JP4990297B2 - Grape peel / seed lactic acid bacteria fermented product and medicine using the same - Google Patents
Grape peel / seed lactic acid bacteria fermented product and medicine using the same Download PDFInfo
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- JP4990297B2 JP4990297B2 JP2008556104A JP2008556104A JP4990297B2 JP 4990297 B2 JP4990297 B2 JP 4990297B2 JP 2008556104 A JP2008556104 A JP 2008556104A JP 2008556104 A JP2008556104 A JP 2008556104A JP 4990297 B2 JP4990297 B2 JP 4990297B2
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- lactic acid
- acid bacteria
- seed
- grape skin
- lactobacillus
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Description
本発明は、ブドウの果皮及び種子の少なくともいずれかを利用した、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を治療、改善、又は予防するための医薬及び食品、並びに、皮膚の美白・老化防止のための医薬及び食品に関する。 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.
メタボリックシンドロームとは、内臓脂肪の蓄積により引き起こされる、肥満、糖尿病、高血圧、高脂血症等の生活習慣病が重なった状態をいい、この状態が続くと動脈硬化が進行し易くなり、心筋梗塞や脳卒中を起こす危険性が増加する。最近の厚生労働省の調査によると、メタボリックシンドロームとその予備群は中高年世代で2,000万人近いことが示され、中高年男性の2人に1人はメタボリックシンドロームかその予備軍であると考えられている。
メタボリックシンドロームを引き起こす原因は、過栄養、運動不足、ストレス過剰、高脂肪食過剰摂取等の生活習慣に基づく内臓脂肪の蓄積と考えられている。この内臓脂肪の蓄積により、血中グルコース量や血中中性脂肪量、血圧等を調節するインスリンに対する感受性が低下し、インスリン抵抗性と呼ばれる状態を作り出し、肥満、糖尿病、高血圧、高脂血症や、動脈硬化等を引き起こす。
インスリン抵抗性を引き起こす因子としては、マクロファージや肥大肥満細胞から産生される腫瘍壊死因子−α(TNF−α)が考えられており、インスリンのシグナル伝達は、このTNF−αにより阻害されることが示されている(例えば、非特許文献1参照)。これに対し、脂肪細胞から産生されるアディポネクチンは、TNF−αに拮抗的な作用を示し、インスリンに対する感受性を増強させることが知られている(例えば、非特許文献2参照)。更に近年、高脂肪食による肥満などの環境因子により、アディポネクチンの欠乏が惹起され、インスリンに対する感受性が低下することが示され、アディポネクチンは糖尿病やメタボリックシンドロームの病態評価の指標、及び治療のターゲット分子として期待されている(例えば、非特許文献3参照)。
これらのことから、メタボリックシンドロームの治療、改善、又は予防においては、内臓脂肪、血中グルコース、血中中性脂肪などの肥満、糖尿病誘導因子を減少させ、一方で、アディポネクチンなどの抗糖尿病作用、抗メタボリックシンドローム作用を有する因子を誘導することが重要であると考えられる。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.
また、体内に炭水化物が摂取されると、消化管においてα−アミラーゼ、α−グルコシダーゼによって消化され、最終的に単糖類であるグルコースに分解されて、腸管膜上の繊毛から吸収される。吸収されたグルコースは血中に移行し、一時的に過血糖症状が起こる。II型糖尿病患者においては、インスリンの作用がはたらかず、高血糖値が維持され、グルコースによる血管障害が惹起されて、神経障害、網膜症、白内障など重篤な合併症が引き起こされる。
これに対して、α−アミラーゼ、α−グルコシダーゼ等の消化酵素の働きを阻害し、グルコースの生成を抑制することで、摂取された糖類は吸収されることなく体外へ排出され、炭水化物摂取による血糖値上昇は抑制される。
肥満遺伝子であるAy遺伝子を導入したマウスであるKKAyマウスは、II型糖尿病モデルマウスとして用いられており、肥満、インスリン抵抗性、高中性脂肪血症など、メタボリックシンドロームの症状をきたすことが知られている。また、メタボリックシンドロームの症状をきたした時には、血中アディポネクチンは著明に減少することが確認されており、更に、遺伝子組換えアディポネクチンの補給により、インスリン抵抗性や高中性脂肪血症が改善されることが報告されている(例えば、非特許文献2参照)。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).
このようなII型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を治療、改善、又は予防する方法としては、従来から様々な方法が提唱されているが、前記したようにメタボリックシンドローム及びその予備群の患者数は非常に多く、そのため、より効果的、かつ安全な治療、改善、又は予防方法が、未だ求められているのが現状である。 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.
一方で、皮膚の美白や老化防止を望む人の数も、女性を中心として非常に多い。皮膚の老化は、加齢に加えて、炎症に起因するところが大きく、皮膚の炎症は肌の硬化、しわ形成、はり・つやの喪失の原因と考えられている。炎症の原因としては紫外線によるものが一般的であるが、近年、メイラード反応により生成する終末糖化産物群(Advanced Glycation End Products;AGE)の関与が注目されている。メイラード反応はタンパク質と糖の非酵素的反応であり、生成されたAGEはマクロファージなどの炎症性細胞の細胞膜レセプター(RAGE)を介して活性化し、TNF−αなどの炎症性サイトカインの産生を誘導する。タンパク質の非酵素的糖化は通常の老化により進行するが、特に前記したようなメタボリックシンドロームなど、血糖値が高い人ではその進行が速く、皮膚老化促進の原因のひとつとなっている(例えば、非特許文献4参照)。 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).
また、紫外線を浴びた皮膚では、ケラチノサイトからαメラノサイト刺激ホルモン(α−MSH)と呼ばれる物質が産生され、α−MSHはメラノサイトにはたらきメラニン合成酵素であるチロシナーゼを活性化させる。メラノサイト内にあるチロシンは、チロシナーゼの作用によりドーパ(DOPA)を経てドーパキノン(DOPAquinone)に変換され、ユーメラニン(Eumelanin)やフェオメラニン(Pheomelanin)などのメラニンが生成されて皮膚への沈着が起こり、肌のくすみの原因となる(例えば、非特許文献5参照)。 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).
AGEによって誘導される炎症性サイトカインの産生を抑制し、細胞内チロシナーゼの活性阻害によりメラニン生成を抑制する物質は、皮膚の美白、老化防止において優れた効果を発揮することが考えられ、このような物質が求められているのが現状である。 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.
本発明は、前記従来における諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、ブドウの果皮及び種子の少なくともいずれかを利用し、有用なブドウ果皮・種子乳酸菌発酵物を提供することを目的とする。また、前記ブドウ果皮・種子乳酸菌発酵物を利用し、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を効果的、かつ安全に治療、改善、又は予防することが可能な医薬及び食品、並びに、皮膚の美白・老化防止に効果的、かつ安全な医薬及び食品を提供することを目的とする。 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.
前記課題を解決するため、本発明者らは鋭意検討した結果、以下のような知見を得た。即ち、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させたブドウ果皮・種子乳酸菌発酵物が、優れた血糖値上昇抑制作用、血中トリグリセリド(中性脂肪)上昇抑制作用、肥満抑制作用、血中アディポネクチン上昇作用等を有しており、そのため、前記ブドウ果皮・種子乳酸菌発酵物を利用することにより、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を、効果的、かつ安全に治療、改善、又は予防することができるという知見である。また、前記ブドウ果皮・種子乳酸菌発酵物は、メイラード反応により生成した終末糖化産物群(AGE)に起因するTNF−α産生を抑制する作用、チロシナーゼ活性抑制作用、メラニン合成抑制作用、紫外線による皮膚炎症を抑制する作用等も有しており、そのため、皮膚の美白・老化防止にも効果的であるという知見である。 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.
近年、植物において、果汁よりも、果皮や種子に多量のフラボノイド類が含まれていることが報告され、これらの未利用農産物が注目され始めている。フラボノイド類は酵素と結合し、その活性を阻害することが知られており、過食等によって引き起こされるメタボリックシンドロームの予防、治療を対象とした機能性食品として開発が行われている。しかしながら、既存のフラボノイド類は経口投与したときの吸収面で難点があり、その効果は抽出物、精製物においても十分発揮できていないのが現状である。
本発明者らは、まず、ブドウ果皮及び種子の粉末(ブドウ果皮・種子乾燥パウダー)との培養で好適に増殖する乳酸菌のスクリーニングを行い、適した乳酸菌を選択した。そして、この乳酸菌を用いてブドウ果皮・種子乾燥パウダーを発酵処理することにより、消化酵素(アミラーゼ、グルコシダーゼ)に対する阻害活性が上昇し、かつ、糖負荷マウスに経口投与したときの過血糖症状抑制効果についてもより有効な物質(ブドウ果皮・種子乳酸菌発酵物)が得られることを見出した。更に、得られたブドウ果皮・種子乳酸菌発酵物は、肥満に伴い糖尿病の症状を発症するモデルマウスにおいても、著明な血糖値上昇抑制作用、血中トリグリセリド(中性脂肪)上昇抑制作用、肥満抑制作用等が認められること、また、抗糖尿病因子、抗メタボリックシンドローム因子として注目されているアディポネクチンを顕著に上昇させる作用を有していることを見出した。また、本発明者らは更に、前記ブドウ果皮・種子乳酸菌発酵物は、ウシ血清アルブミン(BSA)とリボースで生成したAGEにより誘導されたマクロファージからのTNF−α産生を抑制できること、B16メラノーマ細胞の細胞内チロシナーゼ活性を抑制し、α−MSH刺激によるメラニン合成を抑制できること、マウスへの経口投与により紫外線による皮膚炎症を抑制できることをも見出し、本発明の完成に至った。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.
本発明は、本発明者らによる前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
<1> ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られることを特徴とするブドウ果皮・種子乳酸菌発酵物である。
<2> 乾燥した状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られる前記<1>に記載のブドウ果皮・種子乳酸菌発酵物である。
<3> 微粉(パウダー)化された状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られる前記<1>から<2>のいずれかに記載のブドウ果皮・種子乳酸菌発酵物である。
<4> 乾燥粉末(乾燥パウダー)の状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られる前記<1>から<3>のいずれかに記載のブドウ果皮・種子乳酸菌発酵物である。
<5> 乳酸菌として、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ラクトバチル キタサトニス(Lactobacillus kitasatonisi)、ラクトバチル ロイテリ(Lactobacillus reuteri)、ラクトバチル ジョンソニ(Lactobacillus jhonsonii)、ラクトバチルス アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス ブレビス(Lactobacillus brevis)、ラクトバチルス コリニホルミス(Lactobacillus coryniformis)、ラクトバチルス カルバタス(Lactobacillus curvatus)、ラクトバチルス デルブリュキィ(Lactobacillus delbrueckii)、ラクトバチルス ファルシミナス(Lactobacillus farciminus)、ラクトバチルス ラムノサス(Lactobacillus rhamnosus)、ペディオコッカス ダムノサス(Pediococcus damnosusu)、ペディオコッカス ハロフィラス(Pediococcus halophilus)、ペディオコッカス パルバラス(Pediococcus parvulus)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus)、ビフィドバクテリウム アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム ビフィダス((Bifidobacterium bifidus)、ラクトコッカス クレモリス(Lactococcus cremoris)、ラクトコッカス ラクティス(Lactococcus lactis)、リューコノストック エノス(Leuconostoc oenos)、リューコノストック ラクティス(Leuconostoc lactis)、リューコノストック メセンテロイデス(Leuconostoc mesenteroides)、及び、エンテロコッカス フェカリス(Enterococcus faecalis)からなる群より選択される少なくともいずれかを使用して得られる前記<1>から<4>のいずれかに記載のブドウ果皮・種子乳酸菌発酵物である。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> メタボリックシンドローム関連症状を治療又は予防するための医薬であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含むことを特徴とする医薬である。
<7> 乾燥した状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<6>に記載の医薬である。
<8> 微粉(パウダー)化された状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<6>から<7>のいずれかに記載の医薬である。
<9> 乾燥粉末(乾燥パウダー)の状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<6>から<8>のいずれかに記載の医薬である。
<10> 乳酸菌として、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ラクトバチル キタサトニス(Lactobacillus kitasatonisi)、ラクトバチル ロイテリ(Lactobacillus reuteri)、ラクトバチル ジョンソニ(Lactobacillus jhonsonii)、ラクトバチルス アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス ブレビス(Lactobacillus brevis)、ラクトバチルス コリニホルミス(Lactobacillus coryniformis)、ラクトバチルス カルバタス(Lactobacillus curvatus)、ラクトバチルス デルブリュキィ(Lactobacillus delbrueckii)、ラクトバチルス ファルシミナス(Lactobacillus farciminus)、ラクトバチルス ラムノサス(Lactobacillus rhamnosus)、ペディオコッカス ダムノサス(Pediococcus damnosusu)、ペディオコッカス ハロフィラス(Pediococcus halophilus)、ペディオコッカス パルバラス(Pediococcus parvulus)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus)、ビフィドバクテリウム アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム ビフィダス((Bifidobacterium bifidus)、ラクトコッカス クレモリス(Lactococcus cremoris)、ラクトコッカス ラクティス(Lactococcus lactis)、リューコノストック エノス(Leuconostoc oenos)、リューコノストック ラクティス(Leuconostoc lactis)、リューコノストック メセンテロイデス(Leuconostoc mesenteroides)、及び、エンテロコッカス フェカリス(Enterococcus faecalis)からなる群より選択される少なくともいずれかを使用して得られるブドウ果皮・種子乳酸菌発酵物を含む前記<6>から<9>のいずれかに記載の医薬である。
<11> II型糖尿病の治療又は予防に用いられる前記<6>から<10>のいずれかに記載の医薬である。
<12> 高脂血症の治療又は予防に用いられる前記<6>から<10>のいずれかに記載の医薬である。
<13> 肥満の治療又は予防に用いられる前記<6>から<10>のいずれかに記載の医薬である。
<14> 血中グルコース濃度を低下させる作用を有する前記<6>から<10>のいずれかに記載の医薬である。
<15> 血中トリグリセリド濃度を低下させる作用を有する前記<6>から<10>のいずれかに記載の医薬である。
<16> 腹腔内脂肪重量を低下させる作用を有する前記<6>から<10>のいずれかに記載の医薬である。
<17> 血中アディポネクチン濃度を上昇させる作用を有する前記<6>から<10>のいずれかに記載の医薬である。<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> 皮膚の美白・老化防止のための医薬であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含むことを特徴とする医薬である。
<19> 乾燥した状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<18>に記載の医薬である。
<20> 微粉(パウダー)化された状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<18>から<19>のいずれかに記載の医薬である。
<21> 乾燥粉末(乾燥パウダー)の状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<18>から<20>のいずれかに記載の医薬である。
<22> 乳酸菌として、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ラクトバチル キタサトニス(Lactobacillus kitasatonisi)、ラクトバチル ロイテリ(Lactobacillus reuteri)、ラクトバチル ジョンソニ(Lactobacillus jhonsonii)、ラクトバチルス アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス ブレビス(Lactobacillus brevis)、ラクトバチルス コリニホルミス(Lactobacillus coryniformis)、ラクトバチルス カルバタス(Lactobacillus curvatus)、ラクトバチルス デルブリュキィ(Lactobacillus delbrueckii)、ラクトバチルス ファルシミナス(Lactobacillus farciminus)、ラクトバチルス ラムノサス(Lactobacillus rhamnosus)、ペディオコッカス ダムノサス(Pediococcus damnosusu)、ペディオコッカス ハロフィラス(Pediococcus halophilus)、ペディオコッカス パルバラス(Pediococcus parvulus)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus)、ビフィドバクテリウム アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム ビフィダス((Bifidobacterium bifidus)、ラクトコッカス クレモリス(Lactococcus cremoris)、ラクトコッカス ラクティス(Lactococcus lactis)、リューコノストック エノス(Leuconostoc oenos)、リューコノストック ラクティス(Leuconostoc lactis)、リューコノストック メセンテロイデス(Leuconostoc mesenteroides)、及び、エンテロコッカス フェカリス(Enterococcus faecalis)からなる群より選択される少なくともいずれかを使用して得られるブドウ果皮・種子乳酸菌発酵物を含む前記<18>から<21>のいずれかに記載の医薬である。
<23> メイラード反応により生成した終末糖化産物群(AGE)に起因するTNF−α産生を抑制する作用を有する前記<18>から<22>のいずれかに記載の医薬である。
<24> チロシナーゼ活性抑制作用を有する前記<18>から<22>のいずれかに記載の医薬である。
<25> メラニン合成抑制作用を有する前記<18>から<22>のいずれかに記載の医薬である。
<26> 紫外線による皮膚炎症を抑制する作用を有する前記<18>から<22>のいずれかに記載の医薬である。<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> メタボリックシンドローム関連症状を改善又は予防するための食品であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含むことを特徴とする食品である。
<28> 乾燥した状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<27>に記載の食品である。
<29> 微粉(パウダー)化された状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<27>から<28>のいずれかに記載の食品である。
<30> 乾燥粉末(乾燥パウダー)の状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<27>から<29>のいずれかに記載の食品である。
<31> 乳酸菌として、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ラクトバチル キタサトニス(Lactobacillus kitasatonisi)、ラクトバチル ロイテリ(Lactobacillus reuteri)、ラクトバチル ジョンソニ(Lactobacillus jhonsonii)、ラクトバチルス アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス ブレビス(Lactobacillus brevis)、ラクトバチルス コリニホルミス(Lactobacillus coryniformis)、ラクトバチルス カルバタス(Lactobacillus curvatus)、ラクトバチルス デルブリュキィ(Lactobacillus delbrueckii)、ラクトバチルス ファルシミナス(Lactobacillus farciminus)、ラクトバチルス ラムノサス(Lactobacillus rhamnosus)、ペディオコッカス ダムノサス(Pediococcus damnosusu)、ペディオコッカス ハロフィラス(Pediococcus halophilus)、ペディオコッカス パルバラス(Pediococcus parvulus)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus)、ビフィドバクテリウム アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム ビフィダス((Bifidobacterium bifidus)、ラクトコッカス クレモリス(Lactococcus cremoris)、ラクトコッカス ラクティス(Lactococcus lactis)、リューコノストック エノス(Leuconostoc oenos)、リューコノストック ラクティス(Leuconostoc lactis)、リューコノストック メセンテロイデス(Leuconostoc mesenteroides)、及び、エンテロコッカス フェカリス(Enterococcus faecalis)からなる群より選択される少なくともいずれかを使用して得られるブドウ果皮・種子乳酸菌発酵物を含む前記<27>から<30>のいずれかに記載の食品である。
<32> II型糖尿病の改善又は予防に用いられる前記<27>から<31>のいずれかに記載の食品である。
<33> 高脂血症の改善又は予防に用いられる前記<27>から<31>のいずれかに記載の食品である。
<34> 肥満の改善又は予防に用いられる前記<27>から<31>のいずれかに記載の食品である。
<35> 血中グルコース濃度を低下させる作用を有する前記<27>から<31>のいずれかに記載の食品である。
<36> 血中トリグリセリド濃度を低下させる作用を有する前記<27>から<31>のいずれかに記載の食品である。
<37> 腹腔内脂肪重量を低下させる作用を有する前記<27>から<31>のいずれかにに記載の食品である。
<38> 血中アディポネクチン濃度を上昇させる作用を有する前記<27>から<31>のいずれかに記載の食品である。<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> 皮膚の美白・老化防止のための食品であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含むことを特徴とする食品である。
<40> 乾燥した状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<39>に記載の食品である。
<41> 微粉(パウダー)化された状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<39>から<40>のいずれかに記載の食品である。
<42> 乾燥粉末(乾燥パウダー)の状態のブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含む前記<39>から<41>のいずれかに記載の食品である。
<43> 乳酸菌として、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ラクトバチル キタサトニス(Lactobacillus kitasatonisi)、ラクトバチル ロイテリ(Lactobacillus reuteri)、ラクトバチル ジョンソニ(Lactobacillus jhonsonii)、ラクトバチルス アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス ブレビス(Lactobacillus brevis)、ラクトバチルス コリニホルミス(Lactobacillus coryniformis)、ラクトバチルス カルバタス(Lactobacillus curvatus)、ラクトバチルス デルブリュキィ(Lactobacillus delbrueckii)、ラクトバチルス ファルシミナス(Lactobacillus farciminus)、ラクトバチルス ラムノサス(Lactobacillus rhamnosus)、ペディオコッカス ダムノサス(Pediococcus damnosusu)、ペディオコッカス ハロフィラス(Pediococcus halophilus)、ペディオコッカス パルバラス(Pediococcus parvulus)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus)、ビフィドバクテリウム アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム ビフィダス((Bifidobacterium bifidus)、ラクトコッカス クレモリス(Lactococcus cremoris)、ラクトコッカス ラクティス(Lactococcus lactis)、リューコノストック エノス(Leuconostoc oenos)、リューコノストック ラクティス(Leuconostoc lactis)、リューコノストック メセンテロイデス(Leuconostoc mesenteroides)、及び、エンテロコッカス フェカリス(Enterococcus faecalis)からなる群より選択される少なくともいずれかを使用して得られるブドウ果皮・種子乳酸菌発酵物を含む前記<39>から<42>のいずれかに記載の食品である。
<44> メイラード反応により生成した終末糖化産物群(AGE)に起因するTNF−α産生を抑制する作用を有する前記<39>から<43>のいずれかに記載の食品である。
<45> チロシナーゼ活性抑制作用を有する前記<39>から<43>のいずれかに記載の食品である。
<46> メラニン合成抑制作用を有する前記<39>から<43>のいずれかに記載の食品である。
<47> 紫外線による皮膚炎症を抑制する作用を有する前記<39>から<43>のいずれかに記載の食品である。<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.
本発明によれば、前記従来における諸問題を解決し、前記目的を達成することができ、ブドウの果皮及び種子の少なくともいずれかを利用し、有用なブドウ果皮・種子乳酸菌発酵物を提供することができる。更に、前記ブドウ果皮・種子乳酸菌発酵物を利用し、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を効果的、かつ安全に治療、改善、又は予防することが可能な医薬及び食品、並びに、皮膚の美白・老化防止に効果的、かつ安全な医薬及び食品を提供することができる。 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.
(ブドウ果皮・種子乳酸菌発酵物)
本発明のブドウ果皮・種子乳酸菌発酵物は、ブドウの果皮及び種子の少なくともいずれか(果皮・種子)を乳酸菌で発酵させて得られることを特徴とする。なお、本明細書中において、前記ブドウ果皮・種子乳酸菌発酵物を、「乳酸菌発酵ブドウ果皮・種子」等と称することがある。(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.
<ブドウ>
前記ブドウとしては、ブドウ科(Vitaceae)ブドウ属(Vitis)に属する植物であれば特に制限はなく、目的に応じて適宜選択することができ、例えば、ヨーロッパ・ブドウ(ヴィティス・ヴィニフェラ(Vitis vinifera))、アメリカ・ブドウ(ヴィティス・ラブルスカ(Vitis labrusca))、ヴィティス・アムレンシス(Vitis amurensis)、ヴィティス・コイネティアエ(Vitis coignetiae)、これらの交雑種などが挙げられる。前記ブドウの品種としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、甲州(Koshu)、シャルドネ(Chardonnay)、ミュスカ(Muscat)、ソーヴィニヨン・ブラン(Sauvignon Blanc)、セミヨン(Semillon)、リースリング(Riesling)、シルヴァナアー(Silvaner)、ミューラ・トゥルガウ(Muller−Thurgau)、ケルナー(Kerner)、ゲヴュルツトラミネール(Gewurztraminer)、シャスラ(Chasselas)、シュナン・ブラン(Chenin Blanc)、セミヨン(Semillon)、デラウェア(Delaware)、バッカス(Bacchus)、ピノ・グリ(Pinot Gris)、ピノ・プラン(Pinot Blanc)、フルミント(Furminnt)、ミュスカデ(Muscadet)、ミュスカデル(Muscadelle)、リースリング(Riesling)、カベルネ・ソーヴィニヨン(Cabernet Sauvignon)、マスカット・ベリーA(Muscat Bailey A)、ネッビオーロ(Nebbiolo)、グルナッシュ(Grenache)、メルロー(Merlot)、シラー(Syrah)、ネグロアマーロ(Negroamaro)、サンジョヴェーゼ(Sangiovese)、タナ(Tannat)、プリミティーボ(Primitivo)、ブルネエッロ(Brunello)、カベルネ・フラン(Cabernet Franc)、ガメイ(Gamay)、コンコード(Concord)、サンジョヴェーゼ(Sangiovese)、サンソー(Cinsaut)、ジンファンデル(Zinfandel)、セルベル(Seibel)、ツヴァイゲルトレーベ(Zweigeltrebe)、テンプラニーリョ(Tempranillo)、バラベーラ(Barbera)、ピノ・ノワール(Pinot Noir)、ピノ・ムニエ(Pinot Meunier)、プティ・ヴェルドー(Petit Verdot)、マルヴァジア(Malvasia)、マルベック(Malbec)、ムールヴェードル(Mourverder)、巨峰(kyoho)などが挙げられる。<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.
<果皮・種子>
前記果皮・種子としては、前記したブドウの果皮及び種子の少なくともいずれかであれば、特に制限はなく、目的に応じて適宜選択することができ、前記果皮・種子の状態としては、生の状態であってもよいし、乾燥した状態であってもよい。また、前記果皮・種子のサイズとしては、前記果皮及び種子の少なくともいずれかを採取したそのままの状態であってもよいし、所望の大きさに裁断された状態であってもよいし、微粉(パウダー)化された状態であってもよい。中でも、前記果皮・種子は、後述する乳酸菌を作用させ易くする観点から、乾燥した状態であることが好ましく、また、微粉(パウダー)化された状態であることが好ましい。すなわち、前記果皮・種子は、乾燥粉末(乾燥パウダー)の状態であることが特に好ましい。前記果皮・種子の乾燥粉末(乾燥パウダー)を作製する方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、公知の手法を用いて行うことができる。前記果皮・種子の乾燥粉末(乾燥パウダー)の平均粒子径としては、特に制限はなく、目的に応じて適宜選択することができるが、例えば、10〜200メッシュが好ましい。前記平均粒子径が、10メッシュ未満であると、発酵物の活性が弱いことがある。一方、前記平均粒子径が前記特に好ましい範囲内であると、強い活性を有する発酵物が得られる点で、有利である。
また、前記ブドウの果皮・種子としては、その入手方法に制限はなく、目的に応じて適宜選択することができるが、例えば、ワインやジュース等の製造過程で果汁を絞った後のブドウの残り滓を使用することが、コスト性に優れ、かつ、産業廃棄物を減らすことのできる点で、望ましいと考えられる。
また、前記ブドウの果皮・種子としては、前記ブドウの果皮を単独で使用してもよいし、前記ブドウの種子を単独で使用してもよいし、前記ブドウの果皮と種子とを併用してもよい。
ここで、前記ブドウ果皮・種子乳酸菌発酵物の原料としては、その全てが必ずしも前記したブドウの果皮及び種子の少なくともいずれかからなる必要はなく、その少なくとも一部に前記したブドウの果皮及び種子の少なくともいずれかが含まれていればよい。例えば、前記ブドウ果皮・種子乳酸菌発酵物の原料としては、前記ブドウの果皮・種子以外に、前記ブドウの果実、果汁、葉、茎などが適宜含まれていてもよい。前記ブドウの果皮・種子と、前記ブドウの果皮・種子以外との使用量比としては、特に制限はなく、目的に応じて適宜選択することができる。<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.
<乳酸菌>
前記乳酸菌としては、発酵によって乳酸を産生する細菌であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、ラクトバチルス属(Lactobacillus)、ペディオコッカス属(Pediococcus)、ビフィドバクテリウム属(Bifidobacterium)、ラクトコッカス属(Lactococcus)、リューコノストック属(Leuconostoc)、エンテロコッカス属(Enterococcus)に属する細菌などが挙げられる。前記乳酸菌の種としても、特に制限はなく、目的に応じて適宜選択することができるが、中でも、前記したブドウの果皮・種子を炭素源として、効率よく増殖を行うことのできる種を使用することが好ましい。このような好ましい種としては、例えば、ラクトバチルス属として、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ラクトバチル キタサトニス(Lactobacillus kitasatonisi)、ラクトバチル ロイテリ(Lactobacillus reuteri)、ラクトバチル ジョンソニ(Lactobacillus jhonsonii)、ラクトバチルス アシドフィラス(Lactobacillus acidophilus)、ラクトバチルス ブレビス(Lactobacillus brevis)、ラクトバチルス コリニホルミス(Lactobacillus coryniformis)、ラクトバチルス カルバタス(Lactobacillus curvatus)、ラクトバチルス デルブリュキィ(Lactobacillus delbrueckii)、ラクトバチルス ファルシミナス(Lactobacillus farciminus)、ラクトバチルス ラムノサス(Lactobacillus rhamnosus);ペディオコッカス属として、ペディオコッカス ダムノサス(Pediococcus damnosusu)、ペディオコッカス ハロフィラス(Pediococcus halophilus)、ペディオコッカス パルバラス(Pediococcus parvulus)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus);ビフィドバクテリウム属として、ビフィドバクテリウム アドレセンティス(Bifidobacterium adolescentis)、ビフィドバクテリウム ビフィダス((Bifidobacterium bifidus);ラクトコッカス属として、ラクトコッカス クレモリス(Lactococcus cremoris)、ラクトコッカス ラクティス(Lactococcus lactis);リューコノストック属として、リューコノストック エノス(Leuconostoc oenos)、リューコノストック ラクティス(Leuconostoc lactis)、リューコノストック メセンテロイデス(Leuconostoc mesenteroides);エンテロコッカス属として、エンテロコッカス フェカリス(Enterococcus faecalis)などが挙げられる。中でも、前記乳酸菌の種としては、ラクトバチルス プランタラム(Lactobacillus plantarum)が、効率よく増殖し、得られた発酵物の活性が強い点で、特に好ましい。<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.
<発酵>
前記発酵は、前記したブドウの果皮・種子を少なくとも一部に含む原料を、前記乳酸菌を用いて発酵させることにより行うことができる。前記発酵の方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記原料に、前記乳酸菌を添加し、培養する方法などが挙げられる。より具体的には、例えば、後述する実施例に記載の方法により前記発酵を行うことができる。<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.
前記発酵時の乳酸菌の使用量としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記原料10gに対して、1012CFU以上が好ましく、1013CFU以上が特に好ましい。前記乳酸菌の使用量が、1012CFU未満であると、発酵物の活性が弱くなることがある。一方、前記乳酸菌の使用量が前記特に好ましい範囲内であると、強い活性を有する発酵物が得られる点で、有利である。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.
前記発酵時の温度としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、10〜40℃が好ましく、17〜30℃が特に好ましい。前記温度が、10℃未満であると、菌の増殖が悪くなることがあり、40℃を超えると、菌が生育できなくなることがある。一方、前記温度が前記特に好ましい範囲内であると、強い活性を有する発酵物が得られる点で、有利である。 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.
前記発酵時間としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、48〜144時間が好ましく、72時間が特に好ましい。前記発酵時間が、48時間未満であると、発酵物の活性が弱いことがある。一方、前記発酵時間が、前記特に好ましい範囲内であると、強い活性を有する発酵物が得られる点で、有利である。 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.
前記発酵により、本発明のブドウ果皮・種子乳酸菌発酵物を得ることができる。なお、前記ブドウ果皮・種子乳酸菌発酵物は、後述する実施例に示されるように、未発酵のブドウ果皮・種子とは異なる成分からなるものであり、そのため、新規の組成物であるということができる。
前記ブドウ果皮・種子乳酸菌発酵物は、そのままで、又は適宜加工することによって、様々な用途に使用することが可能である。例えば、後述する実施例に示されるように、前記ブドウ果皮・種子乳酸菌発酵物は、優れた血糖値上昇抑制作用、血中トリグリセリド(中性脂肪)上昇抑制作用、肥満抑制作用、血中アディポネクチン上昇作用等を有しており、そのため、前記ブドウ果皮・種子乳酸菌発酵物は、例えば、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を治療、改善、又は予防することを目的とした、後述する本発明の第1の医薬及び食品の有効成分として、好適に利用可能である。また、前記ブドウ果皮・種子乳酸菌発酵物は、メイラード反応により生成した終末糖化産物群(AGE)に起因するTNF−α産生を抑制する作用、チロシナーゼ活性抑制作用、メラニン合成抑制作用、紫外線による皮膚炎症を抑制する作用等にも優れ、そのため、前記ブドウ果皮・種子乳酸菌発酵物は、例えば、皮膚の美白・老化防止を目的とした、後述する本発明の第2の医薬及び食品の有効成分としても、好適に利用可能である。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.
(医薬)
本発明の第1の医薬は、メタボリックシンドローム関連症状を治療又は予防するための医薬であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含み、必要に応じて適宜その他の成分を含む。
また、本発明の第2の医薬は、皮膚の美白・老化防止のための医薬であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含み、必要に応じて適宜その他の成分を含む。
なお、本明細書中において、前記「第1の医薬」及び前記「第2の医薬」を単に「医薬」と総称することがある。(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”.
<ブドウ果皮・種子乳酸菌発酵物>
前記ブドウ果皮・種子乳酸菌発酵物は、ブドウの果皮及び種子の少なくともいずれか(果皮・種子)を乳酸菌で発酵させて得られるものであり、前記「ブドウ」、「果皮・種子」、「乳酸菌」、「発酵」としては、前記した本発明のブドウ果皮・種子乳酸菌発酵物の項目に記載した通りである。したがって、前記医薬におけるブドウ果皮・種子乳酸菌発酵物としては、前記した本発明のブドウ果皮・種子乳酸菌発酵物を好適に使用することができる。
前記医薬中の前記ブドウ果皮・種子乳酸菌発酵物の含有量は、特に制限はなく、目的に応じて適宜選択することができ、また、前記医薬は、前記ブドウ果皮・種子乳酸菌発酵物そのものであってもよい。<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.
<その他の成分>
前記その他の成分としては、特に制限はなく、本発明の効果を損なわない範囲内で、目的に応じて適宜選択することができ、例えば、医薬的に許容され得る担体などが挙げられる。前記担体としては、特に制限はなく、例えば、後述する前記医薬の剤型等に応じて適宜選択することができる。また、前記医薬中の前記その他の成分の含有量としても、特に制限はなく、目的に応じて適宜選択することができる。<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.
<剤型>
前記医薬の剤型としては、特に制限はなく、例えば、所望の投与方法に応じて適宜選択することができ、例えば、経口固形剤(錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等)、経口液剤(内服液剤、シロップ剤、エリキシル剤等)、注射剤(溶液、懸濁液、用事溶解用固形剤等)、坐剤、軟膏剤、貼付剤、ゲル剤、クリーム剤、外用散剤、スプレー剤、吸入散剤などが挙げられる。<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.
前記経口固形剤としては、例えば、前記ブドウ果皮・種子乳酸菌発酵物に、賦形剤、更には必要に応じて結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等の添加剤を加え、常法により製造することができる。
前記賦形剤としては、例えば、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸などが挙げられる。前記結合剤としては、例えば、水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドンなどが挙げられる。前記崩壊剤としては、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖などが挙げられる。前記滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコールなどが挙げられる。前記着色剤としては、例えば、酸化チタン、酸化鉄などが挙げられる。前記矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。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.
前記経口液剤としては、例えば、前記ブドウ果皮・種子乳酸菌発酵物に、矯味・矯臭剤、緩衝剤、安定化剤等の添加剤を加え、常法により製造することができる。
前記矯味・矯臭剤としては、例えば、白糖、橙皮、クエン酸、酒石酸などが挙げられる。前記緩衝剤としては、例えば、クエン酸ナトリウムなどが挙げられる。前記安定化剤としては、例えば、トラガント、アラビアゴム、ゼラチンなどが挙げられる。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.
前記注射剤としては、例えば、前記ブドウ果皮・種子乳酸菌発酵物に、pH調節剤、緩衝剤、安定化剤、等張化剤、局所麻酔剤等を添加し、常法により皮下用、筋肉内用、静脈内用等の注射剤を製造することができる。
前記pH調節剤及び前記緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウムなどが挙げられる。前記安定化剤としては、例えば、ピロ亜硫酸ナトリウム、EDTA、チオグリコール酸、チオ乳酸などが挙げられる。前記等張化剤としては、例えば、塩化ナトリウム、ブドウ糖などが挙げられる。前記局所麻酔剤としては、例えば、塩酸プロカイン、塩酸リドカインなどが挙げられる。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.
前記坐剤としては、例えば、前記ブドウ果皮・種子乳酸菌発酵物に、ポリエチレングリコール、ラノリン、カカオ脂、脂肪酸トリグリセリド等の公知の坐剤製剤用担体と、必要に応じてツイーン(TWEEN:登録商標)等の界面活性剤などを加えた後、常法により製造することができる。 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.
前記軟膏剤としては、例えば、前記ブドウ果皮・種子乳酸菌発酵物に、公知の基剤、安定剤、湿潤剤、保存剤等を配合し、常法により混合し、製造することができる。
前記基剤としては、例えば、流動パラフィン、白色ワセリン、サラシミツロウ、オクチルドデシルアルコール、パラフィンなどが挙げられる。前記保存剤としては、例えば、パラオキシ安息香酸メチル、パラオキシ安息香酸エチル、パラオキシ安息香酸プロピルなどが挙げられる。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.
前記貼付剤としては、例えば、公知の支持体に前記軟膏剤としてのクリーム剤、ゲル剤、ペースト剤等を、常法により塗布し、製造することができる。前記支持体としては、例えば、綿、スフ、化学繊維からなる織布、不織布、軟質塩化ビニル、ポリエチレン、ポリウレタン等のフィルム、発泡体シートなどが挙げられる。 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.
<投与>
前記医薬の投与対象としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、マウス、ラット、ハムスター、イヌ、ネコ、ウシ、ブタ、サルなどが挙げられる。
また、前記医薬の投与方法としては、特に制限はなく、前記医薬の剤型等に応じて適宜選択することができ、例えば、経口投与、注射による投与などが挙げられる。
また、前記医薬の投与量としては、特に制限はなく、投与対象である患者の年齢、体重、性別、症状等に応じて適宜選択することができるが、ヒト成人1日あたり、有効成分である前記ブドウ果皮・種子乳酸菌発酵物の量として、50mg〜5g程度が好ましい。
また、前記医薬の投与頻度としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記1日あたりの投与量を、1日に1回で投与してもよいし、複数回に分けて投与してもよい。
また、前記医薬の投与時期としても、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記医薬は、治療的に投与されてもよいし、予防的に投与されてもよい。<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. .
<対象>
前記第1の医薬の治療又は予防対象としては、特に制限はなく、「メタボリックシンドローム関連症状」の中から、目的に応じて適宜選択することができる。ここで、メタボリックシンドロームとは、一般に、内臓脂肪の蓄積によってインスリン抵抗性の状態が起こり、これによりII型糖尿病(高血糖)、高血圧、高脂血症(例えば、高コレステロール血症、高トリグリセリド血症)、肥満等の、動脈硬化が引き起こされるやすくなる要因が一患者内に複数存在するようになった状態をいう。例えば、2005年4月に日本内科学会総会において公表された日本における基準では、内臓脂肪の蓄積を反映するウエスト周囲径が、男性で85cm以上、女性で90cm以上の患者(内臓脂肪面積100cm2以上に相当)の中で、(1)血清脂質異常(トリグリセリド値150mg/dL以上、又はHDLコレステロール値40mg/dL未満)、(2)血圧高値(最高血圧130mmHg以上、又は最低血圧85mmHg以上)(3)高血糖(空腹時血糖値110mg/dL以上)の3項目のうち2項目以上にあてはまる場合をメタボリックシンドロームと診断すると規定されている。
したがって、前記医薬の治療又は予防対象となる「メタボリックシンドローム関連症状」としては、前記メタボリックシンドロームに関連して起こり得る症状であれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、メタボリックシンドロームの要因となる内臓脂肪蓄積、インスリン抵抗性といった状態や、これらの状態から導かれるII型糖尿病(高血糖)、高血圧、高脂血症(例えば、高コレステロール血症、高トリグリセリド血症)、肥満等の症状、また、これらの症状が進行した結果導かれる動脈硬化、心筋梗塞、脳卒中などが挙げられる。前記医薬は、本発明の前記ブドウ果皮・種子乳酸菌発酵物を少なくとも含むので、優れた血糖値上昇抑制作用、血中トリグリセリド上昇抑制作用、肥満抑制作用、血中アディポネクチン上昇作用等を有しており、したがって、前記した各種メタボリックシンドローム関連症状の中でも、II型糖尿病(高血糖)、高脂血症(特に高トリグリセリド血症)、肥満の治療又は予防に、特に好適である。<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.
また、前記第2の医薬は、皮膚の美白・老化防止を目的とした医薬であり、したがって、前記医薬の対象となる症状としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、皮膚の炎症、硬化、色素沈着、しわ形成、はり・つやの喪失、日焼け、かさつき、シミ、ソバカス、にきび、ふきでものなどが挙げられる。前記医薬は、本発明の前記ブドウ果皮・種子乳酸菌発酵物を少なくとも含むので、メイラード反応により生成した終末糖化産物群(AGE)に起因するTNF−α産生を抑制する作用、チロシナーゼ活性抑制作用、メラニン合成抑制作用、紫外線による皮膚炎症を抑制する作用等に優れ、そのため、皮膚の美白・老化防止に効果的である。 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.
(食品)
本発明の第1の食品は、メタボリックシンドローム関連症状を改善又は予防するための食品であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含み、必要に応じて適宜その他の成分を含む。
また、本発明の第2の食品は、皮膚の美白・老化防止のための食品であって、ブドウの果皮及び種子の少なくともいずれかを乳酸菌で発酵させて得られるブドウ果皮・種子乳酸菌発酵物を含み、必要に応じて適宜その他の成分を含む。
なお、本明細書中において、前記「第1の食品」及び前記「第2の食品」を単に「食品」と総称することがある。(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”.
<ブドウ果皮・種子乳酸菌発酵物>
前記ブドウ果皮・種子乳酸菌発酵物は、ブドウの果皮及び種子の少なくともいずれか(果皮・種子)を乳酸菌で発酵させて得られるものであり、前記「ブドウ」、「果皮・種子」、「乳酸菌」、「発酵」としては、前記した本発明のブドウ果皮・種子乳酸菌発酵物の項目に記載した通りである。したがって、前記食品におけるブドウ果皮・種子乳酸菌発酵物としては、前記した本発明のブドウ果皮・種子乳酸菌発酵物を好適に使用することができる。
前記食品中の前記ブドウ果皮・種子乳酸菌発酵物の含有量は、特に制限はなく、目的に応じて適宜選択することができ、また、前記食品は、前記ブドウ果皮・種子乳酸菌発酵物そのものであってもよい。<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.
<その他の成分>
前記その他の成分としては、特に制限はなく、本発明の効果を損なわない範囲内で、目的に応じて適宜選択することができ、例えば、各種食品原料などが挙げられる。前記食品原料としても、特に制限はなく、例えば、後述する前記食品の種類等に応じて適宜選択することができる。また、前記食品中の前記その他の成分の含有量としても、特に制限はなく、目的に応じて適宜選択することができる。<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.
<食品の種類>
前記食品の種類としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ゼリー、キャンディー、チョコレート、ビスケット、グミ等の菓子類;柿の種、おかき等のおつまみ類;緑茶、紅茶、コーヒー、清涼飲料等の嗜好飲料;原料乳、ヨーグルト、アイスクリーム等の乳製品;野菜飲料、果実飲料、ジャム類等の野菜・果実加工品;スープ等の液体食品;パン類、麺類等の穀物加工品;各種調味料;ハンバーグ、コロッケ、かまぼこ、さつま揚げ等の肉・魚介類加工品;などが挙げられる。これらの食品の製造方法としては、特に制限はなく、例えば、通常の各種食品の製造方法に応じて、適宜製造することができる。
また、前記食品は、例えば、錠剤、顆粒剤、カプセル剤等の経口固形剤や、内服液剤、シロップ剤等の経口液剤として製造されたものであってもよい。前記経口固形剤、経口液剤の製造方法は、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記した医薬の経口固形剤、経口液剤の製造方法にならい、製造することができる。<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.
前記食品の投与(摂取)形態としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記した本発明の医薬の投与形態(投与対象、投与方法、投与量、投与時期)に準じて、適宜投与(摂取)することができる。
また、前記食品の改善又は予防対象としても、特に制限はなく、前記した本発明の医薬の治療又は予防対象と同様に適宜選択することができる。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.
前記第1の食品は、本発明の前記ブドウ果皮・種子乳酸菌発酵物を少なくとも含むので、優れた血糖値上昇抑制作用、血中トリグリセリド上昇抑制作用、肥満抑制作用、血中アディポネクチン上昇作用等を有しており、したがって、前記食品は、II型糖尿病(高血糖)、高脂血症(特に高トリグリセリド血症)、肥満等のメタボリックシンドローム関連症状を改善又は予防する目的で摂取される機能性食品として、特に有用である。
より具体的には、例えば、メタボリックシンドローム関連症状を引き起こし易くする原因の一つであると考えられる高脂肪食の調理時又は摂取時に、前記食品を調味料として用いることにより摂取する態様や、高脂肪食の摂取前又は摂取後に、前記食品を錠剤やカプセル剤型のサプリメントとして摂取する態様などが好適であると考えられる。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.
また、前記第2の食品は、本発明の前記ブドウ果皮・種子乳酸菌発酵物を少なくとも含むので、メイラード反応により生成した終末糖化産物群(AGE)に起因するTNF−α産生を抑制する作用、チロシナーゼ活性抑制作用、メラニン合成抑制作用、紫外線による皮膚炎症を抑制する作用等に優れ、そのため、皮膚の美白・老化防止を目的として摂取される機能性食品として、特に有用である。
より具体的には、例えば、前記食品を錠剤やカプセル剤型のサプリメントとして摂取する態様などが好適であると考えられる。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.
[効果]
本発明によれば、様々な用途に好適なブドウ果皮・種子乳酸菌発酵物を提供することができ、更に、前記ブドウ果皮・種子乳酸菌発酵物を利用した、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状を治療、改善又は予防することが可能な医薬及び食品、並びに、皮膚の美白・老化防止に効果的、かつ安全な医薬及び食品を提供することができる。
本発明のブドウ果皮・種子乳酸菌発酵物、並びに前記ブドウ果皮・種子乳酸菌発酵物を含む本発明の医薬及び食品は、従来は廃棄されていたブドウの果皮や種子を有効利用できることから、環境性の観点からも優れたものであるということができる。また、本発明のブドウ果皮・種子乳酸菌発酵物は、抽出、精製等したものではなく、より自然食品に近い。そのため、前記ブドウ果皮・種子乳酸菌発酵物を含む本発明の医薬及び食品は、前記したようなメタボリックシンドローム関連症状の治療、改善、予防効果や、皮膚の美白・老化防止効果に優れるのみならず、安全性にも極めて優れたものであるということができる。[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.
以下に実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
(実施例1:至適乳酸菌のスクリーニング)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の作製に好適な乳酸菌を選択するため、以下のようにして至適乳酸菌のスクリーニングを行った。
甲州ブドウの果皮及び種子を粉砕し、ブドウ果皮・種子乾燥パウダーを作製し、1質量%になるようにPBS 50mlに懸濁後、表1に示す各乳酸菌を1×106CFU/mlの濃度になるよう接種した。37℃、48時間培養後、培養液を回収し、pHをpHメーターで測定した。
結果を表1に示す。各菌の添加前のpH7.0に対し、いずれの乳酸菌の添加培養後においてもpHの減少が認められた。中でも、ラクトバチルス プランタラム(Lactobacillus plantarum)、ラクトバチルス サリバリウス(Lactobacillus salivalius)、ラクトバチルス アミロボラス(Lactobacillus amylovorus)、ラクトバチルス カゼイ(Lactobacillus casei)、ペディオコッカス ペントサセウス(Pediococcus pentosaceus)の添加培養後においては顕著なpHの減少が認められ、これらの乳酸菌はブドウ果皮及び種子を炭素源としてよく増殖することが示された(表1)。
なお、以降の実施例において、乳酸菌発酵ブドウ果皮・種子の検体の作製は、特に記す場合を除き、Lactobacillus plantarum(KU146)を用いて行った。(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.
(実施例2:添加乳酸菌量の検討)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の作製に好適な乳酸菌の添加量を、以下のようにして検討した。
甲州ブドウの果皮及び種子を粉砕し、ブドウ果皮・種子乾燥パウダーを作製し、10質量%になるようにPBS 20mlに懸濁後、図1に表示する各量の乳酸菌を添加して、5日間、37℃で培養した。得られた各検体の生理活性について、α−グルコシダーゼ酵素阻害活性を指標に検討した。α−グルコシダーゼ活性の測定は酵素基質法にて行った。すなわち、蒸留水で10mg/mlの濃度に調整した各検体10mlに、0.07U/mlのαグルコシダーゼ溶液(シグマ社)50mlを加え、5分間反応させた後、5mMのp−nitrophenyl−a−d−glucopyranoside(ナカライテスク株式会社)溶液50mlを加えた。5分間反応させた後、波長405nmで吸光度を測定した。
結果を図1に示す。測定値は検体の代わりに蒸留水で行ったときの値(control)を100%とし、controlに対する%を縦軸に示した。添加乳酸菌109CFU/mlの濃度から酵素阻害活性が認められ、1011CFU/mlでプラトーに達していた(図1)。(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).
(実施例3:培養時間の検討)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の作製に好適な培養時間を、以下のようにして検討した。
甲州ブドウの果皮及び種子を粉砕し、ブドウ果皮・種子乾燥パウダーを作製し、10質量%になるようにPBS 20mlに懸濁後、1011CFU/mlの乳酸菌を添加して、1日から6日間、37℃で培養した。得られた各検体の生理活性について、α−グルコシダーゼ酵素阻害活性を指標に検討した。α−グルコシダーゼの活性測定については実施例2と同様に行った。
結果を図2に示す。測定値は検体の代わりに蒸留水で行ったときの値(control)を100%とし、controlに対する%を縦軸に示した。培養2日目から強い酵素阻害活性が認められ、3日目以降プラトーに達していた(図2)。(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).
[検体作製法]
実施例1〜3の結果から、以降の実施例におけるブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の検体の作製は、特に記す場合を除き、以下の方法で行った。
甲州ブドウの果皮及び種子を粉砕し、ブドウ果皮・種子乾燥パウダーを作製し、10質量%になるようにPBSに懸濁後、乳酸菌(Lactobacillus plantarum(KU146))を1×1011CFU/mlの濃度になるよう接種した。37℃、48時間(2日間)培養後、培養液を回収し、80℃、2時間熱処理した。得られた検体は蒸留水で希釈し、各実施例における所望の濃度に調整した。[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.
(実施例4:アミラーゼ酵素阻害作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のアミラーゼ酵素阻害作用を、以下のようにして検討した。
α−アミラーゼ活性の測定は市販のアミラーゼテストワコー(和光純薬工業株式会社)を参考に行った。すなわち、蒸留水で2.5mg/mlの濃度に調整した各検体20mlに、7U/mlのα−アミラーゼ溶液(和光純薬工業株式会社)50mlを加え、5分間反応させた後、基質緩衝液(4%デンプン溶液)50mlを加えた。7.5分間反応させた後、発色液(0.01N ヨウ素液)50ml、及び蒸留水150mlを加え、波長450nmで吸光度を測定した。なお、使用した各検体の詳細は以下の通りである。
1群(無処理ブドウ果皮・種子粉末):前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダーを検体として使用した(比較対照群)。
2群(ブドウ果皮・種子+乳酸菌(発酵)):前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子を検体として使用した(実験群)。
3群(ブドウ果皮・種子+乳酸菌(未発酵)):前記[検体作製法]において、生きた乳酸菌の代わりに、80℃、2時間の熱処理で殺菌した乳酸菌の死菌を接種し、作製したものを検体として使用した(比較対照群)。
4群(乳酸菌のみ):前記[検体作製法]において使用した乳酸菌(Lactobacillus plantarum(KU146))のみを検体として使用した(比較対照群)。
結果を図3に示す。測定値は検体の代わりに蒸留水で行ったときの値(control)を100%とし、controlに対する%を縦軸に示した。図3の結果より、乳酸菌発酵ブドウ果皮・種子検体(2群)は、多糖類から2糖類に変換する酵素であるアミラーゼの活性を効果的に抑制したことが判る。すなわち、乳酸菌発酵ブドウ果皮・種子の摂取によって、食事によって取り込まれた多糖類は、グルコースに変換されることなく体外へ排出されることが考えられる。このことから、乳酸菌発酵ブドウ果皮・種子は、食後の血糖値の上昇を抑制することができ、糖尿病の予防や治療に有効であると判定された。一方、乳酸菌発酵ブドウ果皮・種子以外の検体ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例5:グルコシダーゼ酵素阻害作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のグルコシダーゼ酵素阻害作用を、以下のようにして検討した。
α−グルコシダーゼ活性の測定は酵素基質法にて行った。すなわち、蒸留水で10mg/mlの濃度に調整した各検体10mlに、0.07U/mlのα−グルコシダーゼ溶液(シグマ社)50mlを加え、5分間反応させた後、5mMのp−nitrophenyl−a−d−glucopyranoside(ナカライテスク株式会社)溶液 50mlを加えた。5分間反応させた後、波長405nmで吸光度を測定した。なお、使用した各検体の詳細は実施例4と同様である。
結果を図4に示す。測定値は検体の代わりに蒸留水で行ったときの値(control)を100%とし、controlに対する%を縦軸に示した。図4の結果より、乳酸菌発酵ブドウ果皮・種子検体(2群)は、2糖類からグルコースに変換する酵素であるグルコシダーゼの活性を効果的に抑制したことが判る。すなわち、乳酸菌発酵ブドウ果皮・種子の摂取によって、食事によって取り込まれた2糖類は、グルコースに変換されることなく体外へ排出されることが考えられる。このことから、乳酸菌発酵ブドウ果皮・種子は、食後の血糖値の上昇を抑制することができ、糖尿病の予防や治療に有効であると判定された。一方、乳酸菌発酵ブドウ果皮・種子以外の検体ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例6:スクロース負荷による過血糖抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のスクロース負荷による過血糖抑制作用を、以下のようにして検討した。
6週齢、雄性、ICRマウス(日本SLC株式会社)を、20時間絶食させた後、蒸留水に懸濁した各検体100mg/kgを、胃ゾンデを用いて強制経口投与した。更に、蒸留水に溶解したスクロース(和光純薬工業株式会社)を4g/kgで経口投与し、投与30、60、90分後にヘマトクリット毛細管(テルモ株式会社)を用い、眼底静脈より採血を行った。血液は2,500rpm、10分間遠心分離後、血清を回収し、グルコースCIIテストワコー(和光純薬工業株式会社)を用いてグルコース濃度を測定した。なお、使用した各検体の詳細は実施例4と同様である。また、コントロールとしては各検体の代わりに蒸留水のみを投与した。
結果を図5に示す。コントロール群では、2糖類であるスクロースは、体内の酵素であるグルコシダーゼによりグルコース分解され、腸管より吸収され、投与30分後では急激な血中グルコースの上昇が認められた。一方で、スクロースと同時に乳酸菌発酵ブドウ果皮・種子検体を投与することにより、血中でのグルコースの上昇は緩やかとなり、投与30分、90分後では有意な血中グルコース濃度の抑制が認められた(図5、―○―)。このことから、乳酸菌発酵ブドウ果皮・種子は、食後の血糖値の上昇を抑制することができ、糖尿病の予防や治療に有効であると判定された。一方、乳酸菌発酵ブドウ果皮・種子以外の検体ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例7:デンプン負荷による過血糖抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のデンプン負荷による過血糖抑制作用を、以下のようにして検討した。
6週齢、雄性、ICRマウス(日本SLC株式会社)を、20時間絶食させた後、蒸留水に懸濁した各検体100mg/kgを、胃ゾンデを用いて強制経口投与した。更に、蒸留水に溶解したポテトデンプン(シグマ社)を4g/kgで経口投与し、投与30、60、90分後にヘマトクリット毛細管(テルモ株式会社)を用い、眼底静脈より採血を行った。血液は2,500rpm、10分間遠心分離後、血清を回収し、グルコースCIIテストワコー(和光純薬工業株式会社)を用いてグルコース濃度を測定した。なお、使用した各検体の詳細は実施例4と同様である。また、コントロールとしては各検体の代わりに蒸留水のみを投与した。
結果を図6に示す。コントロール群では、多糖類であるデンプンは、体内の酵素であるアミラーゼ、及びグルコシダーゼによりグルコース分解され、腸管より吸収され、投与30分後では急激な血中グルコースの上昇が認められた。一方で、デンプンと同時に乳酸菌発酵ブドウ果皮・種子検体を投与することにより、血中でのグルコースの上昇は緩やかとなり、投与30分、60分、90分後において有意な血中グルコース濃度の抑制が認められた(図6、―○―)。このことから、乳酸菌発酵ブドウ果皮・種子は、食後の血糖値の上昇を抑制することができ、糖尿病の予防や治療に有効であると判定された。一方、乳酸菌発酵ブドウ果皮・種子以外の検体ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例8:オリーブオイル負荷による血中トリグリセリド(TG)上昇抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のオリーブオイル負荷による血中トリグリセリド(TG)上昇抑制作用を、以下のようにして検討した。
6週齢、雄性、ICRマウス(日本SLC株式会社)を、20時間絶食させた後、蒸留水に懸濁した各検体100mg/kgを、胃ゾンデを用いて強制経口投与した。検体投与30分後、オリーブオイル(和光純薬工業株式会社)を5ml/kgで経口投与し、オリーブオイル投与90分後に各マウスより血液を回収した。血液は2,500rpm、10分間遠心分離後、血清を回収し、トリグリセリド濃度をトリグリセライドE−テストワコー(和光純薬工業株式会社)を用いて測定した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子(実験群)、及び、前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダー(無処理ブドウ果皮・種子;比較対照群)を使用した。また、コントロールとしては検体の代わりに蒸留水のみを投与した。
結果を図7に示す。コントロール群では、オリーブオイル投与90分後において血中TG濃度の著明な上昇が認められた。一方で、オリーブオイル投与前に乳酸菌発酵ブドウ果皮・種子検体を投与することにより、有意な血中TG濃度の抑制が認められた(図7)。このことから、乳酸菌発酵ブドウ果皮・種子は、食後の血中TG濃度の上昇を抑制することができ、高脂血症の予防や治療に有効であると判定された。一方、無処理のブドウ果皮・種子ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例9:II型糖尿病マウスを用いた抗糖尿病作用)
II型糖尿病モデルマウスとして用いられ、かつ、内臓脂肪蓄積、インスリン抵抗性、肥満、高血糖、高トリグリセリド血症等のメタボリックシンドローム関連症状をきたすことが知られているKKAyマウスを用い、本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のメタボリックシンドローム関連症状に対する効果を、以下のようにして検討した。
5週齢、雄性、KKAy/TaJclマウス(日本クレア株式会社)を用い、蒸留水に懸濁した各検体150mg/kgを、胃ゾンデを用いて1日2回、強制経口投与した。週5回、5週間投与を行い、最終投与翌日に各マウスより血液、及び腹腔内脂肪を回収した。血液は2,500rpm、10分間遠心分離後、血清を回収し、グルコース及びトリグリセリド濃度を、グルコースCIIテストワコー(和光純薬工業株式会社)及びトリグリセライドE−テストワコー(和光純薬工業株式会社)を用いて測定し、更にアディポネクチン濃度を、CircuLexマウスアディポネクチンELISAキット(CircuLex;CycLex社)を用いて測定した。腹腔内脂肪は重量を測定した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子(実験群)、及び、前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダー(無処理ブドウ果皮・種子;比較対照群)を使用した。また、コントロールとしては検体の代わりに蒸留水のみを投与した。
結果を図8A〜図8Fに示す。
図8Aに9週齢マウスの外観を示す。コントロール群のマウスでは外観の顕著な肥満化が認められたのに対して、乳酸菌発酵ブドウ果皮・種子検体投与群のマウスでは肥満化は認められなかった。
図8Bに体重増加について示す。コントロール群においては実験開始時(5週齢)に比べ、5週間後(9週齢)では著明な体重増加が認められた。一方、乳酸菌発酵ブドウ果皮・種子検体投与群では、9週齢において、コントロール群と比べ、有意な体重増加の抑制が認められた。
図8Cに9週齢マウスにおける腹腔内脂肪重量について示す。コントロール群と比べ、乳酸菌発酵ブドウ果皮・種子検体投与群のマウスは有意な腹腔内脂肪重量増加の抑制が認められた。
図8Dに血清中トリグリセリド(TG)濃度について示す。コントロール群においては実験開始時(5週齢)に比べ、5週間後(9週齢)では著明な血清中トリグリセリド濃度の増加が認められた。一方、乳酸菌発酵ブドウ果皮・種子検体投与群では、9週齢において、コントロール群と比べ、有意な血清中トリグリセリド濃度増加の抑制が認められた。
図8Eに血清中グルコース濃度について示す。コントロール群においては実験開始時(5週齢)に比べ、5週間後(9週齢)では著明な血清中グルコース濃度の増加が認められた。一方、乳酸菌発酵ブドウ果皮・種子検体投与群では、9週齢においてもグルコース濃度の増加は全く認められず、コントロール群と比べ、有意な血清中グルコース濃度増加の抑制が認められた。
図8Fに9週齢マウスにおける血清中アディポネクチン濃度について示す。コントロール群と比べ、乳酸菌発酵ブドウ果皮・種子検体投与群のマウスでは、有意なアディポネクチン濃度の上昇が認められた。
肥満に伴い糖尿病を発症し、かつ各種メタボリックシンドローム関連症状を示すKKAyII型糖尿病モデルマウスにおいて、乳酸菌発酵ブドウ果皮・種子検体を投与することにより、肥満の抑制、血中トリグリセリド上昇の抑制、血糖値上昇の抑制が認められ、更に、抗メタボリックシンドローム因子として注目されているアディポネクチンについては上昇が認められた。これらの結果から、本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)は、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状の予防や治療に有効であると判定された。一方、無処理のブドウ果皮・種子ではこれらの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る(図8B〜8F)。(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).
(実施例10:脂肪細胞からのアディポネクチン産生誘導作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の脂肪細胞からのアディポネクチン産生誘導作用を、以下のようにして検討した。
2×105個の脂肪細胞前駆細胞(3T3−L1;大日本住友製薬)を24穴プレートに播種し、脂肪前駆細胞培養用培地(大日本住友製薬株式会社)を用い、37℃炭酸ガス培養器で3日間培養した。培養後、脂肪細胞分化培地(大日本住友製薬株式会社)に培地交換し、さらに3日間培養して脂肪細胞に分化させた。分化後、脂肪細胞培養用培地(大日本住友製薬株式会社)に培地交換し、7日間培養して実験に使用した。
分化させた脂肪細胞のプレートの各穴に0.1、1mg/mlの検体を添加し、24時間37℃炭酸ガス培養器で培養した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子を使用した。また、コントロールには同量の精製水を添加した。培養後、プレートを1,000rpm、5分間遠心後、上清を回収し、上清中に含まれるアディポネクチン量をELISAキット(アディポネクチン マウスEIAキットt;MBL社)を用いて測定した。グラフには各群3穴で行った平均値と標準誤差を示した。
結果を図9に示す。乳酸菌発酵ブドウ果皮・種子を添加して培養することにより、用量依存的に脂肪細胞からのアディポネクチン産生量を増加させることができ、1mg/mlの用量では有意な増加作用が認められた(図9)。メタボリックシンドローム因子として注目されているアディポネクチン産生を誘導する作用を有することから、乳酸菌発酵ブドウ果皮・種子は、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状の予防や治療に有効であることが判る。(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.
(実施例11:TNF−αによるアディポネクチン産生抑制に対する拮抗作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のTNF−αによるアディポネクチン産生抑制に対する拮抗作用を、以下のようにして検討した。
前記実施例10と同様にして分化させた脂肪細胞のプレートの各穴に、1mg/mlの検体及び各濃度のTNF−αを添加し、24時間37℃炭酸ガス培養器で培養した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子を使用した。また、コントロールにはTNF−α、及び検体の代わりに同量の精製水を添加した。培養後、プレートを1,000rpm、5分間遠心後、上清を回収し、上清中に含まれるアディポネクチン量をELISAキットを用いて測定した。
結果を図10に示す。脂肪細胞をTNF−αで処理することにより、アディポネクチン産生量の低下が認められた。一方、乳酸菌発酵ブドウ果皮・種子の添加により、TNF−α処理によるアディポネクチン産生量低下に対する有意な抑制作用が認められた(図10)。メタボリックシンドローム因子として注目されているアディポネクチンの産生量低下を抑制する作用を有することから、乳酸菌発酵ブドウ果皮・種子は、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状の予防や治療に有効であることが判る。(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.
(実施例12:AGE刺激マクロファージからのTNF−α産生抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のAGE刺激マクロファージからのTNF−α産生抑制作用を、以下のようにして検討した。
10週齢、雄性C57BL/6マウス(日本SLC株式会社)の腹腔内に10%チオグリコレート(ディフコ社)を投与し、3日後に腹腔細胞を回収した。細胞をRPMI培地(ギブコ社)で洗浄し、10%牛胎児血清(FCS)を含むRPMI培地に懸濁して2x106cells/mlの濃度に調整した。48穴プレートの各穴に細胞液500μlを加え、37℃炭酸ガス培養器で2時間培養後、下記のようにして作製した終末糖化産物群(AGE)50μl及び各濃度の検体を添加し、24時間37℃炭酸ガス培養器で培養した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子(実験群)、及び、前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダー(無処理ブドウ果皮・種子;比較対照群)を使用した。また、コントロールの穴には検体の代わりに同量の精製水を加えた。培養終了後、細胞上清を回収し、上清中に含まれるTNF−α量をELISAキット(BD社)を用いて定量した。グラフにはAGE無添加の吸光度を1としたときのコントロール及び検体処理の値を示した。
−終末糖化産物群(AGE)の作製−
J.V.Valenciaらの方法(非特許文献 Analytical Biocemi.2004;324:68−78.)に従い行った。すなわち、10%牛血清アルブミン(BSA;AP社)、及び10%D(−)リボース(和光純薬工業株式会社)を等量混合し、37℃で14日間反応させ、メイラード反応によりAGEを作製した。AGEの生成は吸光度計により波長600nmの吸光度を測定して確認した。
結果を図11に示す。AGE刺激により、腹腔細胞からのTNF−α産生誘導の増加が認められた。一方、乳酸菌発酵ブドウ果皮・種子の添加により、有意なTNF−α産生抑制作用が認められた(図11)。糖とタンパク質とのメイラード反応により生成したAGEによる炎症サイトカインの産生誘導に対し、乳酸菌発酵ブドウ果皮・種子による抑制効果が認められたことから、乳酸菌発酵ブドウ果皮・種子が、AGE炎症による皮膚老化に対し、予防的効果を発揮できることが示された。一方、無処理のブドウ果皮・種子ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。
(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.
(実施例13:B16メラノーマ細胞の細胞内チロシナーゼ活性抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のB16メラノーマ細胞の細胞内チロシナーゼ活性抑制作用を、以下のようにして検討した。
B16メラノーマ細胞を、10%FCSを含むダルベッコ変性MEM培地(DMEM;ギブコ社)に懸濁して、1x105cells/mlの濃度に調整し、6穴プレートの各穴に細胞液3mlを加え、37℃炭酸ガス培養器で1時間培養した。培養後、各濃度の検体を加え、さらに2日間培養後、リン酸緩衝液(PBS)で細胞表面を洗い、100nMフェニルメチルスルフォニルフルオリド(PMSF;和光純薬工業株式会社)、及び1%トリトンX(ICN社)を含んだリン酸緩衝液300μlを加え、凍結融解して細胞を破壊した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子(実験群)、及び、前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダー(無処理ブドウ果皮・種子;比較対照群)を使用した。コントロールの穴には検体の代わりに同量の精製水を加えた。細胞破壊液を回収し、1,2000rpmで10分間遠心分離後、上清80μlに2mg/mlのL−3−(3,4−ジヒドロフェニル)アラニン(L−DOPA;東京化成工業株式会社)20μlを加え、室温で1日間反応させた後、L−DOPAからチロシナーゼにより返還されたメラニン色素量を、波長450nmで吸光度を測定して定量した。グラフには細胞破壊液の代わりにPBSとL−DOPAを混合したときの値を1としたときのコントロール及び検体処理の値を示した。各群それぞれ3穴で行った平均値と標準誤差で示した。
結果を図12に示す。細胞内チロシナーゼによりL−DOPAが返還され、メラニン色素生成が認められた。一方、乳酸菌発酵ブドウ果皮・種子を添加して培養した細胞では有意なメラニン色素の生成抑制作用が認められ、細胞内チロシナーゼの活性が抑制されていることが確認された(図12)。このことから、乳酸菌発酵ブドウ果皮・種子は、メラノサイトの細胞内チロシナーゼの活性を阻害することにより、メラニン色素の生成を抑制し、美白効果を発揮できることが示された。一方、無処理のブドウ果皮・種子ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例14:B16メラノーマ細胞のメラニン合成抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)のB16メラノーマ細胞のメラニン合成抑制作用を、以下のようにして検討した。
B16メラノーマ細胞を、10%FCSを含むDMEM培地に懸濁して、1x105cells/mlの濃度に調整し、6穴プレートの各穴に細胞液3mlを加え、37℃炭酸ガス培養器で1時間培養した。培養後、10μg/mlのαMSH(SIGMA社)、及び各濃度の検体を加え、さらに5日間培養した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子(実験群)、及び、前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダー(無処理ブドウ果皮・種子;比較対照群)を使用した。コントロールの穴には検体の代わりに同量の精製水を加えた。培養後、リン酸緩衝液で細胞表面を洗い、0.25%トリプシンで細胞を剥がして回収し、2N NaOH 300μlを加え、90℃で30分間処理して細胞を破壊した。12,000rpmで10分間遠心分離後、上清を回収し、細胞内メラニン色素量を、波長450nmで吸光度を測定して定量した。グラフの値は、αMSH無添加の吸光度を1としたときのコントロール及び検体処理の値を示した。各群それぞれ3穴で行った平均値と標準誤差で示した。
結果を図13に示す。αMSH刺激により、約4.5倍のメラニン合成量の増加が認められた。一方、乳酸菌発酵ブドウ果皮・種子の添加により、有意なメラニン量の抑制作用が認められた(図13)。このことから、乳酸菌発酵ブドウ果皮・種子は、メラニン色素の生成を抑制することにより、美白効果を発揮できることが示された。一方、無処理のブドウ果皮・種子ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例15:紫外線照射マウスにおける皮膚炎症抑制作用)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の紫外線(UVB)照射マウスにおける皮膚炎症抑制作用を、以下のようにして検討した。
10週齢、雄性BALB/cマウス(日本SLC株式会社)の背部体毛を剃毛して600mJ/cm2に相当するUVBを照射した。照射1日後、1%エバンスブルー色素(和光純薬工業株式会社)200μlを静脈内投与して、炎症部位に漏出した色素量を以下の方法で定量した。すなわち、エバンスブルー投与30分後、1cm2の背部皮膚を切り取り、1N 水酸化カリウム液0.25mlに24時間浸して溶解した。3N リン酸溶液0.125mlで中和後、アセトン0.325mlを加え、色素を抽出した。2,500rpm、10分間遠心後、アセトン層を回収し、波長595nmで吸光度を測定して色素量を定量した。検体は300mg/kgの用量で、紫外線照射4日前から照射30分前まで1日1回、計5回経口投与した。なお、検体としては、前記[検体作製法]により作製された乳酸菌発酵ブドウ果皮・種子(実験群)、及び、前記[検体作製法]において使用したブドウ果皮・種子乾燥パウダー(無処理ブドウ果皮・種子;比較対照群)を使用した。また、コントロール群には検体の代わりに同量の精製水を投与した。グラフには1群4匹で行ったときの、紫外線非照射のマウスの値を1としたときのコントロール及び検体投与マウスの値を示した。
結果を図14に示す。紫外線照射により、局所におけるエバンスブルー色素の漏出が認められ、皮膚の炎症が確認された。一方、乳酸菌発酵ブドウ果皮・種子を経口投与したマウスにおいては、有意な色素漏出の抑制作用が認められた(図14)。このことから、乳酸菌発酵ブドウ果皮・種子は経口投与において、紫外線による皮膚炎症を抑制できることが示された。一方、無処理のブドウ果皮・種子ではこの作用は認められず、乳酸菌によるブドウ果皮・種子の発酵が重要であることが判る。(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.
(実施例16:乳酸菌発酵によるブドウ果皮・種子の成分変化の解析)
本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)の成分を、以下のようにして検討した。
図15に示した方法で分離・分画することにより、乳酸菌発酵ブドウ果皮・種子検体の分析用試料を調製した。具体的には、甲州の果皮及び種子10gから得られた乾燥粉末(乾燥パウダー)1gをPBS10mlに懸濁し、各乳酸菌(L.plantarum(KU146)、L.plantarum(KU551)、L.salivalius(Yu13))を1×1011CFU/mlの濃度になるよう接種し、37℃、48時間(2日間)発酵させた後の培養液をろ過し、ろ液と残渣に分けた。ろ液は、n−ヘキサンで3回抽出してn−ヘキサン画分を得た(2.3mg)。更に水層は、酢酸エチルで3回抽出して酢酸エチル画分を得た(11.1mg)。同様に、クロロホルム画分(2.0mg)、n−ブタノール画分(40.0mg)、水画分(257.0mg)を得た。一方、残渣は熱メタノールで3回抽出し、メタノール画分(80.0mg)を得た。
得られた画分をTLC及びHPLCを用いて分析した結果、乳酸菌発酵による成分変化はn−ヘキサン画分ではみられなかったが、酢酸エチル画分(図16A、図16B)、クロロホルム画分(図17A、図17B)、n−ブタノール画分(図18A、図18B)、メタノール画分(図19A、図19B)では認められた。なお、TLC及びHPLCの分析条件は図16A〜図19B中に示した通りである。より具体的には、乳酸菌発酵ブドウ果皮・種子の各画分において、以下のような成分変化が認められた。
酢酸エチル画分では、TLCクロマトグラムにおいて、Rf値 0.10〜0.20のスポット(図16A中、矢印)の消失が確認され(図16A)、HPLCクロマトグラムにおいて、tR 6min付近のピークの減少、tR 8min、13min付近のピークの増大が確認された(図16B)。
クロロホルム画分では、TLCクロマトグラムにおいて、Rf値 0.42に黄色スポット(図17A中、矢印、濃いスポット部分)とRf値 0.02に赤紫色スポット(図17A中、矢印、濃いスポット部分)が新たに出現した(図17A)。HPLCクロマトグラムにおいて、tR 4.5、6−7、11min付近に新たなピークが確認された(図17B)。
n−ブタノール画分では、TLCクロマトグラムにおいて、Rf値 0.20〜0.25の褐色スポット(図18A中、矢印、濃いスポット部分)の大きさが減少した(図18A)。HPLCクロマトグラムにおいて、tR 6min付近のピークが増大し(10%)、26min付近のピークが増大した(1%)(図18B)。
メタノール画分では、TLCクロマトグラムにおいて、Rf値 0.25付近の褐色スポット(図19A中、矢印、濃いスポット部分)の大きさが減少した(図19A)。HPLCクロマトグラムにおいて、tR 5min付近に新たなピークが確認された(図19B)。
本実施例16の結果から、各乳酸菌発酵ブドウ果皮・種子(図16A〜図19B中、KU146、KU551、Yu13)では、その成分が、無処理のブドウ果皮・種子(図16A〜図19中、甲州)から大きく変化していることが示された。本発明のブドウ果皮・種子乳酸菌発酵物(乳酸菌発酵ブドウ果皮・種子)は、このような成分変化が起こった結果、実施例1〜11で示されたようなメタボリックシンドローム関連症状に対する優れた治療、改善、予防効果、並びに、実施例12〜15で示されたような皮膚の美白・老化防止効果を奏することができるようになると考えられる。(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.
本発明のブドウ果皮・種子乳酸菌発酵物、並びに前記ブドウ果皮・種子乳酸菌発酵物を利用した本発明の医薬及び食品は、例えば、II型糖尿病、高脂血症、肥満等のメタボリックシンドローム関連症状の治療、改善又は予防や、皮膚の美白・老化防止に、好適に利用可能である。 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.
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WO2021059986A1 (en) | 2019-09-25 | 2021-04-01 | 株式会社村田製作所 | Collagen production enhancer, pharmaceutical, cosmetic, and method for producing collagen production enhancer |
KR20210152384A (en) * | 2020-06-08 | 2021-12-15 | 주식회사 디캔트 | Fermenting method of by-product produced from alcoholic beverages with an increased content of catechins |
WO2021251508A1 (en) * | 2020-06-08 | 2021-12-16 | 김상욱 | Method for extracting extract of liquor production by-product and composition for whitening, wrinkle improvement and anti-aging comprising the extract according to the method |
KR102466755B1 (en) | 2020-06-08 | 2022-11-16 | 주식회사 디캔트 | Fermenting method of by-product produced from alcoholic beverages with an increased content of catechins |
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