JP5872725B1 - Dipeptidyl peptidase IV inhibitory composition derived from bonito - Google Patents
Dipeptidyl peptidase IV inhibitory composition derived from bonito Download PDFInfo
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- JP5872725B1 JP5872725B1 JP2015127871A JP2015127871A JP5872725B1 JP 5872725 B1 JP5872725 B1 JP 5872725B1 JP 2015127871 A JP2015127871 A JP 2015127871A JP 2015127871 A JP2015127871 A JP 2015127871A JP 5872725 B1 JP5872725 B1 JP 5872725B1
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Abstract
【課題】ジペプチジルペプチダーゼＩＶ（ＤＰＰＩＶ）阻害作用を有する、経口摂取して安全性の高い鰹だし又は鰹だし粕由来の組成物の提供。【解決手段】鰹だし由来の又は鰹だし粕由来のＤＰＰＩＶ阻害組成物。以下の工程を含む、鰹だし粕由来のジペプチジルペプチダーゼＩＶ阻害組成物の製造方法。１）鰹節を６０℃〜１００℃の熱水で抽出し、更に遠心分離によって上澄を取り除き、鰹だし粕を得る工程；２）前記鰹だし粕を、至適条件下、特定の群より選択される少なくとも１種の菌株由来のプロテアーゼであって食品工業用途のプロテアーゼによって酵素分解し、さらに遠心分離によって清澄液を得る工程；３）前記清澄液を精製する工程；及び４）エタノール濃度１０％、２５％、５０％及び９９．５％のステップワイズグラジエントによる溶出を行う工程【選択図】図１[Problem] To provide a dip stock or a composition derived from bonito stock which has a dipeptidyl peptidase IV (DPPIV) inhibitory action and is highly safe when taken orally. A DPPIV-inhibiting composition derived from or brewed from bonito. A method for producing a dipeptidyl peptidase IV-inhibiting composition derived from soup stock, comprising the following steps: 1) A step of extracting bonito with hot water at 60 ° C. to 100 ° C. and further removing the supernatant by centrifugation to obtain a soup stock; 2) selecting the soup stock from a specific group under optimum conditions A protease derived from at least one strain that is enzymatically degraded by a protease for food industry use, and further obtaining a clarified liquid by centrifugation; 3) a step of purifying the clarified liquid; and 4) an ethanol concentration of 10% , 25%, 50% and 99.5% stepwise gradient elution steps [selection] Figure 1
Description
本発明は、ジペプチジルペプチダーゼＩＶ（ＤＰＰＩＶ）阻害組成物であって、従来から食材として用いられてきた安全性の高い鰹だし、又はその鰹だし粕由来の組成物、及びそれらの製造方法に関する。 The present invention relates to a dipeptidyl peptidase IV (DPPIV) -inhibiting composition, which has been used as a food and has a high safety, or a composition derived from the soup stock, and a method for producing them.
近年、全世界において糖尿病が爆発的に増加している。日本では糖尿病患者６００万人、その予備軍は１２００万人〜１５００万人といわれている。糖尿病疾患によって、高血糖が長く続くことにより血管が徐々に障害を受け、さまざまな臓器に異常が生じる。
この糖尿病疾患の三大合併症として、糖尿病性腎症、糖尿病性網膜症、糖尿病性神経症が以前から知られているが、近年では、糖尿病疾患によって、動脈硬化症発症のリスクが高くなることも知られている。
そして糖尿病としては「膵β細胞の破壊的病変でインスリンの欠乏が生じて起こる」Ｉ型糖尿病と「膵β細胞の機能異常によるインスリン分泌能低下と肝、筋、脂肪組織等の標的臓器におけるインスリン感受性低下が併発することによって発症する」ＩＩ型糖尿病が存在する。
昨今、激増する糖尿病はＩＩ型に由来するものであり、糖尿病の９０〜９５％を占めていると考えられている。このＩＩ型糖尿病は「生活習慣病」といわれているように、ストレス、肥満、運動不足による基礎代謝能低下と、それに加えて、高カロリー食摂取等、現代型社会生活によって引き起こされている。
In recent years, diabetes has exploded in the whole world. In Japan, it is said that there are 6 million people with diabetes, and 12 to 15 million reserves. Due to diabetic disease, blood vessels are gradually damaged due to long-lasting hyperglycemia and abnormalities occur in various organs.
As the three major complications of diabetic disease, diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy have been known for some time, but in recent years, the risk of developing arteriosclerosis is increased by diabetic disease. Is also known.
Diabetes mellitus is "type I diabetes caused by insulin deficiency due to destructive lesions of pancreatic beta cells" and "insulin secretion in target organs such as liver, muscle, adipose tissue, etc. There is type II diabetes, which is manifested by a concomitant decline in sensitivity.
Recently, the rapidly increasing diabetes is derived from type II and is considered to account for 90 to 95% of diabetes. This type II diabetes is caused by modern social life such as stress reduction, obesity, decline in basic metabolic capacity due to lack of exercise, and intake of high-calorie foods, as it is said to be a “lifestyle-related disease”.
このような糖尿病に関する研究分野において、消化管ホルモンであるインクレチンが注目されつつある。インクレチンはインスリン分泌を増強する消化管ホルモンの総称で、ＧＩＰ（グルコース依存性インスリン分泌ポリペプチド（ｇｌｕｃｏｓｅ−ｄｅｐｅｎｄｅｎｔ ｉｎｓｕｌｉｎｏｔｒｏｐｉｃ ｐｏｌｙｐｅｐｔｉｄｅ））やＧＬＰ−１（グルカゴン様ペプチド−１（ｇｌｕｃａｇｏｎ−ｌｉｋｅ ｐｅｐｔｉｄｅ−１））等が知られている。これらは、膵β細胞に発現する受容体を介したグルコース応答性インスリン分泌を促進し、食後の血糖上昇を抑制する。また、インスリン分泌促進以外に、膵β細胞の保護及び増殖作用といった活性を持っている。
しかしながら、インクレチンの問題点として、体内安定性が挙げられる。すなわち、インクレチンは体内に普遍的に存在するＤＰＰＩＶによって速やかに不活性なものへと分解され、数分間で半減してしまう。そこで、安定なＤＰＰＩＶ阻害剤の開発が進められている（特許文献１、非特許文献１）。
In the research field related to diabetes, incretin, which is a gastrointestinal hormone, is attracting attention. Incretin is a general term for gastrointestinal hormones that enhance insulin secretion. GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) ) Etc. are known. These promote glucose-responsive insulin secretion through a receptor expressed in pancreatic β cells, and suppress postprandial blood glucose elevation. In addition to promoting insulin secretion, it has activities such as protecting and proliferating pancreatic β cells.
However, a problem with incretin is internal stability. That is, incretin is rapidly decomposed into inactive substances by DPPIV, which is universally present in the body, and halved in a few minutes. Therefore, development of a stable DPPIV inhibitor is underway (
他方、製剤あるいは食品として幅広く使用されているコラーゲン及びその熱変性体であるゼラチンは機能特性が高く、加工適性を向上させるため、タンパク質分解酵素を用いて処理したもの、あるいは酸−塩基分解により部分加水分解処理したものを使用することが多い（特許文献２〜４）。また、タンパク質であるため抗原性を有し、アレルギー体質のヒトの摂取には問題がある。そのため、コラーゲンをコラゲナーゼによって、低分子化することにより抗原性をなくしアレルギー患者向けのタンパク質源あるいは輸液製剤成分としての利用が開示されている（特許文献５）。
また、コラゲナーゼによるコラーゲンの分解物の生理活性については、フィブリン凝集阻害活性（特許文献６）、麻酔作用（非特許文献２）が知られている。
On the other hand, collagen, which is widely used as a preparation or food, and gelatin, which is a heat-denatured product thereof, have high functional characteristics and are processed with a proteolytic enzyme in order to improve processability, or partially by acid-base decomposition. A hydrolyzed product is often used (
As for the physiological activity of collagen degradation products by collagenase, fibrin aggregation inhibitory activity (Patent Document 6) and anesthetic action (Non-Patent Document 2) are known.
一方で、ペプチド性のＤＰＰＩＶ阻害剤に関する報告が幾つかある（非特許文献３〜６）。
また、食品由来のペプチドによるＤＰＰＩＶ阻害剤に関する報告もある（特許文献７）。
ところでまた、ＤＰＰＩＶ阻害とは血糖上昇抑制の作用機序の点で異なるが、α−グルコシダーゼを阻害することにより血糖上昇を抑制する物質として、食品由来の豆鼓エキス及び難消化性デキストリン（特許文献８及び特許文献９）を挙げることができる。
On the other hand, there are some reports on peptidic DPPIV inhibitors (Non-Patent
There is also a report on DPPIV inhibitors using food-derived peptides (Patent Document 7).
By the way, although it is different from DPPIV inhibition in the mechanism of action of suppressing blood sugar elevation, as a substance that suppresses blood sugar elevation by inhibiting α-glucosidase, food-derived soybean drum extract and indigestible dextrin (patent document) 8 and Patent Document 9).
しかしながら、化学合成品であるＤＰＰＩＶ阻害剤は非天然物であり、摂取する際の安全性に課題が残る。
ペプチド性のＤＰＰＩＶ阻害剤もまた、食品由来ではない場合、摂取した際の安全性の点で課題が残る。
さらにまた、α−グルコシダーゼ阻害作用を有する物質は、食品由来といえども、摂取後に低血糖などの副作用のおそれがある。
よって、天然成分由来の安全性の高いＤＰＰＩＶ阻害作用を有する物質が望まれていた。
この点、鰹だし、即ち、鰹節を熱水で抽出しただし汁、及び、粕として残存する鰹だし粕は従来から食品として利用されており、安全性の点から優れていると考えられる。
However, DPPIV inhibitors that are chemically synthesized products are non-natural products, and there remains a problem in safety when ingested.
Peptide DPPIV inhibitors also have problems in terms of safety when ingested when not derived from food.
Furthermore, even if the substance having an α-glucosidase inhibitory action is derived from food, there is a risk of side effects such as hypoglycemia after ingestion.
Therefore, a highly safe substance derived from natural ingredients and having a DPPIV inhibitory action has been desired.
In this respect, the soup stock, that is, the koji koji is extracted with hot water, but the soup and the soup stock remaining as the soup has been conventionally used as food and is considered to be excellent from the viewpoint of safety.
よって、本発明は、鰹節由来であって、ＤＰＰＩＶ阻害活性が強くかつ安全性に優れたＤＰＰＩＶ阻害組成物及びその製造方法の提供を課題とする。 Therefore, an object of the present invention is to provide a DPPIV-inhibiting composition that is derived from bonito and has strong DPPIV-inhibiting activity and excellent safety, and a method for producing the same.
即ち、本発明は、鰹節を６０℃〜１００℃の熱水で抽出し、さらに遠心分離によって上澄を得る工程から成る、鰹だし由来のジペプチジルペプチダーゼＩＶ阻害組成物に関する。
一態様において、本発明の組成物は、Ｓｅｒ−Ｇｌｙの配列から成るペプチドを、好ましくは、当該組成物の乾燥物１００ｇ当り０．１乃至１０ｍｇ含有する。
本発明の別の態様は、 Ｇｌｙ−Ｌｅｕ、Ｌｅｕ−Ｐｈｅ、Ｐｈｅ−Ｐｒｏ、Ｍｅｔ−Ｐｒｏ−Ｐｈｅ、Ｌｅｕ−Ｐｒｏ−Ｌｅｕ、Ｍｅｔ−Ｐｒｏ−Ｌｅｕ及びＡｌａ−Ｇｌｙ−Ａｌａ−Ｍｅｔ−Ｐｒｏから成る群より選択される少なくとも１種のペプチドを含有する、鰹だし粕由来のジペプチジルペプチダーゼＩＶ阻害組成物に関する。
好ましくは、前記組成物は、Ｇｌｙ−Ｌｅｕ、Ｌｅｕ−Ｐｈｅ、Ｐｈｅ−Ｐｒｏ、Ｍｅｔ−Ｐｒｏ−Ｐｈｅ、Ｌｅｕ−Ｐｒｏ−Ｌｅｕ、Ｍｅｔ−Ｐｒｏ−Ｌｅｕ及びＡｌａ−Ｇｌｙ−Ａｌａ−Ｍｅｔ−Ｐｒｏから成る群より選択される少なくとも１種のペプチドを、当該組成物の乾燥物に基づき１乃至１０質量％の量で含有する。
本発明のまた別の態様は、
１）鰹節を６０℃〜１００℃の熱水で抽出し、さらに遠心分離によって上澄を取り除き、鰹だし粕を得る工程；
２）前記鰹だし粕を、至適条件下、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｉｚａｅ属、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ属及びＡｓｐｅｒｉｇｉｌｌｕｓ ｎｉｇｅｒ属からなる群より選択される少なくとも１種の菌株由来のプロテアーゼであって食品工業用途のプロテアーゼ、好ましくは中性プロテアーゼによって酵素分解し、さらに遠心分離によって清澄液を得る工程；及び
３）前記清澄液を精製する工程；
を経て得られる、鰹だし粕由来のジペプチジルペプチダーゼＩＶ阻害組成物に関する。
好ましくは、本発明は、清澄液を精製する工程の後に、エタノール濃度１０％、２５％、５０％及び９９．５％のステップワイズグラジエントによる溶出を行う工程をさらに含む。
また、本発明は、上記組成物がα−グルコシダーゼ阻害作用を実質的に有しないことを特徴とする、組成物にも関する。
さらにまた本発明は、上記組成物が食後血糖値上昇抑制作用を有することを特徴とする、組成物に関する。
本発明のさらなる態様は、上記組成物のうちいずれかの組成物を含有することを特徴とする、医薬組成物に関する。
本発明のさらに別の態様は、
以下の工程：
１）鰹節を６０℃〜１００℃の熱水で抽出し、さらに遠心分離によって上澄を取り除き、鰹だし粕を得る工程；
２）前記鰹だし粕を、至適条件下、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｉｚａｅ属、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ属及びＡｓｐｅｒｉｇｉｌｌｕｓ ｎｉｇｅｒ属からなる群より選択される少なくとも１種の菌株由来のプロテアーゼであって食品工業用途のプロテアーゼによって酵素分解し、さらに遠心分離によって清澄液を得る工程； 及び
３）前記清澄液を精製する工程；及び
４）エタノール濃度１０％、２５％、５０％及び９９．５％のステップワイズグラジエントによる溶出を行う工程；
を含むことを特徴とする、鰹だし粕由来のジペプチジルペプチダーゼＩＶ阻害組成物の製造方法にも関する。
That is, the present invention relates to a dipeptidyl peptidase IV-inhibiting composition derived from bonito, which comprises a step of extracting bonito with hot water at 60 ° C. to 100 ° C. and further obtaining a supernatant by centrifugation.
In one embodiment, the composition of the present invention preferably contains 0.1 to 10 mg of a peptide consisting of the Ser-Gly sequence per 100 g of dried product of the composition.
Another aspect of the present invention is the group consisting of Gly-Leu, Leu-Phe, Phe-Pro, Met-Pro-Phe, Leu-Pro-Leu, Met-Pro-Leu and Ala-Gly-Ala-Met-Pro The present invention relates to a dipeptidyl peptidase IV-inhibiting composition derived from soup stock containing at least one peptide selected from the above.
Preferably, the composition comprises Gly-Leu, Leu-Phe, Phe-Pro, Met-Pro-Phe, Leu-Pro-Leu, Met-Pro-Leu and Ala-Gly-Ala-Met-Pro. At least one peptide selected from the above is contained in an amount of 1 to 10% by mass based on the dried product.
Another aspect of the present invention provides:
1) A step of extracting bonito with hot water at 60 ° C. to 100 ° C. and further removing the supernatant by centrifugation to obtain a soup stock koji;
2) The soup stock is a protease derived from at least one strain selected from the group consisting of the genus Aspergillus oryzae, the genus Bacillus licheniformis and the genus Aspergillus niger under optimal conditions, A step of enzymatically degrading with a neutral protease and further obtaining a clarified liquid by centrifugation; and 3) a step of purifying the clarified liquid;
The present invention relates to a dipeptidyl peptidase IV-inhibiting composition derived from bonito soup.
Preferably, the present invention further includes a step of performing elution with a stepwise gradient of ethanol concentrations of 10%, 25%, 50% and 99.5% after the step of purifying the clarified liquid.
The present invention also relates to a composition characterized in that the composition does not substantially have an α-glucosidase inhibitory action.
Furthermore, the present invention relates to a composition characterized in that the composition has a postprandial blood glucose level inhibitory action.
A further aspect of the present invention relates to a pharmaceutical composition characterized in that it contains any of the above compositions.
Yet another aspect of the present invention provides:
The following steps:
1) A step of extracting bonito with hot water at 60 ° C. to 100 ° C. and further removing the supernatant by centrifugation to obtain a soup stock koji;
2) The soup stock is a protease derived from at least one strain selected from the group consisting of Aspergillus oryzae, Bacillus licheniformis and Aspergillus niger under optimal conditions, and is enzymatically degraded by a protease for food industry use And a step of obtaining a clarified liquid by centrifugation; and 3) a step of purifying the clarified liquid; and 4) a step of elution with a stepwise gradient of ethanol concentrations of 10%, 25%, 50% and 99.5%. ;
The present invention also relates to a method for producing a dipeptidyl peptidase IV-inhibiting composition derived from soup stock.
本発明は、安全性の観点から、食材として古くから用いられ、食されている鰹だし、あるいは鰹だし粕由来の成分に着目し、研究を行った結果として、為された。
本発明において用いられる鰹節は、当業者に既知の手法により製造されたものであれば十分であり、もちろん市場に流通しているものでも構わない。
From the viewpoint of safety, the present invention has been made as a result of conducting research by paying attention to ingredients that have been used and eaten for a long time as food ingredients, or ingredients derived from soup stock.
The bonito used in the present invention is sufficient as long as it is manufactured by a method known to those skilled in the art, and of course, it may be one that is distributed in the market.
鰹だしは、例えば、鰹節を適当に削ったものに水を加え、およそ５分間〜６０分間の間、およそ５０℃以上の温度に、好ましくは８０℃〜１００℃の温度に加熱し、熱水で抽出し、その後、遠心分離等によって、上澄として得ることが出来る。遠心分離に替え、又は遠心分離とともに、濾過を行ってもよい。また、鰹だしは、例えば,鰹節を適当に削ったものを、水を沸騰させて火を止め得られた沸騰水に入れ、鰹節が底に沈むまで１〜２分程度、そのまま置き、ふきん等で鰹節を静かに濾すことによって、得ることも出来る。
そして、鰹だしは、上述の鰹節を、水で加熱抽出して得られる水溶性タンパク質、アミノ酸、ペプチドであるということができる。
本発明においては、通常公知の方法により製造しただしを用いることも可能であり、市販品を用いることも可能である。
For example, the soup stock is prepared by adding water to an appropriately shaved bonito and heating it to a temperature of about 50 ° C. or more, preferably 80 ° C. to 100 ° C. for about 5 minutes to 60 minutes. And then obtained as a supernatant by centrifugation or the like. Filtration may be performed in place of or along with centrifugation. In addition, for example, bonito stock is made by appropriately shaving bonito into boiling water obtained by boiling water to stop the fire, and leaving it for about 1 to 2 minutes until the bonito sinks to the bottom. It can also be obtained by gently filtering the bonito.
The soup stock can be said to be a water-soluble protein, amino acid, and peptide obtained by heating and extracting the above-mentioned koji koji with water.
In the present invention, however, it is possible to use a product manufactured by a generally known method, but it is also possible to use a commercially available product.
医薬品又は特定保健用食品として許容され得る規格内にあれば、上述のようにして得られた鰹だしを、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物として、そのまま用いてもよい。もちろん、必要に応じて鰹だしを精製して当該組成物を製造することもできる。
また、鰹だし由来のＤＰＰＩＶ阻害組成物については、だしとの混合物で用いることが可能である。
Soup stock obtained as described above may be used as it is as a DPPIV-inhibiting composition derived from the stock of the present invention as long as it is within the standards acceptable as a pharmaceutical product or food for specified health use. Of course, the composition can be produced by purifying the dashi stock as necessary.
Moreover, about the DPPIV inhibitory composition derived from a dashi stock, it can be used with a mixture with a dashi stock.
こうして得られた鰹だし由来のＤＰＰＩＶ阻害組成物は、活性成分として、セリン−グリシン（Ｓｅｒ−Ｇｌｙ，ＳＧ）の配列から成るペプチドを、当該組成物の乾燥物（水分約５質量％以下）１００ｇ当り０．１乃至１０ｍｇ、好ましくは、１乃至６ｍｇ含有する。当該組成物の乾燥は、例えば噴霧乾燥又は凍結乾燥などにより行われる。 The soup stock-derived DPPIV-inhibiting composition thus obtained contains, as an active ingredient, a peptide comprising a serine-glycine (Ser-Gly, SG) sequence, 100 g of a dried product (water content of about 5% by mass or less). It contains 0.1 to 10 mg, preferably 1 to 6 mg per unit. The composition is dried by, for example, spray drying or freeze drying.
他方、鰹だし粕を原料として得られる本発明のＤＰＰＩＶ阻害組成物は、本発明の鰹だしから成るＤＰＰＩＶ阻害組成物と比較して、数倍のＤＰＰＩＶ活性を有する。
鰹だし粕は、前記遠心分離によって上澄を除去した後に沈澱物として、又は濾過によって残留物として、得ることができる。そして、鰹だし粕を、特定のプロテアーゼを用いて酵素分解することによって、分解後に得られる組成物において、ＤＰＰＩＶ阻害活性が発現することとなる。
On the other hand, the DPPIV-inhibiting composition of the present invention obtained from the soup stock has a DPPIV activity several times that of the DPPIV-inhibiting composition comprising the soup stock of the present invention.
Soup stock can be obtained as a precipitate after removing the supernatant by centrifugation or as a residue by filtration. And, by decomposing dashi stock with a specific protease, DPPIV inhibitory activity is expressed in the composition obtained after the decomposition.
そのようなプロテアーゼは、本発明においては、安全性の観点から、食品工業用途のものが選択される。特に、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｉｚａｅ属、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ属及びＡｓｐｅｒｉｇｉｌｌｕｓ ｎｉｇｅｒ属からなる群より選択される菌株由来のプロテアーゼが好ましい。この中でも特に好ましいのは、
プロテアーゼＭ「アマノ」Ｇ（天野エンザイム社製；Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ属；至適温度４０℃〜６０℃（５０℃）；至適ｐＨ４〜７（ｐＨ６））、
スミチームＬＰ５０Ｄ （新日本化学工業社製；Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ属；至適温度４５℃〜６０℃（５０℃）；至適ｐＨ５〜８（ｐＨ７））、
スミチームＦＰ（新日本化学工業社製；Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ属；至適温度４５℃〜５５℃（５０℃）；至適ｐＨ４〜８（ｐＨ６））、
デナチームＡＰ（ナガセケムテックス社製； Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ属；至適温度４０〜５５℃；至適ｐＨ６〜８（ｐＨ７））、
プロチンＳＤ−ＡＣ１０Ｆ（天野エンザイム社製； Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ属；至適温度４０℃〜６０℃；至適ｐＨ５．５〜９．０（ｐＨ８〜９））、
デナプシン２Ｐ（ ナガセケムテックス社製；Ａｓｐｅｒｉｇｉｌｌｕｓ ｎｉｇｅｒ属；至適温度４０℃から６０℃（５０℃）；至適ｐＨ２〜４（ｐＨ３））
である。
これらプロテアーゼは、本発明の完成のために本発明者らによって選択されたもので、食品工業用途であって安全性が高く、且つ、鰹だし粕を酵素分解したときに、高いＤＰＰＩＶ阻害活性をその酵素分解組成物に付与し得る。
さらには、上記６種のプロテアーゼの中でも、より高いＤＰＰＩＶ阻害活性を付与し得るという観点から、中性プロテアーゼ、即ち、プロテアーゼＭ「アマノ」Ｇ及びスミチームＬＰ５０Ｄ が最も好ましい。
In the present invention, such a protease is selected for use in the food industry from the viewpoint of safety. In particular, a protease derived from a strain selected from the group consisting of the genus Aspergillus oryzae, the genus Bacillus licheniformis, and the genus Aspergillus niger is preferred. Particularly preferred among these
Protease M “Amano” G (manufactured by Amano Enzyme; Aspergillus oryzae;
Sumiteam LP50D (manufactured by Shin Nippon Chemical Industry Co., Ltd .; Aspergillus oryzae; optimum temperature 45 ° C to 60 ° C (50 ° C);
Sumiteam FP (manufactured by Shin Nippon Chemical Industry Co., Ltd .; Aspergillus oryzae; optimum temperature 45 ° C. to 55 ° C. (50 ° C.);
Denateam AP (manufactured by Nagase ChemteX Corporation; genus Aspergillus oryzae; optimum temperature 40-55 ° C; optimum pH 6-8 (pH 7)),
Protin SD-AC10F (manufactured by Amano Enzyme; Bacillus licheniformis genus;
Denapsin 2P (manufactured by Nagase ChemteX Corporation; Aspergillus niger genus;
It is.
These proteases have been selected by the present inventors for the completion of the present invention, are highly safe for food industry use, and have high DPPIV inhibitory activity when enzymatically digested koji dashi. It can be applied to the enzymatic degradation composition.
Further, among the above six proteases, neutral proteases, ie, protease M “Amano” G and Sumiteam LP50D are most preferable from the viewpoint that higher DPPIV inhibitory activity can be imparted.
上記プロテアーゼによる処理条件は、プロテアーゼの特性に合せて適宜選択すればよい。酵素量及び処理時間については、特に限定はないが、酵素量は０．１％〜２％対原料タンパク質である。また、反応時間は２時間から２０時間が好ましい。また、反応温度は、３５℃〜６０℃が好ましい。
なお、プロテアーゼ処理は、加熱等により酵素を失活させることで終了させることができる。また、酵素反応後のｐＨは、その後の市販適用のために中和することが望ましい。
What is necessary is just to select the process conditions by the said protease suitably according to the characteristic of protease. There are no particular limitations on the amount of enzyme and the treatment time, but the amount of enzyme is 0.1% to 2% of the starting protein. The reaction time is preferably 2 to 20 hours. The reaction temperature is preferably 35 ° C to 60 ° C.
The protease treatment can be terminated by inactivating the enzyme by heating or the like. Moreover, it is desirable to neutralize the pH after the enzyme reaction for subsequent commercial application.
プロテアーゼ処理で得られたプロテアーゼ分解物は、そのまま公知の手段で濾過又は遠心分離にかけて、未分解物を除去し、清澄液（上澄）を得る。そして、収率よく高活性のＤＰＰＩＶ阻害活性が得られる観点から、得られた清澄液を濃縮等し、さらに凍結乾燥又は噴霧乾燥することが望ましい。さらに各種の精製方法に供することで、ＤＰＰＩＶの阻害活性をより高めた画分を得ることもできる。樹脂精製法で精製することも好ましい。 The protease degradation product obtained by the protease treatment is directly filtered or centrifuged by a known means to remove the undegraded product and obtain a clarified liquid (supernatant). Then, from the viewpoint of obtaining a highly active DPPIV inhibitory activity with high yield, it is desirable to concentrate the obtained clarified liquid and further freeze-dry or spray-dry it. Furthermore, the fraction which improved DPPIV inhibitory activity more can also be obtained by using for various purification methods. It is also preferable to purify by a resin purification method.
樹脂精製法で使用する樹脂としては、例えば、陽イオン交換樹脂、陰イオン交換樹脂、多孔性樹脂、特殊樹脂（キレート樹脂、合成吸着剤、蛋白分離剤）等が挙げられるが、回収した画分の脱塩処理工程が不要であることから、合成吸着剤を用いるのが好ましい。合成吸着剤としては、例えば、芳香族（スチレン−ビニルベンゼン）系、芳香族系修飾型、アクリル（メタクリル）系等が挙げられるが、これらに限定されるものではない。 Examples of the resin used in the resin purification method include a cation exchange resin, an anion exchange resin, a porous resin, and a special resin (chelate resin, synthetic adsorbent, protein separating agent). It is preferable to use a synthetic adsorbent because the desalting step is unnecessary. Examples of the synthetic adsorbent include, but are not limited to, an aromatic (styrene-vinylbenzene) type, an aromatic modified type, and an acrylic (methacrylic) type.
また、樹脂精製法において、ＤＰＰＩＶ阻害成分を含むプロテアーゼ分解物の樹脂への吸着は、公知の手法により行えばよい。
次いで、吸着したプロテアーゼ分解物の溶離には、酸、アルカリ又は種々の有機溶媒、例えば、メタノール、エタノール、プロパノール、イソプロパノール、ブタノール等の低級アルコールや、酢酸エチル、酢酸ブチル等のエステル類、アセトン等のケトン類を用いることできるが、これらに限定されるものではない。又は、酸、アルカリとの混合溶媒としてもよい。なお、経済性と安全性の点からは、濃度５０％以下のエタノール水溶液又は水を用いて溶離するのが好ましい。樹脂精製法は、バッチ法又はカラム法にて行うことができる。回収した画分は減圧又は限外濾過により濃縮し、さらに必要に応じて溶媒を完全に除去して乾固するか凍結乾燥を行ってもよい。
In the resin purification method, adsorption of protease degradation products containing DPPIV-inhibiting components to the resin may be performed by a known method.
Next, for elution of the adsorbed protease degradation product, acid, alkali or various organic solvents, for example, lower alcohols such as methanol, ethanol, propanol, isopropanol and butanol, esters such as ethyl acetate and butyl acetate, acetone and the like However, it is not limited to these. Or it is good also as a mixed solvent with an acid and an alkali. From the viewpoint of economy and safety, it is preferable to elute using an aqueous ethanol solution or water having a concentration of 50% or less. The resin purification method can be performed by a batch method or a column method. The collected fraction may be concentrated by reduced pressure or ultrafiltration, and if necessary, the solvent may be completely removed to dryness or lyophilization.
好ましくは、プロテアーゼ処理後に得られた清澄液を、精製した後、エタノール濃度１０％、２５％、５０％及び９９．５％のステップワイズグラジエントによる溶出にかける。これによって、ＤＰＰＩＶ阻害性の有効成分である、Ｇｌｙ−Ｌｅｕ、Ｌｅｕ−Ｐｈｅ、Ｐｈｅ−Ｐｒｏ、Ｍｅｔ−Ｐｒｏ−Ｐｈｅ、Ｌｅｕ−Ｐｒｏ−Ｌｅｕ、Ｍｅｔ−Ｐｒｏ−Ｌｅｕ及びＡｌａ−Ｇｌｙ−Ａｌａ−Ｍｅｔ−Ｐｒｏを含有する画分を効率的に得ることができる。 Preferably, the clarified liquid obtained after the protease treatment is purified and then subjected to elution with stepwise gradients of ethanol concentrations of 10%, 25%, 50% and 99.5%. Thereby, the active ingredients of DPPIV inhibition are Gly-Leu, Leu-Phe, Phe-Pro, Met-Pro-Phe, Leu-Pro-Leu, Met-Pro-Leu and Ala-Gly-Ala-Met- A fraction containing Pro can be obtained efficiently.
なお本発明においては、メタノール、エタノール等の有機溶媒沈澱法によって、前記有機溶媒中に前記プロテアーゼ分解物を混合し、沈澱画分と上清画分を分離して、上清画分を回収することでＤＰＰＩＶ阻害の高い画分を得ることもできる。この場合、沈澱画分と上清画分とが分離するまでは有機溶媒混合物を静置することが好ましい。なお、静置温度は低温で行うことが好ましい。また、回収した上清画分は減圧又は限外濾過により濃縮し、さらに必要に応じて溶媒を完全に除去して乾固するか凍結乾燥を行ってもよい。 In the present invention, the protease degradation product is mixed in the organic solvent by an organic solvent precipitation method such as methanol or ethanol, the precipitated fraction and the supernatant fraction are separated, and the supernatant fraction is recovered. Thus, a fraction having high DPPIV inhibition can also be obtained. In this case, the organic solvent mixture is preferably allowed to stand until the precipitate fraction and the supernatant fraction are separated. Note that the standing temperature is preferably low. Further, the collected supernatant fraction may be concentrated by reduced pressure or ultrafiltration, and if necessary, the solvent may be completely removed to dryness or lyophilization.
また、本発明の製造方法によって、鰹節からＤＰＰＩＶ阻害活性の高い組成物を、比較的簡便に且つ効率的に得ることができる。
本発明の製造方法に従い製造され市販されている鰹節若しくはそのだしを、そのまま食してもよいし、若しくは濃縮することによってＤＰＰＩＶ阻害活性の力価をより高めることができる。
Further, by the production method of the present invention, a composition having high DPPIV inhibitory activity can be obtained relatively easily and efficiently from bonito.
The bonito or soup stock produced and marketed according to the production method of the present invention may be eaten as it is, or the potency of DPPIV inhibitory activity can be further increased by concentrating.
一方、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物、又は鰹だし粕由来のＤＰＰＩＶ阻害組成物に含まれる活性成分の抽出及びその精製は、通常公知の方法を用いて行うことができる。
具体的には、例えば、鰹節の組織を粉砕した後、水洗、希塩溶液による抽出、酸あるいはアルカリ溶液による抽出、ペプシン、トリプシンやヒアルロニダーゼ等の酵素による抽出を行い、塩析や透析等の公知の精製手段を施して、活性成分を得ることができる。
On the other hand, the extraction of the active ingredient contained in the DPPIV-inhibiting composition derived from the soup stock or the DPPIV-inhibiting composition derived from the soup stock and purification thereof can be carried out using a generally known method.
Specifically, for example, after crushing bonito tissue, washing with water, extraction with a dilute salt solution, extraction with an acid or alkali solution, extraction with an enzyme such as pepsin, trypsin or hyaluronidase, and the like, such as salting out and dialysis The active ingredient can be obtained by applying the purification means.
上記のとおり本発明のＤＰＰＩＶ阻害組成物は、安全性に優れたものであり、しかもコラゲナーゼ処理されたコラーゲン又はゼラチンの分解物に比べて、高いＤＰＰＩＶ阻害活性を有する。 As described above, the DPPIV inhibitory composition of the present invention is excellent in safety, and has a higher DPPIV inhibitory activity than collagenase-treated collagen or gelatin degradation products.
本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物は、Ｇｌｙ−Ｌｅｕ、Ｌｅｕ−Ｐｈｅ、Ｐｈｅ−Ｐｒｏ、Ｍｅｔ−Ｐｒｏ−Ｐｈｅ、Ｌｅｕ−Ｐｒｏ−Ｌｅｕ、Ｍｅｔ−Ｐｒｏ−Ｌｅｕ及びＡｌａ−Ｇｌｙ−Ａｌａ−Ｍｅｔ−Ｐｒｏから成る群より選択される少なくとも１種のペプチドを含有する。これらペプチドは、当該組成物の乾燥物に基づき、１乃至１０質量％の高濃度で含まれることを特徴とする。そのため、高いＤＰＰＩＶ阻害活性を有し、特定保健用食品の有効成分としてより期待される。さらに、α−グルコシダーゼ阻害作用を実質的に有しないため、摂取後の低血糖などのおそれが低い。なお、α−グルコシダーゼ阻害作用を実質的に有しないとは、α―グルコシダーゼ阻害活性（ＩＣ５０）が、およそ１．５ｍｇ／ｍＬ以上であることを意味する。また、当該組成物の乾燥は、例えば噴霧乾燥又は凍結乾燥などにより行われる。 The DPPIV-inhibiting composition derived from the soup stock of the present invention includes Gly-Leu, Leu-Phe, Phe-Pro, Met-Pro-Phe, Leu-Pro-Leu, Met-Pro-Leu and Ala-Gly-Ala- Contains at least one peptide selected from the group consisting of Met-Pro. These peptides are characterized by being contained at a high concentration of 1 to 10% by mass based on the dried product of the composition. Therefore, it has high DPPIV inhibitory activity and is expected more as an active ingredient of food for specified health use. Furthermore, since it has substantially no α-glucosidase inhibitory action, there is a low risk of hypoglycemia after ingestion. The expression “having substantially no α-glucosidase inhibitory activity” means that the α-glucosidase inhibitory activity (IC50) is about 1.5 mg / mL or more. The composition is dried by, for example, spray drying or freeze drying.
本発明の鰹だし由来の又は鰹だし粕由来のＤＰＰＩＶ阻害組成物は、哺乳動物の糖尿病予防・治療剤として有用である。
また、本組成物のＤＰＰＩＶ阻害作用が、ＤＰＰＩＶ阻害のみであり、α−グルコシダーゼ阻害活性を有しないため、低血糖などの副作用のおそれも低く、安全性は高いと考えられる。
また、本組成物は安全性が高いため、食品、菓子等に有効量を配合することができ、これらの食品、菓子等を継続的に摂取することで糖尿病の発症を予防し得る。
なお且つ、当該組成物はまた、ペプシン、トリプシン又はキモトリプシンといった消化管プロテアーゼに対する耐性が高く、これらプロテアーゼによる処理を受けたとしても、処理前と比較して、そのＤＰＰＩＶ阻害活性値が低下しないという利点を有する。
また、本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物は、一回のプロテアーゼ処理工程のみで上記利点を付与し、且つ活性が高く安全性に優れたＤＰＰＩＶ阻害剤である。
さらにまた、本発明の組成物は、血糖低下効能を有する製薬と比較して、活性がほどほど抑えられているため、副作用のおそれもより低く、特定保健用食品の有効成分としてより適するものといえる。
The DPPIV-inhibiting composition derived from or from a koji stock of the present invention is useful as an agent for preventing or treating diabetes in mammals.
Moreover, since the DPPIV inhibitory action of this composition is only DPPIV inhibition and does not have an α-glucosidase inhibitory activity, the risk of side effects such as hypoglycemia is low, and the safety is considered high.
Moreover, since this composition is highly safe, an effective amount can be mix | blended with foodstuffs, confectionery, etc. Onset of diabetes can be prevented by ingesting these foodstuffs, confectionery, etc. continuously.
Furthermore, the composition is also highly resistant to gastrointestinal proteases such as pepsin, trypsin, or chymotrypsin, and even when subjected to treatment with these proteases, its DPPIV inhibitory activity value does not decrease compared to before treatment. Have
Moreover, the DPPIV inhibitory composition derived from the soup stock of this invention is the DPPIV inhibitor which provided the said advantage only by one protease processing process, was highly active, and was excellent in safety | security.
Furthermore, since the activity of the composition of the present invention is moderately suppressed as compared with a pharmaceutical having a hypoglycemic effect, the risk of side effects is lower, and it can be said that the composition of the present invention is more suitable as an active ingredient of a food for specified health use. .
実施例１．本発明の鰹だし由来のＤＰＰＩＶ阻害組成物の製造
工業的スケールにおいては、鰹節（タンパク質１６０ｋｇ）に加水１０００％後、９５℃±３℃、３５分間で熱水抽出し、下記の比率の本発明に係る鰹だし由来のＤＰＰＩＶ阻害組成物を得た（表１）。また鰹だし粕とのタンパク質比率は、２５対７５であった。
実施例２−１．本発明の鰹だし由来のＤＰＰＩＶ阻害組成物の消化管プロテアーゼ耐性試験
ヒトにおいて効果が発揮するためには、消化管プロテアーゼ耐性が必須である。本発明に係る鰹だし由来のＤＰＰＩＶ阻害組成物の腸管プロテアーゼ耐性を調べるために、表２に示すとおりの条件及び消化酵素にて処理を行った。
即ち、鰹だし由来のＤＰＰＩＶ阻害組成物の消化管プロテアーゼ耐性は、下記表２の方法で試料分取後、ＤＰＰＩＶ阻害活性を測定し阻害率を調べることにより図ることが出来る。
That is, the digestive tract protease resistance of the DPPIV-inhibiting composition derived from bonito can be achieved by measuring the DPPIV-inhibiting activity and examining the inhibition rate after sample collection by the method shown in Table 2 below.
試験例１．ＤＰＰＩＶ阻害活性の測定
鰹だし由来のＤＰＰＩＶ阻害組成物、その消化管プロテアーゼ（ペプシン、トリプシン、キモトリプシン）処理品及び鰹だし粕由来のＤＰＰＩＶ阻害組成物の食品工業用プロテアーゼ分解組成物を、凍結乾燥後、それらの１６ｍｇ、８ｍｇ、４ｍｇ、２ｍｇ、１ｍｇを５０ｍＭトリス塩酸緩衝液（ｐＨ７．５）１ｍＬにそれぞれ溶解し、ＤＰＰＩＶ阻害活性測定のための試料とした。
Test Example 1 Measurement of DPPIV inhibitory activity DPPIV-inhibiting composition derived from broth, its digestive tract protease (pepsin, trypsin, chymotrypsin) treated product and protease-degrading composition for food industry of DPPIV-inhibiting composition derived from broth These 16 mg, 8 mg, 4 mg, 2 mg, and 1 mg were dissolved in 1 mL of 50 mM Tris-HCl buffer (pH 7.5), respectively, and used as samples for measuring DPPIV inhibitory activity.
次に、各試料のＤＰＰＩＶ阻害活性を、ＤＰＰＩＶ阻害活性測定キット（Ｂｉｏ Ｖｉｓｉｏｎ製；Ｋ７８０−１００型）を用いて測定した。ＤＰＰＩＶを直線的な検量線が認められる反応時間の範囲において、所定の濃度に調製後、各Ｗｅｌｌに５０μＬずつ分取し、各試料（凍結乾燥品）を上記の濃度に調製後、２５μＬをＤＰＰＩＶが５０μＬ入った各Ｗｅｌｌに分注し（各試料の各濃度でｎ＝４回測定）、３７℃１０分間反応させた。
次に、疑似サンプル基質(インクレチンホルモン)（Ｈ−Ｇｌｙ−Ｐｒｏ−７−アミノ−４−メチルクマリン）を所定の濃度調整後、２５μＬを分注し、反応時間０〜３０分の間、数回測定し、検量線直線が認められる反応時間間の増加率から、コントロール（阻害物質なし）と、各種試料の測定値から、ＤＰＰＩＶ測定阻害活性（％）を計算し、そのＤＰＰＩＶの５０％阻害活性が認められる各種試料濃度をＩＣ５０値として算出した。また、各試料の各濃度で４回測定し、得られた４回分のＩＣ５０値の平均値を算出し、これをＤＰＰＩＶ阻害活性（％）とした。
Next, the DPPIV inhibitory activity of each sample was measured using a DPPIV inhibitory activity measurement kit (manufactured by BioVision; model K780-100). DPPIV was prepared to a predetermined concentration within the reaction time range in which a linear calibration curve was observed, and 50 μL was dispensed into each well, and each sample (lyophilized product) was adjusted to the above concentration, and 25 μL was added to DPPIV. Was dispensed into each well containing 50 μL (measured n = 4 times at each concentration of each sample) and allowed to react at 37 ° C. for 10 minutes.
Next, after adjusting a predetermined concentration of pseudo sample substrate (incretin hormone) (H-Gly-Pro-7-amino-4-methylcoumarin), 25 μL is dispensed, and the reaction time is 0 to 30 minutes. Inhibition activity (%) of DPPIV was calculated from the measured values of the control (no inhibitor) and the measured values of various samples from the increase rate during the reaction time in which the calibration curve was observed, and 50% inhibition of DPPIV. Various sample concentrations at which activity was recognized were calculated as IC50 values. Moreover, it measured 4 times with each density | concentration of each sample, the average value of the obtained IC50 value for 4 times was computed, and this was made into DPPIV inhibitory activity (%).
なお、ＤＰＰＩＶ阻害測定は、測定条件励起波長３８０ｎｍ、測定波長４６０ｎｍの蛍光マイクロプレートリーダー（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ製）にて測定した。 In addition, the DPPIV inhibition measurement was performed with a fluorescence microplate reader (manufactured by Thermo Fisher) having an excitation wavelength of 380 nm and a measurement wavelength of 460 nm.
実施例２−２．本発明に係る鰹だし由来のＤＰＰＩＶ阻害組成物のＤＰＰＩＶ阻害活性
上記実施例２−１で調製した鰹だし由来のＤＰＰＩＶ阻害組成物のＤＰＰＩＶ阻害活性を、上記試験例１の測定方法で測定した。
結果を下記表３に示す。
The results are shown in Table 3 below.
上記表３に示すとおり、鰹だし由来のＤＰＰＩＶ阻害組成物それ自体のＤＰＰＩＶ阻害活性値（ＩＣ５０）は、３０４９μｇ／ｍＬであった。
これに対し、ペプシン処理後の阻害活性（力価）は、２８．３％向上し、ペプシン処理後トリプシン・キモトリプシン処理後の阻害活性（ＩＣ５０）は、２５８８μｇ／ｍＬであった。
よって、鰹だし由来のＤＰＰＩＶ阻害組成物のＤＰＰＩＶ阻害活性は、消化管プロテアーゼ処理により低下しないこと、即ち、体内に吸収後も、その活性を発現し得る可能性が示された。
As shown in Table 3 above, the DPPIV inhibitory activity value (IC50) of the persimmon-derived DPPIV inhibitory composition itself was 3049 μg / mL.
In contrast, the inhibitory activity (titer) after pepsin treatment was improved by 28.3%, and the inhibitory activity (IC50) after pepsin treatment and after trypsin / chymotrypsin treatment was 2588 μg / mL.
Therefore, it was shown that the DPPIV inhibitory activity of the DPPIV-inhibiting composition derived from bonito is not reduced by the digestive tract protease treatment, that is, the possibility that the activity can be expressed even after absorption in the body.
実施例３．鰹だし粕由来のＤＰＰＩＶ阻害組成物の調製及びそのＤＰＰＩＶ阻害活性の測定
上記実施例１の表１に示されるとおりの、鰹だし抽出後に得られた鰹だし粕タンパク質を、各種のプロテアーゼを用いて可溶化し、ＤＰＰＩＶ阻害活性をそれぞれ測定した。
鰹だし粕（タンパク質７２％）に水１０倍量（対基質）加え、下記表４に示す各プロテアーゼ（いずれも食品工業用途）の至適ｐＨに調製後、当該プロテアーゼを用いて、５０℃にて１６時間反応させた。そしてその後、９８℃、１５分間でプロテアーゼを失活させ、遠心分離（１５０００回転、５分間）した後、得られた清澄液を、本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物とし、それらのＤＰＰＩＶ阻害活性を測定した。
各種試料の測定値から、ＤＰＰＩＶ測定阻害活性（％）を計算し、そのＤＰＰＩＶの５０％阻害活性が認められる各種試料濃度をＩＣ５０値として算出した。また、各試料の各濃度で４回測定し、得られた４回分のＩＣ５０値の平均値を算出し、これをＤＰＰＩＶ阻害率とした。なお、コントロール（阻害物質なし）のＤＰＰＩＶ測定阻害活性（％）を１００とした。
結果を下記表４に示す。
After adding 10 times the amount of water (to substrate) to koji dashi koji (72% protein) and adjusting to the optimum pH for each protease shown in Table 4 below (both for food industry use), the protease is used to bring it to 50 ° C. For 16 hours. Then, after inactivating the protease at 98 ° C. for 15 minutes and centrifuging (15000 rpm, 5 minutes), the resulting clarified liquid was used as a DPPIV-inhibiting composition derived from the soup stock of the present invention. DPPIV inhibitory activity was measured.
DPPIV measurement inhibitory activity (%) was calculated from the measured values of various samples, and various sample concentrations at which 50% inhibitory activity of DPPIV was observed were calculated as IC50 values. Moreover, it measured 4 times with each density | concentration of each sample, the average value of the obtained IC50 value for 4 times was computed, and this was made into DPPIV inhibition rate. The control (no inhibitory substance) DPPIV measurement inhibitory activity (%) was defined as 100.
The results are shown in Table 4 below.
比較例１．他のプロテアーゼを用いて得られた鰹だし粕由来の組成物のＤＰＰＩＶ阻害活性の測定
用いるプロテアーゼを変更した他は実施例３と同様にして、比較例組成物を得、それらのＤＰＰＩＶ阻害活性を測定した。
結果を下記表５に示す。
The results are shown in Table 5 below.
上記表４及び表５の結果から、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ属、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ属又はＡｓｐｅｒｉｇｉｌｌｕｓ ｎｉｇｅｒ属のいずれかの菌株由来のプロテアーゼで酵素処理して得られる本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物は、その他の菌株由来のプロテアーゼを用いて得られた組成物と比べ、ＤＰＰＩＶ阻害活性がより高いことが判る。その他の菌株を用いた場合には、むしろ、ＤＰＰＩＶ阻害活性が低下したことが明らかとなった。
さらには、上記表４に示されるとおり、本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物の中でも、中性プロテアーゼであるプロテアーゼＭ「アマノ」Ｇ及びスミチームＬＰ５０Ｄを用いて分解した組成物について、ＤＰＰＩＶ阻害活性が特に強いことが判った。
From the results of Table 4 and Table 5 above, the DPPIV-inhibiting composition derived from the bamboo shoots of the present invention obtained by enzymatic treatment with a protease from any strain of the genus Aspergillus oryzae, Bacillus licheniformis or Aspergillus niger, It can be seen that the DPPIV inhibitory activity is higher than the compositions obtained using proteases derived from other strains. When other strains were used, the DPPIV inhibitory activity was rather decreased.
Furthermore, as shown in Table 4 above, among the DPPIV-inhibiting compositions derived from dashi stock of the present invention, the composition decomposed using protease M “Amano” G and Sumiteam LP50D, which are neutral proteases, It was found that the inhibitory activity was particularly strong.
実施例４．実施例３で得られた本発明に係る鰹だし粕由来のＤＰＰＩＶ阻害組成物の濃度依存性についての試験
上記実施例３で得られた本発明に係る鰹だし粕由来のＤＰＰＩＶ阻害組成物の６種について、それらのＤＰＰＩＶ阻害の濃度依存性について試験を行った。
結果を下記表６及び図１に示す。
The results are shown in Table 6 below and FIG.
表６及び図１の結果から、濃度が高くなるほどＤＰＰＩＶ阻害活性が減少する傾向にあり、その阻害活性は、表４の結果を反映していた。 From the results of Table 6 and FIG. 1, the DPPIV inhibitory activity tended to decrease as the concentration increased, and the inhibitory activity reflected the results of Table 4.
また、上記６種の組成物のＤＰＰＩＶ阻害活性値（ＩＣ５０）及び力価を下記表７に示す。なお、最も活性が強かった組成物番号２のスミチームＬＰ５０Ｄを用いて得られた組成物の力価を１００とした。
他方、アンジオテンシン変換酵素（ＡＣＥ）阻害活性を有する鰹だし粕のプロテアーゼＮＹ−１００による酵素分解物のＤＰＰＩＶ阻害値は、５２５．７μｇ／ｍＬであった。その中に存在する強力なＡＣＥ阻害ペプチドＶａｌ−ＴｒｐのＤＰＰＩＶ阻害活性は検出されなかった。精製過程（疎水性樹脂における水溶出画分の除去）で、ＤＰＰＩＶ阻害活性成分（親水性成分）が排除されていることが推察された（表８）。
実施例５．本発明の鰹だし由来のＤＰＰＩＶ阻害組成物のα−グルコシダーゼ阻害活性についての試験
食後血糖上昇抑制作用機構には、２糖類の酵素分解を阻害するα−グルコシダーゼ阻害と難消化性デキストリンよる糖の吸収を遅延させる効果が知られている。
食後の血糖が低い場合は、食事前の摂取による低血糖の副作用が認められているα−グルコシダーゼ阻害活性について、実施例１で得た本発明の鰹だし由来のＤＰＰＩＶ阻害組成物の濃度別に値を測定した（図２）。
図２に示される結果から、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物には、α−グルコシダーゼ活性を阻害する物質は、高濃度においても存在しないことが推察された。
なお、α−グルコシダーゼ阻害活性の測定は、Ｂｅｎａｖｅｎｔｅ−Ｇａｒｃｉａ ｅｔ．ａｌ，Ｊ Ａｇｒｉｃ Ｆｏｏｄ Ｃｈｅｍ，５６（１５），６１８５−２０５（２００８）に従い行った。
Example 5 FIG. Test of α-glucosidase inhibitory activity of DPPIV inhibitory composition derived from Japanese persimmon stock of the present invention The mechanism of action to suppress postprandial blood glucose elevation includes α-glucosidase inhibition that inhibits the enzymatic degradation of disaccharides and absorption of sugars by indigestible dextrins. The effect of delaying is known.
When the postprandial blood glucose level is low, the α-glucosidase inhibitory activity in which the side effect of hypoglycemia due to ingestion before meals is recognized is a value according to the concentration of the DPPIV-inhibiting composition derived from the bonito stock of the present invention obtained in Example 1. Was measured (FIG. 2).
From the results shown in FIG. 2, it was inferred that the DPPIV-inhibiting composition derived from the dashi stock of the present invention does not contain a substance that inhibits α-glucosidase activity even at a high concentration.
The α-glucosidase inhibitory activity was measured using Benavente-Garcia et. al, J Agric Food Chem, 56 (15), 6185-205 (2008).
本発明に係る鰹だし由来の及び鰹だし粕由来のＤＰＰＩＶ阻害組成物が、ＤＰＰＩＶ阻害作用を有する一方で、α−グルコシダーゼ阻害作用を有していないことは、特定保健用食品としては初の機構の組み合わせであり、副作用の非常に少ない優れた組成物であると考えられる（表９）。
これに対して、特定保健用食品の血糖上昇抑制成分である豆鼓エキス（特許文献８）及び難消化性デキストリン（特許文献９）は、α-グルコシダーゼ阻害活性を有するが、ＤＰＰＩＶ阻害活性を有していないことが明らかであり、この観点において、本発明に係る組成物は、これら物質と比べてより優れていると考えられる。
In contrast, bean extract (Patent Document 8) and indigestible dextrin (Patent Document 9), which are components for suppressing blood sugar elevation in foods for specified health use, have an α-glucosidase inhibitory activity but have a DPPIV inhibitory activity. In this respect, the composition according to the present invention is considered to be superior to these substances.
実施例６．本発明に係る鰹だし由来のＤＰＰＩＶ阻害組成物の血糖上昇抑制作用についての試験
上記のとおり本発明に係る鰹だし由来のＤＰＰＩＶ阻害組成物にはＤＰＰＩＶ阻害活性が認められたことから、糖尿病患者及びその境界型糖尿病（糖尿病予備軍）患者にとって、当該本発明の鰹だし由来のＤＰＰＩＶ阻害組成物を摂取することによって、治療及び健康効果が期待される。
しかしながら、当該組成物に認められるにおいや味は比較的変性しやすく、サプリメントとして利用するには適しているとは言い難い。
そこで、本発明に係る当該組成物が凍結乾燥品としても、ヒト体内において、ＤＰＰＩＶ阻害性糖代謝改善作用（血糖上昇抑制作用）を有するか否かを明らかにするために、下記のとおり臨床試験を行った。
Example 6 Test for suppressing blood glucose elevation of DPPIV-inhibiting composition derived from Japanese persimmon according to the present invention As described above, DPPIV-inhibiting composition derived from Japanese persimmon has a DPPIV-inhibiting activity. For those patients with borderline diabetes (pre-diabetes), treatment and health effects are expected by taking the DPPIV-inhibiting composition derived from the soup stock of the present invention.
However, the odor and taste found in the composition are relatively easy to denature and are not suitable for use as a supplement.
Therefore, in order to clarify whether or not the composition according to the present invention has a DPPIV-inhibitory glucose metabolism-improving action (inhibition of blood sugar increase) in the human body even as a lyophilized product, the following clinical test is conducted. Went.
（試験方法）
まず、被験者の空腹時の血糖値を、簡易自己血糖測定装置を用いて測定した。次いで、実施例１の鰹だし由来のＤＰＰＩＶ阻害組成物の凍結乾燥物４．５ｇに水１５０ｍＬを加えたものを摂取し、その３０分後に、米飯２００ｇ（約糖質７０ｇ）、ふりかけ２ｇを摂取し、さらにその後、３０、４５、６０、７５、９０、１２０分及び１５０分の血糖値を経時的に測定した。
別の指定日に、対照群として、実施例１の当該組成物の代わりに白湯を１５０ｍＬ摂取後、米飯２００ｇとふりかけ２ｇを食し、上記と同様に血糖値を測定した。
(Test method)
First, the subject's fasting blood glucose level was measured using a simple self-blood glucose measurement device. Next, ingestion of lyophilized DPPIV-inhibited DPPIV-inhibiting composition of Example 1 with 150 mL of water was taken, and 30 minutes later, 200 g of cooked rice (about 70 g of carbohydrates) and 2 g of sprinkle were ingested. Thereafter, blood glucose levels were measured over time at 30, 45, 60, 75, 90, 120 minutes and 150 minutes.
On another designated day, as a control group, after taking 150 mL of white hot water instead of the composition of Example 1, 200 g of cooked rice and 2 g of sprinkled rice were eaten, and the blood glucose level was measured in the same manner as described above.
（被験者）
被験者は、正常者及び境界型糖尿病（糖尿病予備軍）であり、医師の問診、血液検査で健常と認められた対象者とする。
試験は２日間に分けて行い、１日目（当日）と２日目（翌日又は約１週間後指定）の試験内容は上記のとおりであった。
また、本発明に係る鰹だし由来のＤＰＰＩＶ阻害組成物又は白湯と米飯の摂食前に、空腹時の血糖値を測定することから、当日の朝は絶食とした。前日２１時以降は摂食せず、前日からの摂食リズムが２日間ともに同じようになるようにした。
(subject)
The test subjects are normal subjects and borderline diabetes mellitus (diabetes reserve army), and subjects who are recognized as healthy by medical examinations and blood tests.
The test was divided into two days, and the contents of the test on the first day (current day) and the second day (next day or about one week later) were as described above.
In addition, the fasting blood glucose level was measured before ingestion of the DPPIV-inhibiting composition or white hot water and cooked rice derived from the soup stock according to the present invention. I did not eat after 21:00 on the previous day, and the eating rhythm from the previous day was the same for both days.
（採血及び血糖値の測定）
採血は、各自専用のペン型採血針を用いて指先から行った。通常、採血量は米粒約半分の大きさ程度（約２μＬ）であり、採決後に使い捨ての専用試験紙を装着した簡易自己血糖測定装置を用いて血糖値を測定した。
空腹時血糖値を測定してから摂食後血糖値の経時的測定を完了するまでに約３時間半を要し、各測定の間隔における行動は自由とし、血糖値に大きく影響を及ぼすような運動や活動は控えるように制限した。
(Blood collection and blood glucose level measurement)
Blood collection was performed from the fingertip using a pen-type blood collection needle dedicated to each person. Usually, the amount of blood collected was about half the size of rice grains (about 2 μL), and the blood glucose level was measured using a simple self-blood glucose measuring device equipped with a disposable test paper after the vote.
It takes about three and a half hours to measure blood glucose level after eating after measuring fasting blood glucose level. And restricted activities to refrain.
（結果）
実施例１の組成物の凍結乾燥物を摂取した群の１４名中１２名に糖負荷後３０分から上昇抑制作用が示され、２時間経過後まで、対照群（白湯摂取群）に対して、５％の危険率で有意差が示された（有効率８５．７％）。２名の内、１名は摂取４５分から抑制効果を示し、その後、徐々に正常血糖値に復帰した。１名は変化がなかった。
実施例１の組成物の凍結乾燥物及び白湯摂取後の血糖値の変化を図３に示した。また、下記表１０に実施例１の組成物の凍結乾燥物及び白湯摂取後の血糖降下曲線面積を表した。５％の危険率で群間の有意差が認められた。
また、糖摂取なし且つ実施例１組成物の凍結乾燥物の摂取のみでは、血糖低下作用は示さなかった。
Twelve out of 14 groups ingesting the lyophilized product of the composition of Example 1 showed an increase-inhibiting effect from 30 minutes after sugar loading, and until 2 hours later, with respect to the control group (white water ingestion group), A significant difference was shown with a 5% risk rate (effective rate 85.7%). Of the two, one showed an inhibitory effect from 45 minutes after ingestion, and then gradually returned to normal blood glucose levels. One person did not change.
The change in blood glucose level after ingestion of the freeze-dried product and white water of the composition of Example 1 is shown in FIG. Table 10 below shows the blood glucose lowering curve area after ingestion of the lyophilized product of the composition of Example 1 and white water. There was a significant difference between the groups with a 5% risk rate.
Moreover, the blood glucose lowering effect was not shown only by ingestion of sugar and ingestion of the lyophilized product of Example 1 composition alone.
（結論）
上記臨床試験から、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物と同様にその凍結乾燥品の摂取により、ヒトに対して血糖上昇抑制作用を示すことが明らかとなった。
さらに、摂食前後及び期間中に副作用及び自覚症状もなく、医師の問診においても異常となる所見は得られなかった。
糖摂取なし且つ実施例１組成物の凍結乾燥物の摂取のみでは、血糖低下作用を示さなかったことから、製薬で懸念される低血糖による副作用はなく、正常血糖者及び低血糖者に作用しないと考えられる。
また、その阻害作用が、ＤＰＰＩＶ阻害のみによるものであることが分かっており、トクホの血糖上昇抑制成分である豆鼓エキス（日本サプリメント）、難消化性デキストリン（松谷化学工業）の作用機序のようにα−グルコシダーゼ阻害によるものでないことが明らかであり、この観点から、本発明の鰹だし由来の及び鰹だし粕由来のＤＰＰＩＶ阻害組成物が、特定保健用食品としてより優れた製品となり得ることが示唆される。
(Conclusion)
From the above clinical trials, it was revealed that ingestion of the lyophilized product as well as the DPPIV-inhibiting composition derived from the dashi broth of the present invention exhibits a blood glucose increase inhibitory effect on humans.
Furthermore, there were no side effects and subjective symptoms before and after eating and during the period, and no abnormal findings were obtained in the doctor's inquiry.
No sugar intake and lyophilized product of Example 1 alone did not show a hypoglycemic effect, so there are no side effects due to hypoglycemia, which is a concern for pharmaceuticals, and it does not affect normoglycemic or hypoglycemic people it is conceivable that.
In addition, it is known that the inhibitory action is due only to DPPIV inhibition, and the action mechanism of Tokuho's blood sugar elevation inhibitory component, bean extract (Nippon Supplement), indigestible dextrin (Matsutani Chemical Industry) Thus, it is clear that it is not due to α-glucosidase inhibition, and from this point of view, the DPPIV-inhibiting composition derived from koji stock and koji stock can be a better product as a food for specified health use. Is suggested.
実施例７．鰹だし由来のＤＰＰＩＶ阻害組成物中の活性ペプチドの同定
鰹だしの凍結乾燥品１００ｍｇを水１ｍＬに溶解し、２０ｍＭリン酸緩衝液ｐＨ７．５（１０ｍＬ）で平衡化した固相抽出管（ＳｕｐｅｌＴＭ Ｓｈｅｒｅ Ｃａｒｂｏｎ／ＮＨ２６ｍＬ ＳＰＥ Ｃａｒｔｒｉｄｇｅ（ＳＵＰＥＬＣＯ）に負荷し（得られた画分１）、１００％エタノール（１５ｍＬ）溶出（得られた画分２）後、２Ｍ ＮａＣｌ ５０ｍＭ Ｔｒｉｓ−ＨＣｌ ｐＨ９．０（４ｍＬ）で溶出した（得られた画分３）。
各々の画分についてＤＰＰＩＶ阻害活性を測定した結果、コントロールを１００％として、それぞれの阻害活性は、５４、６６、３７％（ｐ＜０．００１）であり、画分３に高い値が得られた。そこで、当該画分３を逆相ＨＰＬＣに供した。条件は下記のとおりである。
ＩｎｔｅｒＳｕｓｔａｉｎＣ１８カラム（４．６×１５０ｍｍ、ＧＬサイエンス）
移動相；Ａ：０．１％ギ酸溶液−Ｂ：アセトニトリル１００％液／Ａ０％−Ｂ１００％／３０ｍｉｎ
また、その溶出曲線を図４に示す。
Example 7 Identification of Active Peptide in DPPIV Inhibiting Composition Derived from Boiled
As a result of measuring DPPIV inhibitory activity for each fraction, the inhibitory activity was 54, 66, and 37% (p <0.001) with the control as 100%, and a high value was obtained in
Interstain C18 column (4.6 x 150 mm, GL Sciences)
Mobile phase; A: 0.1% formic acid solution-B: 100% acetonitrile / A0% -B100% / 30 min
Moreover, the elution curve is shown in FIG.
ピークに１〜５に対応する試料をそれぞれ分取し、ロータリーエバポレーターにて濃縮乾固し、それをＤＰＰＩＶ阻害活性試料とした。阻害活性を測定した結果、分画１にＤＰＰＩＶ阻害活性が認められた。また、分画１は、濃度依存的にＤＰＰＩＶ阻害が増加したことも分かった。
この分画１について、ＬＣ／ＭＳ／ＭＳによる分析とｄｅ ｎｏｖｏ ｓｅｑｕｅｎｃｅ解析を行った。
サンプル:かつおだし分画１
カラム：ＡＣＱＵＩＴＹ ＵＰＬＣ ＢＥＨ Ｃ１８ｃｏｌｕｍｎ
（１．７μｍ，２．１×５０ｍｍ，Ｗａｔｅｒｓ）
質量分析：Ｘｅｖｏ Ｇ２−ｑＴＯＦ（Ｗａｔｅｒｓ）
スペクトル解析：ＢｉｏＬｙｎｘソフトウェア（Ｗａｔｅｒｓ）
ピークのペプチド配列同定：ｄｅ ｎｏｖｏ ｓｅｑｕｅｎｃｅ
ＬＣ／ＭＳ／ＭＳによる分析の結果を、図５に示した。
Samples corresponding to
This
Sample:
Column: ACQUITY UPLC BEH C18 column
(1.7 μm, 2.1 × 50 mm, Waters)
Mass spectrometry: Xevo G2-qTOF (Waters)
Spectral analysis: BioLynx software (Waters)
Peptide sequence identification: de novo sequence
The results of analysis by LC / MS / MS are shown in FIG.
上記クロマトグラフィーで確認されたピークに相当する画分のｄｅ ｎｏｖｏ ｓｅｑｕｅｎｃｅ解析を行ったところ、当該画分に含まれている物質は、セリン−グリシン（ＳＧ）又はグリシン−セリン（ＧＳ）の配列をもつペプチドであることが推定されたが、さらに高感度（ｐＭ）Ｎ末端アミノ酸配列解析Ｐｒｏｃｉｓｅ４９１−ｃＬＣ（ＡＢＩ）により、当該画分に含まれている物質は、セリン−グリシン（ＳＧ）の配列からなるペプチドであることが決定した。 When de novo sequence analysis of the fraction corresponding to the peak confirmed by the above chromatography was performed, the substance contained in the fraction had the sequence of serine-glycine (SG) or glycine-serine (GS). It was estimated that the substance contained in the fraction was determined from the serine-glycine (SG) sequence by high sensitivity (pM) N-terminal amino acid sequence analysis Procise491-cLC (ABI). Was determined to be a peptide.
合成したＳＧのＤＰＰＩＶ阻害活性（ＩＣ５０）を測定したところ、１５９５μＭであった。
これに対し、本実施例で得られた、上記同定したＳＧの阻害活性（ＩＣ５０）は２５８．７μｇ／ｍＬであり、実施例２−２の鰹だし由来のＤＰＰＩＶ阻害組成物の阻害活性値（３０４９．３μｇ／ｍＬ；上記表３参照のこと）の約１０倍であった。また、鰹だし由来のＤＰＰＩＶ阻害組成物中のＳＧの含有量は３．２６ｍｇ（当該組成物の乾燥物１００ｇ当り）であることから、寄与率は０．０３８％となり、当該組成物中の主要なＤＰＰＩＶ阻害成分の一つと推定された（表１１）。
On the other hand, the inhibitory activity (IC50) of the above-identified SG obtained in this example is 258.7 μg / mL, and the inhibitory activity value of the DPPIV inhibitory composition derived from the bonito stock of Example 2-2 ( 3099.3 μg / mL; see Table 3 above). Moreover, since the content of SG in the DPPIV-inhibiting composition derived from bonito is 3.26 mg (per 100 g of the dried product of the composition), the contribution ratio is 0.038%, which is the main component in the composition. It was estimated to be one of the DPPIV inhibitory components (Table 11).
実施例８．鰹だし粕をスミチームＬＰ５０Ｄにより分解して得た組成物の同定
実施例３及び４の結果から、組成物２（上記表７参照）のＤＰＰＩＶ阻害活性が高かったことを受け、当該組成物中のＤＰＰＩＶ阻害物質の同定を行った。
上記実施例１の表１に示されるとおりの、鰹だし抽出後に得られた鰹だし粕（タンパク質７２％）に水１０倍量（対基質）を加え、スミチームＬＰ５０Ｄの至適ｐＨ７に調節後、５０℃にて１６時間反応させた。そしてその後、９８℃、１５分間で失活させ、遠心分離（１５０００回転、５分間）した後、清澄液を得た（当該清澄液をＮ５と呼ぶ。上記組成物２に相当する）。
Example 8 FIG. Identification of the composition obtained by decomposing the soup stock with Sumiteam LP50D From the results of Examples 3 and 4, the composition 2 (see Table 7 above) had a high DPPIV inhibitory activity. Identification of DPPIV inhibitors was performed.
As shown in Table 1 of Example 1 above, 10 times the amount of water (versus substrate) was added to the soup stock (protein 72%) obtained after the soup stock extraction, and adjusted to the
次に、得られたＮ５について、エタノール濃度１０％、２５％、５０％及び９９．５％のステップワイズグラジエントによるＤＰＰＩＶ阻害活性画分の分離を行った。
水溶出分画と、１０％エタノール溶出分画により、ＤＰＰＩＶ阻害活性を９倍の比率で分け得て、酵素分解物（Ｎ５）のＩＣ５０の５倍高いＩＣ５０＝７３．７１μｇ／ｍＬの画分を得た（以降、この得られた画分をＹ−２画分と呼ぶ）（表１２）。酵素分解物（Ｎ５）からの収率は、２０％量得られ、阻害活性の回収率は、約１００％であった。
The water elution fraction and the 10% ethanol elution fraction can separate the DPPIV inhibitory activity at a ratio of 9 times, and the fraction of IC50 = 73.71 μg / mL, which is 5 times higher than the IC50 of the enzymatic degradation product (N5). Obtained (hereinafter, the obtained fraction is referred to as Y-2 fraction) (Table 12). The yield from the enzymatic degradation product (N5) was 20%, and the recovery rate of the inhibitory activity was about 100%.
また他方、Ｙ−２画分取得の工業スケール精製方法を確立した。
Ｎ５酵素分解物（タンパク質量５ｋｇ）を、疎水性樹脂（充填剤ＳＰ２０７；三菱化学）１００Ｌを充填し平衡化したカラムに負荷し、下記表１３に記載のタイムコースで精製を行った。
非吸着画分を別取りし、吸着画分を１０％エタノール濃度溶出液で脱着、Ｙ−２画分を得た。その後、４サイクル分を減圧濃縮によりエタノールを除去、噴霧乾燥し、Ｙ−２画分の粉末を得た。
N5 enzyme degradation product (
The non-adsorbed fraction was separated, and the adsorbed fraction was desorbed with a 10% ethanol concentration eluent to obtain a Y-2 fraction. Thereafter, ethanol was removed from the 4 cycles by vacuum concentration and spray-dried to obtain a powder of the Y-2 fraction.
上記表１２中の、Ｎ５酵素分解物の各分画におけるＤＰＰＩＶ阻害活性とＹ−２画分のＩＣ５０値の算出式を、図６に示した。 The calculation formula of DPPIV inhibitory activity in each fraction of N5 enzyme degradation product and IC50 value of Y-2 fraction in Table 12 above is shown in FIG.
また、Ｙ−２画分の凍結乾燥物について、ＬＣ／ＭＳ／ＭＳによる分析とｄｅ ｎｏｖｏ ｓｅｑｕｅｎｃｅ解析を、下記の条件を設定して、行った。得られたクロマトグラムを、図７に示す。
サンプル：Ｙ−２画分の凍結乾燥物１０ｍｇ／ｍＬ
カラム：ＡＣＱＵＩＴＹ ＵＰＬＣ Ｍ−Ｃｌａｓｓ ＨＳＳ Ｔ３ ｃｏｌｕｍｎ
(１．８μｍ，０．７５×１５０ｍｍ，Ｗａｔｅｒｓ)
質量分析：Ｘｅｖｏ Ｇ２−ｑＴＯＦ（Ｗａｔｅｒｓ）
スペクトル解析：ＢｉｏＬｙｎｘソフトウェア（Ｗａｔｅｒｓ）
ピークのペプチド配列同定：ｄｅ ｎｏｖｏ ｓｅｑｕｅｎｃｅ
Moreover, about the freeze-dried product of the Y-2 fraction, the analysis by LC / MS / MS and the de novo sequence analysis were performed by setting the following conditions. The obtained chromatogram is shown in FIG.
Sample: Lyophilized product of Y-2
Column: ACQUITY UPLC M-Class HSS T3 column
(1.8μm, 0.75x150mm, Waters)
Mass spectrometry: Xevo G2-qTOF (Waters)
Spectral analysis: BioLynx software (Waters)
Peptide sequence identification: de novo sequence
得られた１５のピークについて（図７）、それぞれにＭＳスペクトルのｄｅ ｎｏｖｏ ｓｅｑｕｅｎｃｅ解析を行った。その結果、１８種のペプチド配列が同定された。
これらペプチドについて、上記試験例１に記載されているのと同様の方法によって、ＤＰＰＩＶ阻害活性を測定し、その内、阻害活性が最も高かった７種のペプチドについて、さらに同定した。当該７種のペプチド配列及びＤＰＰＩＶ阻害活性を下記表１４に示す。
About these peptides, DPPIV inhibitory activity was measured by the method similar to that described in Test Example 1 above, and among them, seven peptides having the highest inhibitory activity were further identified. The seven peptide sequences and DPPIV inhibitory activity are shown in Table 14 below.
また、上記７種のペプチドの、Ｙ−２画分に対するＭＳ面積当たりの寄与率を求めた。寄与率は、当該７種のペプチドで約４０．０９〜４８．９５％であった。その中で、ＭＰＦ、ＬＰＬ及びＭＰＬの３種のトリペプチドは、３３．５２〜４０．９３％の寄与率を示した（表１５）。
また、Ｙ−２画分の乾燥物１００ｍｇ中の、７種のペプチドの含有量は、下記表１６に記載のとおりであった。
α−グルコシダーゼ阻害活性、ＤＰＰＩＶ阻害活性及び参照としてＡＣＥ（アンジオテンシン変換酵素）阻害活性について、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物及び本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物と、特許文献８及び９に記載の組成物とを対比した。結果を下記表１７に示す。表中、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物をＡとし、及び本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物をＹ−２とする。表中の％は、阻害率を表す。
表１７より、α−グルコシダーゼ阻害活性を有しない本発明の組成物は、低血糖などのおそれがより低いといえる。
Regarding α-glucosidase inhibitory activity, DPPIV inhibitory activity and ACE (Angiotensin Converting Enzyme) inhibitory activity as a reference, DPPIV inhibitory composition derived from the bonito of the present invention, DPPIV inhibitory composition derived from the bonito of the present invention, and patent literature Contrast with the compositions described in 8 and 9. The results are shown in Table 17 below. In the table, the DPPIV-inhibiting composition derived from the soup stock of the present invention is designated as A, and the DPPIV inhibiting composition derived from the soup stock of the present invention is designated as Y-2. % In the table represents the inhibition rate.
From Table 17, it can be said that the composition of the present invention having no α-glucosidase inhibitory activity has a lower risk of hypoglycemia or the like.
本発明において得られた本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物（Ｙ−２画分）は、飲料として、そのままでも又は種々の調味料との混合物としても、摂取できる。 The DPPIV-inhibiting composition (Y-2 fraction) derived from the soup stock obtained in the present invention can be ingested as a beverage or as a mixture with various seasonings.
（本発明の鰹だし由来のＤＰＰＩＶ阻害組成物及び鰹だし粕由来のＤＰＰＩＶ阻害組成物の飲食料及び医薬への応用について）
本発明の飲食用組成物（飲食料）は、表１６に示されたペプチドの一種以上を、１回の摂取量として０．００１ｍｇ〜１００ｍｇ添加して製造される。
本発明の鰹だし由来のＤＰＰＩＶ阻害組成物及び鰹だし粕由来のＤＰＰＩＶ阻害組成物は、乾燥させることによって、取り扱いが容易で安定な固体ないし粉末形態とすることができ、当該形態の水への溶解性もよい。また、胃腸管からの吸収もよい。したがって、食品への添加の時期、及び方法に特別の制限はなく、粉末状、溶液状、懸濁液状等として、食品製造の原料段階、中間工程、最終工程に、食品分野で慣用の方法で添加することが可能である。
飲食品の形態としては、固形状、半流動状、流動状などを挙げることができる。固形状食品としては、シート状、タブレットやカプセルなどの錠剤、顆粒粉末などの形態の一般食品及び健康食品が挙げられる。半流動状食品としては、ペースト状、ゼリー状、ゲル状などの、また、流動状食品としては、ジュース、清涼飲料、茶飲料、ドリンク剤などの形態の一般食品及び健康食品が挙げられる。飲食物を栄養ドリンクや調味料として、本発明の組成物を継続して摂取することにより、血糖の上昇を抑制することも可能である。
(Regarding the application of the DPPIV-inhibiting composition derived from Japanese persimmon stock and the DPPIV-inhibiting composition derived from Japanese persimmon stock to foods and beverages)
The composition for eating and drinking (food and beverage) of the present invention is produced by adding 0.001 mg to 100 mg of one or more of the peptides shown in Table 16 as a single intake.
The dried dashi-derived DPPIV inhibitor composition and the dried dashi-derived DPPIV inhibitor composition of the present invention can be made into a solid or powder form that is easy to handle and stable by drying. Good solubility. Absorption from the gastrointestinal tract is also good. Therefore, there are no particular restrictions on the timing and method of addition to food, and it can be used as a powder, solution, suspension, etc., in the raw material stage, intermediate process, and final process of food production by a method commonly used in the food field. It is possible to add.
Examples of the form of food and drink include solid, semi-fluid, and fluid. Examples of the solid food include general foods and health foods in the form of sheets, tablets such as tablets and capsules, and granular powders. Examples of the semi-fluid food include pastes, jellies, and gels. Examples of the liquid food include general foods and health foods in the form of juices, soft drinks, tea drinks, and drinks. It is also possible to suppress an increase in blood sugar by continuously ingesting the composition of the present invention using a food or drink as an energy drink or a seasoning.
本発明によるＤＰＰＩＶ阻害剤である形の医薬組成物は、本発明のペプチドを、上記飲食用組成物と同様の量で含有する。本発明の医薬組成物は、患者のＤＰＰＩＶを阻害し、例えば血糖上昇抑制作用を発揮させるために、高血糖症状の方に食前に投与するとよく、あるいは本発明の医薬組成物の有効成分は天然物である鰹だし又は鰹だし粕由来であることから、継続して安全に使用することもできる。本発明の医薬組成物により血糖上昇を予防することができる。医薬組成物の形態は、錠剤、カプセル剤、顆粒剤、シロップ等の経口投与剤が好ましい。液剤は、用時溶解できる乾燥固体であってもよい。 The pharmaceutical composition in the form of a DPPIV inhibitor according to the present invention contains the peptide of the present invention in the same amount as the above-mentioned composition for eating and drinking. The pharmaceutical composition of the present invention may be administered to a hyperglycemic symptom before meals in order to inhibit DPPIV of a patient, for example, to exert an action to suppress an increase in blood glucose, or the active ingredient of the pharmaceutical composition of the present invention is natural Since it is derived from the soup stock or soup stock soup, it can be used safely continuously. An increase in blood sugar can be prevented by the pharmaceutical composition of the present invention. The form of the pharmaceutical composition is preferably an oral administration agent such as a tablet, capsule, granule or syrup. The liquid agent may be a dry solid that can be dissolved at the time of use.
配合例．
本発明の鰹だし粕由来のＤＰＰＩＶ阻害組成物（Ｙ−２画分）の乾燥物（粉末品）を原料素材として用い、当該乾燥物１００ｍｇを配合した機能性食品を得た。加工食品として飲料、錠剤、スープ等にも用いられる。
Ｙ−２画分を配合した食品素材の配合割合を下記表１８に示した。
Ｙ−２画分の錠果・錠剤・カプセル製造について、Ｙ−２画分、食品素材を計量、混合・造粒・打錠・コート工程後、ＤＰＰＩＶ阻害活性、ペプチド量を測定して製品化できる。
Formulation example.
Using a dried product (powder product) of the DPPIV-inhibiting composition (Y-2 fraction) derived from the dried koji stock of the present invention as a raw material, a functional food containing 100 mg of the dried product was obtained. Used as processed foods in beverages, tablets, soups, etc.
The blending ratio of the food material blended with the Y-2 fraction is shown in Table 18 below.
For Y-2 fraction tablets, tablets, and capsules, measure Y-2 fraction, food ingredients, mix, granulate, tablet, and coat, then measure DPPIV inhibitory activity and peptide amount to commercialize it can.
鰹節、本発明の鰹だし由来のＤＰＰＩＶ阻害組成物（鰹だし由来と略記）及び鰹だし粕由来のＤＰＰＩＶ阻害組成物（Ｙ−２画分）の各素材について、ＤＰＰＩＶ阻害活性と質量から換算表を示した。
（考察）製薬と比較した、特定保健用食品の成分としての本発明のペプチドの適用性について
効能重視の製薬と異なり、特定保健用食品はあくまでも食品であって、同様の効能を有する当該製薬と同等の効き目を必要としない。むしろ、安全性の点から、特定保健用食品の効き目は、製薬よりも１０００〜１００００倍ほど弱いことが望ましい。
現在、我が国で承認されているＤＰＰＩＶ阻害薬は７つあるが、いずれも製薬として強力なｎＭレベルで効果を発揮する。その強力な効果の反面、副作用のおそれも懸念される。
これに対し、本発明の７種のペプチドを含有する組成物（Ｙ−２画分）は、効果がμＭレベルであり、製薬の力価よりも１０００倍程度弱いことから、効き過ぎず、特定保健用食品の成分として、妥当である。今後の動物試験及び臨床試験で十分にその効果が証明されると考えられる。
(Discussion) The applicability of the peptide of the present invention as a component of a food for specified health use compared with a pharmaceutical product Unlike a pharmaceutical product with emphasis on efficacy, a food product for specified health use is only a food product and has the same efficacy Equivalent effect is not required. Rather, from the viewpoint of safety, it is desirable that the effectiveness of the food for specified health use is about 1000 to 10,000 times weaker than that of pharmaceuticals.
At present, there are seven DPPIV inhibitors approved in Japan, all of which are effective as pharmaceuticals at powerful nM levels. Despite its powerful effects, there are concerns about possible side effects.
On the other hand, the composition containing the seven peptides of the present invention (Y-2 fraction) has an effect on the μM level and is about 1000 times weaker than the pharmaceutical titer. Appropriate as a component of health food. Future animal studies and clinical studies are expected to prove their effects.
Claims (7)
Ｇｌｙ−Ｌｅｕ、Ｌｅｕ−Ｐｈｅ、Ｐｈｅ−Ｐｒｏ、Ｍｅｔ−Ｐｒｏ−Ｐｈｅ、Ｌｅｕ−Ｐｒｏ−Ｌｅｕ、Ｍｅｔ−Ｐｒｏ−Ｌｅｕ及びＡｌａ−Ｇｌｙ−Ａｌａ−Ｍｅｔ−Ｐｒｏを、当該組成物の乾燥物に基づき１乃至１０質量％の量でそれぞれ含有し、且つα−グルコシダーゼ阻害活性（ＩＣ５０）が１．５ｍｇ／ｍＬ以上である、前記鰹だし粕由来のジペプチジルペプチダーゼＩＶ阻害組成物。 A dipeptidyl peptidase IV-inhibiting composition derived from rice cake,
Gly-Leu, Leu-Phe, Phe-Pro, Met-Pro-Phe, Leu-Pro-Leu, the Met-Pro-Leu and Ala-Gly-Ala-Met- Pro, 1 based on the dry matter of the composition The dipeptidyl peptidase IV-inhibiting composition derived from the soup stock which has an α-glucosidase inhibitory activity (IC50) of 1.5 mg / mL or more, each contained in an amount of 10 to 10% by mass .
１）鰹節を６０℃〜１００℃の熱水で抽出し、さらに遠心分離によって上澄を取り除き、鰹だし粕を得る工程；
２）前記鰹だし粕を、至適条件下、Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｉｚａｅ属、Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ属及びＡｓｐｅｒｉｇｉｌｌｕｓ ｎｉｇｅｒ属からなる群より選択される少なくとも１種の菌株由来のプロテアーゼであって食品工業用途のプロテアーゼによって酵素分解し、さらに遠心分離によって清澄液を得る工程；及び
３）前記清澄液を精製する工程；
を含むことを特徴とする、前記鰹だし粕由来のジペプチジルペプチダーゼＩＶ阻害組成物の製造方法。 Gly-Leu, Leu-Phe, Phe-Pro, Met-Pro-Phe, Leu-Pro-Leu, Met-Pro-Leu and Ala-Gly-Ala-Met-Pro based on the dried product of the composition 1 A method for producing a dipeptidyl peptidase IV inhibitory composition derived from tsujidashi koji, each of which is contained in an amount of 10 to 10% by mass, and the α-glucosidase inhibitory activity (IC50) is 1.5 mg / mL or more,
1) A step of extracting bonito with hot water at 60 ° C. to 100 ° C. and further removing the supernatant by centrifugation to obtain a soup stock koji;
2) The soup stock is a protease derived from at least one strain selected from the group consisting of Aspergillus oryzae, Bacillus licheniformis and Aspergillus niger under optimal conditions, and is enzymatically degraded by a protease for food industry use And a step of obtaining a clarified liquid by centrifugation; and 3) a step of purifying the clarified liquid;
A method for producing a dipeptidyl peptidase IV-inhibiting composition derived from the soup stock.
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| JP7068942B2 (en) * | 2018-06-25 | 2022-05-17 | サンスター株式会社 | Tripeptide-containing composition |
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