KR20200145941A - A food composition for prevention or improvement of diseases related to tight junction protein comprising Lactobacillus casei HY2782 as an effective compotent - Google Patents

Abstract

The present invention relates to a food composition for the prevention or alleviation of diseases related to tight junction protein caused by fine dust, and containing Lactobacillus casei HY2782 as an active ingredient. The Lactobacillus casei HY2782 has efficiency of adjusting OCLN and RUNX1 gene expression in human lung cells and RUNX1 gene expression in human colon cells, and thus can be used as an active ingredient contained in a food composition, such as fermented milk and health functional food, for the prevention or alleviation of diseases related to tight junction protein caused by fine dust.

Description

A food composition for prevention or improvement of diseases related to tight junction protein comprising Lactobacillus casei HY2782 as an effective compotent }

The present invention relates to a food composition for the prevention or improvement of diseases related to adhesion junction proteins by fine dust containing Lactobacillus casei HY2782 as an active ingredient, and more specifically, OCLN and RUNX1 of human lung cells. In food compositions for the prevention or improvement of diseases related to tight junction proteins by fine dust containing Lactobacillus casei HY2782 as an active ingredient, which has the effect of regulating gene expression and expression of RUNX1 gene in human colon cells. About.

Probiotics are'probiotics that have a beneficial effect on the host animal by improving the intestinal flora', starting with what Fuller defined in 1989, and published in 2001,'probiotics that benefit the health of the host when consumed in sufficient amounts. The FAO/WHO definition of'living microorganism' is widely used. In addition, in 1999, Salminen et al. defined it as'a microbial preparation or a component of a microorganism having a beneficial effect on the host', and there is an interpretation that expanded the range of probiotics from live cells to dead cells. As research results and scientific data that human intestinal microbes, including probiotics, have an important effect on human health, increase, consumers' awareness of probiotics has increased, and accordingly, the demand for probiotic products is gradually increasing. 11 species of Lactobacillus registered by the Ministry of Food and Drug Safety ( L. acidophilus, L. casei, L. gasseri, L. delbruekii subsp. bulgaricus, L. helveticus, L. fermentum, L. paracasei, L. plantarum, L. plantarum, L. reuteri, L. rhamnosus, L. salivarius ) and Lactococcus 1 species ( Lc. lactis ), Enterococcus 2 species ( E. faecium, E. faecalis ), Streptococcus 1 species ( Streptococcus ) the S. thermophilus), Bifidobacterium (Bifidobacterium) 4 jong (B. bifidum, B. breve, B. longum, B. animalis subsp. was announced as probiotics with respect to the 19 kinds of strains to lactis) many companies are probiotics Research and sell products.

RUNX is a runt-related transcription factor, and as a new tumor suppressor whose cancer suppressing activity has been recently identified, many studies are being conducted on various cancers. RUNX has a runt domain called PEBP2/CBF (polyoma virul enhancer binding protein 2/core binding factor), is a signal transduction target of the TGF-β superfamily and plays an important role in mammalian development.

The RUNX family consists of RUNX1 (PEBP2aB/CBFA2/AML1), RUNX2 (PEBP2aB/CBFA1/AML3) and RUNX3 (PEB2aC/CBFA3/AML2). These three RUNX families play an important role in the normal development and differentiation process and in the oncogenesis process. RUNX1 (Runt-related transcription factor 1), which plays an important role in the hematopoietic process, is the most frequent part of chromosome translocation in leukemia, and accounts for about 30% of the causes of acute leukemia. In particular, according to recent Miao research results, it has been reported that overexpression of the RUNX1 gene increases the expression of tight junction proteins such as ZO-1, occludin, and claudin-5. The recovery of the reduction in gene expression of RUNX1 due to fine dust, etc. plays an important role in maintaining a healthy state continuously by stabilizing the expression of ZO-1, occludin and claudin-5, which are the close junction proteins.

Epithelial cells constitute the mucosal barrier and provide an important interface between the external environment and mucosal and submucosal tissues and extracellular compartments. One of the most important functions of mucosal epithelial cells is to measure and regulate mucosal permeability. In this context, epithelial cells create selective permeable barriers between different physiological compartments. Selective permeability is the result of the regulated transport of molecules through the cytoplasm [transcellular pathway] and the regulated permeability of the intercellular space [paracellular pathway].

Intercellular junctions between epithelial cells are known to be involved in both maintenance and regulation of epithelial barrier function and cell-cell adhesion. The tight junction (TJ) of epithelial and endothelial cells is particularly important for cell-cell junctions that regulate the permeability of pericellular pathways, and also divides the cell surface into apical and basolateral compartments. The tight junction forms a continuous circumferential intercellular contact between epithelial cells and creates a regulated barrier to the pericellular movement of water, solutes and immune cells. It also provides a second type of barrier that contributes to cell polarity by limiting the exchange of membrane lipids between the apical and basolateral membrane regions.

Numerous proteins, including both integral and peripheral plasma membrane proteins, have been identified as being involved in tight junctions. The understanding of the current state of the complex structures and interactive functions of these proteins is limited. Among the many proteins involved in epithelial conjugation, it can act in the physiological regulation of the epithelial conjugation

Several categories of trans-epithelial membrane proteins have been identified. These include a number of junctional adhesion molecules (JAMs) and other tight junction (TJ) related molecules named Occludin (OCLN), claudins and zonulins.

Fine dust is simply classified comprehensively according to particle size. Fine dust less than 10μg is called "PM10 (thoracic particles)", less than 2.5μm is called "PM2.5 (fine particles)", and less than 0.1μm is called "UFP (ultrafine particles)", 2.5 Between ~10μm is called "PM10-2.5 (coarse particles)". In general, the combustion of fossil fuels from industrial activities is the main source of PM2.5, and other factors such as heating, cooking, indoor activities, and biotic or inanimate factors are also the main sources of specific areas. According to a recent study, fine dust induces oxidative stress through free radicals, and through this inflammatory reaction, it can cause or worsen existing ischemic heart disease, including myocardial infarction, heart failure, arrhythmia, and stroke. It turned out to be. In particular, long-term exposure can increase mortality from mild to related diseases, and reducing the concentration of fine dust can conversely reduce the mortality associated with cardiovascular disease. In addition, social interest is increasing in Korea due to fine dust, so it is necessary to prepare urgent measures for fine dust based on domestic and foreign data.

Therefore, the present inventors improved Lactobacillus case i (HY2782) by controlling the expression of OCLN and RUNX1 genes in human lung cells due to fine dust and the expression of RUNX1 gene in human colon cells, thereby improving adhesion-related protein-related diseases. The present invention was completed by discovering that it has the effect of.

Republic of Korea Patent Publication No. 10-2017-0129718 (2017.11.27)

The present invention is in close contact by fine dust containing Lactobacillus casei HY2782 as an active ingredient, which has the effect of regulating the expression of OCLN and RUNX1 genes in human lung cells and RUNX1 gene in human colon cells. It is an object to provide a food composition for preventing or improving protein-related diseases.

In order to achieve the above object, the present invention uses Lactobacillus casei HY2782, which has an effect of regulating the expression of OCLN and RUNX1 genes in human lung cells and expression of RUNX1 gene in human colon cells, as an active ingredient. It characterized in that it provides a food composition for preventing or improving diseases related to the adhesion junction protein by containing fine dust.

Hereinafter, the present invention will be described in detail.

Lactobacillus casei ( Lactobacillus casei ) HY2782 from which the prophage of the present invention has been removed is a method such as Shimizu-Kadota (Document: 1982, Appl. Environ. Microbiol. 43(6), p.1284), and the separation process is as follows.

Lactobacillus casei ( Lactobacillus casei ) YIT9018 in MRT liquid medium (literature: 1969. Nippon Nogei Kagaku Kaishi. 43(5). p.311) was cultured and washed until the beginning of the log phase, and mutagenic substances were treated and washed at 37°C. Incubated for 30 minutes at. This was centrifuged, washed with a phosphate solution (pH 7.0), suspended in a new MRT liquid medium, and then spread on Rogosa agar plate medium (Document: 1962. J. Infect. Dis. 110, p.258). Incubated for 48 hours at 30 ℃. Among the cultured cells, cells that can be grown at 37°C and whose growth is inhibited at 42°C by the reprika-plating method (Reference: 1982, Molecular cloning, a laboratory manual. Cold spring harbor laboratory) can be found. It was separated into a temperature sensitive mutant strain. The isolated 570 temperature-sensitive mutant strains were heat-treated at 42°C for 30 minutes and incubated at 30°C for 24 hours, followed by a soft-agar layer method (Reference: 1959. Interscience publisher Inc. New York). After the heat treatment, the number of latent phages increased significantly after the heat treatment by measuring the number of phages, and they were separated into thermoinducible mutants. The isolated heat-causing mutant strain was inoculated into a new MRT liquid medium containing antisera that can inactivate latent phages, incubated until the beginning of the logarithmic phase, and then heat-treated at 42°C for 30 minutes. It was plated and cultured at 37°C to isolate 240 strains forming colonies. The isolated strains were used as indicator bacteria and cultured by placing the upper layer of Lactobacillus casei YIT9018 culture medium on a soft-agar layer method, and then the formation of plaques was investigated to isolate the formation of plaques. In addition, the strains separated by the soft-agar layer method using Lactobacillus casei YIT9029 (Japan Microbiological Industry Research Institute strain deposit No. 5852) as the indicator bacteria were cultured by adding the upper layer of the culture medium. Plaque formation was investigated to isolate strains in which no plaque was formed. In addition, after incubating the isolated strain at 37°C until the beginning of logarithmic phase, heat treatment at 42°C for 30 minutes, and then confirming the presence or absence of plaques by a soft-agar layer method. cured strain). The number of strains thus isolated was 32 strains. By examining the mycological properties of these strains, Lactobacillus casei HY2782 of the present invention having almost the same mycological properties with the parent strain Lactobacillus casei YIT9018 was isolated.

The mycological properties of the Lactobacillus casei HY2782 of the present invention isolated in this way are as follows.

(1) Type of bacteria (cultivated for 2 days in 37℃, MRS agar plate medium)

1) Cell type: rod type

2) Gram staining: positive

3) Mobility: None

(2) Type of fungus (cultivated for 2 days in 37℃, MRS agar plate medium)

1) Shape: Round

2) Surface: smooth

(3) Physiological properties

1) Growth temperature: Growth possible growth temperature is 13 to 43 ℃, optimum growth temperature is 33 to 37 ℃

2) Growth pH: Growth possible growth pH is 4.5 to 7.5, optimal pH is 5.0 to 5.5

3) Effect of oxygen: anaerobic pain

4) Catalia:-

5) Gas formation:-

6) Growth at 15℃:-

7) Growth at 45℃: +

8) 16S rDNA analysis

The strain of the present invention was identified by carrying out a molecular genetic method through 16S rDNA analysis. The results of 16S rDNA nucleotide sequence analysis are shown in Table 1 (http://www.ncbi.nlm.nih.gov/blast).

As can be seen in Table 1 below, the 16S rRNA gene of Lactobacillus casei HY2782 of the present invention was found to be 99% identical to the 16S rRNA gene of Lactobacillus casei .

Description Max score Total score Query cover Max
ident Accession Lactobacillus sp. strain KL-1-1 16S ribosomal RNA gene, partial sequence 2723 2723 99 99 KX499357 Lactobacillus sp. strain 13-2 16S ribosomal RNA gene, partial sequence 2723 2723 99 99 KX499356 Lactobacillus casei strain CU 16S ribosomal RNA gene, partial sequence 2723 2723 99 99 KX426048 Lactobacillus casei strain HH7 16S ribosomal RNA gene, partial sequence 2723 2723 99 99 KU587809 Lactobacillus paracasei strain FS3 16S ribosomal RNA gene, partial sequence 2723 2723 99 99 KU315064

According to the 16S rRNA sequence and mycological properties, the strain of the present invention was named Lactobacillus casei HY2782, and was deposited with the Korea Research Institute of Bioscience and Biotechnology (KCTC) as of December 19, 2017 (accession number: KCTC 13438BP).

On the other hand, the food composition containing Lactobacillus casei HY2782, its lysate or its culture as an active ingredient as an active ingredient is food, food additives, beverages, beverage additives, fermented milk, health functional foods, etc. Can be used as When used as food, food additives, beverages, beverage additives, or health functional foods, various foods, fermented milk, meat, beverages, chocolate, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, alcoholic beverages, vitamins It may be a combination drug, alcohol and other health functional foods, but is not limited thereto.

In particular, the fermented milk containing Lactobacillus casei HY2782, its lysate or its culture as an active ingredient as an active ingredient is a combination of freeze-dried powder of probiotics, lactic acid bacteria culture solution and mixed fruit juice syrup in a certain ratio. Then, it is homogeneous at 150bar, cooled to 10℃ or less, and packaged in a container to prepare fermented milk.

In addition, Lactobacillus casei according to the present invention ( Lactobacillus casei ) HY2782, its lysate or a functional beverage containing a culture product thereof as an active ingredient as an active ingredient is a mixed fruit juice syrup, a freeze-dried powder of probiotics and water are combined in a certain ratio It is homogeneous at 150bar, cooled to 10℃ or less, and then packaged in a small container such as a glass bottle or plastic bottle to prepare a functional beverage.

In addition, the health functional food containing Lactobacillus casei HY2782, its lysate or its culture as an active ingredient as an active ingredient, in addition to containing the freeze-dried powder of probiotics, vitamins as nutritional supplements B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid esters, nicotinic acid amide, oligosaccharides, etc. may be added, and other food additives may be added.

Lactobacillus casei HY2782 of the present invention has the effect of regulating the expression of OCLN and RUNX1 genes in human lung cells and RUNX1 gene in human colon cells, so it is caused by fine dust containing it as an active ingredient. It can be used as a food composition such as fermented milk and health functional foods for the prevention or improvement of diseases related to adhesion protein.

1 is a graph confirming the change in the expression of OCLN gene by treatment with fine dust and Lactobacillus casei HY2782 of the present invention through real-time polymerase chain reaction in human lung cancer cells A549 cells.
2 is a graph confirming the change in the expression of the RUNX1 gene by treatment with fine dust and Lactobacillus casei HY2782 of the present invention through real-time polymerase chain reaction in human lung cancer cells, A549 cells.
Figure 3 is a graph confirming the change in the expression of the RUNX1 gene by the treatment of fine dust and Lactobacillus casei HY2782 of the present invention through real-time polymerase chain reaction in human normal colon cell line (CCD-18Co).

Hereinafter, the present invention will be described in more detail through examples. However, the following examples do not limit the scope of the present invention, and conventional changes by those skilled in the art are possible within the scope of the technical idea of the present invention.

<Example 1>

Lactobacillus Casey ( Lactobacillus casei ) HY2782 Manufacture of freeze-dried powder

After culturing Lactobacillus casei HY2782 in MRS broth medium, the probiotic concentrate obtained by centrifuging the culture solution at 8,000 rpm for 15 minutes has 10% by weight of skim milk powder that is pressurized as a coating agent and cryoprotectant. The contained aqueous solution was mixed in a weight ratio of 1:1, and then frozen at -70°C for 6 hours to prepare a lyophilized powder.

<Example 2>

Lactobacillus Casey ( Lactobacillus casei ) Manufacture of fermented milk containing HY2782 as an active ingredient

Lactobacillus culture broth is a mixture of 95.36% by weight of crude milk and 4.6% by weight of skim milk powder (or mixed milk powder). (Brix ⁰ ) was mixed to be about 16.3 to 16.5%. After the mixing, UHT heat treatment (sterilization at 135° C. for 2 seconds), after cooling to an appropriate temperature, Streptococcus thermophilus and lactose degrading enzyme (Valley laboratory, USA) were added by 0.02% by weight, respectively, and It was prepared by incubating for 6 hours so that the total number of lactic acid bacteria in the BCP medium was 5.0X10 ⁸ cfu/ml or more, the titratable acidity was 0.89~0.91%, and the pH was 4.55~4.65.

Mixed fruit syrup 13% by weight of liquid fructose, 5% by weight of white sugar, 10.9% by weight of mixed fruit juice concentrate 56Brix ⁰ , 1.0% by weight of pectin, 0.1% by weight of fresh fruit mix essence and 70% by weight of purified water were mixed by stirring at 35°C. It was prepared by cooling after UHT heat treatment (sterilization at 135°C for 2 seconds).

69.5% by weight of the lactic acid bacteria culture solution and 0.1% by weight of Lactobacillus casei HY2782 freeze-dried powder of Example 1 and 30.4% by weight of the mixed fruit juice syrup were combined, homogenized at 150bar, and then cooled to 10°C or less. A fermented milk containing Lactobacillus casei HY2782 as an active ingredient was prepared.

<Example 3>

Lactobacillus Casey ( Lactobacillus casei ) Manufacture of functional beverage containing HY2782 as an active ingredient

Mixed fruit juice syrup contains 13% by weight of liquid fructose, 2.5% by weight of white sugar, 2.5% by weight of brown sugar, 56Brix ⁰ 10.9% by weight of mixed fruit juice concentrate, 1.0% by weight of pectin, 0.1% by weight of fresh fruit mix essence and 70% by weight of purified water at 35°C. After stirring and mixing at, UHT heat treatment (sterilization at 135° C. for 2 seconds) and then cooled to prepare.

In addition, 30.4% by weight of the mixed fruit juice syrup prepared by the above method and 0.1% by weight of Lactobacillus casei HY2782 lyophilized powder of Example 1 and 69.5% by weight of purified water were combined and homogenized at 150 bar, and then 10°C. After cooling to the following, a functional beverage containing Lactobacillus casei HY2782 of the present invention was prepared by packing it in a small container such as a glass bottle or a PET bottle.

<Example 4>

Lactobacillus Casey ( Lactobacillus casei ) Manufacture of health functional food containing HY2782 as an active ingredient

Nutritional supplements (vitamins B ₁ , B ₂ , B ₅ , B ₆ , E and acetic acid esters, nicotinic acid amide) and oligosaccharides in 0.1% by weight of the Lactobacillus casei HY2782 freeze-dried powder of Example 1 above. Lactobacillus casei of Example 1 ( Lactobacillus casei ) HY2782 10 parts by weight based on 100 parts by weight of freeze-dried powder was added and mixed in a high-speed rotary mixer. 10 parts by weight of sterile 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 dryer at 50° C. and then passed through 12-14 mesh to uniformly prepare granules. The granules prepared as described above were extruded in appropriate amounts to form tablets or powders, or filled in hard capsules to produce hard capsule products.

<Test Example 1>

1-1. Lactobacillus casei ( Lactobacillus casei ) HY2782 Preparation of live cells

The Lactobacillus casei ( Lactobacillus casei ) HY2782 was inoculated into MRS liquid medium and cultured at 37°C for 18-20 hours. In order to confirm the correct number of viable bacteria, the lactic acid bacteria that had been cultured were diluted decimally and cultured at 37° C. for 2 to 3 days using MRS agar medium to measure the number of bacteria.

When applied to the cell experiment, the medium used for cell culture was added and diluted based on the number of bacteria obtained previously, and used as live cells.

1-2. Culture of A549 cells, human lung cancer cells

A549 cells, which are human lung cancer cells, were purchased from Korea Cell Line Bank (Seoul) and used. In RPMI medium (Roswell Park Memorial Institute medium) supplemented with 10% heatinactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, human lung cancer cells, A549 cells, and RPMI were added in a weight ratio of 1:4. It was subcultured once every 2-3 days (culture conditions: 37°C incubator supplied with 5% CO ₂ ).

That is, after removing the RPMI medium for passage and washing once with 4 ml of PBS, 1 ml of Trypsin-EDTA Solution 1X (1XTE, Sigma) was treated and placed in the incubator for 10 to 15 minutes. After confirming that the adhesion of the human lung cancer cells, A549 cells, was removed, 4 ml of RPMI medium was added to recover the human lung cancer cells, A549 cells. The recovered human lung cancer cells, A549 cells, were centrifuged at 1,200 rpm for 3 minutes, and then the supernatant was carefully removed, and 1 ml of RPMI medium was added to release the cell pellet. Human lung cancer cells A549 cells and RPMI medium were diluted in a weight ratio of 1:4, put in a 100Ø cell culture dish, mixed well, and cultured in an incubator for 2-3 days before use.

1-3. RNA extraction from A549 cells, human lung cancer cells treated with fine dust

The human lung cancer cells of Test Example 1-2, A549 cells, were inoculated into 96 wells at 2×10 ⁴ cells per well, cultured for 24 to 48 hours, and washed once with RPMI medium without FBS.

The Lactobacillus casei ( Lactobacillus casei ) HY2782 live cells of Test Example 1-1 were dissolved in RPMI medium without FBS at a concentration of 1x10 ⁶ CFU/ml, and washed once with RPMI medium without FBS. Each well was treated with human lung cancer cells, A549 cells, and at the same time, ERM-CZ100 (polycyclic aromatic hydrocarbons, Sigma) and ERM-CZ120 (heavy metals, Sigma) were each present at a concentration of 200 μg/ml. 400 μg/ml At the concentration of each well, A549 cells, which are human lung cancer cells, were treated.

Then, the Lactobacillus casei ( Lactobacillus casei ) HY2782 live cells and fine dust-treated human lung cancer cells A549 cells were cultured for 20 to 24 hours, washed twice with PBS, and then easy-spin ^™ lysis 1 ml of buffer (iNtRON, USA) was added and A549 cells, which are human lung cancer cells, were lysed. RNA was extracted from the lysed human lung cancer cells, A549 cells, using the easy-spin ^™ [DNA free] Total RNA Extraction Kit (Invitrogen, USA). RNA purity and degree of degradation were confirmed with an ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).

Meanwhile, RNA was extracted in the same manner as described above, except that only fine dust was treated in human lung cancer cells, A549 cells, in order to further clarify the cause of the change in gene expression.

In addition, in order to further clarify the cause of the change in gene expression, RNA was extracted in the same manner as described above, except that A549 cells, which are human lung cancer cells, were not treated with anything.

1-4. Analysis of gene expression in A549 cells, human lung cancer cells treated with fine dust using real-time polymerase chain reaction

From each of the isolated RNAs of Test Example 1-3, cDNA was synthesized using an Omniscript reverse transcription kit (Qiagen, Germany). RNA expression was performed using a Taqman gene expression master mix (Taqman, USA), and analyzed and quantified by QuantStudio 6 Flex Real-Time PCR System (Applied biosystems, USA). Human-derived primers used for this were GAPDH (Hs03929097_g1), OCLN (Hs00170162_m1), and RUNX1 (Hs01021970_m1), which were ordered and used through Tagman. The fold change in gene expression was calculated based on the Ct (treshhold cycle), an index of gene expression.

The results are shown in Fig. 1 (OCLN gene expression) and Fig. 2 (RUNX1 gene expression).

As can be seen in FIG. 1, based on gene expression 1 of the normal group that did not treat any human lung cancer cells, A549 cells, in the group treated with only fine dust (fine dust) in the human lung cancer cells A549 cells, OCLN ( Occludin) gene expression increased 2.1 times, but in the group (HY2782) treated with fine dust and Lactobacillus casei HY2782 live cells in human lung cancer cells A549 cells at the same time (HY2782), the OCLN gene expression decreased again to 1.4 times.

As can be seen in FIG. 2, based on gene expression 1 of the normal group in which nothing was treated with human lung cancer cells A549 cells, the group treated with only fine dust in human lung cancer cells A549 cells (fine dust) was RUNX1 ( Runt-related transcription factor 1) gene expression increased 3.4 times, but RUNX1 gene expression was 2.5 times in the group (HY2782) treated with fine dust and Lactobacillus casei HY2782 live cells in human lung cancer cells, A549 cells. It decreased again by double.

<Test Example 2>

2-1. Culture of human-derived normal colon cells (CCD-18Co)

CCD-18Co cells, a normal human colon cell line, were purchased from Korea Cell Line Bank (Seoul) and used. In RPMI medium (Gibco, USA) supplemented with 10% FBS (fetal bovine serum), 10 U/mL penicillin, and 100 μg/mL streptomycin, human normal colon cell line (CCD-18Co) and RPMI in a weight ratio of 1:4 It was used by subculture once every 2-3 days (culture conditions: 37 ℃ incubator supplied with 5% CO ₂ ).

That is, after removing the RPMI medium for passage and washing once with 4 ml of PBS, 1 ml of Trypsin-EDTA Solution 1X (1XTE, Sigma) was treated and placed in the incubator for 10 to 15 minutes. After confirming that the adhesion of the human normal colon cell line (CCD-18Co) had fallen, 4 ml of RPMI medium was added to recover the human normal colon cell line (CCD-18Co). The recovered human normal colon cell line (CCD-18Co) was centrifuged at 1,200 rpm for 3 minutes, then the supernatant was carefully removed, and 1 ml of RPMI medium was added to release the cell pellet. Human normal colon cell line (CCD-18Co) and RPMI medium were diluted in a weight ratio of 1:4, put in a 100 ㆈ cell culture dish, mixed well, and cultured in an incubator for 2-3 days and used.

2-2. RNA extraction from fine dust-treated human normal colon cell line (CCD-18Co)

The human normal colon cell line (CCD-18Co) of Test Example 2-1 was inoculated into 96 wells at 2x10 ⁴ cells per well, cultured for 24 to 48 hours, and washed once with DMEM medium without FBS. gave.

Dissolve the live cells of Lactobacillus casei HY2782 of Test Example 1-1 in DMEM medium without FBS at a concentration of 1x10 ⁵ CFU/ml, and washed once with DMEM medium without FBS. Each well was treated with a human normal colon cell line (CCD-18Co), and at the same time, ERM-CZ100 (polycyclic aromatic hydrocarbons, Sigma) and ERM-CZ120 (heavy metals, Sigma) were each present at a concentration of 100 μg/ml. It was treated with the human normal colon cell line (CCD-18Co) in each well at a concentration of 200 μg/ml.

Then, the Lactobacillus casei ( Lactobacillus casei ) HY2782 live cells and fine dust-treated human normal colon cell line (CCD-18Co) was cultured for 20 to 24 hours, washed twice with PBS, and then easy- 1 ml of spin ^™ lysis buffer (iNtRON, USA) was added and the human normal colon cell line (CCD-18Co) was dissolved. RNA was extracted from the lysed human normal colon cell line (CCD-18Co) using easy-spin ^™ [DNA free] Total RNA Extraction Kit (Invitrogen, USA). RNA purity and degree of degradation were confirmed with an ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).

Meanwhile, RNA was extracted in the same manner as above, except that only fine dust was treated in human normal colon cell line (CCD-18Co) in order to further clarify the cause of the change in gene expression.

In addition, in order to further clarify the cause of the change in gene expression, RNA was extracted in the same manner as described above, except that nothing was treated with the human normal colon cell line (CCD-18Co).

2-3. Gene expression analysis in human normal colon cell line (CCD-18Co) treated with fine dust using real-time polymerase chain reaction

From each of the isolated RNAs of Test Example 2-2, cDNA was synthesized using an Omniscript reverse transcription kit (Qiagen, Germany). RNA expression was performed using a Taqman gene expression master mix (Taqman, USA), and analyzed and quantified by QuantStudio 6 Flex Real-Time PCR System (Applied biosystems, USA). Human-derived primers used for this were GAPDH (Hs03929097_g1) and RUNX1 (Hs01021970_m1), which were ordered and used through Tagman. The fold change in gene expression was calculated based on the Ct (treshhold cycle), an index of gene expression.

The results are shown in Fig. 3 (RUNX1 gene expression).

As can be seen in Figure 3, based on gene expression 1 of the normal group that did not treat anything on CCD-18Co cells, which are human normal colon cells, the group treated with only fine dust on CCD-18Co cells, which are human normal colon cells ( In fine dust), RUNX1 (Runt-related transcription factor 1) gene expression increased 15.1 times, but the experimental group treated with fine dust and Lactobacillus casei HY2782 live cells simultaneously in CCD-18Co cells, normal human colon cells. In (HY2782), the expression of RUNX1 gene decreased again to 5.8 fold.

When the experimental results of FIGS. 1 to 3 above are summarized, Lactobacillus casei HY2782 of the present invention is OCLN (Occludin) and RUNX1 (Runt-related transcription factor) which are increased due to fine dust in human lung cells. 1) Efficacy in improving intracellular regulatory abnormalities of OCLN and RUNX1 genes caused by fine dust by reducing gene expression and reducing RUNX1 (Runt-related transcription factor 1) gene expression, which increases due to fine dust in human normal colon cells It was confirmed that it has.

Korea Research Institute of Bioscience and Biotechnology KCTC13438BP 20171219

Claims (7)

Food composition for preventing or improving tight junction protein related diseases by fine dust containing Lactobacillus casei HY2782 (accession number: KCTC 13438BP) as an active ingredient.
The method according to claim 1,
The tight junction protein-related disease is any one selected from the group consisting of lung damage, bowel damage, skin damage, pneumonia, enteritis, dermatitis, and leaky intestinal syndrome. Food composition for preventing or improving related diseases.
The method according to claim 1 or 2,
The Lactobacillus casei HY2782 (accession number: KCTC 13438BP) is a tight junction protein by reducing the expression of OCLN (Occludin) and RUNX1 (Runt-related transcription factor 1) genes in human lung cancer cells. ) Food composition, characterized in that to prevent or improve related diseases.
The method according to claim 1 or 2,
The Lactobacillus casei HY2782 (accession number: KCTC 13438BP) reduces the expression of the RUNX1 (Runt-related transcription factor 1) gene in human normal colon cells, thereby reducing tight junction protein related diseases. Food composition, characterized in that to prevent or improve.
The method according to claim 1 or 2,
The food composition, characterized in that it has any one formulation selected from fermented milk, health functional food, functional beverage.
The method of claim 3,
The food composition, characterized in that it has any one formulation selected from fermented milk, health functional food, functional beverage.
The method of claim 4,
The food composition, characterized in that it has any one formulation selected from fermented milk, health functional food, functional beverage.

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