JPWO2014091988A1 - Soymilk fermented extract and hypocotyl fermented extract - Google Patents

Abstract

It aims at providing the fermented extract useful as raw materials, such as cosmetics, a pharmaceutical, and food-drinks. By the method comprising fermenting soymilk or soybean hypocotyl with lactic acid bacteria, mixing the fermented product with an organic solvent, and removing the solid content, the isoflavone aglycone and polyamine are contained, and the keratinocyte activating action, A soymilk fermented extract or hypocotyl fermented extract having at least one of anti-aging effects such as skin fibroblast activation, antioxidant and anti-glycation effects is obtained.

Description

  The present invention relates to a fermented soymilk extract containing a high content of isoflavone aglycone and polyamine, and in particular, has a low sugar content and a cell activation effect, an antioxidant effect, an anti-glycation effect and a moisturizing effect on epidermal keratinocytes and skin fibroblasts. It is related with the soymilk fermented extract which has anti-aging effects, such as. The present invention also relates to a hypocotyl fermented extract obtained by fermenting soybean hypocotyls with lactic acid bacteria, in particular, high content of isoflavone aglycone and polyamine, cell activation action against epidermal keratinocytes and skin fibroblasts, The present invention relates to a hypocotyl fermented extract having an anti-aging action such as an oxidizing action and an anti-glycation action.

  In recent years, in the cosmetics industry, the demand for natural and organic cosmetics has been increasing as the naturalism of customers has increased. Various plant-derived functional materials have been developed to meet the needs of the cosmetic market. And in order to give further new added value to these plant natural materials, utilization of microbial fermentation technique attracts attention. For example, soy milk contains high-quality proteins, isoflavone compounds, and the like, and further, it is expected to exhibit various physiological effects by producing soy peptide, isoflavone aglycone, and the like by fermentation. Regarding the cosmetic effects of materials and extracts obtained by microbial fermentation of soybeans and soy milk, for example, whitening action by suppressing melanin production (for example, see Patent Document 1), moisturizing action by improving hyaluronic acid production ability (for example, patents) Literature 2, Non-Patent Literatures 1 and 2), hair-growth effect (for example, see Patent Literature 3) by activation of an estrogen receptor α agonist (for example, see Patent Literature 4), and the like have been reported.

  The soymilk fermented extract is a quasi-drug raw material standard (hereinafter sometimes referred to as “Kotohara regulations”) 2006 (March 31, 2006, from the medicinal diet No. 0331030, Ministry of Health, Labor and Welfare, Ministry of Health, Labor and Welfare) "Liquid" (component code 532101). According to Sotohara, the soymilk fermented solution is “an extract obtained by adding ethanol to a culture solution obtained by fermenting soymilk obtained from soybean seeds with Lactobacillus delbrueckii (L. delbrueckii) and filtering. Includes 0.01 to 0.04% nitrogen when quantified. "If it satisfies this, it can be said that safety is confirmed from the viewpoint of actual use as long as this is satisfied. In addition, L. delbrueckii is a lactic acid bacterium that has been used for a long time in the production of yogurt, and is considered to be highly safe from experience.

  Isoflavone compounds contained in soybean are known to have physiological activities such as estrogenic activity, anti-estrogenic activity, and antioxidant activity, and their usefulness has attracted attention. When soybean is powdered as it is and the isoflavone compound is measured, the presence of 12 types of isoflavone compounds is confirmed. These are based on three types of daidzein, genistein, and glycitein (these are called isoflavone aglycones) (Chemical Formula 1).

  Most of the isoflavone compounds contained in soybeans are isoflavone glycosides (daidine, genistin, glycitin) having these as basic skeletons, and compounds in which these isoflavone glycosides are further modified by acetylation or malonylation It exists as (Chemical formula 2).

  Among these isoflavone compounds, isoflavone aglycone is generally considered to have a high pharmacological action because it is closer to the structure of estrogen than isoflavone glycoside. Moreover, since the hydrophobicity is higher than that of the isoflavone glycoside, it can be expected that the permeability to the skin is high. For example, with respect to genistein, it has been shown that application may have an effect of preventing skin aging (see Non-Patent Document 3, for example).

  However, the isoflavone compound itself has a small amount existing in nature, and particularly the amount of isoflavone aglycone is extremely small.

  It is also known that soybeans contain polyamines. Polyamine is a generic term for aliphatic hydrocarbons having two or more primary amino groups, and is a natural product that exists universally in the living body, and more than 20 types of polyamines have been found. Typical polyamines include putrescine, spermidine, spermine and cadaverine.

  The main physiological functions of polyamines are as follows: (1) Stabilization and structural change of nucleic acid by interaction with nucleic acid (2) Promoting action on various nucleic acid synthesis systems (3) Activation of protein synthesis system (4) Cell membrane (5) Elimination of active oxygen (6) Promotion of cell growth (7) Antiallergic action is known (for example, see Patent Document 5 and Non-Patent Documents 4 and 5) ).

  Here, a conventional technique for fermenting soybean raw materials with lactic acid bacteria will be described. It has been reported that fermented soy milk rich in isoflavone aglycone can be obtained by using lactic acid bacteria having a β-glucosidase activity, Bifidobacterium, etc. as a method for degrading isoflavone glycosides into isoflavone aglycones ( For example, see Patent Document 6 and Non-Patent Document 6-11). However, polyamines obtained after these fermentations have not been confirmed. In particular, the lactic acid bacteria L. used in the production of the soymilk fermenting bacteria specified in the outer regulations. Regarding delbrueckii, there are particularly few reports on conversion to isoflavone aglycone in soy milk (see, for example, Non-Patent Document 6-11). For example, in Non-Patent Documents 6 and 7, L. Due to the co-culture of delbrueckii and other lactic acid bacteria, L. It is difficult to say that the conversion is made by delbrueckii alone. In Non-Patent Document 8, L., which is an enteric bacterium of birds. Although delbrueckii analogues have been reported to convert genistin to genistein in a synthetic medium, the strain has not been fully identified.

  In Non-Patent Document 9, L. delbrueckii subsp. Although conversion to isoflavone aglycone by bulgaricus alone has been reported, the isoflavone content of the soymilk used for fermentation (reconstituted liquid with soybean isolate protein) is small (total amount 42 mg / L, 103 μM), and the resulting isoflavone aglycone is about 24 mg. / L (about 90 μM). About the nonpatent literature 10 and 11, the fermented liquor of high isoflavone aglycone content is obtained by fermenting soymilk, without adding saccharides. Non-Patent Document 12 reports that when glucose is added to soy milk and fermented with lactic acid bacteria, the isoflavone glycoside is converted to the aglycon when a specific strain is used. However, the amount of polyamine has not been confirmed for any of these fermentations.

  In addition, as a conventional technology for fermenting soybean raw materials with microorganisms, it is also reported that by inoculating koji mold with soybeans, isoflavone glycosides are decomposed into isoflavone aglycones by the enzyme activity of koji molds, and foods with high isoflavone aglycone content can be obtained. (For example, see Patent Document 7).

  In addition, soybean hypocotyls, which are the parts that become buds and roots when germinating soybeans, are rich in micronutrients such as proteins and oligosaccharides, as well as isoflavones and saponins. It has been reported that a fermented product containing equol is obtained by fermenting with a specific Lactococcus d'Albier having (Patent Document 8).

About the manufacturing method of a polyamine, it is known that it will obtain by adding an acid from soybean seeds, soybean germ, soybean milk, etc. (for example, refer to patent documents 5, 9, and 10). Moreover, the report regarding the component which accelerates | stimulates the production | generation of the polyamine by intestinal bacteria is made (for example, refer patent document 11).
Furthermore, it is reported that the amount of polyamine is increased by fermenting a mixture of soy milk and okara with lactic acid bacteria as compared to the mixture before fermentation (Non-patent Document 13). However, by fermenting only soy milk with lactic acid bacteria No increase in the amount of extracted polyamine has been reported. In addition, it has not been reported to produce a hypocotyl fermented extract containing high polyamine by fermenting soybean hypocotyl with lactic acid bacteria.

  Next, the cell activation effect of epidermal keratinocytes will be described. Skin is a tissue composed of epidermis and dermis, and functions as a barrier that protects the body from physical or chemical stress from the outside. The epidermis existing outside the skin consists of a basal layer, a spiny layer, a granule layer, and a stratum corneum, and is mainly composed of keratinocytes. The keratinocytes that have divided in the basement membrane move to the upper layer through differentiation and maturation, reach the outer layer of the horny layer, and then drop off and repeat turnover (metabolism) to form the epidermis.

  The epidermis turnover is involved in the maintenance of skin barrier ability, moisture content, melanin excretion and the like. For this reason, epidermal keratinocyte activators are used for improving skin roughness, moisturizing effect, skin dullness improving effect, whitening effect, and skin texture improvement. As for the soymilk fermented extract, the hair-growth effect by estrogen receptor α agonist activation has been reported (for example, see Patent Document 3), but remarkable cell activation against epidermal keratinocytes by isoflavone aglycone and polyamine-rich fermented soymilk fermented extract. No effect has been reported so far.

  Next, the cell activation effect of skin fibroblasts will be described. Skin fibroblasts are cells that exist in the dermis and produce collagen, elastin, hyaluronic acid, and the like, and are known to contribute to forming a dermal structure by using collagen as a fiber bundle. When these fibroblasts are damaged or decreased by aging or UV exposure, regular fibers cannot be produced, and skin aging such as wrinkles and sagging occurs. Therefore, an agent having an activating effect on skin fibroblasts is useful as an active ingredient of a skin external preparation, particularly a skin external preparation for preventing aging. However, no significant cell activation effect on skin fibroblasts by isoflavone aglycone and soymilk fermented extract containing a high amount of polyamine has been reported so far.

  Next, the antioxidant action will be described. The skin is an organ located in the outermost layer of the living body, and is an organ that is greatly affected by the external environment. The most important of these is the presence of ultraviolet rays. In the presence of ultraviolet rays, reactive oxygen species are generated and converted into various radical species in the living body, which has the positive effect of sterilization, elimination of invading substances into the body, etc., but due to its high reactivity, the epidermis Attacks hyaluronic acid, collagen, elastin, and fibroblasts, which are constituents of cells and tissues, and the dermis, and negative effects are caused by their deterioration, reduction, or degeneration. Specific examples include drying due to a decrease in moisturizing power, wrinkles due to a decrease in elasticity and flexibility, formation of sagging, rough skin due to an inflammatory reaction, dullness due to unevenness and decreased blood flow, and the like. Therefore, an agent having an action of eliminating active oxygen species is useful as an active ingredient of a skin external preparation, particularly a skin external preparation for preventing aging. However, the antioxidant effect of isoflavone aglycone and polyamine-rich soymilk fermented extract has not been reported so far.

  Next, the anti-glycation action will be described. Carbohydrates are very important as energy sources in living organisms including humans. However, carbohydrates are known to cause glycation reactions with proteins. In the saccharification reaction, the first stage is a non-enzymatic reaction between a carbonyl group of a carbohydrate and an amino group such as a protein, and finally a final glycation product (hereinafter referred to as “AGEs”) from a Schiff base through an Amadori compound. Is a series of reactions to form. Due to the saccharification reaction, the protein is modified non-enzymatically by sugar, and as a result, denaturation of the protein, cross-linking between proteins, and the like occur, resulting in a decrease in protein function. The glycation reaction not only causes direct damage by modifying and changing the structure of extracellular matrix constituent proteins such as collagen, but also affects cellular responses by being recognized by receptors with glycated proteins as ligands. Bring.

  Extracellular matrix components such as collagen account for the majority of the dry weight in tissues such as bone and skin. Therefore, for example, when collagen is glycated and abnormally crosslinked, osteoporosis, osteoarthritis, etc. occur in bone and cartilage tissue, and elasticity in skin, dullness due to yellowing, etc. occur. . Furthermore, since abnormally cross-linked collagen and the like are less susceptible to degradation by collagenase or the like, expression of collagenase or the like is induced, and problems such as degradation to normal collagen occur.

  For this reason, if the saccharification reaction is suppressed in some form, that is, the formation of AGEs can be suppressed, or the degradation of AGEs can be promoted, it is effective in preventing or improving skin elasticity and dullness. Expected to be.

  Conventionally, N-phenacyl thiazolium bromide is known as a compound having an anti-glycation action (see, for example, Non-Patent Document 14). However, this compound has a safety problem and is not suitable as a pharmaceutical or a topical skin preparation. Moreover, as an ingredient derived from a natural product having an anti-glycation action, for example, an extract from olive is known (see, for example, Patent Document 12). However, the provision of a substance having an anti-glycation action is not sufficient, and the development and provision of a further new anti-glycation action substance is strongly demanded. Furthermore, although the soybean hot water extract and isoflavone are also reported to have an anti-glycation effect (see, for example, Patent Document 13), the production of AGEs cannot be completely suppressed using these, and the effect Is not enough. On the other hand, no anti-glycation action by isoflavone aglycone and polyamine-rich soymilk fermented extract has been reported so far.

  Next, the moisturizing action will be described. The amount of moisture in the stratum corneum is closely related to the condition of the skin, and in order to maintain a healthy bare skin, it is necessary for the stratum corneum to retain appropriate moisture. In addition, the stratum corneum has a barrier function that prevents foreign matter from entering the skin and transpiration of moisture necessary for the living body. When the stratum corneum barrier function is lowered, the stratum corneum water content is lowered with an increase in the transdermal water transpiration amount, and rough skin and various aging symptoms are caused. Therefore, an agent having an action of increasing the amount of moisture in the stratum corneum or an agent having an action of enhancing the function of the stratum corneum is useful as an active ingredient of an external preparation for skin, particularly an external preparation for preventing aging. However, the moisturizing action by isoflavone aglycone and polyamine-rich fermented soymilk fermented extract has not been reported so far.

Japanese Patent No. 2804312 Japanese Patent No. 3184114 JP 2012-176931 A JP 2006-070146 A JP 2009-234939 A JP 2001-340059 A Japanese Patent No. 3014145 Japanese Patent No. 5030790 JP 2012-006954 A JP 2012-46544 A JP 2012-102054 A JP 2001-122758 A JP 2012-2119014 A

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There are three problems with fermented soymilk extracts currently on the market.
The first problem is that the content of the isoflavone compound, which is a component particularly expected as a physiologically active substance contained in soybean, is extremely small. In fact, commercially available fermented soymilk extract has a low isoflavone compound content (0 to 120 μM) and a lower isoflavone aglycone content (0 to 60 μM, conversion rate to isoflavone aglycone 0 to 50%). From such a background, a fermented soymilk extract having a high content of isoflavone aglycone is strongly demanded.

  The second problem is that the content of polyamine, which is a component expected as another physiologically active substance, is low. The total amount of polyamine in the soymilk fermented extract actually marketed is 10 to 15 μM, and therefore a soymilk fermented extract having a high polyamine content is strongly demanded. The reason for the very low polyamine content of commercially available soymilk fermented extract is that the solubility of polyamine is low in ethanol used in production. Therefore, in order to produce a fermented soymilk extract having a high polyamine content, it is necessary to improve the concentration of the polyamine in the fermentation broth and optimize the concentration of the organic solvent used for extraction.

  A third problem is that carbohydrates such as glucose remain. It has been reported that oligosaccharides contained in soybeans are sucrose (sucrose, 5%), stachyose (4%), raffinose (1.1%), and verbus course (trace) (for example, Fumio Yamauchi et al. Ed., “Soy Science”, Asakura Shoten, 1993, p. 49), in lactic acid bacteria fermentation using soybean raw materials such as soy milk, depending on the type of lactic acid bacteria, there is a shortage of carbohydrates. When sugars such as glucose are added to compensate for this, it is considered that undigested sugars remain in the fermentation broth. However, these sugars cause stickiness when blended as a cosmetic raw material, and in the case of reducing sugars, they may become a raw material for the Maillard reaction and promote skin aging (for example, Japanese Patent Application Laid-Open No. 2011-2011). -102270). Therefore, a fermented soymilk extract in which saccharides such as glucose do not remain has been demanded by means such as utilizing lactic acid bacteria that grow well even without addition of glucose.

  This invention is made | formed in view of the said situation, and makes it a subject to provide a fermented extract useful for the use to cosmetics, a pharmaceutical, food / beverage products etc. which solve one or more of the said subjects.

As a result of intensive studies to solve the above problems, the present inventors performed lactic acid bacteria fermentation of soy milk without supplementing carbohydrates, and by extracting this, isoflavone aglycone and polyamine are highly contained, A fermented soymilk extract with a low glucose content is obtained, which further has anti-aging effects such as cell activation, antioxidant, anti-glycation and moisturizing effects on epidermal keratinocytes and skin fibroblasts. I found it. In addition, fermenting soybean hypocotyls with lactic acid bacteria and extracting them yields a hypocotyl fermented extract containing a high content of isoflavone aglycone and polyamine, which further promotes cell activation against epidermal keratinocytes and skin fibroblasts. It has been found that it has an anti-aging action such as an action, an antioxidant action and an anti-glycation action, and has completed the present invention.
That is, the present invention relates to the following.

[1]
The following steps (A), (B) and (C):
(A) a step of fermenting soy milk with lactic acid bacteria;
(B) a step of mixing the fermented product obtained in step (A) and an organic solvent; and (C) a step of removing the solid content produced in step (B);
A soymilk fermented extract obtained by a method comprising
The following (i), (ii) and (iii):
(I) The content of the isoflavone compound is 150 μM or more;
(Ii) conversion rate from isoflavone glycoside to isoflavone aglycone in step (A) is 50% or more; and (iii) polyamine content is 20 μM or more;
Satisfaction soymilk extract.
[2]
(Iv) The fermented soymilk extract according to [1], wherein the sum of the contents of glucose and sucrose is 3.0 mg / mL or less.
[3]
Next (v) and (vi):
(V) Sum of glucose and sucrose content (mg / mL): isoflavone compound content (μM) not more than 0.1; and (vi) Sum of glucose and sucrose content (mg / mL): polyamine Content (μM) of 0.3 or less;
The fermented soymilk extract according to [1] or [2], which satisfies at least one of the following.
[4]
The soymilk fermented extract according to any one of [1] to [3], wherein the lactic acid bacterium in the step (A) is Lactobacillus delbrueckii.
[5]
Prior to step (A), the soymilk fermented extract according to any one of [1] to [4], which is obtained by a method comprising a step of (P) performing pre-culture with soymilk.
[6]
The following steps (A ′), (B ′) and (C ′):
(A ′) a step of fermenting soybean hypocotyls with lactic acid bacteria;
(B ′) a step of mixing the fermented product obtained in step (A ′) with an organic solvent; and (C ′) a step of removing solids produced in step (B ′);
Hypocotyl fermented extract obtained by a method comprising:
[7]
The hypocotyl fermented extract according to [6], which contains 100 μM or more of glycitein.
[8]
The anti-aging agent containing the fermented extract in any one of [1]-[7].
[9]
The anti-aging agent according to [8], which has one or more of a skin keratinocyte activation effect, a skin fibroblast activation effect, an antioxidant effect, an anti-glycation effect and a moisturizing effect.
[10]
Cosmetics, a skin external preparation, a pharmaceutical, or food-drinks containing the fermented extract in any one of Claims [1]-[7].
[11]
A method for producing a polyamine, which comprises fermenting soy milk with lactic acid bacteria and then extracting the polyamine.
[12]
A method for producing a polyamine, comprising fermenting a soybean hypocotyl with lactic acid bacteria and then extracting the polyamine.
[13]
A method for producing isoflavone aglycone or polyamine, comprising a step of fermenting a soybean hypocotyl with lactic acid bacteria Lactobacillus delbrueckii.
[14]
Soy milk delbrueckii subsp. lactis JCM 1105, L. delbrueckii subsp. lactis JCM 1148, L.L. delbrueckii subsp. bulgaricus JCM 1001, L. delbrueckii subsp. lactis JCM 1010 and L. delbrueckii subsp. A fermented soymilk product obtained by fermentation with a lactic acid bacterium selected from delbrueckii JCM 1012.
[15]
A hypocotyl fermented product obtained by fermenting soybean hypocotyl with lactic acid bacteria Lactobacillus delbrueckii.

  According to the present invention, it is possible to provide a soymilk fermented extract containing a high content of isoflavone aglycone and polyamine, and in particular, it has a low sugar content and has a cell activation effect and an antioxidant effect on epidermal keratinocytes and skin fibroblasts. The fermented soymilk extract exhibiting anti-aging effects such as anti-glycation action and moisturizing action can be provided. In addition, according to the present invention, a hypocotyl fermented extract obtained by fermenting soybean hypocotyls with lactic acid bacteria can be provided, in particular, high content of isoflavone aglycone and polyamine, and epidermal keratinocytes and skin fibers. It is possible to provide a hypocotyl fermented extract having an anti-aging action such as a cell activation action, an antioxidant action and an anti-glycation action on blast cells. The fermented soymilk extract and hypocotyl fermented extract of the present invention are useful for cosmetics, pharmaceuticals, and foods and drinks, and particularly useful for cosmetics.

It is a figure which shows the cell activation effect | action in the epidermal keratinocyte of a fermented extract. It is a figure which shows the cell activation effect | action in the skin fibroblast of a fermented extract. It is a figure which shows the antioxidant action (DPPH radical scavenging activity) of a fermented extract. It is a figure which shows the antioxidant action (DPPH radical scavenging activity) of an isoflavone sample. It is a figure which shows the anti-glycation effect of a fermented extract. It is a figure which shows the anti- glycation effect | action of an isoflavone sample. It is a figure which shows the antioxidant action (cytoprotective action) of fermented soymilk extract. It is a figure which shows the moisturizing effect (horny layer water content increasing effect) of the soymilk fermented extract. It is a figure which shows the moisturizing effect (horny layer barrier function enhancement effect) of fermented soymilk extract.

  Hereinafter, the present invention will be described in detail. The following description may be made based on representative embodiments of the present invention, but the present invention is not limited to such embodiments.

In the present specification, the “fermented soymilk extract” means the following steps (A), (B) and (C):
(A) a step of fermenting soy milk with lactic acid bacteria;
(B) refers to an extract obtained by a method comprising a step of mixing the fermented product obtained in step (A) with an organic solvent; and (C) a step of removing the solid content produced in step (B). The liquid obtained by removing the solid content in the step (C), a concentrated liquid obtained by concentrating the liquid, or a solid obtained by drying the liquid.

  In the present specification, “soy milk” refers to various known soy milks, and includes, for example, unprepared soy milk, prepared soy milk, soy milk beverages, and the like. It can be prepared from one or more parts of soybean seed endosperm, hypocotyl (also referred to as germ) and skin (hereinafter referred to as soybean raw material), and each is extracted from one or more parts and mixed appropriately. You can also A suspension obtained by appropriately pulverizing and immersing a soybean raw material or a slurry obtained by immersing a soybean raw material and then wet pulverizing it can be used as a fermentation raw material as the soy milk of the present invention. Furthermore, the soy milk of the present invention also includes so-called soy bean prepared by dissolving, suspending, or homogenizing powdered soy beans or whole-grain soybeans in water, and soy milk made from these soy milk. In addition, soy milk of the present invention includes so-called protein beverages that have soy protein as a main component and have a soy milk-like flavor.

  Soybean varieties, brands, and colors are not particularly limited, and for example, domestic soybeans, US soybeans such as IOM, genetically modified soybeans, or genetically non-recombinant soybeans can be used. Green soybeans, which are immature seeds of soybeans, can also be used. When obtaining a soymilk fermented extract containing a high amount of isoflavone aglycone, soymilk having a total content of isoflavone compounds of isoflavone aglycone and isoflavone glycoside (including acetyl glycoside and malonyl glycoside) of 150 μM or more is preferably used. .

  In this specification, lactic acid bacteria will not be specifically limited if it is a microbe which produces lactic acid from saccharides by fermentation. In particular, Lactobacillus bacteria are preferably used, and are described in the outer standard of the soymilk fermentation broth. delbrueckii is particularly preferably used. L. As for delbrueckii, there is no particular limitation on the subspecies. delbrueckii subsp. lactis, L.L. delbrueckii subsp. bulgaricus, L .; delbrueckii subsp. delbrueckii and the like.

  From the viewpoint of obtaining a soymilk fermented extract having a low glucose and sucrose content and a soymilk fermented extract having a high polyamine content, L. delbrueckii subsp. lactis JCM 1105, L. delbrueckii subsp. lactis JCM 1148, L.L. delbrueckii subsp. bulgaricus JCM 1001, L. delbrueckii subsp. lactis JCM 1010 and L. delbrueckii subsp. Bacteria selected from delbrueckii JCM 1012 are preferably used. From the viewpoint of obtaining a fermented soymilk extract having a high isoflavone aglycone content, a lactic acid bacterium having a high conversion rate from an isoflavone glycoside to isoflavone aglycone described below is preferably used. delbrueckii subsp. Lactis JCM 1105 or 1148 strain is particularly preferably used.

  The fermentation conditions for the lactic acid bacteria in the step (A) are not particularly limited as long as the lactic acid bacteria grow and the soymilk is fermented, and can be appropriately selected according to the type of the lactic acid bacteria to be used. Soymilk fermentation can be confirmed by an increase in the amount of lactic acid in the fermented product.

  From the viewpoint of obtaining a soymilk fermented extract having a high isoflavone aglycone content, fermentation conditions in which the conversion rate of isoflavone glycosides in soymilk to isoflavone aglycone is 50% or more, particularly 55% or more are preferred. Here, the conversion rate of isoflavone aglycone is calculated by calculating the molar concentration of various isoflavone compounds before and after fermentation, [(isoflavone glycoside before fermentation) − (isoflavone glycoside after fermentation)] / (before fermentation). It can be calculated as (isoflavone glycoside) × 100 (%).

  The soy milk used for fermentation can be added with saccharides, nitrogen source, etc., if necessary, pH can be adjusted, and can be sterilized by heating or the like. In one aspect, from the viewpoint of obtaining a fermented soymilk extract with a low content of carbohydrates (particularly, reducing sugars used as a raw material for the Maillard reaction), it is preferable to use soymilk without addition of sugars, particularly reducing sugars, for fermentation. Such saccharides include: all monosaccharides including, for example, glucose, fructose and glyceraldehyde, reducing sugars such as maltose-type disaccharides or oligosaccharides including lactose, arabinose and maltose; and, for example, by hydrolysis, such as sucrose Non-reducing sugars that are reducing sugars are exemplified. In one embodiment, it is particularly preferable to use soy milk to which no glucose is added for fermentation.

  The lactic acid bacteria to be inoculated may be pre-cultured in any medium, and can be cultured in, for example, plant-derived raw material medium (soy milk, plant-derived peptone, etc.), yeast extract, various synthetic media (MRS medium, etc.), etc. . In one embodiment, pre-culture can be performed using soy milk from the viewpoint of simplifying the production process and avoiding the use of a medium containing animal-derived materials. The culture conditions for pre-culture are not particularly limited as long as lactic acid bacteria are increased.

  Fermentation temperature can be suitably selected according to the kind of lactic acid bacteria, and although it does not specifically limit, it can carry out at 10-50 degreeC, Preferably it can carry out at 20-40 degreeC. About pH during culture | cultivation, it may leave as it is or may be controlled with an acid or an alkali. Various carbon sources and nitrogen sources can also be fed. Any conditions such as aerobic conditions, anaerobic conditions, stirring culture, and stationary culture can be selected depending on the type of lactic acid bacteria to be used. L. lactic acid bacteria When delbrueckii is used, the culture is preferably performed under anaerobic conditions.

  Fermentation time can be suitably selected according to the kind of lactic acid bacteria, and although it does not specifically limit, fermentation can be performed for 6 to 72 hours, 12 to 60 hours simply. For example, since lactic acid can serve as an indicator of the progress of lactic acid bacteria fermentation, the culture can be stopped depending on the concentration of lactic acid. For example, the culture is stopped when the lactic acid concentration reaches 0.1 to 7 mg / mL. can do.

  In addition, from the viewpoint of obtaining a fermented soymilk extract with a low sugar content, in the fermentation process, it is confirmed that the sugar has been reduced by analyzing the sugar in the culture solution, and cultured in a state where the sugar is consumed. Is preferred, because a soymilk fermented extract with less sugar content is obtained. Examples of the saccharide include glucose, fructose, sucrose, and maltose. The method for analyzing these carbohydrates is not particularly limited, but is preferably a phosphate-phenylhydrazine method (a method in which a saccharide is separated by a column and then reacted with a phosphate-phenylhydrazine reagent to detect it after fluorescence derivatization. ) Or a method using a biosensor.

  You may adjust the pH of fermented material as needed after completion | finish of fermentation. For example, the pH lowered due to the production of lactic acid may be appropriately adjusted to neutral with alkali. After completion of fermentation, the formed card may be destroyed by stirring.

  The obtained fermented product may be mixed with an organic solvent in the step (B) as it is, but from the viewpoint of efficiently removing the high molecular protein in the step (B), heat sterilization may be performed after completion of the fermentation. desirable. The heating condition is not particularly limited as long as the solid content is sufficiently formed. For example, the heating is performed at 60 to 120 ° C. for 1 to 120 minutes, and conveniently at 80 to 110 ° C. for 20 to 60 minutes. Can be implemented.

  In the next step (B), the obtained fermented product is mixed with an organic solvent. Thereby, desired components, such as an isoflavone compound, can be extracted in the organic solvent from the obtained fermented material. As the organic solvent, a polar solvent miscible with water is preferably used. In consideration of application to cosmetics, those having high safety are preferable, and ethanol, butylene glycol, and the like are particularly preferably used. Especially, since ethanol is described in the outer standard of a soymilk fermentation liquid, it is used especially preferably. Moreover, ethanol is also preferably used when blending fermented soymilk extract with food and drink and oral medicine. Regarding the addition amount and concentration of the organic solvent, particularly if the solid content is generated and desired components such as isoflavone compounds can be extracted, and as a result, a fermented soymilk extract containing the desired components can be obtained. Although it is not limited, the quantity and density | concentration which solid content produces | generates and isolate | separates from a clear extract are preferable, for example, the organic solvent of the quantity of 5-90% (v / v) can be added with respect to a culture solution. . For example, in the case of ethanol, 15% (v / v) or more, preferably 50% (v / v) or more of ethanol can be added to the culture solution. After the addition, operations such as heating, cooling, and standing can also be performed.

  Subsequently, the process (C) which removes the solid content produced | generated at a process (B) is performed. Step (C) can be performed using any method that can remove solids, and for example, a method of separating the supernatant by standing or centrifuging, a method of filtering, or the like can be used.

  In addition to the above steps, additional steps may be performed depending on the purpose. For example, from the viewpoint of obtaining a fermented extract having a high content of isoflavone compounds (particularly isoflavone aglycone), inclusion of desired components such as isoflavone compounds can also be carried out by adding cyclodextrin or the like. The addition of cyclodextrin can be performed at any stage as long as it is performed prior to the removal of the solid content in the step (C). For example, before the step (A), during the fermentation in the step (A), Any one or more of after step (A) and before step (B), during mixing with the organic solvent in step (B), and after step (B) and before step (C) It can be done at this stage. As the cyclodextrin, β-cyclodextrin or γ-cyclodextrin can be used, and β-cyclodextrin can be preferably used for the purpose of inclusion of an isoflavone compound.

In the present specification, the “hypocotyl ferment extract” means the following steps (A ′), (B ′) and (C ′):
(A ′) a step of fermenting soybean hypocotyls with lactic acid bacteria;
(B ′) a step of mixing the fermented product obtained in step (A ′) with an organic solvent; and (C ′) a step of removing solids produced in step (B ′);
For example, a liquid obtained by removing the solid content in the step (C ′), a concentrated liquid obtained by concentrating this, or a solid obtained by drying the liquid may be used.

  In this specification, the soy hypocotyl refers to a soy hypocotyl (also referred to as germ), which is not only a raw soy hypocotyl but also subjected to any pretreatment such as degreasing, roasting, dipping, and heat treatment. Including.

For fermentation in the step (A ′), the soybean hypocotyl itself can be used, or it may be pulverized. From the viewpoint of further improving the extraction efficiency, a suspension of soybean hypocotyl or its pulverized product suspended in water or a hypocotyl extract obtained by extracting soybean hypocotyl or its pulverized product with water and removing the solid content It can also be used.
From the viewpoint of easily obtaining a hypocotyl fermented extract having a high content of desired components (particularly isoflavone aglycone), an immersion product obtained by immersing soybean hypocotyl or a pulverized product thereof in water, and this immersion product was further heated. You may use a processed material etc. for fermentation of a process (A '). Without being bound by theory, it is believed that these treatments promote the conversion of isoflavone glycosides into isoflavone aglycones and the conversion of isoflavone malonyl glycosides into isoflavone glycosides in soybean hypocotyls. It is done.

  The variety, brand, and color of the soybean from which the soybean hypocotyl is derived are not particularly limited, and any soybean can be used in the same manner as mentioned for the soy milk.

  Steps (A ′), (B ′) and (C ′) are the same as steps (A), (B) and (C), respectively, except that soybean hypocotyl is used instead of soy milk. Can do.

  The soymilk fermented extract or hypocotyl fermented extract thus obtained (hereinafter also simply referred to as “fermented extract”) can be used as it is as a raw material for cosmetics, external preparations for skin, pharmaceuticals and foods and drinks. Further, it can be appropriately diluted with water, an organic solvent or the like, or a preservative such as paraben or phenoxyethanol can be added. Further, for example, the organic solvent can be distilled off by heating or decompression, the components can be concentrated, and pulverization can be performed by freeze drying or spray drying. The powdered product can be dissolved, dispersed or suspended in water or an organic solvent.

  The fermented extract of the present invention preferably has a high content of isoflavone compounds from the viewpoint of exhibiting various actions. The isoflavone analysis can be performed using a known method capable of analyzing various isoflavone compounds, and preferably an HPLC method is used.

  In the present specification, the isoflavone compound refers to daidzein, daidzin, acetyldaidine, malonyldaidine, genistein, genistin, acetylgenistin, malonylgenistin, glycitein, glycitin, acetylglycitin and malonylglycitin. Among these, it is more preferable that the contents of daidzein, genistein and glycitein, which are isoflavone aglycones, are particularly large from the viewpoint of exerting various actions. The concentration of the isoflavone compound contained in the fermented extract of the present invention is preferably as high as possible from the viewpoint of exhibiting various actions, and preferably contains 150 μM or more of the isoflavone compound. For example, in the case of fermented soymilk extract, isoflavone aglycone is preferably 75 μM or more, and more preferably 150 μM or more.

  In the case of a hypocotyl fermented extract, the isoflavone aglycone content is preferably 550 μM or more, more preferably 700 μM or more, and further preferably 1000 μM or more. From the viewpoint of exhibiting a high anti-glycation effect, the glycitein content is particularly preferably 100 μM or more, and more preferably 200 μM or more.

  The fermented extract of the present invention preferably has a high polyamine content from the viewpoint of exhibiting various effects. The polyamine analysis can be performed using a known method capable of analyzing various polyamines such as an HPLC method and an enzymatic method, and the HPLC method is preferably used. Specifically, Novella-Rodriguez S.M. Based on the above method (J. Agric. Food Chem., 48, 5117-5123 (2000)), polyamines can be analyzed using HPLC.

  In the present specification, the polyamine is not particularly limited as long as it is an aliphatic hydrocarbon having two or more primary amino groups, and examples thereof include putrescine, spermidine, spermine, and cadaverine. The concentration of the polyamine contained in the fermented extract of the present invention is preferably as high as possible from the viewpoint of exhibiting various effects, and the total of putrescine, spermidine and cadaverine, which are the main polyamines in the fermented extract, is preferably 20 μM or more, and 30 μM. More preferably, it is more preferably 40 μM or more. In particular, in the case of hypocotyl fermented extract, the total of spermidine and cadaverine is preferably 310 μM or more, and more preferably 400 μM or more.

  In addition, the fermented extract of the present invention may have the above content by adding each of these components itself (raw powder etc.) to the above isoflavone compound, isoflavone aglycone and polyamine. Since it is expensive, it is desirable that it is a fermented extract satisfying the above content without adding such from the viewpoint of economy. In addition, as shown also in the below-mentioned Example, the present inventors confirmed that the fermented extract of this invention shows a high effect (an antioxidant action, an anti-glycation action, etc.) compared with an isoflavone compound alone. ing.

  About the carbohydrate contained in the obtained fermented extract, it is a raw material for the Maillard reaction that causes aging, and it causes stickiness when applied to the skin when blended in cosmetics, so less is preferable .

  Examples of the saccharide include glucose, fructose, sucrose, and maltose. The method for analyzing these carbohydrates is not particularly limited, but is preferably a phosphate-phenylhydrazine method (a method in which a saccharide is separated by a column and then reacted with a phosphate-phenylhydrazine reagent to detect it after fluorescence derivatization. ) Or a method using a biosensor.

  The fermented extract of the present invention preferably has a saccharide content of 4.0 mg / mL or less, more preferably 3.0 mg or less, and even more preferably 1.5 mg / mL or less. In particular, the sum of the contents of glucose and sucrose is preferably 3.0 mg / mL or less, and more preferably 1.5 mg / mL or less.

  In one embodiment, the fermented extract of the present invention comprises a sum of glucose and sucrose contents (mg / mL): an isoflavone compound content (μM), that is, a sum of glucose and sucrose contents (mg / mL) / The content (μM) of the isoflavone compound is preferably 0.1 or less, more preferably 0.05 or less, further preferably 0.015 or less, and particularly preferably 0.01 or less. preferable.

  In one embodiment, the fermented extract of the present invention contains isoflavone aglycone, and sum of glucose and sucrose content (mg / mL): isoflavone aglycone content (μM), that is, sum of glucose and sucrose content. The content (μM) of (mg / mL) / isoflavone aglycone is preferably 0.2 or less, more preferably 0.1 or less, and particularly preferably 0.01 or less.

  In one embodiment, the fermented extract of the present invention comprises the sum of glucose and sucrose content (mg / mL): the content of polyamine (μM), that is, the sum of glucose and sucrose content (mg / mL) / polyamine. The content (μM) is preferably 0.3 or less, more preferably 0.25 or less, further preferably 0.1 or less, and particularly preferably 0.05 or less.

  Although it does not specifically limit about the other component of the fermented extract of this invention, About liquid nitrogen content, it is preferable that it is 0.01 to 0.04% of an outer original rule description.

  The fermented extract of the present invention may be suspended or transparent, and the color is preferably pale yellow, pale brown or colorless, for example. The fragrance is not particularly limited, but a fragrance having a small soybean odor and a refreshing fragrance of lactic acid bacteria fermentation is preferable.

  The fermented extract of the present invention, as shown in the examples described later, exhibits one or more of cell activation action, antioxidant action, anti-glycation action and moisturizing action on epidermal keratinocytes and dermal fibroblasts, In one embodiment, it preferably exhibits at least one of cell activation and anti-glycation effects on epidermal keratinocytes and dermal fibroblasts.

  The cell activation activity of epidermal keratinocytes is prepared by adding 0.01 to 50% of the fermented extract of the present invention to a commercially available normal human epidermal keratinocytes (Kurabo Co., Ltd.) with respect to the medium. This can be used to determine whether to promote cell growth. When the sample has an activation activity of epidermal keratinocytes, the activity when no sample is added is 100%. For example, the activation activity of epidermal keratinocytes when a sample is added is 110% or more. Sure.

  The cell activation activity of skin fibroblasts was prepared by adding 0.01 to 50% of the fermented extract of the present invention to commercially available normal human skin fibroblasts (manufactured by Kurabo) to prepare a culture solution. This can be used to determine whether to promote cell growth. The case where the sample has a fibroblast activation activity refers to a case where the activation activity of the fibroblast when the sample is added is 110% or more, assuming that the activity when the sample is not added is 100%.

  Antioxidant action (DPPH radical scavenging activity) can be measured by adding the fermented extract of the present invention to DPPH radical, which is pseudo-active oxygen, and determining whether to eliminate DPPH radical. The case where the sample has an antioxidant action means that the DPPH radical elimination rate when the sample is added is 10% or more when the DPPH radical elimination rate without addition of the sample is 0%.

  Antioxidant action (cytoprotective action) is prepared by adding 0.01-50% of the fermented extract of the present invention to a commercially available normal human epidermal keratinocyte (Kurabo Co., Ltd.) with respect to the medium. This can be used to determine whether to reduce cell damage due to exposure to reactive oxygen species. The case where the sample has a cytoprotective action refers to the case where the cell viability when the sample is added is increased by 10% or more with respect to the cell viability when the sample is not added under the condition where the active oxygen is exposed.

  The anti-glycation action can be measured by adding the fermented extract of the present invention to a mixed solution of glucose and human serum albumin and using this to determine whether to suppress the production of AGEs. The case where the sample has an anti-glycation effect refers to the case where the AGEs production inhibitory activity when the sample is added is 10% or more, for example, when the sample-free AGEs production inhibitory activity is 0%.

  The moisturizing effect is determined by measuring the amount of stratum corneum moisture and the amount of transdermal water transpiration after applying the skin lotion containing 0.01 to 50% of the fermented extract of the present invention to humans. It can be measured by confirming the decrease in the amount. In the case of having a sample moisturizing effect, the moisture content of the stratum corneum increases by 10% or more compared to the case where the sample cost-containing lotion is applied by applying the sample-containing lotion, or the transdermal moisture transpiration amount is 10 If it decreases by more than%.

  The present invention also relates to an anti-aging agent comprising the fermented extract of the present invention. By confirming that the skin keratinocyte activation action, skin fibroblast activation action, antioxidant action, anti-glycation action and moisturizing action are one or more of the above, the anti-aging agent has the anti-aging action. Can be confirmed.

  The present invention also relates to a cosmetic and a skin external preparation containing the fermented extract of the present invention. The cosmetic and external preparation for skin of the present invention can be prepared by blending the fermented extract of the present invention with various types of base materials known as normal cosmetic and external preparation for skin according to conventional methods. The blending amount of the fermented extract is preferably 0.01 to 50% by mass with respect to the total amount from the viewpoints of effects and stability.

  It does not specifically limit as a form of cosmetics and a skin external preparation, For example, arbitrary dosage forms, such as a milky lotion, cream, aqueous solution, a pack, can be selected.

  In the cosmetic and skin external preparation of the present invention, in addition to the above-mentioned essential components, ingredients blended in normal cosmetics and skin external preparations, such as oils, powders, purified water, polymer compounds, gelling agents, ultraviolet rays Absorbers, ultraviolet scattering agents, antioxidants, dyes, preservatives, fragrances, and cosmetic ingredients can be appropriately selected and used within a range not impairing the effects of the present invention.

When the fermented extract of the present invention is used as a pharmaceutical product, a pharmaceutical preparation can be prepared by adding a pharmaceutically acceptable excipient.
The pharmaceutical preparation is not particularly limited, but is externally used for tablets, capsules, granules, fine granules, powders, liquids, syrups, chewable, troches, etc., ointments, gels, creams, patches, etc. The dosage form may be an agent, injection, sublingual, inhalant, eye drop, suppository and the like. Moreover, the pharmaceutical of this invention can also be provided as a kit containing the fermented extract of this invention.

  The fermented extract of this invention can be mix | blended with food-drinks from a viewpoint of ingestion ease. Examples of the food and drink include supplements, foods for specified health use, functional nutritional foods, health foods, functional foods, health supplements, and normal foods and drinks. The shape of the food and drink includes liquids such as juices, soft drinks, drinks, teas, solids such as biscuits, tablets, granule powders, powders and capsules, semi-fluid forms such as pastes, jellies, soups, seasonings, dressings, etc. Is mentioned.

  The present invention is also characterized in that the soy milk is fermented with lactic acid bacteria and then the polyamine is extracted, and the method for producing polyamines, and the soybean hypocotyl is fermented with lactic acid bacteria and then the polyamines are extracted. The present invention also relates to a method for producing a polyamine. Thereby, a polyamine can be produced without the addition of an acid. The method can be carried out with reference to the above description relating to the fermented extract of the present invention. For example, the fermentation of soy milk with lactic acid bacteria and the fermentation of soybean hypocotyls with lactic acid bacteria are carried out by performing the above steps (A) and (A ′). Can be done with reference. Extraction of the polyamine from the fermented product can be performed using a technique known to those skilled in the art, for example, referring to the steps (B) and (C) and the steps (B ′) and (C ′). be able to.

  The present invention also relates to a method for producing an isoflavone aglycone or polyamine, comprising a step of fermenting soybean hypocotyls with the lactic acid bacterium Lactobacillus delbrueckii. The method can be carried out with reference to the above description regarding the fermented extract of the present invention.

  The present invention also provides soy milk with L. delbrueckii subsp. lactis JCM 1105, L. delbrueckii subsp. lactis JCM 1148, L.L. delbrueckii subsp. bulgaricus JCM 1001, L. delbrueckii subsp. lactis JCM 1010 and L. delbrueckii subsp. The present invention also relates to a soymilk fermented product obtained by fermentation with a lactic acid bacterium selected from delbrueckii JCM 1012 and a hypocotyl fermented product obtained by fermenting a soy hypocotyl with a lactic acid bacterium Lactobacillus delbrueckii. These fermented products can be obtained by referring to the above description of the fermented extract of the present invention, for example, referring to the above steps (A) and (A ′).

  Hereinafter, the present invention will be described more specifically by way of examples, comparative examples, analysis examples, and test examples (sometimes simply referred to as “examples”). However, the technical scope of the present invention is not limited by these examples.

[Production of fermented soymilk extract]
[Example 1-1]
<Preparation of fermented soymilk extract in which pre-culture was performed in MRS medium>
The soymilk fermented extract was prepared by the method shown below.

  Various types of L.A. delbrueckii (obtained from RIKEN) was inoculated into MRS medium and pre-cultured at 37 ° C. for 24 hours. The culture medium was collected by centrifugation, and then the 10-fold concentrated bacterial solution was inoculated into non-adjusted soy milk (manufactured by Kikkoman Soy Foods) and anaerobically cultured at 37 ° C. for 48 hours. After completion of the culture, the mixture was heated at 100 ° C. for 30 minutes, and then ethanol was added to a content of 50% (v / v) and stirred. The added solution was allowed to stand at 4 ° C. for 24 hours, and then insoluble matters were removed by centrifugation and filtration to obtain a clear fermented soymilk extract.

[Example 1-2]
<Preparation of fermented soymilk extract that has been pre-cultured with unadjusted soymilk>
The soymilk fermented extract was prepared by the method shown below.
L. delbrueckii JCM 1105 was inoculated into unconditioned soymilk and pre-cultured at 37 ° C. for 24 hours. The culture medium was collected by centrifugation, and then the 10-fold concentrated bacterial solution was inoculated into unadjusted soymilk (manufactured by Kikkoman Soy Foods) and cultured at 37 ° C. for 48 hours. After completion of the culture, the mixture was heated at 100 ° C. for 30 minutes, and then ethanol was added to a content of 50% (v / v) and stirred. The added solution was allowed to stand at 4 ° C. for 24 hours, and then insoluble matters were removed by centrifugation and filtration to obtain a clear fermented soymilk extract. L. Also when delbrueckii JCM 1148 was used for the same operation, a clear fermented soymilk extract was obtained.

[Comparative Example 1]
<Preparation of soymilk extract (non-fermented)>
Unadjusted soymilk (Kikkoman Soy Foods Co., Ltd.) was heated at 100 ° C. for 30 minutes, and then ethanol was added to a content of 50% (v / v), followed by centrifugation and filtration to obtain a clear soymilk extract. (Non-fermented) was obtained.
[Example 2]
<Preparation of hypocotyl fermented extract>
The hypocotyl fermented extract was prepared by the method shown below.
Purified water was added to the hypocotyl crushed with a mill at a weight of 16% and immersed at room temperature for 5 hours. Next, after heat treatment at 100 ° C. for 2 hours, centrifugation and heat sterilization treatment were performed, and the obtained liquid was used as a hypocotyl extract. L. delbrueckii subsp. lactis JCM 1105 or L. delbrueckii subsp. lactis JCM 1148 (obtained from RIKEN), inoculated into MRS medium, pre-cultured at 37 ° C. for 24 hours, centrifuged after culturing the culture medium and inoculated into hypocotyl extract at 37 ° C. Cultured for 72 hours. After completion of the culture, heat at 100 ° C. for 30 minutes, add ethanol to 50% (v / v), stir, let stand at 4 ° C. for 24 hours, and then remove insoluble matter by centrifugation and filtration. As a result, a clear hypocotyl fermented extract was obtained.

[Comparative Example 2]
<Preparation of hypocotyl extract (non-fermented)>
The hypocotyl extract prepared in Example 2 is heated at 100 ° C. for 30 minutes, and then ethanol is added to a content of 50% (v / v), followed by centrifugation and filtration to obtain a clear hypocotyl extract. (Non-fermented) was obtained.

[Various sample analysis]
[Analysis Example 1]
(A) Quantitative analysis of isoflavone All samples were filtered with a PTFE 0.45 μm filter, and then subjected to HPLC (Tosoh Corp., LC-8020) under the following conditions to separate and detect isoflavone glycosides and isoflavone aglycones. . As the HPLC column, YMC-Triart C18 (manufactured by YMC, TA12S03-0546WT, 50 × 4.6 mm ID, particle diameter 3 μm) was used. For elution, 5% and 95% of eluent A (acetonitrile: purified water: formic acid = 10: 90: 0.1) and eluent B (acetonitrile: purified water: formic acid = 60: 40: 0.1), respectively. A linear gradient starting with the containing solution and ending with 35% eluent A and 65% eluent B was used. The flow rate was 2.0 mL / min, and the absorbance at 254 nm was measured using a UV detector. The isoflavone content in the sample was calculated by determining the peak area of each isoflavone from the standard solution and the HPLC chart of the sample. As a standard compound, a purified product of isoflavone compound manufactured by Wako Pure Chemical Industries, Ltd. (Daidin, glycitin, genistin, daidzein, glycitein, genistein, 6 "-O-acetyldaidine, 6" -O-acetylglycitin, 6 "-O- Acetylgenistin, 6 "-O-malonyldaidine, 6" -O-malonylglycitin and 6 "-O-malonylgenistin) were used.

The conversion rate from isoflavone glycoside to isoflavone aglycone is calculated by calculating the molar concentration of isoflavone glycoside (including malonyl glycoside) before and after fermentation, and [(isoflavone glycoside before fermentation)-(fermentation Later isoflavone glycoside)] / (isoflavone glycoside before fermentation) × 100 (%).
Table 1 shows the concentrations of various isoflavones in the samples of the examples and comparative examples and the commercially available soymilk fermentation broth.

  About the soybean milk fermented extract prepared in Example 1-1, there existed a difference in the presence or absence of aglyconization according to the kind of lactic acid bacteria to be used. L. delbrueckii subsp. lactis JCM 1105, L. delbrueckii subsp. lactis JCM 1148 and L. delbrueckii subsp. When Bulgaricus JCM 1001 was used, the reduction of isoflavone glycosides, daidzin and genistin, and the formation of isoflavone aglycones, daidzein and genistein, were observed, and a fermented soymilk extract containing high isoflavone aglycone content was obtained. The concentration of these isoflavone aglycones was higher than the soymilk fermentation broth already on the market. Moreover, since the aglyconization of isoflavone was recognized also in the soymilk fermented extract prepared in Example 1-2, not only the MRS medium but also non-adjusted soymilk can be used for the pre-culture of the soymilk fermented extract. Became clear.

  On the other hand, L. delbrueckii subsp. lactis JCM 1010 and L. delbrueckii subsp. In the soymilk fermented extract prepared using delbrueckii JCM 1012, aglyconization of isoflavones was not observed.

  Further, the hypocotyl fermented extract prepared in Example 2 is also described in L.P. delbrueckii subsp. lactis JCM 1105 and L. delbrueckii subsp. When lactis JCM 1148 was used, compared with the hypocotyl extract (non-fermented) prepared in Comparative Example 2, the reduction of isoflavone glycosides daidin, glycitin and genistin and isoflavone aglycone daidzein, glycitein and An increase in genistein was observed. The isoflavone aglycone content of the hypocotyl fermented extract was even higher than the fermented soymilk extract of Example 1-1 prepared using the same lactic acid bacteria for fermentation. In particular, glycitein, which was shown to have a particularly large contribution to the AGEs production inhibitory activity in Test Example 4 described later, contained a large amount of 200 μM or more.

[Analysis Example 2]
(B) Quantitative analysis of polyamines All samples were filtered through a PTFE 0.45 μm filter, and then Novella-Rodriguez S. et al. , J .; Agric. Food Chem. , 48, 5117-5123 (2000) et al., Polyamines were analyzed using HPLC [column: Nova Pack C ₁₈ , 3.9 × 150 mm, particle size 4 μm (manufactured by Waters)].
Table 2 shows the concentrations of various polyamines in the samples of Examples and Comparative Examples and the commercially available soymilk fermented extract.

  The concentration of putrescine, spermidine and cadaverine in the fermented soymilk extract prepared in Example 1-1 was found to be about 2.0 to 2.5 times higher than the soymilk extract (non-fermented) of Comparative Example 1. It was. The concentration of these polyamines was higher than the soymilk fermentation broth already on the market.

  Moreover, the concentration of putrescine, cadaverine and spermidine in the hypocotyl fermented extract prepared in Example 2 is about 1.4 to 3.0 times higher than the hypocotyl extract (non-fermented) of Comparative Example 2. I understood. The concentration of spermine was very small compared to the measurement limit or less than putrescine, cadaverine and spermidine. The polyamine content of the hypocotyl fermented extract was even higher than that of the soymilk fermented extract, and in particular, contained very large amounts of spermidine and cadaverine.

[Analysis Example 3]
(C) Analysis of total nitrogen content, pH, glucose, sucrose and lactic acid content In addition, the table shows the total nitrogen content, pH, glucose, sucrose and lactic acid content of samples of each example and comparative example and commercially available soymilk fermentation broth. It was shown in 3. The methods used for each analysis are as follows:

  In the soymilk fermented extract and the hypocotyl fermented extract, a decrease in pH and lactic acid production were observed compared to the soymilk extract (non-fermented) and hypocotyl extract (non-fermented). Moreover, the total nitrogen amount in the soymilk fermented extract was around 0.040, and the total nitrogen amount in the hypocotyl fermented extract was around 0.110. The total content of glucose and sucrose in the fermented soymilk extract and hypocotyl fermented extract was significantly lower than that of the commercially available soymilk fermented liquid, and all were 1.5 mg / mL or less.

Further, based on the results of the above analysis examples 1 to 3, the results of calculating the sugar mass with respect to the amount of isoflavone or polyamine for the samples of the examples and comparative examples and the commercially available soymilk fermentation liquid are shown in Tables 4 and 5 below. Show. In addition, the content of each component in Tables 4 and 5 was calculated as follows:
(A1) Isoflavone compound: Total amount (μM) of nine isoflavones described in Table 1
(A2) Isoflavone aglycone: total amount (μM) of daidzein, glycitein and genistein listed in Table 1
(B) Polyamine: Total amount (μM) of putrescine, spermidine and cadaverine described in Table 2
(C) Carbohydrate: Total amount (mg / mL) after converting glucose and sucrose contents shown in Table 3 to μM

[Evaluation of effect of prototype]
[Test Example 1] Evaluation of cell activation effect of fermented extract on epidermal keratinocytes Among the fermented extracts prepared in Example 1-1 and Example 2, L. delbrueckii subsp. lactis JCM 1105 and L. delbrueckii subsp. About the fermented extract prepared using lactis JCM 1148 and the extract (non-fermented) prepared in Comparative Examples 1 and 2, the cell activation effect in epidermal keratinocytes was evaluated by the method described below. All samples were adjusted to pH 7.4 ± 0.05 using 1N sodium hydroxide, and then sterilized by filtration with a 0.20 μm filter.

Normal human epidermal keratinocytes [manufactured by Kurabo Industries, product name NHEK (NB)] are seeded in a 96-well microplate so as to be 5 × 10 ³ cells per well, at 37 ° C. in a carbon dioxide concentration of 5 vol%. Cultured for 48 hours. As a seeding medium, HuMedia-KG2 medium (Kurabo) was used. Subsequently, it replaced | exchanged for the sample culture solution prepared to each density | concentration using the HuMedia-KB2 (made by Kurabo Industries) culture medium, and also culture | cultivated for 48 hours. Each sample culture solution was prepared so that the final ethanol concentration was 1%.

  After completion of the culture, the number of viable cells was measured using Cell Counting Kit-8 (manufactured by Dojin Chemical Co., Ltd.). For the evaluation, in addition to the sample culture solution, HuMedia-KG2 was used as a positive control, and 1% ethanol-containing HuMedia-KB2 was used as a blank for each sample.

  The cell activation effect evaluation results in the epidermal keratinocytes are shown in FIG. 1 as relative values [Index (%)] with the cell activation effect in the blank with no sample taken as 100. No significant epidermal cell activation effect was observed in the blank ethanol 1% and the soymilk extract (non-fermented) of Comparative Example 1, whereas in the soymilk fermented extract of Example 1-1, L. delbrueckii subsp. lactis JCM 1105 and L. delbrueckii subsp. In both samples fermented with lactis JCM 1148, 124%, a significant cell activation effect was observed. In addition, the hypocotyl fermented extract of Example 2 is also described in L.P. delbrueckii subsp. 152% of the sample fermented with lactis JCM 1105, L. delbrueckii subsp. In the samples fermented with lactis JCM 1148, a significant cell activating effect was observed at 151% in all samples. In addition, it showed a high effect compared to the hypocotyl extract (non-fermented) of Comparative Example 2, and the effect was exhibited by fermentation. A tendency to increase was observed. As is clear from FIG. 1, it was confirmed that the fermented extract of the present invention has a very strong cell activation effect on normal human epidermal keratinocytes.

[Test Example 2] Evaluation of cell activation effect of fermented extract in skin fibroblasts For the same sample as in Test Example 1, the cell activation action in skin fibroblasts was evaluated by the method shown below. All samples were adjusted to pH 7.4 ± 0.05 using 1N sodium hydroxide, and then sterilized by filtration with a 0.20 μm filter.

Normal human skin fibroblasts [manufactured by Kurabo Industries, product name NHDF (NB)] are seeded in a 96-well microplate so as to be 2 × 10 ³ cells per well, at 37 ° C. in a carbon dioxide concentration of 5 vol%. Cultured for 48 hours. As the seeding medium, 10% FBS-containing DMEM medium (manufactured by Life Technology) was used. Subsequently, it replaced | exchanged for the sample culture solution prepared to each density | concentration using FBS free DMEM culture medium, and also culture | cultivated for 48 hours. Each sample culture solution was prepared so that the final ethanol concentration was 1%.

  After completion of the culture, the number of viable cells was measured using Cell Counting Kit-8 (manufactured by Dojin Chemical Co., Ltd.). For the evaluation, in addition to the sample culture solution, 10% FBS-containing DMEM medium was used as a positive control, and 1% ethanol-containing FBS-free DMEM medium was used as a blank for each sample.

  The cell activation effect evaluation results in skin fibroblasts are shown in FIG. 2 as relative values [Index (%)] with the cell activation effect in the blank with no sample taken as 100. In the ethanol 1% blank and the soymilk extract of Comparative Example 1 (non-fermented), the dermal fibroblast activation effect was not observed, whereas in the soymilk fermented extract of Example 1-1, L. delbrueckii subsp. 113% of the sample fermented with lactis JCM 1105, L. delbrueckii subsp. In the samples fermented with lactis JCM 1148, 117%, a significant cell activation effect was observed, and a tendency for the action to be enhanced by fermentation was observed. In addition, the hypocotyl fermented extract of Example 2 is also described in L.P. delbrueckii subsp. 132% for the sample fermented with lactis JCM 1105; delbrueckii subsp. In the sample fermented with lactis JCM 1148, a strong cell activation effect was observed at 137%, and the effect of the hypocotyl fermented extract was higher than that of the fermented soymilk extract of Example 1-1. As is clear from FIG. 2, it was confirmed that the fermented extract of the present invention has a very strong cell activation effect on normal human skin fibroblasts.

[Test Example 3] Evaluation of Antioxidant Action (DPPH Radical Scavenging Activity) of Fermented Extract For the same sample as in Test Example 1 and commercially available soymilk fermented extract, the antioxidant action (DPPH radical scavenging activity) was evaluated by the following method. did. All samples were adjusted to pH 7.4 ± 0.05 using 1N sodium hydroxide, and then sterilized by filtration with a 0.20 μm filter.

40 μL of sample was added to each 96-well microplate. Thereto, 80 μL each of 0.1 M phosphate buffer (pH 5.5) and 0.15 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) methanol solution were added and mixed well. And left in the dark for 1 hour. After standing, the absorbance at 517 nm derived from DPPH radical was measured. When the absorbance when no sample is added is (A) and the absorbance when a sample is added is (B), the DPPH radical elimination rate is defined by the following equation.
DPPH radical elimination rate = {1- (B) / (A)} × 100
Trolox was used as a positive control having an antioxidant effect, and 50% ethanol was used as a blank for each sample.

  The evaluation results of the antioxidant action (DPPH radical scavenging activity) are shown in FIG. The DPPH radical scavenging activity in the soymilk extract of Comparative Example 1 (non-fermented) was 54%, whereas in the soymilk fermented extract of Example 1-1, L. delbrueckii subsp. 76% for the sample fermented with lactis JCM 1105; delbrueckii subsp. A DPPH radical scavenging activity as high as 69% was observed in the sample fermented with lactis JCM 1148, and the tendency of the action to be enhanced by fermentation was observed. The DPPH radical scavenging activity in the hypocotyl extract (non-fermented) of Comparative Example 2 was 77%, whereas in the hypocotyl fermented extract of Example 2, L. delbrueckii subsp. 82% for the sample fermented with lactis JCM 1105, L. delbrueckii subsp. In the sample fermented with lactis JCM 1148, a very high DPPH radical scavenging activity of 80% was observed, and a tendency for the action to be enhanced by fermentation was observed. Moreover, the effect of the hypocotyl fermented extract was higher than that of the fermented soymilk extract of Example 1-1. On the other hand, the DPPH radical scavenging activity was hardly observed in the soymilk fermented extracts manufactured by Company A and Company B. As is clear from FIG. 3, it was confirmed that the fermented extract of the present invention has a very strong antioxidant effect.

  The results of evaluating the antioxidant activity of the isoflavone preparation by the same method are shown in FIG. In Example 1-1. delbrueckii subsp. lactis JCM 1105 and L. delbrueckii subsp. A high DPPH radical scavenging activity was not observed even in the 200 μM isoflavone preparation, which is more than the isoflavone contained in the fermented soymilk fermented with lactis JCM 1148. From this, it was considered that most of the DPPH radical scavenging activity of the fermented extract was exhibited by active ingredients other than isoflavones. Therefore, the fermented extract of the present invention contains a component having high antioxidant ability other than isoflavone in addition to isoflavone widely known as an antioxidant substance, and exhibits a very strong antioxidant action. Was confirmed.

[Test Example 4] Evaluation of anti-glycation effect of fermented extract About the same sample as Test Example 3, the anti-glycation effect was evaluated by the method shown below. All samples were adjusted to pH 7.4 ± 0.05 using 1N sodium hydroxide, and then sterilized by filtration with a 0.20 μm filter.

Add 500 μL of 0.1 M PBS, 20 μL of 40 mg / mL human serum albumin, 100 μL of 2 M glucose solution, 360 μL of distilled water and 20 μL of sample to a 1.5 mL microtube, mix well, and incubate at 60 ° C. for 40 hours did. At the same time, each reaction blank was incubated with distilled water added instead of the glucose solution. As a positive control, a sample added with distilled water instead of the sample was incubated, and as a positive control blank, a sample added with distilled water instead of the positive control glucose solution was incubated. After incubation, the test solution was dispensed into a 96-well plate, and fluorescence was measured at an excitation wavelength of 370 nm and a fluorescence wavelength of 440 nm. When the fluorescence in the case of sample addition is (A), the fluorescence of the reaction blank is (B), the fluorescence of the positive control is (C), and the fluorescence of the positive control blank is (D), the AGEs production inhibitory activity is Defined.
AGE generation inhibitory activity = {1- (AB) / (CD)} × 100
In addition, aminoguanidine was used as a positive control having AGEs production inhibitory activity.

  The anti-glycation effect evaluation results of the fermented extract are shown in FIG. The AGEs production inhibitory activity in the soymilk extract (non-fermented) of Comparative Example 1 was 17%, whereas in the soymilk fermented extract of Example 1-1, L. delbrueckii subsp. 30% for the sample fermented with lactis JCM 1105, L. delbrueckii subsp. In the sample fermented with lactis JCM 1148, a high AGEs production inhibitory activity of 36% was observed, and a tendency for the action to be enhanced by fermentation was observed. In addition, the AGEs production inhibitory activity in the hypocotyl extract (non-fermented) of Comparative Example 2 was 81%, whereas in the hypocotyl fermented extract of Example 2, L. delbrueckii subsp. lactis JCM 1105 and L. delbrueckii subsp. In both samples fermented with lactis JCM 1148, a very high AGEs production inhibitory activity of 100% was observed, and the tendency of the action to be enhanced by fermentation was observed, similar to the fermented soymilk extract. Moreover, the effect of the hypocotyl fermented extract was higher than that of the fermented soymilk extract of Example 1-1. On the other hand, the AGEs production inhibitory activity was not recognized in the soymilk fermented extracts manufactured by Company A and Company B. As is clear from FIG. 5, it was confirmed that the fermented extract of the present invention has a very strong anti-glycation action.

  Similarly, the results of evaluating the anti-glycation effect of the isoflavone preparation are shown in FIG. About daidzin, daidzein, genistin, and genistein contained in fermented soymilk extract, L. of Example 1-1. delbrueckii subsp. lactis JCM 1105 and L. delbrueckii subsp. A strong anti-glycation effect comparable to that of a soymilk fermented extract was not observed even in a 200 μM sample higher than the concentration contained in the fermented soymilk fermented with lactis JCM 1148. From this, it was considered that the anti-glycation effect of the fermented soymilk extract may have a synergistic effect due to the combination of isoflavones, or may be exhibited by an active ingredient other than isoflavones. In addition, since a very remarkable AGEs production inhibitory action was observed in the glycitein 200 μM preparation, it is considered that the anti-glycation action of the hypocotyl fermented extract shown in FIG. 5 may be mainly exerted by glycitein. It was.

[Test Example 5] Evaluation of antioxidant effect (cytoprotective action) of fermented soymilk extract Among the fermented soymilk extracts prepared in Example 1-1, L. delbrueckii subsp. For the soymilk fermented extract prepared using lactis JCM 1148 and the soymilk extract (non-fermented) prepared in Comparative Example 1, the antioxidant action (cytoprotective action) was evaluated by the method described below. All samples were adjusted to pH 7.4 ± 0.05 using 1N sodium hydroxide, and then sterilized by filtration with a 0.20 μm filter.

Normal human epidermal keratinocytes [manufactured by Kurabo Industries, product name NHEK (NB)] are seeded in a 96-well microplate so as to be 10 × 10 ³ cells per well, at 37 ° C. in a carbon dioxide concentration of 5 vol%. Cultured for 24 hours. As a seeding medium, HuMedia-KG2 medium (Kurabo) was used. Subsequently, it replaced | exchanged for the sample culture solution adjusted to each density | concentration using HuMedia-KB2 (made by Kurabo Industries) culture medium, and also culture | cultivated for 24 hours. Each sample culture solution was prepared so that the final ethanol concentration was 1%. Twenty-four hours after the change to the sample culture medium, tert-butyl hydroxide (t-BHP) was exposed to a final concentration of 0.5 mM and cultured for 2 hours. Next, each well was thoroughly washed with HBSS (−), and then further cultured in HuMedia-KB2 medium for 22 hours.

  After completion of the culture, the cell viability was calculated using Cell Counting Kit-8 (manufactured by Dojindo). In addition to the sample culture solution, Trolox was used as a positive control, DMSO was used as a Trolox blank, and 1% ethanol-containing HuMedia-KB2 medium was used as a blank for each sample.

  The evaluation results of the antioxidant effect (cytoprotective effect) are shown in FIG. The cell viability of the soymilk extract (non-fermented) of Comparative Example 1 was 14%, whereas the fermented soymilk extract of Example 1-1 had a high cell viability of 48%. A tendency to enhance the protective effect was observed. As is clear from FIG. 7, it was confirmed that the fermented soymilk extract of the present invention has a very strong antioxidant effect.

[Test Example 6] Evaluation of moisturizing action of fermented soymilk extract Among the fermented soymilk extracts prepared in Example 1-1, L. delbrueckii subsp. About the soymilk fermented extract prepared using lactis JCM 1148, the moisturizing effect in human in vivo was evaluated by the method shown below. All samples were adjusted to pH 7.4 ± 0.05 using 1N sodium hydroxide, and then sterilized by filtration with a 0.20 μm filter.

  Using the soymilk fermented extract, two types of lotions were prepared based on the formulation in Table 6 and used as test samples.

  A 3 cm square test site was taken on the inner side of the forearm of the subject, and the test sample was applied three times a day for 3 days. The stratum corneum moisture content and transdermal moisture transpiration were measured 8 to 10 hours after the final application of the test sample. A highly sensitive stratum corneum film thickness / moisture meter ASA-MX3 was used for the measurement of the stratum corneum moisture content, and Vaposcan AS-VT100RS was used for the measurement of the transdermal moisture transpiration amount (both manufactured by Asahi Biomed).

  The evaluation results of the moisturizing effect of the fermented soymilk extract are shown in FIGS. In all three subjects, the stratum corneum moisture content increased by 10% or more by application of the soymilk fermented extract-containing lotion. Moreover, the tendency for the transdermal moisture transpiration amount to decrease by 10% or more by application of the soymilk fermented extract-containing lotion was observed. From this, it was confirmed that the fermented soymilk extract has a stratum corneum moisture content increasing action and a stratum corneum barrier function enhancing action, and exhibits a moisturizing action in human in vivo.

  This application includes Japanese Patent Application No. 2012-269928 filed on December 11, 2012, Japanese Patent Application No. 2013-137901 filed on July 1, 2013, and July 17, 2013. This application claims priority based on Japanese Patent Application No. 2013-148391 filed on the same day, the contents of which are incorporated herein by reference.

  According to the present invention, it is possible to provide a soymilk fermented extract containing a high content of isoflavone aglycone and polyamine, and in particular, it has a low sugar content and has a cell activation effect and an antioxidant effect on epidermal keratinocytes and skin fibroblasts. The fermented soymilk extract exhibiting anti-aging effects such as anti-glycation action and moisturizing action can be provided. In addition, according to the present invention, a hypocotyl fermented extract obtained by fermenting soybean hypocotyls with lactic acid bacteria can be provided, in particular, high content of isoflavone aglycone and polyamine, and epidermal keratinocytes and skin fibers. It is possible to provide a hypocotyl fermented extract having an anti-aging action such as a cell activation action, an antioxidant action and an anti-glycation action on blast cells. The soymilk fermented extract and hypocotyl fermented extract of the present invention have industrial applicability in the fields of cosmetics, pharmaceuticals, and foods and drinks.

Claims (15)

The following steps (A), (B) and (C):
(A) a step of fermenting soy milk with lactic acid bacteria;
(B) a step of mixing the fermented product obtained in step (A) and an organic solvent; and (C) a step of removing the solid content produced in step (B);
A soymilk fermented extract obtained by a method comprising
The following (i), (ii) and (iii):
(I) The content of the isoflavone compound is 150 μM or more;
(Ii) conversion rate from isoflavone glycoside to isoflavone aglycone in step (A) is 50% or more; and (iii) polyamine content is 20 μM or more;
Satisfaction soymilk extract.   (Iv) The fermented soymilk extract according to claim 1, wherein the sum of the contents of glucose and sucrose is 3.0 mg / mL or less. Next (v) and (vi):
(V) Sum of glucose and sucrose content (mg / mL): isoflavone compound content (μM) not more than 0.1; and (vi) Sum of glucose and sucrose content (mg / mL): polyamine Content (μM) of 0.3 or less;
The soymilk fermented extract according to claim 1 or 2, satisfying at least one of the following.   The fermented soymilk extract according to any one of claims 1 to 3, wherein the lactic acid bacterium in the step (A) is Lactobacillus delbrueckii.   Prior to step (A), the soymilk fermented extract according to any one of claims 1 to 4, which is obtained by a method comprising a step of (P) performing pre-culture with soymilk. The following steps (A ′), (B ′) and (C ′):
(A ′) a step of fermenting soybean hypocotyls with lactic acid bacteria;
(B ′) a step of mixing the fermented product obtained in step (A ′) with an organic solvent; and (C ′) a step of removing solids produced in step (B ′);
Hypocotyl fermented extract obtained by a method comprising:   The hypocotyl fermented extract according to claim 6, comprising glycitein in an amount of 100 µM or more.   The anti-aging agent containing the fermented extract of any one of Claims 1-7.   The anti-aging agent according to claim 8, which has one or more of a skin keratinocyte activation effect, a skin fibroblast activation effect, an antioxidant effect, an anti-glycation effect and a moisturizing effect.   Cosmetics, a skin external preparation, a pharmaceutical, or food-drinks containing the fermented extract of any one of Claims 1-7.   A method for producing a polyamine, which comprises fermenting soy milk with lactic acid bacteria and then extracting the polyamine.   A method for producing a polyamine, comprising fermenting a soybean hypocotyl with lactic acid bacteria and then extracting the polyamine.   A method for producing isoflavone aglycone or polyamine, comprising a step of fermenting soybean hypocotyl with Lactobacillus delbrueckii.   Soy milk delbrueckii subsp. lactis JCM 1105, L. delbrueckii subsp. lactis JCM 1148, L.L. delbrueckii subsp. bulgaricus JCM 1001, L. delbrueckii subsp. lactis JCM 1010 and L. delbrueckii subsp. A fermented soymilk product obtained by fermentation with a lactic acid bacterium selected from delbrueckii JCM 1012.   A hypocotyl fermented product obtained by fermenting soybean hypocotyl with lactic acid bacteria Lactobacillus delbrueckii.