KR101670955B1 - Feed for farming-fish and Farming-fish farmed using that - Google Patents

Abstract

The present invention relates to a feed for aquaculture and to aquaculture farmed using the same, and more particularly, to a feed system for aquaculture that incorporates a mixed probiotic agent capable of replacing antibiotics used in aquaculture will be

Description

Technical Field [0001] The present invention relates to a feed composition for fish farming,

The present invention relates to a feed for aquaculture and to aquaculture farmed using the same, and relates to an auxiliary feed for aquaculture that can replace antibiotics used in fish culture.

Aquaculture is focused on mass production through high density breeding to cover the growing consumption of aquatic products. However, the anthropogenic high-density aquaculture system exposed the aquatic organisms to various forms of stress (high density rearing, physical disability, and water quality deterioration) (Wendelaar Bonga, 1997)

Antibiotics and various kinds of chemicals are used after disease occurrence rather than prevention of aquaculture as fish diseases. Abuse of such drugs not only reduces the immune response by giving stress to the fish, but also increases the resistance to environmental pollution and pathogens (Park, 2004), and the possibility of affecting the human body. For this reason, the government has recently regulated the use of antibiotics except for veterinarian prescriptions for both fish feeds and animal feeds on July 1, 2011 (Ministry of Agriculture, Forestry and Fisheries, 2011) Management of inflow water and effluent, etc., to enhance the sanitation management conditions of the farms for the production of safe aquaculture products.

As the use of antibiotics in combination feeds is restricted, it is necessary to develop natural feed additives to replace antibiotics. In addition, studies on feed additives of eco-friendly materials for substituting antibiotics are actively under way by the consumers' distrust of the stability of aquatic foods and the policy of orienting organic and organic fisheries.

Probiotic agents are defined as live, discontinued or mixed strains that, when ingested by an animal or human, improve the characteristics of endogenous native microorganisms and thereby have a beneficial effect on the host. It is known that lactic acid and acetic acid, which are fermentation products of these strains, inhibit the production of carcinogens and acidify the intestines, thereby lowering the growth of harmful bacteria and increasing the survival rate of lactic acid bacteria relatively (Lee, 2008). It has been reported that probiotics have a positive impact on the health of aquaculture when fed through feed or feed (Ai et al., 2011), improving the feed utilization of aquaculture fish, controlling the distribution of microorganisms in the intestinal tract, (2002), Nayak et al., 2002), and that the antagonistic action of the pathogens, such as Bacillus subtilis, is positively affecting the antagonistic action of pathogens (Balcar et al., 2006; Irianto and Austin, 2002; Kesarcodi-Watson et al. 2010; Wang et al., 2008). According to recent studies, it has been reported that the combination probiotics can increase a broader range of disease resistance (Chapman, 2014) because of the species specificity that inhibits only certain harmful bacteria depending on the nature of the useful strain.

Recently, development of a feed additive for fish culture or a feed for aquaculture using a strain having an antibacterial activity by replacing the antimicrobial agent has been continued. For example, a technique using a strain of Bacillus genus KR-OF1 has been known, Although green tea extracts have been developed for feed for fish farming, there is a problem in that they can not be used to replace antibiotics.

Korean Patent Publication No. 10-2014-0091988 (Disclosure Date: Jul. 23, 2014) Korean Patent Laid-Open No. 10-2012-0138181 (Publication date: December 24, 2012)

As a result of efforts to develop a feed for aquaculture that can replace antibiotics, the present inventors have found that when a mixed strain of a specific strain is introduced into a feed as a probiotic agent, the immunity of trout and other aquaculture fishes is improved, And it is possible to cultivate environmentally friendly and sustainable organic aquaculture. Thus, the present invention has been completed. That is, the present invention provides feed for aquaculture fish and aquacultured fish cultured using the same.

The present invention relates to a feed for aquaculture fish, comprising: a basic feed; And a combination probiotic agent.

In the present invention, the above-mentioned multiprobiotic agent is selected from the group consisting of Bacillus licheniformis , Bacillus subtilis, subtilis ), Paenibacillus polymyxa ), Aspergillus oryzae , and Sacchromyces cerevisiae .

In addition, the above-mentioned compounding probiotics can be used in a concentration of 1 × 10 ⁷ to 8 × 10 ⁸ cfu / kg of Bacillus licheniformis, 5 × 10 ⁷ to 1 × 10 ⁹ cfu / kg of Bacillus subtilis, to ^{7 ~ 1 × 10 9 cfu /} ㎏, Aspergillus duck chair bacteria ^{1 × 10 8 ~ 5 × 10} 9 cfu / ㎏ , and saccharose three Levy cyano bacteria ^{1 × 10 8 ~ 5 × 10} 9 cfu / ㎏ Mrs in the And the like.

The feed for aquaculture of the present invention may include 0.1 to 5 parts by weight of the combined probiotic for 100 parts by weight of the base feed.

In the feed for aquaculture fish of the present invention, the basic feed includes fish meal, soybean meal, wheat gluten meal, blood meal, squid liver powder, wheat flour, fish oil oil, vitamin premix and mineral premix, and cellulose.

In the feed for aquaculture fish of the present invention, the basic feed comprises 25 to 35% by weight of fish meal, 8 to 15% by weight of soybean meal, 5 to 10% by weight of wheat gluten, 4 to 8% % Of wheat flour, 20 to 30 wt% of wheat flour, 8 to 14 wt% of fish oil, 1 to 3 wt% of vitamin premix, 1 to 3 wt% of mineral premix, and 0.1 to 1 wt% of cellulose.

In addition, in the basic feed for aquaculture of the present invention, the vitamin premix may contain 250 to 350 mg / kg of ascorbic acid, 120 to 120 mg / dl of calcium pantothenate per kg of vitamin pre- 180 mg / kg, choline bitate 2,800-3,300 mg / kg, inositol 120-180 mg / kg, menadione 3-10 mg / kg, Niacin 100-200 mg / 10 to 30 mg / kg of pyridoxine hydrochloride, 20 to 50 mg / kg of Rivoflavin, 5 to 30 mg / kg of thiamine mononitrate, dl-α-tocopherol acetate (dl- kg of folic acid, 2 to 10 mg / kg of biotin, 2 to 10 mg / kg of folic acid, and 100 to 300 mg / kg of retinyl acetate. And a concentration of 0.01 to 0.5 mg / kg of cobalamin.

In addition, in the basic feed, which is a feed ingredient for aquaculture fish of the present invention, the mineral premix contains 400 to 500 mg / kg of NaCl, 1200 to 1500 mg / kg of MgSO ₄ .H ₂ O, ZnSO ₄ .H ₂ O 200 to 250 mg / kg, Fe-Citrate 250 to 350 mg / kg, MnSO ₄ 0.005 to 0.050 mg / kg, FeSO ₄ 0.0100 to 0.1000 mg / kg, CuSO ₄ 0.00010 to 0.00100 mg / kg , 0.0001 to 0.0050 mg / kg of calcium iodate, 0.00050 to 0.00500 mg / kg of MgO and 0.00005 to 0.00100 mg / kg of NaSeO ₃ .

In the feed for aquaculture of the present invention, the aquaculture may be a trout or a flounder.

In addition, the present invention relates to a method for culturing fish, and is characterized in that the fish are cultured in the various types of fish for aquaculture described above.

In addition, the present invention relates to aquaculture fish, which is characterized in that it is a fish cultured according to the above-described various forms of aquaculture and / or aquaculture method.

In addition, the above-described aquaculture fish may be one species selected from trout, halibut, catfish or eel, preferably trout or halibut, more preferably rainbow trout.

The feed for aquaculture fish containing the compound probiotics of the present invention can act as a competitive inhibitor for promoting the secretion of secretory enzymes, organic acids, vitamins and non-toxic antibacterial substances in the intestines of aquaculture fish to prevent adherence of the intestinal mucous membranes of fishes, The effect of improving growth, feed intake and nonspecific immune response can be replaced by existing antibiotics because it can enhance growth and immunity.

FIG. 1 is a photograph of a pilot plant used for breeding rainbow trout performed in the embodiment of the present invention. FIG.

Rainbow Trout, a kind of trout, is a cold-water fish species imported from the United States in 1965, has been established as an early seed production technology, has a strong resistance to diseases, and is easy to manage because of its light-temperate fish species. It is a species. Trout farming has been steadily growing since the 1970s. In 2013, 3,390 tons of 20,461 tonnes of domestic freshwater fish farming are produced, followed by eels and catfish (KOSTAT, 2014).

Probiotic agents play an important role in maintaining the balance of normal intestinal flora by inhibiting the growth of pathogenic bacteria by enzymes, organic acids, vitamins and non-toxic antimicrobial substances secreted by microorganisms by ingesting living microbial cells. Therefore, in order to stabilize the intestines of the breeding animals in general, beneficial microorganisms are mixed in the feed. The beneficial microorganisms mainly used are Lactobacillus, Bifidobacterium, Clostridium, and Bacillus. These microorganisms are known to have the effect of inhibiting the death and proliferation of harmful microorganisms in the intestines by producing lactic acid, which is widely used for the purpose of improving digestive power and increasing immunity.

The present invention relates to a method of introducing such a probiotic agent as a component of aquaculture feed, wherein the aquaculture feed of the present invention is characterized by including a complex probiotic agent introducing a specific bacterium.

The present invention relates to a feed for aquaculture fish, comprising: a basic feed; And a combination probiotic agent.

The combined probiotic, which is one of the components of the present invention, is Bacillus licheniformis licheniformis , Bacillus subtilis, subtilis ), Paenibacillus polymyxa ), Aspergillus Mrs into oryzae) and Saccharomyces Celebi cyano bacteria (Sacchromyces cerevisiae , and preferably all of five species.

The combined probiotics were prepared from 1 × 10 ⁷ to 8 × 10 ⁸ cfu / kg of Bacillus licheniformis, 5 × 10 ⁷ to 1 × 10 ⁹ cfu / kg of Bacillus subtilis, 5 × 10 6 to ^{7 ~ 1 × 10 9 cfu /} ㎏, Aspergillus duck chair bacteria ^{1 × 10 8 ~ 5 × 10} 9 cfu / ㎏ , and saccharose three Levy cyano bacteria ^{1 × 10 8 ~ 5 × 10} 9 cfu / ㎏ Mrs in the And preferably 1 × 10 ⁸ to 6 × 10 ⁸ cfu / kg of Bacillus licheniformis, 1 × 10 ⁸ to 5 × 10 ⁸ cfu / kg of Bacillus subtilis, ^{× 10 7 ~ 5 × 10 8} cfu / ㎏, Aspergillus fungus in duck chair ^{8 × 10 8 ~ 5 × 10} 8 cfu / ㎏ and MRS three Levy cyano bacteria as saccharose ^{5 × 10 8 ~ 3 × 10} 9 cfu / Kg. ≪ / RTI >

The feed for aquaculture of the present invention may include 0.1 to 5 parts by weight, preferably 0.3 to 2 parts by weight, of the combined probiotic microbial agent per 100 parts by weight of the base feed, If it is contained in an amount of less than 1 part by weight, there may be a problem that the antimicrobial activity effect or the like is lowered, and even if the amount is more than 2 parts by weight, there is no increase in the antibacterial activity effect due to an increase in the amount of use.

In the feed for aquaculture fish of the present invention, the basic feed includes fish meal, soybean meal, wheat gluten meal, blood meal, squid liver powder, wheat flour, fish oil oil, vitamin premix and mineral premix, and cellulose.

The basic diet includes 25 to 35% by weight of fish meal, 8 to 15% by weight of soybean meal, 5 to 10% by weight of wheat gluten, 4 to 8% by weight of blood, 3 to 7% by weight of squid liver powder, 20 to 30% Preferably 8 to 14% by weight of fish oil, 1 to 3% by weight of vitamin premix and 1 to 3% by weight of mineral premix and 0.1 to 1% by weight of cellulose, preferably 28 to 32% by weight of fish meal, 10 to 12% % Of wheat gluten, 6 to 8 wt% of wheat gluten, 5 to 7 wt% of blood components, 5 to 6 wt% of squid liver powder, 23 to 27 wt% of wheat flour, 10 to 13 wt% of fish oil, 1.5 to 2.5 wt% 1.5 to 2.5% by weight of cellulose and 0.2 to 0.8% by weight of cellulose.

Also, the vitamin premix may contain 250 to 350 mg / kg of ascorbic acid, 120 to 180 mg / kg of dl-calcium pantothenate, 2 to 800 mg / kg of choline bitate per kg of vitamin pre- 3,000 mg / kg, inositol 120 to 180 mg / kg, menadione 3 to 10 mg / kg, Niacin 100 to 200 mg / kg, pyridoxine hydrochloride 10 to 30 mg / kg 20 to 50 mg / kg of riboflavin, 5 to 30 mg / kg of thiamine mononitrate, 100 to 300 mg / kg of dl-α-tocopherol acetate, 2 to 10 mg / kg of retinyl acetate, 0.5 to 5 mg / kg of biotin, 2 to 10 mg / kg of folic acid and 0.01 to 0.5 mg / kg of cobalamin .

In addition, the mineral premix are mineral premix 1kg NaCl 400 ~ 500 mg / kg , MgSO 4 · H 2 O 1200 ~ 1500 mg / kg, ZnSO 4 · H 2 O 200 ~ 250 mg / kg, Fe- citrate (Fe per -Citrate) 250 ~ 350 mg / kg , MnSO 4 0.005 ~ 0.050 mg / kg, FeSO 4 0.0100 ~ 0.1000 mg / kg, CuSO 4 0.00010 ~ 0.00100 mg / kg, calcium EO date (Calciumiodate) 0.0001 ~ 0.0050 mg / kg, MgO 0.00050 to 0.00500 mg / kg and NaSeO ₃ 0.00005 to 0.00100 mg / kg.

In the feed for aquaculture of the present invention, the aquaculture fish may be one selected from trout, halibut, catfish or eel, preferably trout or halibut, more preferably a rainbow trout among the trout.

The present invention also relates to a method for culturing fish with the above-described feed for aquaculture, comprising the step of culturing the fish of the present invention at a temperature of 15 ± 0.5 ° C., which is about 3 to 3.5% Feed can be fed and raised.

As described above, the feed for aquaculture of the present invention and the fish cultured by the above-described aquaculture method can greatly increase growth and immunity.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by the Examples described below, and should be construed to facilitate understanding of the present invention.

[ Example ]

Preparation Example  One

(1) As shown in Table 1 below, standard strains were distributed from KCTC (Korea Research Institute of Bioscience and Biotechnology).

Sacchromyces cerevisiae KCTC 7906 Aspergillus ory KCTC 6292 Lactobacillus acidophilus KCTC 3171 Paenibacillus polymyxa KCTC 1761 Lacdtobacillus plantarum subsp . plantarum KCTC 3103

(2) Next, a culture medium for β-glycan (strain producing β-glucan) was prepared as shown in Table 2 below.

division content Sucrose 100g NH ₄ Cl 4.2 g 4.2 g KH ₂ PO ₄ 1.0 g MgSO ₄ .7H ₂ O 0.4 g Mineral solution.  10ml Anilline blue 2.0 g Agar 15g Distilled water 1,000ml (1) Mineral solution Component: FeSO ₄ 5 g, MnSO ₄ 2 g, CoCl ₂ 1 g, ZnCl _{2 2} g in 0.1 M HCl 1 L

Next, the roots of the swimming roots were taken from the rhizosphere soil and air dried. After the soil samples were placed in sterilized physiological saline and subjected to heat treatment (80, 1 hr) and not heat-treated, the plates were plated on the selective medium and then cultured in an incubator . Thereafter, the bacterium integrated into the colony of the indicator aniline blue was selected as the? -Glycan producing strain (see FIG. 1).

(3) Identification of isolated strains

Primary separation The strains were separated by pure separation and submitted to the Institute for Identification (Solgent) 16S rRNA gene experiment methods.

① Bacterial DNA isolation

An appropriate amount of cells was subjected to DNA isolation using a solgent purification bead.

② Polymerase chain reaction (PCR)

PCR was performed using universal primer 27F (5'-AGA GTT TGA TCC TGG CTC AG-3 ') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3') and Solgent EF-Taq, PCT 9700 (ABI) PCR machine was used.

③ Next, PCR product purify was performed using the solgent PCR purification kit.

(4) Next, electrophoresis confirmation (1% agarose gel) was performed, and the gene sequence obtained by sequencing was compared with the standard strain.

At this time, as the gene fitness of DC-HRP01 of FIG. 1, Bacillus horikoshii , and its gene sequence is shown in Fig.

In addition, DC-HRP02 is a Bacillus sp. ABR4, and DC-NHRP01 as Bacillus megaterium .

Here, it is assumed that the strain belonging to the strain which can be used as a probiotic agent and belonging to the strain registered as a constituent (Ministry of Agriculture, Forestry and Fisheries Notice No. 2011-125, Appendix 3) is Bacillus megaterium which is DC-NHRP01 and only this isolate is added. The remaining strains, DC-HRP01 and DC-HRP02, will be further analyzed to examine applications for registration of superior quality components.

(4) Self-contained strain

Bacillus with excellent protein and lipidolytic activity subtillus (Bacillus subtilis), and Bacillus licheniformis were added.

The microorganisms to be tested with the probiotics are Sacchromyces cerevisiae KCTC 7906, Aspergillus oryzae KCTC 6292, Lactobacillus acidophillus KCTC 3171, Paenibacillus polymyxa KCTC 1761, Lacdtobacillus plantarum subsp. plantarum KCTC 3103, Bacillus megaterium, Bacillus subtillus, and Bacillus licheniformis .

In addition, the strain was stored in a vial containing 10% skim milk for sterilization, sterilized by adding sterilized pure sterilized strain to three sterilized plots, and sterilized glycerol and liquid culture. Were stored in -70 ° C deep freezer.

Preparation Example  2: Composite probiotics ( probiotics ) Strain Selection

(One) Culture medium  Ready

The growth media of the seeds for the probiotics were Sacchromyces cerevisiae KCTC 7906 was grown in YM medium (Yeast extract 3.0 g, Malt extract 3.0 g, peptone 5.0 g, dextrose 10.0 g, Distilled water 1.0 L), Aspergillus oryae KCTC 6292 contains malt extract medium (malt extract 20.0 g, peptone 5.0 g, distilled water 1.0 L), lactic acid bacteria Lactobacillus acidophillus KCTC 3171 and Lacdtobacillus plantarum subsp . plantarum KCTC 3103 is MRS medium (peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 20.0g, tween 80 1.0ml, K 2 HPO 4 2.0g, sodium acetate 5.0g, triammonium citrate 2.0g, MgSO 4 · H ₂ O, distilled water 1.0 L) was used, and Paenibacillus polymyxa KCTC 1761, Bacillus megaterium , Bacillus subtillus , and Bacillus licheniformis were used in Nutrient medium (beef extract 3.0 g, peptone 5.0 g, distilled water 1.0 L).

(2) Culture method

Cultivation of probiotics was carried out in a test tube containing 10 ml of each culture medium as described above. The bacteria were shake cultured in a shaking incubator at 30 DEG C and 170 rpm for 36 hours. Yeast and fungi were cultured in a shaking incubator at 25 DEG C and 150 rpm And cultured for 3 days.

(3) Growth activity investigation

1) Heat resistance investigation

To measure the heat resistance of the probiotics, 50 ml of the culture broth grown in the liquid medium was centrifuged (4,000 rpm, 20 minutes) to discard the supernatant, and 10 ml of phosphate buffer (pH 7.2) was added to the precipitated pellet. Prepared.

The heat treatment was performed in a high pressure autoclave at 80 for 1 hour and viable microbial counts were then cultured in BL agar medium for 30 to 48 hours.

Bacterium Before sterilization
(CFU / ml) Population after sterilization
(CFU / ml) Survival rate (%) Sacchromyces cerevisiae KCTC 7906 2.5 x 10 ⁹ 1.8 x 10 ⁷ 0.7 Aspergillus oryae KCTC 3.0 × 10 ⁹ 3.0 × 10 ⁶ 0.1 Lactobacillus acidophillus KCTC 3171 1.5 x 10 ⁹ 3.0 × 10 ⁶ 0.2 Lacdtobacillus plantarum subsp. plantarum KCTC 3103 2.0 × 10 ⁹ 6.0 × 10 ⁶ 0.3 Paenibacillus polymyxa KCTC 1761 2.0 × 10 ⁹ 1.0 × 10 ⁶ 5.2 Bacillus megaterium DCP01 6.5 × 10 ⁹ 4.1 × 10 ⁹ 62.6 Bacillus subtillus 8.0 × 10 ⁹ 7.4 × 10 ⁹ 93.4 Bacillus licheniformis 6.0 × 10 ⁹ 4.6 × 10 ⁹ 75.5

Heat Resistance from Sacchromyces cerevisiae KCTC 7906, Aspergillus oryae KCTC 6292, Lactobacillus acidophillus KCTC 3171 and Lacdtobacillus plantarum subsp . Plantarum KCTC 3103 was weak, and Paenibacillus polymyxa KCTC 1761 was inferior in traceability. Bacillus sp. megaterium , Bacillus subtillus , and Bacillus Licheniformis showed high heat resistance.

2) Acid resistance  Research

To investigate the acid resistance, 0.4 g NaCl and conc. HCl (1.4 ml) was placed in a 200 ml volumetric flask, and 2 ml of the culture solution was added to artificial gastric juice mixed with distilled water. The number of viable microorganisms before and after the stirring for 4 hours was counted using a BL agar medium, Respectively.

Bacterium Pre-reaction population
(CFU / ml) Population after reaction
(CFU / ml) Survival rate (%) Sacchromyces cerevisiae KCTC 7906 1.1 x 10 ⁸ 2.8 x 10 ⁷ 25.2 Aspergillus oryae KCTC 1.5 × 10 ⁸ 3.4 × 10 ⁷ 22.5 Lactobacillus acidophillus KCTC 3171 7.5 × 10 ⁷ 6.7 x 10 ⁷ 89.5 Lacdtobacillus plantarum subsp. plantarum KCTC 3103 1.0 × 10 ⁸ 8.2 × 10 ⁷ 82.1 Paenibacillus polymyxa KCTC 1761 1.0 × 10 ⁸ 8.3 × 10 ⁷ 83.2 Bacillus megaterium DCP01 3.3 x 10 ⁸ 2.3 × 10 ⁷ 68.2 Bacillus subtillus 4.0 x 10 ⁸ 1.6 x 10 ⁸ 41.2 Bacillus licheniformis 3.0 × 10 ⁸ 2.7 × 10 ⁸ 75.5

In the acid resistance test, lactic acid bacteria showed high acid resistance of over 80%.

(3) Inhibitory activity against pathogenic microorganisms

To verify the antimicrobial effect of pathogenic microorganisms on intestinal pathogens, the antimicrobial effect was verified using the Agar diffusion assay (Agar Paper Disc Diffusion Method).

Bottom agar (MRSa) and then hardened by the first frequency divider to produce a Top agar (TSB + 0.7% Agarose ) pathogens diluent (Escherichia coli K99- Escherichia coli K88- coli Escherichia coli and Salmonella enteritidis - chicken diarrhea jangyeomgyun each dilution 1 × 10 ^-2) by the frequency divider, each cakes over the bottom agar plate (plate). A paper disc containing seeds of each seed was placed on a hardened plate and cultured at 37 ° C for 12 hours. If a clear zone is formed after incubation, the clear zone formed on the plate is measured and quantified. If the transparent cores are not measurable - (no effect), if it is smaller than 15 mm, + (effect is weak), if 15 to 25 mm it is ++ (good effect) ++++ (very large effect) and the results are shown in Table 5 below.

Bacterium Intestinal pathogenic microorganism Escherichia coli K99 Escherichia coli K88 Salmonella enteritidis Sacchromyces cerevisiae KCTC 7906 + + + Aspergillus oryae KCTC + + + Lactobacillus acidophillus KCTC 3171 +++ ++ ++ Lacdtobacillus plantarum subsp. plantarum KCTC 3103 +++ ++ +++ Paenibacillus polymyxa KCTC 1761 ++ +++ +++ Bacillus megaterium DCP01 + + + Bacillus subtillus ++ ++ + Bacillus licheniformis ++ ++ ++

All of the strains showed antimicrobial activity against intestinal pathogenic microorganisms, and lactic acid bacteria showed excellent antimicrobial activity.

From the above results, it was found that the acidic and antibacterial activity of lactic acid bacteria was high and the heat resistance was superior to that of bacillus producing heat resistant spores. Thus, Bacillus strains have a high survival effect on thermal lethality when producing feeds using probiotics It is expected that the microorganism itself will play a role in the intestinal tract when the feedstuff production process is carried out because the survival residual effect of lactic acid bacteria, yeast and fungi is low. Therefore, introduction of the prebiotic concept was considered to be necessary to quantitatively control populations so as to help the function of the intestines in the animal gut.

Therefore, as the microorganism strain for the probiotic agent, Sacchromyces cerevisiae KCTC 7906, Aspergillus oryza KCTC 6292, Lactobacillus acidophillus KCTC 3171 and Lacdtobacillus plantarum subsp . plantarum KCTC 3103, Paenibacillus polymyxa KCTC 1761 and its self-retaining bacteria, Bacillus subtillus And Bacillus licheniformis In addition, Bacillus megaterium DCP01 exhibited the required functionality as a probiotic agent, and was selected as a bacterium for probiotics.

Preparation Example  3: Production of complex probiotics

Among the microorganism strains, Bacillus licheniformis bacteria licheniformis , Bacillus subtilis, subtilis), Bacillus penny poly dynamic dead cells (Paenibacillus polymyxa), the fungus Aspergillus duck chair (Aspergillus oryzae and Sacchromyces cerevisiae were mixed at a weight ratio of 1: 1: 1: 1: 1 to prepare a combined probiotic. The bacterial counts of the combined probiotics were as shown in Table 6 below.

division Number of bacteria (cfu / kg) Bacillus subtilis
( Bacillus subtilis ) 2.8 × 10 ⁸ Bacillus licheniformis
( Bacillus lichemifomis ) 3.4 × 10 ⁸ Penny Bacillus polymicrobacteria
( Paenibacillus polymyxa ) 3.5 × 10 ⁸ Aspergillus oryzae
( Aspergillus oryzae ) 1.6 × 10 ⁹ Saccharomyces cerevisiae
( Sacchromyces cerevisiae ) 1.1 × 10 ⁹

Example  1: Production of feed for aquaculture fish

0.5 parts by weight of the complex probiotics prepared in Preparation Example 3 were mixed with 100 parts by weight of the basic feed having the composition shown in Table 7 below to prepare a feed for aquaculture fish (hereinafter referred to as MP ₂ ). The pellet was extruded by a pelletizer, dried naturally in a well-ventilated place (48 hours), uniformly pelletized using a 1,500 μm and 2,000 μm sieve to fit the size of the test sample, Lt; RTI ID = 0.0 > -20 C. < / RTI >

Ingredients Content (% by weight) Fish meal, herring ⁽¹⁾ 29.6 Soybean meal ⁽²⁾ 11.0 Wheat gluten meal ⁽¹⁾ 7.3 Blood meal ⁽¹⁾ 6.0 Squid liver powder ⁽¹⁾ 5.5 Wheat flour ⁽¹⁾ 24.3 Fish oil, menhaden ⁽³⁾ 11.8 Vitamin premix ⁽⁴⁾ 2.0 Mineral premix ( ⁵⁾ 2.0 Cellulose ⁽⁶⁾ 0.5 (One): Suhyup feed C. Uiryeong, Korea
(2): The feed Co. Goyang, Korea
(3): Jeil feed Co. Hamman, Korea
(4): Contains (as mg / kg in diets): Ascorbic acid, 300; dl-Calcium pantothenate, 150; Choline bitate, 3000; Inositol, 150; Menadion, 6; Niacin, 150; Pyridoxine · HCl, 15; Rivoflavin, 30; Thiamine mononitrate, 15; dl -? - Tocopherol acetate, 201; Retinyl acetate, 6; Biotin, 1.5; Folic acid, 5.4; Cobalamin, 0.06
(5): Contains (as mg / kg in diets): NaCl, 437.4; _{MgSO 4 · H 2 O, 1379.8} ; ZnSO ₄ .H ₂ O, 226.4; Fe-Citrate, 299; MnSO _4, 0.016; FeSO _4, 0.0378; CuSO _4, 0.00033; Calciumiodate, 0.0006; MgO, 0.00135; NaSeO _3, 0.00025
(6): Sigma-Aldrich Korea Yongin, Korea

Comparative Example  1 ~ Comparative Example  3

Comparative Example 1 (control, cont) was prepared as the basic feedstuff of Table 7, and as Comparative Example 2, a mixture of antibiotic (Oxytetracycline; OTC, 0.5 part by weight) and 100 parts by weight of the base feed of Table 7 , OTC) was prepared. As Comparative Example 3, 100 parts by weight of the above basic feed and 100 parts by weight of a third-party complex ( Clostridium butyricum , Bacillus licheniformis and Bacillus subtilis were mixed at the same weight ratio) to prepare feed (MP ₁ ). Then, all the feed sources were uniformly mixed, extruded by a pelletizer, dried naturally in a well-ventilated place (48 hours), and the particle size was adjusted to 1,500 μm and 2,000 μm sieve , Sealed, and stored at -20 DEG C while being refrigerated.

Manufacturing example  : Breeding of rainbow trout

The rainbow trout was purchased from a fish farm located in Sangju, Kyungpook province, and transported to the Pusan National University Feed and Nutrition Research Institute. After preliminary breeding, rainbow trout, 5.98 ± 0.12 g (mean ± SD), of the average fish body was housed in a 50 L rectangular water tank shown in FIG. 3 and randomly placed in each experimental group. Air stones were installed for sufficient oxygen supply and the average water temperature was maintained at 15 ± 0.5 ℃. Feeding amount of 1 day was supplied 3 ~ 3.5% of body weight twice a day during the whole experimental period, and breeding experiment was carried out for a total of 8 weeks.

All the experimental results were analyzed by ANOVA (Analysis of Variance) test with SAS program and the significant difference (P <0.05) between the mean values was tested with Least Significant Difference (LSD).

Experimental Example  1: Biometry

In all experiments, weights and body weights were measured every 2 weeks and weight gain,%, feed efficiency,% specific growth rate,% / day, protein efficiency ratio ,%), And survival rate (%). The calculation formulas of the above measurement items are as shown in the following equations (1) to (5), respectively.

[Equation 1]

Weight gain (WG,%) = (final weight - initial weight) x 100% / (initial weight)

&Quot; (2) &quot;

Feed efficiency (FE,%) = (wet weight gain / dry feed intake) × 100%

&Quot; (3) &quot;

Daily growth rate (SGR,%) = (log _e final weight - log _e initial weight) x 100% / (days of the experiment)

&Quot; (4) &quot;

Protein conversion efficiency (PER) = (wet weight gain / protein intake)

&Quot; (5) &quot;

Survival rate (%) = (total number of fish - number of dead fish) × 100% / (total number of fish)

After the last 8 weeks of rearing, the growth rate, feed efficiency, daily growth rate, protein conversion efficiency, and survival rate were measured, and the results are shown in Tables 8 and 4 to 8, respectively.

division Diets Pooled
SEM ⁽¹⁾ Comparative Example 1
(Cont) Comparative Example 2
(OTC) Comparative Example 3
(MP ₁ ) Example 1
(MP ₂ ) Growth rate (%) 210.89 ^b 224.56 ^ab 227.26 ^ab 238.41 ^a 3.79 Feed efficiency (%) 95.12 97.89 99.46 98.35 0.79 Daily growth rate (%) 2.52 ^b 2.61 ^ab 2.63 ^ab 2.71 ^a 0.02 Protein conversion efficiency 0.43 0.44 0.45 0.44 0.01 Survival rate (%) 91.7 91.7 95.0 91.7 1.44 (One) : Pooled standard error of mean: SD / √ n

(P <0.05), but OTC, MP ₁ , and MP ₂ were significantly higher than those of control And There was no significant difference (P> 0.05) in the experimental period when MP ₂ was supplied.

Experimental Example  2 : Full body  General compositional analysis

The water was dried by the atmospheric pressure heating method (135 ° C, 2 hours) according to AOAC (Association of Official Analytical Chemists, 2000) method using randomly extracted and pulverized whole bodies for each tank. The crude protein was analyzed by Kjeldahl nitrogen determination method (N × 6.25), and the query was analyzed by direct method. The crude fat samples were lyophilized for 12 hours and analyzed by Soxhlet extraction using soxtec system 1046 (Tacator AB, Sweden). The results are shown in Table 9 below.

division Diets Pooled
SEM ⁽¹⁾ Comparative Example 1
(Cont) Comparative Example 2
(OTC) Comparative Example 3
(MP ₁ ) Example 1
(MP ₂ ) moisture
(Moisture, wt%) 73.65 74.01 71.05 73.08 0.44 protein
(Protein, wt%) 18.04 18.51 18.85 18.31 0.16 Lipid
(Lipid, wt%) 7.85 7.42 7.77 7.58 0.16 Ash (% by weight) 1.73 1.75 1.71 1.78 0.01 (1): Pooled standard error of mean: SD / √n

From the results of Table 9, it was confirmed that the nutrients were almost similar.

Experimental Example  3: Blood measurement experiment

The blood component of fish can be used as a dimension of nutrition, health condition and stress interpretation, and it changes according to the deficiency of essential nutrients of feed or the habitat environment and growth of the fish species. Blood components were examined to evaluate the effect on physiological state.

After 8 weeks of breeding experiment, the test fish were fasted for about 24 hours until the blood was collected and then 4 rats were randomly selected for each tank. The blood was syringe (1 mL) Were collected. Glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose and total cholesterol were purchased from a commercial kit (Kit), and the serum was separated by centrifugation (3,000 rpm for 10 minutes) (DRI-CHEM 4000i-Fuji Dri-Chem Slide- 3150 (Minato-ku, Tokyo, Japan)). The results are shown in Tables 10 and 9 to 12, respectively.

division Diets Pooled
SEM ⁽³⁾ Comparative Example 1
(Cont) Comparative Example 2
(OTC) Comparative Example 3
(MP ₁ ) Example 1
(MP ₂ ) GOT (U / L) ⁽¹⁾ 20.3 21.3 19.0 21.0 0.46 GPT (U / L) ⁽²⁾ 20.0 22.0 19.3 21.3 0.55 Glucose (mg / dL) 37.3 ^a 33.3 ^ab 34.6 ^ab 32.3 ^b 0.79 T-Cholesterol (mg / dL) 123.3 118.6 116.0 121.3 1.51 (1): Glutamic oxaloacetic transaminase. One unit is defined as the amount of enzyme causing the transamination of 1.0 μmol of L-aspartate per minute at 25 ° C and pH 7.4
(2): Glutamic pyruvic transaminase. One unit is defined as the amount of enzyme causing the transamination of 1.0 μmol of L-alanine per minute at 25 ° C and pH 7.4
(3): Pooled standard error of mean: SD / √n

GOT, GPT, and total cholesterol of plasma were not significantly different in all experimental periods (P> 0.05) in the results of Table 10 and FIGS. 9 to 12. However, the content of glucose was significantly lower in Example 1 than in Comparative Example 1 (P <0.05), and there was no significant difference between Comparative Example 2, Comparative Example 3 and Example 1 (P > 0.05).

Experimental Example  4: Nonspecific Immune Response Experiment

(One) SOD  Active measurement experiment

SOD assay kit (Sigma-Aldrich, 191600) was used to separate the sera from each experimental fish. The percent inhibition of the enzyme was calculated by WST-1 (Water Soluble Tetrazolium dye) and xanthine oxidase according to the manufacturer's instructions. Each sample was incubated in an incubator at 37 ° C for 20 minutes, and absorbance was measured at a wavelength of 450 nm (absorbance at a wavelength for measuring the color of WST-1 reacted with active oxygen) . The inhibition rates were expressed as SOD activity units per mg protein, and the results are shown in the following Table 1 and FIG.

(2) Lysozyme ( Lysozyme ) Active measurement experiment

To each sample, 0.1 ml of serum and 0.05 ml of sodium phosphate buffer (pH 6.2) were mixed with 2 ml of suspension suspending Micrococcus lysodeikticus (0.2 mg / ml). Then, the reaction was measured at a temperature of 20 ° C at a light absorbance of 530 nm at 0.5 minute and 4.5 minutes by a spectrophotometer. The active unit of lysozyme was defined as the amount of enzyme that showed a decrease in absorbance of 0.001 per minute. The measurement results were obtained, and the results are shown in Table 1 and FIG.

division Diets Pooled
SEM ⁽¹⁾ Comparative Example 1
(Cont) Comparative Example 2
(OTC) Comparative Example 3
(MP ₁ ) Example 1
(MP ₂ ) SOD (% inhibition) 37.70 ^b 44.49 ^ab 50.47 ^a 48.58 ^a 1.97 Lysozyme (U / ml) 0.83 ^b 1.17 ^a 1.13 ^a 1.13 ^a 0.05 (1): Pooled standard error of mean: SD / √n

Macrophages increase reactive oxygen species (ROS) by hydrolyzing bacteria and viruses that enter the body. SOD activity is a typical antioxidant enzyme that catalyzes the conversion of peroxide ions into oxygen and hydrogen peroxide. Lysozyme is part of an endogenous defense mechanism that is a mycotic bacterium originating from lymphocytes that exhibits properties such as antibiotics (Ingram, et al., 2003) 1980). In the SOD activity experiment of Table 11, Comparative Example 3 and Example 1 were significantly higher than Comparative Examples 1 and 2 (P < 0.05). However, no significant difference was observed in the experimental period in which the comparative examples 3 and ₁₂ were supplied (P> 0.05).

Comparing Example 2, Comparative Example 3 and Example 1 were significantly higher than Comparative Example 1 (P < 0.05). However, there was no significant difference between the Comparative Example 2, the Comparative Example 3 and the Example 1 (P> 0.05).

Through the above examples and experimental results, it can be seen that the feed of the present invention containing the compound probiotic agent It has been confirmed that it can enhance the growth and immunity of cultured fish and can be effective as an antibiotic substitute.

Claims (10)

Basic feed; And
Bacillus licheniformis , Bacillus subtilis , Paenibacillus polymyxa , Aspergillus oryzae , and Saccharomyces cerevisiae can be used as the antioxidants (for example , Bacillus licheniformis , Bacillus subtilis , Paenibacillus polymyxa , Aspergillus oryzae , Sacchromyces cerevisiae );
And 0.1 to 5 parts by weight of the compounding probiotics based on 100 parts by weight of the foundation feed,
The composite probiotics are Bacillus Lai Kenny Po Ms bacteria ^{1 × 10 7 ~ 8 × 10} 8 cfu / ㎏, Bacillus subtilis seugyun ^{5 × 10 7 ~ 1 × 10} 9 cfu / ㎏, Penny Bacillus poly dynamic dead cells 5 × 10 ⁷ ~ 1 × 10 ⁹ cfu / ㎏, Aspergillus duck character in bacteria ^{1 × 10 8 ~ 5 × 10} 9 cfu / ㎏ , and saccharose in MRS three Levy cyano bacteria ^{1 × 10 8 ~ 5 × 10} 9 containing cfu / ㎏ And a feed for aquaculture.
delete delete The method according to claim 1,
Wheat gluten meal, blood meal, squid liver powder, wheat flour, fish oil, vitamin premix, and mineral premix (such as starch, wheat gluten meal, premix) and cellulose.
5. The method of claim 4,
25 to 35 wt% of fish meal, 8 to 15 wt% of soybean meal, 5 to 10 wt% of wheat gluten, 4 to 8 wt% of blood meal, 3 to 7 wt% of squid liver powder, 20 to 30 wt% of wheat flour, 1 to 3% by weight of a vitamin premix, 1 to 3% by weight of a mineral premix and 0.1 to 1% by weight of a cellulose.
The method according to claim 4, wherein the vitamin premix
Ascorbic acid 250-350 mg / kg, dl-Calcium pantothenate 120-180 mg / kg, choline bitate 2,800-3,300 mg / kg, Inositol 120 (Pyridoxine HCl) 10 to 30 mg / kg, Rivoflavin 20 to 50 mg / kg, Niacin 100 to 200 mg / kg, kg of thiamine mononitrate, 5 to 30 mg / kg of dl-α-Tocopherol acetate, 100 to 300 mg / kg of retinol acetate, 2 to 10 mg / kg, biotin 0.5 to 5 mg / kg, folic acid 2 to 10 mg / kg, and cobalamin 0.01 to 0.5 mg / kg.
5. The method according to claim 4, wherein the mineral premix comprises
NaCl 400 ~ 500 mg / kg, MgSO 4 · H 2 O 1200 ~ 1500 mg / kg, ZnSO 4 · H 2 O 200 ~ 250 mg / kg, Fe- citrate (Fe-Citrate) 250 ~ 350 mg / kg, _{MnSO 4 0.005 ~ 0.050 mg / kg} , FeSO 4 0.0100 ~ 0.1000 mg / kg, CuSO 4 0.00010 ~ 0.00100 mg / kg, calcium EO date (Calciumiodate) 0.0001 ~ 0.0050 mg / kg, MgO 0.00050 ~ 0.00500 mg / kg and NaSeO ₃ 0.00005 to 0.00100 mg / kg. &Lt; / RTI &gt;
The feed for aquaculture fish according to any one of claims 1 and 4 to 7, wherein the cultured fish is trout or flounder.
A method of growing fish with a feed for aquaculture according to any one of claims 1 and 4 to 7.
Aquaculture fish cultivated with feed for aquaculture of any one of paragraphs 1 and 4 to 7.

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