As described above, by using the flagellated alga Chrysecodinium of the present invention as a feed, zooplankton, which is a feed for marine fish, can be efficiently cultivated. In addition, DH lacking in zooplankton
Since A can be strengthened, it is not necessary to administer fish oil or the like in addition to zooplankton to compensate for the lack of DHA.

According to the culture method of the present invention, DH
It is possible to obtain the flagellated chrysocodinium having a further increased A content, and it is possible to extract a DHA-rich oil from the chrysecodinium obtained by culturing.

[Brief description of drawings]

FIG. 1 is a graph showing changes over time in the number of rotifers in the culture of rotifers fed with the flagellated chrysocodinium of the present invention.

FIG. 2 is a graph showing changes over time in the growth and survival rate of Artemia in the culture of Artemia that was fed with the flagellate Chrysecodinium of the present invention.

FIG. 3 is a graph showing changes over time in cell density and DHA production when high-density culture of chrysocodynium spices was performed.

FIG. 4 is a graph showing the effect of EPA and DHA on changes in fish weight in the development of juvenile striped horse mackerel.

FIG. 5 is a graph showing the effect of EPA and DHA on the change in survival rate in rearing juvenile striped horse mackerel.

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Claims (7)

[Claims] 1. A feed for zooplankton containing a flagellated alga Chrysecodinium. 2. A method for culturing a flagellated alga Chrycecodinium, which comprises using a medium containing at least one nitrogen compound selected from the group consisting of glutamic acid and salts thereof and yeast extract as a nitrogen source. 3. The culture method according to claim 2, wherein a medium containing alcohols as a carbon source is used. 4. The culture method according to claim 3, wherein the culture is performed at 20 ° C. or lower. 5. Extraction from flagellated alga Chrycecodinium cultured in a medium containing at least one nitrogen compound selected from the group consisting of glutamic acid and salts thereof and yeast extract as a nitrogen source. A method for producing an oil high in docosahexaenoic acid. 6. The method according to claim 5, wherein a medium containing alcohols as a carbon source is used. 7. The production method according to claim 6, wherein the culture is performed at 20 ° C. or lower.