KR101312377B1 - Lactobacillus johnsonii HY7041 having anti-inflammatory functions and products containing thereof as effective component - Google Patents

Abstract

The present invention relates to Lactobacillus johnsonii HY7041 having an anti-inflammatory effect and a product containing the same as an active ingredient, the pharmaceutical composition, fermented milk, health function for the anti-inflammatory effect as well as the treatment and prevention of inflammatory diseases. It can be used as food.

Description

Lactobacillus johnsonii HY7041 having anti-inflammatory functions and products containing based as effective component}

The present invention relates to Lactobacillus johnsonii HY7041 having an anti-inflammatory effect and a product containing the same as an active ingredient, and more particularly, to induce immune tolerance by inducing the formation of Foxp3 + Tregs to mediate various Th-1 mediations. The present invention relates to Lactobacillus johnsonii HY7041 having an anti-inflammatory effect by preventing or treating an inflammatory disease, and a pharmaceutical composition, fermented milk, a beverage and a dietary supplement containing the same as an active ingredient.

Recently, various roles of lactic acid bacteria have been studied, and therapeutic effects of inflammatory diseases such as colorectal inflammatory disease, atopic dermatitis, and diabetes, including rheumatoid arthritis, have been revealed. Intake of Lactobacillus casei was effective in inhibiting arthritis in animal models of rheumatoid arthritis mice induced with type 2 collagen (Life Science, 1998; 63 (8): 635-644) and arthritis using Lewis rats. Intake of Lactobacillus GG in animal models has been reported to be effective in inhibiting arthritis (Journal of Nutrition, 2004; 134 (8): 1964-1969). In addition, the mechanism of inhibition of rheumatoid arthritis of Lactobacillus casei has been revealed. In contrast, the expression of IL-10 (Interleukin-10), an anti-inflammatory cytokine, is increased in CD4 T cells, whereas TNF-α ( Inflammatory cytokine expression, such as Tumor necrosis factor-α) and IL-1β (Interleukin-1β), has been reported to be inhibited (Molecular Immunology, 2008; 46 (1): 172-180).

The present inventors induce the formation of CD4 + Foxp3 + Tregs, which are immunoregulatory / inhibitory T cells, in mouse mesenteric lymphocyte cells targeting various lactic acid bacteria to develop probiotics that can prevent or treat diseases caused by inflammatory reactions. In addition, Lactobacillus johnsonii HY7041 has the greatest efficacy in the process of selecting lactic acid bacteria that can increase the expression of anti-inflammatory cytokine IL-10 and lower the expression of the representative inflammatory cytokine IL-12. It has been found to have completed the present invention.

An object of the present invention is to provide Lactobacillus johnsonii HY7041 having an anti-inflammatory effect that can prevent or treat various inflammatory diseases and pharmaceutical compositions, fermented milk, beverages and health functional foods containing the same as an active ingredient. do.

In order to achieve the above object, the present invention induces the formation of CD4 + Foxp3 + Tregs, increases the expression of IL-10, a representative anti-inflammatory cytokine, and simultaneously reduces the expression of IL-12, a representative inflammatory cytokine, by colitis. Providing Lactobacillus johnsonii HY7041 having an anti-inflammatory effect to prevent and treat various inflammatory diseases such as arthritis, and pharmaceutical compositions, fermented milk, beverages, and health functional foods containing the same as an active ingredient. It features.

Hereinafter, the present invention will be described in detail.

In order to isolate the strain according to the present invention, healthy adult feces were placed in an MRS liquid medium containing 0.02% sodium azide and cultured at 37 ° C for 24 hours, and then 10 μl of platinum The culture broth was plated on an EMS agar medium containing 0.02% sodium azide and cultured at 37 ° C for 48 hours. Each of the thus formed single colonies was used separately. The isolates were cultured on MRS agar plate at 37 ° C for 48 hours. The size and morphology of the isolated strains were observed by optical microscopy through Gram stain (Cowan, 1974), and the biochemical characteristics Were identified and identified by Bergey's Mannual of Systematic Bacteriology, Bergey's Mannual of Determinative Bacteriology and 16S rDNA genetic analysis after investigating the characteristics of strains according to the methods of Cowan and Steel (1984) and Macfaddin (1984).

The characteristics of the novel lactic acid bacteria isolated according to the present invention are as follows.

1) Type of bacteria: Bacillus

2) Colony type: It has circular convex ridge and the surface is somewhat rough.

3) Optimum growth temperature: 37 ~ 40 ℃

4) Mobility: None

5) Gram staining: positive

6) Catalase: negative

7) Oxygen Influence: Fame Anaerobic

6) Growth at 15 ℃:-

7) Growth at 45 ℃: +

8) Sugar Fermentation Experiment and Identification

Amigdalin:-

Arabian:-

Cellobiose: +

Esculin: +

Fructose: +

Galactose: +

Glucose: +

Gluconate:-

Lactose: +

Maltose: +

Mannitol:-

Mannose: +

Melegitos:-

Melibiose:-

Rafinos: +

Ribose:-

Rhamnose:-

Raised:-

Sorbitol:-

Sucrose: +

Trehalose: +

Xylose:-

9) 16S rDNA Analysis

Molecular genetic methods through 16S rDNA analysis were performed to identify new strains of the present invention.

PCR using 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3') primers using the genomic DNA isolated from the new strain of the present invention cultured in MRS medium as a template. Reaction ((95 ℃, 3min), 30cycles (95 ℃, 30sec; 50 ℃, 30sec; 72 ℃, 90sec), 72 ℃, 10min] to obtain 1.45kbp of amplification product and then purified The results of sequencing analysis through sequencing reactions are described in the sequence list (SEQ No. 1). Based on the nucleotide sequence, BLAST search results (http://www.ncbi.nlm.nih.gov/blast), Lactobacillus johnsonii ( Lactobacillus johnsonii ) strain as shown in Table 1 could be identified.

Based on the genetic, morphological, physiological and growth characteristics of the lactic acid bacteria as described above, the lactic acid bacteria of the present invention was named Lactobacillus johnsonii HY7041 and was deposited on June 10, 2011 by the Korea Research Institute of Bioscience and Biotechnology. Accession number: KCTC 11958BP).

Meanwhile, the pharmaceutical composition containing Lactobacillus johnsonii HY7041 having an anti-inflammatory effect of the present invention as an active ingredient may be used alone or in combination with pharmaceutical excipients according to the methods commonly used pharmaceutically. It can be used by formulating it in the form of tablets, capsules and the like.

In the case of humans, a typical daily dose may range from 1 to 30 mg / kg body weight, and may be administered in single or divided doses. However, the actual dosage should be determined in light of various relevant factors such as route of administration, age, sex, weight, health status and severity of the disease.

Of course, since the pharmaceutical composition of the present invention has little toxicity and side effects, it can be safely used even when taken for a long time.

In addition, fermented milk containing Lactobacillus johnsonii HY7041 having an anti-inflammatory effect of the present invention as an active ingredient is 150 bar by combining a lactobacillus culture solution, Lactobacillus johnsonii HY7041 and a mixed juice syrup at a predetermined ratio. After homogeneous in and cooled to 10 ℃ or less and then packaged in a container to prepare.

In addition, a beverage containing Lactobacillus johnsonii HY7041 having an anti-inflammatory effect of the present invention as an active ingredient is a blended juice syrup, Lactobacillus johnsonii HY7041 and water at a constant ratio of 150 bar in combination After homogeneous, the mixture is cooled to 10 ° C. or less, and then packaged in a small packaging container such as a glass bottle or a plastic bottle.

In addition, a health functional food containing Lactobacillus johnsonii HY7041 having an anti-inflammatory effect of the present invention as an active ingredient, in addition to containing the Lactobacillus johnsonii HY7041 as a nutritional supplement component vitamin B1 , B2, B5, B6, E and acetate esters, nicotinic acid amides, oligosaccharides and the like may be added, and other food additives may be added.

Lactobacillus johnsonii HY7041 according to the present invention is the expression of Foxp3 (Forkhead box p3) transcriptional regulator that can induce immune tolerance in regulatory T cells (Regulatory T cells) Pharmaceutical composition having a prophylactic or therapeutic function of inflammatory diseases because it is very effective in increasing the expression of IL-10, a representative anti-inflammatory cytokine, and at the same time reducing the expression of the representative inflammatory cytokine, IL-12, It can be used for the functional food.

1 is a graph showing the results of measuring the ratio of Foxp3 + population through flow cytometry after co-culture of mouse mesenteric lymphocyte-derived immune cells and various lactic acid bacteria at a ratio of 1:10.
Figure 2 is a graph showing the results of measuring the expression level of the anti-inflammatory cytokine IL-10 present in the medium while co-culturing the mesenteric lymphocyte-derived immune cells and various lactic acid bacteria in a ratio of 1:10 of the mouse.
Figure 3 is a graph showing the results of measuring the expression level of inflammatory cytokine IL-12 present in the medium while co-culturing the mesenteric lymphocyte-derived immune cells and various lactic acid bacteria in a ratio of 1:10 of the mouse.
4 is a graph showing a result of comparing and calculating the ratio of the amount of IL-10 expression of FIG. 3 and the amount of IL-12 expression of FIG. 4.

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 >

Lactobacillus john sony ( Lactobacillus johnsonii ) Lyophilized powder production including HY7041

The Lactobacillus johnsonii HY7041 of the present invention is a liquid medium containing Proteose peptone # 3 for food ingredients, Yeast Extract, Beef Extract, and glucose, and cultured at 37 ° C. for about 16 hours, followed by centrifugation of the culture solution. And washed with sterile saline solution and then dispersed in sterile oil. Freeze-drying again yielded about 10 ¹¹ cfu bacteria per gram (g) of lyophilized powder. This lyophilized powder was used as an immunomodulatory and potentiating material.

Meanwhile, Lactobacillus johnsonii HY7041 of the present invention may be provided in a lyophilized powder form or culture form as described above.

<Example 2>

Lactobacillus john sony ( Lactobacillus johnsonii ) Preparation of a pharmaceutical composition containing HY7041 as an active ingredient

Although an example of the preparation of a pharmaceutical composition containing Lactobacillus johnsonii HY7041 of the present invention as an active ingredient is described, the present invention is not intended to limit the present invention but is only intended to be described in detail.

Manufacture of tablets

Lactobacillus johnsonii of Example 1 ( Lactobacillus johnsonii ) 100mg of lyophilized powder, including corn starch 100mg, lactose 100mg and polyvinylpyrrolidone 97mg homogeneously mixed and granulated by wet granulation method 2 mg of magnesium stearate was added and mixed, and the tablets were compressed to 400 mg.

Preparation of capsules

Lactobacillus johnsonii of Example 1 ( Lactobacillus johnsonii ) 100mg of lyophilized powder including HY7041, corn starch 100mg, lactose 100mg, magnesium stearate 2mg and then completely mixed hard according to the manufacturing method of the capsule Capsules were prepared by filling gelatin capsules.

<Example 3>

Lactobacillus john sony ( Lactobacillus johnsonii ) Preparation of Fermented Milk Containing HY7041 as an Active Ingredient

The method for producing fermented milk consisting of lactic acid bacteria culture medium, Lactobacillus johnsonii of the present invention, HY7041, and mixed fruit juice syrup 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.

Next, the mixed fruit juice syrup was prepared by mixing 13 wt% of liquid fructose, 5 wt% of white sugar, 10.9 wt% of mixed juice concentrate 56Brix ^o , 1.0 wt% of pectin, 0.1 wt% of fresh fruit mix essence and 70 wt% of purified water at 30-35 캜 Mixed with stirring, heat-treated with UHT (sterilized at 135 ° C for 2 seconds), and cooled.

Then, the mixture was cultured at 150 bar and homogenized at 150 bar by lactic acid bacteria culture solution 69.5% by weight and 0.1% by weight of the lyophilized powder including Lactobacillus johnsonii HY7041 and 30.4% by weight of the mixed juice syrup. It was cooled to below to prepare a fermented milk containing Lactobacillus johnsonii HY7041 as an active ingredient.

<Example 4>

Lactobacillus john sony ( Lactobacillus johnsonii ) Preparation of Functional Drink Containing HY7041 as an Active Ingredient

Lactobacillus johnsonii of the present invention ( Lactobacillus johnsonii ) HY7041 and a method for producing a functional beverage consisting of mixed juice syrup 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.

And homogeneous at 150 bar by combining 30.4% by weight of the mixed juice syrup prepared by the above method and 0.1% by weight of the lyophilized powder including Lactobacillus johnsonii HY7041 and 69.5% by weight of the remaining purified water. After cooling to 10 ° C. or less, it was packaged in a small packaging container such as a glass bottle or a plastic bottle to prepare a functional beverage containing Lactobacillus johnsonii HY7041 as an active ingredient.

<Example 5>

Lactobacillus john sony ( Lactobacillus johnsonii ) Preparation of health functional food containing HY7041 as an active ingredient

Lactobacillus johnsonii ( Lactobacillus johnsonii ) HY7041 of the lyophilized powder containing 0.1% by weight of the nutritional supplements (vitamins B1, B2, B5, B6, E and acetate esters, nicotinic acid amide) and oligosaccharides 1 part by weight of 100 parts by weight of the lyophilized powder including Lactobacillus johnsonii HY7041 was added and mixed in a high speed rotary mixer. To 100 parts by weight of the mixture, 10 parts by weight of sterilized purified water was added and mixed to form 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 >

Lactobacillus john sony ( Lactobacillus johnsonii ) Measurement of Foxp3 + Population Growth of HY7041

Immunity tolerance plays an important role in the treatment and prevention of various immune diseases. Cells that play an important role in immune tolerance are known as T cells, and the regulatory T cells with CD4 + Foxp3 + traits are representative. The immunoregulatory T cells specifically expressed Foxp3 (Forkhead box p3) transcriptional regulators in the cell, and thus GFP (Green Fluorescent Protein) fluorescent labeling proteins were labeled on Foxp3 transcriptional regulators. Mesenteric lymph nodes were isolated from 6-8 week-old Foxp3-GFP KI mice using transgenic mice (Foxp3-GFP KI mice), and then mesenteric lymphocytes and various lactic acid bacteria were co-cultured at a ratio of 1:10. After co-culture, the expression level of Foxp3 transcriptional regulator was measured by measuring GFP + (Foxp3 +) signal using flow cytometer.

Lactobacillus johnsonii HY7041, Bifidobacterium ( Bifidobacterium ) , L. casei ( L. casei ) was used as the lactic acid strain.

In addition, mesenteric lymphocytes of normal mice were used as negative control for transgenic mice (Foxp3-GFP KI mice).

In this test, PBS (Phosphate buffered saline) was a non-treated control group, and LPS (Lipopolysaccharide) was used as a positive control group.

The results are shown in Fig.

As can be seen in Figure 1, compared to Bifidobacterium ( Lifidobacterium ) , L. casei lactic acid bacteria Lactobacillus johnsonii HY7041 lactic acid bacteria of the present invention is about 11% Foxp3 + population Increasing the expression of Foxp3 transcriptional regulators can be seen to increase the most.

&Lt; Test Example 2 &

Lactobacillus john sony ( Lactobacillus johnsonii ) HY7041 Measurement of IL-10 Expression Increase

IL-10 (Interleukin-10) is a representative anti-inflammatory cytokine along with Tumor growth factor-β (TGF-β), which is secreted from Th2 cells (Helper T Cell 2) to release Th1 cells (Helper T Cell 1). It is known to suppress the secretion of IFN-γ (Interferone-γ). Cells expressing IL-10 include macrophage, dendritic cells, CD8 + T cells, B cells, mast cells, and the like.

In this study, to investigate the effect of lactic acid strains on the expression of IL-10, mesenteric lymph nodes were isolated from Foxp3-GFP KI mice in the same manner as in Test Example 1, and then co-cultured with mesenteric lymphocytes and various lactic acid bacteria and then secreted into the medium. IL-10 expression level was quantified by ELISA method.

Lactobacillus johnsonii HY7041, Bifidobacterium ( Bifidobacterium ) , L. casei ( L. casei ) was used as the lactic acid strain.

Phosphate buffered saline (PBS) was used as an untreated control in this test.

The results are shown in Fig.

As can be seen in Figure 2, compared to Bifidobacterium ( Lifidobacterium ) , L. casei lactic acid bacteria Lactobacillus Johnii HY7041 lactic acid strain of the present invention is the most expression of IL-10 You can see that it increases a lot.

<Test Example 3>

Lactobacillus john sony ( Lactobacillus johnsonii ) Determination of Inflammatory Cytokine IL-12 Expression of HY7041

IL-12 (Interleukin-12), known as a representative inflammatory cytokine, is mainly produced in antigen presenting cells when foreign pathogenic substances in cells caused by viral infections invade, and play an important role in causing and developing Th1 immune responses. do. IL-12 induces the production of IFN-γ and TNF-α in Th1 cells and inhibits the development of Th2 cells. Therefore, lactic acid bacteria that reduce or inhibit the production of IL-12 can be said to have a strong anti-inflammatory effect.

In this test, mesenteric lymph nodes were isolated from Foxp3-GFP KI mice in the same manner as in Test Example 1, followed by co-culture of mesenteric lymphocytes and various lactic acid bacteria, and the amount of IL-12 secreted into the medium was quantified by ELISA.

Lactobacillus johnsonii HY7041, Bifidobacterium ( Bifidobacterium ) , L. casei ( L. casei ) was used as the lactic acid strain. PBS was used as an untreated control in this test.

The results are shown in Fig.

As can be seen in Figure 3, Lactobacillus johnsonii HY7041 lactic acid strain of the present invention compared to the Bifidobacterium ( Lifidobacterium ) , L. casei lactic acid strains, the expression of IL-12 It can be seen that the most suppressed.

<Test Example 4>

Lactobacillus john sony ( Lactobacillus johnsonii ) Expression of IL-10 / IL-12 in HY7041

In order to select the lactic acid strain that inhibits the expression of inflammatory cytokine IL-12 while increasing the expression of IL-10, an anti-inflammatory cytokine, the amount of IL-10 expressed in Test Example 2 was measured by IL-12. The expression ratio of IL-10 / IL-12 divided by the expression amount of was measured.

The results are shown in Fig.

As can be seen in Figure 4, compared to Bifidobacterium ( Lifidobacterium ) , L. casei lactic acid strain Lactobacillus johnsonii HY7041 lactic acid strain of the present invention shows the most excellent efficacy I could confirm it.

Korea Biotechnology Research Institute KCTC11958BP 20110610

Attach an electronic file to a sequence list

Claims (12)

Lactobacillus has anti-inflammatory effects johnsonii ) HY7041 (Accession Number: KCTC 11958BP).
The method of claim 1,
The strain is Lactobacillus johnsonii ( Lactobacillus johnsonii ) HY7041 having anti-inflammatory efficacy by increasing the expression of Foxp3 (Forkhead box p3) transcriptional regulator of Regulatory T cells (Tregs).
The method of claim 1,
The strain is Lactobacillus johnsonii HY7041 having anti-inflammatory effect by increasing the expression of anti-inflammatory cytokine IL-10 (Interleukin-10).
The method of claim 1,
The strain is Lactobacillus johnsonii HY7041 which has anti-inflammatory effect by reducing the expression of inflammatory cytokine IL-12 (Interleukin-12).
Lactobacillus johnsonii of claim 1 ( Lactobacillus johnsonii ) A pharmaceutical composition for preventing or treating anti-inflammatory, characterized in that it contains as an active ingredient.
Lactobacillus of any one of claims 2 to 4 ( Lactobacillus johnsonii ) Anti-inflammatory preventive or therapeutic pharmaceutical composition comprising HY7041 as an active ingredient.
Lactobacillus johnsonii of claim 1 ( Lactobacillus johnsonii ) Fermented milk, characterized in that it contains as an active ingredient.
Lactobacillus of any one of claims 2 to 4 ( Lactobacillus johnsonii ) Fermented milk characterized by containing HY7041 as an active ingredient.
A beverage comprising Lactobacillus johnsonii HY7041 as claimed in claim 1 as an active ingredient.
Lactobacillus of any one of claims 2 to 4 ( Lactobacillus johnsonii ) A beverage characterized by containing HY7041 as an active ingredient.
Paragraph 1 Lactobacillus John Sony (Lactobacillus johnsonii ) A dietary supplement comprising HY7041 as an active ingredient.
Lactobacillus of any one of claims 2 to 4 ( Lactobacillus johnsonii ) A dietary supplement comprising HY7041 as an active ingredient.

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