JPH06271470A - Immunostimulating agent for fish - Google Patents

Abstract

PURPOSE:To obtain the subject stimulating agent containing a scarcely water-soluble low-molecular chitin fine powder having a specific viscosity and a specific average particle diameter as an active component, effective for reducing the administration dose of antibiotics, etc., and improving the problem of residual chemicals and useful for producing fish having high quality. CONSTITUTION:Chitin known as a constituent component of the cell wall of crustaceans such as shrimp and crab, insects such as beetles and cricket and fungi such as shiitake mushroom is dissolved by adding concentrated hydrochloric acid. The solution is stirred for several tens min to several hr and the reaction product is washed with water, neutralized and washed under centrifugal force to obtain a scarcely water-soluble low-molecular chitin fine powder having a viscosity of 30-5cp (measured by dissolving in a lithium chloride solution) and an average particle diameter of <=10mum. The chitin is used as an active component of the objective immunostimulating agent.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention has a viscosity of 30 to 5 cp.
An immunostimulant for fish for oral administration, characterized in that it is a water-insoluble fine particle low molecular weight chitin having an average particle diameter of 10 μm or less (as measured by a viscosity based on a lithium chloride solution dissolution method), as an active ingredient. Regarding

[0002]

2. Description of the Related Art In recent years, as the aquaculture industry has flourished, a variety of aquaculture fish species and aquaculture forms have increased, and aquaculture production has increased. Since cultured fish are generally exposed to stress unlike fish that grow in the natural environment, the immune system of the fish itself is also reduced, and due to the reasons such as the acquisition of drug resistance of fish disease bacteria, Deterioration of the disease situation for cultured fish has become a problem, for example, streptococcal disease of yellowtail, nodular disease, nocardiosis, vibrio disease of Thailand, iridvirus infection, bacterial kidney disease of salmon, trout of trout. Vibrio disease, infectious hematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN), eel paracolo disease, fin erythema, red spot disease, carp perforation, and prawn vibrio disease.

As a countermeasure against these diseases, especially against bacterial diseases, drugs such as antibiotics have been used for a long time. However, the emergence of resistant bacteria against various drugs and the large amount of drug residues in fish and the like are large. Since it is a social problem, in order to reduce the administration of antibiotics and produce high quality fish, it is necessary to improve the fish from nutritional and immunological standpoints, that is, to restore the biological defense ability and There has been a demand for a safe functional feed that can produce healthy fish by augmentation.

[0004] In general, in order to restore and enhance the biological defense ability, the action of humoral immunity is also an important factor in the biological defense mechanism along with a series of increased activities (phagocytosis, treatment, sterilization) in eliminating foreign substances from leukocytes. That is, complement itself plays a protective role in the early stages of infection,
More importantly, increased activity of humoral immunity can trigger increased activity of cell-mediated immunity. It is known that when the complement activity is increased, the complement component is adsorbed on the surface of a foreign substance such as a bacterium to facilitate the recognition of the foreign substance to leukocytes (opsonin effect), and thus the foreign substance can be eliminated more effectively. ing.

Although specific immunity also exists in the fish immune system, when compared with mammals, there is only one type of specific antibody (Ig M class), and the induction period until antibody production or antibody In the duration, there are large variations among individuals, and the secondary immune response of the antibody-producing cells to the specific antigen is weak. Also, both primary and secondary immune responses have a short duration. In addition, the direct influence of environmental temperature (Immunology in the veterinary field Modern Publishing P40
1 to P402), which suggests that nonspecific immunity is more important than specific immunity in the immune system of fish, which is phylogenetically much lower than mammals. There is.

At present, few substances have been found to have an activating effect on the fish immune system. For example, glucan (JP-A-2-
218615, JP-A-2-256620), glycyrrhizin (JP-A-2-250832), egg white (JP-A-3-251537), FK-
565 (Japanese Patent Application Laid-Open No. 63-233923) and the like are known as substances having an activating effect on the fish immune system, but since the oral administration method is most suitable for aquaculture, these immunostimulating substances are It was difficult to put to practical use.

Further, chitin is contained as a constituent of the cell wall of crustaceans such as shrimp and crab, insects such as beetles and crickets, and shiitake mushrooms and fungi, and supports organisms like celluloses in the plant kingdom. N-acetyl-, which has an acetylated amino group.
It is a high molecular polysaccharide in which D-glucosamine is β-1,4 linked. Usually, as a method for producing chitin, insoluble chitin is obtained by deproteinizing crab and shrimp shells containing a large amount of chitin with sodium hydroxide solution and then decalcifying with hydrochloric acid solution. The standard for chitin is not generally clear, and it is often the case that chitin is one that is physically insoluble in dilute acid. Further, by treating chitin with a strong inorganic acid, β-1,4 bonds are hydrolyzed, and chitins having different molecular weights can be obtained, the solution viscosity is lowered and the molecular weight is also reduced (Food Chemical, P10- P91 (1
986-11)).

Further, chitin having about 6 or less N-acetylglucosamine bound thereto is called chitin oligosaccharide, which is a very low molecular weight compound and is water-soluble. The immunological efficacy of chitin against mammals (Japanese Patent Publication No. 61-55487 and Japanese Patent Publication No. 62-61927) has been confirmed. However, in the case of high molecular weight chitin, the immunopotentiating ability against mammals is not recognized so much, and the low molecular weight chitin oligosaccharide having six N-acetylglucosamines bound is effective against the antitumor activity and the infection defense ability. It has the highest immunostimulatory activity compared to other chitin oligosaccharides (“application of chitin and chitosan” P175-P193, (1990) Gihodo Publishing) (Food Chemical, P32 (1986)), and deacetylation degree. 70% of chitosan has antitumor activity,
Having high immunostimulatory activity against infection defense (V
accine, vol. 2, 93-99 (Mar. 19
84), Carbohydrate Research
h, 146, 251-258 (1986)), but these are only when chitin is administered by injection to a mammal.

At present, krill and the like are fed as feed materials to fish such as yellowtail and Thailand, but this is the administration for the purpose of utilizing the useful protein of krill meal as the feed, and in this case. In the above, the administration is merely as chitin in the state in which the outer shell is constituted. Especially in Thailand, a large amount of krill containing astaxanthin is fed to improve the body color tone. As described above, although chitin is contained in the feed and constantly administered to fish in the farm, there is no report on the resistance to disease, that is, the protective effect against infection. Furthermore, when chitin was administered to rainbow trout at a laboratory level in an amount of 100 mg / kg body weight, no results showing excellent immunostimulatory action were obtained,
It is the extent to which the phagocytic ability and chemiluminescent ability of white blood cells are partially increased (Fisheries research, March 1992 Vol. 1).
1 No. 2 P67, Midori Shobo Publishing Co., Ltd.).

[0010]

At present, many attempts have been made as immunostimulants from the physiological standpoint of fish, but in the fishery industry, the oral administration method, which is particularly convenient for administration, is effective and inexpensive. Since it is necessary, few have been put to practical use. Furthermore, since it is desired to develop a substance that is safe and has an excellent immunostimulatory effect on fish due to residual problems of antibiotics, etc., a function that has a more effective immunostimulatory effect on oral administration to fish. The purpose is to provide specific chitin.

[0011]

Under the circumstances, the inventors of the present invention have earnestly studied, and as a result, in the clay measurement by the lithium chloride solution dissolution method described below, 30 to 5 cp.
In addition to the low water-soluble fine particle low molecular weight chitin having a specific property of having an average particle diameter of 10 μm or less,
Oral administration in fish markedly increased complement activity, activation of cell-mediated immunity and humoral immunity was confirmed, and further excellent protection against infection was confirmed, and it has an effect as an immunostimulant for oral administration to fish. I found that.

The present invention has been completed based on the above findings, and has a viscosity of 30 to 5 cp (provided that the viscosity is measured by a solution method of a lithium chloride solution) and an average particle diameter is 10 μm or less. An orally-administered fish immunostimulant, which comprises soluble particulate low-molecular-weight chitin as an active ingredient. First, the poorly water-soluble particulate low-molecular-weight chitin in the present invention has a viscosity of 30 to 5 cp and an average particle diameter of 10 μm or less as determined by a lithium chloride solution dissolution method. Soluble means very slightly soluble or insoluble in water. Further, the lithium chloride solution dissolution method in the viscosity measurement of the present invention means, for example, adding poorly water-soluble fine particle low molecular weight chitin to a 0.5% acetic acid aqueous solution at a ratio of 0.5% and stirring with a stirrer for 24 hours. , Filtration, washing with water,
After drying, 0.4 g of the dried product was used, 40 g of N, N-dimethylacetamide and 40 g of N-methyl-2-pyrrolidone.
and 8 g of lithium chloride were dissolved, and the viscosity (cp) was measured by a B-type viscometer (Tokyo Keiki) under the condition of a measurement temperature of 20 ° C. Further, in the case of poorly water-soluble fine particle low molecular weight chitin having a viscosity of 30 cp or more and an average particle size of 10 μm or more, the desired immunostimulatory effect cannot be expressed, and the viscosity is 30 cp or more and the average particle size is Those having a viscosity of 10 μm or less cannot express the desired immunostimulatory effect, and those having an extremely low molecular weight with a viscosity of 5 cp or less cannot express the target immunostimulatory effect.

In the production of the poorly water-soluble fine particle low molecular weight chitin of the present invention, for example, commercially available chitin is dissolved by adding concentrated hydrochloric acid, and after the dissolution, the reaction is carried out by stirring for several tens minutes to several hours. After the completion of the reaction, washing with water or neutralization, centrifugal washing, and those having an average particle size of 10 μm or more are subjected to a treatment for miniaturization by ultrasonic waves or the like to obtain the above-mentioned poorly water-soluble fine particle low molecular weight chitin. You can

The dosage of poorly water-soluble fine particle low molecular weight chitin as an immunostimulant varies depending on the type and size of fish.
Usually, in the case of oral administration, it may be 1 mg or more per 1 kg of fish body weight per day, preferably 10 mg to 100 mg. Further, the administration means of the poorly water-soluble fine particle low molecular weight chitin of the present invention is not particularly limited as long as it can be orally fed as it is or mixed with a fish feed such as a raw feed or a mixed feed, and simply In adjusting the fish feed by adding the poorly water-soluble particulate low-molecular-weight chitin of the present invention, the poorly water-soluble particulate low-molecular-weight chitin, which is a fish immunostimulant, and other known feed ingredients for fish are added or mixed. It may be a fish feed, for example, calcium carbonate, calcium phosphate, calcium sulfate, wheat flour, starch, dextrin, feed yeast and other raw materials for feed and fish meal, cereals, plaques, dilute by mixing with meal, Alternatively, it may be used by adding it to a premix of vitamins, minerals or the like or a fat or oil for feed. In the case of raw bait, an adhering agent such as sodium alginate and guar gum may be used at the same time.

Further, examples of the fish targeted by the present invention include seawater-cultured fish such as yellowtail, yellowtail, Thailand and coho salmon, and freshwater cultured fish such as eel, sweetfish, trout and carp. It can be added to the compound feed and administered. Regarding the timing of administration, it is better to continue administration for a certain period before infection for the purpose of disease prevention, but cultured fish are often over-cultured at a certain place, and they are always exposed to stress, Since it is considered that the immunity is also lowered, it is preferable to administer the drug all the time.

[0016]

Reference Example The method for preparing chitin 1 to 9 used in the examples will be described below. (1) Preparation of chitin 1 Chitin PSH (purchased from Yaizu Suisan Chemical Co., Ltd.) was used as a control product. (2) Adjustment of chitin 2 Chitin PSH was suspended in distilled water at a ratio of 0.5%, and an ultrasonic crusher (UD-201, Tomi Seiko Co., Ltd.) was used for 15 minutes under the conditions of an output of 160 W and an oscillation frequency of 20 KHz. It was treated and used as a control product.

(3) Preparation of chitin 3 Chitin PSH was suspended in distilled water at a ratio of 0.5%, and an ultrasonic crusher (UD-201, Tomi-Seiko Co., Ltd.) was used under the conditions of an output of 160 W and an oscillation frequency of 20 KHz. And treated for 35 minutes as a control product. (4) Preparation of chitin 4 10 g of chitin 1, 14 ml of concentrated hydrochloric acid (36%) and 86 ml of distilled water were mixed and reacted in a boiling water bath for 3 hours. After completion of the reaction, centrifugal washing with distilled water (5000 rpm 1
(0 minutes) was repeated 3 times to give a control product.

(5) Preparation of chitin 5 Chitin 4 was suspended in distilled water at a ratio of 0.5%, and output 16 with an ultrasonic disintegrator (UD-201, Tomi Seiko Co., Ltd.).
It was treated for 5 minutes under the conditions of 0 W and an oscillation frequency of 20 KHz, and used as a control product. (6) Adjustment of chitin 6 Chitin 4 was suspended in distilled water at a ratio of 0.5%, and output 16 with an ultrasonic disintegrator (UD-201, Tomi Seiko Co., Ltd.).
It is processed for 30 minutes under the condition of 0 W and oscillation frequency of 20 KHz,
It was used as a control product.

(7) Preparation of chitin 7 Chitin oligosaccharide (purchased from Yaizu Suisan Chemical Co., Ltd.)
Was used as a sample. (8) Adjustment of chitin 8 10 g of chitin 1 and 50 ml of concentrated hydrochloric acid (36%) were mixed, dissolved at 40 ° C, stirred for 2.5 hours while maintaining at 40 ° C, and 50 ml of distilled water was added to stop the reaction. And neutralize with sodium carbonate (powder), and centrifuge with distilled water (5000
r. p. m. 10 minutes) was repeated 3 times to give a control product.

(9) Preparation of chitin 9 Chitin 8 was suspended in distilled water at a ratio of 0.5%, and output 16 with an ultrasonic disintegrator (UD-201, Tomi-Seiko Co., Ltd.).
It was treated for 3 minutes under the conditions of 0 W and an oscillation frequency of 20 KHz, and used as a control product. (10) Preparation of chitin 10 Chitin 8 was suspended in distilled water at a rate of 0.5%, and output 16 with an ultrasonic disintegrator (UD-201, Tomi Seiko Co., Ltd.).
It is processed for 30 minutes under the condition of 0 W and oscillation frequency of 20 KHz,
The poorly water-soluble fine particle low molecular weight chitin of the present invention was used.

(11) Preparation of chitin 11 10 g of chitin 1 and 150 ml of concentrated hydrochloric acid (36%) were mixed, dissolved at room temperature, stirred for 2 hours at room temperature, and distilled water 100 was added.
0 ml was added to stop the reaction, and centrifugal washing with distilled water (5,0
00r. p. m. 10 minutes) was repeated 3 times to obtain the poorly water-soluble fine particle low molecular weight chitin of the present invention.

Physical properties of these chitins (viscosity, average particle size)
Is shown in Table 1.

[0023]

[Table 1]

To measure the viscosity, each chitin was added to a 0.5% acetic acid aqueous solution at a ratio of 0.5%, stirred with a stirrer for 24 hours, filtered, washed with water and dried. 4 g of N, N-dimethylacetamide 40 g, N-methyl-2
-Dissolved in a mixed solution of 40 g of pyrrolidone and 8 g of lithium chloride, and measured the viscosity (cp) with a B-type viscometer (Tokyo Keiki) under the condition of a measurement temperature of 20 ° C. In addition, chitin oligosaccharide (chitin 7) was treated with 0.5% acetic acid in water,
It was dissolved in the above mixed solution and the viscosity was measured. In addition, the particle size was not measured due to its water solubility.

The average particle diameter was measured using a particle counter KL-10 type (Rion Co., Ltd.).

[0026]

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

[0027]

[Example 1] Immunostimulatory effect of various chitin oral administrations on Magoi A total of 12 groups were prepared with 5 Magoi having an average body weight of 50 g as one group. Different in viscosity and average particle size shown in Table 1 11
Chitin of each type was adsorbed and mixed with water to the carp growing pellets (5P) for daily dietary fish feed so as to give a dose of 10 mg per Kg of the test fish. Each adjusted feed was fed continuously for 7 days, and after the feeding was completed, the immune activity of the test fish in each section was measured. The breeding conditions during the test were heat retention and running under running water (water temperature 24 ° C to 25 ° C).

(Method for adjusting carp serum) Blood was collected from the tail of the test fish in each section using a Nipro syringe (3 ml, 21 G), and the obtained blood was collected at 3,000 r.p.m. p. m. After centrifugation for 15 minutes, the separated serum was tested. (Method for adjusting white blood cells derived from carp kidney) The kidneys were taken out from each test fish, transferred into ice-cold calcium ion- and magnesium ion-free Hanks buffer solution, homogenized, and then filtered using a glass wheel to obtain. Pe for cell suspension
rcoll (specific gravity = 1.075 g / ml),
1,300 r. p. m. After centrifuging for 15 minutes, the white blood cell layer separated at the boundary surface was taken out, and 3.0 x with Hank's buffer solution containing calcium ions and magnesium ions.
It was adjusted to 10 ⁶ / ml and used for the test.

Phagocytosis assay method; Labtech chamber- (4
Leukocyte suspension (3.
0 × 10 ⁶ / ml) 100 μl and L-15 medium (GIB
CO) 900 μl was added, and the mixture was cultured at 25 ° C. for 2 hours to remove non-adherent cells and obtain glass-adherent cells. Further, latex particles (GIBCO: 0.81 μm) 100 μl
And 900 μl of L-15 medium were added and reacted at 25 ° C. for 1 hour for phagocytosis. The supernatant was discarded, washed well, fixed with methanol and stained with Giemsa. The phagocytosis rate was measured by a microscope based on the following formula.

[0030]

[Equation 1]

Complement value (ACH50) measurement method: Using serum obtained from test fish in each section, Yano et al. (Journal of the Fisheries Society of Japan 5
4 (6), 1049-1054 (1988)). That is, 0.1% gelatin-veronal buffer (pH = 7.5) containing 10 mM EGTA and 10 mM MgCl ₂ (10 mM EGTA.M
test serum diluted with g.GVB) 0.5, 0.4, 0.
The same buffer solution was added to 3, 0.2 and 0.1 ml respectively.
Add 1, 0.2, 0.3, 0.4 ml to make the total volume 0.5 m
l, and rabbit red blood cell suspension (2 × 10 ⁸ Cels)
0.2 ml (ls / ml) was added and the mixture was reacted at 15 ° C. for 1.5 hours. 0.1% gelatin containing 10 mM EDTA-
Veronal buffer (pH = 7.5) (10 mM EDT
A ・ GVB) was added to stop the reaction by adding 2.8 ml, 3000
r. p. m. After centrifuging at 5 minutes, the absorbance of the supernatant (wavelength = 41
The hemolysis degree (y) was determined from 4 nm). Serum dilution ratio (x) is plotted on the horizontal axis of the log-log graph and y / (100-
y) is plotted, and from the obtained straight line, y = 50 (50%
Hemolysis), that is, the dilution ratio on the vertical axis 1 is calculated, and ACH
The value was 50 (unit / ml).

The results are shown in Table 2.

[0033]

[Table 2]

As shown in Table 2, as compared with the control group, chitin 1, 2, 3, 4, 5, 6, 7, 8 and 9 showed little increase in phagocytic rate of carp leukocytes, and chitin. 9 has a slightly higher complement value than the control group. On the other hand, the leukocytes of the carp, which was administered with chitins 10 and 11 of the present invention, showed a high increase in phagocytosis rate, and similarly with respect to complement activity, chitins 10 and 11 also showed the highest increase in activity. Is. This chitin 10 has a viscosity of 2
4 cp, average particle size 2.3 μm, and chitin 1
No. 1 is a poorly water-soluble fine particle low molecular weight chitin having a viscosity of 17 cp and an average particle diameter of 7.5 μm, and an effective immunostimulatory effect for oral cell-mediated immunity and cell-mediated immunity was observed.

[0035]

Example 2 Opsonin Effect of Oral Administration of Various Chitins on Magoi A total of 12 groups were prepared with 5 magois having an average body weight of 50 g as one group. Eleven types of chitin having different viscosities and average particle sizes shown in Table 1 were adjusted according to Example 1 so that the dose was 10 mg per Kg of the test fish. 7 of each adjusted feed
According to Example 1 from the test fish in each ward,
Serum was separated and 5 volumes of the obtained serum were added to 1 volume of latex particles, and the mixture was reacted at room temperature for 1 hour to perform opsonization. The phagocytosis rate of untreated Magoi glass-attached leukocytes for each opsonin latex particle was determined according to Example 1 and used as an opsonin index. The breeding conditions during the test period were incubation and running under running water (water temperature 24 ° C to 25 ° C).

The results are shown in Table 3.

[0037]

[Table 3]

The results are shown in Table 3, and compared with the control group, chitin 1, 2, 3, 4, 5, 6, 7, 8
And 9 showed similar values, and no opsonin effect was observed. On the other hand, a high activity increase was observed for chitins 10 and 11, and a significantly high activity increase was observed by administration of poorly water-soluble fine particle low molecular weight chitin having a viscosity of 24 cp, an average particle size of 2.3 μm, and a viscosity of 17 cp and an average particle size of 7.5 μm. Recognized,
An immunostimulatory effect on the opsonin effect was observed.

[0039]

Example 3 Poorly water-soluble fine particle low-molecular-weight chitin dosage: A total of 4 groups were prepared, with 5 magois weighing about 50 g as one group. Chitin 1 for carp feed (Nippon Formula Feed Co., Ltd.)
0, that is, a viscosity of 24 cp and an average particle diameter of 2.3 μm, a poorly water-soluble fine particle low molecular weight chitin administered at 0, 1, 10, 1
Water was adsorbed, mixed, and adjusted to a weight of 00 mg / Kg. Each adjusted feed was fed to Magoy for 7 days, and after the feeding, serum and leukocytes were obtained from the test fish in each section by the method shown in Example 1, and the phagocytosis rate and complement value were measured. The breeding conditions were heat retention and running under running water (water temperature 24 ° C to 25 ° C).

The results are shown in Table 4.

[0041]

[Table 4]

The results are shown in Table 4, and in the low water-soluble fine particle low molecular weight chitin administration group, an increase in the phagocytic rate of leukocytes and the complement value in serum was recognized as compared with the control group in which no addition was made. , Especially water-insoluble fine particles low molecular weight chitin concentration 10
A remarkable increase in activity was observed at mg / Kg body weight and 100 mg / Kg body weight.

[0043]

Example 4 Effect of Oral Administration of Various Chitins on Magoi, Infection Protective Effect, 10 magois having an average body weight of 50 g were treated as one group, and 9 groups in total were prepared. Different in viscosity and average particle size shown in Table 1 11
Chitin of each type was adjusted according to Example 1 so that the dose was 10 mg per Kg of the test fish. Each adjusted feed was fed continuously for 7 days, and Aeromona was added to the test fish in each ward.
S. hydrophila was inoculated intraperitoneally with a bacterial amount of 1.0 × 10 ⁷ CFU / tail, and the survival rate was determined from the survival number 7 days after infection. The breeding conditions during the test period were incubation and running under running water (water temperature 24 ° C to 25 ° C).

The results are shown in Table 5.

[0045]

[Table 5]

The results are shown in Table 5. As shown in Table 5, in the control group, all test fish died on the second day after infection, whereas in chitin 1, 2 and 4, 100% mortality and chitin 3 were observed. ,
Mortality rate of 90% for 5 and 7, 80% for chitin 6 and 8
, And chitin 9 showed 70% mortality, and no infection protective effect was observed. On the other hand, the mortality rate was 30% in the administration of chitin 10, that is, a viscosity of 24 cp and an average particle diameter of 2.3 μm, and chitin 11, that is, a viscosity of 17 cp and an average particle diameter of 7.5 μm, which are poorly water-soluble fine particles of low molecular weight chitin. , The protective effect of infection was confirmed.

[0047]

[Example 5] Immunostimulatory effect of various chitins on rainbow trout A total of 3 groups including 10 rainbow trouts having an average body weight of 100 g as one group.
I prepared a ward. Chitin 1 and chitin 11 having different viscosities and average particle sizes shown in Table 1 were applied to a Nohsan compound feed for rainbow trout cultivation (Nohsan Co., Ltd.) at 10 per Kg of fish weight.
Each feed prepared by adsorbing and mixing with water so as to give a mg dose was orally administered for 7 consecutive days. After completion of the administration, 5 fish were randomly picked from each section and the immunoreactivity was measured. The breeding conditions during the breeding period are running under running water (water temperature 14.
5 ° C.).

The results are shown in Table 6.

[0049]

[Table 6]

The results are shown in Table 6, and as to chitin 1, that is, chitin having a high molecular weight and a larger average particle diameter than the control group, no increase in phagocytosis rate and complement value was observed. On the other hand, with respect to chitin 11, that is, low water-soluble fine particle low molecular weight chitin having a viscosity of 17 cp and an average particle diameter of 7.5 μm, a marked increase in phagocytosis rate and complement value was observed.

[0051]

[Example 6] Field evaluation test of poorly water-soluble fine particle low molecular weight chitin for rainbow trout A total of 2 rainbow trouts with a body weight of about 100 g were treated as one group.
A ward is prepared, and a dosage of poorly water-soluble fine particle low molecular weight chitin having a viscosity of 20 cp and an average particle size of 6.8 μm in a rainbow trout-growing Noh San compounded feed (Noh Sun Co., Ltd.) having a feed amount of about 1.2%. Was adjusted to be 10 mg / Kg of body weight by adsorbing and mixing with water. Feeding a diet containing poorly water-soluble fine particles low molecular weight chitin to rainbow trout for 30 days
The effect on complement titer and mortality due to Vibrio anguillarum infection was investigated. Regarding complement value, pH of 10 mM EGTA / Mg / GVB was 7.
0, the reaction temperature was measured at 20 ° C. The breeding conditions are kept in running water in a concrete tank (water temperature: 17.3 ° C-
18.6 ° C).

The results are shown in Table 7.

[0053]

[Table 7]

As shown in Table 7, the test group showed an improvement in phagocytosis rate, complement value, and decrease in mortality rate as compared with the control group, and the effectiveness of the poorly water-soluble fine particle low molecular weight chitin against rainbow trout was confirmed. It was Moreover, there was no problem in the weight of the test fish at the end of the test, and there was a tendency for the weight to increase.

[0055]

[Example 7] Field evaluation test of poorly water-soluble low-molecular-weight chitin for yellowtail A total of 2 groups were prepared with 1 group of 5000 juvenile yellowtail fish weighing about 180 g, and the test group had a viscosity of 17 cp and an average particle size of 2.7 µm. The poorly water-soluble fine particle low molecular weight chitin of was mixed and fed to a raw sardine feed with a feed mix for fish farming Hamachi Moist N (Marubeni Feed Co., Ltd.) and Vitamin Premix (Mitaka Pharmaceutical Co., Ltd.) so that a dose of 10 mg / Kg body weight was obtained. . The control group was fed under the same conditions except for chitin. The feeding period was about 1 month from the beginning of June to the beginning of July, and a total of 25 feedings were conducted to examine the effects on the phagocytosis rate, complement value (5 samples in each group), and death rate due to nodule disease. It was Regarding complement value, 10 mM EGTA / Mg
-The pH of GVB was measured at 7.0 and the reaction temperature was 20 ° C. The water temperature during the test is 21.0 ° C to 2 ° C.
It was 4.8 ° C.

The results are shown in Table 8.

[0057]

[Table 8]

As shown in Table 8, in the test group, the phagocytic ability and the complement value were higher than those in the control group, the mortality rate was decreased, and the efficacy of the poorly water-soluble fine particle low molecular weight chitin against yellowtail was observed. Was recognized. In addition, there was no problem in the weight of the test fish at the end of the test.

[0059]

INDUSTRIAL APPLICABILITY The immunostimulant for orally administered fish containing the poorly water-soluble fine particle low-molecular weight chitin of the present invention as an active ingredient remarkably increases complement activity in oral administration and effectively activates fish immunity. In order to reduce the administration of antibiotics, improve the residual problem, and produce high quality fish, which imparts the effect of preventing infection, from the viewpoint of nutritional and immunological aspects of fish. It is possible to provide a functional feed having a more effective immunostimulatory effect in oral administration to fish, which can produce healthy fish by improving the above, that is, restoring and enhancing the biological defense ability.

Claims (2)

[Claims] 1. A poorly water-soluble fine particle low molecular weight chitin having a viscosity of 30 to 5 cp (provided that the viscosity is measured by a lithium chloride solution dissolution method) and an average particle diameter of 10 μm or less is used as an active ingredient. An immunostimulant for fish for oral administration. 2. The immunostimulant for oral administration according to claim 1, wherein the poorly water-soluble fine particle low molecular weight chitin is contained in a dosage amount of at least 1 mg / Kg body weight or more.