JP7544498B2 - Composition for activating skin cells and topical skin preparation - Google Patents

Description

IPOD FERM BP-10879

The present invention relates to a composition for activating skin cells and a topical skin preparation that utilizes a fermentation product of yeast using a culture obtained by culturing lactic acid bacteria in a medium containing milk components as a raw material.

It is known that cultures of lactic acid bacteria and yeast have useful properties such as moisturizing effects on the skin, and development of cosmetic materials using these microorganisms is underway. For example, Patent Document 1 discloses a skin moisturizer made from the residual liquid obtained by inoculating and culturing lactic acid bacteria in a medium containing milk components, and separating insoluble substances from the resulting culture. Meanwhile, Patent Documents 2 and 3 describe the removal or suppression of the fermentation odor of lactic acid bacteria fermentation products used as cosmetic materials by heating under reduced pressure or treating with carbonates, etc. Furthermore, Patent Documents 4 and 5 describe that the culture obtained by culturing lactic acid bacteria in a medium containing milk components can be further fermented with yeast to suppress the fermentation odor and impart a rose-like or fruit-like scent.

Special Publication No. 2-40643 Special Publication No. 2-24247 Patent No. 4512265 Patent No. 6449334 JP 2017-212894 A

However, it was not known that the culture obtained by culturing lactic acid bacteria in a medium containing milk components had the effect of activating skin cells.

The object of the present invention is therefore to provide a composition for activating skin cells, which has an excellent effect of activating skin cells and is easy to use in the form of cosmetics and the like because it uses materials derived from natural products. It is also an object of the present invention to provide an external skin preparation that utilizes the composition.

The inventors conducted extensive research to achieve the above-mentioned objective, and discovered that a fermented product obtained by culturing lactic acid bacteria in a medium containing milk components and then treating the culture with yeast has an excellent effect of activating skin cells, which led to the completion of the present invention.

In other words, in a first aspect, the present invention provides a composition for activating skin cells, which contains a fermentation product of yeast using a culture obtained by culturing lactic acid bacteria in a medium containing milk components as a raw material.

In the above composition, the yeast is preferably one or more species selected from the group consisting of Wickerhamomyces pijperi, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

In the above composition, it is preferable that the lactic acid bacteria produce metabolites derived from milk components when cultured in the milk component-containing medium, and that the raw material for fermentation by the yeast contains the metabolites derived from milk components.

In the above composition, the lactic acid bacteria is preferably Streptococcus thermophilus.

On the other hand, in a second aspect, the present invention provides the use of the above composition as an external preparation for the skin.

According to the present invention, a fermented product is obtained by further treating a culture obtained by culturing lactic acid bacteria in a medium containing milk components with yeast, and is therefore suitable for use as a composition for activating skin cells. In addition, the fermented product is easily usable as a naturally derived material in the form of cosmetics and other products that can be used on a daily basis at the discretion of the general consumer.

[Existence of impossible or impractical circumstances]
The composition of the present invention includes a culture of lactic acid bacteria and a fermentation product of yeast, but there are circumstances that make it impossible or almost impractical to directly identify them by their structure or characteristics. That is, the composition or skin topical agent provided by the present invention has an excellent effect of activating skin cells, but the fermentation product involved in the effect contains a wide variety of components in a complex manner, and completely analyzing and identifying these components or identifying specific components necessary for activating skin cells requires considerable trial and error and experiments, which imposes significantly excessive economic expenditure and time.

1 is a graph showing the results of investigating the effect of lactic acid bacteria fermentation broth (LF) or yeast fermentation broth 1 (YW1) on the adhesion ability of normal human epidermal keratinocytes to the bottom of a culture plate in Test Example 1. 1 is a graph showing the results of investigating the effect of lactic acid bacteria fermentation broth (LF) or yeast fermentation broth 1 (YW1) on the proliferation ability of normal human epidermal keratinocytes after adhesion to the bottom of a culture plate in Test Example 1. 1 is a graph showing the results of investigating the effects of yeast fermentation liquids 2 to 6 (YW2, YW3, YW4, YK, or YS) on the proliferation ability of normal human epidermal keratinocytes after adhesion to the bottom of a culture plate in Test Example 2.

There are no particular limitations on the lactic acid bacteria used in the present invention, so long as the culture is compatible with cosmetic materials, etc. For example, microorganisms belonging to the genus Streptococcus, such as Streptococcus thermophilus; microorganisms belonging to the genus Lactobacillus, such as Lactobacillus casei, Lactobacillus mali, Lactobacillus acidophilus, Lactobacillus delbrueckii ssp. bulgaricus, and Lactobacillus helveticus; microorganisms belonging to the genus Lactobacillus, such as Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. Microorganisms belonging to the genus Lactococcus such as Enterococcus cremoris, microorganisms belonging to the genus Enterococcus such as Enterococcus faecalis, microorganisms belonging to the genus Bifidobacterium such as Bifidobacterium breve, Bifidobacterium bifidum, and Bifidobacterium longum, microorganisms belonging to the genus Pediococcus such as Pediococcus acidilactici and Pediococcus pentosaceus, microorganisms belonging to the genus Pediococcus such as Weissella vilidescens and Weissella confusa Examples of suitable lactic acid bacteria include microorganisms belonging to the genus Weissella, such as Bacillus confusa. Among these, preferred examples include microorganisms belonging to the genus Streptococcus, more specifically Streptococcus thermophilus. Microorganisms belonging to the genus Bifidobacterium, sometimes called bifidobacteria, are included in the lactic acid bacteria of the present invention. Lactic acid bacteria may be used alone or in combination of two or more types. That is, treatment with two or more different types of lactic acid bacteria may be performed sequentially, or two or more different types of lactic acid bacteria may be used in combination at the same time, or these treatments may be combined.

The yeast used in the present invention is not particularly limited as long as the fermented product is compatible with cosmetic materials, etc. For example, the genera Wickerhamomyces, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Wickerhamia, Pichia, Eremascus, Endomyces, Nadsonia, Saccharomycodes, Hanseniaspora, Candida, Galactomyces, Torulaspora, Lodderomyces, Wingea, Endomycopsis, and the like may be used. Examples of microorganisms include those belonging to the genera Endomycopsis, Hansenula, Pachysolen, Citeromyces, Debaryomyces, Schwanniomyces, Dekkera, Saccharomycopsis, Lipomyces, Spermophthora, Eremothecium, Crebrothecium, Ashbya, Nematospora, Metschnikowia, and Coccidiascus. Among them, preferred examples include microorganisms belonging to the genus Wickerhamomyces, Kluyveromyces, and Saccharomyces, and more specifically, Wickerhamomyces pijperi, Kluyveromyces marxianus, Saccharomyces cerevisiae, and the like. In addition, those that were classified as Pichia pijperi in the old classification but can be newly classified as Wickerhamomyces pijperi are included in the Wickerhamomyces pijperi of the present invention. The yeast may be used alone or in combination of two or more kinds. That is, treatment with two or more different types of yeast may be performed sequentially, treatment with two or more different types of yeast may be performed simultaneously, or these treatments may be combined.

In the present invention, lactic acid bacteria are cultured in a medium containing milk components to obtain a culture product. Examples of milk components include raw animal milk such as cow's milk, goat's milk, and sheep's milk, heated milk, skim milk powder, whole milk powder, fresh cream, whey, and whey protein. Lactic acid bacteria may be cultured using only milk components, and the medium in which the lactic acid bacteria are cultured may contain other components other than milk components. The content of milk components in the medium is preferably 0.1 to 100% by mass, more preferably 1 to 30% by mass, and even more preferably 2 to 5% by mass, calculated on a dry matter basis. If the content of milk components is within this range, components for yeast fermentation, such as metabolites derived from milk components, described below, are sufficiently produced. As other components, sugars such as glucose, galactose, lactose, and fructose, or salts such as sodium, potassium, and magnesium are preferred from the viewpoint of the growth and proliferation of lactic acid bacteria. Lactic acid bacteria can be cultured by contacting the bacteria with a culture medium as in the usual manner, and is not particularly limited. However, from the viewpoint of productivity, it is preferable to obtain a culture of lactic acid bacteria by liquid culture in a jar fermenter or culture tank.

The culture temperature for lactic acid bacteria is not particularly limited, but is preferably 30 to 45°C, more preferably 37 to 42°C, from the viewpoint of sufficiently producing components for yeast fermentation, described later, such as metabolites derived from milk components. The culture time is preferably 12 hours or more, more preferably 18 to 24 hours. Examples of the culture method include agitation culture, static culture, shaking culture, neutral culture, etc. From the obtained culture, it is preferable to separate and remove the bacterial cells by a separation means such as centrifugation or filter filtration, and use the resulting supernatant for yeast fermentation, described later. The culture supernatant of lactic acid bacteria obtained in this manner usually contains sugars such as lactose and galactose, amino acids, lactic acid, proteins, etc.

In the present invention, the lactic acid bacteria culture obtained as described above is used as a raw material to obtain a fermented product by yeast. Fermentation can be performed by contacting yeast with a fermentation source as in the usual manner, and is not particularly limited, but in terms of productivity, it is preferable to obtain the fermented product by yeast by liquid culture in a jar fermenter or culture tank. In that case, the yeast can be cultured only with the above-mentioned lactic acid bacteria culture, or the liquid medium in which the lactic acid bacteria is cultured may contain other components other than the above-mentioned lactic acid bacteria culture. The content of the above-mentioned lactic acid bacteria culture in the liquid medium is typically, for example, preferably 1 to 100 v/v% in volume terms, more preferably 10 to 100 v/v%, and even more preferably 50 to 100 v/v%, when a culture supernatant of the lactic acid bacteria is used. If the content of the lactic acid bacteria culture is within this range, sufficient components for achieving the effects of the present invention, such as decomposition products of the lactic acid bacteria culture, are produced. As other ingredients, from the viewpoint of yeast growth and proliferation, sugars such as glucose, galactose, lactose, and fructose, and salts such as sodium, potassium, and magnesium are preferred.

The fermentation temperature by yeast is not particularly limited, but from the viewpoint of yeast growth and proliferation, 15 to 30°C is preferable, and 20 to 30°C is more preferable. The fermentation time is preferably 8 hours or more, and 24 to 32 hours is more preferable. As described above, the fermentation mode is preferably liquid culture, in which case examples include agitation culture, static culture, shaking culture, and neutral culture.

The obtained fermented product may be subjected to heat sterilization or freeze-drying together with the yeast cells as is, or any other treatment may be performed as long as the effects of the present invention are not impaired. However, it is preferable to separate and remove the cells by a separation method such as centrifugation or filtration. The supernatant of the obtained fermented product may be diluted with an appropriate solvent, freeze-dried, concentrated by distillation, or any other treatment may be performed as long as the effects of the present invention are not impaired.

In the present invention, the fermented product obtained as described above is used to act on the skin, thereby activating the cells of the skin to which it is applied. The amount to be applied to the skin is arbitrary, but typically, when yeast is cultured in liquid and the fermented product is obtained as the culture supernatant, the amount is preferably 1 to 1000 μg/ ^cm2 , and more preferably 150 to 300 μg/ ^cm2 , calculated as the dry matter.

For the purpose of application to the skin as described above, the fermented product of the present invention may be used as an external skin preparation as it is, or may be incorporated in the manufacturing process of the external skin preparation. Specifically, it may be suitably used as a cosmetic preparation or an ingredient thereof in the form of, for example, a milky lotion, cream, cleansing, massage, sunscreen, makeup base, cream foundation, etc. Note that the term "cosmetics" as used herein includes drugs, quasi-drugs, and cosmetics as defined in the Law Concerning the Assurance of Quality, Effectiveness, and Safety of Drugs, Medical Devices, etc.

The external preparation for skin may take the form of a pack, mask, gel, etc., in which the component to be applied to the skin is supported on a suitable base material. In other words, it can be suitably used as the component to be applied to the skin in such an external preparation for skin.

In any non-limiting embodiment of the present invention, the above-mentioned topical skin preparation may be labeled with any functionality, such as being used to provide a moisturizing effect, a firming effect, or a gloss effect. Here, the stratum corneum, which constitutes the outermost layer of the epidermis, which is the barrier structure of the skin, is composed of proteins such as keratin, intercellular lipids such as ceramide, and the like. That is, the stratum corneum is composed of epidermal keratinocytes that proliferate in the basal layer inside the epidermis, which then migrate toward the outside of the epidermis while differentiating (keratinizing) into the spinous layer and the granular layer, and finally undergo cell death. It can be said that metabolism is always occurring in order to maintain healthy skin. Therefore, it is believed that by activating the skin cells involved in such metabolism, the functionality of the epidermis, which is the barrier structure of the skin, is maintained or improved, and thus effects such as moisturizing, gloss, and firmness can be obtained.

The following examples are provided for more detailed explanation, but the scope of the present invention is not limited to these examples.

1. Test Sample (1) Phosphate buffered saline (hereinafter referred to as "PBS" or "Cont") was used as the first sample.

(2) As the second sample, a culture supernatant of Streptococcus thermophilus YIT2084 strain (FERM BP-10879, date of deposit: August 18, 2006) was prepared. Specifically, the above strain was cultured in a medium containing 3% by mass of skim milk powder, and the culture supernatant was filtered through a 0.45 μm filter for use. Analysis of this culture supernatant showed that it contained 1.1% by mass of lactose, 0.4% by mass of lactic acid, and had a pH of 4.0. Hereinafter, the second sample will be referred to as "lactic acid bacteria fermentation liquid" or "LF."

(3) As a third sample, a culture supernatant of Wickerhamomyces pijperi YIT8095 strain ("NBRC1290 strain" was obtained from the National Institute of Technology and Evaluation (NITE) and used in this example) was prepared. Specifically, glucose was added to the lactic acid bacteria fermentation liquid previously prepared in (2) above to a final concentration of 0.3% by mass to prepare a lactic acid bacteria fermentation liquid-containing medium, and 1 L of this medium was prepared in a 2 L jar fermenter. Separately, a preculture liquid of the above strain was prepared using a similar lactic acid bacteria fermentation liquid-containing medium containing 1% by mass of glucose, and the preculture liquid was inoculated into the above lactic acid bacteria fermentation liquid-containing medium placed in a 2 L jar fermenter at an amount of 0.3% by mass, and cultured for 24 hours under conditions of agitation speed of 100 rpm, 20°C, and no pH adjustment, and the culture supernatant was filtered through a 0.45 μm filter for use. Hereinafter, the third sample will be referred to as "yeast fermentation liquid 1" or "YW1."

(4) As the fourth sample, a culture supernatant of Wickerhamomyces pijperi YIT12779 strain ("NBRC1887 strain" was obtained from the National Institute of Technology and Evaluation (NITE) and used in this example) was prepared. Specifically, the same culture as in (3) above was performed, and the culture supernatant was filtered through a 0.45 μm filter before use. Hereinafter, the fourth sample is referred to as "yeast fermentation liquid 2" or "YW2".

(5) As the fifth sample, a culture supernatant of Wickerhamomyces pijperi YIT12780 strain ("NBRC102058 strain" was obtained from the National Institute of Technology and Evaluation (NITE) and used in this example) was prepared. Specifically, the same culture as in (3) above was performed, and the culture supernatant was filtered through a 0.45 μm filter before use. Hereinafter, the fifth sample is referred to as "yeast fermentation liquid 3" or "YW3".

(6) As the sixth sample, a culture supernatant of Wickerhamomyces pijperi YIT12781 strain ("NBRC102059 strain" was obtained from the National Institute of Technology and Evaluation (NITE) and used in this example) was prepared. Specifically, the same culture as in (3) above was performed, and the culture supernatant was filtered through a 0.45 μm filter before use. Hereinafter, the sixth sample is referred to as "yeast fermentation liquid 4" or "YW4".

(7) As the seventh sample, a culture supernatant of Kluyveromyces marxianus YIT12612 strain ("NBRC0260 strain" was obtained from the National Institute of Technology and Evaluation (NITE) and used in this example) was prepared. Specifically, the same culture as in (3) above was performed, and the culture supernatant was filtered through a 0.45 μm filter before use. Hereinafter, the seventh sample is referred to as "yeast fermentation liquid 5" or "YK".

(8) As the eighth sample, a culture supernatant of Saccharomyces cerevisiae YIT8264 ^T strain (NBRC10217 strain obtained from the National Institute of Technology and Evaluation (NITE) and used in this example) was prepared. Specifically, the same culture as in (3) above was carried out, and the culture supernatant was filtered through a 0.45 μm filter before use. Hereinafter, the eighth sample is referred to as "yeast fermentation liquid 6" or "YS".

2. Cells Used Normal Human Epidermal Keratinocytes (Kurabo Industries, Ltd.) (hereinafter referred to as "NHEK") were purchased and used.

The media used were "Humedia-KB2" (Kurabo Industries, Ltd.) (hereafter referred to as "KB2 medium"), a basal medium for epidermal keratinocytes, and "HuMedia-KG2" (Kurabo Industries, Ltd.) (hereafter referred to as "KG2 medium"), a medium for proliferation of epidermal keratinocytes. The culture was carried out under conditions of 37°C and 5% _CO2 atmosphere.

[Test Example 1]
In Test Example 1, lactic acid bacteria fermentation liquid (LF) and yeast fermentation liquid 1 (YW1) were compared as test samples.

NHEK were seeded on a 24-well plate at 3 x ¹⁰⁴ cells/ ^cm2 , and 0.5 ml of KG2 medium supplemented with lactic acid bacteria fermentation liquid (LF) or yeast fermentation liquid 1 (YW1) at a concentration of 0.5 v/v%, 1 v/v%, or 2 v/v% was added to each well. After 24 hours of culture, the cells were washed once with PBS, fixed with ethanol, and stained with Janus green to determine the number of cells adhering to the bottom of the culture plate.

1-2. Cell proliferation NHEK were suspended in KG2 medium to 3 x ¹⁰⁴ cells/ ^cm2 , seeded on a 24-well plate, and cultured for 24 hours. After washing once with PBS, 0.5 ml of KB2 medium containing lactic acid bacteria fermentation liquid (LF) or yeast fermentation liquid 1 (YW1) at a concentration of 0.5 v/v%, 1 v/v%, or 2 v/v% was added, and the cells were cultured for another 24 hours. After the culture, the cells were stained with Janus Green in the same manner as in 1-1 above to determine the cell number.

As a result, as shown in Figure 1, when each fermentation liquid was added to the medium, the number of cells that adhered to the bottom of the culture plate (number of adhered cells) increased compared to when no fermentation liquid was added. Also, as shown in Figure 2, when each fermentation liquid was added to the medium, the number of cells that proliferated after adhering to the bottom of the culture plate (number of reached cells) increased compared to when no fermentation liquid was added. Furthermore, in terms of both cell adhesion and proliferation, the effect of yeast fermentation liquid was more pronounced than that of lactic acid bacteria fermentation liquid.

From the above, it has become clear that cultures obtained by cultivating lactic acid bacteria in a medium containing milk components, and fermented products obtained by fermenting the cultures with yeast, have the effect of activating skin cells. It has also become clear that the effect of activating skin cells can be further enhanced by subjecting the medium containing milk components to a fermentation process with yeast in addition to treatment with lactic acid bacteria.

[Test Example 2]
The test samples "yeast fermentation liquid 2 (YW2), yeast fermentation liquid 3 (YW3), yeast fermentation liquid 4 (YW4), yeast fermentation liquid 5 (YK), and yeast fermentation liquid 6 (YS)" were examined for their effects on normal human epidermal keratinocytes.

2-1. Cell proliferation The effect on the proliferation ability of normal human epidermal keratinocytes after adhesion to the bottom surface of a culture plate was examined in the same manner as in "1-2. Cell proliferation" of Test Example 1. The concentration of each fermentation solution added to the liquid medium was 0.5 v/v %.

As shown in Figure 3, other strains of Wickerhamomyces pijperi, as well as Kluyveromyces marxianus and Saccharomyces cerevisiae, were also found to have an effect of activating skin cells, and as in Test Example 1, the degree of activation was more pronounced than that of the lactic acid bacteria fermentation liquid.

Claims (1)

A composition for activating skin cells, comprising a fermentation product of yeast using a culture obtained by culturing lactic acid bacteria in a medium containing a milk component as a raw material,
The yeast is Wickerhamomyces pijperi YIT8095 strain,
The lactic acid bacterium is Streptococcus thermophilus,
the skin cells are epidermal keratinocytes;
A composition for activating skin cells , wherein the activation is an improvement in the migration, adhesion or proliferation ability of the cells .

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