KR100568036B1 - Bifidobacterium strain activating intestinal functions and potentiating immune functions, and probiotics containing the same - Google Patents

Abstract

The present invention is a novel Bifidobacterium long gum ( Bifidobacterium) that acts to activate the intestinal function and enhance immune function longum ) SPM1205 (Accession No .: KCTC 10630BP) and probiotics containing the same.

Description

Bifidobacterium strain activating intestinal functions and potentiating immune functions, and probiotics containing the same}

1 shows the cell morphology of Bifidobacterium spp. Isolated from feces of healthy adults 20-30 years old;

2 shows the results of fructose-6-phosphate phosphoketolase test for identifying Bifidobacterium;

3 shows genome fingerprints of Bifidobacterium spp. Using microbial uniprimer 1;

4 shows genome fingerprints of Bifidobacterium spp. Using microbial uniprimer 2;

5 shows 16s rRNA identification and BLAST search results of Bifidobacterium longgum;

6 is a graph showing the effect of Bifidobacterium strains on the corss presentation of exogenous antigen (p-OVA);

7 is a graph showing the effect of Bifidobacterium strain supernatant on NO production;

8 is a graph showing TNF-α production by Bifidobacterium strain supernatant;

9 is a photograph showing macrophage morphological changes caused by Bifidobacterium spp.

10 is a graph showing the weight change of rats due to probiotic administration;

11 is a photograph showing the distribution of microorganisms in the intestine due to probiotic administration;

12 is a graph showing the number of bacteria in feces after probiotic administration.

The present invention is a novel Bifidobacterium long gum ( Bifidobacterium) that acts to activate the intestinal function and enhance immune function longum ) SPM1205 and to probiotics containing the same.

Probiotics adhere to the intestinal mucosa of the host, are easy to cultivate, must be nontoxic and non-pathogenic to the host, have a beneficial effect on the host, produce useful enzymes or physiological end products for use by the host, survive for a long time, It must be able to withstand HCl in the host stomach and bile salts of the small intestine. Suit probiotic agents that are used include Lactobacillus ash FIG pillar's (Lactobacillus acidophilus), Lactobacillus bifidus (Lactobacillus bifidus), Streptococcus faecalis (Streptococcus faecalis), Streptococcus passive help (Streptococcus faecium), Bacillus subtilis (Bacillus subtilis ), Bacillus licheniformis , Bacillus polyfermenticus , Bacillus mesentericus , Saccharomyces cerevisiae , Clostridium butytricum butyricum ). However, probiotics that have beneficial effects such as strengthening the immune function of the human body have not been developed yet.

Bifidobacterium is a non-pathogenic Gram-positive, anaerobic bacterium that maintains the balance of intestinal microflora, improves lactose resistance, decreases serum cholesterol levels, increases vitamin synthesis, inhibits carcinogenesis, enhances immune function, and prevents cancer. It is known to have a beneficial effect. Table 1 summarizes the characteristics of Bifidobacterium.

Characteristics of Bifidobacterium Cell morphology High variable; Characterized by an amphora-like cell colony Colony form Soft, convex, creamy to white, shiny and pale Gram dyeing Gram-positive Aerobic hyperplasia No growth in the presence of O ₂ Optimum growth temperature 37 ~ 41 ℃ Optimum pH for First Growth 6.5-7.0 Fermentation products Acetic acid Glucose breakdown Glucose is proprietary and characteristically degraded by fructose-6-phosphate shunt habitat Feces of infants, adults and suckling calves; Human vagina

The present inventors have isolated new Bifidobacterium strains from human feces, found strains that have beneficial effects on the human body such as activating intestinal function and enhancing immune function, and completed the present invention.

It is therefore an object of the present invention to provide a novel Bifidobacterium long gum SPM1205 and a probiotic containing the same, which show intestinal function activation and immune function enhancing action.

The present invention relates to Bifidobacterium longum SPM1205 (Accession No .: KCTC 10630BP), which has intestinal function activation and immune function enhancing action.

The present invention also relates to a probiotic containing said Bifidobacterium strain. The probiotic according to the present invention can be used as a formal or immune enhancing agent.

Hereinafter, the present invention will be described in detail.

We collected feces from middle-aged, healthy Korean adults with normal eating habits and selected obligate anaerobic Bifidobacterium strains from selection media. Several Bifidobacterium strains were also purchased from ATCC and KCTC. The inventors conducted a Bifidobacterium RAPD (Random Amplified Polymorphic DNA) pattern analysis using a Uniprimer kit, and identified specific probes of Bifidobacterium strains. In order to confirm the physiological characteristics of Bifidobacterium, pH-dependent proliferation and inhibition of growth and glycosylation at temperature were investigated. In order to confirm immune cell activation by Bifidobacterium, the effects of cross-presentation of exogenous antigens and the effects on NO production were evaluated. In addition, mouse macrophage lines RAW 264.7 cells were treated with selected Bifidobacteria, TNF-α production level results were tested and morphological changes of macrophages were observed. In addition, in order to test the suitability and safety of Bifidobacteria, weight comparison and hematological and histological examinations of the animals were performed and microbial distribution in the intestine was observed.

As a result of the above experiments, the present inventors discovered a new Bifidobacterium strain, Bifidobacterium longgum SPM1205, which acts to activate the intestinal function of the human body and at the same time enhance the immune function.

Bifidobacterium longgum SPM1205 of the present invention was deposited on May 10, 2004 at the Korea Institute of Bioscience and Biotechnology, 52 Ueo-dong, Yuseong-gu, Daejeon under the Treaty of Budapest, and was assigned accession number KCTC 10630BP.

Hereinafter, the present invention will be described in detail by way of examples, which are intended to aid the understanding of the present invention, but are not intended to limit the scope of the present invention in any way.

Example  One: Bifidobacterium Strain  Isolation, Culture and Identification

Bifidobacterium was isolated from feces of healthy adults aged 20-30. To maintain anaerobic conditions, samples were used from BBL's anaerobic sample collection & transport system and used within a 24-hour limit.

As a selection medium for Bifidobacterium, 5% sheep blood was added to BL broth (Blood-Liver, Nissue), and GAM (general anaerobic medium, Nissue) was used as a growth medium. Skim milk broth was used as a medium for maintaining bacterial strains.

Anaerobic culture of Bifidobacterium was carried out using a Bactron Anaerobic Chamber (Sheldon Manufacturing Inc).

In order to count the viable cells, in the bifidobacteria culture bacteria BL agar medium (5% sheep blood was added) and were incubated under anaerobic conditions for 48 hours at 37 ℃ were counted for viable cells with 10 ^{4-10 5} concentrations. Bifidobacteria were selected in the form of light brown, milky white and tan brown with a reddish brown central portion of 0.5 mm to 2.0 mm. When viewed under the microscope, the V, Y, X forms of Bifidobacterium, which show typical Bifidobacterium morphology, were selected. The cell morphology of Bifidobacterium spp. Is shown in FIG.

Bifidobacterium spp. Was also identified using the fructose-6-phosphate phsphoketolase test. The results are shown in FIG. In FIG. 2, reddish violet is a positive result and yellow is a negative result.

Bifidobacterium strains isolated from healthy adults are as shown in Table 2 below.

Bifidobacterium Strain Isolated from Healthy Adults number gender age Diet Number of bifidus isolates Strain One M 26 Omnivorous nd 2 F 21 Omnivorous 3 ^ａ  SPM0211, SPM0212, SPM0214 3 F 22 vegetarian 1 ^a  SPM0308 4 M 33 Omnivorous nd 5 M 21 Omnivorous nd 6 F 20 vegetarian nd 7 M 25 Omnivorous nd 8 F 22 Omnivorous nd 9 M 25 Omnivorous nd 10 M 25 Omnivorous 1 ^a  SPM1005 11 F 22 vegetarian nd 12 F 22 Omnivorous 4 ^ａ  SPM1204, SPM1205, SPM1206, SPM1207 13 M 24 Omnivorous 3 ^ａ  SPM1307, SPM1308, SPM1309 14 M 24 Omnivorous nd 15 F 22 Omnivorous nd 16 M 20 vegetarian 7 ^a  SPM1601, SPM1602, SPM1603, SPM1604, SPM1605, SPM1606, SPM1608

nd: no detection a: number of strains isolated from fecal sample

Example  2: Bifidobacterium spp . From  Genome DNA  Manufacturing and purification ( PCR - RAPD )

After removing the supernatant by centrifuging the bacteria obtained in Example 1 at 5,000 rpm for 10 minutes, the precipitate was suspended by adding 12% sucrose and 1 ml of 25 mM Tris-HCl (pH 8.0). This procedure was repeated two more times and finally 400 μl of this buffer was added and finally resuspended. Subsequently, mutanolysine (Sigma, St. Louis, MO; 100 µg) and lysozyme (Boehringer Mannheim Canada, Laval, QC, Canada; 200 µg) were added to the bacterial solution, and reacted at 37 ° C for 2 hours. After the reaction was completed, 100 µl of 10% sodium dodecyl sulfate (SDS), 200 µl of 250 mM EDTA (pH 8.0) and 1 µg of protease K (Boehringer Mannheim) were added thereto, and reacted at 37 ° C. for 2 hours. . After the reaction, 200 μl of CTAB / NaCl solution (10% hexadecyltrimethyl ammonium bromide in 0.7M NaCl) and 240 μl of 5 M NaCl were added and the mixture was left at 65 ° C. for 20 minutes. Again, the mixture was treated with 1.4 ml of chloroform / isoamyl alcohol in a 24: 1 ratio to remove the protein, and then 1.0 ml of cold isopropanol was added to precipitate the genomic DNA, the impurities were removed with 70% ethanol and dried, 200 Μl of deionized water was added to lyse the genomic DNA.

Polymerase chain reaction and genetic fingerprinting were performed to identify the genetic characteristics of the Bifidobacterium spp. The polymerase chain reaction included a total of 30 containing 2.5 units of Taq DNA polymerase, 1.5 mM MgCl ₂ , 0.25 mM dNTPs, 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 10 pmol primer and 200 ng genomic DNA. It was performed in μl volume. The amplification reaction was performed using an amplifier of Perkin Elmer, and primers for performing gene fingerprint analysis were purchased from Serene Biosciences (Korean Patent Application No. 97-16981) (Table 3).

Nucleotide Sequence of the SRILS Microbial UniPrimers ™ Kit Used primer Sequence (5 '→ 3') Microbial Uniprimer 1 5’-ATCCAAGGTCCGAGACAACC-3 ’ Microbial Uniprimer 2 5’-CCCAGCAACTGATCGCACAC-3 ’ Microbial Uniprimer 3 5’-GTGTGCGATCAGTTGCTGGG-3 ’ Microbial Uniprimer 4 5’-AGGACTCGATAACAGGCTCC-3 ’

The reaction conditions consisted of 40 cycles of 30 seconds at 94 ° C, 1 minute at 40-50 ° C, and 2 minutes at 72 ° C. After the polymerase chain reaction was completed, the amplification products were separated by electrophoresis for 5 hours on a 2% agarose gel, and the results were read by ethidium bromide staining. In this experiment, the same experiment was repeated twice to investigate the reproducibility of the genetic fingerprinting method.

The result of polymerase chain reaction analysis using microbial uniprimer 1 is shown in FIG. 3 (lane M1: size marker 1 kb ladder; lane 1: Bifidobacterium) catenulatum KCTC 3221; Lane 2: B. infantis KCTC 3127; Lane 3: B. breve KCTC 3220; Lane 4: B. longum KCTC 3128; Lane 5: B. KCTC 3273; Lane 6: B. infantis KCTC 3368; Lane 7: B. KCTC 3271; Lane 8: B. gallicum KCTC 3277; Lane 9: Lactobacillus ruminus KCTC 10615BP SPM0211; Lane 10: SPM0214; Lane 11: B. pseudocatenulatum KCTC 10616BP SPM1204; Lane 12: B. longum SPM1205; Lane 13: B. longum SPM1206; Lane 14: SPM1207; Lane 15: SPM1307; Lane 16: SPM1601; Lane M2: size marker 100 bp ladder). In the polymerase chain reaction, Lactobacillus ruminus KCTC10615BP SPM0211, B. pseudocatenulatum KCTC 10616BP SPM1204, and B. longum SPM1205 showed different band patterns from the investigated strains.

The result of polymerase chain reaction analysis using microbial uniprimer 2 is shown in FIG. 4 (lane M1: size marker 1 kb ladder; lane 1: Bifidobacterium) catenulatum KCTC 3221; Lane 2: B. infantis KCTC 3127; Lane 3: B. breve KCTC 3220; Lane 4: B. longum KCTC 3128; Lane 5: B. KCTC 3273; Lane 6: B. infantis KCTC 3368; Lane 7: B. KCTC3271; Lane 8: B. gallicum KCTC3277; Lane 9: Lactobacillus ruminus KCTC10615BP; Lane 10: SPM0214; Lane 11: B. pseudocatenulatum KCTC 10616BP SPM1204; Lane 12: B. longum SPM1205; Lane 13: B. longum SPM1206; Lane 14: SPM1207; Lane 15: SPM1307; Lane 16: SPM1601; Lane M2: size marker 100 bp ladder). In the polymerase chain reaction, Lactobacillus ruminus KCTC10615BP SPM0211 and B. pseudocatenulatum KCTC10616BP SPM1204 showed a different band pattern from other strains studied, but B. longum SPM1205 showed a similar band pattern to SPM1207.

However, Lactobacillus ruminus KCTC 10615BP SPM0211, B. pseudocatenulatum KCTC 10616BP SPM1204, and B. longum SPM1205 showed specific band patterns.

5 shows the results of 16s rRNA identification and BLAST search of Bifidobacterium long gum SPM1205.

Experimental Example  One: Bifidobacterium spp Characterization of.

PH dependent proliferation of Bifidobacterium spp. And inhibition of proliferation at temperature (75 ° C.) are shown in Tables 4 and 5.

PH-dependent Growth of Bifidobacterium spp. Strain / pH 3 4 5 6 7 8 10 SPM0211 - + + + + + + SPM0212 - + + + + + + SPM0214 - - + + + + + SPM0308 - - + + + + + SPM1005 - + + + + + + SPM1204 - - + + + + + SPM1205 - - + + + + + SPM1206 - - + + + + + SPM1207 - - + + + + + SPM1307 - - + + + + + SPM1308 - - + + + + + SPM1309 - - + + + + + SPM1601 - - + + + + + SPM1602 - - + + + + + SPM1603 - - + + + + + SPM1604 - - + + + + + SPM1605 - - + + + + + SPM1606 - - + + + + + SPM1608 - - + + + + + B. longum KCTC3128 - - + + + + + B. bifidum KCTC3202 - - + + + + + B. breve KCTC3220 - - + + + + + B. catenulatum KCTC3221 - - + + + + + B. gallicum KCTC3277 - - + + + + +

Inhibition of growth at temperature (75 ° C) Strain / hour 5 minutes 10 minutes 20 minutes 30 minutes SPM0211 - - - - SPM0212 - - - - SPM0214 - - - - SPM0308 - - - - SPM1005 - - - - SPM1204 + - - - SPM1205 - - - - SPM1206 - - - - SPM1207 - - - - SPM1307 - - - - SPM1308 + - - - SPM1309 - - - - SPM1601 + - - - SPM1602 + + + - SPM1603 - - - - SPM1604 - - - - SPM1605 - - - - SPM1606 - - - - SPM1608 - - - - B. longum KCTC3128 - - - - B. bifidum KCTC3202 - - - - B. breve KCTC3220 - - - - B. catenulatum KCTC3221 - - - - B. gallicum KCTC3277 - - - -

In addition, the glycosylation test results of Bifidobacterium long gum SPM1205 are shown in Table 6.

Glycolytic Activity of Bifidobacterium Longgum SPM1205 No. Party Bifidobacterium longgum SPM1205 0 contrast - One L-Arabinose - 2 D-ribose - 3 Xylose - 4 Galactose + 5 Fructose - 6 Mannos - 7 Mannitol - 8 Sorbitol - 9 Salinity - 10 Cellobiose - 11 Maltose - 12 Lactose - 13 Melibiose + 14 Saccharose - 15 Trehalose - 16 Inulin - 17 Melezitos - 18 Raffinos - 19 Starch - 20 Gluconate -

Experimental Example  2: Bifidobacterium spp Evaluation of cross presentation

The same dose (10 mg / ml) of p-OVA was added to a culture of DC 2.4 cells and incubated for 2 hours, followed by different supernatants of Bifidobacteria (ATCC3127, ATCC3128, ATCC3220, ATCC3221, ATCC3277, ATCC3368, SPM0214, SPM1204, SPM1205, SPM1206, SPM1207, SPM1601, SPM1602, SPM1606, SPM1608). The amount of cross-presented OVA peptide was then measured by B3Z T hybridoma activation assay. The results are shown in FIG. The result is the mean ± standard deviation of three independent experiments. As can be seen from FIG. 6, the supernatant of Bifidobacteria was found to enhance the cross-presentation of exogenous antigen (p-OVA).

In addition, the cross-presentation promoting effect of DC 2.4 of Bifidobacterium spp. Is shown in Table 7. In Table 7, DC 2.4 cells (4 × 10 ⁴ cells / well) were collected from p-OVA (1 mg / well) and various Bifidobacterium spp. It shows the% enhancement activity when incubated with the supernatant. 100% positive control was measured with p-OVA (1 mg / well) alone.

Cross-presentation Enhancement Effect of Bifidobacterium spp. By DC 2.4 Strain activation% SPM0214 SPM1204 SPM1205 SPM1206 SPM1207 SPM1601 SPM1602 SPM1606 SPM1608 B. infantis ATCC3127 B. longum ATCC3128 B. breve ATCC3220 B. catenulatum ATCC3221 B. gallicum ATCC3277 B. infantis ATCC3368 118.4 131.7 127.6 120 125.1 122.7 124.4 120.6 116.3 128.8 111.2 123.9 113.9 112 115.7

Experimental Example  3: Bifidobacterium Strain Supernatant  Impact on NO Production

The effects on NO production of the supernatant of ATCC bifidobacteria strains (15707, 27919) and bifidobacteria strains (SPM1204, SPM1205) in RAW 264.7 cells were investigated. 6 wells per group were used and 200 μl of cells were added to each well. Plates were then incubated overnight and 100 μl from the surface of each well was transferred to a new plate. Accumulated nitrite concentration was measured overnight after stimulation with Griess reagent for 10 minutes at room temperature and read with an ELISA reader at 540 nm. NO production was determined by nitrite accumulation using this method. The results are shown in Figure 7 as the mean ± standard deviation of three independent experiments.

If a thorn gets stuck in the hand, the thorn enters the body. At this time, mast cells in the body help secrete histamine and cytokines. In other words, it tells us that something is wrong with our body. Blood vessels around the finger create gaps between the blood vessels that allow other immune cells to reach the site. Through these gaps, macrophages run to eat bacteria and damaged cells. Subsequently, immune cells arrive and go through this process to the therapeutic stage. The swelling of the area when the thorns are stuck in the hand is due to this reaction. In most cases, inflammation is a helper that helps our body fight against various disease-causing bacteria or viruses. At this time, nitric oxide (NO) is used to create gaps between blood vessels so that immune cells can quickly move to inflammation sites. Therefore, it can be concluded that the bifidus strain used in this experiment had an immune function activation.

Experimental Example  4: Bifidobacteria Strain In supernatant  by TNF -α production

TNF-α production by supernatants of several Bifidobacteria strains in mouse macrophage lines was measured with increasing concentrations of Bifidobacteria (2.5, 5 and 10 ml). RAW 264.7 cells (5 × 10 ⁵ cells / ml) were incubated for 48 hours with some Bifidobacterial supernatant. TNF-α bioassays were measured by their proliferation in TNF-α sensitive cell line, WEHI-16. The results are shown in Figure 8 as the mean ± standard deviation of three independent experiments.

Macrophage function can be summarized in nine ways:

1) Healing damaged tissue by producing growth factors of fibroblast and vascular endothelial cells

2) destruction of tumor cells by activated macrophages (action of TNF-α),

3) Destroys microorganisms, antigens, dead tissues and red blood cells by phagocytosis in natural immunity,

4) regulate the inflammatory response by secreting substances or cytokines involved in the inflammatory response,

5) secretion of blood coagulation or complement proteins, angiogenesis factors,

6) antigen processing and presentation in the acquired immune response,

7) acts as effector cells in humoral immune responses,

8) the ability to remove antigens from delayed type hypersensitivity,

9) Regulates the immune response by secreting cytokines that regulate the action of immune cells.

From the function of the macrophages as described above, it can be seen that the bifidus secrete a substance or cytokine (TNF-α, IL1β) involved in the inflammatory response during macrophage treatment to regulate the inflammatory response.

Experimental Example  5: Bifidobacteria spp Changes in macrophage morphology by

Macrophage morphological changes were observed for Bifidobacterium spp. RAW cells were cultured on cover slips in the presence of different concentrations of medium (a), LPS (10 ng / ml: b), SPM1205 (3 μl / ml: c), ATCC25962 (3 μl / ml: d). Cells were immobilized and stained with Diff-quick and observed under x400 optical microscope. The results are shown in FIG.

In general, macrophages are known to have a smooth and rounded shape in dormant state, and when activated, the cell size increases and the cell surface changes roughly. Therefore, it was confirmed that the macrophages are activated when the bifidus strain is treated in the macrophage line.

Experimental Example  6: Probiotic  Dress Effect and Safety Assessment

1) weight comparison

Figure 10 (SB6: B. Longgum SPM1205; DUOLAC: Probiotics developed by Cell Biotech, and mixed four Lactobacillus strains and one Bifidobacterium strain for each experimental animal group One formulation). It was found that there was no significant difference in weight gain in each group (p> 0.05), and thus there was no change in growth pattern due to probiotic administration.

2) Heematology

Experimental animals were anesthetized with diethyl ether and opened, and blood was collected from the heart. The collected blood was immediately placed in EDTA-treated bottles and some in normal bottles to prevent blood clotting. The results are shown in Table 8.

Blood biochemical measurements of rats fed different probiotics contrast Bifidobacterium longgum SPM 1205 DUOLAC S-GOT (IU / l) 98 ± 13 103 ± 11  108 ± 15 S-GPT (IU / l) 45 ± 11 46 ± 13  40 ± 7 Total Protein (g / dl) 6.4 ± 0.2 6.12 ± 0.3 6.05 ± 0.3 Albumin (g / dl) 2.76 ± 0.1 2.78 ± 0.09 2.77 ± 0.14 Total cholesterol (mg / dl) 74 ± 8 69 ± 7 78 ± 12

As shown in Table 8, there was no difference in hematologic findings in the present probiotic-administered group, from which the safety of the probiotic was confirmed.

3) Histology

Experimental animals in each group were anesthetized with diethyl ether, and collected immediately after taking blood samples, and washed with physiological saline and weighed. The results are shown in Table 9.

Comparison of organ weights between control and probiotic groups Control Bifidobacterium longgum SPM 1205 DUOLAC kidney 2.25 g 2.35 g 2.35 g Zira 0.55 g 0.6 g 0.6 g liver 11.6 g 13 g 13.8 g

As shown in Table 9, the appearance and weight of organs in each group were confirmed without any significant difference.

4) Distribution of microorganisms in the intestinal tract

After the experiment, the colon parts of each experimental animal were cut and the intestine contents were simply stained and observed under a microscope. The results are shown in Figure 11 (a: control; b: B. long gum SPM 1205 administration group; c: DUOLAC administration group). As shown in FIG. 11, in the case of the control group, the distribution of the microbial group of the probiotic-administered group was simple and mainly bacilli, compared to the various and complicated types of bacteria. This is thought to be a change in intestinal flora caused by probiotic administration.

In addition, the number of bacteria in feces after probiotic administration was measured, and the results are shown in FIG. 12.

In view of the above results, it can be concluded that B. long gum SPM 1205 of the present invention may exhibit constipation, diarrhea improvement effect, etc. by the maintenance of beneficial intestinal bacteria.

According to the present invention, there is provided a Bifidobacterium strain having a beneficial effect on the human body such as activating intestinal function and enhancing immune function, and a probiotic containing the same.

Claims (3)

Bifidobacterium longum SPM1205 (Accession Number: KCTC 10630BP), which acts to activate intestinal function and enhance immune function.  Probiotic containing a strain according to claim 1. The probiotic according to claim 2, which is used as a formal or immune enhancing agent.

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