JP2946122B2 - Red tide control feed - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a feed for controlling red tide.
More specifically, it is non-toxic to fish and shellfish,
The present invention relates to a feed containing a microorganism that specifically kills plankton constituting red tide.

[0002]

BACKGROUND OF THE INVENTION The occurrence of red tide has severely damaged the fish farming industry. The red tide is caused by the abnormal occurrence of phytoplankton due to nutrients that have accumulated wastewater containing nitrogen compounds, phosphorus compounds and other nutrients through rivers into the sea, especially the coastal and inland seas. In fish farms in closed sea areas, food is over-administered, and its feed residues promote water pollution and eutrophication, causing red tides.

[0003] It is known that the main plankton that constitutes the red tide that causes fishery damage is algae such as phytoplankton diatoms and dinoflagellates, and in particular, high-grade fish such as yellowtail, which is the center of farmed fish, is one of algae. It is known that the red tide caused by algae belonging to Chattonella such as Chattonella antiqua causes serious damage.

As a conventional countermeasure against red tide, reduction of nutrient concentration by water quality regulation has been widely carried out, but as other countermeasures, modification of sea bottom mud by clay spraying method or killing of algae by chemical method or the like. Various types of red tide damage improvement measures such as suspension of activities and their combination have been studied,
Attempted.

[0005] For example, a method of spraying a red tide treating agent comprising a clay mineral and an inorganic acid (Japanese Patent Publication No. 63-24486) uses an aluminum-type clay mineral powder of displaceable aluminum having a specific base substitution capacity as a red tide treating agent. Method (Japanese Unexamined Patent Publication No. Sho 59-18365), a method of spraying a substance having an algicidal activity such as an alkali metal halide or hexazinone (Japanese Patent Publication No. Sho 56-4)
No. 9309 and Japanese Patent Publication No. 60-3361) have been proposed. Further, as a fish feed, a feed containing taurine, which has an effect of enhancing the resistance of fish to red tide plankton, has been proposed.

[0006]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a means for solving the problem of red tide caused by excessively administered food residues from the side of the feed to be administered.

[0007]

Means for Solving the Problems As a result of diligent research, the present inventors have cultivated a microorganism belonging to a novel Pseudomonasasstutzeri isolated from submarine mud in Ariake Sea and Hakata Bay and sediment in a sewage treatment plant. The liquid has a specific high lethal activity against Châtonella antica, has a growth inhibitory activity, but has no toxicity to fish, and has the same effect on microorganisms belonging to known Pseudomonas stutzeri. Confirmed that it was completely harmless, and completed the present invention.

[0008] That is, the present invention provides a red tide control feed comprising a culture solution of a microorganism belonging to Pseudomonas stutzeri, which exhibits a red tide control effect, or contains a processed product thereof.

[0009]

DETAILED DESCRIPTION OF THE INVENTION In the feed of the present invention, cells belonging to Pseudomonas stutzeri are used.

As the cells belonging to Pseudomonas stutzeri, there are known Pseudomonas stutzeri I.
FO3773 (Deposited number of Osaka Fermentation Research Institute, same hereafter)
(Psuedomonas stutzeri IFO 3773), Pseudomonas
Stutzeli IFO 12510 (Psuedomonas stutze
ri IFO 12510), Pseudomonas stazzelli IF
O12695 (Psuedomonas stutzeri IFO12695), Pseudomonas stutzeri IFO13596 (Psue
domonas stutzeri IFO 13596), Pseudomonas stutzeri IFO 14165
FO 14165) can be used, but the microorganisms A41, B47 and MM4 belonging to Pseudomonas stutzeri newly isolated from the seabed mud collected by the present inventors from the Ariake Sea and Hakata Bay, and the sediment sludge of the Fukuoka Sewage Treatment Plant. Strains are preferably used.

Hereinafter, a method for isolating these novel cells, characteristics, and a culture method will be described. Separation and culture method of bacterial cells 1 g of submarine mud collected from the Ariake Sea and Hakata Bay and 10 g of sediment sludge from a sewage treatment plant in Fukuoka City were added to 10 ml of ES broth medium and incubated at 25 ° C. for 48 hours.
The culture solution was applied to an SN nutrient agar plate medium (Difco Beef Extract 5 g, polypeptone 10 g, sodium chloride 30 g, agar 20 g, purified water 1000 ml, pH 8.5) and cultured at 30 ° C. for 2 days.

[0012] Strains were isolated from the colonies formed on the medium, and the algicidal activity of these strains against Châtonella antica was measured by the method described below. One strain showing strong algicidal activity was selected for each strain. . The obtained A41 strain, B4
Table 1 shows the bacteriological properties of the seven strains and the MM4 strain.

[0013]

[Table 1]

Based on the above mycological properties, the present inventors identified all three strains as belonging to Pseudomonas stutzeri, and identified the A41 strain collected from the Ariake Sea as Pseudomonas stutzeri A41, Hakata Bay. B47 strain collected from Pseudomonas stutzeri B
47. The MM4 strain collected from the sewage treatment plant was named Pseudomonas stutzeri MM4. In addition, these strains have been deposited with the Japan Fine Art Institute on February 19, as deposit numbers 12015, 12016, and 12017, respectively.

The strain can be cultured in a liquid medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known nutrients conventionally used for culturing bacteria can be used. For example, glucose, succinic acid, lactic acid and the like can be used as a carbon source, and glutamic acid, ammonium sulfate and the like can be used as a nitrogen source. Others
If necessary, inorganic salts such as magnesium sulfate and potassium hydrogen phosphate can be added, but for sodium chloride, it is preferable to add 2.0 to 3.0%.

As for the culture method, liquid culture under aerobic conditions, particularly submerged culture, is most suitable as in the known method for culturing bacteria.
0 ° C. The cultivation time is not particularly limited, but in order to use it for red tide control feed, it is preferably 3 to 6 hours after the start of cultivation.
The culture after day is preferred.

Algicidal Activity of Various Microorganisms The algicidal activity of various microorganisms against Chatonella antica was examined. As a result, microorganisms belonging to Pseudomonas stutzeri generally exhibited relatively high lethal effect.
Among them, A41 strain, B47 strain and MM4 strain were found to have particularly high lethal effect. Table 2 shows the test results using five microorganisms such as Escherichia coli as controls.

[0018]

[Table 2]

The algicidal activity against Chatonella antica was determined by the method of Ishio et al. (Nippon Suisan Gakkaishi, 54 , 11).
75 to 1181 (1988)) and evaluated as the lowest lethal concentration (LLC). That is, the culture medium of each strain (the dilution rate of the culture solution itself is set to 100%) is sequentially diluted with a 96-well tissue culture plate, and the ES medium containing about 100 chatonella antica per well is contained. And add
LLC was determined as the concentration (dilution ratio%) of the culture solution in which Antica was killed.

The ES medium used here was 70%
It consists of 100 ml of seawater and 2 ml of an ES concentrate having the following composition.

ES concentrated solution NaNO ₃ 350 mg Sodium glycerophosphate 50 mg Tris 0.5 mg Fe (as Fe-EDTA) 25 mg P metal 2.5 ml Vitamin B ₁₂ 10 μg Vitamin B ₁ 0.5 mg Biotin 5 μg pH 8.1 H ₂ O 100 ml Shatonella used here -Antica is 1 per day
It can be obtained by culturing at 25 ° C. in an ES medium while irradiating with light (fluorescent lamp (6000 lux) for 2 hours, and culturing for 2 weeks gives a culture solution of about 10 ⁴ cells / ml. And the concentration of Chatonella antica were measured by counting under a microscope.

In addition, it was confirmed in a later-described experiment using medaka that the microorganism belonging to Pseudomonas stutzeri has no toxicity to fish. Therefore, it is considered non-toxic to cultured fish such as hamachi and Thailand.

Pseudomonas stattze against fish
A re three aquarium filled with one animal killifish average body length 5cm to effect artificial seawater 100ml of where, A41, B47, and culture of MM4 and (those that have been cultured for 3 days in SN medium) 0.5 ml, was respectively added, Twenty-four hours later, the killifish in each aquarium were still alive.

From the above, it can be seen that the culture solution of Pseudomonas stutzeri does not show any toxicity to Medaka.

Red Tide Control Feed The red tide control feed of the present invention is obtained by mixing a culture of microorganisms belonging to Pseudomonas stutzeri with a known feed conventionally used for fish cultivation. In this case, the culture to be used may be the culture solution itself, or a concentrate of the culture solution or a processed product obtained by drying or pulverizing the culture solution may be used. In addition, when the known feed mixed with the culture is a feed containing a large amount of water, in addition to mixing, the microorganisms are directly inoculated into the feed and then cultured to increase the cell concentration. Red tide control feed can also be used. As a feed that can be used in such a method, there is a raw feed such as sardine, saury, etc., which is pulverized, heat-sterilized, inoculated, and incubated at 25 to 30 ° C. for 2 to 6 days. It can be used as a red tide control feed easily.

The thus produced red tide control feed usually contains microbial cells at a rate of 10 ⁹ to 10 ¹⁰ cells / g.

The feed of the present invention is characterized in that the feed is fed only to the fish cultivated in the water area where the red tide, especially the red tide caused by Chattonella Antica, feeds the required amount of the feed, and the water in the water area is not affected by the Chattonella Antica. Is to die.
This is thought to be due to the fact that Pseudomonas stutzeri in the feed elutes into seawater, selectively killing Chatonella antica and causing coagulation and sedimentation. On the other hand, fish that consume it are effective as feed and have no toxicity.

Another characteristic of the feed of the present invention is that
It is unknown what effect it is, but the fish that ingested it gains resistance to the red tide. Has the advantage of being kept to a minimum.

When the feed of the present invention is administered to fish cultured in a sea area where red tide does not occur, the occurrence of red tide can be prevented. In addition, if this feed is administered to fish cultured in the sea area where red tide is already occurring, it will not only help control the red tide itself, but also prevent damage such as fish death due to the already generated red tide. The feed of the invention is very useful for the aquaculture industry, which is severely affected by red tide.

[0030]

As described above, the feed containing the strain belonging to Pseudomonas stutzeri, particularly the three new strains A41, B47 and MM4, according to the present invention, is a plankton composed of red tide, and is particularly suitable for fish. Shows selectively high lethal activity and agglutinative sedimentation action against harmful Chatonella antica, and has no toxicity to fish. In addition, fish that have consumed them have an advantage that they have resistance to Châtonella antica and are less likely to die due to red tide. Therefore, by administering to farmed fish, it is possible to effectively protect or prevent cultured fish, especially high-grade fish such as yellowtail (hamachi) while controlling or preventing red tide in the cultured sea area, and increase productivity.

[0031]

EXAMPLES The feed of the present invention will be described below with reference to examples. Example 1 Fresh sardines were washed with tap water and minced with an electric mixer (Model MX-V350, manufactured by Matsushita Electric Industrial Co., Ltd.). 5 g of the sardine meat was placed in a 100-ml Ehrenmeyer flask, heat-sterilized in an autoclave at 121 ° C. for 20 minutes, inoculated with a loopful of A41 strain, and incubated at 25 ° C. for 7 days. A feed containing the A41 strain was prepared. As a control, a diet containing only the minced sardine meat, which did not contain the A41 strain, and a culture solution (LLC = 0.5%) obtained by culturing the A41 strain in an SN medium for 3 days were prepared.

Immediately after 10 medaka fish were placed in 1000 ml of artificial seawater supplemented with 10 ⁴ cells / ml of Châtonella antica, 5 g of the above-mentioned three kinds of feeds were added, and in addition to the case where no feed was given. And observed. Table 3 shows the results.

[0033]

[Table 3]

When a culture solution of the A41 strain and sardine meat inoculated with the A41 strain were added as feeds,
Antica was killed, and the water, which had become cloudy due to the free-living fungi of Chatuna Antica, became clean. At this time, 1
No toxic effects were observed in killifish for more than a week. Also, when the culture solution of the A41 strain was placed in water containing only the killifish, no toxicity was found in the killifish.

When sterilized minced sardine was fed as a feed, it had no lethal effect on Chatonella antica, and all the killifish died only one hour after the start of the experiment.

Example 2 Fresh sardines were washed with tap water and minced with an electric mixer (Model MX-V350, manufactured by Matsushita Electric Industrial Co., Ltd.). 5 g of the sardine meat was placed in a 100-ml Ehrenmeyer flask, heat-sterilized in an autoclave at 121 ° C. for 20 minutes, inoculated with one loopful of B47 strain, and incubated at 25 ° C. for 7 days. A feed containing the B47 strain was prepared. As a comparative control, a feed containing only the minced sardine meat treated in the same manner but not containing the B47 strain was prepared.

Immediately after 10 killifishes were placed in 1000 ml of artificial seawater to which 10 ⁴ cell / ml of Shatonella antica had been added, 5 g of the above-mentioned two kinds of feeds were added, and in addition to the case where no feed was given. And observed. Table 4 shows the results.

[0038]

[Table 4]

When sardine meat inoculated with the B47 strain was added as a feed, chatonella antica was killed, and water turbid due to floating live bacteria of chatonella antica was cleaned. At this time, no toxic effects were observed in the killifish for more than one week.

In addition, when minced sardine meat was fed as a feed, it did not show any lethal effect on Châtonella antica, and all the killifish killed only one hour after the start of the experiment.

Example 3 Fresh sardines were washed with tap water and minced with an electric mixer (Model MX-V350, manufactured by Matsushita Electric Industrial Co., Ltd.). 5 g of the sardine meat was placed in a 100 ml Erlenmeyer flask, heat-sterilized in an autoclave at 121 ° C. for 20 minutes, inoculated with one loopful of MM4 strain, and incubated at 25 ° C. for 7 days. Feed containing MM4 strain was prepared. As a control, a minced sardine-only feed containing no MM4 strain was prepared.

5 g of the above two kinds of feed per 10 animals is 1
Feeding once a day, the medaka was reared for 7 days. This is 10
Ten animals were placed in 1000 ml of artificial seawater supplemented with ⁴ cells / ml of Châtonella antica, and the progress was observed. Table 5 shows the results.

[0043]

[Table 5]

In the case where the feed containing the MM4 strain was given beforehand, most of the animals survived even after 24 hours.
On the other hand, when a feed containing no MM4 strain was given, all died one hour later.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ identification code FI C12R 1:38) (58) Investigated field (Int.Cl. ⁶ , DB name) A01N 63/02 A01N 63/00 BIOSIS (DIALOG) )

Claims (1)

(57) [Claims] 1. Pseudomonas stutzer, which has a red tide control action
A red tide controlling feed comprising a culture solution of a microorganism belonging to i) or a processed product thereof.