KR101426276B1 - The products containing probiotic Lactobacillus plantarum HY7712 having activity preventing from the atherogenic legion formation and foam cell formation, which caused heart disease and stroke as effective component - Google Patents

Abstract

The present invention relates to a product containing Lactobacillus plantarum HY7712 having an activity of inhibiting foam cell formation and vascular lesion formation which induces heart disease and stroke as an active ingredient, and Lactobacillus plantarum HY7712 Inhibits oxidative damage in human macrophages, inhibits LDL receptor and scavanger receptors CD36, SR-A and SR-B1 expression, promotes ABCA1 and ABCG1 expression, inhibits macrophage foam cell formation, A pharmaceutical composition having an effect of inhibiting the expression of macrophage-adhered substances and inhibiting vascular lesion formation, thereby having an effect of inhibiting foam formation and vascular lesion formation which cause heart disease and stroke, a health functional food, a functional beverage, and a fermentation product And the like.

Description

[0001] The present invention relates to a product containing, as an active ingredient, Lactobacillus plantarum HY7712 having an activity to inhibit foam cell formation and vascular lesion formation which cause heart disease and stroke, and to provide a product containing, as an active ingredient, Lactobacillus plantarum HY7712 having an atherogenic legion formation and foam cell formation, which cause heart disease and stroke as effective component}

TECHNICAL FIELD The present invention relates to a product containing Lactobacillus plantarum HY7712 having an activity of inhibiting foam formation and vascular lesion formation which induces heart disease and stroke as an active ingredient. More particularly, the present invention relates to a product containing human macrophages (THP- 1 cells) inhibited the expression of LDL receptor and scavenger receptors CD36 (Cluster of Differentiation 36), SR-A (Scavenger Receptor A) and SR-B1 (Scavenger Receptor B1), which are involved in the absorption of cholesterol, Inhibition of foam cell formation by promoting ABCA1 and ABCG1 expression, which are ATP-binding cassette subfamily involved in cholesterol release, and inhibition of vascular lesion formation in inducing vascular lesions in ApoE ^{- / -} mice And inhibiting the expression of adventitious macrophages in blood vessels to inhibit foam cell formation and vascular lesion formation leading to heart disease and stroke, A pharmaceutical composition containing Plantarum HY7712 as an active ingredient, a fermented milk, a beverage, a health functional food, and the like.

Macrophages present in the tissues are immune system cells that absorb both LDL (low density lipoprotein) and oxidized LDL (oxidized LDL) recpetor. Vascular lesions caused by various factors are closely related to bubble cell formation. The initial mechanism of vascular lesion formation is that the monocyte circulating in the blood is moved to the inner wall of the blood vessel by the inflammatory reaction, , And the converted macrophages receive cholesterol and accumulate cholesterol and turn into foam cells. When lesions are present in the blood vessels, the blood vessels lose their elasticity and the lumens thicken, resulting in blood flow disorders, which occur mainly in the coronary arteries and the cerebral arteries, resulting in heart disease and stroke (Hansson GK and Libby P. 2006. Immunology 6: 508-519).

Oxidative stress plays an important role in the pathogenesis of cardiovascular disease. LDL-cholesterol is produced in the liver and is transported to the blood, which is normally absorbed into the blood vessel wall. It is able to migrate back into the bloodstream, but LDL-cholesterol, which is oxidatively stressed, such as active oxygen, can not escape from the blood and remains in the blood vessel wall. When macrophage absorbs it, macrophages differentiate into foam cells. Foam cells continuously absorb LDL-cholesterol, which eventually ruptures in the walls of the blood vessels. The blood vessel walls lose their elasticity and become hardened. (Glass CK and Witztum JL 2001. Cell (104): 503-516). Therefore, inhibiting the absorption of cholesterol into the intravascular macrophages, promoting the release of absorbed cholesterol, inhibiting the oxidation of LDL-cholesterol, and inhibiting the formation of foam cells in the intravascular macrophages, It is very important to treat.

On the other hand, lactic acid bacteria which are abundant in traditional fermented foods such as kimchi, play a role in decomposing fibrous and complex proteins into important nutritional components in symbiosis with the digestive system of the human body, and maintaining intestinal pH as acidic to produce E. coli, ( Chlostridum sp.) As well as to suppress the growth of diarrhea and constipation, as well as vitamin synthesis, blood cholesterol degradation and plays a role. Especially lactic acid bacteria can bind strongly to the mucous membrane and epithelial cells of the intestines, which helps a lot of the action. In particular, Lactobacillus plantarum is one of the most widely distributed lactic acid bacteria in nature, separated mainly from kimchi, pickles, tomatoes, milk, cheese, and butter. Kimchi has a high fermentation and when the acid resistance and in biliary river bacteria mainly grow, with the antimicrobial activity of the Puja Solarium (Fusarium), Pseudomonas (Pseudomonas), it has an antioxidant and anti-inflammatory effect is a useful strain.

On the other hand, Korean Patent Laid-Open No. 10-2007-00078010 (entitled Lactobacillus plantarum K8 inhibiting cholesterol absorption and improving blood lipids by improving blood lipids) discloses ABCA1 expression, LDL inhibitor, LDL Receptor, CD36, SR-A1, and SR-B1 as an invention relating to Lactobacillus plantarum K8 inhibiting cholesterol absorption, improving HDL lipoprotein in the blood and lowering LDL lipoprotein and improving blood lipid Inhibition of macrophage infiltration by ABCG1 and ABCA1 and inhibition of macrophage formation by apoE ^{- / -} mice induced angiogenesis in macrophages, The physiological activity will be different from that of Lactobacillus plantarum HY7712 of the present invention, which has the effect of inhibiting foam cells and inhibiting lesion formation.

Therefore, the inventors of the present invention have found that Lactobacillus plantarum HY7712 macrophage cells of novel patent application No. 2011-0030183 (a novel Lactobacillus plantarum HY7712 having immunity enhancement and antioxidative activity and products containing it as an active ingredient) To inhibit the formation of foam cells and to inhibit vascular adhesion molecules of macrophages, and thus it has been found to be effective in inhibiting vascular lesions which are caused in cardiovascular and cerebrovascular diseases to cause heart disease and stroke. Respectively.

Korean Patent Laid-Open No. 10-2007-00078010 (Title of the invention: Prevention of arteriosclerosis by inhibition of cholesterol absorption and improvement of blood lipids Lactobacillus plantarum K8, published on July 30, 2007)

The present invention inhibits the expression of LDL receptor and scavanger receptors CD36, SR-A and SR-B1 in human macrophages (THP-1 cells), inhibits oxidative damage and inhibits the expression of ABCA1, ABCG1 Inhibition of foam cell formation by promoting expression and inhibition of lesion formation and vascular endothelial cell adhesion during induction of vascular lesions in ApoE ^{- / -} mice, resulting in cardiovascular and cerebrovascular diseases, A pharmaceutical composition containing Lactobacillus plantarum HY7712 having an anti-vascular lesion formation-inducing activity as an active ingredient, a fermented milk, a beverage, a health functional food, and the like.

In order to achieve the above object, the present invention provides a method for inhibiting the formation of foam cells of macrophages and inhibiting the expression of macrophage adhesion molecules in vascular endothelial cells, thereby inhibiting vascular lesions that cause heart disease and stroke. A pharmaceutical composition containing Lanta Room HY7712 as an active ingredient, a fermented milk, a beverage, and a health functional food.

Hereinafter, the present invention will be described in detail.

Lactobacillus plantarum HY7712 according to the present invention is a novel strain of Lactobacillus plantarum isolated and identified from kimchi. As a result of 16S rRNA sequence analysis for identification and classification of microorganisms, Lactobacillus plantarum KW30 ( Lactobacillus plantrum KW30 ) Showed the highest homology with Lactobacillus plantarum, and the microorganism was identified as Lactobacillus plantarum and designated as Lactobacillus plantarum HY7712. The microorganism was named Lactobacillus plantarum HY7712, Deposited on Feb. 23, 2011 (Accession No. KCTC 11874BP). The nucleotide sequence of the 16S rRNA gene of Lactobacillus plantarum HY7712 is shown in SEQ ID NO. 1.

The Lactobacillus plantarum HY7712 of the present invention is a facultive anaerobe capable of growing under both aerobic and anaerobic conditions. It is spore-free and has no motility. The cell type is bacterium. More specific physiological characteristics of Lactobacillus plantarum HY7712 were analyzed according to the conventional methods in the related art, and the results are shown in Table 1.

Morphology and physiology, biochemical characteristics Morphology Bacteria (Rod) Motility - Spore - Catalase - Homo-Hetero Fermentation In-situ heterotrophic fermentation Growth at 15 ℃ + Proliferation at 45 ° C - Proliferation in 3% NaCl + Anaerobic proliferation + Glucose utilization CO ₂ production - Fermentation characteristics D-arabinose - Riboze + D-xylose - Galactose + D-glucoside + D-fructose + D-mannooz + Sorbitol + Amigalline + Esculin + Salincin + Selovioz + Malooz + Lactooz + Melibiose + Shukrooz + Trehalose + Melijito Oz + D-rainozo + Gluconate +

  +: Positive, -: negative response

Our bodies are exposed to numerous oxidative impairments, naturally occurring as noxious and reactive oxygen. This active oxygen reacts with lipids, proteins, sugars, and DNA constituents of the cell to cause destruction, thereby causing various diseases such as cancer, brain disease and heart disease as well as aging. Antioxidants are substances that block the active oxygen, which is an antioxidant that provides naturally occurring antioxidants in the body as well as vitamin E, C, and beta carotene. Lactic acid has antioxidant effects bifidobacteria tumefaciens Infante Tees (Bifidobacterium infantis), bifidobacteria tumefaciens ronggeom (Bifidobacterium longum), Lactobacillus momentum spread (Lactobacillus fermentum), Lactococcus lactis (Lactococcus lactis) ssp. Lactis (lactis), and the like are known, Lactobacillus Planta room HY7712 is Patent Application No. 2011-0030183 (title of the invention: containing a novel Lactobacillus Planta room HY7712 and this active ingredient with antioxidant activity and immunity enhancement ) Was found to have antioxidant activity.

When we look at the important mechanism of vascular lesion formation, LDL among blood cholesterol is closely related. When blood cholesterol migrates along the blood vessels, LDL is moved in a concentration-dependent manner on the blood vessel wall. The LDL absorbed from subcutaneous veins undergoes oxidative LDL formation. Monocytes moving along the blood vessels enter the blood vessel walls to absorb the oxidized LDL, and vascular lesions begin to form from these processes. The monocyte-colony stimulating factor (M-CSF) differentiates into macrophages, and the oxidized LDL is absorbed through the scavenger receptors on the surface of the differentiated macrophages.

In the present invention, Lactobacillus plantarum HY7712, which inhibits LDL oxidation in human macrophages, was selected.

In the present invention, Lactobacillus plantarum HY7712, which inhibits the expression of scavenger receptor CD36, SR-A and SR-B1, which is involved in the absorption of oxidized LDL in human macrophages, was selected.

It has been known that the blood LDL ratio should be lowered in order to prevent vascular lesions which are caused in cardiovascular and cerebrovascular diseases and cause heart disease and stroke, and it is known that the role of macrophages in the outflow of cholesterol in tissues is known to be important . The intracellular accumulation of cholesterol received by the receptor in macrophages is excreted by the HDL interaction with apo lipoprotein by ABCA1, ABCG1, an ATP-bingind cassette subfamily present in macrophages. As such, macrophages excrete cholesterol externally through the interaction of ABC protein with HDL.

In the present invention, Lactobacillus plantarum HY7712 promoting the expression of ABCA1 and ABCG1 involved in cholesterol release in human macrophages was selected.

As described above, the Lactobacillus plantarum HY7712 of the present invention inhibits oxidative damage, inhibits oxidized LDL absorption, inhibits foam cell formation through promotion of cholesterol release, inhibits vascular lesion formation through inhibition of macrophage adhesion molecule expression And can be used as a health functional food, a functional beverage, a fermented product and the like containing the active ingredient as an effective ingredient.

FIG. 1 is a graph showing changes in expression of CD36, SR-A, and SR-B1 as scavenger receptors by macrophage treatment of Lactobacillus plantarum HY7712.
FIG. 2 is a photograph showing the absorption of LDL receptor and LDL by macrophage treatment of Lactobacillus plantarum HY7712. FIG.
FIG. 3 is a graph showing changes in ABCA1 and ABCG1 expression involved in cholesterol efflux by macrophage treatment of Lactobacillus plantarum HY7712. FIG.
FIG. 4 is a graph showing the lipid accumulation result of macrophage treatment of Lactobacillus plantarum HY7712. FIG.
5 is a graph showing the results of measurement of foam cell formation by LDL oxidation by macrophage treatment of Lactobacillus plantarum HY7712.
6 is a graph showing the results of measurement of intravascular macrophage-adhered substance expression of Lactobacillus plantarum HY7712.
7 is a photograph showing the results of measurement of intravascular lesion formation of Lactobacillus plantarum HY7712.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following embodiments are not intended to limit the scope of the present invention, and ordinary variations by those skilled in the art within the scope of the technical idea of the present invention are possible.

≪ Example 1 >

Isolation and Identification of Lactobacillus planta HY7712 from Kimchi

Lactobacillus plantarum according to the present invention was prepared by declining the kimchi broth collected in each region, culturing it on a MRS solid medium containing 1.5% agar, incubating the culture at 37 ° C for 48 hours, Was taken as a loop and inoculated onto fresh MRS solid medium and purely isolated. The purely isolated colonies were inoculated into the MRS liquid medium and cultured at 37 DEG C for 24 hours. Lactobacillus plantarum was used for the experiment.

The morphology and physiological characteristics of the isolated strains were then determined using API 50CH and API 50CHL kits. As a result of the API kit of strain HY7712, Table 1 shows that the strain HY7712 was the same as that of lactobacillus plantarum. In addition, the 16S rRNA gene sequence for identification and classification of lactic acid bacteria was analyzed, and the 16S rRNA gene sequence of the confirmed HY7712 strain was described in the sequence listing (SEQ No. 1). As a result of 16S rRNA gene sequencing analysis, HY7712 strain showed the highest homology with Lactobacillus plantarum standard strain and was identified as Lactobacillus plantarum , designated as Lactobacillus plantarum HY7712, Deposited with the Gene Bank on February 23, 2011 (Accession No .: KCTC 11874BP).

≪ Example 2 >

Preparation of freeze-dried powder containing Lactobacillus planta HY7712

Lactobacillus plantarum HY7712 of Example 1 was inoculated into MRS broth and cultured for 24 hours in a 37 ° C incubator. The supernatant was removed by centrifugation at 5000 rpm for 20 minutes, washed with saline, and then centrifuged at 5000 rpm for 20 minutes to remove the supernatant. The collected cells were mixed with the powder of skim milk powder as a preservative at a ratio of 1: 1 and lyophilized to prepare a powder.

Lactobacillus plantarum HY7712 of the present invention may be provided in the form of a lyophilized powder or a culture as described above.

≪ Example 3 >

Production of a pharmaceutical composition containing Lactobacillus plantarum HY7712 as an active ingredient

Manufacture of liquid agent

100 mg of the lyophilized powder containing Lactobacillus plantarum HY7712 of Example 2, 10 g of isomerized sugar, and 5 g of mannitol were dissolved in purified water in accordance with the usual preparation method, and the lemon flavor was added in an appropriate amount. To adjust the total volume to 100 ml, and the solution was filled in a brown bottle and sterilized to prepare a liquid preparation.

Preparation of capsules

100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed thoroughly with 100 mg of the lyophilized powder containing Lactobacillus plantarum HY7712 of Example 2, and the resulting mixture was thoroughly mixed with hard gelatine capsules To prepare a capsules.

<Example 4>

Production of fermented milk containing Lactobacillus planta HY7712 as an active ingredient

A method for producing a fermented milk containing the lactobacillus plantarum HY7712 of the present invention as an active ingredient is as follows.

The lactic acid bacteria culture solution was prepared by stirring 95.36 wt% of crude oil and 4.6 wt% of skim milk powder (or mixed powdered milk) and measuring specific gravity at 15 ° C from 1.0473 to 1.0475, a titratable acidity of 0.200 to 0.220%, pH of 6.65 to 6.70, of Brix (Brix ^o) were mixed such that the degree of 16.3 ~ 16.5%. After mixing, the mixture was heat-treated with UHT (sterilized at 135 ° C. for 2 seconds), cooled to 40 ° C., added with 0.02% by weight of Streptococcus thermophilus and Lactolytic enzyme (Valley laboratory, USA) The total number of lactic acid bacteria in the medium was 1.0 × 10 ⁹ cfu / ml or more, the titratable acidity was 0.89 to 0.91%, and the pH was 4.55 to 4.65.

Then, mixed juice syrup, liquid fructose, 13 wt.%, Sugar 5 wt.%, Mixed juice concentrate 56Brix ^o 10.9% by weight of pectin, 1.0% by weight, fresh fruit mix Essence 0.1% and purified water 70% by weight at 30 ~ 35 ℃ Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.

Then, a mixture of 69.5% by weight of the culture liquid of lactic acid bacteria and 0.1% by weight of freeze-dried powder containing Lactobacillus plantarum HY7712 of Example 2 and 30.4% by weight of the mixed fruit juice syrup were combined and homogenized at 150 bar, Lactobacillus planta HY7712 as an active ingredient was prepared.

&Lt; Example 5 >

Production of vegetable juice containing Lactobacillus planta HY7712 as an active ingredient

The method for producing vegetable juice containing the lactobacillus plantarum HY7712 of the present invention as an active ingredient is as follows.

Citric acid juice 1 wt.%, Spinach juice 1 wt.%, Lettuce juice 2 wt.%, Celery juice 1 wt.%, Anhydrous citric acid 0.2 wt.%, 0.1% by weight of freeze-dried powder containing Lactobacillus plantarum HY7712 of Example 2 was uniformly mixed in a blender and sterilized at 85 to 97 ° C using a sterilizer to prepare vegetable juice containing Lactobacillus plantarum HY7712 as an active ingredient .

&Lt; Example 6 >

Production of functional beverage containing Lactobacillus plantarum HY7712 as an active ingredient

A method for producing a functional beverage containing the Lactobacillus plantarum HY7712 of the present invention as an active ingredient is as follows.

First, a mixed juice syrup was 13% by weight of liquid fructose, white sugar, 2.5 wt%, brown sugar 2.5% by weight, mixed juice concentrate 56Brix ^o 10.9% by weight of pectin, 1.0% by weight, fresh 0.1% by weight fruit mix essence and purified water 70% The mixture was stirred at 30 to 35 ° C, mixed, and heat-treated by UHT (sterilized at 135 ° C for 2 seconds) and cooled.

Then, 30.4% by weight of the mixed fruit juice syrup prepared by the above method, 0.1% by weight of the lyophilized powder containing the lactobacillus planta HY7712 of Example 2 and 69.5% by weight of the remaining purified water were combined and homogenized at 150 bar, And then packed in a small bottle container such as a glass bottle or a plastic bottle to prepare a functional beverage containing Lactobacillus plantarum HY7712 as an active ingredient.

&Lt; Example 7 >

Production of Health Functional Foods Containing Lactobacillus plantarum HY7712 as an Active Ingredient

(Vitamin B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid ester, nicotinic acid amide) and oligosaccharide were added to 0.1 wt% of the lyophilized powder containing Lactobacillus plantarum HY7712 of Example 2 Was added in an amount of 10 parts by weight based on 100 parts by weight of the lyophilized powder containing Lactobacillus plantarum HY7712 of Example 2 and mixed in a high-speed rotary mixer. 10 parts by weight of sterilized purified water was added to the mixture, mixed and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum drier at 40 to 50 DEG C and then passed through 12 to 14 mesh to prepare uniform granules. The granules thus prepared were extruded in suitable amounts to be purified or powdered or filled into hard capsules to prepare hard capsule products.

&Lt; Test Example 1 >

Effects of Lactobacillus plantarum HY7712 on the Expression of CD36, SR-A and SR-B1

Scavenger receptors such as CD36, SR-A, and SR-B1 are receptors of oxidized LDL and play important roles in macrophages. Therefore, expression of CD36, SR-A and SR-B1 was confirmed in order to confirm the effect of suppressing the absorption of oxidized LDL of Lactobacillus plantarum HY7712, a Kimchi isolate of the present invention.

1-1 . Culture of macrophages

The animal cell used in this study was a human monocyte monocyte cell line, THP-1, which was purchased from the Korean Cell Line Bank (cellbank.snu.ac.kr). For general cell maintenance, cells were cultured in medium containing 85% RPMI 1640 medium (Gibco # 22400) and 15% FBS (Gibco # 16000) and subcultured once a week at a ratio of 1: 4 .

1-2 Preparation of Lactic Acid Bacteria Samples

The Lactobacillus plantarum HY7712 culture broth isolated from kimchi broth collected in each region cultivated in MRS liquid medium was centrifuged and the cells were washed 3 times with PBS. The washed Lactobacillus planta HY7712 cells were killed by boiling in a bath for 15 minutes at 100 ° C and used for the experiment.

Differentiation of 1-3 macrophages

The THP-1 cells were subcultured on a 6-well plate at 1 × 10 ⁶ cells / well and cultured with 100 ng / ml phorbol 12-myristate 13-acetate (PMA, Sigma # p1585) for 72 hours to differentiate into macrophages.

1-4 Measurement of Expression of CD36, SR-A, and SR-B1 in THP-1 Cells by Treatment with Lactobacillus plantarum HY7712

The differentiated macrophages were treated with 10 ⁶ CFU / well of killed Lactobacillus plantarum HY7712 for 24 hours to analyze the expression of genes related to macrophage scavenger receptors CD36, SR-A and SR-B1 .

RNA was isolated from macrophages using TRIZOL reagent (Invitrogen, Carsbad, CA, USA). CDNA was synthesized from the separated RNA using QuantiTect reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantified by real-time PCR using the QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA) and the CFX384 real-time RT-PCR detection system (Bio-Rad, Hercules, CA). Human primer sequences are shown in Table 2. GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) was used as an internal control.

gene Primer sequence Human SR-A For ATT GCC CTT TAC CTC CTC GT Human SR-A Rev ATG AGG TTG GCT TCC ATG TC Human CD36 For AGA TGC AGC CTC ATT TCC AC Human CD36 rev TGG GTT TTC AAC TGG AGG GG Human SR-B1 For CTT CCT CGA GTA CCG CAC Human SR-B1 Rev GAG CCA CGA AGC GAT AGG

The results are shown in Fig.

As can be seen from FIG. 1, it was confirmed that Lactobacillus plantarum HY7712 of the present invention decreased expression of CD36, SR-A, and SR-B1 and inhibited oxidized LDL absorption in THP-1 cells.

&Lt; Test Example 2 &

Measurement of LDL receptor and LDL absorption in THP-1 cells by treatment with Lactobacillus plantarum HY7712

The differentiated macrophages were treated with 10 ⁶ CFU / well of the killed Lactobacillus plantarum HY7712 cells for 24 hours and treated with LDL-DyLight ^™ 549 at 100 μl / well for 4 hours at 37 ° C. The control group treated PBS with differentiated macrophages. After washing with PBS, LDL absorption was measured at excitation and emission wavelengths at 540 nm and 570 nm, respectively. Cells were fixed with Cell-Bassed Assay Fixative Solution to identify LDL receptor, and coated with 100 μl / well of Cell-Bassed Assay Blocking Solution for 30 min. LDL Receptor Primary Antibody. After washing, the cells were incubated with 100 μl / well DyLight ^{TM 488} Conjugated Secondary Antibody. LDL receptor and LDL uptake was measured by fluorescence microscopy at 485 nm and 535 nm, respectively.

The results are shown in Fig.

As can be seen from FIG. 2, it was confirmed that Lactobacillus plantarum HY7712 of the present invention inhibits LDL receptor expression and thus suppresses uptake of LDL.

&Lt; Test Example 3 >

Measurement of ABCA1 and ABCG1 expression levels in THP-1 cells by Lactobacillus plantarum HY7712 treatment

The ATP binding cassette subfamily, such as ABCA1 and ABCG1, is a gene associated with cholesterol release and plays an important role in macrophages. Therefore, the expression of ABCA1 and ABCG1 genes was analyzed to confirm the inhibitory effect of cholesterol on the foam cell formation of Lactobacillus plantarum HY7712, a Kimchi isolate of the present invention.

The differentiated macrophages were treated with 10 ⁶ CFU / well of the killed Lactobacillus plantarum HY7712 cells for 24 hours. RNA was isolated from macrophages using TRIZOL reagent (Invitrogen, Carsbad, CA, USA). CDNA was synthesized from the separated RNA using QuantiTect reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantified by real-time PCR using the QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, CA) and the CFX384 real-time RT-PCR detection system (Bio-Rad, Hercules, CA). Human primer sequences are shown in Table 3. GAPDH was used as an internal control. All experiments were performed with three iterations to calculate the mean and standard deviation per sample.

gene Primer sequence Human ABCG1 For CAA CAG TTT GTG GCC CTT TT Human ABCG1 Rev AGT TCC AGG CTG GGG TAC TT Human ABCA1 For GCC TAC TGC AGC ATC GTG TA Human ABCA1 rev GTC GAA GCT GAC GAA GAA CC

The results are shown in Fig.

As can be seen from FIG. 3, it was confirmed that Lactobacillus plantarum HY7712 of the present invention promotes the expression of ABCA1 and ABCG1, thereby promoting cholesterol release in THP-1 cells.

<Test Example 4>

Measurement of lipid accumulation in THP-1 cells by Lactobacillus plantarum HY7712 treatment

The effect of the Lactobacillus plantarum HY7712 treatment of the present invention on the inhibition of foam cell formation by reduction of lipid accumulation in THP-1 cells was examined.

Differentiated THP-1 cells were treated with 1% oxidized LDL cholesterol (oxLDL) for 24 h to absorb oxLDL into macrophages. Thereafter, the Con group (untreated group) was cultured for 24 hours in the absence of any treatment with the cells, and the HY7712-treated group was treated with 10 ⁶ CFU / well of Lactobacillus plantarum HY7712 per well, Lt; / RTI &gt;

The medium was carefully removed from each well, washed three times with PBS, and fixed with 3.8% 1 ml formaldehyde for 1 hour. Immobilized cells were stained with 1 ml of an oil-red O staining solution (Sigma # O0625) in PBS-T for 1 hour. After removing the dye and washing 3 times with PBS, cells stained with a microscope were observed and treated with 1 ml of isopropyl alcohol for 30 minutes. Decomposed isopropyl alcohol was collected and absorbance was measured at 594 nm.

The results are shown in Fig.

As can be seen in FIG. 4, the HY7712 treated group of the present invention had a lipid accumulation amount of about 20% lower than that of the control group without any treatment. From these facts, it was found that Lactobacillus plantarum HY7712 of the present invention has an effect of inhibiting foam cell formation due to a decrease in lipid accumulation.

&Lt; Test Example 5 >

Measurement of LDL oxidation inhibition by Lactobacillus plantarum HY7712 treatment

The effect of inhibiting LDL oxidation of Lactobacillus plantarum HY7712 of the present invention was investigated.

5-1 Antioxidant Suppression of Lactobacillus plantarum HY7712

The cultured Lactobacillus plantarum HY7712 culture was centrifuged and the cells were washed three times with PBS. The washed Lactobacillus plantarum HY7712 cells were killed by boiling in a bath for 15 minutes at 100 ° C and incubated with H ₂ O ₂ for 24 hours to inhibit antioxidant activity.

5-2. Measurement of lipid accumulation in THP-1 cells by Lactobacillus plantarum HY7712 treatment

Differentiated THP-1 cells were treated with 1% oxidized LDL cholesterol (oxLDL) for 24 hours to allow the cells to absorb oxLDL. Thereafter, the Con group (untreated group) was cultured for 24 hours in the absence of any treatment on the oxLDL-absorbed THP-1 cells, and the Con + H ₂ O ₂ group was incubated with the oxLDL-absorbed THP-1 Cells were further incubated for 24 h with oxidative damage induced by H ₂ O ₂ , In the HY7712 + H ₂ O _2- treated group, oxidative damage was induced in THP-1 cells that had absorbed oxLDL by H ₂ O ₂ , and Lactobacillus plantarum HY7712 in which the antioxidative capacity was inhibited was cultured in 10 ⁶ CFU / well Lt; / RTI &gt; for 24 hours.

The medium was carefully removed from each well, washed three times with PBS, and fixed with 3.8% 1 ml formaldehyde for 1 hour. Immobilized cells were stained with 1 ml of an oil-red O staining solution (Sigma # O0625) in PBS-T for 1 hour. After removing the dye and washing 3 times with PBS, cells stained with a microscope were observed and treated with 1 ml of isopropyl alcohol for 30 minutes. Decomposed isopropyl alcohol was collected and absorbance was measured at 594 nm.

The results are shown in Fig.

As can be seen from FIG. 5, in the Con + H ₂ O ₂ group due to oxidative damage, the lipid accumulation was increased by about 10% as compared with the Con group (untreated group). This indicates that lipid accumulation by oxidative damage increases foam cell formation.

On the other hand the accumulation of lipid HY7712 + H ₂ O ₂ treated group was approximately 10% lipid accumulation was decreased as compared to the group O ₂ + H ₂ Con. This indicates that the Lactobacillus plantarum HY7712 of the present invention inhibits LDL oxidation and oxidative damage and reduces lipid accumulation as compared with the Con + H ₂ O ₂ group in which LDL oxidation is induced by oxidative damage.

From these facts, it was found that the Lactobacillus plantarum HY7712 of the present invention inhibits foam cell formation by inhibiting LDL oxidation and inhibiting oxidative damage, thereby inhibiting foam cell formation.

&Lt; Test Example 6 >

Inhibitory effect of Lactobacillus plantarum HY7712 on intravascular macrophage adhesion

The inhibitory effect of Lactobacillus plantarum HY7712 of the present invention on the expression of macrophage adhesion substance in vascular endometrium was examined.

6-1 Experimental animal breeding and sacrifice

ApoE ^{- / -} 9 week old male mice were purchased from Jackson Labratories (Bar Harbor, ME) and used for one week after being fed with high fat high cholesterol diet. All mice in the experimental group were randomly assigned to 10 mice. The experimental groups were divided into three groups: normal diet group, high fat high cholesterol diet group (HFD + HCD), lactobacillus supplement group (high fat high cholesterol diet + Lactobacillus plantarum HY7712; HFD + HCD + HY7712). The dietary group was used as a control group, the high fat high cholesterol diet group was used as a negative control, and the lactic acid supplement group was used as an experimental group. In the experiment, only high fat high cholesterol diet group and lactobacillus supplement group were compared.

The lyophilized powder of Lactobacillus plantarum HY7712 of Example 1 was mixed with diets such that the lyophilized powder was 5 x 10 ⁹ CFU per g of the dietary weight, and then administered to the mice. The total body weight and feed intake were measured twice a week by feeding the experimental diet for a total of 9 weeks. After 9 weeks of feeding, the animals were fasted for 12 hours, sacrificed after being anesthetized with diethyl ether. Blood was collected from the lower vena cava to analyze the blood of the experimental animals.

The vascular tissues were collected, washed with PBS, weighed, analyzed for gene expression and lesion formation.

6-2. Lactobacillus plantarum HY7712 reduced intravascular macrophage mRNA expression

The expression of genes associated with intravascular macrophage adhesion (VCAM) was analyzed. RNA was isolated from blood vessels using TRIZOL reagent (Invitrogen, Carsbad, CA, USA). CDNA was synthesized from the separated RNA using QuantiTect reverse transcription kit (Qiagen, Valencia, Calif.). RNA expression was quantified by real-time PCR using QuantiTect SYBR Green RT-PCR kit (Qiagen, Valencia, Calif.) And CFX384 Real-time RT-PCR detection system (Bio-Rad, Hercules, Calif. Are shown in Table 4. GAPDH was used as an internal control.

gene Primer sequence Mice VCAM For TAC CAG CTC CCA AAA TCC TG Mice VCAM Rev TCT GCT AAT TCC AGC CTC GT

The results are shown in Fig.

As can be seen from FIG. 6, VCAM expression, which is involved in intravascular macrophage adhesion, was significantly decreased by the intake of Lactobacillus plantarum HY7712 when vascular lesions were induced by high-fat diet-induced high cholesterol diet. From these facts, it was found that Lactobacillus plantarum HY7712 of the present invention has an effect of inhibiting the expression of adherent macrophages.

6-3. Inhibition of vascular lesion formation of Lactobacillus plantarum HY7712

For hematoxylin and eosin (H & E) staining of the isolated blood vessels, blood vessels were washed with PBS and fixed in 10% (v / v) formalin / PBS. The sections were stained with hematoxylin and eosin (H & E) and examined under a microscope.

The results are shown in Fig.

As shown in FIG. 7, the lactic acid bacteria supplement group (HFD + HCD + HY7712) was significantly higher in the high blood cholesterol diet group (HFD + HCD) by the intake of Lactobacillus plantarum HY7712 And inhibited the formation of lesions. From these facts, it was found that Lactobacillus plantarum HY7712 of the present invention has an effect of inhibiting vascular lesion formation which causes heart disease and stroke.

Korea Biotechnology Research Institute KCTC11874BP 20110223

<110> KOREA YAKULT CO., LTD <120> The new Lactobacillus plantarum HY7712 stimulate immunity and          products containing as effective component <130> P11-03 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1031 <212> DNA <213> Lactobacillus plantarum <400> 1 atttattggg cgtaaagcga gcgcaggcgg ttttttaagt ctgatgtgaa agccttcggc 60 tcaaccgaag aagtgcatcg gaaactggga aacttgagtg cagaagagga cagtggaact 120 ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga aggcggctgt 180 ctggtctgta actgacgctg aggctcgaaa gtatgggtag caaacaggat tagataccct 240 ggtagtccat accgtaaacg atgaatgcta agtgttggag ggtttccgcc cttcagtgct 300 gcagctaacg cattaagcat tccgcctggg gagtacggcc gcaaggctga aactcaaagg 360 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc tacgcgaatt 420 ggagggtttc cgcccttcag tgctgcagct aacgcattaa gcattccgcc tggggagtac 480 ggccgcaagg ctgaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 540 gtttaattcg aagctacgcg aagaacctta ccaggtcttg acatactatg caaatctaag 600 agattagacg ttcccttcgg ggacatggat acaggtggtg catggttgtc gtcagctcgt 660 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttattatca gttgccagca 720 ttaagttggg cactctggtg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt 780 caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggat ggtacaacga 840 gttgcgaact cgcgagagta agctaatctc ttaaagccat tctcagttcg gattgtaggc 900 tgcaactcgc ctacatgaag tcggaatcgc tagtaatcgc ggatcagcat gccgcggtga 960 atacgttccc gggccttgta cacaccgccc gtcacaccat gagagtttgt aacacccaaa 1020 gtcggtgggg t 1031

Claims (13)

delete delete delete delete delete delete Vascular disease prevention food containing Lactobacillus plantarum HY7712 (accession no.: KCTC11874BP) which has an activity of inhibiting foam cell formation and inhibiting vascular lesion formation causing heart disease and stroke Composition.
8. The method of claim 7,
The Lactobacillus plantarum HY7712 inhibits oxidative damage in human macrophages, thereby inhibiting foam cell formation and vascular lesion formation leading to heart disease and stroke. A food composition for preventing diseases.
8. The method of claim 7,
The Lactobacillus plantarum HY7712 inhibits the expression of LDL receptors and LDL uptake in human macrophages, thereby inhibiting foam cell formation and vascular lesions that cause heart disease and stroke. Which comprises administering to said mammal a composition for preventing vascular disease.
8. The method of claim 7,
The Lactobacillus plantarum HY7712 inhibits the expression of the scavenger receptors CD36 (Cluster of Differentiation 36), SRA (Scavenger Receptor A) and SR-B1 (Scavenger Receptor B1) in human macrophages Wherein the composition inhibits foam formation and vascular lesion formation that cause heart disease and stroke.
8. The method of claim 7,
The Lactobacillus plantarum HY7712 induces the expression of ABCA1 and ABCG1, which are ATP-binding cassette subfamily in human macrophages, to promote cholesterol release, thereby inhibiting foam cell formation and vascular lesions Of the composition for preventing vascular diseases.
8. The method of claim 7,
The Lactobacillus plantarum HY7712 inhibits the expression of the vascular cell adhesion molecule in the mouse to inhibit foam cell formation and vascular lesion formation leading to heart disease and stroke Wherein the composition is used for the prevention of vascular diseases.
13. The method according to any one of claims 7 to 12,
Wherein the food composition is any one of a functional beverage, a health functional food, and a fermented product.

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