JP2022174972A - Lactic acid bacteria-derived angiotensin-converting enzyme inhibitor - Google Patents

Abstract

To provide an ACE inhibitor derived from Lactococcus lactic acid bacteria with high safety.SOLUTION: An angiotensin-converting enzyme inhibitor contains at least one selected from a viable cell body, a dead cell and extract of Lactococcus lactis N7 strain (accession number: FERM P-18217). There are also provided a skin aging inhibitory composition and a wrinkle improving composition each containing the angiotensin-converting enzyme inhibitor as an active ingredient, and a method for increasing ACE inhibitory activity by using the angiotensin-converting enzyme inhibitor.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to an angiotensin-converting enzyme inhibitor, a skin aging-inhibiting composition and a wrinkle-improving composition containing the inhibitor, and a method for increasing angiotensin-converting enzyme inhibitory activity.

Angiotensin-converting enzyme (hereinafter referred to as "ACE") is present mainly in vascular endothelial cells in the lungs, and acts on angiotensin I to liberate dipeptides from the terminals to produce angiotensin II, which has a strong blood pressure-increasing effect. It is an enzyme that produces ACE also has the action of decomposing and inactivating bradykinin, which has an antihypertensive action.
As described above, ACE produces a pressor peptide (angiotensin II) and degrades and inactivates a hypotensive peptide (bradykinin), resulting in an effect of increasing blood pressure. That is, ACE inhibitors can be inhibited from increasing blood pressure by inhibiting ACE, and various development studies have been made on ACE inhibitors as effective oral antihypertensive agents.
On the other hand, recently, it has been reported that photoaging of the skin is suppressed by inhibiting ACE expressed in the skin with an ACE inhibitor (Non-Patent Document 1), and a wrinkle-improving composition using it has been proposed. (for example, Patent Document 1, etc.).

Many natural and synthetic substances, including snake venom peptides, have been reported as ACE inhibitors. The substance has already been commercialized as an oral antihypertensive agent. However, pharmaceuticals containing synthetic substances as active ingredients have safety problems such as side effects, and antihypertensive agents with high safety are expected, and ACE inhibitors derived from natural products are being studied in various fields.
Certain kinds of lactic acid bacteria are known as probiotics (microorganisms that contribute to maintaining the health of the host when taken orally in appropriate amounts), and various effects have been reported. On the other hand, there is also a report of an external preparation using a lactic acid bacteria cell component (Non-Patent Document 2). Consumers have a good image of lactic acid bacteria, and in addition, ACE inhibition derived from highly safe lactic acid bacteria is being developed for the development of new cosmetic materials that can be prepared at low cost by making them derived from microorganisms rather than synthetic substances. Substance development is required.
So far, it has been reported that certain types of lactic acid bacteria produce peptides exhibiting an ACE inhibitory effect during milk fermentation (eg, Patent Document 2, etc.). The production of these functional peptides is affected by the proteolytic degradation of the strain, and lactic acid bacteria that produce effective peptide species and amounts are limited. was excluded from screening.
Currently, lactic acid bacteria are divided into 44 genera, and some genera have low safety. Among these, a new proposal is desired for an ACE inhibitor derived from Lactococcus lactic acid bacteria, which has been confirmed to be highly safe, and which is not related to the proteolytic degradation of the strain. By screening from those unrelated to the proteolytic degradation of strains, the number of strains that can be screened increases, and effective strains may become easier to find. In addition, proteolytic activity is often influenced by the activity of the proteolytic enzyme of the strain, and the only way to increase the activity of this enzyme is to use a method such as genetic recombination.

JP 2011-148734 A JP 2008-133251 A

Matsuura-Hachiya et al. 2013. Biochem. Biophys. Khmaladze et al. 2019. Exp. Dermatol. Kimoto-Nira et al. 2019. Lett. Appl.

The present invention provides an ACE inhibitor derived from Lactococcus lactic acid bacteria, which is highly safe and widely used as a probiotic, and a skin aging inhibiting composition and wrinkle-improving composition using the inhibitor, and ACE. An object of the present invention is to provide a simple method for increasing inhibitory activity.

As a result of intensive studies to solve the above problems, the inventors of the present invention have found that specific lactic acid bacteria belonging to the genus Lactococcus themselves have high ACE inhibitory activity, thereby solving the above problems. .

Specifically, the gist of the present invention is as follows.
1. An angiotensin-converting enzyme inhibitor containing one or more selected from viable cells, dead cells and extracts of Lactococcus lactis strain N7 (accession number: FERM P-18217).
2.1. A composition for inhibiting skin aging, comprising the angiotensin converting enzyme inhibitor described above as an active ingredient.
3.1. A wrinkle-improving composition containing the angiotensin converting enzyme inhibitor as an active ingredient.
4. A method for increasing angiotensin-converting enzyme inhibitory activity, which comprises culturing and/or heat-treating Lactococcus lactis strain N7 (accession number: FERM P-18217) under a specific carbon source.

Lactic acid bacteria N7 strain belonging to Lactococcus in the present invention has high safety because the cells themselves have high ACE inhibitory activity. The lactic acid bacterium strain N7 in the present invention can be used as a skin aging-inhibiting composition and a wrinkle-improving composition.

1 is a graph showing the test results of the "ACE inhibitory activity confirmation test of lactic acid bacteria N7 strain and H61 strain" in Example 1. FIG. FIG. 10 is an SA-β-Gal-stained microscopic image in Example 5 “Examination of the effect of lactobacillus N7 strain on the cell proliferation ability of normal human dermal fibroblast senescence-induced cells”. Images of non-senescence-induced cells are shown on the left, and senescence-induced cells are shown on the right.

The present invention will be described in detail below.
The present invention provides an ACE inhibitor containing one or more selected from viable cells, dead cells and extracts of Lactococcus lactis strain N7.
The official name of the Lactococcus lactis N7 strain is Lactococcus lactis subspecies lactis biovariety diacetylactis N7 strain, which is deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center. Accession number is FERM P-18217.
The lactic acid bacterium N7 strain in the present invention is a strain having extremely high ACE inhibitory activity among the strains stored in the lactic acid bacterium library of the livestock product research area in the Livestock Research Division, National Agriculture and Food Research Organization. It was selected as
Although it will be described in detail in the examples below, the ACE inhibitory activity of the lactic acid bacterium N7 strain in the present invention has an effect of suppressing the decrease in the survival rate of normal human epidermal keratinocytes due to ultraviolet UV-B irradiation (Non-Patent Document 3). ) is about 1.7 times higher than that of the lactic acid bacterium H61 strain confirmed by the present inventors, so its usefulness is considered to be extremely high.

<Culture conditions>
The culture conditions for the lactic acid bacteria N7 strain in the present invention are not particularly limited, and the culture can be performed under conventionally known conditions.
Examples of the culture medium include a medium added with a necessary nutrient source such as a carbon source, a nitrogen source, or inorganic salts that can be assimilated by the lactic acid bacteria belonging to the genus Lactococcus. Either a natural medium or a synthetic medium may be used as long as it is a medium in which lactic acid bacteria can grow, and a person skilled in the art can appropriately select an appropriate known medium to be used. Glucose, lactose, galactose, fructose, trehalose, sucrose, mannose, blackstrap molasses, etc. can be used as carbon sources, and meat extracts, peptones, yeast extracts, casein hydrolysates, whey protein hydrolysates as nitrogen sources. products, soy protein hydrolysates, etc. can be used. As inorganic salts, phosphates, sodium, potassium, magnesium and the like can be used. Suitable media for culturing lactic acid bacteria include, for example, M17 medium (with added glucose), MRS liquid medium, GYP medium, TYG medium, GAM medium, animal milk, skim milk, and milk whey. preferred.
The lactobacillus N7 strain of the present invention can be improved in ACE inhibitory activity by culturing it in MRS medium (where the carbon source is glucose), which is generally used for culturing lactic acid bacteria, in place of another sugar source. Among others, it has been confirmed by the examples described later that the ACE inhibitory activity is improved by culturing in a medium supplemented with lactose or fructose, as compared with the case where glucose is added.

Culture conditions are not particularly limited as long as lactic acid bacteria can grow.
Although the pH of the medium is not particularly limited, it usually ranges from pH 5 to 7, preferably from pH 6 to 7. In addition, the culture medium and culture apparatus can be used after being sterilized at a low temperature of 100° C. or less.
The culture temperature may be set within the range of the growth temperature of the N7 strain of lactic acid bacteria, preferably within the range of the optimum growth temperature. Although the culture time is not limited, it can be, for example, about 1 to 2 days.
There is no need to block or supply oxygen during culture. The form of culture includes stationary culture, shaking culture, tank culture, and the like.

<Heat treatment>
It has also been confirmed by experimental examples described later that the ACE inhibitory activity of the lactic acid bacterium strain N7 in the present invention is greatly improved in the ACE inhibitory activity of the heat-treated cells after culture compared with the ACE inhibitory activity of the live cells.
Here, heat treatment means, for example, placing an aqueous suspension of bacterial cells in a block heater or hot water bath to keep it warm for a certain period of time. Suitable temperatures for heat treatment include, but are not limited to, 95°C, 100°C, and 121°C.

The lactic acid bacterium N7 strain contained in the ACE inhibitor of the present invention may be a viable cell, a dead cell, or an extract.
The term "extract" as used in the present invention refers to the supernatant obtained by centrifugation of a bacterial cell suspended in a solvent such as water. The fungus suspended in water is centrifuged as it is after heat treatment, and is called "hot water extract". Both are included in the extract of the present invention.
In the examples, all of the substances actually used are extracts, but since the substances contained in the extracts are originally present in the cells, the ACE inhibitory effect can be exerted on both viable and dead cells. It is considered that the substance that has is included.
It is thought that the ACE inhibitory activity of the microbial cells is related to the glycoproteins of the microbial cells, and since sugar components are extracted with hot water, it is thought that the amount of involved components extracted is greater than that of viable cells.

The skin aging-inhibiting composition and wrinkle-improving composition of the present invention can be administered by administering the above-mentioned bacterial cells or extracts as they are or by diluting them with water or the like. Further, as components constituting the anti-wrinkle composition, other components used in external skin preparations and the like can be appropriately blended as needed within a range that does not impair the effects of the present invention. Examples of other components (optional compounding components) include oils, surfactants, powders, coloring materials, water, alcohols, thickeners, chelating agents, silicone agents, antioxidants, UV absorbers, moisturizing agents, Fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizers and the like.

EXAMPLES The present invention will be described below with reference to examples, but the technical scope of the present invention is not limited by these examples.

<Example 1: ACE inhibitory activity confirmation test of lactic acid bacteria N7 strain>
(1) Test specimen Lactic acid bacteria strains N7 and H61 were each cultured overnight at 30°C in MRS medium (Becton, Dickinson and Company, Sparks, MD, USA), and then centrifuged at 5000g for 10 minutes. After the cells were washed twice with 0.85% sodium chloride solution, they were suspended in water and heat-treated at 121° C. for 15 minutes. This sample was freeze-dried to prepare a freeze-dried powder of dead cells, which is widely used industrially, and used as a test sample for measuring ACE inhibitory activity.
2 mg of each test sample was taken and suspended in water. This dead cell aqueous suspension was centrifuged at 13,000 g for 10 minutes, and the resulting supernatant (killed cell water extract) was diluted 10-fold with water. It was subjected to an activity test.
(2) Test method ACE inhibitory activity was measured using an ACE inhibitory activity measurement kit (DOJINDO LABORATORIES, Kumamoto) according to the protocol of the kit.
1. In a 96-well microplate, add 20 μL of sample to sample wells, blank 1 and blank 2 wells.
2. Add 20 μL of the reaction substrate included in the kit to each well.
3. Add 20 μL of water to blank 2 wells.
4. Add 20 μL of Enzyme Reaction to Sample wells and Blank 1 wells.
5. A seal is affixed to the plate, and the plate is kept in a constant temperature bath at 37°C for 1 hour.
6. Add 200 μL of indicator solution to each well.
7. Incubate the plate at room temperature for 10 minutes.
8. Absorbance at 450 nm is measured with a BIO-RAD Model 3550 Microplate Reader (Hercules, Calif.).
[Calculation formula for ACE inhibitory activity]
ACE inhibitory activity (%) = [(A _blank1 -A _sample )/(A _blank1 -A _blank2 )] x 100
A _blank1 : absorbance of water (450 nm)
A _blank2 : Absorbance of reaction substrate A _sample : Absorbance of bacterial sample The test results are shown in FIG.

(3) Results As shown in FIG. 1, the ACE inhibitory activity of the lactic acid bacterium N7 strain in the present invention was 54.3%, while that of the lactic acid bacterium H61 strain was 32.8%. That is, it was revealed that the ACE inhibitory activity of the lactic acid bacterium N7 strain is about 1.7 times higher than that of the lactic acid bacterium H61 strain.
The present inventors confirmed that the lactic acid bacterium H61 strain has an effect of suppressing the decrease in the survival rate of normal human epidermal keratinocytes due to ultraviolet UV-B irradiation. expected to be effective.
As described above, it was confirmed that the N7 strain of lactic acid bacteria in the present invention has high ACE inhibitory activity.

<Example 2: Confirmation test 1 regarding culture conditions and ACE inhibitory activity of lactic acid bacteria strain N7>
For the lactic acid bacteria N7 strain, the MRS culture solution cultured overnight was inoculated into the MRS medium at 0.5% (v / v), and the cells were cultured overnight at 30 ° C. (stationary phase), and the bacteria were cultured for 5.5 hours. Cells (logarithmic phase) and cells cultured overnight at 37° C. were harvested, washed three times with water, and suspended in water to 100 mg/mL (wet cell weight w/v). This bacterial cell aqueous suspension was heat-treated at 95° C. for 15 minutes and centrifuged at 13000 g for 10 minutes. was performed, and ACE inhibitory activity (%) was calculated according to the ACE inhibitory activity calculation formula.

表１中の数値は平均値（標準偏差）を意味する。
表１中の＊は、Ｐ＜０．０５ ｖｓ ｃｏｎｔｒｏｌ（３０℃、定常期、加熱処理有）を意味する。 (3) Results Table 1 shows the test results.

Numerical values in Table 1 mean average values (standard deviation).
* in Table 1 means P<0.05 vs control (30° C., stationary phase, with heat treatment).

From the results in Table 1 above, the ACE inhibitory activity of the lactic acid bacterium N7 strain was higher at a culture temperature of 30°C than at 37°C, higher in stationary phase culture than in logarithmic phase culture, and higher in the case of heat treatment. It was found to be higher than the cells without heat treatment. Therefore, it was clarified that the optimal culture conditions for the N7 strain are overnight culture at 30°C.

<Example 3: Culture condition of lactic acid bacteria strain N7 and confirmatory test 2 regarding ACE inhibitory activity>
(1) Test method An MRS medium (C free MRS) containing no carbohydrate (glucose) was prepared.
This MRS medium (C free MRS) contains 1% proteose peptone, 0.5% yeast extract, 0.1% Tween 80, 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% magnesium sulfate. , 0.005% manganese sulfate, and 0.2% dipotassium phosphate (pH 6.5).
10% solutions of glucose, fructose, lactose and galactose were prepared, filtered through a filter (0.2 μm, manufactured by Advance Co., Ltd.) and sterilized. This solution was added to the MRS medium to a final concentration of 0.5% (v/v). The N7 strain was cultured overnight in 10% skim milk, and one platinum loop thereof was suspended in a 0.85% sodium chloride solution. Incubate overnight. After collecting the cultured cells, the cells were washed with water three times and then suspended in water to a concentration of 100 mg/mL (wet cell weight w/v). This bacterial cell aqueous suspension was heat-treated at 95° C. for 15 minutes and centrifuged at 13000 g for 10 minutes. was performed, and ACE inhibitory activity (%) was calculated according to the ACE inhibitory activity calculation formula.

表２中の数値は平均値（標準偏差）で示す。
表２中の異なる文字間では、有意差あり（Ｐ＜０．０５）を意味する。 (2) Results Table 2 shows the test results.

Numerical values in Table 2 are shown as average values (standard deviation).
Between different letters in Table 2 means significant difference (P<0.05).

From the results in Table 2 above, the ACE inhibitory activity of the lactic acid bacterium N7 strain changes depending on the type of carbon source in the medium, and culturing in a medium supplemented with lactose or fructose is better than the commonly used glucose supplemented medium. It was confirmed that the ACE inhibitory activity was higher than when it was used.

<Example 4: Examination of cytotoxicity of lactobacillus strain N7 to normal human dermal fibroblasts>
(1) Test specimen Lactic acid bacterium strain N7 was cultured overnight at 30°C in MRS medium, collected, washed with water three times, suspended in water, and heat-treated at 121°C for 15 minutes. Did. This bacterial cell aqueous suspension was centrifuged at 13,000 g for 10 minutes, and the supernatant (hot water extract) was collected and freeze-dried to obtain a test sample.
(2) Test Method Normal human dermal fibroblasts were seeded in a 48-well plate at a cell density of 1.0×10 ⁴ cells/well using DMEM medium (5% FBS). After culturing for 24 hours, the above test specimen was replaced with a DMEM medium (5% FBS) containing the concentrations shown in Table 3 below, and cultured for 72 hours. After culturing, the cells were collected by trypsinization, and the number of cells was counted using flow cytometry. The measured values were tested for significance using the Student's t-test.

表３中（１）は、試料未処理細胞に対する有意差を意味する。 (3) Results Table 3 shows the test results.

(1) in Table 3 means a significant difference from sample untreated cells.

As shown in Table 3, when the concentration of the hot water extract of lactic acid bacteria N7 strain was 10 mg/mL, abnormalities in the morphology of normal human dermal fibroblasts were observed. %) was observed, the maximum concentration was set to 5 mg/mL in Example 5 below.
It was also found that addition of 0.04 to 5 mg/mL of the hot water extract of lactic acid bacteria strain N7 does not affect the survival of normal human dermal fibroblasts.

<Example 5: Examination of the effect of lactic acid bacteria N7 strain on the cell proliferation ability of normal human dermal fibroblast senescence-induced cells>
(1) Preparation of Senescent-Induced Cells Freshly prepared DMEM (0% FBS) containing 600 μmol/L hydrogen peroxide was added to normal human dermal fibroblasts and cultured at 37° C. for 1 hour. After culturing for 1 hour, the hydrogen peroxide-containing DMEM (0% FBS) was removed, and DMEM medium (10% FBS) was added. After repeating this operation for 4 days, the cells were cultured in a DMEM medium (10% FBS) for 3 days. The cells obtained here were used as senescence-inducing cells.
Senescence of senescence-induced cells was determined by SA-β-Gal staining.
Specifically, senescence-induced cells and non-senescence-induced cells were seeded in a 48-well plate at a cell density of 5×10 ⁴ cells/well. After culturing for 24 hours, the cells were fixed with PBS containing 3% formaldehyde. pH 6 reaction solution containing 1 mg/mL X-Gal (40 mmol/L citric acid/sodium phosphate, 0.5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L sodium chloride, 2 mmol/L magnesium chloride ) and cultured for 16 hours.
After culturing, microscopic observation was performed to compare the staining intensity of SA-β-Gal, a senescence marker, between the non-senescence-induced cells and the senescence-induced cells, and it was confirmed that the staining intensity increased in the senescence-induced cells compared to the non-senescence-induced cells. .

(2) Evaluation Test of Cell Proliferation Ability Senescence-inducing cells and non-senescence-inducing cells were seeded in a 48-well plate at a cell density of 1.0×10 ⁴ cells/well using DMEM medium (5% FBS). After culturing for 24 hours, in the senescence-inducing cell group, the cells of the lactic acid bacterium N7 strain were heat-treated at 121° C. for 15 minutes and then centrifuged. The medium was changed (5% FBS) and cultured for 72 hours. Non-senescence-induced cells were replaced with DMEM medium (5% FBS) containing no test sample and cultured for 72 hours. After culturing, the cells were collected by trypsinization, and the number of cells was counted using flow cytometry. A significance test was performed using the Student's t-test. Table 4 shows the results.

表４中の（１）は、非老化誘導細胞の試料未処理細胞に対する有意差を意味する。
表４中の（２）は、老化誘導細胞の試料未処理細胞に対する有意差を意味する。

(1) in Table 4 means a significant difference of non-senescence-induced cells to sample untreated cells.
(2) in Table 4 means a significant difference of senescence-induced cells to sample untreated cells.

(3) Results As shown in Table 4, the senescence-induced cells significantly decreased in cell number compared to the non-senescence-induced cells. When 0.5 to 5.0 mg/mL of the hot water extract of lactic acid bacteria strain N7 was added to normal human dermal fibroblast senescence-induced cells, the number of cells significantly increased compared to when it was not added. An increase in proliferative capacity was confirmed.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a highly safe Lactococcus lactobacillus-derived ACE inhibitor. It is also possible to provide a skin aging-inhibiting composition and a wrinkle-improving composition using the same.

Claims (4)

An angiotensin-converting enzyme inhibitor containing one or more selected from viable cells, dead cells and extracts of Lactococcus lactis strain N7 (accession number: FERM P-18217). A composition for inhibiting skin aging, comprising the angiotensin converting enzyme inhibitor according to claim 1 as an active ingredient. A wrinkle-improving composition comprising the angiotensin-converting enzyme inhibitor according to claim 1 as an active ingredient. A method for increasing angiotensin-converting enzyme inhibitory activity, which comprises culturing and/or heat-treating Lactococcus lactis strain N7 (accession number: FERM P-18217) under a specific carbon source.

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