KR20090066410A - Composition for forage additive comprising bacillus subtilis bc1212 - Google Patents

Abstract

A composition for feed additives comprising Bacillus subtilis BC1212 is provided to produce antibiotic with antibacterial activity in order to improve feed efficiency and weight increase. A composition for feed additives contains Bacillus subtilis BC1212(KACC 91141). The strain produces surfactin C. The composition for feed additives comprises 2.8wt% of wheat bran, 4.9wt% of rice bran, 5.1wt% of soybean cake, 0.02wt% of FeSO4, 0.5wt% of MnSO4 and 87.13wt% of water. The composition for feed additives additionally contains one or more enzyme products. The enzyme products are selected from the group consisting of lipase, phytase, amylase, phosphatase, carboxymethylcellulas, xylanase, maltase and converting enzyme. The composition for feed additives additionally contains non-pathogenic microorganisms. The non-pathogenic microorganisms are selected from the group consisting of microorganisms belonging to Lactobacillus, Aspergillus oryzae and Saccharomyces cerevisiae.

Description

Composition for feed containing a Bacillus subtilis BCC1212 strain TECHNICAL FIELD [Composition for Forage Additive Comprising Bacillus subtilis BC1212}

The present invention relates to a feed additive composition, and more particularly to a feed additive composition comprising a microorganism of Bacillus subtilis species that produce antibiotics.

Livestock and the products they produce are an important source of protein for humankind's survival, and the development of livestock raising and utilization is closely linked to improving human health and quality of life. Grain is currently used in many parts of the feed for raising livestock, and the recent rapid increase in the population has increased the demand for grain for food, making it difficult to use grain as a feed source. It is limiting the development of livestock raising. Therefore, the feed industry has made a lot of efforts to maximize the efficiency of the feed and the feed used to replace the grain, the development of feed additive composition to improve the digestive function and absorption rate of animals in the feed industry It has taken an important position. The development of feed additives will not only reduce feed consumption by maximizing feed efficiency by promoting livestock fattening and digestion, but also contribute to improving farm household income levels by enabling high quality livestock production due to rapid growth and fattening. It can be said that the importance is very large in that it can be.

Traditionally, animal feed for pigs and poultry has been used mainly for soybean meal, sesame seeds, wheat flour, rice straw and corn, and recently, alternative products such as peanuts, peas, sugar beets, pulp, grain by-products, animal intestine flour and fishmeal flour. The use of is greatly increased. Animal feed currently used in livestock farms is added with probiotics (living microbial agents) along with amino acids, inorganic dyes, vitamins, antibiotics, enzymes, etc., which help to improve nutrition, improve digestion and absorption, promote growth, and prevent disease. To indicate. However, it is not easy to maintain the health of breeding animals due to diseases of the digestive system or the use of antibiotics for a long time. In addition, long-term use of animal drugs and antibiotics in livestock is limited in their use because of the ingestion of residual drugs by humans causing changes in allergic or normal intestinal flora, and their range of use is also limited and reduced.

In order to improve these problems, microorganisms such as Bacillus natto, lactic acid bacteria, butyric acid bacteria, Lactobacillus bifidus, etc. have been studied and used as probiotics for the prevention and treatment of diseases occurring in farm animals. There are many things that are lacking. Therefore, the development of new probiotics that are not only safer for animals but also show excellent viability in the intestines of animals are required.

Accordingly, the present inventors have continued extensive research on microorganisms that can be used as an animal feed additive, and as a result, the Bacillus subtilis BC1212 strain, which has been isolated by the present inventors as a microbial strain belonging to the Bacillus subtilis species, has been conventionally bred. Unlike microorganisms that are used as microbial additives in animals, it not only produces antifungal and anti-living substances, but also has various enzyme-generating ability, which can be useful as a probiotic for feed addition by increasing the suitability and digestion promoting action of a breeding animal. The present invention was completed by revealing.

An object of the present invention is to provide a composition for feed addition comprising Bacillus subtilis BC1212 strain.

In order to achieve the above object, the present invention provides a composition for feed addition comprising Bacillus subtilis BC1212 strain.

The Bacillus subtilis BC1212 strain belongs to the facultative anaerobic Bacteria family of the genus Bacillus, and produces sulfactin C represented by the antibiotic formula (1).

 The Bacillus subtilis BC1212 strain has been deposited with the Institute of Agricultural Biotechnology on November 8, 2004 (Accession No .: KACC 91141).

Bacillus subtilis BC1212 strain used in the preparation of the feed additive composition of the present invention can be cultured in a liquid medium or a solid medium by a known fermentation technique. As the medium, various mediums such as natural medium, semi-synthetic medium and synthetic medium can be used without limitation. (Iii) glucose, sucrose, dextrin, glycerol, starch, molasses and the like may be used as the carbon source; (Ii) nitrogen sources may include peptone, meat extract, yeast extract, dried yeast, soy cake, urea, thiourea, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds; (Iii) As inorganic salts, phosphates such as magnesium, manganese, potassium, calcium and iron, nitrates, carbonates, chlorides and the like can be used. However, the carbon source, nitrogen source and inorganic salts are not necessarily limited to the above components, in addition, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.

The microorganism is incubated for a suitable period of time at a suitable temperature in accordance with known fermentation techniques, preferably for 12 hours to 5 days at 20 ℃ to 50 ℃, for example. The culture obtained according to the present invention can be used together with the cells isolated or by the culture alone by repeated washing. In addition, the culture medium or separated cells containing the cells can be formulated by lyophilization or spray drying with or without the addition of additives. The formulation thus obtained can be used as a feed additive or feed of the present invention.

In order to prepare a composition for feed addition according to the present invention, the culture medium is formulated directly, or diluents such as wheat flour, starch, textrin, and the like, such as cereals, rice bran and skim rice bran, feed material such as bran, oil and fat-rich seed cake and Can be formulated together. In addition, after washing the culture solution to separate the cells, the isolated cells can be formulated by the method described above. The viable microbial preparation of the obtained Bacillus subtilis BC1212 strain is administered to an appropriate amount of animal in the form of a feed containing it or in the form of a feed additive to gain weight and promote growth. According to a preferred embodiment of the present invention, the composition for feed addition of the present invention is 1 to 20% by weight of bran, 1 to 20% by weight of rice bran, 1 to 20% by weight of soybean meal, FeSO ₄ 0.001 to 0.1% by weight, MnSO ₄ 0.001 to 0.1 wt% and balance water, preferably 2.8 wt% bran, 4.9 wt% rice bran, 5.1 wt% soybean meal, 0.02 wt% FeSO ₄ , 0.05 wt% MnSO ₄ , and balance (87.13 wt%) water. Include.

In addition, the feed composition of the present invention may be in the form of a dry or liquid formulation, it may further comprise one or more enzyme preparation. Enzyme preparations can be added in either dry or liquid form, lipolytic enzymes such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, starches and glycogen Amylase, an enzyme that hydrolyzes an α-1,4-glycoside bond, and phosphatase, cellulose, an enzyme that hydrolyzes an organophosphate ester carboxymethylcellulase to break down cellulose, xylanase to break down xylose, maltase to hydrolyze maltose into two molecules of glucose, and It may be selected and used from the group consisting of sugar producing enzymes such as invertase which hydrolyzes saccharose to form a glucose-fructose mixture.

In addition, the compositions of the present invention may further comprise other non-pathogenic microorganisms in addition to the Bacillus subtilis BC1212 strain. The microorganisms that can be added include the microorganisms of Lactobacillus sp., Which have physiological activity and organic degradability under anaerobic conditions such as cow's stomach, increase the body weight of livestock, increase milk yield, and increase digestive absorption rate of feed. Filaments such as Aspergillus oryzae (Slyter, LLJ Animal Sci. 1976, 43. 910-926) and yeasts such as Saccharomyces cerevisiae (Jhonson, DE et al) J. Anim. Sci., 1983, 56, 735-739; Williams, PEV et al, 1990, 211), and the like.

Raw materials such as peanuts, peas, sugar beets, pulp, grain by-products, animal viscera flour and fishmeal flour, including various grains and soy protein, may be appropriately used. The processing process is a process in which the feed material is compressed to a constant outlet under pressure in a state in which the feed material is filled, and in the case of protein, it is preferable to use an extrusion process in which the availability is increased. Extrusion has the advantages of denaturing proteins through heat treatment, destroying antienzyme factors, and increasing the activity of protease by enzymatic exposure to new sites by molecular structural changes. In addition, in the case of soy protein, the extrusion process improves the digestibility of the protein and inactivates antinutrients such as trypsin inhibitor, one of the inhibitors of protease present in soybean. Increasing digestion can increase the nutritional value of soy protein.

Representative examples of animals in which the composition for feed addition of the present invention can be used are as follows: cattle such as edible cattle, cows, calves, pigs, piglets, sheep, goats, horses, rabbits, dogs, cats and the like; Poultry such as chicks, chickens, domestic chickens, roosters, ducks, geese, turkeys, quails, birds, etc. In addition, since the dosage varies depending on the type of animal to be administered, the age and the type of other feed components, it is difficult to define a constant, but in general, the feed composition of the present invention is a live Bacillus subtilis BC1212 10 per kg of feed. ^{2-10 15} cells, preferably 10 ^4-10 to ¹² cells, more preferably the addition of 10 ⁷ ~10 ¹¹ cells.

According to a preferred embodiment of the present invention, the Bacillus subtilis BC1212 strain used in the feed composition of the present invention has excellent viability even under acidic pH conditions, especially pH 1 similar to gastric acid (see Table 2), It also shows excellent survival and growth properties in bile. From this, it can be seen that the strain can be safely delivered to the large intestine to survive in the stomach and small intestine when used as a probiotic in livestock.

In addition, according to another preferred embodiment of the present invention, the Bacillus subtilis BC1212 strain exhibits enzymatic activity quite different from other strains of Bacillus subtilis species that are conventionally used as probiotics (see FIG. 5). Bacillus subtilis BC1212 has high activity against alkaline phosphatase and acid phosphatase.Alkaline phosphatase is an enzyme secreted from the bacterial wall and plays an important role in bacterial resistance to the external environment. Do it. In addition, acid phosphatase may increase phosphate utilization in feeds as phosphatase. Acid and base phosphatase are enzymes that release phosphorus from phosphorylated organics. Phosphorus is one of the major minerals required by life and is one of the essential factors for energy metabolism (ATP) and kinase cascade in vivo. . The strong phosphatase activity of the Bacillus subtilis BC1212 strain liberates phosphorus from phosphorylated proteins and sugars in the intestine to supply phosphorus in insufficient livestock bodies, and phosphatase is also present between the cell wall and the cell membrane of the BC1212 strain. Absorption of free phosphorus necessary for survival from phosphated proteins and sugars increases the survival rate (PNAS, 2004, 101, 7919-7924; Biochem, J., 1971, 125, 635-641). These results indicate that the strong phosphatase activity of the Bacillus subtilis BC1212 strain provides nutrients that are easily deficient in livestock, and the BC1212 strain itself absorbs large amounts of phosphorus necessary for survival to increase the survival rate. Together with the effect of increasing the production of sulfectin, it has been shown to improve the intestinal environment of the livestock, thus inducing more effective weight gain of the livestock.

In addition, according to another preferred embodiment of the present invention, when feeding the feed containing the strain increases the body weight of the animal compared to the non-added control, the feed efficiency is also slightly higher than the non-added control (see Table 3) . In addition, the total number of anaerobic bacteria in fecal microorganisms does not show a difference, but the number of Bacillus subtilis in feces increases by the feeding of the Bacillus subtilis BC1212 strain, while the number of Escherichia coli decreases (see Table 4). ).

The feed composition of the present invention comprising the Bacillus subtilis BC1212 strain produces sulfectin C having antimicrobial activity unlike microorganisms used for conventional feed addition, and has a different pattern from the conventional strain in the enzyme production capacity. It has excellent feed efficiency and weight gain effect of livestock animals.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.

Example  One: Bacillus Subtilis BC1212  Culture of the strain and Sulfectin  detach

10 g of doenjang was dissolved in 100 ml of sterile distilled water for the isolation of the sulfectin-producing strain, and 10 ml of the dilution obtained by diluting four times by tenths of the doenjang solution as a stock solution was added to the nutrient agar, Difco Co. USA) 1 ml each and incubated for 48 hours at 28 ℃. In order to identify the sulfectin-producing strain in the colonies formed after the culture, the cells were inoculated with a toothpick using a toothpick in a nutritive agar medium containing blood, and then cultured for 48 hours at 28 ° C. In particular, the strain with the largest size of the ring was isolated and used as a strain for sulfectin production. Nutrient agar medium was used as a solid medium for strain storage and preculture, and sulfectin using GYM medium (glucose 4 g, Yeast extract 4 g, Malt extract 10 g, CaCO ₃ 2 g, pH 7.2) as a liquid medium. Produced. In order to confirm the incubation time and the production of sulfectin, preculture was performed in 10 ml of the same medium for 2-3 days, and then pre-cultured at 2.5% in 200 ml medium (500 ml flask) for the main culture. The culture was inoculated and incubated at 28 rpm at 28 rpm for 7-8 days. At this time, the culture was sampled every 12 hours to determine the growth of the strain and the production of sulfectin. The degree of growth was compared by measuring the absorbance (OD ₆₀₀ ) at 600 nm, the sulfectin production was measured using a standard (sigma S3523). As a result, the production of sulfectin and the growth of the strains both increased rapidly from 80 hours of culture and showed a maximum value after 140 hours, but significantly decreased after 180 hours (FIG. 1).

16S rDNA for accurate identification of the strain Identification of strains using genetic analysis method (Micro ID, 411, Genetic Engineering Specialized Entrepreneurship Center, Seoul National University) was performed by arranging the sequences by referring to the secondary structure of the ribosomal RNA, and then calculating the% Similarity value. I completed the identification. The determined number of base sequences is 804 base pairs, having the base sequence set forth in SEQ ID NO: 1. Based on the determined nucleotide sequence, the% Similarity value was obtained by referring to the secondary structure of ribosomal RNA. As a result, the homology of the 16s RNA sequence was 99.75% identical to that of Bacillus subtilis NRRL-B-23049T / AF074975. From this, the inventors named the strain "Bacillus subtilis BC1212" strain belonging to Bacillus subtilis, and deposited on November 8, 2004 with the Institute of Agricultural Biotechnology (Accession Number: KACC 91141).

Example  2: Bacillus Subtilis BC1212  Determination of Antimicrobial Activity of Strains

In order to confirm the antimicrobial activity of the Bacillus subtilis BC1212 strain isolated in Example 1, the culture supernatant obtained by incubating the strain for 24 hours in a nutrient medium (BD Co., USA) and centrifuging the pH using HCl The precipitate was precipitated at about 2 degrees and the precipitate was centrifuged again to discard the supernatant and recover only the precipitate. The obtained precipitate was extracted twice with ethanol to remove water soluble impurities, and only the fractions dissolved in ethanol were recovered.

Next, column chromatography was performed to identify the active substance. First, the obtained ethanol fraction was concentrated on a vacuum evaporator, and then loaded into a reversed-phase C ₁₈ column filled with 5% methanol, eluted with 5% methanol, the same solvent, to separate only the fraction containing the antimicrobial substance. In order to increase the purity again, Sephadex LH-20 column chromatography was performed and eluted with ethanol, and each fraction was determined by LC / MS and NMR to determine the structure of the compound.

As a result, the material exhibiting the antimicrobial activity in the culture supernatant of the Bacillus subtilis BC1212 strain exhibited the separation phase as shown in FIG. 2, and the NMR and LC were purified by purely separating 11 components of the antimicrobial active fraction. As a result of analyzing the structure using / MS, it was confirmed that the sulfactin C (surfactin C) represented by the formula (1). As a result, the material showing the antimicrobial activity in the culture supernatant of the Bacillus subtilis BC3125 strain showed the separation pattern as shown in Figure 2, NMR and LC by pure separation of the 11 components of the antimicrobial activity fraction occupying the highest amount As a result of analyzing the structure using / MS, it was confirmed that the sulfactin C (surfactin C) represented by the formula (1).

In addition, the antiseptic effect of various pathogenic bacteria acting as a disease agent using the separated sulfactin C was measured by the liquid medium dilution method. In order to measure the minimum inhibitory concentration (MIC), add sulfatin to the nutrient medium (BD, USA), dilute it twice, and then develop the bacteria after 24 hours incubation with the final dilution concentration of 2.5 × 10 ⁵ CFU / ml. The presence or absence was visually confirmed, and the minimum concentration at which no bacterial growth was recognized was determined by MIC. As a result, sulfactin C showed excellent antimicrobial activity mainly against Gram-positive bacteria, and showed higher antimicrobial activity than antibiotics, especially antibiotic resistant bacteria known as MRSA (methicillin resistant Staphylococcus aureus) (Table 1). .

Minimum inhibitory concentrations of sulfactin C against various pathogens Type of pathogen Minimum Inhibition Concentration (㎍ / ㎖) SF AMOX COL NFX STM Esherichia coli DE3 ^a > 250 > 250 0.4 0.1 0.1 Esherichia coli KPU20031 ^b > 250 > 250 0.4 0.1 0.1 Esherichia coli KPU20032 ^b > 250 31.3 0.4 0.1 2 Salmonella thyphimurium KPU2003 ^b > 250 > 250 0.4 0.2 2 Staphylococcus aureus KCCM 40511 ^a 7.8 125 > 250 7.8 > 250 Staphylococcus aureus KCCM 41294 ^a 31.3 125 > 250 > 250 > 250 Staphylococcus aureus KCCM 40881 ^a 15.6 31.3 > 250 31.3 > 250 Staphylococcus aureus KCCM 11593 ^a 15.6 <0.1 > 250 7.8 > 250 Staphylococcus aureus KCCM 40510 ^a 15.6 <0.1 > 250 7.8 > 250 Staphylococcus aureus KCCM 11812 ^a 15.6 125 > 250 31.3 > 250 Staphylococcus aureus KPU2003 ^b 15.6 7.8 > 250 7.8 > 250 Staphylococcus epidermis KPU2003 ^b 15.6 7.8 > 250 7.8 > 250

In the above, the antibiotic resistance activity of the microorganisms (a) used in the experiment is as follows:

Staphylococcus aureus KCCM 40511; Penicillin and gentamicin resistance

Staphylococcus aureus KCCM 41294; Tetracycline, streptomycin, and macrolide resistance

Staphylococcus aureus KCCM 40881; Penicillin resistance

Staphylococcus aureus KCCM 11593; Penicillin resistance

Staphylococcus aureus KCCM 40510; Methicillin resistance

Staphylococcus aureus KCCM 11812; Penicillin resistance.

(b) represents a strain isolated from an infected animal, SF is sulfactin C; AMOX is Amoxacillin; COL is colistin; NFX is norfloxacin; And STM represents streptomycin.

Example  3: Bacillus Subtilis BC1212  Cultivation of Strains

Nutrient agar medium (BD, USA) was used as a solid medium for strain storage and seed inoculation, and a minimal medium was used as a liquid medium for ease of separation and purification. The minimum media consisted of 4% glucose, 30 mM KH ₂ PO ₄ , 30 mM Na ₂ HPO ₄ , 50 mM NH ₄ NO ₃ , 0.8 mM MgSO ₄ , 2 mM FeSO ₄ , 0.004 mM EDTA and 0.007 mM CaCl ₂ . , Inorganic salts and metal components were added separately at the time of inoculation. After 2-3 days preculture in the same liquid medium, the strain was inoculated into the culture at a concentration of 2.5%. The culture medium used for the culture included Composition A (1% glucose, 3% corn starch, 1% soybean meal, 0.5% peptone, 0.5% yeast extract, 0.2% CaCO ₃ , 0.01% antifoam, residual water, Unit; wt / wt%) was used at pH 6.75 and cultured for 5 days at 37 ° C. at a culture rotation speed of 140 rpm to obtain Bacillus subtilis BC1212 strain and its culture. Cultures were taken every 24, 48 and 72 hours, respectively, and then the amount of sulfactin C in the medium was measured using HPLC.

In addition, in order to check whether rice bran, wheat buckwheat, soybean meal, waste molasses, etc., which are currently used as raw materials for animal feed, can be used as a carbon source and a nitrogen source, the contents of C, H, O, N, and S of each raw material are automatically adjusted. Analysis was performed using an elemental analyzer (EA1110, CE Instrument), and composition B (2.8% bran, 4.9% rice bran, 5.1% soybean meal, 0.02% FeSO ₄ , 0.05% MnSO after fixing the carbon and nitrogen sources at 2% and 5%, respectively Bacillus subtilis BC1212 strain was cultured using ₄ , remaining water, unit; wt / wt%). The composition B was used to adjust the pH to 6.75, and incubated at 37 ° C. for 5 days at a culture rotation speed of 140 rpm to obtain Bacillus subtilis BC1212 and the culture solution. Cultures were taken every 24, 48 and 72 hours, respectively, and then the amount of sulfactin C in the medium was measured using HPLC.

As a result, the culture of Bacillus subtilis BC1212 using the composition A and composition B culture medium, it was confirmed that the production of a lot of sulfactin C showing a high antibacterial activity in composition B [Fig. 3].

Example  4: Bacillus Subtilis BC1212  Strain pH  And bile resistance

To assess the viability of the Bacillus subtilis BC1212 strain during digestion, the acidic conditions and resistance to bile were examined. In order to evaluate the viability under acidic conditions, the strain was inoculated in a liquid medium and then incubated at 37 ° C. for 12 hours to obtain a concentration of 10 ⁹ to 10 ¹⁰ colony forming units (CFU) / ml. It was made. The cultured bacteria were collected by centrifugation at 6,000 rpm for 10 minutes, and the cells were washed twice with sterile water and concentrated to 1/100 to give a cell number of about 10 ¹⁰ CFU / ml. Inoculated with diluted bacteria in buffer adjusted to pH 1, 2, 3, 4 and 5, incubated at 37 ℃ for 1, 2 and 4 hours, and then diluted with sterile water to trypticase soy agar (trypticase soy agar, The colonies were counted by inoculation in BD, USA). Survival rate (%) was calculated by dividing the number of viable cells after the reaction time by the number of bacteria at the initial inoculation.

As a result, it was found that Bacillus subtilis BC1212 survived relatively stable even under acidic pH conditions (Table 2). PH 1, which is similar to the pH of gastric acid, showed a survival rate of about 64% for 4 hours, and an excellent survival rate of more than 85.7% after 4 hours treatment at pH 2 and above.

Survival of Bacillus subtilis BC1212 Strains at Various Acidic pH Conditions pH % Survival of Bacillus subtilis BC1212 1 hours 2 hours 4 hours One 76.2 70.3 64.0 2 93.0 91.1 85.7 3 100 97.2 98.4 4 99.7 96.8 96.1 5 99.9 95.9 97.9

Next, in order to evaluate bile resistance, inoculated with Bacillus subtilis BC1212 in a medium to which 0.3% ox bile was added to trypticase soy medium and incubated at 37 ° C., using a spectrophotometer at 550 nm wavelength for 12 hours. The degree of growth of the bacteria by the absorbance of was measured.

As a result, the initial growth was inhibited by the treatment of 0.3% bull bile, but after 6 hours, the growth was normal, and it was confirmed that the Bacillus subtilis BC3125 strain showed excellent survival and growth even in bile (FIG. 4).

From the above results, it was confirmed that Bacillus subtilis BC1212 strain can survive in the stomach and small intestine and be delivered to the large intestine when used as a probiotic in livestock.

Example  5: Bacillus Subtilis BC1212  Enzyme Activity Produced by Strains

API ZYM kit (bioMerieux, Montalieu-Vercieu, France) was used to determine the activity of various enzymes secreted by the Bacillus subtilis BC1212 strain. Bacillus subtilis BC1212 strain was inoculated in trypticase soy medium and incubated at 37 ° C. for 24 hours under aerobic conditions, and enzyme activity was measured. In addition, Bacillus subtilis NATO (KCCM12027, Korea microbial strain bank, Korea) was also cultured under the same conditions as above to compare the enzyme activity with Bacillus subtilis NATO, which is conventionally used as a probiotic. . Enzyme activity was relatively evaluated by confirming the color development from 0 to 5 points compared to the control.

As a result, Bacillus subtilis BC1212 exhibited completely different enzymatic activity from Bacillus subtilis NATO conventionally used as a probiotic (FIG. 5). Bacillus subtilis BC1212 showed high activity against alkaline phosphatase and acid phosphatase. Alkaline phosphatase is an enzyme secreted from the bacterial wall and plays an important role in bacterial resistance to the external environment. In addition, acid phosphatase may increase phosphate utilization in feeds as phosphatase.

Example  6: Bacillus Subtilis BC1212  Strain At weaners Gain  effect

Weaning pigs (Landrace × Yorkshire × Durok) purchased a total of 20 four-week-old (average 28-day) pigs from a ranch attached to Chungnam National University and used them in the experiments. It was divided. The control group was fed a feed not containing Bacillus subtilis BC1212, the experimental group was fed for 4 weeks by adding the Bacillus subtilis BC1212 strain isolated in Example 1 at 0.1% per kg feed. In order to measure the productivity and the rate of increase, the weight gain was calculated by measuring the weight of weaning pigs at the time of acquisition and every week, and the feed intake rate was calculated as the cumulative feed intake weekly. Feed efficiency (%) was expressed by weight gain divided by feed intake rate.

Next, fecals of each test group were collected on the last 28 days after the start of the experiment in order to determine the change in the fecal microorganism due to the feeding of Bacillus subtilis BC1212 probiotics. The collected feces were inoculated into the blood medium to check the colony count of the total anaerobic microorganisms in the collected feces. Also, in order to confirm the presence of Bacillus subtilis in feces, the fecal samples of each test group were subjected to polymixin pyruvate egg yolk mannitol bromothymol blue agar. , Oxoid, UK) and incubated for 48 hours at 37 ℃ anaerobic conditions and the number of colonies of Bacillus subtilis was measured. In addition, in order to detect E. coli, a major bacterium that causes diarrhea in weaning pigs, it was inoculated in E. coli differentiating medium, MacConkey medium (BD company, USA), incubated for 24 hours at 37 ° C aerobic condition, and then lactose was decomposed. Colonies were estimated to be E. coli.

As a result, the weight change according to the feeding of Bacillus subtilis BC1212 was increased compared to the control group, and the feed efficiency was also slightly higher in the Bacillus subtilis BC1212 administration group than the control group (Table 3). In addition, the total number of anaerobic bacteria in fecal microorganisms did not show a difference, but the number of Bacillus subtilis in feces increased and the number of Escherichia coli decreased by feeding Bacillus subtilis BC1212 (Table 4).

Changes in Body Weight According to Salary of Bacillus Subtilis BC1212 in Weaning Pigs No addition control Bacillus subtilis BC1212 administration group  Average weight (㎏)     0 days (start of test)  7.92 ± 1.15 7.95 ± 0.73     7 days  9.99 ± 1.25 9.80 ± 0.79    14 days      13.62 ± 1.33 13.49 ± 1.27    21st 17.25 ± 2.02 19.26 ± 1.40    28 days 22.28 ± 1.88 25.47 ± 1.48  Average daily gain (g)     0-7 days 294 ± 70       287 ± 88     8-14 days 520 ± 52 530 ± 141    15-21 days  520 ± 150 540 ± 98    21-28 days  720 ± 140 887 ± 84  Average feed intake (g)     0-7 days 494 413     8-14 days 642 625    15-21 days 735 758    21-28 days 1086 953  Feed efficiency (weight gain / feed intake,%)     1 week 0.77 ± 0.17  0.66 ± 0.21     2 weeks 0.81 ± 0.08  0.85 ± 0.27     3 weeks 0.73 ± 0.21  0.71 ± 0.14     4 Weeks 0.63 ± 0.13  0.93 ± 0.05

Changes in Fecal Microorganisms with Bacillus subtilis BC1212 Supplementation No addition control Bacillus subtilis BC1212 administration group  Total anaerobic bacteria (log CFU / g) 8.39 ± 0.13 8.32 ± 0.09  Bacillus subtilis numbers (log CFU / g) 8.08 ± 0.10 8.20 ± 0.08  Coliform group count (log CFU / g) 8.38 ± 0.02 8.14 ± 0.13

Production Example . Bacillus Subtilis BC1212  Preparation of a composition for feed addition containing a strain

We prepared a composition for feed addition of the present invention comprising Bacillus subtilis BC1212 strain with the composition of Table 5 below.

Composition ratio of feed composition (unit; weight%) BC1212 Enzyme preparation microbe amino acid Etc Preparation Example 1 100 Preparation Example 2 90 10 Preparation Example 3 80 10 10 Preparation Example 4 70 10 10 10 Preparation Example 5 60 15 15 8 2 Preparation Example 6 55 20 15 8 2

In Table 5, the enzyme preparation used a combination of phytase, cellulase, xylanase, maltase, and converting enzyme, and Aspergillus Orissae was used as a non-pathogenic microorganism.

1 is a graph showing the correlation between the growth of the strain and the production of sulfectin, □ represents the growth of the cells, ● represents the sulfectin production.

Figure 2 is a liquid chromatography data on the material showing the antimicrobial activity in the culture supernatant of Bacillus subtilis BC1212 strain.

Figure 3 is a graph comparing the sulfactin C production of Bacillus subtilis BC1212 strain according to the medium composition.

Figure 4 is a graph evaluating the effect of 0.3% bull bile treatment on the growth of Bacillus subtilis BC1212 strain, "control" is the growth curve of Bacillus subtilis BC1212 untreated bull bile, "bull bile 0.3 % "Represents the growth curve of Bacillus subtilis BC1212 treated with 0.3% bull bile.

5 is a graph comparing the enzyme activity of Bacillus subtilis BC1212 strain and Bacillus subtilis NATO (KCCM12027) strain.

<110> B.N.C BIO PARM CO., LTD. <120> Composition for Prevention or Treatment of Osteoporosis          Comprising Rubus Coreanus Miquel Extract <130> 2007-dpa-0151 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 804 <212> DNA <213> Bacillus subtilis BC1212 <220> <221> rRNA (222) (1) .. (804) <223> 16S rDNA sequence of Bacills subtilis BC1212 <400> 1 gaacgctggc ggcgtgccta atacatgcaa gtcgagcgga cagatgggag cttgctccct 60 gatgttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120 tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca aacataaaag 180 gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240 ggctcaccaa ggcaacgatg cgtagccgac ctgagagggt gatcggccac actgggactg 300 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccgcaa tggacgaaag 360 tctgacggag caacgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgtta 420 gggaagaaca agtaccgttc gaatagggcg gtaccttgac ggtacctaac cagaaagcca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tccggaatta 540 ttgggcgtaa agggctcgca ggcggtttct taagtctgat gtgaaagccc ccggctcaac 600 cggggagggt cattggaaac tggggaactt gagtgcagaa gaggagagtg gaattccacg 660 tgtagcggtg aaatgcgtag agatgtggag gaacaccagt ggcgaaggcg actctctggt 720 ctgtaactga cgctgaggag cgaaagcgtg gggagcgaac aggattagat accctggtag 780 tccacgccgt aaacgatgag tgct 804  

Claims (8)

Feed composition comprising Bacillus subtilis BC1212 strain (Accession No .; KACC 91141). The method according to claim 1, The strain is a composition for feed addition, characterized in that to produce sulfactin C represented by the formula (1). [Formula 1]

The method according to claim 1, 2.8% by weight of bran, 4.9% by weight of rice bran, 5.1% by weight of soybean meal, 0.02% by weight of FeSO ₄ , 0.5% by weight of MnSO ₄ , and the remaining amount (87.13% by weight) of water. The method according to claim 1, Feed composition according to claim 1, further comprising at least one enzyme preparation. The method according to claim 4, The enzyme preparation is a composition for feed addition, characterized in that selected from the group consisting of lipase, phytase, amylase, phosphatase, carboxymethylcellase, xylanase, maltase and convertase. The method according to claim 1, Feed composition for addition, characterized in that it further comprises a non-pathogenic microorganism. The method according to claim 6, The microorganism is a microorganism of the genus Lactobacillus, Aspergillus Oriza, Saccharomyces cerevisiae is selected from the group consisting of feed composition. The method according to claim 1, The feed composition further comprises at least one amino acid.

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