JP6976843B2 - Skin condition improving agent - Google Patents

Description

The present invention is a skin condition improving agent containing a lactic acid bacterium of the genus Lactobacillus gasseri derived from the infant intestine having a high ability to promote hyaluronic acid production and a fermented product thereof as an active ingredient, and a skin containing the skin condition improving agent as an active ingredient. Provide medicines for improving the condition and foods and drinks for improving the skin condition.

In recent years, research on skin aging prevention has been widely conducted. Aging is an important factor in the cause of skin aging, but in addition, the effects of dryness, oxidation, ultraviolet rays, etc. are also cited as direct factors related to skin aging. As specific phenomena of skin aging, reduction of mucopolysaccharides such as hyaluronic acid, collagen cross-linking reaction, and cell damage by ultraviolet rays are known.

Among them, hyaluronic acid is widely present in various organs and tissues such as skin, joints, ligaments, vitreous body, brain, lungs, and kidneys in the living body, and especially in the skin, hyaluronic acid accounts for 50% of the whole body. Acid is present (Non-Patent Document 1). It is known as an important factor to keep the skin moisturized due to its high moisturizing property (Non-Patent Document 2).

T. C. Laurent and J. R. Fraser, “Hyaluronan,” FASEB J, Vol.6, pp.2397-2402, 1992 Papakonstantinou E. ,: Hyaluronic acid: A Key molecule in skin aging, Dermatoendocrinol, 4 (3), 253-8, 2012

The present invention is a skin condition improving agent containing a lactic acid bacterium of the genus Lactobacillus gasseri derived from the infant intestine having a high ability to promote hyaluronic acid production and a fermented product thereof as an active ingredient, and a skin containing the skin condition improving agent as an active ingredient. The purpose is to provide condition-improving drugs and skin condition-improving foods and drinks.

The present inventors focused on the fact that hyaluronic acid is indispensable for improving the moisturizing power of the skin, and as a result of screening for a large number of lactic acid bacteria, the lactic acid bacteria of the genus Lactobacillus gasseri derived from the infant intestine, particularly lacto Bacillus gasseri N320 strain (NITE BP-02287) has a high ability to promote hyaluronic acid production. , It has been found that it is effective for improving the skin condition including the reduction of the gloss of the skin, and the present invention has been completed.

That is, the first invention of the present application is an orally administrable skin condition improving agent containing a lactic acid bacterium of the genus Lactobacillus gasseri and a fermented product of the lactic acid bacterium as an active ingredient, and the second invention of the present application is an improvement of the skin condition. Is a skin condition improving agent according to the first invention of the present application, which comprises improving the dry condition of the skin, improving the gloss of the lowered skin, and improving the feeling of firmness of the skin after washing the face. The lactic acid bacterium is Lactobacillus gasseri N320 strain (NITE BP-02287), which is the skin condition improving agent according to the first invention of the present application or the second invention of the present application. Furthermore, the applicant also intends a skin condition improving drug for improving the skin condition containing the lactic acid bacterium of the Lactobacillus gasseri species, or a food or drink for improving the skin condition.

In the present invention, the improvement of the skin condition means that the decrease in the water content of the skin due to drying is improved, and the skin condition is improved both objectively and subjectively.

According to the present invention, there is provided a skin condition improving agent, a drug, or a food or drink that can be orally ingested on a daily basis to improve the dryness of the skin.

FIG. 1 is a diagram comparing the hyaluronic acid production promoting ability of the strain of the present invention and the comparative strain. FIG. 2 is a diagram comparing the proliferation of skim milk medium between the strain of the present invention and the comparative strain. FIG. 3 is a diagram comparing the relative values of the changes in the skin water content of the cheeks from the pre-intake test for the test food and the placebo. FIG. 4 is a diagram comparing the changes in the doctor's findings score (dry cheeks) from the pre-intake examination by the dermatologist for the test food and the placebo. FIG. 5 is a diagram comparing the amount of change in the doctor's finding score (dry upper back) from the pre-intake examination by the dermatologist for the test food and the placebo. FIG. 6 is a diagram comparing the amount of change in the doctor's finding score (dry neck) from the pre-intake examination by the dermatologist for the test food and the placebo. FIG. 7 is a diagram comparing the changes in the skin questionnaire (tightness after cleansing) score from the pre-intake test for the test food and the placebo. FIG. 8 is a diagram comparing the amount of change in the skin questionnaire (facial skin gloss) score from the pre-intake test for the test food and the placebo. FIG. 9 is a diagram comparing the amount of change in the skin questionnaire (bad make-up) score from the pre-intake test for the test food and the placebo. FIG. 10 is a diagram comparing the changes in the skin questionnaire (face dryness) score from the pre-intake test for the test food and the placebo.

Hereinafter, the present invention will be described in detail.
1. 1. Lactobacillus gasseri N320 strain (NITE BP-02287)
The lactic acid bacterium of the present invention is lactobacillus gasseri. In particular, among the lactic acid bacteria belonging to Lactobacillus gasseri, Lactobacillus gasseri N320 strain (NITE BP-02287). The symbol of N320 referred to in the present invention is a number originally assigned to the strain by Nisshin Foods Holdings Co., Ltd., and the Lactobacillus gasseri N320 strain was isolated for the first time by the present inventor.

The Lactobacillus gasseri N320 strain of the present invention has been deposited under the following conditions.
(1) Depositary organization name: National Institute of Technology and Technology Patented Microbial Deposit Center (2) Contact: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture 292-0818 Room 122 (3) Deposit number: NITE BP -02287
(4) Display for identification: N320
(5) Original deposit date: June 14, 2016 (6) Transfer date to deposit based on the Budapest Treaty: April 10, 2017 The mycological properties of the Japanese invention Lactobacillus gasseri N320 strain are shown in the table below. It is as shown in 1 and 2. This bacteriological property is based on the method described in Bergey's manual of systematic bacteriology Vol.2 (1986). Table 1 shows the shape and the like related to this strain, and Table 2 shows the results of saccharification tests using Api 50CH and Api CHL (manufactured by BioMérieux). In Table 2, "+" indicates fermentable and "-" indicates non-fermentable.

2. 2. Hyaluronic acid production promoting ability test The Lactobacillus gasseri N320 strain of the present invention has a high hyaluronic acid production promoting ability as shown in the experimental examples described later. The hyaluronic acid production promoting ability was confirmed by the following test method.

<Preparation of subject (culture solution) used for evaluation of hyaluronic acid production promoting ability>
The subject (culture solution) used for the evaluation of hyaluronic acid production promoting ability was lactic acid bacteria cultured in the MRS medium (Difco Lactobacilli MRS Broth) shown in Table 3 at 37 ° C for 24 hours, sterilized by heating at 95 ° C for 20 minutes, and then sodium hydroxide. It was obtained by adjusting the pH to 7.0 using an aqueous solution.

<Evaluation of hyaluronic acid production promoting ability>
The hyaluronic acid production promoting action of the sample obtained above was evaluated. That is, when normal human adult epidermal keratinized cells were seeded on a 24-well plate at ^{a concentration of 5.0 × 10 4} cells / well and brought into a confluent state, 500 μL containing each sample group (final concentration 1% amount) was added. HuMedia-KG2 culture solution was added. After the addition, the cells were cultured for 72 hours, and then the amount of hyaluronic acid produced was measured by the ELISA method. The unit for measuring the amount of hyaluronic acid produced was ng / ml, and the Hyaluronan assay kit (manufactured by Biochemical Biobusiness) was used for the measurement. A lactic acid bacterium growth medium (MRS) was used as a control example.

3. 3. Proliferative test in skim milk medium <Skim milk medium proliferative test>
The preculture solution was inoculated into a 10% SM (skimmed milk) medium, and after culturing this at 37 ° C for 24 hours, the proliferation in the milk medium was evaluated by the number of lactic acid bacteria.

4. Food and Beverage The lactic acid bacterium of the present invention can be contained in food and drink and used. The lactic acid bacterium of the present invention can be particularly preferably used for dairy products, and for example, fermented milk containing lactic acid bacteria and a lactic acid bacteria beverage containing lactic acid bacteria can be considered. The current ministry ordinance on ingredient standards for milk and dairy products does not specifically specify the number of lactic acid bacteria as an ingredient standard, but fermented milk (non-fat milk solids content of 8.0% or more) and lactic acid bacteria beverages (non-fat milk solids). 1.0 x 10 ^{7 cfu / ml or more is preferable for those with a minute of 3.0% or more, and 1.0 x 10 6} cfu / ml or more is preferable for lactic acid bacteria beverages (those with a non-fat milk solid content of less than 3.0%), such as milk. The above-mentioned number of bacteria can be realized by growing in a fermented liquid or in the form of a final product. In addition to fermented milk containing lactic acid bacteria and lactic acid bacteria beverages containing lactic acid bacteria, it can also be used for dairy products such as butter, processed egg products such as mayonnaise, and sweet breads such as butter cake. Further, it can be suitably used for processed foods such as instant noodles and cookies. In addition to the above, the food product of the present invention may be in a form (for example, powder, granules, capsules, tablets, etc.) formulated by adding appropriate carriers and additives together with the lactic acid bacteria as needed. ..

It is also useful to include the lactic acid bacterium of the present invention in foods for specified health use, dietary supplements, etc. in addition to general beverages and foods.

In addition to foods, the lactic acid bacterium of the present invention is also used in the fields of cosmetics such as cosmetics, pharmaceuticals such as intestinal regulators, daily necessities such as toothpaste, silage, animal feeds, and animal feeds and plant fertilizers such as plant liquid fertilizers. It can be applied.

5. Pharmaceuticals The lactic acid bacteria of the present invention can be used as pharmaceuticals after being extracted and powdered by concentration or separation after fermentation. For example, it is possible to provide an extract in a bottle or the like, or a dry fermented liquid powder in the form of granules, encapsulation, or tableting using an excipient or the like.

Since the lactic acid bacterium of the present invention (Lactobacillus gasseri N320 strain) has a high ability to promote hyaluronic acid production, it is possible to routinely ingest tablets, pharmaceuticals, or foods and drinks containing the lactic acid bacterium and its fermented product as an active ingredient. For example, the dry state of the skin (particularly, cheeks, back, neck, etc.), the feeling of tension after washing the face, and the decrease in the gloss of the skin can be improved.

Hereinafter, examples of the present invention will be shown, but the present invention is not limited to the following examples.
<Test Example 1> Evaluation of ability to promote hyaluronic acid production The ability to promote hyaluronic acid production was evaluated for the Lactobacillus gasseri N320 strain of the present invention and three Lactobacillus gasseri comparative strains (N219, N220, N313) owned by the company. ..

The hyaluronic acid production promoting ability was evaluated by the following procedure. Each of the strain of the present invention and the comparative strain was cultured in the MRS medium (Difco Lactobacilli MRS Broth) shown in Table 3 at 37 ° C for 24 hours, sterilized by heating at 95 ° C for 20 minutes, and then adjusted to pH 7.0 using an aqueous sodium hydroxide solution. By doing so, a subject (culture medium) was obtained.

Separately, normal human adult epidermal keratinized cells were seeded on a 24-well plate at ^{a concentration of 5.0 × 10 4} cells / well, and when the cells were brought into a confluent state, 500 μL containing each sample group (final concentration 1% amount) was added. HuMedia -KG2 culture solution was added. After the addition, the cells were cultured for 72 hours, and then the amount of hyaluronic acid produced was measured by the ELISA method. As a control example (control), a lactic acid bacterium growth medium (MRS) was used. A Hyaluronan assay kit (manufactured by Biochemical Biobusiness Co., Ltd.) was used for the measurement. The unit of the measured value is the amount of hyaluronic acid produced (ng / ml).

As is clear from Table 4 and FIG. 1, the lactic acid bacterium of the present invention (Lactobacillus gasseri N320 strain) has a very strong ability to promote hyaluronic acid production, which is higher than that of other lactic acid bacteria owned by the company. It was confirmed that it had the ability.

<Test Example 2> Skim milk medium growth test A skim milk medium growth test was carried out on the Lactobacillus gasseri N320 strain of the present invention and three Lactobacillus gasseri comparative strains (N219, N220, N313) owned by the company.

Each strain was inoculated into a 10% SM (skimmed milk) medium, cultured at 37 ° C for 24 hours, and then the proliferative property in the skim milk medium was evaluated by the number of lactic acid bacteria.

The results are shown in Table 5 and FIG.

The strain of the present invention (Lactobacillus gasseri N320 strain) has a bacterial count of 22 × 10 ⁷ cfu / ml, which is highly proliferative compared to other comparative strains, and produces fermented milk containing lactic acid bacteria and a lactic acid bacteria beverage containing lactic acid bacteria. It was a level that was not a problem above.

<Test Example 3> Effect test on human subjects
A total of 43 subjects, 22 males and 21 females aged 35 to 59 years, were selected for the effect test. The method of allocating the subjects is as follows.
-Assigned so that the average value of the amount of skin water evaporation on the right cheek of the subject measured at the time of screening was the same for each group as much as possible.
-As for the ages of the subjects, the average values were assigned to each group so as to be as uniform as possible. At that time, the male-female ratio of each group was assigned to be as equal as possible to the male-female ratio at the time of selection.

<Preparation of test food>
As test foods, a test food and a placebo, which are fermented foods of the lactobacillus gasseri N320 strain shown in Table 6, were prepared. This strain contained in the test food is a dead cell.

<Test method>
A placebo-controlled, double-blind study was conducted on the above subjects. First, 43 subjects were divided into 22 test food intake groups and 21 placebo intake groups according to a random number allocation table, and each subject was given 8 test foods (100 ml) once a day. It was ingested for a week (56 days). As a subject's control item
(1) During the test period, it was prohibited to change or add basic cosmetics that are used daily.
(2) Treatment of waste hair at the test site within 2 weeks before observation was prohibited.
(3) The use of bath salts, etc. during the test period was prohibited.
(4) The use of body lotion was prohibited on the day before and on the day of observation.
(5) During the test period, the use of medicines, newly designated quasi-drugs and Chinese herbs was prohibited in principle.
(6) During the test period, in principle, the intake of processed foods with newly specified beneficial effects on the skin was prohibited.
(7) During the test period, new health foods and foods with health claims (foods for specified health use) other than the health foods and foods with health claims (foods for specified health use and foods with food functional claims) that are currently ingested on a daily basis. ) Was prohibited.
(8) During the test period, excessive alcohol intake beyond the daily intake range was prohibited. In addition, alcohol intake on the day before the observation day was prohibited.
(9) During the test period, activities that cause sunburn, such as outdoor sports and artificial ultraviolet irradiation, were prohibited. Care was taken not to expose the subject site to UV rays indoors or outdoors even in daily life. Specifically, care was taken with a hat, clothing, a sunscreen that is normally used, and the like.
(10) Bathing in hot springs for 3 days before the observation day was prohibited.
(11) During the test period, actions aimed at improving skin condition, such as body esthetics and bedrock baths, were prohibited.
(12) Vigorous exercise was prohibited until the measurement on the day of observation was completed.
(13) During the test period, overseas travel and overseas business trips were prohibited.
(14) On the day of observation, ingestion of irritating foods such as spicy foods (curry, chili pepper, tabasco, etc.) was prohibited.
(15) During the test period, the use of "warm underwear, etc." that show a moisturizing effect due to special effects such as far infrared rays was prohibited.
(16) The day before the observation day, the bath was always taken before going to bed, and the bathing before the measurement on the day of the observation was prohibited.
(17) During the test period, shaving, hair removal, and hair removal at the measurement site (right cheek) were prohibited, but this does not apply to beard treatment.

In principle, the above-mentioned subject management items shall be observed, but this shall not apply if there is a medical need or other danger to the safety of life and body. If any matter contrary to the subject management matters arises, the subject will immediately contact the testing organization. The subjects underwent skin water content measurement, doctor's findings by a dermatologist, and a questionnaire survey before, 4 weeks, and 8 weeks after ingestion.

The skin water content was measured at 3 points in a constant temperature and humidity room by shifting the measurement site of the subject by 5 mm using CORNEOMETER CM 825 (Integral Co., Ltd.). Then, the amount of change after 8 weeks of ingestion was evaluated with the average value of the measured water content (μS) before ingestion as 100%.

The doctor's findings by the dermatologist targeted the subject's cheeks, upper back, and neck, and the dry state was "none (no symptoms)": 0, "mild (slight symptoms are seen). ) ”1,“ Moderate (with obvious symptoms) ”: 2,“ Severe (significant symptoms are seen) ”: 3

In the questionnaire survey, the subject said, "I would like to ask you about your skin condition (trouble) that you are currently worried about." About the feeling of tension after washing your face 1. Very worrisome 2. I'm a little worried 3. I can't say either 4. I don't really care 5. It was evaluated as 5 grades, which do not bother me at all.

<Result>
When the skin water content of the cheeks of the subjects was measured before and 8 weeks after ingestion, as shown in FIG. 3, the skin water content (relative value) of the subjects who ingested the test food was that of the subjects who ingested the placebo. Significantly increased compared to the value. The skin water content (relative value) of each of the test food and placebo shown in FIG. 3 excludes the data of subjects suspected of violating the analysis target exclusion criteria and subjects who had long-term adverse events before the test. It is the average value of 21 subjects in the placebo intake group and 22 subjects in the test food intake group.

In addition, as shown in FIGS. 4 and 5, in the evaluation of the doctor's findings regarding dry cheeks and dry upper back, the score of the test food group was compared with the placebo score at 8 weeks of ingestion. Significantly improved. As for the dryness of the neck, as shown in FIG. 6, the score of the test food group was confirmed to improve at 8 weeks after ingestion as compared with the placebo.

When the results of the questionnaire were analyzed for females, as shown in FIGS. 7 to 10, the scores of the test food groups were significantly improved compared to placebo in 8 weeks after ingestion of the feeling of tension after washing the face and the gloss of the skin on the face. .. In addition, the scores of the test food group tended to improve compared to placebo in terms of poor make-up and dry face.

As a result of a placebo-controlled double-blind study for those who are aware of dry skin, those who feel sagging skin, and those who are aware of rough skin, the main component is lactic acid bacteria of Lactobacillus gasseri species and their fermented products. The skin condition improving composition is effective in improving the skin condition, including a decrease in the water content of the skin due to dryness, deterioration of the dry condition of the cheeks, back and neck, a feeling of tension after washing the face, and a decrease in the gloss of the skin. It was confirmed that there was.

Claims (4)

Lactobacillus gasseri species lactic acid bacteria and fermented products of the lactic acid bacteria are active ingredients, and the lactic acid bacteria are Lactobacillus gasseri N320 strain (NITE BP-02287), which is an orally administrable skin condition improving agent. The skin condition improving agent according to claim 1, wherein the improvement of the skin condition includes improvement of the dry condition of the skin, improvement of the decreased gloss of the skin, and improvement of the feeling of tightness of the skin after washing the face. A pharmaceutical product for improving skin condition, which comprises the skin condition improving agent according to claim 1 or 2 as an active ingredient. A food or drink for improving skin condition, which comprises the skin condition improving agent according to claim 1 or 2 as an active ingredient.

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