KR20020094269A - Feed containing beta-glucan to stimulate the growth of fish - Google Patents

Abstract

PURPOSE: Feed obtained by adding beta-glucan produced from microorganism to conventional feed is provided which increases the growth rate of a fish and reduces the death rate thereof against pathogenic bacteria by enhancing immunity. CONSTITUTION: The feed for promotion of fish growth contains 0.01 to 0.1% by weight of beta-glucan produced by Agrobacterium or Alcaligenes faecalis. For example, 0.05% by weight of beta-glucan is fed to a fry of Sebastes schlegeli in an aquarium for 6 weeks and shows an enhanced growth rate.

Description

Feed containing beta-glucan to stimulate the growth of fish

As fishery production technology and high-density breeding technology advance in fish farming, mass production of various kinds of fish such as shellfish rock, flounder, yellowtail, red snapper, sea bream, and self-propelled fish is being produced. Numerous bacterial diseases, viral diseases and parasitic diseases are a big problem in the production and formation of these fish species. In particular, the recent rapid expansion of aquaculture and artificial aquaculture environments such as high-density farming have caused enormous economic losses by causing many diseases that have not been significantly damaged in natural fish.

In order to prevent diseases of farmed fish, a large amount of antibiotics, chemical synthetic drugs, growth hormones and additives have been administered, or a mixture of food and these drugs has been administered. However, the conventional method of administering additives, such as antibiotics, is not only economical in terms of cost, but also accumulates antibiotics in the body of the farmed fish, causing problems in safety and the appearance of resistant bacteria. .

In light of this situation, it is best to prevent fish from becoming diseased rather than treating it when it is diseased. Immunopotentiators have been developed to activate the immune system of the living body to prevent disease outbreaks. Examples of such immunopotentiators are glucans obtained from bacterial cell wall-derived peptide glycans (Japanese Patent Application Laid-Open No. 10-229831), bacteria and yeast. Etc. are known. Most of these glucans are obtained from yeast, and yeast preparations including glucans and mannoproteins extracted from the cell walls of yeast have been shown to increase the body weight by activating the immune system of domestic animals (Korean Patent Publication No. 1999-0063361). Japanese Patent Laid-Open No. 7-51000 discloses a polysaccharide feed additive for aquaculture fish whose β1,3 glucan is produced by the genus Oreobasidium, while adding β1,3 glucan at a concentration of 0.1 to 5%. Is reported to have an effect on weight gain and survival.

In general, yeast cell wall-derived glucan has a disadvantage in that the yeast cell wall is extremely hard and difficult to digest and thus has low utilization rate of glucan in livestock or fish. In order to solve this problem, it is necessary to decompose chemically and biologically, and a process of extracting and refining a low content component is inevitably provided at a high price. Therefore, techniques for efficiently separating these glucans to enhance the effect and aid in the absorption of immune enhancing substances that are difficult to be absorbed have been reported (Korean Patent Publication No. 1999-028736).

In this regard, microorganisms producing betaglucan secreted out of the cell can be easily separated and the production process can be supplied at a relatively low cost, such as a separate process for decomposition. As a microorganism producing beta glucan out of the cell, Oreobashidium strain (JP-A-7-51000) produces beta glucan mixed with beta 1,3 and 1,6 bonds, and Agrobacterium sp. Produces betaglucan of curdlan type with only beta-1,3 binding. When using aquaculture feed composition containing 0.05 to 5% by weight of beta glucan (curdlan) having such a linear structure in fish farming, the survival rate of the immune system is increased and the pathogen attack test is increased (Japanese Patent Publication No. 1998-313794) can be used as a feed additive for aquaculture.

As described above, the existing patent literature or report has emphasized only the positive side of the beta glucan as an immune enhancing substance. However, as a result of the present inventors performing various precise specification experiments, the inventors found that excessive beta glucan inhibited the growth of fish and showed negative results in the immune response.

Therefore, an object of the present invention is to maximize the growth promoting effect of fish and to enhance the immune function to find the optimal concentration of glucan that can protect the living body from invasion or stress of pathogens, and to provide a feed composition for fish using the same.

1 is a graph showing the cumulative mortality of each feed segment after the attack experiment with Edwardsiella tarda ( Edwardella tarda) .

In the present invention to achieve the above object, beta glucan, Especially AgrobacteriumAgrobacterium) Or Alkaliness Packalis (Alcaligenes faecalisBeta glucan produced by the strain was added to fish farming for the purpose of immune boosting. Betaglucan was produced by culturing Agrobacterium in a fermentation broth in which medium was composed of sugar or raw sugar as a carbon source, ammonium chloride as a nitrogen source, and other inorganic salts. In the present invention, the culture solution is concentrated and dried to use powder, and the culture solution itself may be used or purified powder may be used to dry the powder.

The present invention relates to a feed composition for fish comprising 0.01-0.1% by weight of beta glucan produced from Agrobacterium sp.

Hereinafter, the present invention will be described in detail through the result of breeding the larvae of the rockfish (Urug) and flounder (flatfish). The following examples merely illustrate the present invention in detail, and the content of the present invention is not limited by the examples, and may be applied to other fish species or long breeding periods.

First, the beta glucan used in the present invention was produced by culturing in a fermentation broth containing sugar as a carbon source, ammonium chloride as a nitrogen source, and other minerals in Agrobacterium. After fermentation is complete, beta glucan is insoluble.Beta glucan is insoluble in neutral solution, so to separate beta glucan from fermentation broth from cells, the culture medium is basified to dissolve beta glucan, followed by centrifugation or filter. Remove cells. Beta glucan is precipitated by contacting this solution with a water-soluble organic solvent or by neutralizing the solution, followed by centrifugation and drying to obtain a purified powder. Beta glucan can also be used in purified or unpurified form as above. In the embodiment of the present invention was used a powder form purified by the above method.

Example 1: Zigbolak (Urug) Specification Test

Experimental fish were fed two weeks of pre-fishing while feeding bokbolak fry until the start of the experiment. After 2 weeks of preliminary breeding, an average weight of 4.0 grams of juvenile rockfish larvae was randomly placed in a 100 L round water tank with 3 repetitions of feed.

Breeding water was used seawater filtered by a high-speed sand filter (reverse filtration method), each of the experimental tank was flown water flow rate was 2ℓ / min at the beginning of the experiment. The water temperature was 20 ~ 22 ℃ and depended on the natural water temperature during the experiment. The experiment was carried out for 6 weeks.

The concentrations of beta glucan in the experimental feed were added in four concentrations of 0.05%, 0.10%, 0.25% and 0.5% using the control group without glucan.

Effect of Beta Glucan on Growth of Rockball Rocks

Table 1 shows the results of the experiments performed with beta glucan for 6 weeks. Feed group fed 0.05% glucan showed a significantly higher trend than control group, but showed no significant difference compared to feed group fed 0.1%. Feeding 0.25% was similar to the control and showed the lowest growth rate in the feed fed 0.5%.

This suggests that excessive supply of betaglucan inhibits growth.

Control 0.05% 0.1% 0.25% 0.5% Weight gain 385 405 390 380 360

¹ Growth Rate: (Final Weight-Initial Weight) × 100 / Initial Weight.

Nonspecific Immune Response Test and Evaluation

For the analysis of the activity of the head cells, the heads were isolated by randomly catching three fish from each group after the end of the administration of the experimental feed. Phagocytic cells were separated from the head using Percoll gradient centrifuge, and the number of phagocytes was adjusted to 1 × 10 ⁶ cells / ml. Next, 0.7 ml of luminol and 0.4 ml of cells were placed in a cuvette, followed by incubation for 5 minutes, followed by addition of zymosan, followed by chemiluminescent reaction using a luminometer. (CL) response) was measured.

Lysozyme activity was measured using the turbidimetric method of Parry et al. (1965). That is, the serum isolated from each experiment was suspended in 0.05M sodium phosphate buffer (pH 6.2) at a concentration of 0.2 mg / ml Micrococcus lysodeikticus , and then 950 μl of the suspension and serum 50 μl of the mixture was reacted at 25 ° C. for 30 seconds and 4 minutes 30 seconds, and then lysozyme activity was measured using a spectrophotometer (530 nm). The active unit of lysozyme was defined as the amount of enzyme that showed a decrease in absorbance of 0.001 per minute.

Complement Replacement Pathway (ACP) activity was analyzed using sheep red blood cells (SRBC). SRBC was washed three times in gelatin veronal buffer (GVB) containing Mg ²⁺ and EGTA, and adjusted to 2 × 10 ⁸ / ml in the same buffer. The experimental serum was serially diluted with GVB followed by the addition of 100 L of SRBC. The mixture was incubated at 20 ° C. for 90 minutes with occasional shaking, followed by centrifugation at 1600 g at 4 ° C., and the supernatant was measured at 414 nm of the absorbance of the spectrophotometer. ACP (ACH50) activity was calculated according to the degree of hemolysis.

Feeds fed with beta glucan showed a higher tendency in all feeds than control compared to control (ACH50), serum lysozyme (CL) activity, and cranial cell activity (CL). 2).

Peak value of CL (mV) Lysozyme activity (U / ml) ACH50 (U / ml) Control 930 570 36 0.05% 1040 610 45 0.1% 950 610 45 0.25% 1030 600 41 0.5% 660 580 48

Attack experiment by pathogenic microorganism

After the administration of the experimental feed, the attack experiment was carried out using Edwardsiella tarda , which strengthened virulence through the passage of the fish. The experimental strains were incubated for 48 hours at 27 ° C. in TSA medium to which 1.5% NaCl was added, and the bacterial counts were adjusted to 1 × 10 ⁶ cfu / ml with saline, and 0.1 ml was injected into the abdominal cavity of the zophyllolac. The results of the challenge experiments were expressed as cumulative mortality, and the kidneys of the dead individuals were smeared onto TSA medium to isolate Edwardsiella tarda.

In the challenge experiments inoculated with Edward Siela tarda, all feeds treated with glucan showed lower initial mortality than the control. In addition, the feed group added 0.05% glucan showed statistically significantly lower cumulative mortality than the control group (FIG. 1).

Example 2 flounder (flatfish) specification test

Experimental fish was added to the flounder and the concentration of beta glucan in the experimental feed was added at the concentrations of 0.05%, 0.075%, and 0.10% with the control group without beta glucan. Results of growth experiments in which beta glucan was supplied for 6 weeks are shown in Table 3. Feeds fed with 0.075% glucan showed the highest value compared to the control, and overall, the experimental group fed the beta glucan showed higher growth rate than the control.

Control 0.05% 0.075% 0.1% % Increase ¹ 277 285 295 290

¹ Growth Rate: (Final Weight-Initial Weight) × 100 / Initial Weight.

In the present invention, beta glucan was used as a feed additive for fish to increase the growth rate of fish and enhance immunity to reduce mortality even when attacking external pathogens. In particular, when the beta glucan is added at a concentration of 0.01 to 0.2% with respect to the dry feed, the increase rate and the mortality rate also decreased. These results provide a means to increase the economics of aquaculture by increasing the self-defense ability to protect against external diseases by utilizing beta-glucan's immune enhancing ability.

Claims (2)

Feed containing beta glucan for promoting fish growth, characterized in that containing beta glucan 0.01 to 0.1% by weight per dry feed. [Claim 2] The feed of claim 1, wherein the beta glucan is produced from the genus Agrobacterium or Alkali ness facalis strain.