KR101144279B1 - Novel microorganism bacillus subtilis cj1021 and additives for fish feeds containing it - Google Patents

Abstract

The present invention relates to a feed additive for fish containing a novel microbial Bacillus subtilis C # 1021.
The feed additive for fish of the present invention is a novel microbial Bacillus subtilis CJ1021 strain having high germanium accumulation capacity ( Bacillus subtilis CJ1021, Accession No .: KCTC 11547BP) as an active ingredient.
According to the present invention, there is provided a fish feed additive comprising a novel microbial Bacillus subtilis CJ1021 having a high germanium accumulation capacity, by which it is possible to produce fish in which minerals are accumulated when producing fish.

Description

NOVEL MICROORGANISM BACILLUS SUBTILIS CJ1021 AND ADDITIVES FOR FISH FEEDS CONTAINING IT}

The present invention relates to a novel microbial Bacillus subtilis C1022 and a feed additive for fish containing the same.

Feed additives used to improve feed efficiency of fish include antibiotics, vitamins, amino acid feed, mineral feed, enzymes and hormones.

In addition, the feeding of microorganisms such as bacillus has been reported to balance the intestinal microflora, inhibit the growth of pathogenic Escherichia coli, inhibit mutagen and carcinogens, enhance immune responses, and reduce cholesterol levels. .

The medical benefits of Germanium (hereinafter referred to as Ge) were first discovered after the publication of a report in 1930 that the springs of Lourdes, the French-Spanish border, were very effective in treating various diseases. The analysis of the spring waters revealed that the germanium content was very high.

After that, research on organic germanium that can act pharmacologically without remaining in the body has been carried out. Research into the use of organo germanium in the treatment of refractory adult diseases such as cancer, hepatitis, rheumatoid arthritis, skin diseases, and aging is ongoing.

The role of organogermanium in the living body known to date is to promote intracellular oxygen supply (1), to purify blood (2), to promote the release of heavy metals in the body (3), to activate NK cells and macrophage (4), Induction of interferon secretion (5) and regulation of cytotoxic T-Lymphocyte production (Kobayashi 1992).

In the meantime, efforts have been made to mass-produce high concentrations of safe organic germanium in a short time using microorganisms without using plants.

As a result, Nobuhiro et al. Proposed the possibility of producing yeast containing germanium, and Slawson et al. Hung et al. Show that germanium is toxic to microorganisms but can be accumulated by both energy independent passive binding or energy dependent mechanisms in microbial cells. Announced.

The accumulation of organic germanium in the microbial cell was confirmed by electron microscopic analysis that Klapcinska et al. In the Pseudomonas putida cell, the germanium accumulation mainly in the soluble fraction, the majority of which is bound to nucleic acid and protein.

The study using microbial cells was focused on yeast for SCP (single-cell-protein), and the possibility of organo germanium production using this yeast was confirmed by VanDyke et al. When Saccharomyces concentration of GeO _{2 in} culture medium reached 1.0 mg / ml It began by reporting that the growth of cerevisiae is completely inhibited and that microorganisms can adapt to high concentrations of GeO ₂ .

Wei found that yeast can absorb high concentrations of raw germanium, and that more than 95% of the germanium accumulated in yeast cells is organic germanium, and that yeast has the ability to convert inorganic germanium to organic germanium.

However, there are currently no reports on microorganisms having high accumulation capacity of germanium and feed additives for fish containing them.

In the present invention, in order to solve the above problems, there is an object to provide a novel microorganism with a high germanium accumulation capacity.

In addition, there is also an object to produce fish accumulated minerals by providing a feed additive for fish containing the new microorganisms.

The feed additive for fish of the present invention is a novel microbial Bacillus subtilis CJ1021 strain having high germanium accumulation capacity ( Bacillus subtilis CJ1021, Accession No .: KCTC 11547BP) as an active ingredient.

In addition, the novel microbial Bacillus subtilis CJ1021 strain is composed of microorganism cells, concentrates, culture or dried products thereof.

In addition, the novel microbial Bacillus subtilis CJ1021 strain is inoculated in a medium to which organic germanium is added, and then cultured.

According to the present invention, there is provided a novel microbial Bacillus subtilis CJ1021 having a high germanium accumulation capacity and a feed additive for fish containing the same, and when producing the fish, it is possible to produce fish in which minerals are accumulated.

1 is a schematic diagram illustrating the production process of germanium which is a functional mineral.
Figure 2 is a graph showing the change in water temperature (Salinity) in the breeding water tank (Water temperature)
3 is dissolved oxygen (DO) and pH change in breeding tank
Figure 4 is a graph showing the changes in goblet cells in the digestive tract when supplied to the flounder functional minerals.

When described in more detail with respect to the present invention.

In the present invention, it was carried out an experiment that after using a bacillus which hunyang (adaption) the bacillus medium in order to produce an organic germanium by optimizing the fermentation conditions producing bacillus containing an organic germanium in high concentration.

In addition, the produced organic germanium (Ge- bacillus ) was added to fish feed additives to investigate whether it was accumulated on the flounder and its effects on the growth and other fish. The experiment was conducted.

Here, the accumulation of metal by microorganisms is achieved by the following three mechanisms.

1) Metabolism-independent binding or adsorption

It occurs rapidly in extracellular polysaccharides, capsules, and mucus layers.

Bacterial cell walls, envelops, algae, yeast, and fungal cell walls act as metal adsorbents that bind to charged groups caused by increased metal levels.

This process is affected by environmental factors such as pH and ion competition.

2) Metabolism-dependent intracellular uptake

Metabolism-dependent intracellular uptake is inhibited by low temperatures, metabolic inhibitors, and depletion of energy sources.

The rate of metal uptake is influenced by the physiological state of the cell, the nature and the composition of environmental factors in the growth medium.

3) Cotransport of metals with ion-binding siderphores and organic substances

In the present invention with reference to the above three metal accumulation system, the bacillus strains were first isolated from soil, Cheonggukjang, Nuruk, and the like, and metals to accumulate were added to the culture solution, followed by culturing.

Through this process, strains that accumulate well germanium were selected.

Afterwards, the optimization of the carbon and nitrogen sources was carried out using RSM (response surface method) to optimize the medium, and further optimization of pH, incubation temperature and trace elements.

Then, in the actual mass culture, glucose was added to prevent energy depletion, and catechol was added to the medium to investigate the effect on Ge accumulation.

Hereinafter, the present invention will be described in detail with reference to Examples, but these are not intended to limit the scope of the present invention.

Example 1 Strain Screening

1. Experimental Method

The bacillus strain was isolated from soybean, soil, yeast for strains.

Strains were isolated from commercially purchased Cheonggukjang.

18 ml of distilled water was added to 2 g of Cheonggukjang, pulverized with a mixer, and diluted with serial dilution in the medium.

The bacillus was isolated after incubation at 35 ° C. in LB agar (beef extract 3.0 g, peptone 5.0 g, agar 15.0 g, distilled water 1.0 L, pH 6.8).

Nuruk was purchased from commercially available Nuruk, and 2 g of Nuruk was added to 18 ml of distilled water, pulverized with a mixer, and diluted with serial dilution.

The bacillus was isolated after incubation at 35 ° C. in LB agar (beef extract 3.0 g, peptone 5.0 g, agar 15.0 g, distilled water 1.0 L, pH 6.8).

In order to separate bacteria from soil and plants with high Ge content, bacillus was isolated after collecting soil from garlic field in Jeju Island and grass near company in Jeju Island.

Soil samples were heat-treated at 80 ° C. for 30 minutes and then purely separated under aerobic conditions.

Pure isolates were Gram stained to identify rod shaped bacteria that were G +, and then 16Sr rna analysis was commissioned to Solgent for strain analysis.

2. Experimental results

The strain was isolated after pure separation according to the above experimental method, and the following results were obtained (Table 1-Strains and strain names used in the experiment).

number Strain Strain name One. HS-01 Bacillus licheniformis 2 HS-03 Bacillus pumirus 3 HS-09 Bacillus sp . 4 HS-10 Bacillus megaterium 5 CJ-1012 Bacillus cereus 6 CJ-1014 Bacillus cereus 7 CJ-1015 Bacillus coagulans 8 CJ-1021 Bacillus subtilis 9 CJ-1022 Bacillus subtilis 10 CJ-1028 Bacillus subtilis 11 CJ-1021 Bacillus subtilis 12 CJ-1666 Bacillus subtilis 13 CJ-3015 Bacillus subtilis 14 CJ-3135 Bacillus subtilis 15 CJ-3625 Bacillus coagulans 16 CJ-13241 Bacillus subtilis 17 CJ-102 Bacillus subtilis 18 CJ-102 (2) Bacillus licheniformis 19 CJ-103 Bacillus thuringiensis 20 CJ-109 Bacillus cereus 21 CJ-109 (2) Bacillus megaterium 22 CJ-110 Bacillus thuringiensis 23 CJ-118 Bacillus sphaericus 24 CJ-118 (2) Bacillus sphaericus 25 CJ-119 Bacillus sphaericus 26 CJ-136 Bacillus megaterium 27 CJ-150 Bacillus thuringiensis 28 CJ-165 Methylbacillus glycogenes 29 CJ-166 Bacillus amyloliquefaciens 30 CJ-176 Bacillus circulans 31 CJ-210 Bacillus amyloliquefaciens 32 CJ-304 Bacillus licheniformis 33 BNN-21 Bacillus subtilis 34 SH-20 Bacillus amyloliquefaciens 35 HS-25 Bacillus subtilis 36 HS-30 Bacillus licheniformis 37 HS-23 Bacillus subtilis

A total of 37 kinds of Bacillus were obtained as shown in Table 1, and the strains were tested for germanium accumulation.

Example 2 Germanium Accumulation Experiment

1) Check Ge solubilization

In order for the microorganisms to accumulate metal in the body, an experiment was performed to solubilize germanium because minerals must be dissolved in water (FIG. 1).

Germanium-containing compounds, which are well soluble in water, are Germanium dioxide and were investigated using these compounds.

As a method of solubilizing Germanium dioxide, a method known in the existing patent and a method of directly dissolving in water were performed.

cf-1) The method of the existing patent is to dissolve Germanium dioxide in 50 ml of 5N NaOH, neutralize it, and then to accumulate it in the microorganism. While adding as a result it was able to melt up to 7g.

However, neutralization with 5N HCl precipitated again.

cf-2) As a result of experiment by melting directly after distilled water, it was able to melt up to 0.2 g as a result of adding by stirring with magnetic stirrer at 90 ℃.

4 g could be dissolved based on 1 L.

2) Selection of Adaptive Strains

For the selection of bacillus strain having the ability to convert to high concentration organic germanium, the cultivation was carried out as follows.

Germanium was inoculated into a liquid medium containing 500 mg / L of Germanium dioxide and then incubated at 1,000 mg / L with increasing concentration after 24 hours.

At concentrations after 1,000 mg / L, the strains growing rapidly decreased, and the adaptive strains were selected at 1,000 mg / L.

3) Confirmation of industrial medium optimization

① Carbon source selection

In general microorganisms, carbon sources are used as cell components or energy sources.

In general, the most microbial sugars are glucose, fructose, mannose and galactose, such as hexose or sucrose, maltose and starch. I use it.

When two or more kinds of sugars are present in the medium, they are used first.

Therefore, in this experiment, the carbon source was a carbon source selection experiment targeting 2% (wt) of high fructose, sucrose, and glucose.

② Selection of nitrogen source

The nitrogen source is a major medium component necessary for the synthesis of proteins, nucleic acids, and the like of microorganisms.

Thus, in this experiment, soy peptone and yeast extract, which are mainly used in industrial sites, were used.

③ Concentration Optimization of Industrial Medium

In order to observe the interaction between these variables using the concentrations of high fructose and CSL selected in the previous experiments, and to maximize bacillus germanium accumulation, industrial medium was prepared using Response Surface Methodology (RSM). The concentration of was optimized.

The design of the experiment was carried out under 12 conditions including 4 starpoint and 4 center point replicates according to the 23-fractional factorial design as shown in Table 2 below.

The carbon and nitrogen sources, which are the main components of the medium optimization, were set as independent variables X1 and X2, respectively, and the level of each independent variable was performed in the range of -1.414 to +1.414.

In addition, after 48 hours of incubation, each mineral accumulation was performed as a dependent variable (Y) according to the experimental plan of Table 2 below.

This design has the advantage of being able to experiment at five levels for each independent variable with a small number of experiments, and the results can be used to fit a second order polynomial regression model (Table 2).

variables coded value -1.414 -One 0 +1 +1.414 Carbonsource concentration (X1) 1.17 2 4 6 6.83 Nitrogen source concentration (X2) 0.6 One 2 3 3.4

④ Culture temperature, pH optimization

Changes in temperature and pH are important factors in the growth of microorganisms.

In general, microorganisms can survive under a wide range of temperature conditions, but each microorganism has a different optimal growth temperature, and the maximum growth occurs at that temperature.

At temperatures above the optimum growth temperature, microorganisms cause inactivation of metabolic enzymes and growth is inhibited.

In the case of pH, it is generally known that the growth is best at neutral or weak basic pH (pH 6.8-7.4), but the pH should be well managed to maintain the optimum point because it has various optimal pH depending on the characteristics of the bacteria.

⑤ Optimization of trace elements

Minerals are required as essential nutrients, K is enzymatic activity, Ca is involved in various functions, resistant spore heat resistance, Fe acts as a constituent of cytochrome, the former is classified as macronutrient, and micronutrients are Mn, Zn, Co, Mo, Ni , Cu, etc., and are mainly necessary for maintaining a catalyst or protein structure of a bioreaction.

4) Experiment Method

bacillus was accumulated in LB broth (beef extract 3.0 g, peptone 5.0 g, distilled water 1.0 L, pH 6.8) in germanium at 37 ℃, 150 rpm, 48 hours in a medium containing 1,000 mg / L of Germanium dioxide Was carried out.

In the case of LB broth, the amount of the obtained cells was small, which made it difficult to analyze the following. Therefore, the medium was changed to BHI (brain heart infusion).

Using BHI (Brain heart infusion 37.0 g, Agar 15.0 g, Distilled water 1.0 L) medium, the primary culture was incubated by adding 10 ml of medium to a 20 ml volume of test tube, and the secondary culture was a 500 ml flask. 350 ml was incubated with working volume.

The culture was incubated for 48 hours at 37 ℃, 150 rpm.

After 1,000 ml of the culture was obtained, the resultant was centrifuged at 7,000 g for 15 minutes at 4 ° C. using a Hanil SUPRA22K centrifuge.

The obtained cells were centrifuged, washed twice with brine, twice with distilled water, and dried at 60 ° C. for 48 hours.

After drying for 48 hours at 60 ℃ washed three times with primary distilled water and dried again.

After the dry weight measurement, the request was made to the Sampyo Food Safety Institute to confirm the accumulation of Ge.

The detection limit was 0.6 ppm and the minimum dilution factor was 60 times.

5) Experiment result

The results of the experiment are shown in Table 3 (growth of Bacillus strains, germanium accumulation and dry cell weight).

Strain number Strain name Growth Dry cell weight (g) Ge content (mg / kg) HS-01 Bacillus licheniformis ○ 0.145 6,131.80 HS-03 Bacillus pumirus ○ 0.195 6,844.92 HS-09 Bacillus sp. ○ 0.169 5,060.73 HS-10 Bacillus megaterium ○ 0.445 3,466.11 CJ-1012 Bacillus cereus × CJ-1014 Bacillus cereus × CJ-1015 Bacillus coagulans ○ 0.255 5,197.32 CJ-1021 Bacillus subtilis ○ 0.371 11,536.09 CJ-1022 Bacillus subtilis × CJ-1028 Bacillus subtilis ○ 0.388 7,443.18 CJ-1661 Bacillus subtilis × CJ-1666 Bacillus subtilis ○ 0.205 3,353.44 CJ-3015 Bacillus subtilis × CJ-3135 Bacillus subtilis ○ 0.234 9,577.68 CJ-3625 Bacillus coagulans × CJ-13241 Bacillus subtilis ○ 0.171 3,872.46 CJ-102 Bacillus subtilis ○ 0.127 CJ-102 (2) Bacillus licheniformis ○ 0.342 CJ-103 Bacillus thuringiensis ○ 0.336 CJ-109 Bacillus cereus ○ 0.342 CJ-109 (2) Bacillus megaterium ○ 0.600 CJ-110 Bacillus thuringiensis ○ 0.313 CJ-118 Bacillus sphaericus ○ 0.345 CJ-118 (2) Bacillus sphaericus ○ 0.325 CJ-119 Bacillus sphaericus ○ 0.204 CJ-136 Bacillus megaterium ○ 0.473 6,417.67 CJ-150 Bacillus thuringiensis ○ 0.157 CJ-165 Methybacillus glycogenes × CJ-166 Bacillus amyloliquefaciens ○ 0.488 CJ-176 Bacillus circulans × CJ-210 Bacillus amyloliquefaciens ○ 0.16 CJ-304 Bacillus licheniformis ○ 0.14 CJ-136 Bacillus megaterium ○ 0.438 BNN-21 Bacillus subtilis ○ SH-20 Bacillus amyloliquefaciens ○ HS-25 Bacillus subtilis ○ HS-30 Bacillus licheniformis ○ HS-23 Bacillus subtilis ○

As shown in Table 3, CJ-1021 ( Bacillus subtilis ) The dry cell mass was 0.371 g / L and the germanium accumulation was 11,536.09 ppm, which was confirmed to be superior to the previously reported germanium accumulation strain.

Thus, strain CJ-1021, the optimal strain, was deposited with the patent strain in KCTC (Korean Collection for Type Culture) of Korea Research Institute of Bioscience and Biotechnology (Accession Number: KCTC 11547BP).

Example 3 Efficacy Test as a Feed Additive of Fishes of Functional Minerals

1. Experimental Method

(1) Experimental fish and breeding environment

The halibut fry used in the present invention was purchased for mid-term fry at the seedling culture center in Jeju-do and used for experiments after pure breeding for one week in the indoor breeding dong of the Institute of Marine Science and Environment, Cheju National University.

At the beginning of the experiment, the average fish length was 19.4 ± 0.1 cm and body weight was 118.0 ± 0.6 g.

The breeding tank used 16 FRP round tanks (150 cm × 90 cm in diameter) with a conical central drainage system, and each flounder was equipped with 40 flounder fry.

Breeding yield was returned to 1 ton 15-18 times a day, and aeration for sufficient oxygen supply.

The water temperature, dissolved oxygen (DO), pH, and specific gravity were measured every day during the experiment, DO was DO meter (YSI-85), pH was pH meter (Mettlero toledo), and salinity was salinity meter (YSI-85). Was used.

(2) functional mineral feed and growth

Germanium (Ge) is used as a functional mineral, and the organic or inorganic state of minerals (minerals are accumulated in microorganisms. The effects on growth and health were investigated.

In order to investigate the effects of functional mineral types and concentrations, the germanium experimental groups were divided into three groups: Ge bacillus 0.1 mg / kg diet, Ge bacillus 1.0 mg / kg diet and Germanium dioxide 1.0 mg / kg diet.

And the control was supplied only feed without adding minerals.

The method of adding functional minerals was supplied twice a day after immersion in adsorbed feed for each concentration.

The compound feed used in the experiment was an extruded pellet (EP).

(3) growth

Body weight and body weight of experimental fish were measured every three weeks, and the afternoon and the day before the measurement were fasted.

Body weight was measured up to 1.0 g by electronic balance, and the body length was measured up to 1 by a self-made measuring plate.

After the measurement of the fish, the experimental fish were bathed in 100 ppm of HCl-Oxytetracline for 1 hour.

Individuals who died during breeding were removed from time to time, and the survival rate was expressed as a percentage of the surviving individuals every 3 weeks after fish body measurement.

Total weight gain, daily growth rate, daily feeding rate and feed coefficient were calculated to compare the values of each experiment.

Total weight gain, daily growth rate, daily feed rate and feed coefficient were calculated by the following formula.

total weight gain (TWG) = IW-FW

daily growth rate (DGR) = {(ln FW-ln IW) / t} × 100

daily feeding rate (DFR) = (TF × 100) / {(IW + FW) × day fed / 2}

feed coefficient (FC) = TF / WG

BW: body weight FW: final weight

lW: initial weight TF: total feed

TL: total length t: breeding time

WG: weight gain

(4) digestive system

For the observation of the digestive organs, three specimens of each experimental group were sampled and fixed in Bouin's solution at the beginning of each experiment and measured.

After comparative staining of Harry's hematoxylin and 0.5% eosin, the specimens were observed under an optical microscope.

Histological observations of the digestive tract examined the distribution and number of goblet cells in the intestine's mid intestine.

(5) blood analysis

The blood analysis to check the health of the fish, etc. was randomly selected by six animals in each experiment at each measurement.

Experimental fish were anesthetized with 2-phenoxyethanol solution, and blood was collected using a disposable syringe in the tail artery.

After blood collection, centrifugation (5,000 rpm, 15 minutes) was performed to determine the activity of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), protein, glucose, phosphorus, and cholesterol concentrations in blood plasma (Express plus system, Bayer). , USA).

For statistical analysis of all data, ANOVA-test was performed using the SPSS (Version 12.0) program and Duncan's multiple range test was used to test the significance between the means.

2. Experimental results

(1) Breeding environment

During the experimental period, weekly average water temperature, salinity, DO, pH change among environmental factors in the experimental group were investigated.

During the experiment, the breeding water temperature ranged from 20.0 ℃ to 25.8 ℃ and the average water temperature was 22.5 ℃.

Salinity concentration was about 30.2 ~ 33.3 ppt (Fig. 2).

During the experimental period, the DO ranged from 5.6 to 6.9 mg / L and the pH ranged from 8.2 to 8.5 (Fig. 3).

(2) growth and survival rate changes

① First survey – The results of the first survey are shown in Table 4 below (when fed the functional minerals to the flounder ( Paralichthys olivaceus ) at the first measurement, body length, body weight, total weight change and survival rate).

Experimental group Initial Final Survival rate (%) Number
of fish TL
(cm) BW
(g) Number of fish TL
(cm) BW
(g) W.gain
(g) Control 80 19.4 ± 0.1 118.4 ± 1.7 80 19.3 ± 0.1 142.4 ± 2.0 1919 100.0 Ge- Bacillus 0.1 80 19.4 ± 0.1 120.2 ± 1.7 80 19.7 ± 0.1 152.6 ± 2.2 2591 100.0 Ge- Bacillus 1.0 80 19.4 ± 0.1 117.9 ± 1.7 79 19.8 ± 0.1 153.6 ± 2.0 2706 98.75 Germanium dioxide 1.0 80 19.4 ± 0.1 117.1 ± 1.7 80 19.5 ± 0.1 147.4 ± 2.0 2427 100.0

As shown in Table 4, the body length at the start of the experiment was 19.4 ± 0.1 cm, 19.3 ± 0.1 cm in the control at the first measurement.

Ge- bacillus 0.1 mg / kg diet showed 19.7 ± 0.1 cm and Ge- bacillus 1.0 mg / kg diet showed 19.8 ± 0.1 cm and Germanium 1.0 mg / kg diet showed 19.5 ± 0.1 cm. .

In other words, there was no significant growth difference between the control and the functional mineral supplementation in the first measurement (P> 0.05).

The body weight was 118.0 ± 0.1 g at the start of the experiment, and it grew to 142.4 ± 2.0 g in the control group at the first measurement.

Germanium was grown to 152.6 ± 2.2 g of Ge- bacillus 0.1 mg / kg diet, 153.6 ± 2.0 g of Ge- bacillus 1.0 mg / kg diet, and 147.4 ± 2.0 g of Germanium dioxide 1.0 mg / kg diet. .

In the control and functional mineral supplementation groups, there was a significant growth difference between the Ge- bacillus 0.1 mg / kg diet and Ge- bacillus 1.0 mg / kg diet groups (P <0.05).

Survival rate was 100% in the control group at the first measurement, and the survival rate of the functional mineral-added test group was 97.5-100%, similar to the control.

At total weight gain, the other groups showed higher gain than the control.

② Secondary Survey-The results of the Secondary Survey are shown in Table 5 below (the body length, body weight, total weight change and survival rate when the flounder was supplied with functional minerals during the second measurement).

Experimental group Initial Final Survival rate (%) Number
of fish TL
(cm) BW
(g) Number of fish TL
(cm) BW
(g) W.gain
(g) Control 74 19.3 ± 0.1 142.4 ± 2.0 72 22.2 ± 0.1 185.7 ± 2.9 2878.6 97.3 Ge- Bacillus 0.1 74 19.7 ± 0.1 152.6 ± 2.2 73 22.8 ± 0.1 196.9 ± 3.1 3107.6 98.6 Ge- Bacillus 1.0 73 19.8 ± 0.1 153.6 ± 2.0 69 23.2 ± 0.1 204.9 ± 3.1 3028.3 94.5 Germanium dioxide 1.0 74 19.5 ± 0.1 147.4 ± 2.0 74 22.6 ± 0.1 191.7 ± 3.3 3274.6 100.0

As shown in Table 5, the body length of the control at the second measurement was 22.2 ± 0.1.

In the germanium experiment, Ge- bacillus 0.1 mg / kg diet showed 22.8 ± 0.1 cm, Ge- bacillus 1.0 mg / kg diet showed 23.2 ± 0.1 cm and Germanium dioxide 1.0 mg / kg diet showed 22.6 ± 0.1 cm. .

There was a significant difference in growth between the control and Ge- bacillus 1.0 mg / kg diet and Germanium 1.0 mg / kg diet groups (P <0.05).

The control body weight was 185.7 ± 2.9 g at the second measurement.

Germanium was grown to 196.9 ± 3.1 g of Ge- bacillus 0.1 mg / kg diet, 204.9 ± 3.1 g of Ge- bacillus 1.0 mg / kg diet, and 191.7 ± 3.3 g of Germanium dioxide 1.0 mg / kg diet. .

Ge- bacillus in control and functional mineral supplementation There was a significant difference in growth between the 0.1 mg / kg diet and Ge- bacillus 1.0 mg / kg diet groups (P <0.05).

Survival rate was 97.3% in the control group and the survival rate of the functional mineral-added test group was 94.5 ~ 100%, similar to the control.

Total weight gain increased by 2.9 kg in the control at the second measurement, and was similar or higher than that in the control.

(3) feed coefficient, daily growth rate and daily feed rate

① 1st investigation-The 1st investigation in Table 6 (Feed coefficient, Daily growth rate, Daily feeding rate) when the functional minerals were supplied to the flounder during the 1st measurement. The results are shown.

Experimental group Feed coefficient Daily growth rate (%) Daily feeding rate (%) Control 0.94 1.15 1.08 Ge- bacillus 0.1 1.03 1.48 1.53 Ge- bacillus 1.0 1.02 1.58 1.60 Germanium dioxide 1.0 1.03 1.43 1.47

As shown in Table 6, the feed coefficient was 0.94 in the control, 0.94 ~ 1.03 similar to the control in the other control was similar to the control.

The daily growth rate was 1.15% in the control and 1.16 ~ 1.58% in the other experiments, which was higher than the control.

The daily feeding rate of Ge- bacillus 0.1 mg / kg diet and Ge- bacillus The 1.0 mg / kg diet showed 1.53% and 1.60%, respectively, higher than the control.

The rest of the experimental groups showed 1.12 ~ 1.47% higher than the control.

② Secondary survey-Secondary survey in Table 7 (Feed coefficient, Daily growth rate, Daily feeding rate) when the functional minerals were supplied to the flounder during the second measurement. The results are shown.

Experimental group Feed coefficient Daily growth rate (%) Daily feeding rate (%) Control 1.23 1.45 1.78 Ge- bacillus 0.1 1.21 1.46 1.77 Ge- bacillus 1.0 1.34 1.44 1.92 Germanium dioxide 1.0 1.15 1.58 1.81

As shown in Table 7, the feed coefficient was 1.23 in the control, and the feed efficiency was 1.15 in the Germanium dioxide 1.0 mg / kg diet.

In other experiments, the values of 1.21 ~ 1.35 were similar to those of the control.

The daily growth rate was 1.45% in the control and 1.39 ~ 1.58% in the other experiments, showing a similar trend with the control.

The daily feeding rate was 1.78% in the control and 1.92% in the Ge bacillus 0.1 mg / kg diet, which was higher than the control.

The rest of the experimental groups were 1.77 ~ 1.89% similar to the control.

(4) changes in the digestive system

Histological observations of the digestive tract were examined for the distribution of goblet cells in the mucosal folds of the digestive tract, centering on the mid intestine of the intestine.

As a result, it was shown in Figure 4, the number of goblet cells of the midgut mucosa was 196.7 ± 58.7 at the start, 212.3 ± 81.3 at the first measurement.

The number of goblet cells in each experiment ranged from 128.7 ± 23.5 to 222.7 ± 35.2, and there was no significant difference between the control and experimental groups.

(5) blood analysis

As a result of the experiment, Table 8 (plasma AST, ALT, Protein, Glucose, Phosphorus, Cholesterol changes when functional minerals were supplied to flounder) was shown.

Experimental group AST
(U / L) ALT
(U / L) Protein
(g / dL) Glucose
(mg / dL) Phosphorus
(mg / dL) Cholesterol
(mg / dL) Control 25.5 ± 2.3 17.2 ± 6.1 3.9 ± 0.5 71.2 ± 13.3 26.1 ± 2.3 193.3 ± 31.9 Ge- bacillus 0.1 43.1 ± 12.9 42.0 ± 22.4 4.7 ± 0.2 81.1 ± 10.9 20.9 ± 1.2 248.4 ± 28.6 Ge- bacillus 1.0 27.5
± 8.1 78.4 ± 19.6 5.0 ± 0.1 89.6 ± 18.7 25.4 ± 1.1 219.5 ± 31.2 Germanium dioxide 1.0 20.3
± 1.6 57.5 ± 17.2 4.3 ± 0.3 86.1 ± 23.9 23.5 ± 1.2 212.4 ± 37.2

As shown in Table 8, the AST value was 25.5 ± 2.3 U / L in the control group at the first measurement, and 20.3 ± 1.6 ~ 57.9 ± 7.7 U / L in the experimental group containing functional minerals. There was no difference (P> 0.05).

The ALT value was 17.2 ± 6.2 U / L in the control group and 34.3 ± 16.7 ~ 98.4 ± 53.7 U / L in the functional mineral supplementation group (P> 0.05).

Protein (Protein) value was 3.88 ± 0.47 g / dL in the control group, 3.5 ± 0.3 ~ 5.0 ± 0.1 g / dL in the functional mineral added experimental group was not significantly different between the control and the experimental section (P> 0.05).

Glucose value was 71.2 ± 13.3 mg / dL in the control group and 43.1 ± 3.8 to 111.1 ± 12.3 mg / dL in the experimental group with functional minerals (P> 0.05).

Phosphorus value was 26.1 ± 2.3 mg / dL in the control and 19.2 ± 1.5 ~ 28.2 ± 1.8 mg / dL in the experimental group with functional minerals, and there was no significant difference between the control and the experimental group (P> 0.05).

Cholesterol value was 193.4 ± 31.9 mg / dL in the control group and 212.4 ± 37.2 ~ 330.0 ± 44.3 mg / dL in the experimental group containing functional minerals (P> 0.05).

(6) Germanium Stock Result

The concentration of germanium in the feed and flounder conducted in the experiment was requested to the Korea Basic Science Center to check the concentration of germanium in the fish body.

The experimental results are shown in Table 9 below.

Experimental group Feed (ppm) Body (ppm) Control 0.017 0.061 Ge- bacillus 0.1 0.092 0.09 Ge- bacillus 1.0 0.78 0.27 Germanium dioxide 1.0 0.83 0.18

As shown in Table 9, it was confirmed that Ge- bacillus accumulated better than the mineral itself, and also germanium accumulated in the muscle tissue of the body.

As described above, in the present invention, a higher accumulation amount (<10,000 mg / kg) can be reached in the yeast compared with the conventional method of accumulating germanium, and the effect is equivalent in terms of effects, and it is positive in the field test with the replacement of mineral yeast products. Elicited a tendency to be.

Korea Research Institute of Bioscience and Biotechnology KCTC11547BP 20090814

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Claims (6)

Bacillus subtilis CJ1021, Accession No .: KCTC 11547BP, New Microorganism Bacillus subtilis CJ1021 The novel microbial Bacillus subtilis CJ1021 strain ( Bacillus subtilis CJ1021, Accession No .: KCTC 11547BP) having a germanium accumulation ability according to claim 1, comprising a feed additive for fish. The method of claim 2,
The novel microbial Bacillus subtilis CJ1021 strain is characterized in that consisting of the microbial cells, concentrates, culture or dried products thereof,
Feed additives for fish. The method of claim 2,
The novel microbial Bacillus subtilis CJ1021 strain is inoculated in a medium to which the organic germanium is added, characterized in that the culture,
Feed additives for fish. The method of claim 2,
The germanium is characterized in that the accumulation in the muscle tissue of fish,
Feed additives for fish. The fish feed additive of claim 2 is characterized in that it contains 0.1 to 1.0% based on the total weight of the feed,
Fish feed.

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