KR100494808B1 - Bacillus sp. A8 that is produced the polysaccharide, its application or polysaccharide produced from the same strain - Google Patents

Abstract

The present invention relates to Bacillus sp. A8, a strain that produces polysaccharides, to a polysaccharide produced from the strain and its use, and more particularly, to a strain of the new strain Bacillus sp. A8, which produces polysaccharides from soil. KCTC 10433BP], produced from this strain, has a high pH stability and high salts compatability, resulting in constant intestinal action and treatment of digestive disorders in industrial animals (swine, poultry, etc.). Microorganisms that are specific to enhancing effects and promoting recovery, and also inhibit the onset of diseases caused by the death of beneficial organisms by the misuse of antibiotics and the increase of resistance of pathogens due to the increased anti-potency due to the immune activity of high-functional polysaccharides. It relates to sex polysaccharides and their use as feed additives.

Description

A. genus Bacillus, a strain that produces polysaccharides, polysaccharides produced from this strain and uses thereof [Bacillus sp. A8 that is produced the polysaccharide, its application or polysaccharide produced from the same strain}

The present invention relates to Bacillus sp. A8, a strain that produces polysaccharides, to A8 (A8) polysaccharides produced from the strain, and more particularly, to a method for producing water-soluble polysaccharides from soil. Isolates A8 genus B., which is produced from this strain, and has a high pH stability and high salts compatability, resulting in constant intestinal action and digestion of industrial animals (swine, poultry, etc.). It has a specific effect on increasing the therapeutic effect of the disorder and promoting recovery, and also suppresses the onset of disease caused by the death of enteric beneficial bacteria and the increase of resistance of pathogens due to the misuse of antibiotics due to the increase of the anti- potency due to the immune activity of high-functional polysaccharides. Microbial polysaccharides and their use as feed additives.

Livestock raised for a long time in an artificial environment are affected by changes in feed, antibiotic administration, transport and transport, stress, infection by harmful microorganisms, and the balance between intestinal microorganisms is broken, resulting in a decrease in weight gain and digestion efficiency of feed. In addition, lactic acid bacteria, which are currently used as probiotics to inhibit the growth of pathogens such as Escherichia coli and Salmonella and promote livestock growth, must withstand acid resistance and bile resistance, and the density of probiotic itself must also be maintained at a certain level. have.

Therefore, it acts specifically for the improvement of constant intestinal function and treatment of digestive disorders and promotion of recovery, and the disease caused by the death of beneficial organisms and the resistance of pathogens due to the misuse of antibiotics due to the increase of the anti-potency due to the immune activity. It is possible to suppress the onset, and new polysaccharides are needed to stably produce high quality clean livestock products.

Therefore, the present inventors have conducted the study in view of the above, as a result, isolates A8 genus Bacillus A8 from the soil, the stability of a wide pH range and salts compatability with the salt is very high from this strain industry It has specific properties that specifically affect the intestinal constant action of the animal (swine, poultry, etc.) and increase the treatment effect of digestive disorders, and promote recovery. The present invention has been completed by producing microbial polysaccharides that can contribute to the stabilization of the price of industrial animals by stably producing high-quality clean livestock products by killing beneficial intestinal bacteria and increasing the resistance of pathogens. .

Accordingly, an object of the present invention is to provide a new strain Bacillus genus A8 [KCTC 10433BP] and polysaccharides produced from the strain.

In addition, the present invention has another feature in the feed additive containing the polysaccharide.

The present invention is characterized by Bacillus sp. A8 (KCTC 10433BP), which produces polysaccharides.

In addition, the microbial polysaccharide having an average molecular weight of 7 million daltons, consisting of glucose, fucose, glucuronic acid, and galactose produced from the strain, is another feature.

Referring to the present invention in more detail as follows.

The present invention isolates Bacillus sp. A8 from the new strain Bacillus sp. A8, which produces polysaccharides from soil, and is produced from this strain, and has a high pH stability and high salts compatability. Intestinal activity caused by misuse of antibiotics due to the increase in the anti-tumor due to the immune activity of the high-functional polysaccharides and the specific intestinal function of the intestine of the animals (swine, poultry, etc.) The present invention relates to a microbial polysaccharide capable of inhibiting the development of diseases caused by the killing of beneficial bacteria and increased resistance of pathogens and the use of the same as a feed additive.

The new strain according to the present invention was identified as belonging to the genus Bacillus sp. As a result of morphological, physiological and biochemical identification by pure separation from the soil to produce extracellular microbial polysaccharides from the soil. Genus A8 ( Bacillus sp. A8) was named. In addition, the strain was deposited with the Korea Biotechnology Research Institute Gene Bank on February 24, 2003, and the accession number was assigned to KCTC 10433BP. The strain was separated from the soil and finally selected through a series of processes and identified as Bacillus, but is a strain that produces polysaccharides with physical properties that are completely different from similar strains reported so far.

Also, genus Bacillus. The new microbial polysaccharide produced from A8 [KCTC 10433BP] has an average molecular weight of about 7 million daltons, and its components include glucose, fucose, glucuronic acid and galactose. Among the extracellular polysaccharides produced by bacteria, the constituent ratio is 2.96: 1.00: 1.55: 0.58 (molar ratio), and polysaccharides having such a component and molar ratio have not been reported so far, and it has been confirmed that the polysaccharide is a novel polysaccharide. By confirming that the polysaccharide is stable in a wide range of pH and salts compatability is very high, it is specific to the constant intestinal effect of the intestines of industrial animals (swine, poultry, etc.) and to increase the therapeutic effect of digestive disorders and to promote recovery. In addition, it can be useful as a feed additive that can suppress the onset of disease caused by the increase of resistance of pathogens and the death of enteric beneficial bacteria due to the misuse of antibiotics due to the increase of the anti-tumor due to the immune activity of high-functional polysaccharides. It is expected.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of Polysaccharide Producing Strains

Samples were taken from the soil, and 1 g of soil samples were suspended in 0.85% saline to prepare an inoculation sample. Stain 100 µl of the inoculated sample diluted appropriately with physiological saline in the medium of LB (LB, Tryptone 10 g, Yeast extract 5 g, NaCl 10 g / ℓD.W.) And incubate at 30 ° C for 2 to 3 days. It was. Clonin, which secretes mucus as a single colony, was purely separated and aseptically reinoculated into a polysaccharide-producing liquid medium composed of inorganic salts and incubated with a 500 ml Erlenmeyer flask for 3 days. Strains with good viscosity and yield of the resulting polysaccharides were selected.

Example 2: Strain Identification

Morphological characteristics of the strain isolated in Example 1 were observed with an electron microscope [FIG. 1], and biochemical and physiological characteristics are shown in Table 1 below. The strains were 0.5-0.7 μm × 2.0-2.5 μm bacilli [FIG. 1], which were motile and Gram positive. Endogenous spores were formed and in the O / F test, oxidation and fermentation were performed. It produced catalase but no oxidase and showed slow growth under anaerobic conditions. Acids were produced in D-glucose, D-xylose and D-mannitol and gas was produced in glucose. Casein was degraded while gelatin and starch were not degraded, and no indol was formed. It did not grow at 7% NaCl and grew up to 45 ° C. These results are described in Bergey's manual of systematic bacteriology [Peter, H.A.S., S.M. Nicolas, M.E. Sharp and J.G. Holt. 1986., vol. 2. Baltimore, London] and Cowan and Steel of Manual of the identification of medical bacteria [Cowan, N.R., and K.J. Steel. 1965, Cambridge Univ. Press, London. Pp. 215], the isolates were identified as Bacillus, similar to Bacillus coaguran.

Physiological and Biochemical Characteristics of Polysaccharide Producing A8          Character   Strain A8 Bacillus coagulans    Gram / Shape + / R + / R    Spheres + +    Motility + D    Growth in air + +    Growth anaerobically + D    Catalase + +    Oxidase － d    Glucose (acid) + D    O / F /- O / F O / F /-    Sphere shape X X    Sphere position U UVT    Growth at 45 ℃ + +    Growth at 65 ℃ － d    Growth at pH 5.7 + +    Growth in 7% NaCl － －    Utilization of citrate + d    Anaerobic growth in glucose broth + +    Carbohydrates, acid from:             Glucose Arabinose Mannitol Xylose +-++ + Ddd    VP test + d    Starch hydrolysis － +    Nitrate reduction + d    Indole － －    Gelatin hydrolysis － －    Casein hydrolysis + d    Urease － －    LV － －    Lysozyme sensitivity s s

In addition, by sequencing using the 16S rRNA of this strain and comparative analysis of the nucleotide sequence, it was confirmed that the homology with Bacillus subtilis of 99.8%.

From the above results, the isolated strain was identified as mycorrhizal fungus with similar properties to the genus Bacillus, which is genus Bacillus. It was named A8 ( Bacillus sp . A8) and it was deposited on February 24, 2003 to the Gene Bank of Korea Research Institute of Bioscience and Biotechnology and was given accession number KCTC 10433BP.

Example 3: Polysaccharide Purification

In order to produce polysaccharides, a batch culture was performed using a 5 liter fermentor of a Korean fermenter. Medium composition was glucose 30 g / L, NH ₄ NO ₃ 1 g / L, K ₂ HPO ₄ 1.2 g / L, KH ₂ PO ₄ 0.2 g / L, NaCl 0.1 g / L, MgSO _{4 ·} 7H ₂ O 0.1 g / L, MnSO ₄ H ₂ O 0.1 g / L, FeSO ₄ 7H ₂ O was prepared at 0.05 g / L and incubated for 3 days at an initial pH of 7-8, 30 ℃. In order to separate the cells from the culture solution in the fermentor (jar fermentor), the culture solution was diluted 10-fold and centrifuged (12,000 × g) to remove the cells. To the supernatant from which the cells were removed, 95% ethanol was added to be three times higher than that of the supernatant, and the mixture was left at 4 ° C. for 2 hours to separate a polysaccharide having a white precipitate. The polysaccharide thus separated was named A8 (A8). A8 polysaccharide was added to distilled water, and the solution was dissolved in water. The remaining ethanol was completely removed using a reduced pressure evaporator, and then lyophilized to prepare a white powder.

Example 4: Determination of Molecular Weight

To determine the molecular weight of purified A8 polysaccharide, HPLC [Shimdazu Co. JP] PL-GFC 1000Å (8 ㎛, 300 × 7.5 ㎜, Polymer Lab., USA) column attached with a third distilled water as a solvent, the refractive index detector at a flow rate of 1 ㎖ / min at 25 ℃ ) As a result of detection with RID-6A, the average molecular weight of A8 is estimated to be about 7 million daltons (FIG. 2). In FIG. 2, when logY = aX = b, a = −1.2316 and b = 14.9598.r ² = 0.9643.

Example 5: Analysis Per Configuration

0.1 g of A8 polysaccharide was dissolved in 10 ml of 2N trifluoroacetic acid (TFA), and then hydrolyzed at 121 ° C. for 1 hour, and concentrated under reduced pressure at 40 ° C. using a vacuum concentrator to completely remove TFA. Were analyzed by TLC plate (Merck, 5744). As a developing solvent, acetonitrile / water = 85/15 (v / v) was developed and treated with diphenylamine / anilline / phosphoric acid for 5 minutes at 100 to 105 ° C. The composition sugar of A8 was analyzed by comparative analysis with the material. A8 polysaccharide was a heteropolysaccharide composed of glucose, fucose, glucuronic acid and galactose, and the molar ratio of these components was 2.96: 1.00: 1.55: 0.58 [FIG. 3]. Among the extracellular polysaccharides produced by bacteria, polysaccharides having a molar ratio with these components have not been reported so far, and it was confirmed that the polysaccharides are novel polysaccharides.

Example 6 IR Spectrum for Structural Analysis of Polysaccharides

Infrared absorption spectra for structural analysis of A8 polysaccharides, C = O stretching bands around 3700 cm ^-1 to OH groups, 2900 cm ^-1 to CH groups, 1700 cm ^-1 and 1060 cm ^-1 , which are specific absorption bands for polysaccharides And CO stretching bands appear, presumably the carboxyl group of glucuronic acid, a sugar derivative. In addition, CH ₂ bending was observed at 1400 cm ⁻¹ , and the strong absorption bands in the range of 1000 cm ⁻¹ and 1200 cm ⁻¹ showed typical characteristics of all sugar derivatives [FIG. 4].

Example 7 Physical Properties of Polysaccharides

In order to investigate the applicability of A8 polysaccharide as a feed additive, the viscosity of the polysaccharide under various conditions was measured using a Brookfield ammeter DV-III (Brookfield engineering Lab. INC USA). Viscosity measurements were measured from 0.5 rpm to 150 rpm using a spindle SC4-34 (Brookfield model DVIII-rheometer, SC4-34, 30 rpm, RT).

(1) Measurement of viscosity by concentration

The purified A8 polysaccharide was dissolved in distilled water to 1, 2, 3, 4, 5%, and then mounted with a spindle SC4-34 [Brookfield model DVIII-rheometer, SC4-34, 30 rpm, RT] The viscosity was measured from rpm to 150 rpm and is shown in Table 2 below. The apparent viscosities measured at 30 rpm for each concentration were 25, 75, 125, 200, and 280 cP, respectively, and the concentration increase and apparent viscosity of A8 polysaccharides were proportional to each other.

Measurement of viscosity by concentration of A8 polysaccharide density(%) One 2 3 4 5 Apparent Viscosity (cP) 25 75 125 200 280

(2) Viscosity measurement by temperature

In order to investigate the temperature-specific viscosity of the purified A8 polysaccharide, the concentration of A8 polysaccharide was dissolved in 15 ml of distilled water such that the concentration of A8 polysaccharide was 2%, and heat-treated at room temperature (25 ° C.) to 85 ° C. for 30 minutes. The apparent viscosity was measured with spindle SC4-34 at temperature and is shown in Table 3 below. The A8 polysaccharide had an apparent viscosity loss of about 15% up to 50 ° C and a 30% loss up to 85 ° C. A8 polysaccharides showed a decrease in apparent viscosity with temperature. This result is similar to the physical properties of the temperature according to the general polysaccharides.

Temperature Viscosity Measurement of A8 Polysaccharides Temperature (℃) Room temperature (25) 30 50 70 85 Apparent Viscosity (cP) 75 62 60 48 50

(3) Measurement of viscosity and stability by pH

Viscosity measurement for each pH was measured as the concentration of A8 polysaccharide 2% and the apparent viscosity was adjusted by adjusting the pH from 2 to 11 with 1N NaOH and 1N HCl solution. Measurement of the stability of the A8 polysaccharide to pH was adjusted to 2 to 11, and then measured again after 24 hours to measure the stability of the pH is shown in Table 4 below.

A8 polysaccharide showed little change in apparent viscosity over the entire pH range, and the same results were obtained in the stability experiment. This result is expected to increase the efficiency of reaching the intestine by increasing the survival rate in industrial animals (swine, poultry) when used as a beneficial bacteria (lactic acid bacteria) coating of feed additives. In addition, these results are quite characteristic physical properties that other polysaccharides do not have, and can be easily used for various formulations in application.

Measurement of pH and Stability of A8 Polysaccharides by pH by pH pH 2 pH 3 pH 5 pH 7 pH 9 pH 11 0 hours 24 hours 0 hours 24 hours 0 hours 24 hours 0 hours 24 hours 0 hours 24 hours 0 hours 24 hours Apparent Viscosity (cP) 65 66 73 72 71 71 70 71 75 74 70 71

(4) Temperature stability measurement

Viscosity measurement by temperature was a 2% concentration of A8 polysaccharide and heat-treated for 1 hour at a temperature range of 25 ° C (room temperature) to 85 ° C, and the viscosity was measured after cooling to room temperature. The heat treatment at 121 ℃ was treated for 15 minutes using an autoclave, and after cooling to room temperature to measure the viscosity is shown in Table 5 below. A8 polysaccharides were quite stable over the entire temperature range, and the apparent viscosity was stable even after heat treatment with autoclave. This result is a unique physical property of A8 polysaccharide, and as a result of stability to pH, it is expected to increase the survival rate of lactic acid bacteria in the process of applying heat during the formulation of feed additives, and is very easy to formulate various formulations. This is the result of further highlighting.

Temperature stability measurement of A8 polysaccharides Temperature (℃) Room temperature (25) 30 50 70 85 121 Apparent Viscosity (cP) 75 68 69 75 70 67

(5) Salt compatability measurement for salt

In order to measure the compatibility of various salts using a solution of A8 polysaccharide, NaCl, KCl, CaCl ₂ , and FeCl ₃ were added at 5, 10, and 15% concentrations to measure viscosity. Gelatinization of the A8 polysaccharides for monovalent and divalent cations did not change the viscosity even with increasing salt concentration, but in the case of trivalent cations, the concentration tended to decrease with increasing concentration [Fig. 5]. These results show that the salts compatability is very high, so it is specific to the constant intestinal action and the improvement of the treatment of digestive disorders in the intestines of industrial animals (swine, poultry, etc.) and to promote recovery and maintain the intestinal homeostasis. The killing of enterococci can be controlled. In addition, it is expected to reduce the onset of the disease by inhibiting the increase in the resistance of pathogens due to the misuse of antibiotics by increasing the anti-pathology due to the ability to modulate the immune activity of polysaccharides.

Example 7 Review of Applicability as a Feed Additive

As a result of oral administration of the above-mentioned polysaccharides having excellent heat, pH stability and salt luxury to the pig group in question at 0.5% by weight, homeostasis was maintained in the intestine of the livestock to improve diarrhea, ileitis, and epidemic diarrhea. [Table 6].

Therapeutic Effect of Diarrhea by Oral Administration of A8 Polysaccharides Farm area Breeding scale Problem Symptom Result after administration A Anseong 2,000 Fostering Pigs Ileitis Ileitis B Nonsan 1,500 Piglets E. coli + pandemic diarrhea recovery C Acting 1,000 Piglets Cremitis Crematory D Jincheon-gun 1,000 Hog Diarrhea recovery E Gimcheon 1,200 Piglets Escherichia coli + Infectious diarrhea Improvement

As described above, when the feed additive is prepared from the polysaccharide produced from the new strain Bacillus sp. A8 according to the present invention, the intestinal constant effect on the intestines and digestive disorders of industrial animals (swine, poultry, etc.) It is expected to be very useful for livestock industry because it can suppress the death of beneficial intestinal bacteria and increase disease resistance due to misuse of antibiotics due to the increase of therapeutic effect and promotion of recovery and the increase of anti- potency due to immune activity of high functional polysaccharides. .

Figure 1 shows an electron micrograph of A8 strain genus Bacillus.

2 is a molecular weight of the A8 polysaccharide analyzed by a gel permeation column using HPLC [O: A8 polysaccharide, □: 2 million Dalton dextran, △: 580,000 Dalton dextran, ▽: 14.33 million Dalton dextran, ◇: 7.30,000 Dalton dextran, : 1.50,000 Dalton dextran].

Figure 3 is a component analysis of the A8 polysaccharide by TLC [A: glucose, B: galactose, C: glucosamine, D: A8 polysaccharide, E: mixture of standards, F: glucuronic acid, G: Pew Coos].

Figure 4 shows the IR spectrum of the structure of the A8 polysaccharide.

Figure 5 shows the gelatinization of the salt of the A8 polysaccharide produced by the genus A8 Bacillus.

Claims (3)

Bacillus sp. A8 producing polysaccharides [KCTC 10433BP]. Produced from Bacillus sp.A8 [KCTC 10433BP], contains glucose, fucose, glucuronic acid and galactose, and has an average molecular weight of 700 Microbial polysaccharides, characterized in that the mandaltons. A feed additive comprising the polysaccharide of claim 2.