KR100846672B1 - Improved productivity and survival of bacillus polyfermenticus scd by alginate/methyl-cellulose encapsulelation and use thereof - Google Patents

Abstract

The present invention relates to a method for improving the productivity and viability of Bacillus polypermanticus SCD using an alginate / methyl-cellulose capsule and its use. The present invention relates to an alginate / methyl-cellulose as a cell immobilization technique using an encapsulation process. Rhodes capsules directly coat the cells themselves, shortening the time needed to make spores, dramatically increasing the productivity of Bacillus spp., As well as external physical and chemical effects until the Bacillus polyfermentus SCD reaches the intestines. Protecting from the effects, it is effective in prolonging survival by applying to medicines, functional foods and dietary supplements for the treatment of liver disease.

Bacillus polyfermentus SCD, alginate capsules, probiotics, bis fungi, bisroot, methyl-cellulose, acid resistant, bile resistant

Description

IMPROVED PRODUCTIVITY AND SURVIVAL OF BACILLUS POLYFERMENTICUS SCD BY ALGINATE / METHYL-CELLULOSE ENCAPSULELATION AND USE THEREOF}

1 shows a schematic diagram of a giant capsule forming apparatus.

Figure 2 is a photograph of the observation of the alginate capsules under a scanning electron microscope (SEM).

FIG. 3 is a photograph of Bacillus polyfermenticus SCD captured in an alginate lattice hole under a scanning electron microscope. FIG.

Figure 4 is a cross-sectional view of the alginate capsule (A) is a photograph of the surface shape before exposure to artificial gastric juice, (B) is a photograph showing the surface shape after exposure to artificial gastric juice for 3 hours.

5 is a graph showing the survival rate of Bacillus polypermanticus SCD coated with different concentrations of alginate after exposure to artificial gastric juice, (A) 2% alginate capsules, (B) 3% alginate capsules, (C) It shows the survival rate when the film was coated with a 4% alginate capsule (◆: pH 1.5, □: pH 2.5, ◇: pH 7.0).

Figure 6 is a graph showing the survival rate of Bacillus polypermanticus SCD in 3% alginate capsules of various diameters after exposure to artificial gastric juice (pH 1.5) (◇: small capsules (850㎛), ◆: large capsules (2.8) mm)).

FIG. 7 is a photograph showing an alginate / methyl-cellulose capsule showing methyl-cellulose granules in the cavity of the alginate matrix under a scanning electron microscope.

Figure 8 is a photograph of observation of the capsule after freeze-drying under a scanning electron microscope.

FIG. 9 is a photograph showing the Bacillus polypermanticus SCD captured in an alginate / methyl-cellulose lattice lyophilized after exposure to artificial gastric juice for 3 hours under a scanning electron microscope ((A) 10,000 × low magnification Magnification, (B) high magnification of 20,000 times).

10 is a photograph showing a cross-sectional view of a freeze-dried alginate / methyl-cellulose capsule ((A) surface morphology before exposure, (B) surface morphology after exposure for 3 hours in artificial gastric juice).

The present invention relates to a method for improving the productivity and viability of Bacillus Bacillus using an alginate capsule, and more particularly, the present invention is centrifuged after culturing Bacillus Bacillus polypermanticus SCD. The obtained cell suspension was put in different concentrations of sodium-alginate, mixed, and then transferred to a capsule-forming apparatus, whereby the Bacillus polypermanticus SCD was encapsulated by the capsule, thereby producing acid and bile resistance. And a method for improving survival rate and use thereof.

Probiotics, when used in humans or animals in general, refers to growth microorganisms that favorably affect the health of the host by improving the balance of indigenous microorganisms. In recent years, probiotics have been defined as 'living organisms that are beneficial to health in addition to basic nutrition if consumed to some extent.' This underlines the importance of maintaining a sufficient number of living microorganisms in the gut, and these benefits include improving the balance of microorganisms associated with other health effects such as immunomodulation.

Most probiotics belong to the Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, and Lactobacillus sporogenes , which are generally important components of the human gastrointestinal tract and also exist as harmless commercial organisms. The novel probiotics also include other microorganisms such as yeast (eg Saccharomyces boulardii ) and other significantly different bacterial types (eg Clostridium butyricum , Bacillus subtilis , Bacillus polyfermenticus , etc.). The genus Bacillus contains a variety of industrially important species that are commonly used as hosts in the bioindustry. Commercial products produced by today's Bacillus fermentation include enzymes, antibiotics and pesticides. Other products are well known nucleic acids, nucleosides and amino acids which are mainly used as food sweeteners.

Bacillus polyfermenticus SCD (generally called Bacillus bacillus) is known to secrete 20 kinds of enzymes to recycle nutrients, and to synthesize nutrition of vitamins B1, B2 and K to disseminate nutrition. In addition, it has been shown that ingestion of this strain enhances parenteral infection protection and oral immunity, and enhances the host's immune function, inhibits the growth of intestinal microorganisms that produce carcinogens and carcinogens, It is known to inhibit tumorigenesis by generating antitumor substances or antimutagenic substances. In addition, the bispan bacteria is a useful probiotic probiotic that shows excellent effects in the treatment of long-term intestinal diseases because it is a bacterium forming apo, and thus hardly loses activity due to strong resistance to gastric acid or various enzymes until reaching the intestine. . The strain of the present invention was obtained by identifying the bispan bacteria from Binex Co., Ltd. and named as Bacillus polyfermenticus SCD ( B. polyfermenticus SCD) and deposited in the KCCM 10104 microorganism preservation center attached to the Korea spawn association. I received an accession number. The present inventors have conducted a lot of research to always keep the productivity of the Bacillus Bacillus (Bacillus polyfermentus SCD) and the main technology registered as a patent is as follows.

Liquid cultivation if strains of Bacillus polyfermenticus (US Pat. No. 6,010,898)

2. Manufacturing method of bispan bacteria by liquid fermentation (Korea Patent Registration No. 233615)

3. Method of recovering Bispan bacteria from Bispan bacteria culture (Korea Patent Registration No. 233616)

4. Improved productivity of Bisruit mutants (Korea Patent Registration No. 290005)                         

5. Bispan mutant strains with increased productivity of bacteriocin and bacteriocin production method therefrom (Korean Patent Registration No. 320031)

Although Bacillus polypermanticus SCD is currently commercially available in the form of spores, the incubation time and low productivity required to make spores are a problem. Conventional manufacturing methods were used to form spores of the Bacillus spp. (About 48 hours) in order to ensure that the Bacillus sp. Techniques such as the addition of growth promoters or prebiotics and control of packaging conditions, such as starch, monosaccharides, buffers of whey protein and yoghurt mixtures, and control of packaging conditions have been used to improve bacterial survival. There is a limit. Thus, capsule technology has been widely used to protect microorganisms, cells or tissues from environmental physiological degradation and has been used to enhance the survival and delivery of bacterial cultures.

Rao et al. (1989) found that capsules of Bifidobacterium pseudolongum , consisting of cellulose acetate phthalate (CAP), increased the survival of bacteria under artificial gastric acid conditions compared to unencapsulated bacteria. Found. In general, to improve viability, microencapsulate lactic acid bacteria in cross-linked chitosan membranes prepared by emulsification / interfacial polymerization, and some biopolymers and alginates such as gellan gum, carrageenan / bean bean glue. Fix bacteria with alginate.

In particular, the polysaccharide sodium alginate is most widely used as a solid because of mild and simple fixation conditions, and forms gels when contacted with calcium and polyvalent cations. Alginate capsules (or microparticles) are stable at low pH, but are characterized by swelling and collapse or weakening in weak base conditions.

Accordingly, an object of the present invention is to obtain a vegetative cell without forming spores and to microencapsulate the strain so that many Bacillus bacteria can reach the intestine. The present invention provides a method for improving survival rate.

Another object of the present invention is to provide a capsule of optimal conditions for improving the survival rate of Bacillus polyfermentus SCD.

Still another object of the present invention is to provide a pharmaceutical product and a functional dietary supplement comprising Bacillus polyfermentus SCD having an acid resistance and bile resistance as an active ingredient.

The object of the present invention is to incubate Bacillus polyfermenticus SCD, which is a Bacillus bacterium, and then centrifuged, and the obtained cell suspension is mixed with sodium-alginate and methyl-cellulose having different concentrations, followed by capsule-forming. It was achieved by transferring the Bacillus polypermanticus SCD into a capsule to improve the viability of the Bacillus bacteria and at the same time significantly increase the productivity of Bacillus bacteria.

Hereinafter, the configuration and operation of the present invention.

The present invention comprises the steps of culturing Bacillus polyperienticus SCD that is Bacillus; Centrifuging the strain and mixing the resulting cell suspension in a sodium-alginate and methyl-cellulose mixed solution of varying concentrations; Transferring the mixed solution to a capsule-forming apparatus to coat Bacillus polyfermentus SCD into capsules; Investigating the survival rate of the coated Bacillus polypermanticus SCD in artificial gastric juice and artificial bile salt.

The main material of the cell wall used in the present invention is sodium alginate (manufactured by Sigma). Dihydrate calcium chloride (CaCl ₂ · 2H ₂ O) was purchased from Junsei and sodium citrate was purchased from Sigma. Oxbile and pepsin were obtained from Difco Laboratories and Sigma.

To measure the viable cells of Bacillus polypermanticus SCD coated with alginate capsules, capsular bacteria were isolated from the capsules according to the method of Sheu and Marshall (1993). The separated alginate capsules (0.1 g) were washed with sterile saline solution, dissolved in 1 ml of 0.1 M sodium citrate solution for 30 minutes, and then crushed with vortex. The lysate was plated on TSA plates and incubated at 37 ° C. for 12 hours to determine colony forming units (CFU / g). Thereafter, the number of colonies grown as a result of the culture was counted.

In the present invention, microbial encapsulation is obtained by obtaining vegetative cells without forming spores (about 4 to 12 hours of culture time), and thus, viscerium can be reached in the intestine with high survival rate in gastric acid and bile acid. It is characterized by the presence. In the above case, the incubation time is greatly shortened, the productivity is increased by 3 to 4 times or more, and the bispan spores must be germinated in the intestine. There is no advantage.

In order to powder Bacillus polypermanticus SCD coated with the alginate / methyl-cellulose capsules by the above method, a lyophilization method or the like can be used, which is most commonly a drying method for powdering probiotics. The obtained bispan bacterium powder is formulated into a formulation, for example, a tablet, a granule, and the like.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

EXAMPLE

Example 1 Cultivation of Strains

Bacillus polypermenticus SCD ( Bacillus polypermenticus SCD) was obtained from the research and development department of BINEX CO., LTD., Busan, Republic of Korea and stored in a vial-bottle containing 10% glycerol (v / v) at -70 ℃. Prior to encapsulation, Bacillus polyfermentus SCD preserved under the above conditions was transferred to TSB medium (Trypic Soy Broth, Diffco, Inc.) and incubated at 37 ° C. for 12 hours. All cultures were placed in a 500 ml baffle flask (100 ml of practical volume) and performed in TSB medium at 37 ° C. for 12 hours. The culture solution used for the culture was autoclaved at 121 ° C. for 15 minutes.

Example 2: Encapsulation of Bacillus polyperientus SCD

Experimental Example 1 Preparation of Giant Capsule (Extrusion Method)

All glassware and solutions used in the examples of the present invention were sterilized for 15 minutes at 121 ℃. Bacillus polyperientus SCD cultured by the method of Example 1 was obtained in the late exponential growth period, the strain was centrifuged for 15 minutes at 10,000rpm. As a result of the centrifugation, the pellet containing the strain cells was washed twice with 20 ml of normal saline sterilized under the same conditions as the centrifugation, and suspended in 10 ml of sterile saline to approximately 1.0x10 ^{8 to} 1.0x10 ⁹ cells / ml. . To the sterile vinyl bag (Seaward) were added 10 ml of the cell suspension and 80 ml of 2%, 3%, 4% sodium-alginate solution. The mixed solution was mixed thoroughly using a Stomacher 400 laboratory blender (Seaward). The mixture was then transferred to a capsule-forming apparatus and dropped into a sterilized 0.1 M CaCl ₂ solution through a Paster pipette and Tygon tube on a clean bench. After the mixed solution was solidified for 1 hour or more, an alginate capsule was obtained. 1 shows a schematic diagram of a giant capsule forming apparatus.

Experimental Example 2: Preparation of bovine capsule (using emulsification / internal gel formation method)

Small capsules were prepared using a modified method of Sheu and Marshall (1993). Microcrystalline calcium carbonate (5% CaCO ₃ , w / v) was mixed with 3% alginate containing the culture and the mixture was dispersed by high speed crushing for 4 minutes. The fast crushed mixture was dropped into oil. At the end of the dropping, the mixture was vigorously stirred until the mixture was emulsified and creamy. After 5 minutes emulsification, glacial acetic acid was added and stirring continued for 5 minutes until the calcium carbonate dissolved. A 0.1 M calcium chloride solution was quickly introduced along the beaker's wall and observed for phase separation of the oil / water emulsion. The calcium-alginate capsules in the mixture were allowed to separate for 30 minutes from the bottom of the calcium chloride layer. The separated oil layer was discharged and centrifuged at low speed to obtain a capsule, washed once with 0.85% saline and stored at 4 ° C. The capsule was sized using a 850 μm and 1.18 mm steel body.

Microstructural features of lyophilized capsular cells were examined by scanning electron microscopy (FEG-SEM, Hitachi S-4200; Joel). The capsular cells coated with the capsule preparation method were frozen at 4 ° C and lyophilized at -45 ° C for 12 hours using a vacuum tray freeze dryer (manufactured by I1 New Industrial Co., Ltd.). Samples of the capsular cells were placed on sample stubs with adhesive tape attached to both sides. Samples of the capsular cells were coated with Pt-Pb in an ion sputter (ε-1030, manufactured by Hitachi).

As shown in Figures 2 and 3, the capsules prepared in accordance with Experimental Examples 1 and 2 have an average diameter of 2.8 mm, generally have a spherical shape, and many micropores were found. When observed on a high magnification micrograph, Bacillus polyfermentus SCD cells were randomly distributed and distributed in the alginate capsule. The structure of the alginate and alginate / methyl-cellulose capsules was examined under a scanning electron microscope (SEM) after cutting the lyophilized capsule with a razor. As a result, the dried alginate-capsules are about 2.0 mm in size, have a rough spherical shape (FIG. 2) and have a corrugated surface due to the loss of moisture content during the dehydration process (FIG. 4A). Methyl-cellulose appeared in the cavities (FIG. 7), while bacteria were randomly distributed in the alginate matrix (FIG. 3).

Example 3 Survival Experiments of Bacillus Polyfermentus SCD Encapsulated

Experimental Example 1: Survival of Bacillus polyfermentus SCD Coated Under Artificial Gastric Fluid Conditions

The alkaline bacterium Bacillus polyperientus SCD is very sensitive to pH conditions and alginate capsules are known to be stable to gastric juice and degrade at higher pH conditions. Although Bacillus polypermanticus SCD can be protected by capsules with alginate, the survival of Bacillus strains may produce different results depending on the pH of the exposure medium. Therefore, in order to investigate the resistance to artificial gastric juice of Bacillus polyfermentus SCD coated with alginate capsules, the viability of Bacillus polypermanticus SCD was measured after exposure to a solution consisting of hydrochloric acid as a main component. Artificial gastric juice containing pepsin (0.1M HCl with 0.85% NaCl) was prepared by modifying the method of Rao et al. Bacillus polyfermenticus SCD coated with an alginate capsule of diameter 2.8 mm prepared according to the method of Experimental Example 1 was placed in artificial gastric juice adjusted to the following pH: approximately pH 1.5, 2.5, 4.0 and 7.0 (control) . Bacillus strain samples coated with alginate capsules were incubated at 37 ° C. for 3 hours. During the incubation, the Bacillus polyfermenticus SCD samples (0.1 g) loaded into the capsules were collected at 30 minutes intervals, washed with physiological saline, and then sterilized with vortex for 1 hour in 1 ml of 0.1M sodium citrate solution. Melted. Total viable cell number was expressed by CFU and measured by plate counting method. Diluted samples were incubated for 12 hours at 37 ° C. Mean survival of Bacillus polyperientus SCD was measured from samples diluted and plated three or more times each.

As shown in FIG. 5, as a result of examining the survival of Bacillus polypermanticus SCD coated with sodium alginate capsules prepared by varying the concentration of alginate, the number of viable cells was similar regardless of the concentration of alginate at neutral pH. At low pH, it was rapidly reduced. The viable cell count of Bacillus polypermanticus SCD coated with 2% alginate capsules was 7.0x10 ⁶ cfu / ml before exposure to gastric juice, but 1.7x10 ³ cfu / ml after exposure to gastric juice. The viable cell counts of Bacillus polypermanticus SCD coated with 3% alginate capsules decreased from 1.4 × 10 ⁷ cfu / ml to 2.5 × 10 ⁴ cfu / ml after 3 hours of exposure to artificial gastric juice. The viable cell count of Bacillus polypermanticus SCD coated with 4% concentration of alginate capsules was 1.1 × 10 ⁷ cfu / ml before exposure to gastric juice and 2.2 × 10 ⁴ cfu / ml after 3 hours of exposure. Thus, three hours after exposure to the gastric juice, the survival of Bacillus polypermanticus SCD coated with alginate capsules at 2%, 3%, and 4% concentrations was 0.02, 0.18, and 0.19%, respectively. On the other hand, after exposure to strong acid, uncoated Bacillus polyfermentus SCD and capsule-coated Bacillus polyfermentus SCD did not survive effectively under strong acid conditions. Not shown).

Betty and Stephanopoulos, on the other hand, report that the diffusion of materials through capsules containing higher concentrations of alginate is associated with the number and size of pores rather than the size of the capsule. Therefore, in order to examine the effect of capsule size on the survival of the encapsulated Bacillus polypermanticus SCD under artificial gastric juice, an alginate mixture of 3% concentration was prepared according to the capsule preparation method of Experimental Examples 1 and 2 of Example 2. Capsules were prepared.

As shown in FIG. 6, the viable cell number of Bacillus polypermanticus SCD coated with a capsule (small capsule) of 850 μm diameter was initially 4.6 × 10 ⁷ cfu / ml, and 1.0 hours after exposure to artificial gastric juice. x10 ⁴ cfu / ml (0.02% survival). As expected, the mortality of Bacillus polyperienticus SCD coated with larger capsules (2.8 mm) was lower than that of strains coated with small capsules.

Experimental Example 2: Selecting the Capsule Material by Adding Biopolymer

As reported by Betty and Stephanopoulos, the diffusion of materials through alginate capsules is related to the number and size of pores rather than the size of the capsules, so to improve the survival of the encapsulated Bacillus polypermanticus SCD against harmful substances. It is important to devise other ways to reduce the number and size of pores in the alginate matrix or to reduce the diffusion of these harmful substances into the capsule.

Thus, in order to investigate the effect of the number and size of the capsule pores on the survival of the Bacillus polypermanticus SCD coated under acid, capsule preparation according to the capsule preparation method of Experimental Examples 1 and 2 of Example 2 Various biopolymers were added to the cell suspension and mixed together at a final concentration of 0.5% (w / v). The capsule material used and the concentration used above are shown in Table 1.

As shown in Table 1, the survival of Bacillus polyperienticus SCD coated with capsules prepared by adding all the additives to alginate under artificial gastric juice was higher than that of Bacillus polyperienticus SCD coated with capsule containing only 3% concentration of alginate. High. Among capsules prepared with the addition of biopolymers, Bacillus polyfermenticus SCD coated with 3% alginate and 0.5% methyl-cellulose mixed capsules showed the highest survival, especially for artificial gastric juice (Table 2). At pH 1.5, Bacillus polypermanticus SCD coated with alginate capsules was reduced by 80-90%, while Bacillus polypermanticus SCD coated with alginate / methyl-cellulose capsules was reduced by 15-20% (80 % Survival rate). Bacillus polypermanticus SCD coated with alginate capsules mixed with 0.5% methyl-cellulose was very effective in improving bacterial survival for artificial gastric juice.

Therefore, a capsule prepared by mixing methyl-cerulose wood, which is most effective for survival of Bacillus polyfermentus SCD, with 3% concentration of alginate at different concentrations, and 3% concentration Survival of Bacillus polypermanticus SCD coated with capsules made only of alginate was compared. To obtain high viability of Bacillus polypermanticus SCD, viable cell counts were measured 2 hours after exposure to artificial gastric juice, depending on the concentrations of methyl-cellulose of 0.1, 0.3, 0.5 and 0.7%.

As shown in Table 2, the viability of Bacillus polypermanticus SCD coated with alginate / methyl-cellulose increased more compared to cells coated with alginate alone. Survival of Bacillus polypermanticus SCD increased significantly with higher concentrations of methyl-cellulose, but the viable cell count of cells coated with 0.7% concentration of alginate / methyl-cellulose under artificial gastric juice was 0.5%. It was significantly lower than alginate / methyl-cellulose (Table 2). The low viability of Bacillus polypermanticus SCD coated with alginate / methyl-cellulose of 0.7% is likely due to the limited concentration of methyl-cellulose in the polymer in the alginate matrix. As a result, it can be seen that the optimum coating material for improving the survival of Bacillus polypermanticus SCD is 3% alginate mixed with 0.5% methyl-cellulose.                     

Experimental Example 3: Survival of Bacillus polypermanticus SCD coated under artificial bile salt conditions

After passing through the stomach, the microorganism enters the upper intestine where bile is secreted. Gilliland et al. Reported that probiotic bile salt resistance should be observed when cultured in a medium containing at least 0.3% oxal to encourage conditions of the human intestine. In human gastrointestinal tracts, bile concentrations vary, but are known to be around 0.6%. An artificial bile salt solution (pH 7.2) was used to measure the bile salt resistance of Bacillus polypermanticus SCD coated with alginate capsules. Oxal was added to the sterilized filter to a final concentration of 0.6, 0.8 and 1% and the samples were incubated at 37 ° C. for 6 hours. Survival of Bacillus polyperientus SCD was measured three times by plating the appropriate dilution in the same manner as in Experiment 1 of Example 3. Survival was recorded at 2-hour intervals with exposure up to 6 hours. The effect of the capsule parameters on the survival of Bacillus polyperientus SCD exposed to bile salt solution was tested in the same way as in artificial gastric juice.                     

As shown in Table 3, the survival rates of both Bacillus polypermanticus SCD coated with alginate / methyl-cellulose capsules and alginate / methyl-cellulose capsules were both viable at 2 hours after exposure to bile salts. Decreased. However, the viable cell number of the encapsulated Bacillus polyfermentus SCD increased after 4 hours. As a result, both Bacillus polyperienticus SCD coated with alginate capsules and alginate / methyl-cellulose capsules showed higher growth rates than controls under artificial bile salts.

Experimental Example 4: Survival Comparison of Bacillus Polypermanticus SCD Coated with Undried Capsule and Lyophilized Alginate-Cellulose Capsule

Morphological Observation of Alginate Capsules after Lyophilization

Capsules prepared according to the above examples were initially selected under optical microscope to distinguish features such as wrinkles that are artificially produced in the drying and coating steps when irradiated with a scanning electron microscope (SEM). Observation was carried out under a scanning electron microscope (SEM) to know the morphology of the surface and inside of the capsule (methyl-cellulose modified alginate) selected under the light microscope. Samples prepared for observation under SEM were dehydrated with graded ethanol, dried at the critical point, coated with gold in the sputter and tested on a scanning electron microscope (SEM).

As shown in FIGS. 2 and 8, the alginate / methyl-cellulose capsules and the lyophilized alginate / methyl-cellulose capsules were observed under scanning electron microscopy, and the surface morphology of the alginate / methyl-cellulose capsules was Although the overall shape and size did not change, it is modified by the methyl-cellulose molecules (Fig. 8). As shown in FIG. 9, the scanning electron micrograph of the Bacillus polypermanticus SCD captured in the lyophilized alginate / methyl-cellulose lattice after exposure for 3 hours in artificial gastric juice, consequently methyl-cellulose Woods can be seen altering the internal structure and surface of the alginate capsules. Also, as can be seen in FIG. 10B, the surface of the alginate / methyl-cellulose capsule appears to be smooth after exposure to artificial gastric juice for 3 hours.

Resistance test of artificial gastric juice and bile salt of Bacillus polyfermentus SCD coated after lyophilization

To compare the gastric acid resistance of Bacillus polypermanticus SCD, 0.5% methyl-cellulose was added to 3% alginate capsules, and then washed twice with sterile 0.85% saline solution, followed by 2 at -75 ° C. Frozen for hours. The final product of Bacillus polypermanticus SCD coated with alginate / methyl-cellulose capsules was obtained by lyophilization using a lyophilizer (I1 Shin Engineering Co., Korea) under 15 mmtorr pressure at −45 ° C. for 24 hours. 0.1 g of undried capsules and lyophilized alginate / methyl-cellulose capsules in the above manner were placed in 100 ml of artificial gastric juice (pH 1.5, pepsin addition). Each capsule and the gastric juice mixture were incubated at 37 ° C. for 3 hours, and then the survival of Bacillus polyfermentus SCD was measured in the same manner as in Experiment 1 of Example 3, and then statistically compared. In addition, for survival comparison of the bile salts of Bacillus polypermanticus SCD, samples of undried capsules and lyophilized alginate / methyl-cellulose capsules were placed in an artificial bile salt solution containing 0.6% oxgal. Incubated at 37 ° C. for 6 hours. Survival of Bacillus polyfermentus SCD was measured three times by plating the appropriate dilution in the same manner as in Experiment 1 of Example 3.                     

As shown in Table 4, the survival of Bacillus polyperienticus SCD in lyophilized alginate / methyl-cellulose capsules per hour under artificial gastric juice (pH 1.5, pepsin addition) at 37 ° C. was 3 hours in artificial gastric juice. Survival of Bacillus strains after treatment was maintained at 1.2 × 10 5 cfu / g (60% survival). From the above results, gastric juice appears to put microparticles into the surface needle hole, resulting in loss of viability.

In addition, as shown in Table 5, the survival of the Bacillus polypermanticus SCD in the freeze-dried alginate / methyl-cellulose capsules per hour in an artificial bile salt solution at 37 ° C. showed that the freeze-dried alginate / methyl-cellulose The viable cell number of Bacillus polyperientus SCD in the Rose capsule decreased from 2.6x105 to 2.3x105 cfu / ml (90% survival) after 6 hours exposure to 0.6% Oxgal solution.                     

However, as shown in Tables 4 and 5, the survival of Bacillus polypermanticus SCD in undried alginate capsules in artificial gastric juice and bile salt solution showed the survival of strains coated with lyophilized alginate / methyl-cellulose capsules. Compared with the higher survival rate.

As described above through the above Examples and Experimental Examples, the present invention obtains vegetative cells without forming spores (film culture time is about 4 to 12 hours) and coats the stomach with microcapsules. By gaining resistance to and bile acids, the survival rate is increased, which is an excellent effect to reach a large number of bispan bacteria in the intestine. In addition, since the present invention shortens the incubation time much, productivity has an effect of increasing 3 to 4 times or more, and unlike the conventional method in which spores of Bacillus spp have to be germinated in the intestine to act as a probiotic. There is no need to worry about germination of the strain. In addition, the present invention is to produce a pharmaceutical, functional food and dietary supplement for the liver disease treatment with Bacillus polyfermentus SCD coated with capsules as an active ingredient by allowing the Bacillus polyfermentus SCD to reach the target organ safely Excellent effect on the medicine and health food industry is a very useful invention.

Claims (5)

The cultured Bacillus polyfermenticus SCD (KCCM 10104) was centrifuged, the pellet obtained above was washed and suspended with saline, and the cell suspension was washed with 2-4% sodium-alginate and 0.1-0.7. In a% methyl-cellulose mixed solution, mixed, transferred to a capsule-forming apparatus and inoculated in 0.1 M calcium chloride solution to ensure that the Bacillus polyfermentus SCD is encapsulated by the capsule to have acid and bile acid resistance. A method for improving productivity and survival of Bacillus polyfermentus SCD (KCCM 10104). The productivity of Bacillus polypermanticus SCD (KCCM 10104) according to claim 1, wherein the sodium-alginate / methyl cellulose capsule has a rough spherical shape with a diameter of 0.85 to 2.8 mm and a plurality of pores. How to improve your survival rate. delete delete A dietary supplement composition comprising a sodium-alginate / methyl-cellulose capsule of Bacillus polyfermentus SCD (KCCM 10104) according to claim 1 as an active ingredient.

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