JP4879537B2 - Composition having Helicobacter pylori peeling action - Google Patents
Composition having Helicobacter pylori peeling action Download PDFInfo
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- JP4879537B2 JP4879537B2 JP2005267879A JP2005267879A JP4879537B2 JP 4879537 B2 JP4879537 B2 JP 4879537B2 JP 2005267879 A JP2005267879 A JP 2005267879A JP 2005267879 A JP2005267879 A JP 2005267879A JP 4879537 B2 JP4879537 B2 JP 4879537B2
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- Prior art keywords
- whey
- helicobacter pylori
- milk
- polypeptide
- pharmaceutical composition
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- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
本発明は、ヘリコバクター・ピロリ剥離作用を有する組成物に関する。より詳細には、ホエーを加水分解することにより得られる、ヘリコバクター・ピロリ剥離作用を有する組成物に関する。 The present invention relates to a composition having a Helicobacter pylori peeling action. More specifically, the present invention relates to a composition having a Helicobacter pylori exfoliating action obtained by hydrolyzing whey.
ヘリコバクター・ピロリ(Helicobacter pylori;Hp)は、WarrenとMarshallにより胃潰瘍患者の胃から発見されて以来(非特許文献1参照)、世界的に最も感染者数の多い病原性菌の1つであることが明らかになった(非特許文献2参照)。現在では、胃炎(非特許文献3参照)、消化性潰瘍(非特許文献1及び非特許文献4参照)、胃癌(非特許文献5、非特許文献6及び非特許文献7参照)及び胃リンパ腫(非特許文献8参照)の発症に関与していることが示唆されている。 Helicobacter pylori (Helicobacter pylori; Hp) has since been found in stomach ulcer patient by Warren and Marshall (see Non-Patent Document 1), worldwide it is one of the most infected persons a large number of pathogenic bacteria Became clear (see Non-Patent Document 2). Currently, gastritis (see non-patent document 3), peptic ulcer (see non-patent document 1 and non-patent document 4), gastric cancer (see non-patent document 5, non-patent document 6 and non-patent document 7) and gastric lymphoma ( It is suggested to be involved in the onset of non-patent document 8).
Hpを除菌する治療法や医薬品の研究は盛んに行われている。現在は抗生物質投与による治療が主流である。我国でも日本ヘリコバクター学会が「H. pylori感染の診断と治療のガイドライン」を作成し、除菌治療の第一選択薬としてプロトンポンプ阻害薬(lansoprazole)と抗生物質二剤(amoxicillinおよびclarithromycin)を1週間投与する3剤併用療法を推奨している。しかしながら、副作用が14.8〜45.1%の患者で報告され、耐性菌も5〜10%の頻度で発生していることが問題点として論じられている。一方で抗生物質を用いない除菌法も研究されており、その代表としてはプロバイオティクスを用いる方法などが挙げられる(非特許文献9参照)。しかしながら、メカニズムが解明されていないため、医学的治療に応用するには至っていないのが現状である。そこで胃粘膜の構造および性状、さらに粘膜内でのHpの細菌学的特徴を考慮した除菌方法(素材)を開発することが望まれている。 Researches on therapeutic methods and medicines for eradicating Hp are actively conducted. Currently, treatment with antibiotics is the mainstream. In Japan, the Japan Helicobacter Society has prepared “Guidelines for Diagnosis and Treatment of H. pylori Infection”, and 1 proton pump inhibitor (lansoprazole) and two antibiotics (amoxicillin and clarithromycin) are the first choices for eradication Three-drug combination therapy administered weekly is recommended. However, side effects have been reported in 14.8-45.1% of patients, and it has been discussed as a problem that resistant bacteria are occurring at a frequency of 5-10%. On the other hand, sterilization methods that do not use antibiotics have also been studied, and representative examples include methods using probiotics (see Non-Patent Document 9). However, since the mechanism has not been elucidated, it has not yet been applied to medical treatment. Therefore, it is desired to develop a sterilization method (material) in consideration of the structure and properties of the gastric mucosa and the bacteriological characteristics of Hp in the mucosa.
胃粘膜は、表層ムチン層と腺ムチン層が交互に重なる多重構造をしている。表層ムチンは胃酸から胃を保護するために粘膜上皮細胞から常に分泌されるが、腺ムチンは食事摂取の際に胃底腺から胃酸やペプシンを包む形で分泌される。また、表層ムチンは、セリン、トレオニン、プロリンおよびシステインに富む1,300アミノ酸のポリペプチドを基本骨格とするMUC5AC糖タンパク質であることが知られている(非特許文献10参照)。Hpは、胃内の、主として上皮細胞近くの表層ムチン層で生育・増殖しており、新しいムチンが分泌されると押し上げられて胃内腔表面(粘膜層上層)に近づくが、その後上皮細胞側へ移動するといった動態を繰り返していると考えられている(非特許文献11参照)。Hpと胃上皮細胞やそれを覆う胃ムチンとの接着に関する研究も多く行われており、3’−シアリルラクトース(NeuAcα2−3Galβ1−4Glc;3’SL)に代表されるシアル酸を中心に(非特許文献12及び非特許文献13参照)、糖タンパク質や糖脂質の硫酸化オリゴ糖(非特許文献14及び非特許文献15参照)、さらに、基底膜構成成分であるラミニンやコラーゲン等多数が報告されている(非特許文献16参照)。 The gastric mucosa has a multi-layer structure in which the surface mucin layer and the gland mucin layer alternately overlap. Superficial mucin is always secreted from mucosal epithelial cells to protect the stomach from gastric acid, whereas glandular mucin is secreted from the fundus gland in the form of gastric acid and pepsin when ingested. The surface mucin is known to be a MUC5AC glycoprotein having a 1,300 amino acid polypeptide rich in serine, threonine, proline and cysteine as a basic skeleton (see Non-Patent Document 10). Hp grows and proliferates in the stomach, mainly in the surface mucin layer near the epithelial cells. When new mucin is secreted, it is pushed up and approaches the surface of the stomach lumen (upper layer of the mucosal layer). It is thought that the movement of moving to is repeated (see Non-Patent Document 11). Many studies have been conducted on the adhesion between Hp and gastric epithelial cells and gastric mucin covering the same, mainly sialic acid represented by 3'-sialyl lactose (NeuAcα2-3Galβ1-4Glc; 3'SL) (non- Patent Document 12 and Non-Patent Document 13), sulfated oligosaccharides of glycoproteins and glycolipids (see Non-Patent Document 14 and Non-Patent Document 15), and a large number of laminin and collagen as basement membrane constituents have been reported. (See Non-Patent Document 16).
こうした知見を利用して、in vitroにおける上皮細胞(モデル)や胃ムチンへのHp接着抑制作用が、上記の物質(非特許文献17参照)に加えクランベリージュース(非特許文献18及び非特許文献19参照)や牛乳由来の糖タンパク質(非特許文献20参照)などについて報告されている。これらの結果は、Hpと阻害物質を同時に添加して凝集反応を調べるか、プレートに固定化した細胞あるいはムチンに菌体懸濁液と同時に阻害物質を混入して得られたものである(非特許文献20及び非特許文献19参照)。しかしながら、Hp凝集/接着抑制作用があっても、Hp保菌者に対して投与する場合、実際に除菌効果が得られるか不明である。胃ムチンからのHp剥離作用を調べている報告は殆どなく、クランベリージュースを用いた報告(非特許文献18参照)では、Hp接着抑制作用が認められた高分子非透析画分にHp剥離作用が無かったと示されている。 Utilizing such knowledge, in vitro Hp adhesion inhibitory action on epithelial cells (model) and gastric mucin is added to the above substances (see Non-Patent Document 17) and cranberry juice (Non-Patent Document 18 and Non-Patent Document 19). Reference) and milk-derived glycoproteins (see Non-Patent Document 20). These results were obtained by simultaneously adding Hp and an inhibitor and examining the agglutination reaction, or by mixing an inhibitor with cells or mucin immobilized on a plate at the same time as the cell suspension (non-) (See Patent Literature 20 and Non-Patent Literature 19). However, even if there is an Hp aggregation / adhesion inhibitory effect, it is unclear if the sterilization effect is actually obtained when administered to Hp carriers. There are almost no reports investigating Hp peeling action from gastric mucin, and in the report using cranberry juice (see Non-Patent Document 18), the Hp peeling action is observed in the polymer non-dialysis fraction in which Hp adhesion inhibitory action was observed. It is shown that there was no.
さらに、ピロリに対する感染阻止効果に関し、乳汁由来のムチンに定着阻害効果があることが開示されている(特許文献1参照)。しかしながら、記載されている方法は乳脂肪及びカゼイン、さらにはリポ蛋白を除去した後、セファロースカラムで乳汁由来のムチンを分離するものである。また、乳汁由来のムチンの原料としてチーズ等の製造工程で副産物として大量に出るホエーが利用可能であり、価格的にも実用的にも有利であることが記載されているが、乳汁由来のムチンはその調整方法が煩雑なことに加えて乳汁中の含量が極めて少なく(特許文献1において0.01%の収量)、高価な素材となることが予想され、採算性及び実現性には問題がある。 Furthermore, regarding the infection prevention effect against H. pylori, it has been disclosed that mucin derived from milk has an inhibitory effect on colonization (see Patent Document 1). However, the described method is to separate milk-derived mucin on a Sepharose column after removing milk fat and casein, as well as lipoproteins. In addition, it is described that whey that is produced in large quantities as a by-product in the production process of cheese or the like can be used as a raw material for milk-derived mucin, which is advantageous in terms of price and practicality. In addition to its complicated adjustment method, the content in milk is extremely small (0.01% yield in Patent Document 1) and is expected to be an expensive material, and there are problems in profitability and feasibility. is there.
これまで発明者らは、原材料としてバターミルク(即ち、牛乳から分離したクリームを原料とし、バター製造のチャーニング工程中に脂肪塊と分離されて生じる液体)をプロテアーゼ処理することでシアル酸量に関係なく、Hpとの接着性や剥離活性が著しく上昇することを報告している(非特許文献25)。すなわち、プロテアーゼ処理でタンパク質の立体構造を変化させることにより、シアル酸を中心とした糖鎖部位以外にもHpに対して強い親和性を有するペプチドが形成されることを示している。 So far, the inventors have increased the amount of sialic acid by protease treatment of buttermilk (ie, a liquid produced by separating cream from milk and separated from fat mass during the buttering churn process) as a raw material. Regardless, it has been reported that adhesion to Hp and peeling activity are significantly increased (Non-patent Document 25). That is, it is shown that a peptide having a strong affinity for Hp is formed in addition to the sugar chain site centering on sialic acid by changing the three-dimensional structure of the protein by protease treatment.
ホエーはチーズ製造時に多量に生じ、我が国では年間約32万トン(2002年度)が排出されている。ホエーを有効利用する研究も進み、最近では機能性食品素材として様々な用途に使用されつつあるが、その大部分が産業廃棄物として処分されているのが現状である。 Whey is produced in large quantities during cheese manufacture, and approximately 320,000 tons (fiscal year 2002) are discharged annually in Japan. Research on the effective use of whey has also progressed, and recently it has been used as a functional food material for various purposes, but most of it is disposed of as industrial waste.
本発明は、シアル酸が含まれている乳汁ムチン成分とは関わりなく、全ホエータンパク質を単純にプロテアーゼ処理することで、シアル酸を介さずにHpと接着し、さらに胃ムチンに定着したHpを剥離する作用を有する安全で安価な組成物を提供することを可能にする。 In the present invention, regardless of the milk mucin component containing sialic acid, the whole whey protein is simply treated with protease to adhere to Hp without sialic acid, and further to fix Hp fixed on gastric mucin. It makes it possible to provide a safe and inexpensive composition having a peeling action.
本発明は、ヘリコバクター・ピロリ(Hp)の胃粘膜に対する接着抑制効果のみならず、Hp保菌者の胃粘膜からHpを剥離する効果も有する安価な組成物を提供することを目的とする。 An object of the present invention is to provide an inexpensive composition having not only the effect of suppressing the adhesion of Helicobacter pylori (Hp) to the gastric mucosa but also the effect of peeling Hp from the gastric mucosa of Hp carriers.
本発明者らは、ホエーのHpに対する効果について検討を行った。その結果、ホエーをプロテアーゼ処理することにより、Hpへの接着性が向上したホエー分解物が得られることを見出した。本発明はこれらの知見に基づき完成したものである。 The present inventors examined the effect of whey on Hp. As a result, it was found that a whey degradation product having improved adhesion to Hp can be obtained by treating whey with protease. The present invention has been completed based on these findings.
すなわち、本発明は、ホエーまたはその乾燥物の溶液をプロテアーゼ処理することにより得られる分子量1〜20kDaのポリペプチドを含む組成物を提供するものである。
また、本発明は、上記組成物から沈殿物を除去した液体またはその乾燥粉末を有効成分とする、ヘリコバクター・ピロリ接着抑制作用のみならず剥離作用を有する組成物を提供するものである。
That is, the present invention provides a composition comprising a polypeptide having a molecular weight of 1 to 20 kDa obtained by subjecting a solution of whey or a dried product thereof to protease treatment.
The present invention also provides a composition having not only a Helicobacter pylori adhesion inhibitory action but also a peeling action, comprising as an active ingredient a liquid obtained by removing precipitates from the composition or a dry powder thereof.
また、本発明は、上記組成物から沈殿物を除去した液体またはその乾燥粉末を有効成分とする、ヘリコバクター・ピロリ接着抑制作用のみならず剥離作用を有する組成物を提供するものである。 The present invention also provides a composition having not only a Helicobacter pylori adhesion inhibitory action but also a peeling action, comprising as an active ingredient a liquid obtained by removing precipitates from the composition or a dry powder thereof.
さらに、本発明は、ホエーまたはその乾燥物の溶液をプロテアーゼ処理することにより得られる分子量1〜20kDaのポリペプチドを有効成分として含む、消化器潰瘍の予防用または改善用食品を提供するものである。 Furthermore, the present invention provides a food for preventing or ameliorating digestive ulcer, comprising as an active ingredient a polypeptide having a molecular weight of 1 to 20 kDa obtained by treating a whey or a dried product thereof with a protease. .
また、本発明は、ホエーまたはその乾燥物の溶液をプロテアーゼ処理することにより得られる分子量1〜20kDaのポリペプチドを有効成分として含む、消化器潰瘍の予防剤または治療剤を提供する。 The present invention also provides a prophylactic or therapeutic agent for digestive ulcer, comprising as an active ingredient a polypeptide having a molecular weight of 1 to 20 kDa obtained by subjecting a solution of whey or a dried product thereof to protease treatment.
さらに、本発明は、ホエーまたはその乾燥物の溶液をプロテアーゼ処理することにより得られる分子量1〜20kDaのポリペプチドを有効成分として含む、ヘリコバクター・ピロリ感染の予防用または改善用食品を提供する。 Furthermore, the present invention provides a food for preventing or ameliorating Helicobacter pylori infection, comprising as an active ingredient a polypeptide having a molecular weight of 1 to 20 kDa obtained by subjecting a solution of whey or a dried product thereof to protease treatment.
また、本発明は、ホエーまたはその乾燥物の溶液をプロテアーゼ処理することにより得られる分子量1〜20kDaのポリペプチドを有効成分として含む、ヘリコバクター・ピロリ感染の予防剤または治療剤を提供する。 The present invention also provides a preventive or therapeutic agent for Helicobacter pylori infection, comprising as an active ingredient a polypeptide having a molecular weight of 1 to 20 kDa obtained by treating a whey or a dried product thereof with a protease.
本発明の組成物は、胃の粘膜層に感染したHpを剥離する活性が顕著に高い。
本発明の組成物は、ホエー1Lあたりから乾燥重量で2〜8g回収でき、上述の乳汁ムチンと比較すると回収率が非常に高い。また、ホエーパウダーを初発原料とした場合は10〜20%の高収率で調製することができ、上述の採算性の問題を解決するものである。
The composition of the present invention has a remarkably high activity for peeling off Hp infected to the gastric mucosal layer.
The composition of the present invention can recover 2 to 8 g in dry weight from 1 L of whey, and the recovery rate is very high compared to the milk mucin described above. Moreover, when whey powder is used as the starting material, it can be prepared with a high yield of 10 to 20%, which solves the above-mentioned problem of profitability.
したがって、本発明の組成物を用いることにより、Hp感染に対する予防及び/または治療効果を有する安価な食品や医薬品の開発が可能となる。さらに、これらの食品や医薬品は、Hp感染に関連する消化器潰瘍などの疾患の予防及び/または治療に有効である。 Therefore, by using the composition of the present invention, it is possible to develop inexpensive foods and pharmaceuticals having preventive and / or therapeutic effects against Hp infection. Furthermore, these foods and pharmaceuticals are effective for the prevention and / or treatment of diseases such as digestive ulcers associated with Hp infection.
本発明は、非特許文献25に記載のバターミルクとは全く異なる成分であるホエーを原材料としたものである。アミノ酸パターンは理想的で栄養学的に非常に優れていることから、ホエーを用いることで、バターミルク分解物とは異なる栄養価の高いHp接着抑制/剥離物質を得ることができる。 The present invention uses whey, which is a completely different component from buttermilk described in Non-Patent Document 25, as a raw material. Since the amino acid pattern is ideal and very nutritionally superior, it is possible to obtain a high nutritional Hp adhesion-inhibiting / peeling substance different from buttermilk degradation products by using whey.
成分無調整牛乳に由来する牛乳ホエーや、チーズ製造時に生じるホエーをプロテアーゼで加水分解処理することにより、これらを構成するペプチドを主成分とする本発明の組成物を調製することができる。加水分解はプロテアーゼ処理だけでなく、各種の酸やアルカリなどの化学的処理でも行うことができるが、食品に含有させることを考慮するとプロテアーゼ処理が好ましい。加水分解に用いるプロテアーゼとしては、トリプシン、パパイン、パンクレアチン、ペプシンなどが挙げられるが、乳酸菌やその他のプロテアーゼ活性を有する微生物、及びこれらの抽出物を用いることも可能であり、プロテアーゼ活性をもつものである限りこれらに限定されるものではない。 By hydrolyzing milk whey derived from component-unadjusted milk or whey produced during cheese production with a protease, the composition of the present invention comprising as a main component the peptides constituting them can be prepared. Hydrolysis can be carried out not only by protease treatment but also by chemical treatment such as various acids and alkalis. In consideration of inclusion in food, protease treatment is preferred. Examples of proteases used for hydrolysis include trypsin, papain, pancreatin, pepsin, etc., but it is also possible to use lactic acid bacteria and other microorganisms having protease activity, and extracts thereof, and those having protease activity As long as it is, it is not limited to these.
本発明の組成物の主成分であるポリペプチドは、分子量が1〜20kDaであり、好ましくは5〜15kDaであり、更に好ましくは6〜10kDaである。
本発明の組成物は、例えば以下のようにして調製することができる。ホエーまたはホエーパウダー、あるいはホエーを成分として含有するその他の原料を脱イオン水に懸濁し、85〜90℃で加熱殺菌する。懸濁液を冷却後、水酸化ナトリウムあるいは塩酸を添加して、用いるプロテアーゼに至適のpHに調整する。その後、プロテアーゼ粉末、あるいは懸濁液を添加し、至適温度で緩く撹拌しながら反応させる。反応後、水酸化ナトリウムあるいは塩酸でpHを6.8〜7.0に調整し、90℃、10分間の加熱でプロテアーゼを失活させる。冷却後、凍結乾燥し、ホエー加水分解物を得る。プロテアーゼの使用量、反応温度、反応時間などの具体的な反応条件については、当業者であれば容易に設定が可能であろう。このホエー加水分解物中に含まれるポリペプチドは、本発明に精製して用いてもよく、また未精製のまま用いてもよい。
The polypeptide which is the main component of the composition of the present invention has a molecular weight of 1 to 20 kDa, preferably 5 to 15 kDa, more preferably 6 to 10 kDa.
The composition of the present invention can be prepared, for example, as follows. Whey or whey powder or other raw materials containing whey as a component are suspended in deionized water and sterilized by heating at 85 to 90 ° C. After cooling the suspension, sodium hydroxide or hydrochloric acid is added to adjust the pH to the optimum value for the protease used. Thereafter, protease powder or suspension is added, and the reaction is carried out with gentle stirring at the optimum temperature. After the reaction, the pH is adjusted to 6.8 to 7.0 with sodium hydroxide or hydrochloric acid, and the protease is inactivated by heating at 90 ° C. for 10 minutes. After cooling, it is freeze-dried to obtain a whey hydrolyzate. Specific reaction conditions such as the amount of protease used, the reaction temperature, and the reaction time can be easily set by those skilled in the art. The polypeptide contained in this whey hydrolyzate may be used after purification in the present invention, or may be used as it is without purification.
上記のようにして調製した本発明の組成物は、Hp表層成分との結合能が高く、胃粘膜からHpを剥離させる作用を有する。また、Hp剥離活性は、出発材料であるホエーやホエーパウダーには存在せず、プロテアーゼによる分解で初めて活性が発現する。 The composition of the present invention prepared as described above has a high binding ability with the Hp surface layer component and has an action of peeling Hp from the gastric mucosa. Moreover, Hp peeling activity does not exist in whey and whey powder which are starting materials, and the activity appears only after decomposition by protease.
したがって、本発明は、上記ポリペプチドを有効成分とするHp剥離作用を有する組成物を提供するものである。
本発明の組成物は、ホエー加水分解物から遠心分離などにより沈殿物を除去した液体若しくはその乾燥粉末、またはその沈殿物を用いることもできる。
Therefore, the present invention provides a composition having an Hp peeling action comprising the above polypeptide as an active ingredient.
In the composition of the present invention, a liquid obtained by removing a precipitate from a whey hydrolyzate by centrifugation or the like, a dry powder thereof, or a precipitate thereof can also be used.
本発明において、上記ポリぺプチドを有効成分として食品中に含有させることにより、Hp感染や消化器潰瘍の予防・改善用食品として用いることができる。
本発明の消化器潰瘍の予防・改善用食品は、実質的に上記ポリペプチドのみを含む食品であってもよいし、他の栄養補助食品と組み合わせたサプリメントの形であっても良い。また、当該ポリペプチドを既存の食品や飲料等に直接添加したものであってもよい。例えば、ペプチドをガム、キャンディー、ゼリー、グミ、クッキー、ビスケット、チョコレート等の菓子類、ジュース等の清涼飲料、チーズ、バター、ヨーグルト等の乳製品、アイスクリーム、ハム等の農産加工品、ちくわ、はんぺん等の水産加工品、そば、うどん等の麺類、パン、ケーキ等の小麦粉加工品、缶詰または味噌、醤油、塩、こしょう、さとう、人工甘味料等の調味食品等に直接添加することができる。また、ポリペプチドをこれらの食品の加工段階で混合してもよい。ポリペプチドを食品に加工する際は、通常の食品加工方法に基づき行うことができる。また、ポリペプチドを食品に添加・混合する際は、ポリペプチドを粉末、顆粒、細粒等の固形状で用いてもよく、あるいは液状で用いてもよい。本発明の消化器潰瘍の予防・改善用食品は、消化器潰瘍の予防または改善用の特定保健用食品、機能性食品、健康食品としても応用可能である。なお、「機能性食品」とは、生体調節機能をもつ物質を含む食品を意味し、厚生労働省より特定保健用食品として食品毎に個別の許可を得ることにより、機能性を表示することができる。
In the present invention, by containing the above-mentioned polypeptide as an active ingredient in a food, it can be used as a food for preventing or improving Hp infection or digestive ulcer.
The food for preventing / ameliorating gastrointestinal ulcer of the present invention may be a food containing substantially only the above-mentioned polypeptide, or may be in the form of a supplement combined with other nutritional supplements. Moreover, what added the said polypeptide directly to the existing foodstuff, a drink, etc. may be used. For example, peptides such as gum, candy, jelly, gummi, cookies, biscuits, chocolate and other confectionery, juices and other soft drinks, cheese, butter, yogurt and other dairy products, ice cream, ham and other agricultural products, chikuwa, Can be added directly to processed marine products such as hampen, noodles such as buckwheat and udon, processed flour such as bread and cake, canned or miso, soy sauce, salt, pepper, sugar, and artificial foods . Polypeptides may also be mixed during the processing of these foods. When processing a polypeptide into food, it can be performed based on a normal food processing method. In addition, when adding or mixing a polypeptide to a food, the polypeptide may be used in a solid form such as a powder, granule or fine particle, or in a liquid form. The food for preventing / ameliorating gastrointestinal ulcer according to the present invention can also be applied as a food for specified health use, functional food, and health food for preventing or improving gastrointestinal ulcer. “Functional food” means food containing a substance with a bioregulatory function, and the functionality can be displayed by obtaining individual permission for each food as food for specified health use from the Ministry of Health, Labor and Welfare. .
本発明の食品においては、一般に食品100gあたり0.1〜50g程度の範囲でポリペプチドを配合することが好ましい。摂取量は、摂取対象の性別、体重、体質、年齢、他の摂取食品等により異なるが、一般にヒト成人が対象の場合には、ポリペプチド摂取量が1日あたり3〜15gとなる範囲で摂取させることが好ましい。摂取回数は、1週に数回ないし1日1回が適当である。 In the food of the present invention, it is generally preferable to mix the polypeptide in a range of about 0.1 to 50 g per 100 g of food. The intake varies depending on the sex, body weight, constitution, age, other foods to be ingested, etc., but in general, in the case of human adults, the intake of the polypeptide is in the range of 3 to 15 g per day. It is preferable to make it. The number of times of intake is appropriate several times a week to once a day.
また、本発明の組成物は、Hp感染や消化器潰瘍の予防剤又は治療剤などの医薬組成物として用いることもできる。
本発明の消化器潰瘍の予防剤又は治療剤を生体に投与する場合は、経口的に投与する。
The composition of the present invention can also be used as a pharmaceutical composition such as a preventive or therapeutic agent for Hp infection or digestive ulcer.
When the preventive or therapeutic agent for digestive ulcer of the present invention is administered to a living body, it is administered orally.
本発明の組成物を適当な剤型に製剤化して用いてもよい。例えば錠剤、散剤、顆粒剤、細粒剤、丸剤、カプセル剤、トローチ剤、チュワブル剤、液剤、乳剤、懸濁剤、シロップ剤等の製剤で用いることができる。これらの剤型に製剤化するには薬学上許容しうる適当な担体、賦形剤、添加剤等を用いて行うことができる。 The composition of the present invention may be formulated into an appropriate dosage form and used. For example, it can be used in preparations such as tablets, powders, granules, fine granules, pills, capsules, troches, chewables, solutions, emulsions, suspensions and syrups. Formulation into these dosage forms can be performed using appropriate pharmaceutically acceptable carriers, excipients, additives and the like.
また錠剤、丸剤、散剤、顆粒剤、細粒剤、トローチ剤、チュワブル剤等の固形製剤を調製するには、例えば重炭酸ナトリウム、炭酸カルシウム、デンプン、ショ糖、マンニトール、カルボキシメチルセルロース等の担体、ステアリン酸カルシウム、ステアリン酸マグネシウム、グリセリン等の添加剤を加えて常法により行うことができる。 In order to prepare solid preparations such as tablets, pills, powders, granules, fine granules, troches, chewables, carriers such as sodium bicarbonate, calcium carbonate, starch, sucrose, mannitol, carboxymethylcellulose, etc. In addition, an additive such as calcium stearate, magnesium stearate, glycerin and the like can be added and used in a conventional manner.
本発明の組成物を、医薬として使用する場合には、その投与量は、患者の性別、体重、体質、年齢、症状あるいは投与剤型等により異なるが、一般にポリペプチドを有効成分として成人1日当り0.1〜50g、好ましくは3〜15gの範囲で適宜選択することができる。投与回数は、患者の症状あるいは投与剤型等により異なるが、1日1回ないし1日数回が適当である。 When the composition of the present invention is used as a medicine, the dosage varies depending on the sex, body weight, constitution, age, symptom, dosage form, etc. of the patient. It can be suitably selected within the range of 0.1 to 50 g, preferably 3 to 15 g. The number of administrations varies depending on the patient's symptoms or dosage form, but is suitable once a day to several times a day.
Hp接着試験
材料および方法
(1)使用菌株
この試験において使用したHelicobacter pylori ATCC 43504TおよびATCC 43579はAmerican Type Culture Collectionより購入した。
Hp adhesion test
Materials and Methods (1) Strains Used Helicobacter pylori ATCC 43504 T and ATCC 43579 used in this test were purchased from the American Type Culture Collection.
(2)ホエー加水分解物の調製
成分無調整牛乳をpH4.6に調整後、遠心分離して得た牛乳ホエー、クリームチーズ及びカッテージチーズ製造時に生じるホエーを原材料とした。それぞれを85〜95℃で15分間殺菌し、冷却した。それぞれのホエーを2N水酸化ナトリウムあるいは塩酸(ペプシンを使用する場合は濃塩酸)にて至適pH付近に調製し、ホエー1Lあたり2gの各種プロテアーゼ粉末(日本バイコン社製)を添加し、至適温度で24時間緩く撹拌しながら反応させた。反応開始12時間後にpHを再調整し、2gのプロテアーゼ粉末を再添加した。反応後、水酸化ナトリウムあるいは塩酸にてpHを6.8〜7.0に調整し、90℃、10分間の加熱で酵素を失活させた。冷却後、凍結乾燥し、ホエー加水分解物を得た。使用酵素、その至適温度および至適pHを表1に示す。
(2) Preparation of whey hydrolyzate Whey produced during the production of milk whey, cream cheese and cottage cheese obtained by adjusting the non-component milk to pH 4.6 and then centrifuging was used as a raw material. Each was sterilized at 85-95 ° C. for 15 minutes and cooled. Prepare each whey with 2N sodium hydroxide or hydrochloric acid (concentrated hydrochloric acid if pepsin is used) near the optimum pH, and add 2 g of various protease powders (manufactured by Nippon Bicon) per liter of whey. The reaction was allowed to proceed with gentle stirring at temperature for 24 hours. The pH was readjusted 12 hours after the start of the reaction, and 2 g of protease powder was added again. After the reaction, the pH was adjusted to 6.8 to 7.0 with sodium hydroxide or hydrochloric acid, and the enzyme was inactivated by heating at 90 ° C. for 10 minutes. After cooling, it was freeze-dried to obtain a whey hydrolyzate. Table 1 shows the enzyme used, its optimum temperature and optimum pH.
(3)試験方法
各種プロテアーゼによるホエー加水分解物、未処理ホエー、ブタ胃粗ムチンを、それぞれ10mg/mLとなるように0.1M炭酸バッファー(pH9.6)に懸濁/溶解し、96ウェルマイクロプレート(Nunc A/S)に75μL/ウェルずつ加え、37℃で2時間コーティングした。滅菌PBS(pH7.2)250μLにて各ウェルを3回洗浄後、Hp懸濁液を50μL/ウェルずつ添加し、微好気条件下で緩やかに振とうしながら(30rpm)37℃で2時間培養した。滅菌PBSで5回洗浄することにより非接着菌体を除去した。その後、100μLのPBSを添加して激しいピペッティングを行い、接着菌体を剥離し、50μLを5%緬羊脱繊維血液添加Tryptic soy寒天培地に塗抹した。37℃で96時間、微好気培養後、出現コロニー数をカウントし、1ウェル当たりの接着菌数を算出した。
(3) Test method Suspended / dissolved whey hydrolyzate with various proteases, untreated whey, and porcine stomach mucin in 0.1 M carbonate buffer (pH 9.6) to a concentration of 10 mg / mL, respectively. 75 μL / well was added to a microplate (Nunc A / S) and coated at 37 ° C. for 2 hours. Each well was washed 3 times with 250 μL of sterile PBS (pH 7.2), Hp suspension was added at 50 μL / well, and gently shaken under microaerobic conditions (30 rpm) at 37 ° C. for 2 hours. Cultured. Non-adherent cells were removed by washing 5 times with sterile PBS. Thereafter, 100 μL of PBS was added and vigorous pipetting was performed to peel the adherent cells, and 50 μL was smeared on a Tryptic soy agar medium supplemented with 5% sheep defibrillated blood. After microaerobic culture at 37 ° C. for 96 hours, the number of appearing colonies was counted, and the number of adherent bacteria per well was calculated.
なお、ブタ胃ムチンはSigma社より購入したものを用いた。
また、Hp懸濁液は上記の培地上で96時間培養したHpコロニーを綿棒にて回収し、660nmの吸光度が0.4±0.01となるようにPBSに懸濁したものを100倍希釈した調製液である。
The porcine stomach mucin used was purchased from Sigma.
In addition, Hp suspension was obtained by collecting Hp colonies cultured for 96 hours on the above medium with a cotton swab and suspending them in PBS so that the absorbance at 660 nm was 0.4 ± 0.01. Prepared solution.
(4)統計処理
接着性の比較は独立2標本のt−検定にて実施し、p<0.05を統計学的有意とした。なお、統計解析用ソフトはSTATISTICA(デザインテクノロージーズ)を使用した。
(4) Statistical processing Adhesive comparison was performed by t-test of two independent samples, and p <0.05 was considered statistically significant. The statistical analysis software used was STATISTICA (Design Technologies).
結果
各種プロテアーゼ処理されたホエー加水分解物のATCC 43504T及びATCC 43579に対する接着性を、平均接着菌体数として図1に示す(4回平均)。
Results The adhesion of various protease-treated whey hydrolysates to ATCC 43504 T and ATCC 43579 is shown in FIG. 1 as the average number of adherent cells (average of 4 times).
ATCC 43504Tに対する接着性はパンクレアチン分解ホエーが未処理ホエーと比較して約8倍と有意に強い接着性を示した(p<0.05)。ATCC 43579に対する接着性についてみると、トリプシン(p<0.05)、パパイン(p<0.05)、パンクレアチン(p<0.001)、ペプシン(p<0.05)分解ホエーは未処理ホエーと比較して2〜5倍と有意に強い接着性を示した。 The adhesion to ATCC 43504 T was significantly stronger in pancreatin-degrading whey, approximately 8 times compared to untreated whey (p <0.05). As for the adhesion to ATCC 43579, trypsin (p <0.05), papain (p <0.05), pancreatin (p <0.001), pepsin (p <0.05) decomposed whey is untreated Compared to whey, it showed significantly stronger adhesion, 2 to 5 times.
ブタ胃ムチンとの接着性と比較した結果、ATCC 43504Tに対しては、パンクレアチン分解ホエーが有意に強い接着性を示した(p<0.05)。ATCC 43579に対しても、トリプシン(p<0.01)、パパイン(p<0.05)、パンクレアチン(p<0.001)、ペプシン(p<0.01)分解ホエーは有意に強い接着性を示した。 As a result of comparison with adhesion to porcine stomach mucin, pancreatin-degrading whey showed significantly stronger adhesion to ATCC 43504 T (p <0.05). Trypsin (p <0.01), papain (p <0.05), pancreatin (p <0.001), and pepsin (p <0.01) -degraded whey are also significantly stronger against ATCC 43579. Showed sex.
Hp剥離試験
実施例1でホエーをプロテアーゼ処理することによりHpに対し強力な接着性を示す分解物が得られることが確認されたことから、このホエー分解物を用いてHp剥離活性を確認した。
Hp Peeling Test Since it was confirmed that a degradation product exhibiting strong adhesiveness to Hp was obtained by treating protease with whey in Example 1, Hp peeling activity was confirmed using this whey degradation product.
材料および方法
(1)使用菌株
ヘリコバクター・ピロリATCC 43504TおよびATCC 43579はAmerican Type Culture Collectionより購入した。これらの菌株は5%緬羊脱繊維血液添加Tryptic soy 寒天培地(MERCK)に白金耳にて塗抹し、アネロパック・ヘリコ(三菱ガス)を用いた微好気培養にて96時間毎に継代培養した。なお、継代毎にグラム染色による形態観察とカタラーゼ、ウレアーゼおよびオキシダーゼ試験にて純粋培養が行われていることを確認した。
Materials and Methods (1) Strains Used Helicobacter pylori ATCC 43504 T and ATCC 43579 were purchased from the American Type Culture Collection. These strains were smeared on a Tryptic soy agar medium (MERCK) supplemented with 5% sheep defibrinated blood with platinum ears, and subcultured every 96 hours in microaerobic culture using Aneropac Helico (Mitsubishi Gas). . In addition, it was confirmed that pure culture was performed by morphology observation by Gram staining and catalase, urease, and oxidase tests at each passage.
(2)ホエー加水分解物の調製
実施例1と同様の方法でホエー分解物を調製した。プロテアーゼは実施例1でHpに対し高い接着活性が得られたパパインおよびパンクレアチンを用いた。
(2) Preparation of whey hydrolyzate A whey hydrolyzate was prepared in the same manner as in Example 1. As the protease, papain and pancreatin, which obtained high adhesive activity against Hp in Example 1, were used.
(3)試験方法
ブタ胃ムチンを、10mg/mLとなるように0.1M炭酸バッファーに懸濁し、96ウェルマイクロプレートに75μL/ウェルずつ加えて37℃で2時間コーティングした。滅菌PBS(pH7.2)250μLにて各ウェルを3回洗浄後、Hp懸濁液を50μL/ウェルずつ添加し、微好気条件下で緩やかに振とうしながら(30rpm)37℃で2時間培養した。滅菌PBSにて2回洗浄後、PBSに2.5mg/mLの濃度で懸濁/溶解したホエー加水分解物を50μL/ウェル添加し、微好気条件下で緩やかに振とうしながら(30rpm)37℃で1時間培養した。滅菌PBSにて2回洗浄後、100μLのPBSを添加し、激しいピペッティングにて接着菌体を剥離し、50μLを上記の培地に塗抹し、微好気条件にて培養後のコロニー数を計測した。コントロールはホエー加水分解物の代わりにPBSを用いた。各濃度における剥離率は以下の式で算出した。
剥離率(%)=[(A−B)/A]×100
A:コントロールサンプルにおけるマイクロプレート当たりの接着菌体数
B:ホエー分解物添加サンプルにおけるマイクロプレート当たりの接着菌体数
(3) Test method Porcine gastric mucin was suspended in 0.1 M carbonate buffer so as to be 10 mg / mL, and 75 μL / well was added to a 96-well microplate and coated at 37 ° C. for 2 hours. Each well was washed 3 times with 250 μL of sterile PBS (pH 7.2), Hp suspension was added at 50 μL / well, and gently shaken under microaerobic conditions (30 rpm) at 37 ° C. for 2 hours. Cultured. After washing twice with sterile PBS, 50 μL / well of whey hydrolyzate suspended / dissolved in PBS at a concentration of 2.5 mg / mL was added and gently shaken under microaerobic conditions (30 rpm) The cells were cultured at 37 ° C for 1 hour. After washing twice with sterile PBS, add 100 μL of PBS, peel off the adherent cells by vigorous pipetting, smear 50 μL on the above medium, and count the number of colonies after culturing under microaerobic conditions did. As a control, PBS was used instead of whey hydrolyzate. The peeling rate at each concentration was calculated by the following formula.
Peeling rate (%) = [(A−B) / A] × 100
A: Number of adherent cells per microplate in control sample B: Number of adherent cells per microplate in whey degradation product-added sample
(4)統計処理
接着性の比較は独立2標本のt−検定にて実施し、p<0.05を統計学的有意とした。なお、統計解析用ソフトはSTATISTICA(デザインテクノロージーズ)を使用した。
(4) Statistical processing Adhesive comparison was performed by t-test of two independent samples, and p <0.05 was considered statistically significant. The statistical analysis software used was STATISTICA (Design Technologies).
結果
生乳、クリームチーズ及びカッテージチーズ由来のホエー分解物のHp剥離活性を表2に示す。全てのホエーが未処理では活性が無かったのに対し、パンクレアチン又はパパインで加水分解を行うことにより剥離活性を発揮することが判明した。この活性はバターミルク分解物と比較すると劣るものの、乳汁ムチン(特許文献1)とほぼ同等で、カッテージチーズホエー分解物に関しては大きく上回っていた。以上の結果は、ホエー分解物を予防目的以外に治療目的でも使用できる可能性を示している。
Results Table 2 shows the Hp peeling activity of whey degradation products derived from raw milk, cream cheese and cottage cheese. It was found that all whey had no activity when untreated, but exerted peeling activity by hydrolysis with pancreatin or papain. Although this activity was inferior to the buttermilk degradation product, it was almost equivalent to the milk mucin (Patent Document 1) and greatly exceeded the cottage cheese whey degradation product. The above results indicate the possibility that the whey degradation product can be used not only for preventive purposes but also for therapeutic purposes.
性状分析
実施例1および2で、ホエー分解物にHpに対する強力な接着作用及び剥離作用が確認されたが、既にピロリに対し定着阻害作用を示す類似物質として、乳汁ムチンが報告されている(特許文献1参照)。本実施例ではホエー分解物、乳汁ムチン及びバターミルク分解物との違いを明確にするため、以下の試験検討を行った。
Property analysis In Examples 1 and 2, the whey degradation product was confirmed to have a strong adhesive action and exfoliating action against Hp, but milk mucin has already been reported as a similar substance showing an inhibitory action against H. pylori (patent) Reference 1). In this example, the following examination was conducted in order to clarify the difference from the whey degradation product, milk mucin and buttermilk degradation product.
材料および方法
(1)ホエー加水分解物
生乳、クリームチーズホエー及びカッテージチーズホエーから、実施例1と同様の方法でホエー分解物を調製した。性状分析に用いたホエーは実施例1で活性の強かったパパイン及びパンクレアチン処理物である。乳汁ムチンは特許文献1に、バターミルク分解物は非特許文献25にしたがい調製した。
Materials and Methods (1) Whey hydrolyzate A whey hydrolyzate was prepared from raw milk, cream cheese whey and cottage cheese whey in the same manner as in Example 1. The whey used for the characterization was the papain and pancreatin treated product that was highly active in Example 1. The milk mucin was prepared according to Patent Document 1 and the buttermilk degradation product was prepared according to Non-Patent Document 25.
(2)試験方法
ホエー分解物のタンパク質及び糖質の含有量、分子量分布を、ケルダール法、フェノール硫酸法、及びゲルろ過法でそれぞれ測定し、特許文献1に記載の乳汁ムチン及び非特許文献25に記載のバターミルク分解物の成分組成及び分子量と比較した。また、SDSポリアクリルアミドゲル電気泳動(SDS−PAGE)後に銀染色を行い、ペプチドパターンを比較した(図2A)。さらに、SDS−PAGE後にウエスタンブロット法にて膜転写し、ペルオキシダーゼ標識小麦胚芽レクチンと反応させ、糖鎖パターンを比較した(図2B)。
(2) Test method The protein and carbohydrate content and molecular weight distribution of the whey degradation product were measured by the Kjeldahl method, the phenol sulfate method, and the gel filtration method, respectively. The milk mucin described in Patent Literature 1 and Non-Patent Literature 25 And compared with the component composition and molecular weight of the buttermilk degradation product described in 1. Moreover, silver staining was performed after SDS polyacrylamide gel electrophoresis (SDS-PAGE), and the peptide patterns were compared (FIG. 2A). Furthermore, after SDS-PAGE, the membrane was transferred by Western blotting, reacted with peroxidase-labeled wheat germ lectin, and the sugar chain patterns were compared (FIG. 2B).
結果
表3に示すように、ホエー分解物は乳汁ムチンと比較してタンパク質含量が高く、糖質が少ないことが確認された。一方、バターミルク分解物と比較したところ、タンパク質含量が低く、糖質含量が高いことが認められた。また、乳汁ムチン及びバターミルク分解物とは分子量が大きく異なることが確認された(図2A)。
Results As shown in Table 3, it was confirmed that the whey degradation product had a higher protein content and less carbohydrates than milk mucin. On the other hand, when compared with the buttermilk degradation product, it was confirmed that the protein content was low and the sugar content was high. Moreover, it was confirmed that the molecular weight is significantly different from the milk mucin and the buttermilk degradation product (FIG. 2A).
さらに、ペプチドパターン及び糖鎖パターンが、ホエー分解物及びバターミルク分解物とは明らかに相違し、これらは異なる物質であることが確認された。特に、バターミルク分解物はレクチン反応の結果から糖鎖を含んでいることが確認されたのに対し、ホエー分解物は含んでいなかった(図2B)。 Furthermore, the peptide pattern and sugar chain pattern were clearly different from the whey degradation product and the buttermilk degradation product, and it was confirmed that these were different substances. In particular, the buttermilk degradation product was confirmed to contain sugar chains from the results of the lectin reaction, whereas it did not contain whey degradation product (FIG. 2B).
以上より、ホエー加水分解物が強いHp接着性ならびに剥離活性を有することは明らかである。このホエー分解物中に含まれるポリペプチドは、乳汁ムチンなどの既存の抗ピロリ食品素材と比較して、剥離活性はもとより、生産性、熱安定性が高いことから、大量生産および長期保存が可能な機能性素材として大いに期待できる。 From the above, it is clear that the whey hydrolyzate has strong Hp adhesion and peeling activity. Polypeptides contained in this whey degradation product can be mass-produced and stored for a long period of time due to their high productivity and thermal stability as well as exfoliation activity compared to existing anti-pylori food materials such as milk mucin. It can be greatly expected as a functional material.
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JPH04169539A (en) * | 1990-11-01 | 1992-06-17 | Imuno Japan:Kk | Preventive and therapeutic agent for alimetary disease and production thereof |
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