KR20000007167A - Bifidobacterium longum mk-g7 bifidus strain and this use - Google Patents

Abstract

PURPOSE: A novel bifidus strain is provided which is transferred into an intestine in a certain amount to show a profitable effect in the host. CONSTITUTION: Bifidobacterium longum MK-G7 is provided which represents proper physiological activation to Korean, has excellent adaptability to cultivation in the fermented products, cholesterol lowering activity and microphage activation effect and also tolerance to acid, bile, air and mutation.

Description

Bifidobacterium rongum ＭＫ-Ｇ７ Bifidus strain and use thereof

The present invention is a novel Bifidobacterium longum MK-G7 bifidus strain having excellent physiological activities such as excellent culture application ability, acid resistance, bile resistance, oxygen resistance, cholesterol lowering ability, antimutagenicity, macrophage activity, and the like. To its use.

Microbial food enhancers that enhance the desirable balance of intestinal microorganisms and their physiological properties when they are administered to humans or animals in the form of powders or fermented products are known as probiotics (Fuller, R). A review: Probiotics in man and animals.Journal of Applied Bacteriology.66: 365-378; Lee, YK and S. Salminen. 1995.The coming of age of probiotics.Trends in Food Science & Technology. 6: 241 -245; O'Sullivan, MG, G. Thornton, GCO'Sullivan, and JK Collins. 1992. Probiotic bacteria: Myth or reality? Trends in Food Science & Technology. 3: 309-314).

In particular, because bifidus bacteria exist as the most dominant bacteria in the intestines of breastfeeding infants, infants prevent bacterial diarrhea by preventing intestinal proliferation of harmful bacteria from the outside, such as E. coli, and have the ability to prevent various kinds of bacterial diseases. It is known. These bifidus bacteria decrease with age, and it is known that both the detection rate and the number of bacteria decrease, especially in the elderly.

The beneficial health-promoting effects of bifidus bacteria have been reported in various physiological activities such as resistance to infection, anti-cancer and anti-tumor effects, lowering blood cholesterol and intestinal effects (Hentges, DJ 1983. In: Human intestinal microflora in health). and disease.Academic Press.pp.241-261; Hosono, A., R. Wardardo, and H. Otani. 1990. Inhibitory effects of lactic acid bacteria from fermented milk on the mutagenicities of volatile nitrosoamines.Agricultural and Biological Chemistry. 54 (7): 1639-1643). Therefore, it is important to develop excellent modified strains of lactic acid bacteria such as bifidus as probiotics (Klaenhammer, TR 1982. Microbiological considerations in selection and preparation of Lactobacillus strains for use as dietary adjuncts. Journal of Dairy Science) 65: 1339-1349).

Increasingly, applications of bifidus bacteria in the food industry are increasing, but digestive enzymes are secreted from the gastrointestinal and small intestine in which the bifidus bacteria are organized anaerobic bacteria, and when taken orally, in a fasting environment, ie, the pH drops below 3.0. And bile acids inhibit the growth of bifidus bacteria, which leads to a decrease in the number of bacteria reaching the large intestine and a decrease in the intestinal fixation rate (Mitsuoka, T. 1989. Microbes in the intestine.In: Our Lifelong Partners). . pp.1-18).

In fact, the current bifidus bacteria added in the manufacturing process of most lactic acid bacteria fermented milk products such as yogurt has a fundamental problem that is not grown but rather killed. Therefore, it is reported that even if the ingested bifidus lactobacillus fermented milk produced and marketed as described above is ingested, the number of bifidus bacteria that reach and settle in the large intestine is not sufficient to have a beneficial effect on the host (Desjardins, ML, D. Roy, C.Toupin , and J. Golet. 1990. Uncoupling of growth and acids production in Bifidobacterium spp. Journal of Dairy Science. 73: 1478-1484).

In addition, since the lactic acid bacteria currently used in fermented milk products in Korea are strains derived from foreigners, it has been pointed out as a problem that they do not exhibit the physiological activity suitable for Koreans.

The present invention has been devised in view of the problems of the conventional bifidobacteria, the purpose of which is excellent culture application ability in fermented milk products, acid resistance, bile resistance and oxygen resistance, cholesterol lowering ability, antimutagenicity, macrophage activity, etc. It is to provide a novel bifidus strain having excellent physiological activity suitable for Koreans.

1 is a photograph showing the colony characteristics of the bifidus strain according to the present invention,

Figure 2 is a photograph showing the enzyme pattern (using the API ZYM Test Kit) of the bifidus strain according to the present invention,

Figure 3 is a photograph showing the carbohydrate fermentation properties (using the API 50 CHL Test Kit) of the bifidus strain according to the present invention,

4 is an optical micrograph (× 1,500) of the bifidus strain according to the present invention,

5 is a photograph showing the result of PFGE analysis of the bifidus strain according to the present invention,

6 is an electron micrograph (× 12,000) of the bifidus strain according to the present invention,

7 is a graph showing a change in the number of viable cells during the culture of the bifidus strain according to the present invention,

8 is a graph showing the change in viable cell number during storage of the product containing the bifidus strain according to the present invention.

In order to invent the bifidus strain as described above, the present inventors separated over 200 bifidus strains from 100 healthy feces from healthy Korean infants to young adults, and analyzed acid resistance, bile resistance, and acid resistance of the isolated bifidus strains. The best bifidus strains were selected first by comparing the firing.

In order to test the physiological activity of the selected bifidus strains, the cholesterol lowering ability, antimutagenicity, macrophage activating ability, etc. were examined in vitro, and the most physiologically active bifidus strains were identified. And a novel bifidus strain that is completely different from the standard test strain.

Therefore, the present inventors named this new Bifidobacteria strain Bifidobacterium longum MK-G7 and deposited it as KCTC 0488 BP at the Genetic Resource Center Gene Bank of Korea Research Institute of Science and Technology.

Unlike the conventional bifidus strain, the strain according to the present invention is grown alone or in mixed culture of Lactobacillus ( Lactobacilli ) and Streptococci ( Streptococci ), the growth of bifidus strains is not less than 10 ⁸ cfu / ml level, as well as the shelf life of fermented milk Almost no death occurred during the process, and the product applicability was confirmed to be excellent.

Therefore, the strain according to the present invention can be used in a variety of products, such as fermented milk products, baby food, bifidus fortified milk, lactic acid bacteria formulations, dietary supplements, feed additives, pharmaceuticals, cosmetics.

Hereinafter, the present invention will be described in more detail based on Examples. The following examples are provided to facilitate the understanding of the present invention, and the scope of the present invention is not limited to these examples.

Example 1 Separation of Bifidus Strains from Natural Products

Fecal samples were collected from more than 100 healthy Korean adults and infants, and bifidus bacteria were isolated using TP medium, which is a selection medium of bifidus bacteria.

Diarrhea was diluted to 10 ⁻⁸ using anaerobic dilution, then 100 μl of each dilution was plated in TP medium. The composition of the anaerobic diluent used for dilution of feces is shown in Table 1.

Composition of ingredients in anaerobic diluent ingredient content Remarks 0.78% K ₂ HPO ₄ 37.5 ml Salt solution ¹⁾ 37.5 ml Resazurine (0.1%) 1.0 ml L-cysteineHClH ₂ O 0.5 g L-ascorbic acid (25%) 2.0 ml Na ₂ CO ₃ (8%) 50.0 ml Agar 0.5 g Distilled water 860.0 ml 1) Salt solution: prepared by dissolving 0.47 g of K ₂ HPO ₄ , 1.18 g of NaCl, 1.20 g of (NH ₄ ) ₂ SO ₄ , 0.12 g of CaCl ₂ and 0.25 g of MgSO ₄ · H ₂ O in 100 ml of distilled water.

Component composition of the TP medium is shown in Table 2.

Component Composition of TP Medium ingredient content Remarks Trypticase peptone 10.0 g BBL Proteose peptone No. 3 5.0 g Ammonium Sulfate 3.0 g KH ₂ PO ₄ 2.0 g K ₂ HPO ₄ 1.0 g L-cysteineHCl 0.5 g Magnesium sulfate 0.2 g Agar 15.0 g Sterilized 20% TOS Solution ¹⁾ 50.0 ml Sodium propionate (pH 7.0) 50.0 ml Distilled water 1,000 ml 1) TOS: Transgalactooligosaccharide ※ Final pH is adjusted to 7.0

The medium was replaced with nitrogen in an anaerobic jar using a gas pack system and an anaerobic glove box to form an anaerobic environment. Incubated for -72 hours.

In this way, 200 isolates of bifidus strains were first isolated, and then, the subjects were examined for acid resistance, bile resistance, and oxygen resistance of the bifidus strains to select new bifidus strains with excellent viability and product application ability against stress environment. It was named Bifidobacterium longum MK-G7. During the selection process, identification and identification of new bifidus bacteria, acid resistance, bile resistance, and oxygen resistance were performed as follows.

Example 2 Identification of Bifidobacteria

Bifidobacterium longum ( Bifidobacterium longum ) MK-G7 according to the present invention is shown in Figure 1 the colony formed as a result of culturing in the anaerobic jar (anaerobic jar), solid TP medium of Example 1.

Carbohydrate fermentation using the API 50 CHL test kit (API, France) showed a typical carbohydrate fermentation of Bifidobacterium longum . The enzyme pattern of (Fig. 2, 3) was shown.

On the other hand, after gram staining was observed under a microscope to confirm that the strain is a bifidus strain showing a polymorphism (Fig. 4).

Scardovi (Scardovi, V. 1986. Genus Bifidobacterium . In: Bergey's Manual of Systematic Bacteriology . 9th Ed. Vol. 2. Ed.) As a culture medium inoculated by inoculation into BHI (brain heart infusion) liquid and solid medium. F6CTK (fructose-6-phosphate phosphoketolase) experiments were performed according to the method of Sneath, PH, NSMair, MESharpe, and JGHolt.pp. 1418-1434. Williams and Wilkins Publishers. Yellow reaction solution was negative, purple reaction solution was determined to be positive, and acetic acid and lactic acid were produced at a ratio of 3: 2 for the confirmation experiment of bifidus bacteria by gas chromatography.

Example 3 Molecular Biology of Selected Bifidus

(1) 16S rRNA analysis and DNA fingerprinting

① Preparation of RNA

Cells were harvested from 10 ml of bifidus culture and suspended in 0.5 ml of ice-cold TE (pH 7.5) buffer, followed by 0.6 g glass beads (0.1 mm or less), 0.17 ml macaloid clay, 0.5 ml Phenol-chloroform (1: 1), 50 μl 10% SDS were added and vigorously mixed at 4 ° C. for 5 minutes and then centrifuged at 12,000 rpm for 15 minutes.

The supernatant was extracted with phenol-chloroform (1: 1) solution to precipitate RNA for 30 min at -20 ° C with 1/10 fold of 3 M sodium-acetate and 3 folds of 96% ethanol, followed by centrifugation (12,000 rpm, 15 minutes), the precipitated pellet RNA was washed with 70% ethanol. The pellet RNA was then dried at room temperature and suspended in 50 μl TE pH 7.5 buffer.

② Preparation of DNA for Sequence Analysis

Cells were recovered from 10 ml of bifidus culture incubated overnight, washed with 1 ml TES (20 mM Tris-HCl, 0.1 mM Na ₂ EDTA, 10 mM NaCl) buffer and suspended in 100 μl TES buffer. Lysozyme (Lysozyme) and mutanolysine (mutanolysin) was treated for 15-30 minutes at 37 ℃ and dissolved with SDS (sodium dodecyl sulfate) for 5 minutes at 65 ℃.

Thereafter, the mixture was incubated on ice for 1 hour by addition of NaCl, centrifuged at 12,000 rpm for 20 minutes, and DNA was extracted with the same amount of chloroform / isoamyl alcohol. Purified DNA was precipitated with 1/9 times 3M sodium acetate solution and 2 times 96% ethanol and washed with 70% ice-cold ethanol, followed by vacuum drying the DNA sample to PCR (polymerase). chain reaction). Different parts of the 16S rRNA gene were amplified by binding to the primers of Table 3.

Primers for Sequence Analysis name goal location Sequence 616v 16S 8-27 AG (G / A) GTTTGAT (T / C) C (G / T) GGCTAG 612r 16S 969-984 GTAAGGTTCTTCGCGT 610r 16S 515-531 ACCGCGGCTGCTGGCAC 612v 16S 515-531 ACGCGAAGAACCTTAC 630rII 16S 189-206 CA (T / G) AAAGGAGGTGATCC 941r 23S 1529-1545 ACT (G / A / T) AGATGTTTCA (G / C) TTC * Primers are referred to as universal primers * Position means the nucleotide number of 16S rDNA of E. coli corresponding to the oligo.

16S rDNA gene fractions obtained from the PCR mixture were separated by electrophoresis using 1% TBE (89 mM Tris-borate, 2 mM EDTA) buffer. After ethidium-bromide staining and UV irradiation, gels containing DNA fragments were cut to size and purified using a gel extraction kit (QiaGen), and 16S rDNA fragments were analyzed for nucleotide sequence using the same primers. It was.

③ DNA preparation for DNA fingerprinting

1) Insert DNA Preparation

: The recovered cells were suspended in 50 mM EDTA (pH 8.5) and fixed to 1% low melting agarose. Fixed cells were lysozyme treated at 37 ° C. for 3-4 hours to lyse, and then reacted with 1% SDS and 2 mg / ml proteinase K (0.5 M EDTA, pH 8.5) at 50 ° C. overnight. Finally, the fixed cells were washed at least 5 times with 50 mM EDTA (pH 8.5) and then stored in 4 mM in 50 mM EDTA (pH 8.5). The DNA thus prepared was called insertion DNA.

2) Direct Insert DNA Preparation

: A small amount of lyophilized powder was suspended in 50 mM EDTA (pH 8.5) to recover the cells, and washed with the same buffer one or more times to pellet. Cells were again suspended in 50 mM EDTA (pH 8.5) and fixed in 1% low melting agarose. Meanwhile, DNA preparation for DNA fingerprinting was performed as in 1).

(2) RNA dot blotting and hybridization

: 150 μl of 66% formamide was added to 50 μl of RNA solution, denatured at 65 ° C. for 10 minutes, and immediately transferred to ice water. RNA was blotted onto the gene screen plus membrane of minifold (Shleicher & Schuell, SRC 96-D) and then heated at 80 ° C. for 80 minutes. Wet the filter with distilled water, 5 × SSC (20 × SSC = 3M NaCl, 0.3M disodium citrate dihydrate), 20mM NaHPO ₄ , 7% SDS, 10 × Denhardt (50 × Denhardt = 1% bovine serum albumin, 1% polyvinylpyrrolidon, 1% % Ficoll type 400) at 50 ° C overnight to prehybridize.

Oligos were labeled using Pharmacia's Ready to go kit and 5 pmol oligo and 10 μCi [γ- ³² P] -ATP were used for hybridization. The labeled probes were mixed with a small amount of prehybridization buffer and placed in a hybridization tube containing a filter. Hybridization was performed for 2 hours and washed twice for 10 minutes in 2 × SSC, 0.1% SDS. The characteristics, base sequence, hybridization temperature and washing temperature of the oligos used in the experiment are shown in Table 4.

(3) DNA sequence analysis

: The sequencing reaction was performed using a cycle sequencing kit purchased from Perkin Elmer and a dye terminator. The nucleotide sequence of 16S rDNA was determined using an ABI prism 310 automatic DNA sequencer. Used. The similarity between the amplified 16S rDNA of each sample and the 16S rDNA sequence of known bacteria was calculated by the Blast similarity search program (National Institute of Biotechnology Information).

Sequence alignment was performed using a multiple sequence alignment program MSA and finally identified the bifidus species by observing the 16S rDNA moiety already known to possess the target of the species-specific probe after 16S rDNA similarity calculations. Was run (Table 5).

(4) Pulsed field gel electrophoresis (PFGE) analysis

: Insertion DNA equilibrated in TE buffer for 30 minutes at 4 ° C. was equilibrated in appropriate restriction buffer (3-4 hours, 4 ° C.), then the buffer was removed and 20 units of enzyme and 10 μg methylated bovine serum albumin were added per ml. 200 μl of buffer was added and then treated for 15-30 minutes at 4 ° C. and incubated overnight at 37 ° C.

Restriction enzymes were used for SpeI and XbaI (Promega), and the samples were loaded on 1.1% agarose gel, 0.5 × TBE and subjected to PFGE electrophoresis (Switch time: 2-> 30 seconds, run time: 24 hours, 5.3 v / cm , linear ramping, 14 ° C., 0.5 × TBE). Λ-ladder was used as the size standard and a normalization factor was used for image analysis. As a control bacterium, Bifidobacterium bifidum , Bifidobacterium breve , Bifidobacterium longum , and Bifidobacterium lactis , which are commercial bifidus species, were used [FIG. 5. 01: λ-ladder, 02: NF, 03: Bif.lactis (SpeI), 04: Bif.longum MK-G7 dir (SpeI), 05: Bif.longum MK-G7 ON (SpeI), 06: Bif.longum RD-09 dir (SpeI), 07: Bif.longum RD-09 ON (SpeI), 08: Bif.longum dir (SpeI), 09: Bif.longum ON (SpeI), 10: NF, 11: Bif.lactis (XbaI), 12: Bif.longum MK-G7 dir (XbaI), 13: Bif.longum MK-G7 ON (XbaI), 14: Bif.longum RD-09 dir (XbaI), 15: Bif.longum RD- 09 ON (XbaI), 16: Bif.longum dir (XbaI), 17: Bif.longum ON (XbaI), 18: NF. In the above, dir is the case where DNA is directly inserted, ON is the case of overnight culture. The parenthesis is the restriction used.]

(5) Electron microscopy photography of Bifidobacterium longum MK-G7 bifidus strain

: Preprocessed and photographed with an electron microscope as follows (Fig. 6). First, cells of the Bifidobacterium longum MK-G7 bifidus strain were recovered by centrifugation, and then 23 ° C. in 0.1 M Sorensen's phosphate buffer (pH 7.2) containing 4% paraformaldehyde and 2.5% glutaraldehyde. Fixed for time. Re-fixed with the same buffer containing 2.5% glutaraldehyde under the same condition and washed with buffer and pellet pellet clamped at 4 ° C with the same phosphate buffer containing 1% osmium tetroxide (pH 7.2). Treatment for 1 h.

Each concentration was dehydrated using ethanol (30, 50, 70, 80, 90, 100%) and then embedded in a Spurr's medium. Ultrathin sections are cut with a diamond knife and dyed with uranyl acetate and lead citrate, and some are also phosphotunstic acid and chromic acid. As a mixture, the other sites were stained with periodic acid-lead.

Example 4 Measurement of Acid Tolerance and Bile Tolerance of Bifidus Bacteria

To investigate the effect of pH on the growth of bifidus strains, the initial pH of MIF liquid medium (Difco Laboratories, Detroit, MI) supplemented with 0.05% L-cysteine-HCl was first activated with 4N HCl. The OD ₆₀₀ values were measured over time for 24 hours while incubating with 0.1% NaOH and inoculating to 1% after adjusting to 7.0, 5.0, 4.5, and 4.0, respectively.

In order to measure the biliary resistance, the base medium was prepared by adding L-cysteine.HCl to 0.05% of Na ₂ HPO ₄ at 0.05 N and then substituting with nitrogen gas. Bile was added to the base medium at a level of 0, 0.5, 1%, filled with a tube that can be sealed with an aluminum cap, and then sterilized. Control being used in this medium with the embodiments Bifidobacterium strains isolated from 1 and commercially Bifidobacterium strain [Bifidobacterium rongeom (Bifidobacterium longum), Bifidobacterium bipyridinium bushes (Bifidobacterium bifidum), Bifidobacterium inpeon tooth (Bifidobacterium infantis )] Was inoculated at 1% level and incubated in a 37 ° C. incubator. Immediately after inoculation and 100 μl of the culture solution at 1, 3, 8, and 12 hours of culture were taken and titrated in an anaerobic dilution as described above. The diluted solution was plated on BL solid medium (Difco Laboratories, Detroit, MI) and incubated anaerobicly for 36-48 hours in an anaerobic culture, and the number of viable cells was measured.

As a result, the Bifidobacterium longum MK-G7 strain according to the present invention has both acid resistance and bile resistance, and its performance was also excellent.

When comparing the OD ₆₀₀ values at pH 4.0, the Bifidobacterium longum MK-G7 strain of the present invention is 0.11, the commercial strain Bifidobacterium bifidum is 0.01, and the commercial strain Bifidobacterium Bifidobacterium infantis is 0.03, and the commercial strain Bifidobacterium longum is 0.04.Bifidobacterium longum MK-G7 strain selected according to the present invention has the highest acid resistance. It turned out.

On the other hand, when comparing the OD ₆₀₀ value in 0.5% bile added medium, the Bifidobacterium longum MK-G7 strain of the present invention is 0.45, the commercial strain Bifidobacterium bifidum is 0, commercial strain Bifidobacterium inpeon teeth (Bifidobacterium infantis) is 0.49, turns a commercial strain Bifidobacterium rongeom (Bifidobacterium longum) is a 0.37, as Bifidobacterium rongeom (Bifidobacterium longum) within the biliary MK-G7 strains too high lost.

Bifidobacterium rongum according to the present invention (Bifidobacterium longum) MK-G7 strain and commercial strain [Bifidobacterium rongum (Bifidobacterium longum),Bifidobacterium BifidemBifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium infantis)] Was inoculated at a 1% level in milk medium prepared by adding 0.05% L-cysteine.HCl to 10% skim milk. After inoculation, the culture medium was taken every 12 hours while incubating for 72 hours at 37 ° C. incubator and plated on BL solid medium (Difco Laboratories, Detroit, MI) and cultured anaerobicly to measure the number of viable cells.

As described above, the Bifidobacterium longum MK-G7 strain selected according to the present invention shows high viability even when degradation products accumulate in the medium as the growth of the bifidus cells increases. It can be seen.

Example 5 Oxygen Resistance

The bifidus strain isolated in Example 1 was incubated in modified IP liquid medium in which TOS was replaced with 0.5% of fructooligosaccharide and 0.5% galactooligosaccharide for 24 hours, and then diluted in anaerobic dilution to immediately smear on neutral modified TP plate medium. It was. Each was exposed to oxygen overnight in an anaerobic incubator at 37 ° C., and then incubated for 48 hours in the anaerobic incubator. Oxygen-resistant bifidus strains were selected by comparing the number of bacteria in the experimental group exposed to oxygen for 20 hours with respect to the number of microbial cells exposed to oxygen as survival rate and comparing them with each other.

In order to eliminate the effects of oxygen to the maximum, the experimental medium was dispensed in an anaerobic glove box and left in an anaerobic incubator at 37 ° C. for 12 hours to completely remove oxygen. The bifidus strain used in the experiment was diluted 10 times with oxygen-free anaerobic dilution, rapidly smeared in a clean bench, and incubated for 48 hours using an anaerobic culture tank and an anaerobic glove box.

At this time, the smearing time in the aerobic state was limited to less than 10 minutes, the time that the oxygen in the medium is removed in the anaerobic culture tank to less than 1 hour to reduce the time that the bifidus strain is exposed to oxygen to the maximum.

As a result of the above experiments, Bifidobacterium longum MK-G7 according to the present invention was found to have high oxygen resistance with a survival rate of 43%.

Example 6 Antimutagenicity

Mutation inhibitory effect of bifidus bacteria using preincubation test according to the method of Maron, Ames (Maron, DM and BNAmes. 1983. Revised methods for the Salmonella mutagenicity test. Mutation Research. 113: 173-215) Was investigated. As mutagens, direct mutagen NQO and indirect mutagen IQ were used. NQO and IQ were purchased from Wako Chemical Co. (Japan) and Aroclor 1254 was purchased from Chem Service (USA). Meanwhile, DMSO, L-histidine, D-biotin, D-glucose-6-phosphate, NADPH, NADH, etc. were purchased from Sigma Chemical Co. (USA), and nutritional broth, Bacto-agar. And the like were purchased from Difco Chemical Co. (USA).

The S9 fraction was prepared as follows. Aroclor 1254 diluted in corn oil at a concentration of 200 mg / ml was injected intraperitoneally into a body weight of about 200 g (Spraque-Dawley rat, male) at a 500 mg / kg body weight. After 5 days (only water and fasting from 12 hours before dissection) the livers of the mice were collected under sterile experimental conditions and washed several times with cooled KCl. The washed liver transferred to 3 times 0.15M KCl solution was homogenized, and then centrifuged (9,000 x g, 10 minutes) to obtain the supernatant. This S9 fraction was taken in a Nunc tube and stored in a -80 ° C deep freezer as soon as it was taken.

Conducted a strain isolated in Example 1 and was tested using a Bifidobacterium rongeom (Bifidobacterium longum), Bifidobacterium bipyridinium bushes (Bifidobacterium bifidum), Bifidobacterium inpeon tooth (Bifidobacterium infantis) as commercial strains.

The test strain Salmonella typhimurium TA 98 used in the Salmonella typhimurium reversion assay was distributed by Professor Ames (UC Berkeley, USA). Ampicillin resistance (25 μg / ml) was periodically checked to confirm the genetic stability of TA 98 strain and histidine nutritional requirements in minimal glucose agar medium and histidine addition medium. In the Salmonella typhimurium reversion assay, IQ was used as an indirect mutagen that required S9 as a mutagen and NQO was used as a direct mutagen that does not require activation by S9.

Bifidus Strains were incubated for 10-12 hours in BHI (brain heart infusion) medium with 1% glucose. Bifidus cells obtained after centrifugation were washed three times in 20 mM phosphate buffer (pH 7.0) and then lyophilized. To 0.3 ml of 0.2 M phosphate buffer (pH 4.0) solution, 5 µl of IQ (250 µg / ml) dissolved in DMSO as a mutagen, 10 µl of NQO (1 mg / ml), etc., were mixed, and 2 mg of frozen cells were added. Added. No cells were added to the control group. The mixture was shaken at 37 ° C. for 3 hours and then centrifuged to obtain a supernatant.

After pretreatment in this way, 10 μl of NQO and 10 μl of IQ reaction solution were taken, respectively, mixed with 2.3 ml top agar at 45 ° C., and 100 μl of TA 98 strain culture solution and 500 μl of S9 mixture were added. The number of return colonies formed after incubation for 48 hours at 37 ° C. was evenly spread on minimal glucose agar plates.

Mutagenicity (antimutagenicity,%) was calculated by the following equation.

As a result of the above experiments, Bifidobacterium longum MK-G7 according to the present invention showed a 76.5% mutation inhibition ability against NQO and 75.4% against IQ, and the commercial strain Bifidobacterium bifidum ) (0.0% based on the NQO, 63.9% based on IQ), Bifidobacterium inpeon tooth (Bifidobacterium infantis) (78.9% based on the NQO, 80.2% based on IQ), Bifidobacterium rongeom (Bifidobacterium longum) (NQO 11.0% for IQ and 84.7% for IQ).

Example 7 : in vitro cholesterol lowering ability

In order to select the bifidus strain having excellent cholesterol lowering ability in vitro against the isolated bifidus strain was tested as follows. Incubate each bifidus strain for 24 hours in MRS medium containing 0.045% of soluble cholesterol polyoxyethanyl-cholesteryl sebacate and 0.05% of L-cysteine-HCl as an in vitro cholesterol source. After centrifugation (12,000 × g , 4 ° C.), 0.5 ml of the supernatant was added thereto, and 2 ml of 50% KOH and 3 ml of 95% ethanol were added and reacted in a 60 ° C. constant temperature water bath for 10 minutes. After cooling to room temperature, 5 ml of hexane was added and mixed, and 3 ml of distilled water was added and mixed, and the mixture was left at room temperature for 15 minutes to cause layer separation.

After taking 2.5 ml of hexane layer, hexane was evaporated at 60 ° C. using nitrogen gas, and then 4 ml of ο-phthalaldehyde was added to react for 10 minutes, followed by addition of 2 ml of concentrated sulfuric acid. After reacting for another 10 minutes, the OD ₅₅₀ value was measured. The cholesterol lowering ability was calculated by the following equation.

Bifidobacterium longum (Kifidobacterium longum ) MK-G7 bifidus strain of the present invention shows a cholesterol lowering capacity of 12.00%, commercial strain, Bifidobacterium bifidum (15.6%), Bifidobacterium infuntis Bifidobacterium infantis (15.6%) and Bifidobacterium longum (25.3%) were somewhat lower.

Example 8 Macrophage Cell Line Activity

Selection and cultivation of macrophages and TH (T helper) cell lines Optimal cell lines were selected and cultured to determine whether macrophages were activated by administration of bifidus bacteria. Macrophage cell line was used Raw 264.7 and TH (T helper) cell line was used EL $ cell line. These cells were passaged in Dulbecco's modified eagle medium (DMEM) with 10% FBS (fetal bovine serum) and cultured in a CO ₂ incubator at 5% CO ₂ concentration.

The preparation of lymphocytes was anesthetized with mice using ether, followed by an open abdominal cavity and aseptic selection of PP (peyer's patch), IEL (intraepithelial lymphocyte), and LP (lamina propria). These are finely cut and homogenized and then red blood cells are destroyed using ammonium chloride isotonic solution.

LP cells were prepared according to the method of Lycke (Lycke, N. 1986. A sensitive method for the detection of specific antibody production in different isotypes from single lamina propria plasma cells.Scandinavian Journal of Immunology. 24: 393-403). Mosley and Klein (Mosley, RL and JR Klein. 1992. A rapid method for isolating murine intestine intraepithelial lymphocytes with high yield and purity.Journal of Immunological Method. 156: 19-26). PP cells were treated with collagenase and prepared using De Simone's method (De Simone, C. 1988. Adherence of specific yogurt microorganisms to human peripheral blood lymphocytes. Microbios. 55: 49-57).

Tumor necrosis factor (TNF) -α, interleukin (IL), IFN (interferon), etc. were investigated to measure the production of immune hormone cytokines according to the activation of macrophages. Cytokine concentrations were measured quantitatively for the kin. On the other hand, quantitative color development was performed using biotinylated streptavidin horse radish peroxidase (HRP).

Gene expression of immunohormone cytokines was determined by mRNA levels of IL-6 and TNF-α following bifidus administration. Reverse transcriptase PCR (polymerase chain reaction) method was used as a high accuracy method. The autoradiographs obtained after hybridization using PCR amplification probes for each cytokine were quantified by video scanning.

The ability of pagocytosis was determined by Bifidobacterium lon gum at a cell concentration ofBifidobacterium longum) MK-G7 bifidus strain was 288.4 ng / ml, commercial strain bifidobacterium bifidum (Bifidobacterium bifidum)312.9 ng / ml, commercial strain Bifidobacterium inflorescences (Bifidobacterium infantis)208.6 ng / ml, commercial strain Bifidobacterium lon gum (Bifidobacterium longum) Was found to be 307.3 ng / ml. TNF-α production capacity was obtained at the cell concentrationBifidobacterium longumMK-G7 bifidus strain is 8.64 ng / ml, commercial strain Bifidobacterium bifidum (Bifidobacterium bifidum)Is 2.43 ng / ml, commercial strain Bifidobacterium inflorescences (Bifidobacterium infantis)2.25 ng / ml, commercial strain Bifidobacterium lon gum (Bifidobacterium longum) Was 2.93 ng / ml. On the other hand, IL-6 production capacity was determined to be Bifidobacterium RON gum at cell concentrationBifidobacterium longum) MK-G7 bifidus strain was 0.60 ng / ml, commercial strain Bifidobacterium bifidum (Bifidobacterium bifidum) Is 1.20 ng / ml, the commercial strain Bifidobacterium inflorescences (Bifidobacterium infantis) Is 0.90 ng / ml, commercial strain Bifidobacterium lon gum (Bifidobacterium longum) Was 0.30 ng / ml. Thus according to the present inventionBifidobacterium longumMK-G7 bifidus strains were superior to commercial strains in pagocytosis and TNF-α production, but IL-6 production ability was evaluated to be somewhat low.

Example 9 : Sole Culture in Milk Medium

In order to evaluate the applicability of Bifidobacterium longum MK-G7 to the production of fermented milk products according to the present invention, the fermentation characteristics of the strain were examined.

Bifidobacterium longum MK-G7 Bifidobacteria strain and Bifidobacterium longum, a commercial strain, Bifidobacterium longum and Bifidobacterium Fermentation characteristics of Bifidobacterium bifidum and Bifidobacterium infantis were compared (Tables 6 and 7).

PH, titratable acidity and viable cell counts in skim milk medium of Bifidobacterium longum MK-G7 bifidus strain Use strain pH Titratable acidity (%) Viable cell count (cfu / ml) MK-G7 bifidus strain 4.41 0.92 4.75 × 10 ⁸ Commercial strain Bifidobacterium bifidum 4.87 0.57 4.06 × 10 ⁸ Commercial strain Bifidobacterium infantis 5.15 0.51 1.77 × 10 ⁸ Commercial strain Bifidobacterium longum 3.78 1.34 9.86 × 10 ⁸

Measurement of Lactic Acid and Acetic Acid Production in Skim Milk Medium of Bifidobacterium longum MK-G7 Bifidus Strain Use strain Lactic Acid Production (mM / ml) Acetic acid production amount (mM / ml) Generation ratio (lactic acid: acetic acid) MK-G7 bifidus strain 47.60 68.45 1: 1.438 Commercial strain Bifidobacterium bifidum 12.52 17.39 1: 1.389 Commercial strain Bifidobacterium infantis - - - Commercial strain Bifidobacterium longum 136.46 48.41 1: 0.355

MK-G7 bifidus strain had good growth rate in cultivated skim milk, and showed proper acidity and viable cell number for product production. Indicated.

Example 10 : Functionality and Product Application of Yogurt Prepared by Mixed Culture

In order to replace the commercial Bifidobacterium infantis ( Bifidobacterium infantis ) of the drink yogurt produced and sold by the present applicant company with the Bifidobacterium longum MK-G7 bifidus strain according to the present invention, sample A was 100% of the conventional commercial bifidus strain. , Sample B is 100% Bifidobacterium longum MK-G7 bifidus strain, Sample C is 50% conventional Bifidobacteria strain + 50% Bifidobacterium longum MK-G7 bifidus strain, Sample D is 90% Bifidobacterium longum MK- 10% of G7 bifidus strains were inoculated to measure pH, titratable acidity, and bifidus bacteria at 4 hour intervals from 0 hours to 24 hours of culture. The remaining manufacturing process and the component blending ratio were the same.

The titratable acidity showed similar acid production in all of the specimens from 0h to 8h in culture, but B and C showed higher acid production than control A from 12h in culture (Table 8). Changes in total lactic acid bacteria showed similar trends in all four samples, and B, C, and D maintained higher bacterial counts than control in the change of non-fidus bacteria (Table 9, 10). .

Change in titratable acidity according to incubation time for the production of fermented milk products of Bifidobacterium longum MK-G7 bifidus strain (%) Incubation time Sample A (existing commercial strain) Sample B (100% of MK-G7 strain) Sample C (50% of MK-G7 strain) Sample D (10% of MK-G7 strain) 0 hours 0.16 0.16 0.16 0.16 2 hours 0.17 0.17 0.17 0.16 4 hours 1.45 0.44 0.44 0.39 6 hours 0.65 0.67 0.64 0.61 8 hours 0.76 0.77 0.76 0.76 10 hours 0.81 0.83 0.83 0.80 12 hours 0.88 0.91 0.87 0.83 24 hours 1.17 1.30 1.22 1.17

Changes in the Number of Viable Cells According to Incubation Time in Preparation of Fermented Milk Products of Bifidobacterium longum MK-G7 Bifidus Strain (cfu / ml) Incubation time Sample A (existing commercial strain) Sample B (100% of MK-G7 strain) Sample C (50% of MK-G7 strain) Sample D (10% of MK-G7 strain) 0 hours 5.50 × 10 ⁶ 5.15 × 10 ⁶ 2.60 × 10 ⁶ 8.30 × 10 ⁵ 2 hours 8.45 × 10 ⁶ 1.32 × 10 ⁷ 7.40 × 10 ⁶ 3.00 × 10 ⁶ 4 hours 7.75 × 10 ⁶ 4.68 × 10 ⁷ 3.17 × 10 ⁷ 4.75 × 10 ⁶ 6 hours 1.27 × 10 ⁷ 8.00 × 10 ⁷ 4.80 × 10 ⁷ 2.06 × 10 ⁷ 8 hours 1.15 × 10 ⁷ 7.35 × 10 ⁷ 5.10 × 10 ⁷ 2.05 × 10 ⁷ 10 hours 8.50 × 10 ⁶ 7.55 × 10 ⁷ 4.65 × 10 ⁷ 1.00 × 10 ⁷ 12 hours 1.10 × 10 ⁷ 6.15 × 10 ⁷ 6.00 × 10 ⁷ 1.30 × 10 ⁷ 24 hours 1.65 × 10 ⁷ 6.50 × 10 ⁷ 4.15 × 10 ⁷ 8.50 × 10 ⁶

Changes in Total Lactic Acid Bacteria in Cultured Bifidobacterium longum MK-G7 Bifidus Strains with Culture Time (cfu / ml) Incubation time Sample A (existing commercial strain) Sample B (100% of MK-G7 strain) Sample C (50% of MK-G7 strain) Sample D (10% of MK-G7 strain) 0 hours 5.55 × 10 ⁵ 5.25 × 10 ⁵ 5.35 × 10 ⁵ 4.30 × 10 ⁵ 2 hours 1.14 × 10 ⁷ 4.85 × 10 ⁶ 4.35 × 10 ⁶ 6.25 × 10 ⁶ 4 hours 2.83 × 10 ⁸ 2.97 × 10 ⁸ 2.84 × 10 ⁸ 2.72 × 10 ⁸ 6 hours 3.10 × 10 ⁸ 4.15 × 10 ⁸ 3.30 × 10 ⁸ 4.10 × 10 ⁸ 8 hours 3.95 × 10 ⁸ 2.95 × 10 ⁸ 4.90 × 10 ⁸ 6.95 × 10 ⁸ 10 hours 4.55 × 10 ⁸ 5.65 × 10 ⁸ 4.85 × 10 ⁸ 3.80 × 10 ⁸ 12 hours 4.50 × 10 ⁸ 3.85 × 10 ⁸ 7.15 × 10 ⁸ 4.05 × 10 ⁸ 24 hours 8.85 × 10 ⁸ 4.60 × 10 ⁸ 4.00 × 10 ⁸ 4.00 × 10 ⁸

In addition, the change in the number of bifidus bacteria from the beginning of culture to 12 hours was much higher in the blank sample B in proportion to the acid production capacity, and the numbers of bifidus bacteria were decreased in the order of B, C, and D in proportion to the inoculation amount. The change in the number of bifidus bacteria at 24 hours of cultivation reached the same rate as the control group except the control.

In conclusion, it is possible to lower the amount of Bifidobacterium longum MK-G7 bifidus strain finally selected in this study to 1/5 compared with the existing commercial bifidus strains. Good bacteria can be maintained.

Example 11 Stability of Strains During Product Storage

The existing commercial bifidus bacteria used in the manufacture of the drink yogurt, which is currently produced and sold by Maeil Dairy Co., Ltd., were replaced with new bioactive Bifidobacterium longum MK-G7 bifidus strains suitable for Koreans to produce finished products.

The final product was refrigerated, stored at room temperature, 37 ℃ to compare the pH, titratable acidity, bifidus bacteria, total lactic acid bacteria, sensory properties for 10 days. The stability of the strain was measured as the number of bifidus bacteria at this time.

Survival experiments of the selected bifidus strains were performed under the existing culture environment. A fermented milk product (10 ⁷ cfu / ml, A) produced by the same method was used as a control except for the use of conventional commercial strains. The selected Bifidobacterium longum MK-G7 bifidus strain was selected as 10 ⁷ cfu / ml (B), 0.5 × 10. Inoculation was carried out at a bacterial count of ⁷ cfu / ml (C) and 10 ⁶ cfu / ml (D). Bifidobacterium bacteria are contrary to the number of bacteria A sample 10 ¹ increased cfu / ml B, C, D samples at 10 ² cfu / ml increase in culture conditions, such as the well the growth of Bifidobacterium longum MK-G7 Bifidobacterium strain achieved after fermentation It can be seen that the loss (Fig. 7).

At 4 ° C., A, B, C, and D samples had no significant effect on viability of bifidus bacteria after 9 days of storage (FIG. 8). At 20 ° C., there was a slight decrease in viability during storage, but a decrease of 0.5 × 10 ¹ cfu / ml. However, at 37 ° C., the survival rate after 6 days of storage was lowered at the same rate, indicating the same level of viable count.

Therefore, Bifidobacterium longum MK-G7 bifidus strain was shown to be slightly lower than A sample strain in acid tolerance in weak acidity, but it was superior to the existing commercial bifidus strain due to the increase in the number of bacteria after fermentation. see.

On the other hand, as the preservation temperature increased, the total number of lactic acid bacteria decreased. At 4 ℃, 10 ⁸ cfu / ml was maintained until 5 days of preservation, but the number of bacteria decreased significantly after that. At 20 ℃ was maintained for 10 ⁸ cfu / ml to 9, preserving, at the time of nine days at 37 ℃ retention was maintained for the number of bacteria 10 ⁷ cfu / ml.

The titratable acidity was similar in all samples and increased slightly with increasing temperature. The titratable acidity increased to 2.0% when stored at 37 ℃ for 6 days. The pH also showed a tendency to decrease with time, which was the most severe at 37 ° C. Indeed, the pH decreased from 4.2 to 3.6 when stored for 2 days at 37 ℃.

According to the present invention, it is possible to obtain a novel bifidus strain having excellent cultivation ability, excellent acid resistance, bile resistance and oxygen resistance, cholesterol lowering activity, antimutagenicity, macrophage activity, etc. and having physiological activity suitable for Koreans.

Claims (2)

Bifidobacterium longum MK-G7 bifidus strain (Gene Bank Strain Deposit No .: KCTC 0488 BP). The strain according to claim 1 used as a lactic acid bacteria fermentation strain.