KR20100128168A - Novel bacterial strains having excellent production potential of conjugated linoleic acid - Google Patents

Abstract

PURPOSE: A novel strain having conjugated linoleic acid(CLA) activity is provided to suppress carcinogen and to reduce atherosclerosis anesis symptom and body fat. CONSTITUTION: A Bifidobacterium breve IB84 strain(KCCCM 10998P) or Bifidobacterium breve IB52 strain(KCCM 10997P) is isolated from infant feces. A probiotics composition or functional fermented milk composition contains the Bifidobacterium breve IB84 strain(KCCCM 10998P) or Bifidobacterium breve IB52 strain(KCCM 10997P). A conjugated linoleic acid(CLA) is converted from linoleic acid by contacting with Bifidobacterium breve IB84 strain or Bifidobacterium breve IB52 strain. The CLA is c9,t11(C18:2) CLA.

Description

Novel Bacterial Strains Having Excellent Production Potential of Conjugated Linoleic Acid

The present invention is a novel Bifidobacterium breb ( Bifidobacterium breb) having excellent production capacity of conjugated linoleic acid. breve ).

Immediately after birth, the human digestive system breaks down quickly due to sterility or bacteria. This occurs during birth and passes through the mother's vagina.E. Coli and enterococci are detected within 24 hours.

Bacteria formed in the digestive tract have a significant impact on the health of the host. Intestinal flora can be largely divided into harmful and beneficial bacteria, and harmful bacteria are harmful to humans by producing toxins, producing carcinogens, and causing intestinal rot, and beneficial bacteria are probiotic such as lactobacilli or Bifidobacteria. It is a strain. These microorganisms are balanced and form intestinal flora by mutual symbiosis or antagonism (Mitsuoka, 2000). Bifidobacteria bacteria, and survive for life from birth as one of the normal intestinal flora, oral, Chairman, colon, vagina, and habitats in a variety of organs, including cervical, colon within the Clostridium oil cake Te Leeum (Eubacterium), ( Clostridium ), Bacteroides , etc., dominant species in the colon, 10 ⁸ -10 ¹¹ per gram of colon content (Mitsuoka. (1978)).

Bifidobacterium has anticancer activity (Reddy et al. (1993); Biffiet et al. (1997); Rowland et al. (1998)), immune regulation (Yasui et al. (1991)), digestive system improvement (Saavedra et al. (1994)) and other beneficial actions have been reported. Recent reports have reported that some of the Bifidobacteria isolated from the human body can metabolize fatty acids to produce conjugated linoleic acid (CLA) (Coakley. (2003)).

CLA is a structural isomer of linoleic acid, which is produced during the process of biohydrogenation of linoleic acid into stearic acid by ruminant microorganisms and can be consumed through ruminant fat or milk. Inhibition of tumor (Lee et al. (1994)), increased immunity (Hayek et al. (1999)), decreased body fat (Park) when ingested cis-9, trans-11 CLA or trans-10, cis-12 CLA et al. (1997); West et al. (1998)) have been reported to have beneficial effects on human health (Belury. (2002); Pariza et al. (2000, 2001)).

Traditionally, Bifidobacteria species have been identified based on cell morphology, fermentation assays, enzyme activity and carbohydrate availability. Counting of Bifidobacteria from various environments is achieved by plating on selective media. Substances in the Bifidobacteria selective medium are antibiotics, propionic acid and oligosaccharides. Unfortunately, such phenotypes have a low reproducibility depending on various strains and culture conditions. Recently, molecular biological methods have been used to identify microorganisms.

Recently, molecular biology techniques have been widely used as reliable research methods to identify strain levels. Such molecular biological techniques can be used for identification, but are limited by a wide range of specific fingerprinting databases. In conclusion, the molecular biology approach will be more useful if a database for this has been built up with the increasing variety of molecular biology techniques (Ventura et al. (2004)).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to overcome the limitation of the ability to convert only triglyceride form of linoleic acid (LA) into CLA, which is effective only for free fatty acids, which has been pointed out as a problem of conjugated linoleic acid (CLA) producing active strains reported to date. A polite research effort was made. As a result, by separating the Bifidobacterium breve strain having a high CLA production activity, the present invention was completed.

Accordingly, an object of the present invention is to provide a Bifidobacterium breb IB84 strain (KCCM 10998P) or a Bifidobacterium brb IB52 strain (KCCM 10997P) having CLA production activity and isolated from infant feces.

Another object of the present invention is to provide a probiotic composition comprising a Bifidobacterium breb IB84 strain or a Bifidobacterium breb IB52 strain.

It is another object of the present invention to provide a functional fermented milk composition comprising a Bifidobacterium breb IB84 strain or a Bifidobacterium breb IB52 strain.

It is still another object of the present invention to provide a method for converting linoleic acid to conjugated linoleic acid comprising contacting a bifidobacterium breb IB84 strain or a bifidobacterium breb IB52 strain to linoleic acid.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides Bifidobacterium breb IB84 strain (KCCM 10998P) or Bifidobacterium breb IB52 strain (KCCM 10997P) having CLA production activity and isolated from infant feces.

According to the present invention, it was confirmed that two strains among the 36 Bifidobacterium breb colonies isolated from infant feces have excellent CLA conversion ability. This is called “ Bifidobacterium breve BI52 ”and“ Bifidobacterium breve BI84 ”was deposited on March 31, 2009 to the Korea Center for Microbiological Conservation (KCCM) and was assigned accession numbers KCCM 10997P (IB52) and KCCM 10998P (IB84), respectively.

As used herein, the term “CLA producing activity” refers to the ability of a microorganism to convert linoleic acid to conjugated linoleic acid in vivo or ex vivo. According to the present invention, the Bifidobacterium breb IB52 shows a CLA conversion similar to that of the Bifidobacterium breb LMC 220 found to have a high CLA conversion rate, and the Bifidobacterium breb IB84 shows a higher conversion rate than the LMC 220. .

According to another aspect of the present invention, the present invention provides a probiotic composition comprising a Bifidobacterium breb IB84 strain or a Bifidobacterium breb IB52 strain. As used herein, the term “probiotics” refers to food supplements of live bacteria that contribute to the health of the host organism. The probiotic composition of the present invention may be prepared as a fermented milk product, but may also be prepared in the form of granules, powders and the like.

Although the method of administering the composition of the present invention is not particularly limited, it may be prepared by pills, tablets, or the like, orally administered, or may be prepared in powder or granule form and added to food. The composition of the present invention can be used by itself, for example, when used as a medicine, without formulating powder of each component, but powders, granules, fine granules, tablets, dragees, capsules, tablets, enteric It can be formulated in the form of a coating agent or the like. As the diluent, excipients, binders, disintegrating agents and the like used in general pharmaceutical preparations are used, and colorants, stabilizers, preservatives, lubricants and the like can be added. When the food composition of the present invention is used as a food, it can be used without formulating the powder of each component as it is, but other plant fibers, oligosaccharides, grains, vitamins, etc. may be added, or a flavor, a coloring agent, a spice agent, or the like may be used. It is also possible to form and process it into a form suitable for ingestion by addition. Moreover, as a food additive, it can also add and use it for another foodstuff.

According to another aspect of the invention, the present invention provides a functional fermented milk composition comprising a Bifidobacterium breb IB84 strain or a Bifidobacterium breb IB52 strain.

The production of functional fermented milk using the Bifidobacterium breb IB84 strain or the Bifidobacterium breb IB52 strain can be carried out according to conventional fermentation processes known in the art.

A variety of products can be made by adding ingredients typically added to beverages prepared according to the invention. Functional fermented milk beverages of the present invention may include flavoring agents or natural carbohydrates as additional ingredients. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As the flavoring agent, natural flavoring agents (e.g., taumartin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.) can be used.

According to another aspect of the present invention, the present invention provides a method for converting linoleic acid to conjugated linoleic acid comprising contacting bifidobacterium breb IB84 strain or bifidobacterium breb IB52 strain with linoleic acid. to provide.

The term "contact" of the present invention encompasses all methods of providing an environment for producing conjugated linoleic acid from linoleic acid by the strain of the present invention, in addition to the method of inoculating and culturing the strain of the present invention with a linoleic acid in a medium.

When the strain of the present invention is cultured by inoculating the medium with linoleic acid, the microorganism can be cultured according to a conventional method in the art using a suitable culture solution. The culture preferably comprises a carbon source, a nitrogen source and an osmotic pressure regulator.

In the step of culturing the microorganism of the present invention, the culture temperature is preferably 20-37 ° C, more preferably 23-37 ° C, and most preferably approximately 37 ° C.

In the step of culturing the microorganism of the present invention, Tween80 may be added to neutralize the antimicrobial activity of free linoleic acid and increase the solubility of fatty acids in the medium, and the amount of Tween80 added is preferably 2-5% by weight. More preferably 5% by weight.

According to a preferred embodiment of the invention, the CLA in the process of the invention is c9, t11 (C18: 2) conjugated linoleic acid. CLA has several isomers depending on the position of the double bond in the alkyl chain and the conformation of the double bond, and cis-9, trans-11 isomers and trans-10, cis-12 isomers are the main objectives of the study. As shown in Figure 10 below, the CLA produced by the strain isolated from infant feces in the present invention are mostly cis-9, trans-11 isomer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention is Cifidobacterium breb IB84 strain or Bifidobacterium breb IB52 strain having CLA production activity and isolated from infant feces, probiotics composition comprising them, functional fermented milk composition and linoleic acid using them Provides a way to switch to

(b) The strain of the present invention can be usefully used in the production of functional foods and beverages having effects such as inhibiting carcinogens, reducing atherosclerosis symptoms, reducing adverse effects of immune action and reducing body fat.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experiment method

Bifidobacteria Selection  Establishment of conditions

To select the Bifidobacteria required for the study from feces of infants, each selection medium was used to compare their selectivity. The medium was based on BL (glucose blood liver broth) medium (Difco, USA) to enhance its selectivity, and medium and TOS (NPNL) solution and BS solution, respectively, added with NPNL (nalidixic acid, paramomycin, neomycin sulfate and lithium chloride) solutions. A medium in which TOS was replaced with pullulan based on transgalatooligosaccharide) -propionic acid agar (Ya-kult Honsha, Japan) was used.

Test strains Lactobacillus Planta Room (L. plantarum) KCTC 3108 ^T, Bifidobacterium Brenna probe KCTC 3220 ^T, Bifidobacterium Brenna probe KCTC 3419, Bifidobacterium Brenna probe KCTC 5081, Bifidobacterium Animal the lease (B. animalis) KCTC 3219 ^T, Enterococcus par faecalis (E. faecalis) 2 ilgan anaerobically in the second series 37 ℃ after spotting on each selective media with respect to the modified MRS broth for 7-2 IE The culture was compared with the size of crack formation.

Composition of TOS Medium ingredient Content per liter Trypticase Peptone 10.0 g Yeast extract 1.0 g KH ₂ PO ₄ 3.0 g K ₂ HPO ₄ 4.8 g (NH ₄ ) ₂ SO ₄ 3.0 g MgSO ₄ 0.2 g L-cysteine 0.5 g Sodium propionate 15 g Trans-galactooligosaccharide 10 g Bacto Baby 15 g

The TOS medium was sterilized at 115 ° C. for 15 minutes.

Composition of BL medium ingredient Content per liter Lab-Lemco (Oxoid) 3.0 g Peptose peptone no. 3 (Difco 10.0 g Tripty Case (BBL) 5.0 g Phytone (BBL) 3.0 g Yeast Extract (Difco) 5.0 g Liver Extract Solution 150.0 ml Glucose 10.0 g Soluble starch 0.5 g Solution A 10.0 ml Solution B 10.0 ml Tween 80 1.0 g L-cysteine HCl 0.5 g

Liver extract solution: 10 g (Difco) liver extract powder was extracted with 170 ml distilled water at 50-60 ℃ for 1 hour, boiled for 10 minutes and filtered.

Solution A: 25 g of KH ₂ PO _{4 and} 25 g of K ₂ HPO ₄ were added to 250 ml distilled water.

Solution B: 0.5 g of FeSO ₄ · 7H ₂ O, 10 g of MgSO ₄ · 7H ₂ O, 0.337 g of MnSO ₄ , and 0.5 g of NaCl were added to 250 ml of distilled water.

Composition of Anaerobic Diluent ingredient Content per liter KH ₂ PO ₄  4.5 g Na ₂ HPO ₄  6.0 g L-cysteine 0.5 g Tween 80  0.5 g

The dilution was sterilized for 15 minutes at 115 ℃.

 Composition of NPNL Solution ingredient content Lithium chloride 6 g Nalidix acid 0.03 g Neomycin Sulfate 0.2 g Paromomycin 0.4 g Distilled water 100 ml

Filtered (0.45 μm), 5 ml / 100 ml BL medium

Composition of BS solution ingredient content Sodium propionate 30 g Paromomycin sulfate 0.1 g Neomycin sulfate 0.4 g Lithium chloride 6 g Distilled water 100 ml

Filtered (0.45 μm), 5 ml / 100 ml BL medium

Bifidobacteria  Growth conditions and strain selection

Fecal samples of 12 healthy infants (expressed as AL) of 14 to 100 days of age were used as infant fecal samples used in the present invention. All samples were refrigerated immediately after collection and transported to the laboratory for microbial testing within 12 hours. Separation of Bifidobacteria from the collected feces was performed according to the method of Mitsuoka (1980). After mixing the feces uniformly, dilute to 10 ⁶ ~ 10 ⁸ cfu / ml using anaerobic microbial diluent, and plate 100 μl of the diluted sample on the prepared TOS agar plate onto an anaerobic jar system (Anaerobic jar system, BBL, USA) was incubated for 48 hours at 37 ℃.

The isolated Bifidobacteria were cultured at 37 ° C. for 18-24 hours using a modified MRS culture medium, and were used in the experiment after sufficiently increasing vitality through two or more passages before the experiment. Bifidobacteria, after pure separation and identification, were cultured in modified MRS culture for 18 hours, centrifuged at 10,000 Ⅹg for 10 minutes, and the supernatant was replaced with 10% sterile reduced skim milk containing 25% glycerol. deep freezer) was used for the experiment.

Bifidobacteria  Sympathy

1.Duplex polymerase chain reaction

Five randomly selected colonies grown on TOS agar plates were mixed well with 50 μl of Lyse-NGo (Pierce, USA), and then the thermocycler was used.

Treated under the conditions of to obtain the template DNA.

Based on genus-specific primers based on the xfp gene encoding F6PPK, an enzyme produced only by Bifidobacteria, and where Bifidobacterium Breb differs from other species in the 16S rRNA gene. PCR was carried out using a species-specific primer designed.

Samples with the expected size of the PCR product were selected, single colonies were taken, and PCR was performed in the same manner as above to isolate the fungi that form a desired size band (PCR product).

         Thermal Cycling Program for Treatment cycle Temperature (℃)  time  One  65  30 sec  2  8  30 sec  3  65  90 sec  4  97 180 sec  5  8  60 sec  6  65 180 sec  7  97  60 sec  8  65  60 sec  9  80  fixing

2. Sequencing

  1) 16S rDNA partial sequencing

Colony PCR was performed using single colonies obtained by streaking the isolated strains on TOS agar plates. After template DNA was taken using Lyse-N-Go, PCR was performed using universal primer pairs (27F, 1492R) that amplify 16S rDNA moieties. The PCR product of about 1.5 kb was cleaned using a PCR purification kit (Qiagen, USA), and Macrogen was commissioned for sequencing with 27F and 1492R primer pairs. The obtained sequence was identified by BLAST (Basic Local Alignment Search Tool) search.

2) Sequencing of F6PPK Encoding Genes

PCR was performed using genus specific primers (GP 109, GP 111) based on the xfp gene encoding F6PPK, an enzyme produced only by Bifidobacteria, and the PCR product of the desired size was transferred to pGEM-T easy vector (Promega, USA) and then transformed into E. coli JM109 (Takara, Japan) and plated on LAX agar plate and incubated overnight at 37 ℃. After incubation, white colonies were selected and inoculated in LB culture medium containing 50 ㎍ / ml ampicillin, and cultured at 150 rpm at 37 ° C for 8 hours to make plasmid using miniprep kit (Qiagen, USA). Macrogen Co., Ltd. was sequencing with SP6 and T7 primer pairs. The obtained sequences were compared using the clustal W program.

3) Sequencing of Bile Hydrolase (BSH) Encoding Genes

Several strains with different precipitation sizes were selected by streaking in 2 mM GDCA and 2 mM TDCA, and PCR was performed using primers (GP145, GP146) designed for genes producing BSH, and PCR products of desired size were pGEM-T. After ligating to an easy vector, E. coli JM109 was transformed and plated on a LAX agar plate and incubated overnight at 37 ° C. After incubation, white colonies were selected, inoculated in LB culture medium containing 50 ㎍ / ml ampicillin, incubated at 37 rpm for 150 hours at 150 rpm, and then made a plasmid using miniprep kit. Sequencing was commissioned with SP6 and T7 furimer pairs. The obtained sequences were compared using the clustal W program.

Oligonucleotide Sequences Used primer         Explanation                  order GP-107 BiBRE-1: forward_16S rRNA
B.breve    5'-CCGGATGCTCCATCACAC-3 ' GP-108 BiBRE-2: reverse_16S rRNA
B.breve    5'-ACAAAGTGCCTTGCTCCCT-3 ' GP-109 XFP-F1: Forward_F6PPK
Bifidobacterium    5'-TGGCAGTCCAACAAGCTC-3 ' GP-111 XFP-R1: Forward_F6PPK
Bifidobacterium    5'-TAGGAGCTCCAGATGCCGTG-3 ' GP-116 27F    5'-AGAGTTTGATCCTGGCTCAG-3 ' GP-120 1492R    5'-GGTTACCTTGTTACGACTT-3 ' GP-145 BBSHA-F1: Forward    5'-ATGTGCACTGGTGTCCGTTTC-3 ' GP-146 BBSHA-R1: Reverse    5'-TCATCGGGCGACGCTGCTG-3 '

conditions of duplex PCR Initial degeneration
(Initial denaturation) denaturalization
(denaturation) Loosening
(Annealing) expansion
(Extention) Last expansion Temperature (℃)    95    95    50    72   72  4 time   15:00   1:00   0:30   1:30  10:00 fixing

PCR Conditions for 16S rRNA Amplification Initial degeneration
(Initial denaturation) denaturalization
(denaturation) Loosening
(Annealing) expansion
(Extention) Last expansion Temperature (℃)   95   95   55   72  72 4 time   15:00   1:00   0:45   2:00  10:00 fixing

 Composition of LB Medium    ingredient Content per liter   Trypton  10 g Yeast extract  5 g Sodium chloride   10 g

The composition of LAX baby   ingredient Content per liter   Trypton 10 g Yeast extract  5 g Sodium chloride 10 g   ITPG Stock Solution (100 mM)   1 ml   X-Gal Stock Solution (20 mg / ml)   2 ml  Ampicillin Stock Solution (50 mg / ml)   1 ml

Sterilized at 121 ° C for 15 minutes before adding ITPG, X-Gal and Ampicillin

3. Fast PFGE (pulsed field gel electrophoresis)

The fast PFGE method was performed according to Briczinski and Roberts (2006). After incubating the test strain overnight, 2 ml was taken and centrifuged (14000 × g, 4 ℃, 10 min) to recover the cell precipitate. After washing once with 2 ml of 100 mM Tris, 100 mM EDTA, pH 7.6, it was suspended using 600 μl of the same buffer and 160 μl was transferred to a new tube. 40 μl of lysozyme (100 mg / ml) and 10 μl of proteinase K (20 mg / ml) were added and mixed with 1.6% agarose containing 0.1% SDS in the same volume to recycle plug mold ( Re-usable plug mold (Bio-rad, USA) was dispensed. Lysis plugs 55 ° in 0.5 M EDTA, 1% sarkosyl buffer (pH 9.0) containing 2 ml of lysozyme (4 mg / ml) and mutanolisin (200 units / ml). C, which occurs when incubated for 90 minutes. Thereafter, incubated in fresh sarcosyl buffer containing proteinase K (0.5 mg / ml) at 55 ° C. for 60 minutes. After rinsing at 50 ° C. for 15 minutes using sterile distilled water preheated to 50 ° C., shaking culture was performed at 75 rpm for 15 minutes using 10 mM Tris, 1 mM EDTA buffer (pH 7.6) preheated at 50 ° C. Washed three times. Lysis finished plug was cut to comb size and then treated for 2 hours at 37 ℃ using a 200 μl restriction digest mixture (50 units of Xba I).

Electrophoresis was performed using 1% agarose gel using 0.5 × TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0). Lambda ladders (Bio-rad) were used as molecular size markers. The switch time was electrophoresed at 6 V / cm, 14 ° C. for 18 hours using CHEF DR-III (Bio-rad).

After electrophoresis, the gel was stained with EtBr (ethidium bromide) (0.4 mg / L; Promega) for 1 hour, decolorized with distilled water for 2 hours, and the band pattern was observed using a UV trans-illuminator.

Bifidobacteria Observation of Morphological Characteristics of

1. Observation of optical microscope

Samples were incubated anaerobically at 37 ° C. for 48 hours in BL agar and then single colonies formed were selected. In order to observe the general optical microscope, the strain was stained in slide glass and stained with crystal violet for 1 minute. After fixing with iodine for 30 seconds, it was decolorized with 95% ethyl alcohol and stained with safranin for 1 minute, and observed by a general optical microscope.

 2. Scanning electron microscope

The cultured sample was taken and pre-fixed in 2.5% glutaraldehyde solution (0.1 M phosphate buffer, pH 7.4, 4 ° C.) for 2 hours, washed three times for 10 minutes with buffer solution (0.1 M phosphate buffer), 1% It was post-fixed with OsO (Osmium tetroxide) (0.1 M phosphate buffer, pH 7.4, 4 ° C) for 1 hour. After washing three times for 10 minutes with the same buffer (0.1M phosphate buffer), each treated for 10 minutes with 50% -70% -90% -95% ethanol, three times each 10 minutes with 100% ethanol. After the dehydration treatment, the sample was replaced with isoamyl acetate and dried in a critical point drying process using liquid carbon dioxide (HCP-2, Hitachi, Japan), and then fixed on an aluminum stub to fix an ion coating machine (E-1030, Hitachi, Japan) was coated with Pt-Pd particles at a thickness of about 5-10 nm and observed at an acceleration voltage of 10 kV using a field emission scanning electron microscope (S-4700, Hitachi, Japan).

Bifidobacteria Of physiological properties of

1. Carbohydrate availability

The isolated strain confirmed as Bifidobacterium was confirmed carbohydrate utilization ability using API 50 CHL (Bio-marieux, France).

 Subsequently, the separated samples were passaged twice in a BL culture medium to increase vitality, and then the cells were recovered by centrifugation at 10,000 Ⅹg for 10 minutes. The recovered cells were washed twice in anaerobic dilution and the turbidity was adjusted to 4 Macfarland. 2 ml of suspension was added to 5 ml of BCP medium and then injected into 100 μl of each chapel. The completed kit was observed by incubating for 48 hours at 37 ℃ using an anaerobic incubator.

2. Production of Bile Acidase (BSH)

After streaking the test strain in mMRS agar containing 2 mM GDCA, 2 mM TDCA, 0.035% CaCl ₂ , and 0.05% cysteine, respectively, and incubated for 48 hours at 37 ° C. using an anaerobic culture apparatus, colony formation and precipitation around colonies Formation was confirmed.

Conjugate Linoleic acid ( CLA ) producing ability

1. Spectrophotometric assay

Screening using standard gas liquid chromatography (GLC) consumes considerable time and effort and is limited to selecting a large number of strains, so screening strains that produce CLA is primarily screened using a spectrophotometer.

The test strain was inoculated with 1% of the culture solution in mMRS medium containing 0.5 mg / ml of linoleic acid and 2% (w / v) Tween80 and incubated at 37 ° C. for 24 hours under anaerobic conditions. 1 ml of the culture solution was transferred to a new tube, centrifuged at 20,800 × g for 1 minute, and 500 μl of hexane was mixed with 500 μl of the supernatant except the precipitate, followed by vortexing for 2 minutes. At this time, 100 μl of the extracted hexane layer and 900 μl of methyl alcohol were mixed well, and the absorbance was measured at 233 nm using a spectrophotometer (smart plus SP-1900PC, Youngwoo, Korea).

2. Quantitative analysis of CLA using gas chromatography

The test strain was inoculated with 1% in mMRS medium containing 0.5 mg / ml of linoleic acid and 2% (w / v) Tween80 and incubated under anaerobic conditions at 37 ° C for 24 hours to transfer 0.5 ml to a microtube. 20 μl of the stock solution in which 100 mg of internal standard (C _{17 : 0} ) was dissolved in 8 ml of hexane was added to the microtube and mixed well. 0.5 ml of hexane was added thereto, followed by tilting for 10 minutes, and then allowed to stand for 30 minutes, followed by centrifugation to separate the hexane layer. The hexane layer from which the fatty acids were extracted was transferred to a new tube. The hexane extraction process was repeated four times, collected in one glass tube, and the solvent was evaporated at 60 ° C. using nitrogen gas.

Fatty acid was methylated by treatment at 60 ° C. for 30 minutes using 1.0% HCl dissolved in methanol. After adding 2 ml of saturated sodium chloride, 1 ml of hexane was added to extract fatty acid methyl esters (FAMEs) and transferred to a microtube. The solvent was evaporated using nitrogen gas and then 100 μl of hexane was added to concentrate the FAMEs. CLA methyl esters were analyzed using GC (Varian STAR 3400, USA).

 Conditions for Conjugated Linoleic Acid Analysis by Capillary Gas Chromatography      device     Varian STAR 3400      Colum SP ^TM -2560 (100 m × 0.2 mm, thickness 0.2 μm) Temperature programming     180 ° C, 40 min-3 ° C / min-230 ° C, 30 min   Injection temperature     250 ℃  Detector temperature     280 ℃     Detector     FID (flame ionization detector)

Experiment result

Bifidobacteria Selection  Establishment of conditions

Medium to which NPOS and BS solution were added and TOS to propionic acid agar based on BL medium (Difco) to enhance its selectivity for selective separation of Bifidobacteria As a result of comparing the selectivity using the result, it was confirmed that the medium with TOS and TOS replaced with pullulan has better selectivity with respect to the Bifidobacteria than the medium with NPNL and BS solution added to the BL medium. To check whether Lactobacillus and Enterococcus, microorganisms present in feces, were inhibited, spotted several strains in the laboratory on the medium and observed the results after 48 hours of cultivation. It has been shown to inhibit but some Bifidobacteria were also inhibited in pullulan medium. In the case of infant feces, Bifidobacteria were shown as dominant species compared to Enterococcus and Lactobacillus.

 Comparison of Selection Medium of Bifidobacterium Breb Strain TOS Baby Pullulan Baby Lactobacillus plantarum KCTC 3108 ^T + x Bifidobacterium breb KCTC 5081 +++ +++ Bifidobacterium breb KCTC 3419 +++ x Bifidobacterium breb KCTC 3220 ^T +++ +++ Bifidobacterium Animals KCTC 3219 ^T ++ x Enterococcus paecalis IE 7-2 ++ x

Colony size, +++: 5mm, ++: 3mm-5mm, +: 3mm

Bifidobacteria  Sympathy

1.Duplex polymerase chain reaction

Bifidobacterium brebs have species-specific properties that produce CLA (Coakley et al. (2003); Oh et al. (2003); Rosberg-cody et al. (2004)). In this study, it is necessary to identify Bifidobacterium brebs because biochemical methods require a lot of time and effort to identify species at the biochemical method. B. breve was isolated using a rapid and accurate Duplex PCR technique to identify species.

Preliminary experiments with Bifidobacterium spp. Possessed in the laboratory showed that all Bifidobacterium species formed bands at about 600bp and 950bp in PCR using two pairs of genus-specific primers. In addition, PCR using species-specific primers confirmed that only B. breve produced a 250bp PCR product (FIG. 1).

In order to optimize Duplex PCR, the species-specific primer and two pairs of genus-specific primers were put together using the Bifidobacterium breb and PCR was performed. It was decided to use primer pairs (GP107, GP108, GP109, GP111) that form a nonspecific band just below the species-specific PCR product, which is 950 bp, forming a band at 250 bp and 950 bp (FIG. 2A).

Five randomly selected colonies grown on TOS agar plates were subjected to PCR using genus-specific primers (GP107, GP108) and species-specific primers (GP109, GP111). After picking them, a single colony was taken and PCR was carried out in the same manner as described above to isolate the fungi forming a band (PCR product) of a desired size (Fig. 2B).

The established Duplex PCR technique was used to preselect 36 Bifidobacterium brebs isolated from 2 of 400 isolates Bifidobacterium obtained from 12 infant feces.

Many researchers report that there are more Bifidobacteria in the colon of breast-fed infants than bottle-fed infants. Mitsou et al. (2007) argue that Bifidobacterium longgum and Bifidobacterium breb dominate the feces of breastfeeding healthy infants. Although it was reported that it occupies about 10%, the study was able to isolate Bifidobacterium brebs only in feces of 2 out of 12 infants, of which only 1 out of 40 randomly obtained fungi were found. One was identified as Leeum Breb and the other one was identified as Fidobacterium breb in a total of 40 colonies, indicating that the frequency of appearance and the proportion of total Bifidobacterium differ significantly from host to host.

2. Sequencing

  1) 16S rDNA partial sequencing

Bifidobacterium isolated by Duplex PCR Sequencing of Breb's 16S rDNA gene showed that all strains were Bifidobacterium as shown in Table 10. Breb was shown to show high homology. All samples that produced a PCR product of the desired size through Duplex PCR were obtained from Bifidobacterium. It was found to be Breb.

Stackebarandt and Goebel (1994) say that if the sequence of 16S rRNA genes is at least 97% identical, they can be considered identical. Bifidobacterium Breb The similarity between ATCC 15700 ^T and the 16S rDNA sequence of the isolated strain was consistent with 1374 (98%) of 1388 nucleotides for BB5 and 1379 (99%) of 1388 nucleotides for IB84. (Figures 3 and 4).

Isolated Bifidobacterium Comparing the sequences using clustal W to see the differences between Breb strains, all strains except BB5 showed the same 16S rDNA sequence.

Bifidobacterium Due to the high homology of DNA sequences among genera, it is difficult to identify up to strain stages by 16S rDNA sequencing alone (Ventura et al. (2004)), but multiplex PCR (Bonjoch et al. (2004) using 16S rDNA genes is possible. (Kwon et al. (2005)) and DGGE (Satokari et al. (2001)) have been studied to identify Bifidobacterium, and the experimental results are convenient and accurate for the identification of species level. It was found to be a method.

Partial Sequencing and BLAST Search of 16S rDNA Strain 16S rDNA partial sequencing  Homology% Strain BB5 99-100 Bifidobacterium Breb IB5 99-100 Bifidobacterium Breb IB15 99-100   Bifidobacterium breb IB17 99-100 Bifidobacterium Breb IB25 99-100 Bifidobacterium Breb IB28 99-100 Bifidobacterium Breb IB30 99-100 Bifidobacterium Breb IB33 99-100 Bifidobacterium Breb IB36 99-100 Bifidobacterium Breb IB38 99-100 Bifidobacterium Breb IB39 99-100 Bifidobacterium Breb IB52 99-100 Bifidobacterium Breb IB53 99-100 Bifidobacterium Breb IB59 99-100 Bifidobacterium Breb IB61 99-100 Bifidobacterium Breb IB90 99-100 Bifidobacterium Breb IB91 99-100 Bifidobacterium Breb IB94 99-100 Bifidobacterium Breb IB96 99-100 Bifidobacterium Breb

2. Sequencing of F6PPK Encoding Genes

The PCR product PCR was cloned into pGEM-T easy vector (Promega) using genus-specific primers (GP 109, GP 111) based on the xfp gene encoding F6PPK, an enzyme that is specific to Bifidobacterium genus. . After making the plasmid and comparing the sequence obtained by sequencing the macrogen with SP6 and T7 primer pairs using clustal W, the IB sample was matched with Bifidobacterium brev ATCC 15700 ^T and the BB5 sample was 6 nucleotides. Showed a difference (FIG. 5).

Rossello-Mora and Amman (2001) reported that 16S rRNA gene sequences do not distinguish clear differences between species. Berthoud et al. (2005) identified Bifidobacterium using the xfp gene. Bifidobacterium thermophilum (B. thermophilum ), BP was betting that Te Solarium written or drink pilrum (B. thermacidophilum), Bifidobacterium boum (B. boum) All except BP is also more accurate than when 16S rDNA sequencing method to identify the bacteria. However, comparing the nucleotides of Bifidobacterium breb ATCC 15700 ^T with the samples used in this study, it was confirmed that the 16S rDNA sequencing method was more discriminating than the xfp gene sequencing method.

3. Sequencing BSH-Encoding Genes

PCR products obtained by PCR using primers (GP145, GP146) designed for genes that produce BSH by streaking in 2 mM GDCA and 2 mM TDCA were selected. Promega). The resulting white colonies were made of plasmids, and the nucleotide sequences were compared using the sequences obtained by requesting sequencing to Macrogen Co., Ltd. with SP6 and T7 primer pairs. As a result, 2-5 nucleotides were confirmed to be different.

 Kim et al. (2004a, b) reported that three types of BSH were purified in Bifidobacterium, resulting in differences in biochemical and molecular molecular properties. In this study, several nucleotides were found to differ according to the size of precipitate around the colony.

3. Fast pulsed field gel electrophoresis (PFGE)

To see the difference between strains of Bifidobacterium breb isolated from infant feces, the result of restriction enzyme treatment using Xba I, which is known as an appropriate restriction enzyme to measure the gene size of Bifidobacterium, is shown in FIG. appear.

Briczinski and Roberts (2006) reported no difference between the existing PFGE protocol, which required 5-7 days to complete the experiment, compared to the faster protocol that completed in 24 hours. In this study, we performed PFGE using a fast protocol. As with the 16S rDNA sequencing results, the same PFGE patterns were observed for the isolates from the same infant.

Bifidobacteria  Observation of Morphological Characteristics

Gram staining of experimental strain cells formed after incubating anaerobicly at 37 ° C. for 48 hours in BL agar was as shown in FIG. 7, and the results observed with a scanning electron microscope are shown in FIG. 8. Although optical microscopy alone is difficult to see the typical morphology of Bifidobacteria, scanning electron microscopy shows typical rod-shaped, Y-shaped, and club-like shapes.

Bifidobacteria  Observation of Physiological Characteristics

1. Carbohydrate availability

The isolated strain confirmed as Bifidobacterium was confirmed carbohydrate utilization ability using API 50 CHL (Bio-marieux).

Experimental results As shown in Table 13, the Bifidobacterium breb-type strains and the isolated samples BB5 and IB84 were found to have different carbohydrate utilization. In particular, IB84 showed a tendency to use a large number of carbohydrates under anaerobic conditions.

2. Production of Bile Acidase (BSH)

Strains isolated from infant feces were passaged twice to increase activity and streaked in mMRS medium containing 2 mM GDCA and 2 mM TDCA are shown in Table 14.

Kim et al. (2004b) found that almost all Bifidobacterium has BSH activity. It was confirmed that it is a general characteristic appearing in strains, BSH was separated and purified from various strains and classified into three different types according to their characteristics. In this study, the BSH activity test by Dashkevicz and Feighner (1989) method showed that white precipitates occurred around colonies in almost all Bifidobacterium isolates, and colony formation and sedimentation degree were different according to each strain. Thus, the BSH activity is a general characteristic of Bifidobacterium strains, and the degree of activity and resistance to bile acids are strain-specific characteristics with differences between strains.

   Carbohydrate Fermentation of Bifidobacteria
carbohydrate     Bifidobacteria strains ATCC 15700 BB5 IB84 Glycerol  Erythritol     D-arabinose w     L-arabinose   D-Ribose + + +   D-Xylose   L-Xylose   D-Adonitol   Methyll-βD-xylpyranoside   D-galactose + + +   D-glucose + + +   D-fructose + + +   D-Mannose w + +     L-sorbose   L-Rhamnose  Dulcitol     Inositol D-mannitol + + +   D-sorbitol + + +   Methyl-αD-mannopyranoside w

* w: weak

Continuation of Table 15a
carbohydrate           Bifidobacteria strains  ATCC15700   BB5   IB84  Methyl-αD-Glucopyranoside w + +    N-acetylglucosamine w w +  Amigdalin w + Arbutin w +    Esculin Citrate + + +   Salinity w + +     D-cellobiose + w +   D-maltose + + +   D-lactose + + +     D-Melibiose + +   D-Saccharus + +     D-trehalose w +   Inulin w     D-Melezitose w   D-Raffinose + + Amidodon +    Glycogen w +    Xylitol +     Genthiobiose +   D-Turanos w +   D-Rixos   D-tagatose D-fucose L-fucose w +  D-Arabitol  L-Arabitol Potassium Gluconate 2-ketogluconate potassium 5-ketogluconate potassium

* w: weak

Degradation of Bile Salts (2 mM) by Bifidobacterium Breb Isolated from Infant Feces in Bile Salt-MRS Agar Strain GDCA 2 mM TDCA 2 mM Strain GDCA 2 mM TDCA 2 mM BB5 +++ +++ IB54 +++ +++ IB7 ++ +++ IB55 ++ +++ IB13 + +++ IB59 +++ +++ IB14 ++ +++ IB60 +++ +++ IB26 + - +++ IB61 +++ +++ IB28 + +++ IB62 +++ +++ IB29 + +++ IB65 +++ +++ IB30 + +++ IB67 +++ +++ IB31 ++ +++ IB68 +++ +++ IB32 +++ +++ IB70 +++ +++ IB33 +++ +++ IB75 +++ +++ IB38 +++ +++ IB79 +++ +++ IB46 +++ +++ IB80 +++ +++ IB51-1 ++ ++ IB82 +++ +++ IB51-2 +++ +++ IB84 +++ +++ IB52 +++ +++

     halo size, +++: mm, ++: 3mm-5mm, +: mm

Conjugate Linoleic acid ( CLA ) producing ability

1. Confirmation of CLA production by spectrophotometric assay

In order to confirm whether the isolated strain produces CLA, the isolated strain was incubated for 24 hours after 1% inoculation in the mMRS medium containing 0.5 mg / ml of linoleic acid, and extracted with hexane, and then confirmed the production of CLA using a spectrophotometer. . As a result, all Bifidobacterium brebs isolated from infant feces produced CLA, but three Bifidobacterium breves and Bifidobacterium breves were distributed by the Korean Bacterial Institutions (Bio Resource Center). 2003 did not appear to produce CLA (FIG. 9).

As a result of the experiment using spectrophotometer, each strain showed different CLA production capacity, and some strains isolated from infant feces than LMC 220 from dairy products reported to have high CLA production capacity showed higher O.D.

Pariza and Yang (1999) reported that CLA can be detected using a spectrophotometer, and Barret et al. (2007) used the method to quickly and accurately isolate CLA-producing strains. In this study, CLA-producing strains were successfully screened successfully using spectrophotometry, and fatty acid analysis using GC was required for more accurate quantitative analysis.

2. Result of quantitative analysis of CLA using gas chromatography

In order to find strains with excellent CLA production ability, strains with high OD values were selected for spectrophotometric analysis and analyzed using gas chromatography (GC). Analysis was performed using SP ^™ -2560 (100 m × 0.2 mm, 0.2 μm thickness) colum. Prior to analyzing the selected strains, the time for FAME to pass through the collum was measured using linoleic acid, conjugated methyl ester (Sigma, USA).

The methylation and GC analysis of fatty acids were carried out according to the method of Jung et al. (2006). Fermentation broths of each strain incubated for 0 and 24 hours in mMRS broth added 0.5 mg / ml of LA were incubated for 24 hours after 1% inoculation, and fatty acids were extracted using hexane and methylated to perform GC analysis.

All Bifidobacterium brebs isolated from infant feces could be seen in the chromatogram of the conversion of linoleic acid to conjugated linoleic acid (FIG. 10), with each conversion as shown in FIG. 11. Since linoleic acid is a hydrophobic material, when the linoleic acid stock solution is prepared, the tween 80 content is adjusted to 2% and 5%, and the difference in solubility is tested. Free linoleic acid inhibits the growth of CLA producing strains (Jiang et al. (1998); Kim et al. (2000); Rainio et al. (2001)), while tween 80 neutralizes the antimicrobial activity of free linoleic acid and in liquid medium. It has been reported to increase the solubility of fatty acids (Jiang et al. (1998); Rainio et al. (2001)). As a result of comparing the CLA conversion rate by adjusting the content of tween 80 in this study, it was confirmed that the conversion rate increased by about 0.061 in the medium containing 5% tween 80 than in 2%.

Comparing the isolates with LMC 220 strains with high CLA conversion rates, IB52 and IB84 showed similar conversion rates, and IB84 showed a smaller conversion rate than LMC 220.

Screening of CLA-converted strains using spectrophotometer and GC showed a difference in production, but the production strains were consistent. When the number of strains to be compared is large, using spectrophotometry as the primary screening technique has been found to be efficient.

Bifidobacterium brebs have been reported to have species-specific properties that produce CLA. Coakley et al. (2003) confirmed the CLA production capacity by separating Bifidobacterium from human feces and Bifidobacterium breb and B. dentium had the highest CLA productivity, Oh et al. (2003) confirmed the production of CLA in Bifidobacterium breb and B. pseudocatenulatum . Of the 400 Bifidobacterium isolates from the infant feces used in this study, only Bifidobacterium breve strains were found to have the ability to produce CLA.

1 is a diagram showing the results of agarose gel electrophoresis of the PCR product. Lane M is 1kb of ladder, Lane 1 is B. adolescentis , Lane 2 is B. bifidum , Lane 3 is Bifidobacte Leeum breather bracket, No. 4 lane Bifidobacterium Infante Tees (B. infantis), 5 lane is ronggum Bifidobacterium (B. longum), 6 lane is Bifidobacterium Carthage press ratum (B. catenulatum Lane 7 is B.pseudo-catenulatum , lane 8 is B. dentium , lane 9 is Bifidobacterium suis ( B. suis ), lane 10 represents the band of Bifidobacterium animalis ( B. animalis ).

2A-B show detection of Bifidobacterium brebs by duplex PCR. The conditions of duplex PCR were established as follows (FIG. 2A):

Lane M: 1 kb ladder; Lane 1: bifidobacterium-specific PCR product (923 kb) and bifidobacterium breb-specific PCR product (250 bp); Lane 2: bifidobacterium-specific PCR product (582 bp) and bifidobacterium breb-specific PCR product (250 bp); Lane 3: bifidobacterium breb-specific PCR product (250 bp); Lane 4: bifidobacterium-specific PCR product (923 kb); And lane 5: bifidobacterium-specific PCR product (582 kb).

Figure 2b is a result of electrophoresis confirming that the Bifidobacterium Breb, M lane is 1kb ladder and lanes 1 to 15 are Bifidobacterium breb separated from infant feces.

Figure 3 is a comparison of the 16S rDNA sequence between BB5 and ATCC 15700 ^T.

4 is a comparison of 16S rDNA sequences between IB84 and ATCC 15700 ^T.

Figure 5 is a comparison of the sequence of the F6PPK gene ( xfp ) between BB5, IB84 and ATCC 15700 ^T.

6 shows the PFGE pattern of total DNA of Bifidobacterium strains cleaved by Xba I. The band of each lane is as follows.

Lane M: lambda ladder; Lane 1: BB5; Lane 2: IB26; Lane 3: IB51-1; Lane 4: IB55; Lane 5: IB 62; Lane 6: LMC 220; Lane 7: Bifidobacterium Breb 2003; Lane 8: Bifidobacterium Breb KCTC 3220; Lane 9: Bifidobacterium Breb KCTC 3419; Lane 10: Bifidobacterium Breb KCTC 5081; And lane 11: Bifidobacterium longuem BBL.

7 is a diagram showing the cell morphology of Bifidobacteria.

8 is a diagram showing the results of observing the cell morphology of Bifidobacteria under a scanning electron microscope.

9A-B are graphs showing the results of screening CLA production of Bifidobacterium brebs using spectrophotometry.

10 is a diagram showing a chromatogram of the analysis of conjugated linoleic acid using gas chromatography.

FIG. 11 is a graph showing the conversion rate of conjugated linoleic acid (cis-9, trans-11 isomer) when the Bifidobacterium breb strain was cultured in an MRS culture for 24 hours. Linoleic acid was added as a 50 mg / ml stock solution containing 2% (volume) of tween 80 and filtered and sterilized through a 0.45 μm filter.

12 is a graph comparing the CLA conversion rate according to the tween 80 content.

Claims (5)

Conjugated linoleic acid (Conjugated Linoleic Acid: CLA) produced has an activity isolated from infant feces Bifidobacterium breather bracket (Bifidobacterium breve ) IB84 strain (KCCM 10998P) or Bifidobacterium breve IB52 strain (KCCM 10997P). Bifidobacterium breb breve) IB84 strain (KCCM 10998P) or Bifidobacterium breather bracket (Bifidobacterium breve ) A probiotic composition comprising an IB52 strain (KCCM 10997P). Bifidobacterium breb breve) IB84 strain (KCCM 10998P) or Bifidobacterium breather bracket (Bifidobacterium breve ) Functional fermented milk composition comprising IB52 strain (KCCM 10997P). BP in linoleic gambling te Breda Solarium bracket (Bifidobacterium breve) IB84 strain (KCCM 10998P) or Bifidobacterium breather bracket (Bifidobacterium breve ) A method for converting linoleic acid to conjugated linoleic acid comprising contacting an IB52 strain (KCCM 10997P). 5. The method of claim 4 wherein the conjugated linoleic acid is c9, t11 (C18: 2) conjugated linoleic acid.

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