KR102174589B1 - Development of dietary synbiotics as an antibiotic replacer in Japanese eel - Google Patents

Abstract

The present invention relates to a feed additive composition for aquaculture fish containing Bacillus strain ( Bacillus ) and MOS (mannan oligosaccharide) as an active ingredient and a feed composition comprising the composition.

Description

Development of dietary synbiotics as an antibiotic replacer in Japanese eel}

The present invention relates to a feed additive composition for aquaculture fish containing Bacillus strain ( Bacillus ) and MOS (mannan oligosaccharide) as an active ingredient and a feed composition comprising the composition.

Eel is a representative freshwater fish species that is the No. 1 freshwater aquaculture production in Korea (11,067 tons/300 billion won, fisheries information portal). In 2017, 11,067 tons were produced, an increase of about 12% compared to last year due to steady increase in demand and increased stocking of eel. As researches on eel seed production technology development, compound feed development, and breeding technology development were actively conducted, It is expected to increase. However, in most eel farms, side effects such as diseases appear due to stress from high-density farming, food competition, and contamination of breeding water.

Until recently, strong antibiotics have been used to solve the problem of high-density farming, but in Korea in 2011 due to the problem of residual antibiotics in farmed fish and shellfish, frequent disease outbreaks due to the use of raw feed and high-density farming, and the stability of aquaculture products. From January 1, the use of antibiotics for all animal feeds, including fish feed, for purposes other than prescriptions for treatment by veterinarians and fisheries disease managers was regulated by law. However, many farms still use antibiotics indiscriminately.

Therefore, the development of feed additives that can replace antibiotics is a very urgent situation. Accordingly, in order to replace antibiotics, in the present invention, probiotics were set through screening for indigenous bacteria in the eel intestine for the first time in Korea, and prebiotics that enhance the activity were selected to increase the growth of eel and feed efficiency In addition to killing, we have developed synbiotics that have good effects on improving disease resistance and intestinal function and can replace antibiotics.

Probiotics inhibit the reproduction of harmful bacteria in the intestine, increase digestion and absorption, increase immunity, and improve body weight gain. In addition, it has shown many effects in various fish and terrestrial animals through strengthening of non-specific immune function, antioxidant action, and removal of intestinal toxins. Prebiotics are a type of sugar and are a nutrient that helps the survival and growth of prebiotics in the gut. By establishing useful concentrations of probiotics and prebiotics selected through previous experiments and developing synbiotics, it is believed that it will greatly contribute as a feed additive for fish farming that can replace antibiotics.

1. Korean Patent Registration No. 10-1536901

An object of the present invention is to provide a feed additive composition for aquaculture fish comprising Bacillus strain ( Bacillus ) and MOS (mannan oligosaccharide) as active ingredients.

Another object of the present invention is to provide a feed composition for farmed fish comprising the feed additive composition.

Another object of the present invention is to provide a method for breeding farmed fish comprising the step of feeding the feed additive composition or the feed composition to farmed fish.

Another object of the present invention is to provide a method for increasing immunity against fish pathogenic bacteria by feeding the feed additive composition or the feed composition to farmed fish.

In order to achieve the above object, the present invention provides a feed additive composition for aquaculture fish containing Bacillus strain ( Bacillus ) and MOS (mannan oligosaccharide) as active ingredients.

In one embodiment of the present invention, the Bacillus strain may be Bacillus subtilis .

In one embodiment of the present invention, the concentration of the Bacillus strain may be 0.5 to 1 × 10 ⁷ CFU / g.

In an embodiment of the present invention, the concentration of the MOS may be 1 to 10 g/kg, and preferably 5 g/kg.

In one embodiment of the present invention, the feed additive may be to inhibit the proliferation of pathogenic bacteria in the body.

In one embodiment of the present invention, the fish may be any one selected from the group consisting of eel, freshwater eel, sea eel, yellowtail, red snapper, flounder, flounder, and eel, but is not limited thereto.

In addition, the present invention provides a feed composition for aquaculture fish comprising the feed additive composition.

In one embodiment of the present invention, the feed additive may be included in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total feed composition.

In addition, the present invention provides a method for breeding farmed fish comprising the step of feeding the feed additive composition or the feed composition to farmed fish.

In addition, the present invention provides a method for increasing the immunity against fish pathogenic bacteria by feeding the feed additive composition or the feed composition to farmed fish.

In an embodiment of the present invention, the fish pathogenic bacteria are Edwardsiella tarda, Streptococcus iniae, Vibrio anguillarium, Vibrio campbellii, Vibrio harveyi (Vibrio harveyi), Vibrio furnisii (Vibrio furnisii) and Streptococcus parauberis (Streptococcus parauberis) may be any one or more selected from the group consisting of.

The feed additive composition according to the present invention includes a Bacillus strain concentration of 0.5 to 1×10 ⁷ CFU/g, and a MOS concentration of 1 to 10 g/kg, thereby increasing the feed efficiency of fish to improve growth. In addition, it can be usefully used in the breeding of farmed fish by the effect of inhibiting the proliferation of pathogenic bacteria in the body and increasing immunity to fish pathogenic bacteria.

1 shows the results of H&E staining of the intestinal tissues of eel for each experimental section (scale bar: 100 μm; original magnifiation × 40).
Figure 2 shows the results of measuring Lysozyme activity and MPO activity in order to find out the non-specific immune response in the blood of eel by experiment.
Figure 3 shows the results of measuring the mRNA expression levels of IgM _H and Hsp70 genes in order to determine whether the expression of the immune gene in the intestinal tissue of the eel for each experiment.
Figure 4 shows the results of measuring the cumulative survival rate different from the attack of the eel Vibrio by experimental section.

The term of the present invention, probiotics, is a generic term for living microorganisms that benefit the human body when ingested in an appropriate amount, and refers to bacteria that benefit our body. Most of the probiotics known to date are lactobacilli. Lactobacillus and beneficial bacteria, which are probiotics, survive in the stomach and bile acids in the body, reach the small intestine, and multiply and settle in the intestine. It has beneficial effects on health in the settled intestine, and these probiotics must be non-toxic and non-pathogenic.

The term of the present invention, prebiotics, refers to substances that help improve the intestinal environment by serving as a nutrient source of probiotics as an indigestible component that helps the growth of beneficial bacteria in the intestine. Prebiotics often consist of carbohydrates like oligosaccharides, and most of them exist in the form of dietary fiber.

The term of the present invention, synbiotics, refers to a food ingredient or dietary supplement in which probiotics and prebiotics are combined in a synergism form.

In the present invention, as will probiotics on the cultured fish feed additive composition comprising SynBio ticks including a MOS (mannan oligosaccharide) as, prebiotics contains Bacillus strain (Bacillus) as an active ingredient.

In the feed additive composition of the present invention, auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, anti-fungal enzymes, and microbial preparations in the form of other live bacteria; Grains such as crushed or crushed wheat, oats, barley, corn and rice; Vegetable protein feeds, such as those based on rapeseed, soybeans and sunflowers; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Dried ingredients consisting of sugar and dairy products, such as various milk powders and whey powder; Main components such as lipids, for example animal fats and vegetable fats, optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease prevention agents may additionally be included.

The feed additive composition of the present invention may be in the form of a powder or liquid formulation, and may include an excipient for feed addition. Examples of excipients for feed addition may include calcium carbonate, powder, zeolite, jade flour, or rice bran, but are not limited thereto.

The microbial strain used in the preparation of the feed additive composition of the present invention may be cultured in a liquid medium or a solid medium by a known fermentation technique. As a medium, various mediums such as natural medium, semi-synthetic medium and synthetic medium may be used without limitation. As a carbon source, glucose, sucrose, starch, molase, starch syrup, maltose, and the like can be used. As the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean meal, ammonium salt, nitrate salt and other organic or inorganic nitrogen-containing compounds may be used. As the inorganic salt, phosphates such as magnesium, manganese, potassium, calcium, and iron, carbonates, and chlorides may be used. However, the carbon source, the nitrogen source, and the inorganic salt are not necessarily limited to the above components, and in addition, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.

The strain is cultured for a suitable period at an appropriate temperature according to known fermentation techniques, and may be preferably cultured at 20°C to 50°C for 10 hours to 5 days, and at 40°C to 50°C for 1 to 5 days It may be cultured. The microbial culture solution obtained according to the present invention may be used with cells separated by washing or concentration after centrifugation or as a culture solution alone, preferably freeze-dried or without adding an additive to the entire culture solution containing the active ingredient. It can be spray-dried and formulated. The formulation thus obtained can be used as a feed additive or feed of the present invention.

The feed additive composition of the present invention may be administered to fish alone or in combination with other feed additives in an edible carrier. In addition, the feed additive composition may be easily administered as a top dressing or directly mixed with the feed or separately from the feed, in a separate oral formulation, or in combination with other ingredients. Typically, alone as well known in the art You can use a daily intake or divided daily intake.

Animals to which the feed additive of the present invention can be used may be fish such as eel, freshwater eel, sea eel, yellowtail, red bream, flounder, flounder, and eel, preferably, but is not limited thereto.

The feed additive may be blended in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total feed composition, preferably 0.1 to 1 part by weight, but is not limited thereto.

In addition, the present invention provides a method for increasing the immunity against fish pathogenic bacteria by feeding the feed to fish. When the feed of the present invention is fed to fish, the bioavailability of the active ingredients contained in the feed increases, thereby increasing the weight gain of the fish, and the immunity against fish pathogenic bacteria may be increased because an antibacterial substance is included. In particular, by being used for fish such as eels, it is possible to improve the growth rate and survival rate.

The fish pathogenic bacteria are Edwardsiella tarda, Streptococcus iniae, Vibrio anguillarium, Vibrio campbellii, Vibrio harveyi, Vibrio harveyi, Vibrio harveyi Si (Vibrio furnisii) and Streptococcus parauberis (Streptococcus parauberis) may be any one or more selected from the group consisting of, but is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Experimental method

1.1. Experimental fish and breeding management

The experimental fish were transported to the Feed and Nutrition Research Institute of Pukyong National University, and 20 fry eel with an average fish weight of 9.0 ± 0.11 g were randomly placed in 3 repetitions for each experimental section, and breeding experiments were conducted for 8 weeks. An air stone was installed to supply oxygen to each tank for sufficient oxygen supply, and the average water temperature was maintained at 27.6 ± 0.5℃. Feed was supplied twice a day, at 2-3% of the fish's weight.

1.2. Experimental feed and experimental design

Fish meal and wheat gluten were used as protein sources for the experimental feed, corn starch as a carbohydrate source, and fish oil and soybean oil as lipid sources. ) Was used. In order to confirm the addition ratio of synbiotics in the feed of fry probiotics eels, Bacillus subtilis strain was 0 (BS ₀ ), 0.5×10 ⁷ CFU/g diet (BS _0.5 ) Or 1×10 ⁷ CFU/g diet (BS ₁ ), and MOS (mannan oligosaccharide) 0 (M ₀ ) or 5 g/kg diet (M ₅ ) added to the basic feed, the total amount of feed is 7 Dogs (BS ₀ M ₀ (CON), BS ₀ M ₅ , BS _0.5 M ₀ , BS _0.5 M ₅ , BS ₁ M ₀ , BS ₁ M ₅ ) were prepared. All feed sources were evenly mixed and then extruded with a pellet maker. After drying at room temperature for two days, the particle size was uniformed using a sieve, sealed, and stored frozen at -20°C.

1.3. Body measurement

Fish are measured after the end of the experiment, fasting for 24 hours to measure the growth rate, and then measuring the total weight to measure the weight gain (%), feed efficiency (%), and specific growth rate (%/day). , Protein efficiency ratio, and survival rate (%) were investigated. The calculation formula of the measurement items is as follows.

Weight gain (%) = [(final wt.-initial wt.) × 100] / initial wt.

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

Specific growth rate (%/day) = [(log _e finalwt.-log _e initialwt.)×100] / days

Protein efficiency ratio = (wet weight gain / protein intake)

Survival rate (%) = [(total fish-dead fish) × 100] / total fish

1.4. Intestinal histological analysis

After fixed embedding in a fish body, fixed in Bouin's solution for a certain period of time, embedding in paraffin after washing and dehydration, and then successively sectioned to a thickness of 4-6 µm to make tissue specimens. For observation with an optical microscope, a paraffin cloth After the sample was prepared using each method, Mayer Hematoxylin & Eosin staining was performed.

1.5. Non-specific immunoassay

1.5.1. Lysozyme activity

Micrococcus lysodeikticus (0.2 mg/ml) was suspended in 0.1 ml of serum isolated from fish in each experimental section and 2 ml of suspension in 0.05M sodium phosphate buffer (pH 6.2). The reaction was measured at 0.5 minutes and 4.5 minutes at the absorbance of 530 nm of the spectrophotometer at 20°C. The active unit of Lysozyme was defined as the amount of enzyme showing a decrease in absorbance of 0.001 per minute.

1.5.2. Myeloperoxidase (MPO) activity

MPO activity was measured according to Quade and Roth (1997). After diluting 20µl serum in 96 cells with HBSS without Ca ²⁺ or Mg ²⁺ , TMB (3,3',5,5'-tetramethylbenzidine hydrochloride, 20mM, Sigma-Aldrich, USA) and H ₂ O ₂ ( 5mM) 35 μl was added. The color change reaction was stopped by adding 35µl of 4M sulfuric acid after 2 minutes, and finally, absorbance was measured at 450nm in a microplate reader.

1.6. Immune gene expression analysis

In order to measure the expression of immune genes in the experimental fish, total RNA was isolated from liver tissue according to Invitrogen's protocol using TRIzol Reagent (Invitrogen, USA). Thereafter, Total RNA was synthesized from mRNA to cDNA using Firststrand cDNA Synthesis Kit for RT-PCR (Roche, USA), and then primers for Hsp70 (heat shock protein 70) and IgM _H (immunoglobulin heavy chain) were used. Gene expression was checked, and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primers used are shown in Table 1 below.

direction Primer sequence HSP 70 forward 5'-CCA TCC TGA CCA TCG AAG AC-3' reverse 5'-GTT CTC TTG GCC CTC TCA CA-3' IgMH forward 5'-CGG TTC TTC TGA CAA TCG-3' reverse 5'-TCG GGC ACA GTA ATA CAC-3' GAPDH forward 5'-GTC TGG AGA AAC CTG CTA-3' reverse 5'-ACC TGG TGT TCT GTG TAT C-3'

1.7. Attack test

In the attack test by Vibrio bacteria, V. anguillarum KCCM 40293 was incubated in TSB (Tryptic Soy Broth) at 26° C. for 24 hours and suspended in sterile distilled water at 5×10 ⁷ CFU/ml. Death was recorded after intraperitoneal injection of 0.1 ml of the suspension into the experimental fish randomly selected for each tank. In order to confirm the cause of death of dead fish, kidneys were collected from dead fish every day and cultured in tryptic soy agar (TSA) to confirm the presence of Vibrio bacteria.

1.8. Statistical processing

ANOVA (analysis of variance) test was performed for all data using the SAS program, and the significant difference ( P <0.05) between the means was tested with the least significant difference (LSD).

Example 2. Effect of increasing the growth and feed efficiency of eels according to complex treatment of Bacillus strains (probiotics) and MOS (prebiotics)

In previous studies, the intestines of eels were screened by 16S rRNA sequence analysis to select indigenous bacteria in the intestines. Among the Bacillus subtilis and L. plantarum strains that have antibacterial activity in vitro . The Bacillus subtilis strain, which showed a growth enhancing effect, was selected as a probiotic, and in the present invention, an effective concentration for the Bacillus subtilis strain to be added to the eel feed was confirmed (Lee et al., 2017). , Comparative evaluation of dietary probiotics Bacillus subtilis WB60 and Lactobacillus plantarum KCTC3928 on the growth performance, immunological parameters, gut morphology and disease resistance in Japanese eel, Anguilla japonica , Fish & Shellfish Immunology, 61, 201-210). In addition, an experiment was conducted to confirm the effective concentration of eel feed by adding MOS (mannan oligosaccharide) as a prebiotic.

First, the present inventors added the Bacillus subtilis strain as 0 (BS ₀ ), 0.5×10 ⁷ CFU/g diet (BS _0.5 ) or 1×10 ⁷ CFU/g diet (BS ₁ ), and MOS (mannan oligosaccharide) ) Was added in combination with 0 (M ₀ ) or 5 g/kg diet (M ₅ ), and the effect on the growth and feed efficiency of eel was investigated. For comparative evaluation with the antibiotic OTC treatment group most commonly used in aquaculture, fry eel with an average fish weight of 9 ± 0.11 g were randomly placed in 3 repetitions of 20 for each experimental group, and a breeding experiment was conducted for 8 weeks.

As a result, in terms of the growth and feed efficiency of eel, the experimental group in which 0.5×10 ⁷ CFU/g or 1.0×10 ⁷ CFU/g Bacillus subtilis strain and 5 g/kg MOS were added in combination did not treat anything. Compared to the control group (CON), the weight gain (WG), feed efficiency (FE), specific growth rate (SGR), and protein efficiency ratio (PER) were all significantly increased compared to the control group (CON). (Table 2: Eel growth results by experimental section). Moreover, it was confirmed that the experimental group in which Bacillus subtilis strain and MOS were added in combination showed higher gain rate, feed efficiency, daily growth rate and protein conversion efficiency than the experimental group treated with Bacillus subtilis strain alone or MOS alone ( Table 2).

Therefore, when the probiotic Bacillus subtilis strain is added to the feed at 0.5×10 ⁷ CFU/g to 1.0×10 ⁷ CFU/g, and the prebiotic MOS is added to the feed at 5 g/kg, eel growth It was confirmed that there is a remarkable effect on.

Diets CON BS _0.5 M ₀ BS ₁ M ₀ BS ₀ M ₅ BS _0.5 M ₅ BS ₁ M ₅ OTC IBW(g) ² 8.88±0.15 8.97±0.09 9.09±0.08 9.14±0.12 8.99±0.09 8.92±0.11 9.06±0.13 FBW(g) ³ 17.4±0.42 ^c 17.8±0.50 ^bc 18.4±0.11 ^b 18.2±0.42 ^bc 18.7±0.14 ^a 18.5±0.39 ^a 18.1±0.51 ^bc WG(%) ⁴ 95.9±2.11 ^c 97.8±4.46 ^bc 102±1.18 ^b 99.4±2.90 ^bc 108±2.12 ^a 108±2.36 ^a 100±2.98 ^bc FE (%) ⁵ 79.9±2.00 ^c 81.5±3.72 ^bc 85.3±0.98 ^b 82.9±3.30 ^bc 89.9±1.77 ^a 89.9±1.97 ^a 83.4±2.48 ^bc SGR
(%/day) ⁶ 1.40±0.02 ^c 1.42±0.05 ^bc 1.47±0.01 ^b 1.44±0.03 ^bc 1.52±0.02 ^a 1.52±0.02 ^a 1.54±0.03 ^bc PER ⁷ 1.60±0.04 ^c 1.63±0.07 ^bc 1.71±0.02 ^b 1.66±0.05 ^bc 1.80±0.04 ^a 1.80±0.04 ^a 1.67±0.05 ^bc

¹ Values are means from triplicate groups of fish where the values in each row with different superscripts are significantly different ( P< 0.05)

² Initial body weight(IBW)

³ Final body weight(FBW)

⁴ Weight gain(WG, %) = [(final wt.-initial wt.) × 100] / initial wt.

⁵ Feed efficiency ratio(FE, %) = (wet weight gain / dry feed intake) × 100

⁶ Specific growth rate(SGR, %) = [(log _e finalwt.-log _e initialwt.)×100] / days

⁷ Protein efficiency ratio(PER) = (wet weight gain / protein intake)

Example 3. Effect of improving bowel function by complex treatment of Bacillus strain (probiotics) and MOS (prebiotics)

The present inventors added Bacillus subtilis strain as 0 (BS ₀ ), 0.5×10 ⁷ CFU/g diet (BS _0.5 ) or 1×10 ⁷ CFU/g diet (BS ₁ ), and MOS (mannan oligosaccharide) An experiment was conducted to check whether there is an effect on improving intestinal function by adding a compound with 0 (M ₀ ) or 5 g/kg diet (M ₅ ). The effect on the improvement of intestinal function was confirmed by measuring the vili length and muscular layer thickness, which can represent the development of the intestine.

As a result, in the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which 0.5×10 ⁷ CFU/g or 1.0×10 ⁷ CFU/g of Bacillus subtilis strain and 5 g/kg of MOS were added in combination Compared to the untreated control group (CON), it was confirmed that there is an effect of remarkably improving intestinal function by having a longer fine ridge length and a thicker barrier thickness (Fig. 1 and Table 3).

In addition, the experimental group treated with only Bacillus subtilis strain or only MOS (BS _0.5 M ₀ , BS ₁ M ₀ and BS ₀ M ₅ ) also had the effect of increasing the length and barrier thickness compared to the control (CON), but this is similar to the positive control treated with OTC, and Bacillus subtilis strain and MOS It was a lower level compared to the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) to which the compound was added. That is, in the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which Bacillus subtilis strain and MOS were added in combination, the intestinal function was significantly improved compared to the experimental group treated with only Bacillus subtilis strain or only MOS. It was confirmed that there is (Fig. 1 and Table 3).

Therefore, if the probiotic Bacillus subtilis strain was added to the feed at 0.5×10 ⁷ CFU/g to 1.0×10 ⁷ CFU/g, and the prebiotic MOS was added to the feed at 5 g/kg, the eel It was confirmed that it has the effect of improving intestinal function.

Diets CON BS _0.5 M ₀ BS ₁ M ₀ BS ₀ M ₅ BS _0.5 M ₅ BS ₁ M ₅ OTC Villi length (㎛) 507±61.4 ^c 640±62.0 ^b 712±64.2 ^ab 697±77.9 ^b 825±86.2 ^a 835±45.0 ^a 670±101 ^b Muscular layer thickness (㎛) 151±32.5 ^c 292±9.75 ^a 304±10.8 ^a 222±8.29 ^b 327±42.6 ^a 332±30.3 ^a 205±11.2 ^b

Example 4. Effect of improving disease resistance by complex treatment of Bacillus strain (probiotics) and MOS (prebiotics)

The present inventors added Bacillus subtilis strain as 0 (BS ₀ ), 0.5×10 ⁷ CFU/g diet (BS _0.5 ) or 1×10 ⁷ CFU/g diet (BS ₁ ), and MOS (mannan oligosaccharide) After complex addition with 0 (M ₀ ) or 5 g/kg diet (M ₅ ), an experiment to confirm whether the effect of improving disease resistance was performed by analysis of Lysozyme activity and MPO (Myeloperoxidase) activity in the blood of eel I did.

As a result, in the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which 0.5×10 ⁷ CFU/g or 1.0×10 ⁷ CFU/g of Bacillus subtilis strain and 5 g/kg of MOS were added in combination It was confirmed that Lysozyme activity and MPO activity were significantly increased compared to the untreated control group (CON) (FIG. 2).

In addition, the experimental group treated with only Bacillus subtilis strain or only MOS (BS _0.5 M ₀ , In the case of BS ₁ M ₀ and BS ₀ M ₅ ), Lysozyme activity and MPO activity tended to increase compared to the control (CON), but Bacillus subtilis strain and MOS were added in combination (BS _0.5 M ₅ , BS ₁ M ₅ ) was a lower level. In particular, in the MPO activity, the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which Bacillus subtilis strain and MOS were added in combination was significantly increased compared to the experimental group treated with only Bacillus subtilis strain or MOS alone. Became (Fig. 2).

In addition, the present inventors performed an experiment to analyze the expression level of the immune gene in the intestinal tissue of the eel. As a result, in the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which 0.5×10 ⁷ CFU/g or 1.0×10 ⁷ CFU/g of Bacillus subtilis strain and 5 g/kg of MOS were added in combination It was confirmed that the expression levels of Hsp70 (heat shock protein 70) and IgM _H (immunoglobulin heavy chain) were increased compared to the untreated control (CON) (FIG. 3). In particular, the expression level of IgM _H was increased by about 2.2 times compared to the control (CON), and was increased by about 1.4 to 1.7 times compared to the experimental group treated with only Bacillus strain or only MOS (FIG. 3).

Therefore, if the probiotic Bacillus subtilis strain was added to the feed at 0.5×10 ⁷ CFU/g to 1.0×10 ⁷ CFU/g, and the prebiotic MOS was added to the feed at 5 g/kg, the eel It was confirmed that there is an effect of improving disease resistance.

Example 5. Effect of increasing survival from attack of Vibrio bacteria by complex treatment of Bacillus strain (probiotics) and MOS (prebiotics)

The present inventors added Bacillus subtilis strain as 0 (BS ₀ ), 0.5×10 ⁷ CFU/g diet (BS _0.5 ) or 1×10 ⁷ CFU/g diet (BS ₁ ), and MOS (mannan oligosaccharide) After complex addition to 0 (M ₀ ) or 5 g/kg diet (M ₅ ), an experiment was performed to confirm the survival of the eel from the attack by the Vibrio bacteria.

As a result, in the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which 0.5×10 ⁷ CFU/g or 1.0×10 ⁷ CFU/g of Bacillus subtilis strain and 5 g/kg of MOS were added in combination It was confirmed that the survival rate was increased by about 2.5 times compared to the untreated control group (CON) (FIG. 2).

In addition, the experimental group treated with only Bacillus subtilis strain or only MOS (BS _0.5 M ₀ , In the case of BS ₁ M ₀ and BS ₀ M ₅ ), the survival rate tended to increase compared to the control (CON), but the experimental group in which Bacillus subtilis strain and MOS were added in combination (BS _0.5 M ₅ , BS ₁ M _It was lower than ₅ ). In other words, in the experimental group (BS _0.5 M ₅ , BS ₁ M ₅ ) in which Bacillus subtilis strain and MOS were added in combination, compared to the experimental group treated with Bacillus subtilis strain or only MOS, it survived the attack on Vibrio bacteria. It was confirmed that there is an increasing effect

Therefore, when the probiotic Bacillus subtilis strain is added to the feed at 0.5×10 ⁷ CFU/g to 1.0×10 ⁷ CFU/g, and the prebiotic MOS is added to the feed at 5 g/kg, Vibrio bacteria It was confirmed that there is an effect of increasing the survival of eels against the attack by.

Claims (14)

As a feed additive composition for eel for antibiotic replacement of pathogenic bacteria comprising Bacillus subtilis at a concentration of 0.5 to 1×10 ⁷ CFU/g and MOS (mannan oligosaccharide) at a concentration of 5 g/kg as active ingredients ,
Wherein the pathogenic bacteria is Vibrio Anguilla Solarium (Vibrio anguillarium), Vibrio Kamp Valley (Vibrio campbellii), halbeyi Vibrio (Vibrio harveyi) and Vibrio Furnish when (Vibrio furnisii) would in the composition. delete delete delete delete delete delete As a feed composition for eel for replacing antibiotics of pathogenic bacteria comprising the feed additive composition of claim 1,
Wherein the pathogenic bacteria is Vibrio Anguilla Solarium (Vibrio anguillarium), Vibrio Kamp Valley (Vibrio campbellii), halbeyi Vibrio (Vibrio harveyi) and Vibrio Furnish when (Vibrio furnisii) would in the composition. The method of claim 8,
The feed additive composition, characterized in that contained in an amount of 0.01 to 10 parts by weight based on 100 parts by weight of the total feed composition. delete delete delete A method for increasing the survival of eel from fish pathogenic bacteria comprising the step of feeding the feed additive composition of claim 1 or the feed composition of claim 8 to the eel,
The method of the pathogenic bacteria is Vibrio Anguilla Solarium to the (Vibrio anguillarium), Vibrio Kamp Valley (Vibrio campbellii), halbeyi Vibrio (Vibrio harveyi) and Vibrio Furnish when (Vibrio furnisii).

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