JP4990297B2 - Grape peel / seed lactic acid bacteria fermented product and medicine using the same - Google Patents

Description

  The present invention relates to a pharmaceutical and food for treating, ameliorating, or preventing metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity and the like, and skin using at least one of grape skin and seed The present invention relates to medicines and foods for whitening and preventing aging.

Metabolic syndrome is a condition in which lifestyle-related diseases such as obesity, diabetes, hypertension, and hyperlipidemia caused by visceral fat accumulation overlap, and if this condition continues, arteriosclerosis tends to progress and myocardial infarction And the risk of having a stroke increase. A recent survey by the Ministry of Health, Labor and Welfare shows that the metabolic syndrome and its reserves are nearly 20 million in the middle-aged generation, and one in two middle-aged men is considered to be the metabolic syndrome or its reserve army. ing.
The cause of metabolic syndrome is considered to be visceral fat accumulation based on lifestyle such as overnutrition, lack of exercise, excessive stress, excessive intake of high fat diet. This accumulation of visceral fat reduces the sensitivity to insulin that regulates blood glucose, blood triglyceride, blood pressure, etc., creating a state called insulin resistance, obesity, diabetes, hypertension, hyperlipidemia And cause arteriosclerosis.
As a factor causing insulin resistance, tumor necrosis factor-α (TNF-α) produced from macrophages and hypertrophic mast cells is considered, and insulin signaling may be inhibited by TNF-α. It is shown (for example, refer nonpatent literature 1). In contrast, adiponectin produced from adipocytes is known to exhibit an antagonistic action on TNF-α and enhance sensitivity to insulin (see, for example, Non-Patent Document 2). In recent years, it has been shown that adiponectin deficiency is caused by environmental factors such as obesity due to high-fat diet, and sensitivity to insulin is reduced. It is expected (see, for example, Non-Patent Document 3).
From these facts, in the treatment, improvement or prevention of metabolic syndrome, obesity such as visceral fat, blood glucose, blood neutral fat and the like, decrease diabetes-inducing factors, while antidiabetic action such as adiponectin, It is considered important to induce a factor having an anti-metabolic syndrome action.

In addition, when carbohydrates are taken into the body, they are digested by α-amylase and α-glucosidase in the digestive tract and finally decomposed into glucose, which is a monosaccharide, and absorbed from cilia on the intestinal membrane. The absorbed glucose is transferred into the blood, and hyperglycemic symptoms occur temporarily. In type II diabetic patients, the action of insulin does not work, high blood glucose levels are maintained, vascular injury due to glucose is induced, and serious complications such as neuropathy, retinopathy, and cataract are caused.
In contrast, by inhibiting the action of digestive enzymes such as α-amylase and α-glucosidase and suppressing the production of glucose, the ingested saccharide is excreted out of the body without being absorbed. The increase in value is suppressed.
The KKA ^y mouse, which is a mouse introduced with the ^Ay gene, which is an obesity gene, is used as a model mouse for type II diabetes, and may cause symptoms of metabolic syndrome such as obesity, insulin resistance, and hypertriglyceridemia. Are known. In addition, it has been confirmed that blood adiponectin is markedly reduced when symptoms of metabolic syndrome occur. Furthermore, supplementation with recombinant adiponectin improves insulin resistance and hypertriglyceridemia. (For example, refer nonpatent literature 2).

  As a method for treating, improving, or preventing such metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity and the like, various methods have been proposed conventionally. The number of patients in the reserve group is very large, and therefore there is still a need for more effective and safe treatment, improvement or prevention methods.

  On the other hand, the number of people who want to whiten skin and prevent aging is very large, especially among women. Skin aging is largely caused by inflammation in addition to aging, and skin inflammation is considered to be the cause of skin hardening, wrinkle formation, and loss of burs and gloss. Inflammation is generally caused by ultraviolet rays, but in recent years, the involvement of advanced glycation end products (AGE) produced by Maillard reaction has attracted attention. The Maillard reaction is a non-enzymatic reaction between protein and sugar, and the generated AGE is activated through the cell membrane receptor (RAGE) of inflammatory cells such as macrophages and induces the production of inflammatory cytokines such as TNF-α. . Non-enzymatic glycation of proteins progresses with normal aging, but is particularly rapid in people with high blood glucose levels, such as the metabolic syndrome described above, and is one of the causes of accelerated skin aging (for example, (See Patent Document 4).

  In the skin exposed to ultraviolet rays, a substance called α-melanocyte stimulating hormone (α-MSH) is produced from keratinocytes, and α-MSH acts on melanocytes and activates tyrosinase, which is a melanin synthase. Tyrosine in the melanocyte is converted to dopaquinone (DOPAquinone) through the action of tyrosinase, and melanin such as eumelanin and pheomelanin is generated and deposited on the skin. It causes dull skin (for example, see Non-Patent Document 5).

  A substance that suppresses the production of inflammatory cytokines induced by AGE and suppresses the production of melanin by inhibiting the activity of intracellular tyrosinase is considered to exhibit excellent effects in skin whitening and anti-aging, such as There is a current demand for substances.

J. et al. Biol. Chem. 2001; 276: 41245-41254. Nature Med. 2001; 7: 941-946. Nature Med. 2002; 277: 25863-25866. Diabetologia. 2001; 44: 129-146. J. et al. Clin. Invest. 1994; 93 (5): 2258-2262.

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide a useful grape skin / seed lactic acid bacteria fermented product using at least one of grape skin and seed. In addition, a medicament capable of effectively and safely treating, improving, or preventing metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity, and the like using the grape skin / seed lactic acid bacteria fermented product An object of the present invention is to provide foods and effective and safe medicines and foods for skin whitening and aging prevention.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, a grape skin / seed lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed with lactic acid bacteria has an excellent blood glucose level increase inhibitory effect, blood triglyceride (neutral fat) increase inhibitory effect, obesity inhibitory effect, It has an effect of increasing blood adiponectin, etc., so by using the grape skin / seed lactic acid bacteria fermented product, metabolic syndrome related symptoms such as type II diabetes, hyperlipidemia, obesity, etc. are effective, and It is the knowledge that can be safely treated, improved, or prevented. The grape skin / seed lactic acid bacteria fermented product has the effect of suppressing TNF-α production, tyrosinase activity inhibitory effect, melanin synthesis inhibitory effect, and skin inflammation caused by ultraviolet rays due to the terminal glycation end product group (AGE) produced by Maillard reaction It is a finding that it is also effective for whitening the skin and preventing aging.

In recent years, plants have been reported to contain a larger amount of flavonoids in the skin and seeds than in fruit juice, and these unused agricultural products have begun to attract attention. Flavonoids are known to bind to enzymes and inhibit their activity, and are being developed as functional foods for the prevention and treatment of metabolic syndrome caused by overeating and the like. However, existing flavonoids have difficulty in absorption when administered orally, and the effect is not sufficiently exhibited in extracts and purified products.
The present inventors first screened for lactic acid bacteria that proliferate favorably in culture with grape skin and seed powder (grape skin / seed dried powder), and selected suitable lactic acid bacteria. And, by inhibiting fermentation of grape peel and seed dry powder using this lactic acid bacterium, the inhibitory activity against digestive enzymes (amylase, glucosidase) is increased, and the effect of suppressing hyperglycemia when administered orally to sugar-loaded mice It was also found that a more effective substance (grape peel / fermented lactic acid bacteria) can be obtained. Furthermore, the obtained fermented grape skin / seed lactic acid bacteria is a remarkable inhibitory effect on blood glucose level elevation, blood triglyceride (neutral fat) elevation, and obesity even in model mice that develop diabetes symptoms with obesity. It has been found that an inhibitory action and the like are observed, and that it has an action of remarkably increasing adiponectin which is attracting attention as an anti-diabetic factor and anti-metabolic syndrome factor. In addition, the present inventors further said that the fermented grape skin / seed lactic acid bacteria can suppress TNF-α production from macrophages induced by AGE produced by bovine serum albumin (BSA) and ribose, The inventors have also found that the intracellular tyrosinase activity can be suppressed, melanin synthesis by α-MSH stimulation can be suppressed, and that skin inflammation due to ultraviolet rays can be suppressed by oral administration to mice, and the present invention has been completed.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A grape skin / seed lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds with lactic acid bacteria.
<2> The grape skin / seed lactic acid bacteria fermented product according to <1>, which is obtained by fermenting at least one of grape skins and seeds in a dried state with lactic acid bacteria.
<3> Fermented grape skin / seed lactic acid bacteria according to any one of <1> to <2>, obtained by fermenting at least one of grape skin and seeds in a fine powder (powder) state with lactic acid bacteria It is.
<4> The grape skin / seed lactic acid bacteria fermented product according to any one of <1> to <3>, obtained by fermenting at least one of grape skin and seed in a dry powder (dried powder) state with lactic acid bacteria It is.
As <5> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactobacac) llus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus, Pediococcus halophilus, Pediococcus parvulus, Pediococcus pe Tosaseus (Pediococcus pentocaseus), Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium bifidus), Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecis The obtained using at least one selected from the group consisting of lis) <1> is a grape skin, seed lactobacillus fermentation product as claimed in any one of <4>.

<6> A medicament for treating or preventing metabolic syndrome-related symptoms, comprising a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed with lactic acid bacteria It is a medicine.
<7> The medicament according to <6>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dried state with lactic acid bacteria.
<8> Any one of <6> to <7>, including a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds in a fine powder (powder) state with lactic acid bacteria It is the medicine described.
<9> Any one of <6> to <8>, including a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds in a dry powder (dried powder) state with lactic acid bacteria It is the medicine described.
As <10> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus jonsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactoba) illus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus), Pediococcus halophyllus, Pediococcus parvulus, Pediococcus Pentiococcus pentoaceus, Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium coficus) Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecalis A medicament according to any one of <9> from the <6> containing grape skin, seed lactobacillus fermentation product obtained by using at least one selected from the group consisting of alis).
<11> The medicament according to any one of <6> to <10>, which is used for treatment or prevention of type II diabetes.
<12> The medicament according to any one of <6> to <10>, which is used for treatment or prevention of hyperlipidemia.
<13> The medicament according to any one of <6> to <10>, which is used for treatment or prevention of obesity.
<14> The medicament according to any one of <6> to <10>, which has an action of reducing blood glucose concentration.
<15> The medicament according to any one of <6> to <10>, which has an action of reducing blood triglyceride concentration.
<16> The medicament according to any one of <6> to <10>, which has an action of reducing intraperitoneal fat weight.
<17> The medicament according to any one of <6> to <10>, which has an action of increasing blood adiponectin concentration.

<18> A medicine for whitening the skin and preventing aging, comprising a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed with lactic acid bacteria is there.
<19> The medicament according to <18>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dried state with lactic acid bacteria.
<20> In any one of the above items <18> to <19>, comprising a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds in a powdered state with lactic acid bacteria It is the medicine described.
<21> Any one of the items <18> to <20>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dry powder (dry powder) state with lactic acid bacteria It is the medicine described.
As <22> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus jonsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactoba) illus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus), Pediococcus halophyllus, Pediococcus parvulus, Pediococcus Pentiococcus pentoaceus, Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium coficus) Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecalis Wherein comprising at least grape skin, seed lactobacillus fermentation product obtained by using one selected from the group consisting of alis) from <18> is a medicament according to any one of <21>.
<23> The pharmaceutical according to any one of <18> to <22>, which has an action of suppressing TNF-α production caused by a terminal glycation end product group (AGE) generated by a Maillard reaction.
<24> The medicament according to any one of <18> to <22>, which has a tyrosinase activity inhibitory action.
<25> The medicament according to any one of <18> to <22>, which has an action of inhibiting melanin synthesis.
<26> The medicament according to any one of <18> to <22>, which has an action of suppressing skin inflammation caused by ultraviolet rays.

<27> A food for improving or preventing metabolic syndrome-related symptoms, comprising a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed with lactic acid bacteria It is food.
<28> The food according to <27>, including a fermented grape skin / seed lactic acid bacterium obtained by fermenting at least one of grape skin and seeds in a dried state with lactic acid bacteria.
<29> In any one of the above items <27> to <28>, comprising a fermented grape skin / seed lactic acid bacterium obtained by fermenting at least one of grape skin and seed in a fine powder (powder) state with lactic acid bacteria It is the described food.
<30> Any one of the above <27> to <29>, including a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds in a dry powder (dry powder) state with lactic acid bacteria It is the described food.
As <31> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus jonsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactoba) illus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus), Pediococcus halophyllus, Pediococcus parvulus, Pediococcus Pentiococcus pentoaceus, Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium coficus) Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecalis Wherein comprising at least grape skin, seed lactobacillus fermentation product obtained by using one selected from the group consisting of alis) from <27> is a food product according to any one of <30>.
<32> The food according to any one of <27> to <31>, which is used for improving or preventing type II diabetes.
<33> The food according to any one of <27> to <31>, which is used for improving or preventing hyperlipidemia.
<34> The food according to any one of <27> to <31>, which is used for improving or preventing obesity.
<35> The food according to any one of <27> to <31>, which has an action of decreasing blood glucose concentration.
<36> The food according to any one of <27> to <31>, which has an action of decreasing blood triglyceride concentration.
<37> The food according to any one of <27> to <31>, which has an action of reducing intraperitoneal fat weight.
<38> The food according to any one of <27> to <31>, which has an action of increasing blood adiponectin concentration.

<39> A food for whitening the skin and preventing aging, comprising a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds with lactic acid bacteria is there.
<40> The food according to <39>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dried state with lactic acid bacteria.
<41> In any one of the above items <39> to <40>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a powdered state with lactic acid bacteria It is the described food.
<42> Any one of <39> to <41>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dry powder (dried powder) state with lactic acid bacteria It is the described food.
As <43> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus jonsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactoba) illus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus), Pediococcus halophyllus, Pediococcus parvulus, Pediococcus Pentiococcus pentoaceus, Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium coficus) Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecalis Wherein comprising at least grape skin, seed lactobacillus fermentation product obtained by using one selected from the group consisting of alis) from <39> is a food product according to any one of <42>.
<44> The food according to any one of <39> to <43>, which has an action of suppressing TNF-α production caused by a terminal glycation end product group (AGE) generated by a Maillard reaction.
<45> The food according to any one of <39> to <43>, which has a tyrosinase activity inhibitory action.
<46> The food according to any one of <39> to <43>, which has a melanin synthesis inhibitory action.
<47> The food according to any one of <39> to <43>, which has an action of suppressing skin inflammation caused by ultraviolet rays.

  According to the present invention, it is possible to solve the conventional problems and achieve the object, and to provide a useful fermented grape skin / seed lactic acid bacteria using at least one of grape skin and seed. Can do. Furthermore, using the grape skin / seed lactic acid bacteria fermented product, a drug capable of effectively and safely treating, improving or preventing metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity and the like It is possible to provide a food and a pharmaceutical and food effective and safe for skin whitening and aging prevention.

FIG. 1 is a graph showing the results of examining the optimum amount of lactic acid bacteria when producing a fermented grape skin / seed lactic acid bacterium of the present invention (lactic acid fermented grape skin / seed) in Example 2. FIG. 2 is a graph showing the results of examining the optimum culture time when producing a fermented grape skin / seed lactic acid bacteria (lactic acid fermented grape skin / seed) of the present invention in Example 3. FIG. 3 is a graph showing the results of comparing the amylase enzyme activity inhibiting ability of each sample in Example 4. FIG. 4 is a graph showing the results of comparing the glucosidase enzyme activity inhibiting ability of each sample in Example 5. FIG. 5 is a graph showing the results of comparing the blood glucose level increase inhibiting ability of each specimen according to the sucrose load test in Example 6. FIG. 6 is a graph showing the results of comparing the blood glucose level increase inhibiting ability of each specimen by the starch load test in Example 7. FIG. 7 is a graph showing the results of comparison of blood triglyceride (TG) elevation inhibitory ability of each specimen according to the olive oil load test in Example 8. FIG. 8A is a photographic image showing the difference in appearance of mice (9 weeks old) in each group in Example 9. FIG. 8B is a graph showing differences in weight gain of mice in each group in Example 9. FIG. 8C is a graph showing the difference in intraperitoneal fat weight of mice (9 weeks old) in each group in Example 9. FIG. 8D is a graph showing the difference in serum triglyceride (TG) concentration of each group of mice in Example 9. FIG. 8E is a graph showing the difference in the serum glucose concentration increase of each group of mice in Example 9. FIG. 8F is a graph showing the difference in serum adiponectin concentration of each group of mice (9 weeks old) in Example 9. FIG. 9 is a graph showing the adiponectin production inducing ability of lactic acid bacteria fermented grape skin / seed specimens in Example 10. FIG. 10 is a graph showing the ability of lactic acid bacteria fermented grape skin and seeds to antagonize adiponectin production inhibition by TNF-α in Example 11. FIG. 11 is a graph showing the results of comparing the ability of each sample to suppress TNF-α production from AGE-stimulated macrophages in Example 12. FIG. 12 is a graph showing the results of comparing the ability of B16 melanoma cells to inhibit intracellular tyrosinase activity in Example 13 in Example 13. FIG. 13 is a graph showing the results of comparing the melanin synthesis inhibiting ability of B16 melanoma cells of each specimen in Example 14. FIG. 14 is a graph showing the results of comparison of the skin inflammation suppression ability of each specimen in ultraviolet-irradiated mice in Example 15. FIG. 15 is a diagram showing a method for separating and fractionating a specimen of fermented grape skin / seed lactic acid bacteria (lactic acid fermented grape skin / seed) according to the present invention in Example 16. 16A is a diagram showing TLC results of an ethyl acetate fraction (EtOAc fr) in Example 16. FIG. FIG. 16B is a diagram showing the HPLC result of the ethyl acetate fraction (EtOAc fr) in Example 16. FIG. 17A is a diagram showing TLC results of a chloroform fraction (CHCl ₃ fr) in Example 16. FIG. 17B is a diagram showing the HPLC result of the chloroform fraction (CHCl ₃ fr) in Example 16. FIG. 18A is a diagram showing a TLC result of an n-butanol fraction (n-BuOH fr) in Example 16. FIG. 18B is the figure which showed the HPLC result of the n-butanol fraction (n-BuOH fr) in Example 16. FIG. 19A is a diagram showing a TLC result of a methanol fraction (MeOH fr) in Example 16. FIG. 19B is a diagram showing the HPLC result of the methanol fraction (MeOH fr) in Example 16.

(Grape skin and fermented lactic acid bacteria)
The grape skin / seed lactic acid bacteria fermented product of the present invention is obtained by fermenting grape skin and / or seeds (fruit skin / seed) with lactic acid bacteria. In the present specification, the fermented grape skin / seed lactic acid bacteria may be referred to as “lactic acid bacteria fermented grape skin / seed” or the like.

<Grape>
The grape is not particularly limited as long as it is a plant belonging to the Vitaceae genus (Vitis), and can be appropriately selected according to the purpose. For example, European grape (Vitis vinifera) ), American grapes (Vitis labrusca), Vitis amenrensis, Vitis coigneiae, and hybrids thereof. The grape varieties are not particularly limited and may be appropriately selected according to the purpose. For example, Koshu, Chardonnay, Muscat, Sauvignon Blanc, Semillon ( Semilon, Riesling, Silvaner, Muller-Thurgau, Kerner, Gewurztraminer, Chassela n, Chin Semi B ), Delaware, Bacchus, Pinot Gris, Pinot Plan (Pinot Blank), Fulmint (Furminnt), Muscadet, Muscadelle, Riesling, Cabernet Sauvignon, Muscat berry A (Muscat Ai b) Grenache, Merlot, Syrah, Negroamaro, Sangiovese, Tannat, Primitivo, Brunelet, Brunelet (Gamay), Concord (Co ncord, Sangiovese, Sansou (Cinsaut), Zinfandel, Seibel, Zweigeltrebe, Tempranillo, Barbano (Barino) Examples include Pinot Meunier, Petit Verdot, Malvasia, Malbec, Mourverder, and Kyoho.

<Fruits and seeds>
The skin and seeds are not particularly limited as long as they are at least one of the grape skins and seeds described above, and can be appropriately selected according to the purpose. It may be in a dry state. In addition, the size of the pericarp / seed may be a state in which at least one of the pericarp and seed is collected, or may be in a state of being cut into a desired size, or a fine powder ( It may be in a powdered state. Among them, the pericarp / seed is preferably in a dry state, and preferably in a fine powder state from the viewpoint of facilitating the action of lactic acid bacteria described later. That is, it is particularly preferable that the pericarp / seed is in a dry powder (dry powder) state. There is no restriction | limiting in particular as a method of producing the dry powder (dry powder) of the said skin and seed, According to the objective, it can select suitably, For example, it can carry out using a well-known method. There is no restriction | limiting in particular as an average particle diameter of the dry powder (dry powder) of the said skin and seed, Although it can select suitably according to the objective, For example, 10-200 mesh is preferable. When the average particle size is less than 10 mesh, the activity of the fermented product may be weak. On the other hand, when the average particle diameter is within the particularly preferable range, it is advantageous in that a fermented product having strong activity can be obtained.
The grape skins and seeds are not limited in the method of obtaining them and can be appropriately selected according to the purpose. For example, the remaining grapes after squeezing the fruit juice in the production process of wine or juice. It is considered desirable to use firewood because it is cost effective and can reduce industrial waste.
Moreover, as the grape skin and seed, the grape skin may be used alone, the grape seed may be used alone, or the grape skin and seed may be used in combination. Also good.
Here, the raw material of the fermented product of the grape skin / seed lactic acid bacteria does not necessarily have to be composed of at least one of the grape skin and seed described above, and at least a part of the grape skin and seed described above. It is sufficient that at least one of them is included. For example, the grape skin / seed lactic acid bacteria fermented material may appropriately include the grape fruit, fruit juice, leaves, stems, etc. in addition to the grape skin / seed. There is no restriction | limiting in particular as usage-amount ratio with respect to the said fruit peel and seed, and the said grape peel and seed, It can select suitably according to the objective.

<Lactic acid bacteria>
The lactic acid bacterium is not particularly limited as long as it is a bacterium that produces lactic acid by fermentation, and can be appropriately selected according to the purpose. For example, Lactobacillus, Pediococcus, Bibi Examples include bacteria belonging to the genus Bifidobacterium, the genus Lactococcus, the genus Leuconostoc and the genus Enterococcus. The seeds of the lactic acid bacteria are not particularly limited and can be appropriately selected according to the purpose. Among them, the seeds that can be efficiently propagated using the grape skins and seeds as a carbon source are used. It is preferable. Such preferred species include, for example, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivalus (Lactobacillus salivalus), Lactobacillus s. (Lactobacillus kitatatonisi), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus johnsonii (Lactobacillus jhonsonii), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Fal'cie Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus); Bae As the genus Diococcus, Pediococcus damnosus, Pediococcus halophilus, Pediococcus parva Pediococcus parvulus, Pediococcus pentosaceus; Bifidobacterium, Bifidobacterium adolescentis, Bifidobacteria bifida Lactococcus cremoris, Lactococcus lactis; as the genus Leuconostoc, Leuconostoc oenos, Leuconostoc lactis, Leuconostoc lactis As a genus of Enterococcus, Enterococcus faecalis (Enterococcus faecalis) and the like can be mentioned as Nostock mesenteroides (Leuconostoc mesenteroides). Among them, Lactobacillus plantarum is particularly preferable as a seed of the lactic acid bacterium because it can proliferate efficiently and the activity of the obtained fermented product is strong.

<Fermentation>
The fermentation can be performed by fermenting a raw material containing at least a part of the above-described grape skin and seed using the lactic acid bacteria. There is no restriction | limiting in particular as the method of the said fermentation, According to the objective, it can select suitably, For example, the method etc. which add and culture | cultivate the said lactic acid bacteria to the said raw material are mentioned. More specifically, for example, the fermentation can be performed by the method described in Examples described later.

There is no restriction | limiting in particular as the usage-amount of the lactic acid bacteria at the time of the said fermentation, According to the objective, it can select suitably, For example, 10 < ¹² > CFU or more is preferable with respect to the said raw material 10g, and 10 < ¹³ > CFU or more is especially preferable. . When the amount of the lactic acid bacterium used is less than 10 ¹² CFU, the activity of the fermented product may be weakened. On the other hand, when the amount of the lactic acid bacteria used is within the particularly preferred range, it is advantageous in that a fermented product having a strong activity can be obtained.

  There is no restriction | limiting in particular as temperature at the time of the said fermentation, According to the objective, it can select suitably, For example, 10-40 degreeC is preferable and 17-30 degreeC is especially preferable. If the temperature is less than 10 ° C, the growth of the bacteria may be deteriorated, and if it exceeds 40 ° C, the bacteria may not be able to grow. On the other hand, when the temperature is within the particularly preferable range, it is advantageous in that a fermented product having strong activity can be obtained.

  There is no restriction | limiting in particular as said fermentation time, According to the objective, it can select suitably, For example, 48-144 hours are preferable and 72 hours are especially preferable. When the fermentation time is less than 48 hours, the activity of the fermented product may be weak. On the other hand, when the fermentation time is within the particularly preferable range, it is advantageous in that a fermented product having strong activity can be obtained.

By the fermentation, the fermented grape skin and seed lactic acid bacteria of the present invention can be obtained. The grape skin / seed lactic acid bacteria fermented product is composed of a component different from unfermented grape skin / seed, as shown in the examples described later, and therefore, it is a novel composition. it can.
The grape skin / seed lactic acid bacteria fermented product can be used for various purposes as it is or by being appropriately processed. For example, as shown in the examples described later, the grape skin / seed lactic acid bacteria fermented product has an excellent inhibitory effect on blood glucose level elevation, blood triglyceride (neutral fat) elevation inhibitory effect, obesity inhibitory effect, blood adiponectin elevation Therefore, the fermented grape skin / seed lactic acid bacteria is intended to treat, ameliorate, or prevent metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity, etc. Thus, it can be suitably used as an active ingredient of the first medicament and food of the present invention described later. The grape skin / seed lactic acid bacteria fermented product has an effect of suppressing TNF-α production, tyrosinase activity inhibitory effect, melanin synthesis inhibitory effect, and skin inflammation caused by ultraviolet rays caused by the group of advanced glycation end products (AGE) produced by Maillard reaction. For example, the fermented grape skin / seed lactic acid bacteria is also effective as an active ingredient of the second medicament and food of the present invention described later for the purpose of whitening the skin and preventing aging, for example. Can be suitably used.

(Medicine)
A first medicament of the present invention is a medicament for treating or preventing a metabolic syndrome-related symptom, wherein a grape skin / seed lactic acid bacteria fermented product obtained by fermenting grape skin and / or seeds with lactic acid bacteria is provided. Including other components as necessary.
The second medicament of the present invention is a medicament for whitening the skin and preventing aging, wherein a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds with lactic acid bacteria is used. Including other components as necessary.
In the present specification, the “first drug” and the “second drug” are sometimes simply referred to as “medicine”.

<Fermented grape skin and seed lactic acid bacteria>
The grape skin / seed lactic acid bacteria fermented product is obtained by fermenting at least one of grape skins and seeds (fruit skin / seed) with lactic acid bacteria. "Fermentation" is as described in the above-mentioned item of fermented grape skin / seed lactic acid bacteria of the present invention. Therefore, the above-described fermented grape skin / seed lactic acid bacteria of the present invention can be preferably used as the fermented grape skin / seed lactic acid bacteria in the pharmaceutical.
The content of the grape skin / seed lactic acid bacteria fermented product in the medicament is not particularly limited and can be appropriately selected according to the purpose. The medicament is the grape skin / seed lactic acid bacteria fermented product itself. May be.

<Other ingredients>
There is no restriction | limiting in particular as said other component, In the range which does not impair the effect of this invention, it can select suitably according to the objective, For example, a pharmacologically acceptable carrier etc. are mentioned. There is no restriction | limiting in particular as said carrier, For example, it can select suitably according to the pharmaceutical dosage form etc. which are mentioned later. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said pharmaceutical, According to the objective, it can select suitably.

<Dosage form>
The pharmaceutical dosage form is not particularly limited, and can be appropriately selected according to a desired administration method. For example, oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), Oral solutions (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for erection, etc.), suppositories, ointments, patches, gels, creams, external powders, sprays And inhalable powders.

Examples of the oral solid preparation include, for example, the above-described fermented grape skin and seed lactic acid bacteria, excipients, and additives such as binders, disintegrants, lubricants, coloring agents, flavoring and flavoring agents as necessary. And can be produced by a conventional method.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. It is done. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The oral solution can be produced by a conventional method, for example, by adding additives such as a flavoring / flavoring agent, a buffering agent, and a stabilizer to the grape skin / seed lactic acid bacteria fermented product.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

As the injection, for example, a pH regulator, a buffer, a stabilizer, a tonicity agent, a local anesthetic, and the like are added to the grape skin / seed lactic acid bacteria fermented product. And intravenous injections can be produced.
Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

  Examples of the suppository include a known suppository formulation carrier such as polyethylene glycol, lanolin, cacao butter, fatty acid triglyceride, and the like, and a Tween (TWEEN: registered trademark) as necessary. After adding a surfactant, etc., it can be produced by a conventional method.

The ointment can be produced, for example, by mixing a known base, stabilizer, wetting agent, preservative and the like into the grape skin / seed lactic acid bacteria fermented product and mixing them by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

  As the patch, for example, a cream, gel or paste as the ointment can be applied to a known support by a conventional method. Examples of the support include woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane and other films made of cotton, suf, and chemical fibers, and foam sheets.

<Administration>
There is no restriction | limiting in particular as an administration subject of the said pharmaceutical, According to the objective, it can select suitably, For example, a human, a mouse | mouth, a rat, a hamster, a dog, a cat, a cow, a pig, a monkey etc. are mentioned.
Moreover, there is no restriction | limiting in particular as the administration method of the said pharmaceutical, According to the dosage form etc. of the said pharmaceutical, it can select suitably, For example, oral administration, administration by injection, etc. are mentioned.
The dosage of the drug is not particularly limited and can be appropriately selected according to the age, weight, sex, symptom, etc. of the patient to be administered, and is an active ingredient per day for a human adult. The amount of the grape skin / seed lactic acid bacteria fermentation product is preferably about 50 mg to 5 g.
Further, the frequency of administration of the medicament is not particularly limited and can be appropriately selected according to the purpose. For example, the daily dose may be administered once a day, Multiple doses may be administered.
Moreover, there is no restriction | limiting in particular also as the administration time of the said pharmaceutical, According to the objective, it can select suitably, For example, the said pharmaceutical may be administered therapeutically or may be administered prophylactically. .

<Target>
There is no restriction | limiting in particular as a treatment or prevention target of said 1st pharmaceutical, According to the objective, it can select suitably from "metabolic syndrome related symptoms". Here, metabolic syndrome generally means that insulin resistance is caused by visceral fat accumulation, which causes type II diabetes (hyperglycemia), hypertension, hyperlipidemia (eg hypercholesterolemia, hypertriglyceridemia). Symptom), obesity, and the like, a condition where arteriosclerosis is likely to be caused is present in a patient. For example, according to Japanese standards published at the General Meeting of the Japan Internal Medicine Society in April 2005, patients whose waist circumference reflecting the accumulation of visceral fat is 85 cm or more for men and 90 cm or more for women (visceral fat area of 100 cm ² or more (1) Serum lipid abnormality (triglyceride value of 150 mg / dL or more, or HDL cholesterol value of less than 40 mg / dL), (2) High blood pressure (maximum blood pressure of 130 mmHg or more, or minimum blood pressure of 85 mmHg or more) (3 ) It is stipulated that a metabolic syndrome is diagnosed when two or more of three items of hyperglycemia (fasting blood glucose level 110 mg / dL or more) are applied.
Therefore, the `` metabolic syndrome related symptoms '' to be treated or prevented by the medicine is not particularly limited as long as it is a symptom that can occur in connection with the metabolic syndrome, and can be appropriately selected according to the purpose. For example, conditions such as visceral fat accumulation and insulin resistance that cause metabolic syndrome, type II diabetes (hyperglycemia), hypertension, hyperlipidemia (eg hypercholesterolemia, hypertriglyceridemia) derived from these conditions Symptom), symptoms such as obesity, and arteriosclerosis, myocardial infarction, stroke, etc., which are derived as a result of progression of these symptoms. Since the medicament contains at least the fermented grape skin and seed lactic acid bacteria of the present invention, it has excellent blood glucose level increase inhibitory effect, blood triglyceride increase suppressive effect, obesity suppressive effect, blood adiponectin increase effect, etc. Therefore, among the various metabolic syndrome-related symptoms described above, it is particularly suitable for the treatment or prevention of type II diabetes (hyperglycemia), hyperlipidemia (particularly hypertriglyceridemia), and obesity.

  The second medicine is a medicine for the purpose of whitening the skin and preventing aging. Therefore, the symptom to be the subject of the medicine is not particularly limited and can be appropriately selected according to the purpose. Examples include inflammation of the skin, hardening, pigmentation, wrinkle formation, loss of beam and gloss, sunburn, roughness, spots, buckwheat, acne, and wipes. Since the medicament contains at least the fermented product of the grape skin and seed lactic acid bacteria of the present invention, it has an effect of suppressing TNF-α production, tyrosinase activity inhibiting effect, melanin, caused by the terminal glycation end product group (AGE) produced by Maillard reaction It is excellent in the action of suppressing synthesis, the action of suppressing skin inflammation caused by ultraviolet rays, and the like, and is therefore effective in whitening the skin and preventing aging.

(Food)
A first food of the present invention is a food for improving or preventing a metabolic syndrome-related symptom, and a grape skin / seed lactic acid bacteria fermented product obtained by fermenting grape skin and / or seeds with lactic acid bacteria. Including other components as necessary.
The second food of the present invention is a food for whitening the skin and preventing aging, wherein a fermented grape skin / seed lactic acid bacterium obtained by fermenting at least one of grape skin and seed with lactic acid bacteria is used. Including other components as necessary.
In the present specification, the “first food” and the “second food” may be simply referred to as “food”.

<Fermented grape skin and seed lactic acid bacteria>
The grape skin / seed lactic acid bacteria fermented product is obtained by fermenting at least one of grape skins and seeds (fruit skin / seed) with lactic acid bacteria. "Fermentation" is as described in the above-mentioned item of fermented grape skin / seed lactic acid bacteria of the present invention. Therefore, the above-described fermented grape skin / seed lactic acid bacteria of the present invention can be suitably used as the fermented grape skin / seed lactic acid bacteria in the food.
The content of the grape skin / seed lactic acid bacteria fermented product in the food is not particularly limited and can be appropriately selected according to the purpose, and the food is the grape skin / seed lactic acid bacteria fermented product itself. May be.

<Other ingredients>
There is no restriction | limiting in particular as said other component, In the range which does not impair the effect of this invention, it can select suitably according to the objective, For example, various food raw materials etc. are mentioned. There is no restriction | limiting in particular as said food raw material, For example, it can select suitably according to the kind etc. of the said foodstuff mentioned later. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said foodstuff, According to the objective, it can select suitably.

<Type of food>
The type of the food is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include confectionery such as jelly, candy, chocolate, biscuit, and gummi; snacks such as persimmon seeds and rice cakes; green tea Taste drinks such as tea, coffee and soft drinks; Dairy products such as raw milk, yogurt and ice cream; Vegetable and fruit processed products such as vegetable drinks, fruit drinks and jams; Liquid foods such as soup; Bread and noodles Processed cereals such as: various seasonings; processed meat and seafood such as hamburger, croquette, kamaboko, fried satsuma; and the like. There is no restriction | limiting in particular as a manufacturing method of these foodstuffs, For example, according to the manufacturing method of various normal foodstuffs, it can manufacture suitably.
Moreover, the said foodstuff may be manufactured as oral liquid preparations, such as oral solid preparations, such as a tablet, a granule, a capsule, and an internal use liquid preparation, a syrup preparation, for example. The method for producing the oral solid preparation and oral liquid preparation is not particularly limited and can be appropriately selected according to the purpose. For example, the preparation can be produced in accordance with the above-described method for producing an oral solid preparation for oral medicine or oral liquid preparation. it can.

The form of administration (intake) of the food is not particularly limited and may be appropriately selected depending on the purpose. For example, the form of administration of the medicament of the present invention (administration subject, administration method, dose, administration time) ) And can be administered (taken) as appropriate.
Moreover, there is no restriction | limiting in particular as said food improvement or prevention object, It can select suitably similarly to the above-mentioned medical treatment or prevention object of this invention.

Since the first food contains at least the fermented product of the grape skin / seed lactic acid bacteria of the present invention, it has excellent blood glucose level increase inhibitory effect, blood triglyceride increase suppressive effect, obesity suppressive effect, blood adiponectin increase effect and the like. Therefore, the food is a functional food taken for the purpose of improving or preventing metabolic syndrome-related symptoms such as type II diabetes (hyperglycemia), hyperlipidemia (particularly hypertriglyceridemia), obesity, etc. As particularly useful.
More specifically, for example, an aspect of ingesting the food by using the food as a seasoning during cooking or ingestion of a high-fat food, which is considered to be one of the causes of easily causing metabolic syndrome-related symptoms, An embodiment in which the food is ingested as a tablet or capsule-type supplement before or after ingestion of a fat food is considered suitable.

In addition, since the second food contains at least the fermented product of grape skin and seed lactic acid bacteria according to the present invention, an action of suppressing TNF-α production caused by a terminal glycation end product group (AGE) generated by Maillard reaction, tyrosinase It has excellent activity-inhibiting action, melanin synthesis-inhibiting action, action to suppress skin inflammation caused by ultraviolet rays, etc., and is therefore particularly useful as a functional food taken for the purpose of whitening the skin and preventing aging.
More specifically, for example, an embodiment in which the food is ingested as a tablet or capsule supplement is considered suitable.

[effect]
According to the present invention, fermented grape skin / seed lactic acid bacteria suitable for various applications can be provided, and further, the fermented grape skin / seed lactic acid bacteria can be used for type II diabetes, hyperlipidemia, obesity. It is possible to provide pharmaceuticals and foods that can treat, ameliorate, or prevent metabolic syndrome-related symptoms such as the above, and effective and safe pharmaceuticals and foods for skin whitening / aging prevention.
Since the grape skin / seed lactic acid bacteria fermented product of the present invention and the pharmaceutical and food of the present invention containing the grape skin / seed lactic acid bacteria fermented product can effectively use the grape skins and seeds that have been disposed of conventionally, It can be said that it is excellent also from a viewpoint. In addition, the fermented grape skin / seed lactic acid bacteria of the present invention is not extracted, purified, or the like, and is closer to a natural food. Therefore, the medicament and food of the present invention containing the grape skin and seed lactic acid bacteria fermented product are not only excellent in the treatment, improvement, prevention effect of metabolic syndrome-related symptoms as described above, and skin whitening / antiaging effect, It can be said that it is extremely excellent in safety.

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

(Example 1: Screening of optimal lactic acid bacteria)
In order to select a lactic acid bacterium suitable for producing a fermented grape skin / seed lactic acid bacterium (lactic acid bacterium fermented grape skin / seed) of the present invention, screening for an optimal lactic acid bacterium was performed as follows.
Koshu grape skin and seeds were crushed to produce grape skin / seed dry powder, suspended in 50 ml of PBS to 1% by mass, and each lactic acid bacterium shown in Table 1 at a concentration of 1 × 10 ⁶ CFU / ml I was inoculated to be. After culturing at 37 ° C. for 48 hours, the culture solution was collected and the pH was measured with a pH meter.
The results are shown in Table 1. A decrease in pH was observed after the addition of any lactic acid bacterium to pH 7.0 before the addition of each bacterium. Among them, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivalus (Lactobacillus salivaus), Lactobacillus amylovus (Lactobacillus amylovus), Lactobacillus coaceus (Lactobacillus paceca) It was shown that these lactic acid bacteria proliferate well using grape skin and seeds as carbon sources (Table 1).
In the following examples, the preparation of lactic acid bacteria fermented grape skin / seed specimens was performed using Lactobacillus plantarum (KU146), unless otherwise specified.

(Example 2: Examination of amount of added lactic acid bacteria)
The addition amount of lactic acid bacteria suitable for producing the grape skin / seed lactic acid bacteria fermented product of the present invention (lactic acid bacteria fermented grape skin / seed) was examined as follows.
Koshu grape skin and seeds are crushed to produce grape skin / seed dry powder, suspended in 20 ml of PBS to 10% by mass, and then each amount of lactic acid bacteria shown in FIG. 1 is added for 5 days. And cultured at 37 ° C. The physiological activity of each sample obtained was examined using α-glucosidase enzyme inhibitory activity as an index. The α-glucosidase activity was measured by the enzyme substrate method. Specifically, 50 ml of a 0.07 U / ml α-glucosidase solution (Sigma) was added to 10 ml of each specimen adjusted to a concentration of 10 mg / ml with distilled water, reacted for 5 minutes, and then 5 mM p-nitrophenyl-a-. 50 ml of d-glucopyranoside (Nacalai Tesque) solution was added. After reacting for 5 minutes, the absorbance was measured at a wavelength of 405 nm.
The results are shown in FIG. The measured value was 100% of the value (control) when measured with distilled water instead of the sample, and the vertical axis represents% relative to control. Enzyme inhibitory activity was observed from the concentration of added lactic acid bacteria 10 ⁹ CFU / ml, reaching a plateau at 10 ¹¹ CFU / ml (FIG. 1).

(Example 3: Examination of culture time)
The culture time suitable for producing the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) of the present invention was examined as follows.
Koshu grape skin and seeds were crushed to produce a grape skin / seed dry powder, suspended in 20 ml of PBS to 10% by mass, 10 ¹¹ CFU / ml of lactic acid bacteria were added, and from 1 to 6 Cultured at 37 ° C. for one day. The physiological activity of each sample obtained was examined using α-glucosidase enzyme inhibitory activity as an index. The α-glucosidase activity was measured in the same manner as in Example 2.
The results are shown in FIG. The measured value was 100% of the value (control) when measured with distilled water instead of the sample, and the vertical axis represents% relative to control. A strong enzyme inhibitory activity was observed from the second day of culture, and reached a plateau after the third day (FIG. 2).

[Sample preparation method]
From the results of Examples 1 to 3, the preparation of the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) in the following examples was carried out by the following method, unless otherwise specified.
Koshu grape skin and seeds were crushed to produce a grape skin / seed dry powder, suspended in PBS to 10% by mass, and then lactic acid bacteria (Lactobacillus plantarum (KU146)) was added at 1 × 10 ¹¹ CFU / ml. Inoculated to a concentration. After culturing at 37 ° C. for 48 hours (2 days), the culture solution was collected and heat-treated at 80 ° C. for 2 hours. The obtained specimen was diluted with distilled water and adjusted to a desired concentration in each example.

(Example 4: Inhibitory effect on amylase enzyme)
The amylase enzyme inhibitory action of the fermented grape skin / seed lactic acid bacteria of the present invention (lactic acid fermented grape skin / seed) was examined as follows.
The α-amylase activity was measured with reference to a commercially available amylase test Wako (Wako Pure Chemical Industries, Ltd.). That is, 50 ml of 7 U / ml α-amylase solution (Wako Pure Chemical Industries, Ltd.) was added to 20 ml of each specimen adjusted to a concentration of 2.5 mg / ml with distilled water, and reacted for 5 minutes. 50 ml (4% starch solution) was added. After reacting for 7.5 minutes, 50 ml of coloring solution (0.01N iodine solution) and 150 ml of distilled water were added, and the absorbance was measured at a wavelength of 450 nm. Details of each sample used are as follows.
Group 1 (untreated grape skin / seed powder): The grape skin / seed dried powder used in the [Specimen Preparation Method] was used as a specimen (Comparative Control Group).
Group 2 (grape skin / seed + lactic acid bacteria (fermented)): Lactic acid bacteria fermented grape skin / seed prepared by the above [Sample preparation method] was used as a sample (experimental group).
Group 3 (Grape skin / seed + lactic acid bacteria (unfermented)): In the above [Sample preparation method], inoculated with dead bacteria of lactic acid bacteria sterilized by heat treatment at 80 ° C. for 2 hours instead of living lactic acid bacteria The sample was used as a specimen (comparison control group).
Group 4 (only lactic acid bacteria): Only the lactic acid bacteria (Lactobacillus plantarum (KU146)) used in the above [Sample preparation method] were used as samples (comparison control group).
The results are shown in FIG. The measured value was 100% of the value (control) when measured with distilled water instead of the sample, and the vertical axis represents% relative to control. From the results of FIG. 3, it can be seen that the lactic acid bacteria fermented grape skin / seed sample (group 2) effectively suppressed the activity of amylase, an enzyme that converts polysaccharides to disaccharides. That is, it is conceivable that polysaccharides taken in by meals are discharged from the body without being converted into glucose by ingesting lactic acid bacteria fermented grape peels and seeds. From this, it was determined that lactic acid bacteria fermented grape peels and seeds can suppress an increase in blood glucose level after meals and are effective in the prevention and treatment of diabetes. On the other hand, this effect is not observed in samples other than lactic acid bacteria fermented grape skin and seeds, indicating that fermentation of grape skin and seeds by lactic acid bacteria is important.

(Example 5: Glucosidase enzyme inhibitory action)
The glucosidase enzyme inhibitory action of the fermented grape skin / seed lactic acid bacteria of the present invention (lactic acid fermented grape skin / seed) was examined as follows.
The α-glucosidase activity was measured by the enzyme substrate method. That is, 50 ml of 0.07 U / ml α-glucosidase solution (Sigma) was added to 10 ml of each specimen adjusted to a concentration of 10 mg / ml with distilled water, reacted for 5 minutes, and then 5 mM p-nitrophenyl-a. -50 ml of d-glucopyranoside (Nacalai Tesque) solution was added. After reacting for 5 minutes, the absorbance was measured at a wavelength of 405 nm. The details of each sample used are the same as in Example 4.
The results are shown in FIG. The measured value was 100% of the value (control) when measured with distilled water instead of the sample, and the vertical axis represents% relative to control. From the results of FIG. 4, it can be seen that the lactic acid bacteria fermented grape skin / seed specimen (group 2) effectively suppressed the activity of glucosidase, an enzyme that converts disaccharides into glucose. That is, it is conceivable that disaccharides taken in by meals are excreted outside the body without being converted into glucose by ingesting lactic acid bacteria fermented grape peels and seeds. From this, it was determined that lactic acid bacteria fermented grape peels and seeds can suppress an increase in blood glucose level after meals and are effective in the prevention and treatment of diabetes. On the other hand, this effect is not observed in samples other than lactic acid bacteria fermented grape skin and seeds, indicating that fermentation of grape skin and seeds by lactic acid bacteria is important.

(Example 6: hyperglycemia suppression effect by sucrose load)
The hyperglycemia suppression effect by the sucrose load of the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) of the present invention was examined as follows.
Six-week-old, male, ICR mice (Japan SLC Co., Ltd.) were fasted for 20 hours, and then 100 mg / kg of each sample suspended in distilled water was forcibly orally administered using a stomach tube. Furthermore, sucrose (Wako Pure Chemical Industries, Ltd.) dissolved in distilled water was orally administered at 4 g / kg, and blood was collected from the fundus vein using a hematocrit capillary tube (Terumo Corporation) 30, 60, and 90 minutes after administration. . The blood was centrifuged at 2,500 rpm for 10 minutes, serum was collected, and glucose concentration was measured using Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd.). The details of each sample used are the same as in Example 4. As a control, only distilled water was administered instead of each sample.
The results are shown in FIG. In the control group, sucrose, which is a disaccharide, was glucose-degraded by glucosidase, an enzyme in the body, and was absorbed from the intestinal tract, and a rapid increase in blood glucose was observed 30 minutes after administration. On the other hand, by administering the lactic acid bacteria fermented grape peel and seed specimen simultaneously with sucrose, the increase in glucose in the blood was moderated, and a significant suppression of blood glucose concentration was observed 30 minutes and 90 minutes after administration. (Figure 5,-○-). From this, it was determined that lactic acid bacteria fermented grape peels and seeds can suppress an increase in blood glucose level after meals and are effective in the prevention and treatment of diabetes. On the other hand, this effect is not observed in samples other than lactic acid bacteria fermented grape skin and seeds, indicating that fermentation of grape skin and seeds by lactic acid bacteria is important.

(Example 7: Hyperglycemia suppression effect by starch load)
The hyperglycemia inhibitory effect by the starch load of the fermented grape skin / seed lactic acid bacteria (lactic acid fermented grape skin / seed) of the present invention was examined as follows.
Six-week-old, male, ICR mice (Japan SLC Co., Ltd.) were fasted for 20 hours, and then 100 mg / kg of each sample suspended in distilled water was forcibly orally administered using a stomach tube. Furthermore, potato starch (Sigma) dissolved in distilled water was orally administered at 4 g / kg, and blood was collected from the fundus vein using a hematocrit capillary tube (Terumo Corporation) 30, 60, and 90 minutes after administration. The blood was centrifuged at 2,500 rpm for 10 minutes, serum was collected, and glucose concentration was measured using Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd.). The details of each sample used are the same as in Example 4. As a control, only distilled water was administered instead of each sample.
The results are shown in FIG. In the control group, starch, which is a polysaccharide, was glucose-degraded by amylase and glucosidase, which are internal enzymes, and was absorbed from the intestinal tract, and a rapid increase in blood glucose was observed 30 minutes after administration. On the other hand, by administering the lactic acid bacteria fermented grape peel and seed specimen at the same time as the starch, the rise in glucose in the blood is moderated, and a significant suppression of blood glucose concentration can be achieved 30 minutes, 60 minutes and 90 minutes after administration. It was recognized (FIG. 6,-○-). From this, it was determined that lactic acid bacteria fermented grape peels and seeds can suppress an increase in blood glucose level after meals and are effective in the prevention and treatment of diabetes. On the other hand, this effect is not observed in samples other than lactic acid bacteria fermented grape skin and seeds, indicating that fermentation of grape skin and seeds by lactic acid bacteria is important.

(Example 8: Blood triglyceride (TG) increase suppression action by olive oil load)
The inhibitory effect on blood triglyceride (TG) elevation by the olive oil load of the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) of the present invention was examined as follows.
Six-week-old, male, ICR mice (Japan SLC Co., Ltd.) were fasted for 20 hours, and then 100 mg / kg of each sample suspended in distilled water was forcibly orally administered using a stomach tube. 30 minutes after sample administration, olive oil (Wako Pure Chemical Industries, Ltd.) was orally administered at 5 ml / kg, and blood was collected from each mouse 90 minutes after olive oil administration. The blood was centrifuged at 2,500 rpm for 10 minutes, the serum was collected, and the triglyceride concentration was measured using Triglyceride E-Test Wako (Wako Pure Chemical Industries, Ltd.). In addition, as a sample, the lactic acid bacteria fermented grape skin / seed (experimental group) prepared by the [Sample Preparation Method] and the grape skin / seed dry powder (untreated grape skin / seed used in the [Sample Preparation Method]) Seed; control group). As a control, only distilled water was administered instead of the specimen.
The results are shown in FIG. In the control group, a significant increase in blood TG concentration was observed 90 minutes after the olive oil administration. On the other hand, significant suppression of blood TG concentration was observed by administering the lactic acid bacteria fermented grape skin / seed specimen before administration of olive oil (FIG. 7). From this, it was determined that lactic acid bacteria fermented grape peels and seeds can suppress an increase in blood TG concentration after a meal and are effective in the prevention and treatment of hyperlipidemia. On the other hand, this effect is not observed in untreated grape skins and seeds, indicating that fermentation of grape skins and seeds by lactic acid bacteria is important.

(Example 9: Anti-diabetic action using type II diabetic mice)
The present invention uses KKA ^y mice that are used as type II diabetes model mice and are known to cause metabolic syndrome-related symptoms such as visceral fat accumulation, insulin resistance, obesity, hyperglycemia, and hypertriglyceridemia. The effects of fermented grape skin and seed lactic acid bacteria (lactic acid bacteria fermented grape skin and seed) on metabolic syndrome-related symptoms were examined as follows.
Using 5-week-old, male, KKA ^y / TaJcl mice (CLEA Japan, Inc.), 150 mg / kg of each sample suspended in distilled water was orally administered by gavage twice a day using a stomach tube. The administration was performed 5 times a week for 5 weeks, and blood and intraperitoneal fat were collected from each mouse on the day after the final administration. The blood is centrifuged at 2,500 rpm for 10 minutes, and then the serum is collected, and glucose and triglyceride concentrations are determined using glucose CII test Wako (Wako Pure Chemical Industries, Ltd.) and triglyceride E-test Wako (Wako Pure Chemical Industries, Ltd.). The adiponectin concentration was further measured using a CircuLex mouse adiponectin ELISA kit (CircuLex; CycLex). Intraperitoneal fat was weighed. In addition, as a sample, the lactic acid bacteria fermented grape skin / seed (experimental group) prepared by the [Sample Preparation Method] and the grape skin / seed dry powder (untreated grape skin / seed used in the [Sample Preparation Method]) Seed; control group). As a control, only distilled water was administered instead of the specimen.
The results are shown in FIGS. 8A to 8F.
FIG. 8A shows the appearance of a 9-week-old mouse. In the control group of mice, markedly obese appearance was observed, whereas in the lactic acid bacteria fermented grape skin / seed sample administration group, obesity was not observed.
FIG. 8B shows weight gain. In the control group, a significant increase in body weight was observed after 5 weeks (9 weeks of age) compared to the time when the experiment was started (5 weeks of age). On the other hand, in the lactic acid bacteria fermented grape skin / seed sample administration group, significant suppression of weight gain was observed at 9 weeks of age compared to the control group.
FIG. 8C shows the intraperitoneal fat weight in 9-week-old mice. Compared with the control group, mice in the lactic acid bacteria fermented grape skin / seed sample administration group showed a significant suppression of increase in intraperitoneal fat weight.
FIG. 8D shows the serum triglyceride (TG) concentration. In the control group, a significant increase in serum triglyceride concentration was observed after 5 weeks (9 weeks of age), compared to when the experiment was started (5 weeks of age). On the other hand, in the lactic acid bacteria fermented grape peel / seed sample administration group, a significant increase in serum triglyceride concentration was observed at 9 weeks of age, compared to the control group.
FIG. 8E shows the serum glucose concentration. In the control group, a significant increase in serum glucose concentration was observed after 5 weeks (9 weeks of age) compared to when the experiment was started (5 weeks of age). On the other hand, in the lactic acid bacteria fermented grape skin / seed sample administration group, no increase in glucose concentration was observed even at 9 weeks of age, and a significant suppression of increase in serum glucose concentration was observed compared to the control group.
FIG. 8F shows the serum adiponectin concentration in 9-week-old mice. Compared with the control group, a significant increase in the adiponectin concentration was observed in the mice in the lactic acid bacteria fermented grape peel / seed sample administration group.
In KKA ^y type II diabetes model mice that develop diabetes associated with obesity and exhibit various metabolic syndrome-related symptoms, administration of lactic acid bacteria fermented grape peel and seed specimens suppresses obesity, suppresses blood triglyceride elevation, Suppression of the increase in value was observed, and further, an increase was observed for adiponectin, which has been attracting attention as an anti-metabolic syndrome factor. From these results, the fermented grape skin / seed lactic acid bacteria (lactic acid fermented grape skin / seed) of the present invention are effective for the prevention and treatment of metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity and the like. It was judged. On the other hand, these effects are not observed in untreated grape skin / seed, and it is understood that fermentation of grape skin / seed by lactic acid bacteria is important (FIGS. 8B to 8F).

(Example 10: Adiponectin production inducing action from adipocytes)
The adiponectin production inducing action from adipocytes of the fermented grape skin / seed lactic acid bacteria (lactic acid fermented grape skin / seed) of the present invention was examined as follows.
2 × 10 ⁵ adipocyte progenitor cells (3T3-L1; Sumitomo Dainippon Pharma Co., Ltd.) are seeded in a 24-well plate, and cultured at 37 ° C. carbon dioxide using a culture medium for adipose precursor cells (Dainippon Sumitomo Pharma Co., Ltd.). Cultivated in a vessel for 3 days. After the culture, the medium was replaced with an adipocyte differentiation medium (Dainippon Sumitomo Pharma Co., Ltd.), and further cultured for 3 days to differentiate into adipocytes. After differentiation, the medium was changed to an adipocyte culture medium (Dainippon Sumitomo Pharma Co., Ltd.) and cultured for 7 days for use in experiments.
0.1 and 1 mg / ml specimens were added to each well of the differentiated adipocyte plate and cultured in a 37 ° C. carbon dioxide incubator for 24 hours. In addition, as a specimen, lactic acid bacteria fermented grape skin and seeds produced by the above-mentioned [Sample Preparation Method] were used. In addition, the same amount of purified water was added to the control. After culturing, the plate was centrifuged at 1,000 rpm for 5 minutes, the supernatant was collected, and the amount of adiponectin contained in the supernatant was measured using an ELISA kit (adiponectin mouse EIA kit t; MBL). The graph shows the average value and standard error performed with 3 holes in each group.
The results are shown in FIG. By adding and cultivating lactic acid bacteria fermented grape peels and seeds, the amount of adiponectin produced from adipocytes can be increased in a dose-dependent manner, and a significant increasing effect was observed at a dose of 1 mg / ml (FIG. 9). ). Lactic acid bacteria fermented grape peels and seeds are effective in preventing and treating metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity, etc. I know that there is.

(Example 11: Antagonistic effect on inhibition of adiponectin production by TNF-α)
The antagonistic effect of TNF-α on the inhibition of adiponectin production by the fermented grape skin / seed lactic acid bacteria of the present invention (lactic acid fermented grape skin / seed) was examined as follows.
A 1 mg / ml sample and each concentration of TNF-α were added to each well of a plate of adipocytes differentiated in the same manner as in Example 10, and cultured in a 37 ° C. carbon dioxide incubator for 24 hours. In addition, as a specimen, lactic acid bacteria fermented grape skin and seeds produced by the above-mentioned [Sample Preparation Method] were used. For control, TNF-α and the same amount of purified water were added instead of the specimen. After culturing, the plate was centrifuged at 1,000 rpm for 5 minutes, the supernatant was recovered, and the amount of adiponectin contained in the supernatant was measured using an ELISA kit.
The results are shown in FIG. Adiponectin production was decreased by treating adipocytes with TNF-α. On the other hand, the addition of lactic acid bacteria fermented grape skin and seeds showed a significant inhibitory effect on the decrease in adiponectin production by TNF-α treatment (FIG. 10). Lactic acid bacteria fermented grape peels and seeds prevent or treat metabolic syndrome-related symptoms such as type II diabetes, hyperlipidemia, obesity, etc. It turns out to be effective.

(Example 12: Inhibition of TNF-α production from AGE- stimulated macrophages)
The suppressive action of TNF-α production from AGE- stimulated macrophages of the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) of the present invention was examined as follows.
Ten week-old male C57BL / 6 mice (Japan SLC Co., Ltd.) were intraperitoneally administered with 10% thioglycolate (Difco), and 3 days later, peritoneal cells were collected. The cells were washed with RPMI medium (Gibco), suspended in RPMI medium containing 10% fetal calf serum (FCS), and adjusted to a concentration of 2 × 10 ⁶ cells / ml. After adding 500 μl of cell solution to each hole of 48-well plate and culturing in a 37 ° C. carbon dioxide incubator for 2 hours, 50 μl of terminal glycation product group (AGE) prepared as follows and each concentration sample were added. The cells were cultured in a carbon dioxide incubator for 37 hours. In addition, as a sample, the lactic acid bacteria fermented grape skin / seed (experimental group) prepared by the [Sample Preparation Method] and the grape skin / seed dry powder (untreated grape skin / seed used in the [Sample Preparation Method]) Seed; control group). The same amount of purified water was added to the control hole instead of the specimen. After completion of the culture, the cell supernatant was collected, and the amount of TNF-α contained in the supernatant was quantified using an ELISA kit (BD). The graph shows the control and sample treatment values when the absorbance without AGE addition is taken as 1.
-Production of advanced glycation end products (AGE)-
J. et al. V. This was performed according to the method of Valencia et al. (Non-patent Document Analytical Biochemistry. 2004; 324: 68-78.). That is, 10% bovine serum albumin (BSA; AP company) and 10% D (-) ribose (Wako Pure Chemical Industries, Ltd.) are mixed in equal amounts and reacted at 37 ° C. for 14 days to produce AGE by Maillard reaction. did. The generation of AGE was confirmed by measuring the absorbance at a wavelength of 600 nm with an absorptiometer.
The results are shown in FIG. An increase in induction of TNF-α production from peritoneal cells was observed by AGE stimulation. On the other hand, the addition of lactic acid bacteria fermented grape skin and seeds showed a significant TNF-α production inhibitory effect (FIG. 11). Lactic acid bacteria fermented grape skin and seeds have been shown to suppress the production of inflammatory cytokines by AGE produced by Maillard reaction between sugar and protein. On the other hand, it was shown that a preventive effect can be exhibited. On the other hand, this effect is not observed in untreated grape skins and seeds, indicating that fermentation of grape skins and seeds by lactic acid bacteria is important.

(Example 13: Inhibition of intracellular tyrosinase activity of B16 melanoma cells)
The inhibitory effect on the intracellular tyrosinase activity of B16 melanoma cells of the fermented grape skin / seed lactic acid bacteria (lactic acid fermented grape skin / seed) of the present invention was examined as follows.
B16 melanoma cells were suspended in Dulbecco's modified MEM medium (DMEM; Gibco) containing 10% FCS, adjusted to a concentration of 1 × 10 ⁵ cells / ml, 3 ml of cell solution was added to each well of a 6-well plate, and 37 The cells were cultured for 1 hour in a carbon dioxide gas incubator. After culturing, samples at various concentrations were added, and after further culturing for 2 days, the cell surface was washed with phosphate buffer (PBS), 100 nM phenylmethylsulfonyl fluoride (PMSF; Wako Pure Chemical Industries, Ltd.), and 1% Triton. 300 μl of a phosphate buffer containing X (ICN) was added and freeze-thawed to destroy the cells. In addition, as a sample, the lactic acid bacteria fermented grape skin / seed (experimental group) prepared by the [Sample Preparation Method] and the grape skin / seed dry powder (untreated grape skin / seed used in the [Sample Preparation Method]) Seed; control group). The same amount of purified water was added to the control hole instead of the specimen. The cell lysate was collected and centrifuged at 12,000 rpm for 10 minutes, and then 2 μg / ml L-3- (3,4-dihydrophenyl) alanine (L-DOPA; Tokyo Chemical Industry Co., Ltd.) 20 μl in 80 μl of the supernatant. After reacting at room temperature for 1 day, the amount of melanin returned from L-DOPA by tyrosinase was quantified by measuring the absorbance at a wavelength of 450 nm. The graph shows the control and sample treatment values when the value when PBS and L-DOPA are mixed instead of the cell disruption solution is 1. Each group was shown as an average value and standard error performed with 3 holes.
The results are shown in FIG. L-DOPA was returned by intracellular tyrosinase, and melanin pigment formation was observed. On the other hand, in cells cultured with the addition of lactic acid bacteria fermented grape peels and seeds, a significant melanin pigment production inhibitory action was observed, confirming that the activity of intracellular tyrosinase was suppressed (FIG. 12). From this, it was shown that lactic acid bacteria fermented grape peels and seeds can suppress the production of melanin pigments and exert a whitening effect by inhibiting the activity of intracellular tyrosinase of melanocytes. On the other hand, this effect is not observed in untreated grape skins and seeds, indicating that fermentation of grape skins and seeds by lactic acid bacteria is important.

(Example 14: B16 melanoma cell melanin synthesis inhibitory effect)
The melanin synthesis inhibitory action of B16 melanoma cells of the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) of the present invention was examined as follows.
B16 melanoma cells are suspended in DMEM medium containing 10% FCS, adjusted to a concentration of 1 × 10 ⁵ cells / ml, 3 ml of the cell solution is added to each well of the 6-well plate, and 1 hour in a 37 ° C. carbon dioxide incubator. Cultured. After culturing, 10 μg / ml αMSH (SIGMA) and samples at various concentrations were added, and further cultured for 5 days. In addition, as a sample, the lactic acid bacteria fermented grape skin / seed (experimental group) prepared by the [Sample Preparation Method] and the grape skin / seed dry powder (untreated grape skin / seed used in the [Sample Preparation Method]) Seed; control group). The same amount of purified water was added to the control hole instead of the specimen. After culturing, the cell surface was washed with a phosphate buffer, and the cells were removed by peeling with 0.25% trypsin, and 300 μl of 2N NaOH was added, followed by treatment at 90 ° C. for 30 minutes to destroy the cells. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was collected, and the amount of intracellular melanin was quantified by measuring the absorbance at a wavelength of 450 nm. The values in the graph show the values of control and sample treatment when the absorbance without addition of αMSH is 1. Each group was shown as an average value and standard error performed with 3 holes.
The results are shown in FIG. About 4.5-fold increase in the amount of melanin synthesis was observed by αMSH stimulation. On the other hand, the addition of lactic acid bacteria fermented grape skin and seeds showed a significant inhibitory effect on the amount of melanin (FIG. 13). From this, it was shown that lactic acid bacteria fermented grape peel and seeds can exert a whitening effect by suppressing the production of melanin pigment. On the other hand, this effect is not observed in untreated grape skins and seeds, indicating that fermentation of grape skins and seeds by lactic acid bacteria is important.

(Example 15: Skin inflammation-inhibiting action in UV-irradiated mice)
The skin inflammation inhibitory action in the ultraviolet (UVB) irradiation mouse | mouth of the grape skin and seed lactic-acid-bacteria fermented material (lactic acid bacteria fermented grape skin and seed) of this invention was examined as follows.
A 10-week-old male BALB / c mouse (Japan SLC Co., Ltd.) was shaved on the back hair and irradiated with UVB corresponding to 600 mJ / cm ² . One day after irradiation, 200 μl of 1% Evans Blue dye (Wako Pure Chemical Industries, Ltd.) was intravenously administered, and the amount of dye leaked to the inflamed site was quantified by the following method. That is, 30 minutes after administration of Evans Blue, 1 cm ² of the back skin was cut out and dissolved in 0.25 ml of 1N potassium hydroxide solution for 24 hours. After neutralizing with 0.125 ml of 3N phosphoric acid solution, 0.325 ml of acetone was added to extract the dye. After centrifuging at 2500 rpm for 10 minutes, the acetone layer was collected, and the amount of dye was quantified by measuring the absorbance at a wavelength of 595 nm. The specimen was orally administered at a dose of 300 mg / kg once a day from 4 days before UV irradiation to 30 minutes before irradiation for a total of 5 times. In addition, as a sample, the lactic acid bacteria fermented grape skin / seed (experimental group) prepared by the [Sample Preparation Method] and the grape skin / seed dry powder (untreated grape skin / seed used in the [Sample Preparation Method]) Seed; control group). The same amount of purified water was administered to the control group instead of the specimen. The graph shows the values of the control and sample-administered mice when the value of the mice not irradiated with ultraviolet rays was set to 1 when the test was conducted with 4 animals per group.
The results are shown in FIG. Leakage of Evans blue pigment in the area was observed by ultraviolet irradiation, and skin inflammation was confirmed. On the other hand, a significant inhibitory effect on pigment leakage was observed in mice to which lactic acid bacteria fermented grape skin and seeds were orally administered (FIG. 14). From this, it was shown that lactic acid bacteria fermented grape peels and seeds can suppress skin inflammation caused by ultraviolet rays when administered orally. On the other hand, this effect is not observed in untreated grape skins and seeds, indicating that fermentation of grape skins and seeds by lactic acid bacteria is important.

(Example 16: Analysis of grape skin / seed component change by lactic acid bacteria fermentation)
The components of the grape skin / seed lactic acid bacteria fermented product of the present invention (lactic acid bacteria fermented grape skin / seed) were examined as follows.
Samples for analysis of lactic acid bacteria fermented grape skin and seed specimens were prepared by separation and fractionation by the method shown in FIG. Specifically, 1 g of dry powder (dry powder) obtained from 10 g of Koshu peel and seeds was suspended in 10 ml of PBS, and each lactic acid bacterium (L. plantarum (KU146), L. plantarum (KU551), L. salivavarius (Yu13)) was suspended. )) Was inoculated to a concentration of 1 × 10 ¹¹ CFU / ml, and after fermentation at 37 ° C. for 48 hours (2 days), the culture solution was filtered and separated into a filtrate and a residue. The filtrate was extracted with n-hexane three times to obtain an n-hexane fraction (2.3 mg). Further, the aqueous layer was extracted three times with ethyl acetate to obtain an ethyl acetate fraction (11.1 mg). Similarly, a chloroform fraction (2.0 mg), an n-butanol fraction (40.0 mg), and a water fraction (257.0 mg) were obtained. On the other hand, the residue was extracted three times with hot methanol to obtain a methanol fraction (80.0 mg).
As a result of analyzing the obtained fraction using TLC and HPLC, no component change due to lactic acid bacteria fermentation was observed in the n-hexane fraction, but the ethyl acetate fraction (FIGS. 16A and 16B), the chloroform fraction ( 17A and 17B), n-butanol fraction (FIGS. 18A and 18B), and methanol fraction (FIGS. 19A and 19B). The analysis conditions of TLC and HPLC are as shown in FIGS. 16A to 19B. More specifically, the following component changes were observed in the lactic acid bacteria fermented grape skin and seed fractions.
The ethyl acetate fraction in TLC chromatogram (in FIG. 16A, arrows) spot _{R f} values 0.10 to 0.20 loss is confirmed (FIG. 16A), the HPLC _{chromatogram,} near t R 6min A decrease in peak and an increase in peak around t _R 8 min and 13 min were confirmed (FIG. 16B).
In the chloroform fraction, in the TLC chromatogram, a yellow spot (arrow, dark spot in FIG. 17A) has an R _f value of 0.42, and a reddish purple spot (arrow, dark spot in FIG. 17A) has an R _f value of 0.02. Part) newly appeared (FIG. 17A). In the HPLC chromatogram, new peaks were confirmed around t _R 4.5, 6-7, and 11 min (FIG. 17B).
In the n-butanol fraction, the size of the brown spot (arrow, dark spot portion in FIG. 18A) having an R _f value of 0.20 to 0.25 decreased in the TLC chromatogram (FIG. 18A). In the HPLC chromatogram, the peak near t _R 6 min increased (10%), and the peak near 26 min increased (1%) (FIG. 18B).
In the methanol fraction, the size of the brown spot (arrow, dark spot in FIG. 19A) near the R _f value of 0.25 was reduced in the TLC chromatogram (FIG. 19A). In the HPLC chromatogram, a new peak was confirmed around t _R 5 min (FIG. 19B).
From the result of the present Example 16, in each lactic acid bacteria fermented grape skin / seed (in FIG. 16A to FIG. 19B, KU146, KU551, Yu13), the component is untreated grape skin / seed (in FIG. 16A to FIG. 19). Koshu) showed a big change. The grape skin / seed lactic acid bacteria fermented product of the present invention (lactic acid bacteria fermented grape skin / seed) is an excellent treatment for metabolic syndrome-related symptoms as shown in Examples 1 to 11 as a result of such component changes, It is considered that the improvement, the preventive effect, and the skin whitening / aging prevention effect as shown in Examples 12 to 15 can be achieved.

  The grape skin / seed lactic acid bacteria fermented product of the present invention, and the pharmaceutical and food of the present invention using the grape skin / seed lactic acid bacteria fermented product are, for example, metabolic syndrome related symptoms such as type II diabetes, hyperlipidemia, obesity, etc. It can be suitably used for treatment, improvement or prevention, and skin whitening / aging prevention.

Claims (1)

A medicine for skin whitening and anti-aging of grape skin and seed lactic acid bacteria fermented product obtained by pulverizing and drying grape skin and seeds, fermenting the pulverized product with lactic acid bacteria and heat-treating the grapes, Lactobacillus lactic acid bacteria are added at a concentration of 1 × 10 ¹¹ CFU / ml or more to 20 ml of 10% by weight phosphate buffer of the pulverized product, and 10 to 40 ° C. for 48 hours. pharmaceutical, characterized by comprising obtained by fermentation to 144 hours.