JP4727939B2 - Method for producing low allergenized royal jelly - Google Patents
Method for producing low allergenized royal jelly Download PDFInfo
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- JP4727939B2 JP4727939B2 JP2004107860A JP2004107860A JP4727939B2 JP 4727939 B2 JP4727939 B2 JP 4727939B2 JP 2004107860 A JP2004107860 A JP 2004107860A JP 2004107860 A JP2004107860 A JP 2004107860A JP 4727939 B2 JP4727939 B2 JP 4727939B2
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Images
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- Jellies, Jams, And Syrups (AREA)
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Description
本発明は、低アレルゲン化ローヤルゼリーの製造方法に関する。 The present invention relates to a method for producing a hypoallergenic Royaruzeri over.
従来より、この種の低アレルゲン化ローヤルゼリーとしては、ローヤルゼリーに、糖分解酵素処理及び蛋白質分解酵素処理を施すことにより、実質的に発現されない程度にアレルギー性を低減させた低アレルゲン化ローヤルゼリーが知られている(特許文献1参照)。この低アレルゲン化ローヤルゼリーでは、溶液のpHを6.3〜7.3に調整し、等電点沈澱法を用いて主要なアレルゲンである分子量55kDa蛋白質を含む画分を沈澱除去した後に酵素分解することによってアレルギー反応が抑えられるようになっている。一方、特許文献2には、ローヤルゼリーのプロテアーゼによる分解物を有効成分とする脂質代謝改善剤が開示されている。この脂質代謝改善剤の分解物では、分子量10000以上のものがほとんどなくなって、分子量500〜3000のペプチドが主成分となっている。
本発明は、本発明者らの鋭意研究の結果、ローヤルゼリー中に含まれるアレルギー性を極めて効果的に消失させることができたという知見に基づいてなされたものである。その目的とするところは、ローヤルゼリーが有する有用な生理活性の低下を抑えつつアレルギー性を低減させることが容易な低アレルゲン化ローヤルゼリーの製造方法を提供することにある。 The present invention has been made on the basis of the knowledge that the allergenicity contained in the royal jelly could be eliminated very effectively as a result of intensive studies by the present inventors. And an object thereof is to provide a method for producing easily hypoallergenic Royaruzeri over it possible to reduce the allergic while reduction of useful physiological activities of the royal jelly.
上記の目的を達成するために、請求項1に記載の発明は、ローヤルゼリーに蛋白質分解酵素処理及び糖分解酵素処理を施すことにより、アレルギー性が低減された低アレルゲン化ローヤルゼリーの製造方法であって、pHが6.5〜8.0に調整されたローヤルゼリー希釈液に蛋白質分解酵素としてエンド型中性プロテアーゼ及び糖分解酵素としてβ−マンノシダーゼを同時に添加して行う第1分解工程と、該第1分解工程後のローヤルゼリー希釈液のpHを6.5〜8.0に調整するpH調整工程と、該pH調整工程後のローヤルゼリー希釈液に両酵素処理を行う第2分解工程とを含むことを要旨とする。この構成によれば、基質特異性の高い酵素を用いて製造されることから、ローヤルゼリー由来のペプチド性成分のうち分子量4.5kDaを超える成分が効果的に分解されるとともに、その他の成分に対する影響が少ない。このため、アレルギー性を容易に低減させつつローヤルゼリーの有用な生理活性を低下を抑えることができる。また、ローヤルゼリー由来のペプチド性成分のうちアレルギーを引き起こしやすい成分が効果的に分解されることから、アレルギー性を低減させることが容易となる。さらに、10−ハイドロキシデセン酸が含有されていることから、健康や美容に高い効果を発揮する。 To achieve the above object, the inventions according to claim 1, by performing the protease treatment and glycolytic enzyme treatment in royal jelly, a method of manufacturing the hypoallergenic royal jelly allergic is reduced A first decomposing step of simultaneously adding an endo-type neutral protease as a proteolytic enzyme and β-mannosidase as a saccharolytic enzyme to a royal jelly diluted solution adjusted to a pH of 6.5 to 8.0; A pH adjustment step of adjusting the pH of the royal jelly diluent after the decomposition step to 6.5 to 8.0, and a second decomposition step of performing both enzyme treatments on the royal jelly diluent after the pH adjustment step. The gist. According to this structure, since it is produced using an enzyme having a high substrate specificity, a component exceeding a molecular weight of 4.5 kDa is effectively decomposed among the peptide components derived from royal jelly, and the influence on other components is also achieved. Less is. For this reason, the useful physiological activity of royal jelly can be suppressed while reducing allergenicity easily. Moreover, since the component which is easy to cause allergy among the peptide components derived from a royal jelly is decomposed | disassembled effectively, it becomes easy to reduce allergenicity. Furthermore, since 10-hydroxydecenoic acid is contained, it exhibits a high effect on health and beauty.
本発明によれば、ローヤルゼリーが有する有用な生理活性の低下を抑えつつアレルギー性を低減させることが容易な低アレルゲン化ローヤルゼリーの製造方法を提供することができる。 According to the present invention, it is possible to provide a manufacturing method for easily hypoallergenic Royaruzeri over it possible to reduce the allergic while reduction of useful physiological activities of the royal jelly.
以下、本発明を具体化した実施形態を詳細に説明する。なお、本実施形態では「ローヤルゼリー」を「RJ」と略記する。
RJは、蜜蜂のうち日齢3〜12日の働き蜂が下咽頭腺及び大腮腺から分泌する分泌物を混合して作る乳白色のゼリー状物質である。このRJは、人体に対し好ましい生理活性を持つことが知られている。このRJ中の主な生理活性成分としては、例えば、RJに特有な10−ハイドロキシデセン酸(以下、デセン酸と記載する)等の有機酸類をはじめ、蛋白質、脂質、糖類、ビタミンB類や葉酸、ニコチン酸、パントテン酸等のビタミン類、各種ミネラル類等が挙げられる。このRJの生理活性や薬理作用としては、抗菌作用、免疫増強作用、功腫瘍作用、抗炎症作用、血流量増加作用等が知られている。また、制癌剤の副作用低減や放射線傷害時の延命効果も報告されている。
DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments embodying the present invention will be described in detail. In the present embodiment, “royal jelly” is abbreviated as “RJ”.
RJ is a milky white jelly-like substance made by mixing secretions secreted from the hypopharyngeal gland and the greater vagina by bees aged 3 to 12 days. This RJ is known to have a favorable physiological activity on the human body. The main physiologically active components in the RJ include, for example, organic acids such as 10-hydroxydecenoic acid (hereinafter referred to as decenoic acid) unique to RJ, proteins, lipids, saccharides, vitamin Bs and folic acid. Vitamins such as nicotinic acid and pantothenic acid, and various minerals. As the physiological activity and pharmacological action of RJ, antibacterial action, immunity enhancing action, effective tumor action, anti-inflammatory action, blood flow increasing action and the like are known. In addition, the side effects of anticancer drugs are reduced and the life-prolonging effect at the time of radiation injury is also reported.
本実施形態の低アレルゲン化RJの製造に用いられる原料としては、生RJ又は該生RJを乾燥させて粉末化したRJ粉末が使用される。また、RJの産地は、中国、ブラジル、ヨーロッパ諸国、オセアニア諸国、アメリカ等いずれであってもよい。このRJ中には、生体にアレルギー反応を引き起こす種々のアレルゲンが含有されている。前記RJ中に含まれるアレルゲンとしては、例えば、還元条件下でのSDS−PAGEにて分子量47〜55kDa付近に見られる複数種の蛋白質バンドが挙げられる。このうち、分子量55kDaの蛋白質は、RJ中の主要なアレルゲンである。また、分子量が4.5kDaを越えるペプチド性成分にもアレルゲンとして作用する成分が含まれている。 As a raw material used for manufacturing the allergen-reduced RJ of the present embodiment, raw RJ or RJ powder obtained by drying and pulverizing the raw RJ is used. The production area of RJ may be any of China, Brazil, European countries, Oceania countries, the United States, and the like. This RJ contains various allergens that cause allergic reactions in the living body. Examples of allergens contained in the RJ include multiple types of protein bands that are found in the vicinity of a molecular weight of 47 to 55 kDa by SDS-PAGE under reducing conditions. Of these, a protein with a molecular weight of 55 kDa is a major allergen in RJ. Moreover, the component which acts as an allergen is also contained in the peptidic component having a molecular weight exceeding 4.5 kDa.
実施形態の低アレルゲン化RJは、生RJ由来の有効成分と、生RJ由来のペプチド性成分とを含有しており、経口摂取により体内に吸収されるように構成されている。この低アレルゲン化RJには、前記有効成分及びペプチド性成分以外にも、糖質や脂質等の生RJに由来するその他の成分が含まれていてもよく、その他経口摂取可能な添加物が含まれていても構わない。また、この低アレルゲン化RJには、生RJ由来でない蛋白質が含まれていても構わない。この低アレルゲン化RJの利用形態としては、例えば、液状(ドリンク剤)、凍結乾燥、減圧乾燥又はその他の粉末化手段による粉末状、これに賦形剤を添加した錠剤、カプセル剤、さらに化粧品の基剤を混合したクリームやローション等が挙げられる。 The hypoallergenic RJ of the embodiment contains an active ingredient derived from raw RJ and a peptide component derived from raw RJ, and is configured to be absorbed into the body by oral ingestion. This allergen-reduced RJ may contain other components derived from raw RJ, such as carbohydrates and lipids, in addition to the active ingredient and peptide component, and other ingestible additives. It does not matter. The hypoallergenic RJ may contain a protein that is not derived from raw RJ. Examples of the usage form of this allergen-reduced RJ include, for example, liquid (drink), lyophilized, powdered by drying under reduced pressure or other powdering means, tablets, capsules added with excipients, and cosmetics. Examples include creams and lotions mixed with a base.
前記有効成分は、経口摂取により主として生命活動を活性化させて健康や美容の増進を図る作用を有し、少なくともデセン酸が含まれており、類パロチンやピオプテリン等のRJに特有な成分が含まれているのが好ましい。さらに、この有効成分としては、γ−アミノ酪酸、カルバコール、アセチルコリン等の生理活性物質、ATP等のリン酸化合物、ビタミンB1,B2,B6,B12、ナイアシン、パントテン酸、ニコチン酸、葉酸、ビオチン、ピチオシ、イノシトール等のビタミン類、リン、カルシウム、銅、マンガン、鉄、マグネシウム、亜鉛、タウリン、コリン等のミネラル類、リジン、フェニルアラニン、ロイシン、メチオニン、バリン、スレオニン、トリプトファン等の必須アミノ酸を始めとする各種L−α−アミノ酸が含まれているのが好ましい。なお、この低アレルゲン化RJは、デセン酸を0.18質量%以上含有する調整RJとして製品化されるのが好ましい。 The active ingredient has the effect of activating vital activities mainly by oral ingestion to promote health and beauty, contains at least decenoic acid, and contains ingredients specific to RJ such as parothin and popterin It is preferable. Furthermore, as this active ingredient, physiologically active substances such as γ-aminobutyric acid, carbachol, acetylcholine, phosphate compounds such as ATP, vitamins B1, B2, B6, B12, niacin, pantothenic acid, nicotinic acid, folic acid, biotin, Vitamins such as Pichiosi, Inositol, Minerals such as Phosphorus, Calcium, Copper, Manganese, Iron, Magnesium, Zinc, Taurine, Choline, Essential amino acids such as Lysine, Phenylalanine, Leucine, Methionine, Valine, Threonine, Tryptophan It is preferable that various L-α-amino acids are contained. In addition, it is preferable that this low allergen RJ is commercialized as adjustment RJ containing 0.18 mass% or more of decenoic acid.
前記ペプチド性成分は、生RJに由来するものであり、単純蛋白質、複合蛋白質、ポリペプチド又はオリゴペプチドが挙げられる。このペプチド性成分は、分子量が4.5kDaを越える成分を含有していても構わないが、アレルギー性を低減させるために、分子量が4.5kDa以下の成分のみから構成されているのが好ましい。前記分子量4.5kDa以下の成分のみから構成される低アレルゲン化RJは、還元条件下でのSDS−PAGEを行った後の電気泳動ゲルにおいて、クマシー染色、好ましくは銀染色にて分子量4.5kDaを超える蛋白質バンドが検出されないものである。前記検出されないとは、例えば、生RJについてSDS−PAGEを行う際にクマシー染色にて前記分子量55kDaの蛋白質バンドが明確に視認される蛋白質量と等量(乾燥重量換算又は280nmの吸光度換算)の低アレルゲン化RJを同条件にて電気泳動したとき、分子量4.5kDaを超える蛋白質バンドが視認されないことを意味する。なお、前記分子量4.5kDaを超える蛋白質バンドは、クマシー染色にて視認されないことを意味するが、銀染色にても視認されないのが特に好ましい。また、前記検出されないとは、ゲル濾過カラムを用いた高速液体クロマトグラフィー(HPLC)にて分析したとき、検出波長220nmにおけるクロマトグラムで分子量4.5kDaを越えるピークが検出されないことを意味し、好ましくは分子量4.5kDaを越えるペプチド性成分が検出されないことを意味する。 The peptidic component is derived from raw RJ, and examples thereof include simple proteins, complex proteins, polypeptides, and oligopeptides. This peptidic component may contain a component having a molecular weight exceeding 4.5 kDa, but preferably comprises only a component having a molecular weight of 4.5 kDa or less in order to reduce allergenicity. The allergen-reduced RJ composed only of components having a molecular weight of 4.5 kDa or less is a molecular weight of 4.5 kDa in Coomassie staining, preferably silver staining, in an electrophoresis gel after performing SDS-PAGE under reducing conditions. A protein band exceeding 1 is not detected. For example, when the raw RJ is subjected to SDS-PAGE, it is equivalent to the amount of protein in which the protein band with a molecular weight of 55 kDa is clearly visually recognized by Coomassie staining (in terms of dry weight or in terms of absorbance at 280 nm). It means that when a low allergenized RJ is electrophoresed under the same conditions, a protein band having a molecular weight exceeding 4.5 kDa is not visually recognized. In addition, although the protein band exceeding the said molecular weight 4.5 kDa means that it is not visually recognized by Coomassie dyeing | staining, it is especially preferable not to be visually recognized also by silver dyeing | staining. The term “not detected” means that a peak exceeding a molecular weight of 4.5 kDa is not detected in a chromatogram at a detection wavelength of 220 nm when analyzed by high performance liquid chromatography (HPLC) using a gel filtration column. Means that no peptidic component exceeding a molecular weight of 4.5 kDa is detected.
この低アレルゲン化RJは、ゲル濾過や限外濾過等の分離精製技術、特に分子量に応じて精製する技術を利用して、分子量4.5kDaを越えるペプチド性成分を除去することにより製造されてもよいが、前記有効成分の回収率が高いことからペプチド性成分を酵素分解する処理を行うことにより製造されるのが最も好ましい。前記酵素分解する処理としては、蛋白質分解酵素処理及び/又は糖分解酵素処理が好適に用いられる。この酵素分解する処理は、前記原料(液状)、好ましくは水若しくは緩衝液にて原料を希釈したRJ希釈液に、蛋白質分解酵素及び/又は糖分解酵素を添加して実施される。なお、蛋白質分解酵素処理及び糖分解酵素処理を行う場合には、各処理を別々に行っても、同時に行ってもいずれでもよい。 This allergen-reduced RJ may be produced by removing a peptidic component having a molecular weight of more than 4.5 kDa using a separation and purification technique such as gel filtration or ultrafiltration, particularly a technique for purification according to the molecular weight. Although it is good, since the recovery rate of the active ingredient is high, it is most preferable that it is produced by performing a treatment for enzymatically decomposing the peptide component. As the enzyme decomposing treatment, a proteolytic enzyme treatment and / or a saccharolytic enzyme treatment is preferably used. This enzymatic decomposition treatment is carried out by adding a proteolytic enzyme and / or a glycolytic enzyme to the raw material (liquid), preferably an RJ diluted solution obtained by diluting the raw material with water or a buffer solution. In addition, when performing a proteolytic enzyme treatment and a glycolytic enzyme treatment, each treatment may be performed separately or simultaneously.
蛋白質分解酵素は、ペプチド性成分(蛋白質)のペプチド結合を加水分解し、高分子のペプチド性成分を急速に低分子化(ペプトン化)する酵素である。この蛋白質分解酵素としては、微生物由来又は植物由来のチオールプロテイナーゼ(パパイン、フィチン、プロメライン等)、アスパルティックプロテイナーゼ、セリンプロテイナーゼ(ペプシン、トリプシン、キモトリプシン、パンクレアチン等)、金属プロテイナーゼが挙げられる。また、蛋白質分解酵素は、その至適pHにより酸性プロテアーゼ、中性プロテアーゼ又はアルカリ性プロテアーゼに分類され、基質特異性からエンドプロテアーゼ型(エンド型)又はエクソプロテアーゼ型(エクソ型)に分類される。 Proteolytic enzymes are enzymes that hydrolyze peptide bonds of peptidic components (proteins) and rapidly reduce the molecular weight (peptone) of high-molecular peptidic components. Examples of the proteolytic enzyme include microorganism-derived or plant-derived thiol proteinases (papain, phytin, promeline, etc.), aspartic proteinases, serine proteinases (pepsin, trypsin, chymotrypsin, pancreatin, etc.), and metal proteinases. Proteolytic enzymes are classified into acidic protease, neutral protease or alkaline protease according to their optimum pH, and are classified into endoprotease type (endo type) or exoprotease type (exo type) based on substrate specificity.
これら蛋白質分解酵素のうちRJ中の蛋白質を低分子化させる効果が高いことから、中性プロテアーゼ又はアルカリ性プロテアーゼが好適に用いられる。一方、生RJはpH3.5〜4であることから、蛋白質分解酵素処理を高いpHで行うと多量の塩が生成されて好ましくないことから、中性プロテアーゼが特に好適に用いられる。また、これら蛋白質分解酵素としては、分子量55kDa蛋白質等の高分子を効率よく分解できることから、エンドプロテアーゼ型であるのが好ましい。 Among these proteolytic enzymes, neutral protease or alkaline protease is preferably used because of its high effect of reducing the protein in RJ. On the other hand, since raw RJ has a pH of 3.5 to 4, if protease treatment is performed at a high pH, a large amount of salt is generated, which is not preferable, and neutral protease is particularly preferably used. These proteases are preferably of the endoprotease type because they can efficiently decompose a polymer such as a protein having a molecular weight of 55 kDa.
このような諸条件を満たす蛋白質分解酵素としては、バチルスサブティリス(Bacillus subtilis)由来のエンド型中性プロテアーゼ又はアスペルギルスオリザエ(Aspergillus oryzae)由来のエンド型中性プロテアーゼが特に好適に用いられる。両エンド型中性プロテアーゼは、金属プロテイナーゼに属しており、至適pHが概ね6.5〜7.5の範囲にある。なお、これらの蛋白質分解酵素としては、単独で使用してもよく、2種以上を適宜組合わせて使用してもよい。また、使用される酵素の純度は、特に規定されることはなく、例えば酵素を産出する微生物生産物、植物又は動物からのホモジネートを用いても構わない。 As a proteolytic enzyme satisfying such various conditions, an endo-type neutral protease derived from Bacillus subtilis or an endo-type neutral protease derived from Aspergillus oryzae is particularly preferably used. Both endo-type neutral proteases belong to metalloproteinases, and the optimum pH is generally in the range of 6.5 to 7.5. These proteolytic enzymes may be used alone or in combination of two or more. In addition, the purity of the enzyme used is not particularly limited, and for example, a microbial product producing the enzyme, a homogenate from a plant or an animal may be used.
この蛋白質分解酵素処理は、生RJ中に含まれるアレルゲンの分解を効果的に行うために、蛋白質分解酵素の至適pHにて行われるのが好ましく、蛋白質分解酵素処理中にpHが変動する場合には途中でpH調整を行うように構成されるとよい。このとき、蛋白質分解酵素処理は、蛋白質分解酵素の至適pHに調整された原料又はRJ希釈液に蛋白質分解酵素処理を行う第1分解工程と、該第1分解工程後の原料又はRJ希釈液のpHを前記至適pHに再調整するpH調整工程と、該pH調整工程後の原料又はRJ希釈液に蛋白質分解酵素処理を行う第2分解工程とが実施される。 This proteolytic enzyme treatment is preferably carried out at the optimum pH of the proteolytic enzyme in order to effectively decompose the allergen contained in the raw RJ. When the pH fluctuates during the proteolytic enzyme treatment It is good to comprise so that pH adjustment may be performed on the way. At this time, the proteolytic enzyme treatment includes a first decomposing step of performing proteolytic enzyme treatment on a raw material or RJ diluted solution adjusted to an optimum pH of the proteolytic enzyme, and a raw material or RJ diluted solution after the first decomposing step. A pH adjusting step for re-adjusting the pH to the optimum pH and a second decomposing step for subjecting the raw material or RJ diluent after the pH adjusting step to a proteolytic enzyme treatment are performed.
糖分解酵素は、多糖類や糖蛋白質等に作用して糖鎖を切断する酵素である。この糖分解酵素としては、セルラーゼ、アビセラーゼ、ヘミセルラーゼ、グルコシダーゼ、マンナナーゼ、ガラクトシダーゼ、キシラナーゼ等が挙げられる。これら糖分解酵素のうち、RJのアレルギー性を低減させる効果が高いことから、β−マンノシダーゼを使用することが最も好ましい。なお、これらの糖分解酵素としては、単独で使用してもよく、2種以上を適宜組合わせて使用してもよい。また、使用される酵素の純度は、特に規定されることはなく、例えば酵素を産出する微生物生産物、植物又は動物からのホモジネートを用いても構わない。 A saccharolytic enzyme is an enzyme that acts on polysaccharides, glycoproteins and the like to cleave sugar chains. Examples of the glycolytic enzyme include cellulase, avicellase, hemicellulase, glucosidase, mannanase, galactosidase, xylanase and the like. Among these glycolytic enzymes, β-mannosidase is most preferably used because of its high effect of reducing RJ allergenicity. These glycolytic enzymes may be used alone or in combination of two or more. In addition, the purity of the enzyme used is not particularly limited, and for example, a microbial product producing the enzyme, a homogenate from a plant or an animal may be used.
上記酵素分解する処理は、粘度を低下させて各種酵素反応を円滑に進行させるために、上記原料を所定量の水又は緩衝液で希釈して十分に混合したRJ希釈液を用いるのが好ましい。このとき、原料として生RJを使用する場合には、好ましくは0.2〜1倍量、特に好ましくは0.33倍量の水又は緩衝液で希釈するのが好ましい。希釈するために加えられる水又は緩衝液の量が0.2倍量未満の場合にはRJ希釈液の粘度が高いことから各種酵素反応が円滑に進行せず、逆に1倍量を超える場合には製造のための設備を巨大化する必要があること等から不経済である。一方、原料としてRJ粉末を使用する場合には、前記生RJの場合と同様の理由で、2〜5倍量の水又は緩衝液で希釈するのが好ましい。さらに、前記RJ希釈液のpHを酵素の至適pH付近に調整するのが好ましく、例えばエンド型中性プロテアーゼを用いる場合にはRJ希釈液のpHを好ましくは7.0〜8.0、特に好ましくは7.5に調整するのが好ましい。 In order to reduce the viscosity and smoothly carry out various enzyme reactions, it is preferable to use an RJ diluted solution obtained by diluting the raw material with a predetermined amount of water or a buffer solution and sufficiently mixing the enzyme decomposing treatment. At this time, when raw RJ is used as a raw material, it is preferably diluted with 0.2 to 1 times, particularly preferably 0.33 times the amount of water or buffer. When the amount of water or buffer added for dilution is less than 0.2 times the amount, the enzyme reaction does not proceed smoothly because the viscosity of the RJ dilution is high, and conversely when the amount exceeds 1 time However, it is uneconomical because it is necessary to enlarge the equipment for manufacturing. On the other hand, when RJ powder is used as a raw material, it is preferably diluted with 2 to 5 times the amount of water or buffer for the same reason as in the case of raw RJ. Furthermore, it is preferable to adjust the pH of the RJ dilution to around the optimum pH of the enzyme. For example, when using an endo-type neutral protease, the pH of the RJ dilution is preferably 7.0 to 8.0, particularly It is preferable to adjust to 7.5.
また、この酵素分解する処理において、添加される酵素量は任意に選択することができる。例えば、反応を5時間程度の短時間で終了させたい場合にはRJ固形分1gに対して1〜20mgの酵素を用いればよいが、使用される酵素の純度によっても異なる。一方、例えば酵素反応を一日間かけて行う場合には、酵素量は0.1〜2mg程度でよい。なお、RJ中には数多くの成分が含有されており、酵素に対して阻害的に働く可能性もあることから、通常の基質に対して酵素処理を行う場合の10倍以上使用することが好ましい。但し、これも反応時間との兼ね合いで任意に選択することができる。この酵素分解する処理は至適温度(通常は40〜60℃)で行われるのが好ましい。また、RJと酵素との反応は、アレルゲンと酵素とを接触させやすくするために攪拌しながら行うのがよい。なお、上記処理において、原料又はRJ希釈液中に不溶解物が生成される場合には、適宜遠心又は濾過により不溶解物を除去するのが好ましく、前記不溶解物質中に有用な生理活性成分が含まれている場合には、製品化前に再添加するのが好ましい。さらに、前記酵素分解する処理の終了後、酵素の失活又は除去を行うのが好ましい。 In addition, the amount of enzyme added in this enzymatic decomposition treatment can be arbitrarily selected. For example, when it is desired to complete the reaction in a short time of about 5 hours, an enzyme of 1 to 20 mg per 1 g of RJ solid content may be used, but it varies depending on the purity of the enzyme used. On the other hand, for example, when the enzyme reaction is performed for one day, the amount of the enzyme may be about 0.1 to 2 mg. In addition, since many components are contained in RJ and may act inhibitory to the enzyme, it is preferable to use 10 times or more when performing enzyme treatment on a normal substrate. . However, this can also be arbitrarily selected in view of the reaction time. This enzymatic decomposition treatment is preferably performed at an optimum temperature (usually 40 to 60 ° C.). Further, the reaction between RJ and the enzyme is preferably carried out with stirring in order to make the allergen and the enzyme easy to contact. In the above treatment, when an insoluble matter is generated in the raw material or the RJ diluent, it is preferable to remove the insoluble matter by centrifugation or filtration as appropriate. If it is contained, it is preferably added again before commercialization. Furthermore, it is preferable to inactivate or remove the enzyme after completion of the enzymatic decomposition treatment.
上記実施形態によって発揮される効果について以下に記載する。
・ 実施形態の低アレルゲン化RJは、デセン酸を含有していることから、健康や美容に高い効果を発揮する。さらに、この低アレルゲン化RJには、RJ由来のペプチド性成分のうち分子量4.5kDa以下の成分のみが含有されていることから、アレルギーを引き起こしにくくなっている。即ち、前記RJ由来のペプチド性成分のうち分子量4.5kDaを超える成分は、アレルギーを引き起こす作用があることが本発明者らの鋭意研究によって解明された。
The effects exhibited by the above embodiment will be described below.
-Since allergen-ized RJ of embodiment contains decenoic acid, it exhibits a high effect on health and beauty. Furthermore, since this allergen-reduced RJ contains only components having a molecular weight of 4.5 kDa or less among the peptide components derived from RJ, it is difficult to cause allergies. That is, it has been elucidated by the present inventors that the RJ-derived peptide component having a molecular weight exceeding 4.5 kDa has an effect of causing allergy.
・ 実施形態の低アレルゲン化RJは、RJに対し、作用部位の異なる二種類の酵素である糖分解酵素処理及び蛋白質分解酵素処理を施すことにより、RJ中のほとんど全ての高分子のペプチド性成分を低分子化させ、実質的に発現されない程度にアレルギー性を低減させたものである。さらに、この低アレルゲン化RJでは、前記ペプチド性成分が低分子化されたために、アミノ酸吸収性が著しく向上されている。 -The allergen-reduced RJ of the embodiment is obtained by subjecting RJ to glycolytic enzyme treatment and proteolytic enzyme treatment, which are two types of enzymes having different action sites, so that almost all macromolecular peptide components in RJ Is reduced in molecular weight, and allergenicity is reduced to such an extent that it is not substantially expressed. Furthermore, in this allergen-reduced RJ, the amino acid absorbability is remarkably improved because the peptide component is reduced in molecular weight.
また、本発明者らによる鋭意研究の結果、前記特許文献1のRJに施された処理と同様に、RJに対し、基質に対する作用部位の異なる2種以上の蛋白質分解酵素を作用させた場合でも、酵素の選択によりアレルギー性を確実に低減させることはできなかったことが確認されている。また、RJ中の主要アレルゲンである分子量55kDaの蛋白質を分画し、糖分解酵素処理及び蛋白質分解酵素処理を施した場合でも、RJ中のアレルギー性を完全に除くことはできないことが確認されている。 Further, as a result of intensive studies by the present inventors, even when two or more types of proteolytic enzymes having different action sites on the substrate are allowed to act on RJ, as in the treatment applied to RJ in Patent Document 1. It has been confirmed that allergenicity could not be reliably reduced by enzyme selection. In addition, it was confirmed that allergenicity in RJ could not be completely removed even when a protein with a molecular weight of 55 kDa, which is a major allergen in RJ, was fractionated and subjected to glycolytic enzyme treatment and proteolytic enzyme treatment. Yes.
一方、例えば加水分解酵素又は酸化還元酵素の作用により、アレルギー性が実質的に発現されない程度に低減されたプロポリス製品が開示されている。これに対し、RJに含まれる主要なアレルゲンは、前記従来のプロポリス製品におけるアレルゲンとは異なり、前記加水分解酵素処理及び/又は酸化還元酵素処理では十分に分解され得ないことが本発明者らの鋭意研究の結果によって確認されている。 On the other hand, for example, a propolis product that has been reduced to such an extent that allergenicity is not substantially expressed by the action of a hydrolase or an oxidoreductase is disclosed. On the other hand, the main allergens contained in RJ are different from the allergens in the conventional propolis products and cannot be sufficiently decomposed by the hydrolase treatment and / or the oxidoreductase treatment. It has been confirmed by the results of earnest research.
・ RJ中に含まれる健康増進活性は、デセン酸を始めとしてペプチド性成分、ビタミン類、ミネラル類、アミノ酸等の様々な成分が関係している。このため、ペプチド性成分を除去すると不測の活性低下が引き起こされる懸念があるうえアミノ酸量が激減するため、生RJが有する健康増進活性を十分に引き起こすことができなくなるおそれが高い。従って、アレルゲンを引き起こす成分のみを特異的に分解し、その他の成分には影響を与えない基質特異性の高い低アレルゲン化方法として酵素分解する処理が最も有効である。 -Health promotion activity contained in RJ is related to various components such as decenoic acid, peptidic components, vitamins, minerals and amino acids. For this reason, removal of the peptide component may cause an unexpected decrease in activity, and the amount of amino acids is drastically reduced. Therefore, there is a high possibility that the health promoting activity of raw RJ cannot be sufficiently caused. Therefore, the enzymatic decomposition is most effective as a method for reducing the allergen with high substrate specificity that specifically decomposes only the component causing the allergen and does not affect the other components.
・ 糖分解酵素としてβ−マンノシダーゼを使用することによって、RJのアレルギー性を容易かつ確実に低減させることができる。特に、RJ中の主要なアレルゲンである分子量55kDaの蛋白質は高マンノース型糖鎖を持つことが知られており、それが抗原決定基を構成している可能性が非常に高い。従って、分子量55kDaの蛋白質のペプチド結合を加水分解するともに、前記高マンノース型糖鎖を分解することによってより一層効果的に抗原決定基を消失させることができる可能性が高い。 -By using β-mannosidase as a glycolytic enzyme, the allergy of RJ can be easily and reliably reduced. In particular, a protein with a molecular weight of 55 kDa, which is a major allergen in RJ, is known to have a high mannose-type sugar chain, and it is very likely that it constitutes an antigenic determinant. Therefore, there is a high possibility that the antigenic determinant can be more effectively eliminated by hydrolyzing the peptide bond of the protein having a molecular weight of 55 kDa and decomposing the high-mannose sugar chain.
<<アレルゲンの分解条件の検討>>
RJに含まれる主要なアレルゲンである分子量55kDa蛋白質と、アレルゲンであると疑われる分子量4kDa以上55kDa未満の蛋白質とを効率よく分解するための条件を検討した。
<< Examination of Allergen Degradation Conditions >>
The conditions for efficiently degrading a molecular weight 55 kDa protein, which is a major allergen contained in RJ, and a protein having a molecular weight of 4 kDa or more and less than 55 kDa suspected of being an allergen were examined.
<酵素の選択>
蛋白質分解酵素のみを用いて予備的に行った。
(試験例1):中国産生RJ1000gに純水330mlを加えて均一に成るまで攪拌してRJ希釈液を調製した。このRJ希釈液を炭酸水素ナトリウム又は炭酸カルシウムにてpH7.5に調整した後、100mlずつ9個のビーカーに分取した。各ビーカー内に蛋白質分解酵素1.67gを添加して50℃で16時間反応させた。前記ビーカー内には表1に示されるいずれか一種類の蛋白質分解酵素が添加されている。表1に示される蛋白質分解酵素は、バチルスサブティリス由来のエンド型中性プロテアーゼ(天野エンザイム社製プロテアーゼN)、アスペルギルスオリザエ由来のエンド型中性プロテアーゼ(大和化成社製プロチンP)、アスペルギロペプチダーゼA(盛進社製モルシンF)、ペプシン(和光純薬工業社製)、パンクレアチン(天野エンザイム社製パンクレアチンF)、パパイン(天野エンザイム社製パパインW−40)、プロメライン(天野エンザイム社製プロメラインF)、アクチナーゼ(科研製薬社製アクチナーゼE)、アルカロフィリックプロテアーゼ(東洋紡社製)である。反応終了後、80℃で30分加熱処理して酵素を失活させるとともに殺菌処理をした。
<Enzyme selection>
Preliminary using only proteolytic enzymes.
(Test Example 1): RJ diluted solution was prepared by adding 330 ml of pure water to 1000 g of RJ produced in China and stirring until uniform. The RJ dilution was adjusted to pH 7.5 with sodium bicarbonate or calcium carbonate, and then dispensed into 9 beakers of 100 ml each. 1.67 g of proteolytic enzyme was added to each beaker and reacted at 50 ° C. for 16 hours. Any one type of proteolytic enzymes shown in Table 1 is added to the beaker. The proteolytic enzymes shown in Table 1 are Bacillus subtilis-derived endo-type neutral protease (Protein N manufactured by Amano Enzyme), Aspergillus oryzae-derived endo-type neutral protease (Protin P manufactured by Daiwa Kasei Co., Ltd.), Aspergillo Peptidase A (Morshin F manufactured by Shengshin Co., Ltd.), Pepsin (manufactured by Wako Pure Chemical Industries, Ltd.), Pancreatin (Pancreatin F manufactured by Amano Enzyme), Papain (Papain W-40 manufactured by Amano Enzyme), Promeline (Amano Enzyme) Promeline F), Actinase (Actinase E, Kaken Pharmaceutical Co., Ltd.), Alkalophilic Protease (Toyobo). After completion of the reaction, the enzyme was inactivated by heat treatment at 80 ° C. for 30 minutes and sterilized.
(SDS−PAGEによるRJ由来ペプチド性成分の分解確認1)
試験例1で得られた各サンプルについてSDS−PAGEを行った。各サンプルは、1/4量の200mMトリス塩酸(pH6.8)、250mMジチオスレイトール、5%SDS、22.5%グリセロール及び適量のブロモフェノールブルーを含む5×SDSサンプルバッファーを加えて98℃で2分間加熱した。その後、0.3〜0.5μlについて、厚さ0.45mmの8−25%グラジエントポリアクリルアミドゲルを用い、全自動電気泳動システムPhastSystemTM(アマシャム・ファルマシア・バイオテク社)を使用してSDS−PAGEを行った。なお、分子量マーカーとして。LMW kit E(アマシャム・ファルマシア・バイオテク社)を用いた。電気泳動後のゲルは、クマシーブリリアントブルーR−350でクマシー染色し、30%メタノール及び10%酢酸を含む溶液で脱色した。染色及び脱色されたゲルについて、分子量55kDaの蛋白質バンド及び分子量4kDa以上55kDa未満の蛋白質バンド(表中では>4kDaと記載されている)の分解をそれぞれ目視にて確認した。結果を表1に示す。
(Degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1)
SDS-PAGE was performed on each sample obtained in Test Example 1. Each sample was added with 5 × SDS sample buffer containing 1/4 volume of 200 mM Tris-HCl (pH 6.8), 250 mM dithiothreitol, 5% SDS, 22.5% glycerol and an appropriate amount of bromophenol blue, and 98 ° C. For 2 minutes. Thereafter, about 0.3-0.5 μl, SDS-PAGE was performed using a fully automated electrophoresis system PhastSystem ™ (Amersham Pharmacia Biotech) using an 8-25% gradient polyacrylamide gel having a thickness of 0.45 mm. went. As a molecular weight marker. LMW kit E (Amersham Pharmacia Biotech) was used. The gel after electrophoresis was stained with Coomassie Brilliant Blue R-350 and decolorized with a solution containing 30% methanol and 10% acetic acid. Regarding the stained and decolored gel, the decomposition of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa (described as> 4 kDa in the table) was visually confirmed. The results are shown in Table 1.
<処理条件の検討>
RJ由来のペプチド性成分の分解に好適なpH、反応温度等を検討した。なお、本検討では蛋白質分解酵素及び糖分解酵素を用いて行った。
<Examination of processing conditions>
The pH, reaction temperature, etc. suitable for the decomposition of the RJ-derived peptide component were examined. In this study, a proteolytic enzyme and a glycolytic enzyme were used.
(試験例2):中国産生RJ100gに純水100mlを加えてRJ希釈液を調製した。このRJ希釈液を炭酸水素ナトリウム又は炭酸カルシウムにてpH6.5に調整した後、等電点沈澱法により沈澱画分を分画し、沈澱画分に1.67gのモルシンF及び30mgのβ−マンノシダーゼ(新日本化学工業社製スミチームACH)を同時に添加し、50℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 2): 100 ml of pure water was added to 100 g of Chinese-produced RJ to prepare an RJ dilution. This RJ diluted solution was adjusted to pH 6.5 with sodium hydrogen carbonate or calcium carbonate, and then the precipitated fraction was fractionated by isoelectric point precipitation, and 1.67 g of molsin F and 30 mg of β- Mannosidase (Sumiteam ACH manufactured by Shin Nippon Chemical Industry Co., Ltd.) was added simultaneously and reacted at 50 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例3):中国産生RJ100gに純水100mlを加えてRJ希釈液を調製した。このRJ希釈液をpH6.5に調整した後、1.67gのモルシンF及び30mgのβ−マンノシダーゼを同時に添加し、50℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 3): 100 ml of pure water was added to 100 g of RJ produced in China to prepare an RJ dilution. After adjusting this RJ dilution to pH 6.5, 1.67 g of molsin F and 30 mg of β-mannosidase were added simultaneously and reacted at 50 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例4):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液に1.67gのモルシンF及び30mgのβ−マンノシダーゼを同時に添加し、40℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 4): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. To this RJ dilution, 1.67 g of molsin F and 30 mg of β-mannosidase were simultaneously added and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例5):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液に1.67gのモルシンF及び30mgのβ−マンノシダーゼを同時に添加し、45℃で16時間反応させた後、55℃まで昇温し2時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 5): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. 1.67 g of molsin F and 30 mg of β-mannosidase were simultaneously added to this RJ diluted solution, reacted at 45 ° C. for 16 hours, then heated to 55 ° C. and reacted for 2 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例6):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液に1.83gのモルシンF及び30mgのβ−マンノシダーゼを同時に添加し、40℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 Test Example 6: An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. 1.83 g of molsin F and 30 mg of β-mannosidase were simultaneously added to this RJ dilution, and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例7):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液に1.67gの酸性プロテアーゼ(天野エンザイム社製プロテアーゼM)及び30mgのβ−マンノシダーゼを同時に添加し、40℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 7) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. To this RJ diluted solution, 1.67 g of acidic protease (Protein M manufactured by Amano Enzyme) and 30 mg of β-mannosidase were simultaneously added and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例8):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液に1.67gのプロテアーゼM及び30mgのβ−マンノシダーゼを同時に添加し、40℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 8): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. To this RJ dilution, 1.67 g of protease M and 30 mg of β-mannosidase were simultaneously added, and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例9):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液を炭酸水素ナトリウム又は炭酸カルシウムにてpH6.0に調整し、1.67gのプロテアーゼN及び30mgのβ−マンノシダーゼを同時に添加し、40℃で16時間反応させた。反応終了後、80℃で30分間加熱処理した。 (Test Example 9) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. This RJ diluted solution was adjusted to pH 6.0 with sodium hydrogen carbonate or calcium carbonate, 1.67 g of protease N and 30 mg of β-mannosidase were added simultaneously, and reacted at 40 ° C. for 16 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(SDS−PAGEによるRJ由来ペプチド性成分の分解確認2)
試験例2〜9で得られた各サンプルについて上記試験例1の(SDS−PAGEによるRJ由来ペプチド性成分の分解確認1)と同様にSDS−PAGEを行った。染色及び脱色されたゲルについて、分子量55kDaの蛋白質バンド及び分子量4kDa以上55kDa未満の蛋白質バンドの分解をそれぞれ目視にて確認した。結果を表2に示す。
(Degradation confirmation 2 of RJ-derived peptide component by SDS-PAGE 2)
Each sample obtained in Test Examples 2 to 9 was subjected to SDS-PAGE in the same manner as in Test Example 1 (degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1). Regarding the stained and decolored gel, the degradation of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa were confirmed visually. The results are shown in Table 2.
<pH調整による分解能の検討1>
酵素反応の途中で溶液のpH調整を行って分解能に与える影響を検討した。なお、本検討では蛋白質分解酵素のみを用いて予備的に行った。
<Examination of resolution by pH adjustment 1>
The effect on the resolution was studied by adjusting the pH of the solution during the enzymatic reaction. Note that this study was performed preliminarily using only proteolytic enzymes.
(試験例10):中国産生RJ1000gに純水330mlを加えてRJ希釈液を調製した。このRJ希釈液をpH7.5に調整した後、100mlずつ9個のビーカーに分取した。各ビーカー内に下記表3に示されるいずれか一種類の蛋白質分解酵素1.67gを添加して50℃で2時間反応させることにより第1分解工程を行った。続いて、前記第1分解工程後のRJ希釈液を炭酸水素ナトリウム又は炭酸カルシウムにてpH7.5に再調整することによりpH調整工程を行った後、さらに50℃で14時間反応させることにより第2分解工程を行った。反応終了後、80℃で30分加熱処理した。 (Test Example 10): RJ dilution was prepared by adding 330 ml of pure water to 1000 g of RJ produced in China. The RJ dilution was adjusted to pH 7.5 and then dispensed into 9 beakers of 100 ml each. In each beaker, 1.67 g of any one type of proteolytic enzyme shown in Table 3 below was added and reacted at 50 ° C. for 2 hours to carry out the first decomposition step. Subsequently, after the pH adjustment step was performed by readjusting the RJ diluted solution after the first decomposition step to pH 7.5 with sodium bicarbonate or calcium carbonate, the reaction was further performed at 50 ° C. for 14 hours. Two decomposition steps were performed. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(SDS−PAGEによるRJ由来ペプチド性成分の分解確認3)
試験例10で得られた各サンプルについて上記試験例1の(SDS−PAGEによるRJ由来ペプチド性成分の分解確認1)と同様にSDS−PAGEを行った。染色及び脱色されたゲルについて、分子量55kDaの蛋白質バンド及び分子量4kDa以上55kDa未満の蛋白質バンドの分解をそれぞれ目視にて確認した。結果を表3に示す。
(Degradation confirmation 3 of RJ-derived peptidic component by SDS-PAGE 3)
Each sample obtained in Test Example 10 was subjected to SDS-PAGE in the same manner as in Test Example 1 (degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1). Regarding the stained and decolored gel, the degradation of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa were confirmed visually. The results are shown in Table 3.
<pH調整による分解能の検討2>
酵素反応の途中で溶液のpH調整を行って分解能に与える影響を検討した。なお、本検討では蛋白質分解酵素及び糖分解酵素を用いて行った。
<Examination of resolution by pH adjustment 2>
The effect on the resolution was studied by adjusting the pH of the solution during the enzymatic reaction. In this study, a proteolytic enzyme and a glycolytic enzyme were used.
(試験例11):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液をpH7.5に調整した後、1.67gのプロテアーゼN及び30mgのβ−マンノシダーゼを添加し、50℃で2時間反応させることにより第1分解工程を行った。続いて、前記第1分解工程後のRJ希釈液をpH7.5に再調整することによりpH調整工程を行った後、さらに50℃で14時間反応させることにより第2分解工程を行った。反応終了後、80℃で30分加熱処理した。 (Test Example 11) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting the RJ dilution to pH 7.5, 1.67 g of protease N and 30 mg of β-mannosidase were added and reacted at 50 ° C. for 2 hours to perform the first decomposition step. Subsequently, the RJ diluted solution after the first decomposition step was readjusted to pH 7.5, and then the second decomposition step was performed by further reacting at 50 ° C. for 14 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例12):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液をpH7.5に調整した後、1.67gのプロチンP及び30mgのβ−マンノシダーゼを添加し、50℃で2時間反応させることにより第1分解工程を行った。続いて、前記第1分解工程後のRJ希釈液をpH7.5に再調整することによりpH調整工程を行った後、さらに50℃で14時間反応させることにより第2分解工程を行った。反応終了後、80℃で30分加熱処理した。 Test Example 12: An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting this RJ dilution to pH 7.5, 1.67 g of protin P and 30 mg of β-mannosidase were added, and the first decomposition step was performed by reacting at 50 ° C. for 2 hours. Subsequently, the RJ diluted solution after the first decomposition step was readjusted to pH 7.5, and then the second decomposition step was performed by further reacting at 50 ° C. for 14 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例13):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液をpH7.5に調整した後、1.67gのプロテアーゼNを添加し、50℃で2時間反応させることにより第1分解工程を行った。続いて、前記第1分解工程後のRJ希釈液をpH7.5に再調整することによりpH調整工程を行った後、β−マンノシダーゼ30mgを添加し、さらに50℃で14時間反応させることにより第2分解工程を行った。反応終了後、80℃で30分加熱処理した。 (Test Example 13) An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting this RJ dilution to pH 7.5, 1.67 g of protease N was added and the first decomposition step was performed by reacting at 50 ° C. for 2 hours. Subsequently, after performing the pH adjustment step by readjusting the RJ diluted solution after the first decomposition step to pH 7.5, 30 mg of β-mannosidase was added, and further reacted at 50 ° C. for 14 hours. Two decomposition steps were performed. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例14):中国産生RJ100gに純水33mlを加えてRJ希釈液を調製した。このRJ希釈液をpH7.5に調整した後、1.67gのプロチンPを添加し、50℃で2時間反応させることにより第1分解工程を行った。続いて、前記第1分解工程後のRJ希釈液をpH7.5に再調整することによりpH調整工程を行った後、β−マンノシダーゼ30mgを添加し、さらに50℃で14時間反応させることにより第2分解工程を行った。反応終了後、80℃で30分加熱処理した。 (Test Example 14): An RJ dilution was prepared by adding 33 ml of pure water to 100 g of RJ produced in China. After adjusting this RJ dilution to pH 7.5, 1.67 g of protin P was added, and the first decomposition step was performed by reacting at 50 ° C. for 2 hours. Subsequently, after performing the pH adjustment step by readjusting the RJ diluted solution after the first decomposition step to pH 7.5, 30 mg of β-mannosidase was added, and further reacted at 50 ° C. for 14 hours. Two decomposition steps were performed. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(試験例15):中国産RJ凍結乾燥粉末10gに純水40mlを加えて均一に成るまで攪拌してRJ希釈液を調製した。このRJ希釈液をpH7.5に調整した後、1.67gのプロテアーゼN及び30mgのβ−マンノシダーゼを添加し、50℃で2時間反応させることにより第1分解工程を行った。前記第1分解工程後のRJ希釈液をpH7.5に再調整を行することによりpH調整工程を行った後、さらに50℃で14時間反応させることにより第2分解工程を行った。反応終了後、80℃で30分加熱処理した。 (Test Example 15): 40 ml of pure water was added to 10 g of RJ freeze-dried powder made in China and stirred until uniform to prepare an RJ dilution. After adjusting the RJ dilution to pH 7.5, 1.67 g of protease N and 30 mg of β-mannosidase were added and reacted at 50 ° C. for 2 hours to perform the first decomposition step. The RJ diluted solution after the first decomposition step was readjusted to pH 7.5, and then the pH adjustment step was performed. Then, the second decomposition step was performed by reacting at 50 ° C. for 14 hours. After completion of the reaction, heat treatment was performed at 80 ° C. for 30 minutes.
(SDS−PAGEによるRJ由来ペプチド性成分の分解確認4)
試験例11〜15で得られた各サンプルについて上記試験例1の(SDS−PAGEによるRJ由来ペプチド性成分の分解確認1)と同様にSDS−PAGEを行った。染色及び脱色されたゲルについて、分子量55kDaの蛋白質バンド及び分子量4kDa以上55kDa未満の蛋白質バンドの分解をそれぞれ目視にて確認した。結果を表4に示す。
(Degradation confirmation 4 of RJ-derived peptidic component by SDS-PAGE 4)
Each sample obtained in Test Examples 11 to 15 was subjected to SDS-PAGE in the same manner as in Test Example 1 (degradation confirmation 1 of RJ-derived peptidic component by SDS-PAGE 1). Regarding the stained and decolored gel, the degradation of the protein band having a molecular weight of 55 kDa and the protein band having a molecular weight of 4 kDa or more and less than 55 kDa were confirmed visually. The results are shown in Table 4.
<RJ免疫ラットにおけるIgE抗体応答の検討>
48時間受動皮膚アナフィラキシー(PCA)反応によるRJ特異的IgEの測定
上記実施例1で分子量4kDa以上の蛋白質の分解に顕著な効果が認められた試験例11、12と、効果が認められなかった試験例2、3について、ラットのIgE抗体応答の評価を行った。試験に先立ち、以下のとおりRJに対する抗血清を得た。まず、6週齢の雌性Wistarラットを日本エスエルシー社から購入し、7〜10日間検疫飼育を行った。次に、試験例2,3,11,12の蛋白質濃度をBradfordの方法(M.Bradford,Anal.Biochm.,72,248-254,1976)に基づき、ウシγグロブリンをスタンダードに用いてそれぞれ定量を行った。試験例2,3,11,12又は凍結乾燥RJ粉末3mgをそれぞれ水酸化アルミニウムゲル(アラム・アジュバンド)10mgと混合し、全量が1mlとなるようにエマルジョンを作製した。対照としての卵白アルブミン(OVA)は0.1mgを水酸化アルミニウムゲル10mgと混合し、全量が1mlとなるようにエマルジョンを作製した。各エマルジョンをラット1匹あたり1ml腹腔内投与し、2週間間隔で3回の免疫感作を行った。免疫感作開始から35日後に各群8匹ずつ頚動脈より採血を行った。血液を3000rpmで10分間遠心分離して血餅を取り除き、抗血清を得た。血清は測定まで−40℃にて保存を行った。なお、ラットは全ての飼育期間を通じて、温度23±2℃、湿度55±15%、オールフレッシュ換気回数12回/時、午前8時から12時間照明に条件設定されたセミバリアシステム飼育室内において、PCゲージで5〜6匹ずつ収納し、固形飼料(CE−2、日本クレア)と濾過水道水とを自由摂取させて飼育した。
<Examination of IgE antibody response in RJ immunized rats>
Measurement of RJ-specific IgE by 48-hour passive skin anaphylaxis (PCA) reaction Test Examples 11 and 12 in which significant effects were observed in the degradation of proteins having a molecular weight of 4 kDa or more in Example 1 and tests in which no effect was observed Examples 2 and 3 were evaluated for rat IgE antibody response. Prior to the test, antiserum against RJ was obtained as follows. First, a 6-week-old female Wistar rat was purchased from Japan SLC, and quarantined for 7 to 10 days. Next, based on the method of Bradford (M. Bradford, Anal. Biochm., 72, 248-254, 1976), the protein concentrations of Test Examples 2, 3, 11, and 12 were quantified using bovine γ globulin as a standard. It was. Test Examples 2, 3, 11, 12 or 3 mg of freeze-dried RJ powder were mixed with 10 mg of aluminum hydroxide gel (Alum Adjuband), respectively, to prepare an emulsion so that the total amount was 1 ml. As a control, ovalbumin (OVA) 0.1 mg was mixed with
次に、6週齢の雌性Wistarラット(1群につき2匹)の背部の毛を刈り、各個体より得られたRJ感作抗血清(ラットNo.1〜8)0.1mlをラット背部(8箇所/1匹)に皮内注射した。OVA感作抗血清は、各個体の抗血清を0.1mlずつ混合し、生理食塩水にて1/2系列希釈を行い、抗血清及び抗血清希釈液0.1mlをラット背部に皮内注射した。抗血清注射48時間後に抗原として、生理食塩水1ml中に凍結乾燥RJ粉末、試験例2,3の等量混合物、試験例11,12の等量混合物、又はOVA10mgと、エバンスブルー色素5mgとが含まれるようにそれぞれ混合液を作製し、1匹当たり1mlずつ静脈内投与した。30分後に背部の皮膚を剥離し、抗血清を皮内注射した部位の青染班有無を皮膚内側より観察した。結果を表5に示す。 Next, the hair of the back of 6-week-old female Wistar rats (2 per group) was shaved, and 0.1 ml of RJ-sensitized antiserum (rat No. 1-8) obtained from each individual was placed on the back of the rat ( 8 sites / one animal) were injected intradermally. OVA-sensitized antiserum is mixed with 0.1 ml of each individual antiserum, diluted 1/2 series with physiological saline, and intradermal injection of 0.1 ml of antiserum and antiserum diluted solution into the back of the rat did. 48 hours after the injection of antiserum, lyophilized RJ powder, an equal mixture of Test Examples 2 and 3, an equal mixture of Test Examples 11 and 12, or 10 mg of OVA and 5 mg of Evans Blue dye as an antigen in 1 ml of physiological saline Each liquid mixture was prepared so as to be contained, and 1 ml per mouse was intravenously administered. Thirty minutes later, the skin on the back was peeled off, and the presence or absence of a blue stain at the site where the antiserum was injected intradermally was observed from the inside of the skin. The results are shown in Table 5.
<RJ免疫ラットにおけるIgG抗体応答の検討>
試験に先立ち、以下のとおりRJに対する抗血清を得た。まず、6週齢の雌性Wistarラットを日本エスエルシー社から購入し、7〜10日間検疫飼育を行った。次に、上記実施例2と同様に、試験例11及び12の等量混合物、又は凍結乾燥RJ粉末3mgをそれぞれ水酸化アルミニウムゲル10mgと混合し、全量が1mlとなるようにエマルジョンを作製した。対照としてのOVAも実施例2と同様に0.1mgを水酸化アルミニウムゲル10mgと混合し、全量が1mlとなるようにエマルジョンを作製した。各エマルジョンをラット1匹あたり1ml腹腔内投与し、2週間間隔で3回の免疫感作を行った。免疫感作開始から14日(2回目感作前)、21日、28日(3回目感作前)、35日、42日後に各群8匹ずつ頚動脈より採血を行った。血液を3000rpmで10分間遠心分離して血餅を取り除き、抗血清を得た。血清は測定まで−40℃にて保存を行った。
<Examination of IgG antibody response in RJ immunized rats>
Prior to the test, antiserum against RJ was obtained as follows. First, a 6-week-old female Wistar rat was purchased from Japan SLC, and quarantined for 7 to 10 days. Next, in the same manner as in Example 2 above, an equal mixture of Test Examples 11 and 12 or 3 mg of freeze-dried RJ powder was mixed with 10 mg of aluminum hydroxide gel, respectively, to prepare an emulsion so that the total amount was 1 ml. As a control, OVA was mixed with 10 mg of aluminum hydroxide gel in the same manner as in Example 2 to prepare an emulsion so that the total amount was 1 ml. Each emulsion was intraperitoneally administered 1 ml per rat, and immunized 3 times at 2-week intervals. Blood was collected from the carotid artery 8 animals in each group 14 days (before the second sensitization), 21 days, 28 days (before the third sensitization), 35 days and 42 days after the start of immunization. The blood was centrifuged at 3000 rpm for 10 minutes to remove the clot, and antiserum was obtained. Serum was stored at −40 ° C. until measurement.
次に、凍結乾燥RJ粉末、試験例11及び12の等量混合物、又はOVAをリン酸緩衝液(PBS;137mM NaCl, 8.1mM NaHPO4・12H2O, 2.68mM KCl, 1.47mM KH2PO4)で2mg/mlに調整した抗原液を50μl、96ウェルプレート(Nunc社)に添加した。2時間室温にてインキュベートした後、96ウェルプレート中の抗原液を捨てウェルをPBSにて3回洗浄した。続いて、スキムミルク濃度が5%となるようにPBS−スキムミルク溶液を作製し、96ウェルプレートの1ウェルあたり200μlずつ加え、1時間室温にてインキュベートした後、PBS−スキムミルク溶液を捨て、PBSでウェル内を3回洗浄した。 Next, freeze-dried RJ powder, a mixture of equal amounts of Test Examples 11 and 12, or OVA in phosphate buffer (PBS; 137 mM NaCl, 8.1 mM NaHPO 4 .12H 2 O, 2.68 mM KCl, 1.47 mM KH 2 PO 4 The antigen solution adjusted to 2 mg / ml in 50 μl was added to a 96-well plate (Nunc). After incubating at room temperature for 2 hours, the antigen solution in the 96-well plate was discarded and the wells were washed 3 times with PBS. Subsequently, a PBS-skimmed milk solution was prepared so that the skimmed milk concentration was 5%, 200 μl was added per well of a 96-well plate, incubated for 1 hour at room temperature, the PBS-skimmed milk solution was discarded, and the wells were washed with PBS. The inside was washed 3 times.
次に、前記抗血清をPBSにて1/5に希釈したものを始点として、1/2ずつの系列希釈をしたサンプルをウェルに50μlずつ添加した後、2時間室温でインキュベートした後、ウェル内の抗血清を捨て、0.05% Tween20−PBS溶液でウェルを3回洗浄した。HRP標識抗ラットIgG、IgM、IgA抗体液(CAPPEL社#5574.lot#01809,21mg/ml)をPBSにて2000倍に希釈したものを50μl添加した。ウェル内の抗体液を捨て、0.05% Tween20−PBS溶液でウェルを3回洗浄した。最後に、TMB液(和光)100μlを添加し、30分発色反応を行いマイクロプレートリーダーで450nmにおける吸光度を測定した。抗体価は、非感作血清の450nmにおける吸光度を対照として、抗血清の同波長における吸光度が非感作血清と等価となる最大希釈値とした。結果を表6に示す。 Next, starting with the antiserum diluted to 1/5 in PBS, 50 μl each of the samples diluted in series of 1/2 were added to the wells, incubated for 2 hours at room temperature, The antiserum was discarded and the wells were washed 3 times with 0.05% Tween20-PBS solution. 50 μl of an HRP-labeled anti-rat IgG, IgM, IgA antibody solution (CAPPEL # 5574.lot # 01809, 21 mg / ml) diluted 2000 times with PBS was added. The antibody solution in the wells was discarded, and the wells were washed 3 times with 0.05% Tween20-PBS solution. Finally, 100 μl of TMB solution (Wako) was added, color development reaction was performed for 30 minutes, and the absorbance at 450 nm was measured with a microplate reader. The antibody titer was defined as the maximum dilution at which the absorbance at the same wavelength of the antiserum is equivalent to that of the non-sensitized serum, with the absorbance at 450 nm of the non-sensitized serum as a control. The results are shown in Table 6.
<ヒスタミン放出試験>
上記実施例2と同様の方法にて、凍結乾燥RJ粉末、試験例2,3,11,12及び卵白アルブミン感作抗血清を得た。各抗血清とも等量ずつ混ぜることにより個体間の差を解消した。体重200〜250gの雌性Wistarラットを放血死させ腹腔内にタイロード緩衝液(137mM NaCl, 3.7mM KCl, 1.8mM CaCl2, 1.1mM MgCl2, 11.9mM NaHCO3, 0.4mM NaH2PO4, 5.6mM D-Glucose)10mlを注入し、2分間マッサージ後、ラット腹腔より腹水を回収した。細胞回収液をチューブへ集め、遠心分離(1200rpm,5min)し、ピペットマンにて上清を除いた。さらにチューブへタイロード緩衝液を20ml加え、遠心分離(1200rpm,5min)し、ピペットマンにて上清を除いた。この工程を2回行い、細胞を洗浄した。細胞をタイロード緩衝液5mlに懸濁し、血球計算版にて細胞数の測定を行い、最終細胞数が1×107cells/mlとなるように細胞浮遊液を調整し、24ウェルプレートに0.5mlずつ分注した。
<Histamine release test>
In the same manner as in Example 2, freeze-dried RJ powder, Test Examples 2, 3, 11, and 12 and ovalbumin-sensitized antiserum were obtained. Differences among individuals were eliminated by mixing equal amounts of each antiserum. A female Wistar rat weighing 200-250 g was exsanguinated and injected with Tyrode's buffer (137 mM NaCl, 3.7 mM KCl, 1.8 mM CaCl 2 , 1.1 mM MgCl 2 , 11.9 mM NaHCO 3 , 0.4 mM NaH 2 PO 4 , 5.6 in the abdominal cavity). 10 ml of mM D-Glucose) was injected, and after 2 minutes of massaging, ascites was collected from the rat abdominal cavity. The cell recovery solution was collected in a tube, centrifuged (1200 rpm, 5 min), and the supernatant was removed with Pipetteman. Further, 20 ml of Tyrode buffer was added to the tube, centrifuged (1200 rpm, 5 min), and the supernatant was removed with Pipetteman. This process was performed twice to wash the cells. Suspend the cells in 5 ml of Tyrode's buffer, measure the number of cells with a hemocytometer, adjust the cell suspension so that the final cell count is 1 × 10 7 cells / ml, and add 0 to a 24-well plate. Dispensed 5 ml each.
細胞浮遊液に非感作血清、RJ感作血清のいずれか、又はタイロード緩衝液0.5mlをそれぞれ添加し、CO2インキュベータにて36℃、3時間インキュベートした。24ウェルプレートより細胞浮遊液を回収し、タイロード緩衝液を2ml加えて遠心分離(1200rpm,5min)し、ピペットマンにて上清を除いた。この工程を2回行い、細胞を洗浄した。細胞をタイロード緩衝液0.7mlに懸濁し、24ウェルプレートに移した。細胞浮遊液に凍結乾燥RJ粉末、試験例2,3の等量混合物又は試験例11,12の等量混合物をそれぞれ0.25mg/mlと、タイロード緩衝液0.2mlと、0.1mg/mlホスファチジルセリン(PS溶液)0.1mlとを添加し、CO2インキュベータにて36℃、20分間インキュベートした。氷冷にて反応を停止させた後、24ウェルプレートより細胞浮遊液を回収し、遠心分離(1200rpm,5min)し、ピペットマンにて上清0.8mlを得、ヒスタミン定量用のサンプルとした。ヒスタミン定量には、SPI−bio社のHistamine Enzyme Immunoassay Kitを用い、ELISA法にて測定を行った。結果を表7に示す。 Either non-sensitized serum, RJ-sensitized serum, or 0.5 ml of Tyrode's buffer was added to the cell suspension and incubated at 36 ° C. for 3 hours in a CO 2 incubator. The cell suspension was collected from the 24-well plate, 2 ml of Tyrode buffer was added and centrifuged (1200 rpm, 5 min), and the supernatant was removed with Pipetteman. This process was performed twice to wash the cells. Cells were suspended in 0.7 ml of Tyrode's buffer and transferred to a 24-well plate. In the cell suspension, freeze-dried RJ powder, an equal amount mixture of Test Examples 2 and 3, or an equal amount mixture of Test Examples 11 and 12, respectively, 0.25 mg / ml, Tyrode buffer 0.2 ml, and 0.1 mg / ml 0.1 ml of phosphatidylserine (PS solution) was added and incubated at 36 ° C. for 20 minutes in a CO 2 incubator. After stopping the reaction with ice cooling, the cell suspension was collected from the 24-well plate, centrifuged (1200 rpm, 5 min), and 0.8 ml of the supernatant was obtained with Pipetman, which was used as a sample for histamine determination. Histamine quantification was performed by ELISA using Histamine Enzyme Immunoassay Kit from SPI-bio. The results are shown in Table 7.
<ヒト抗体産生細胞を用いたアレルゲン試験>
ヒト血液を用いたアレルゲン試験を行った。試験に先立ちRJによりアレルギーが起こることが確認されているアレルギー患者YY、アレルギー患者YM、及び健常者AOより血液の提供を受けた。これらの血液は3000rpmで10分間遠心分離して血餅を取り除き、血清とした。血清は測定まで−40℃にて保存を行った。
<Allergen test using human antibody-producing cells>
An allergen test using human blood was performed. Prior to the test, blood was provided from allergic patients YY, allergic patients YM, and healthy persons AO who were confirmed to have allergies by RJ. These blood samples were centrifuged at 3000 rpm for 10 minutes to remove clots and used as serum. Serum was stored at −40 ° C. until measurement.
凍結乾燥RJ粉末、試験例2,3の等量混合物、又は試験例11,12の等量混合物をそれぞれPBSで固形分15mg/mlに調整した抗原液を100μl、96ウェルプレート(Nunc社)に添加した。1時間室温にてインキュベートした後、96ウェルプレート内の抗原液を捨てウェルをPBSにて3回洗浄した。ウシ胎児血清(BSA)濃度が1%となるようにPBS−BSA溶液を作製し、96ウェルプレートの1ウェルあたり200μlずつ加え、1時間室温にてインキュベートした後、PBS−BSA溶液を捨てた。 Freeze-dried RJ powder, equal volume mixture of Test Examples 2 and 3, or equal volume mixture of Test Examples 11 and 12, each adjusted to a solid content of 15 mg / ml with PBS, 100 μl in a 96-well plate (Nunc) Added. After incubating at room temperature for 1 hour, the antigen solution in the 96-well plate was discarded, and the wells were washed 3 times with PBS. A PBS-BSA solution was prepared so that the fetal bovine serum (BSA) concentration was 1%, 200 μl per well of a 96-well plate was added, and incubated at room temperature for 1 hour, and then the PBS-BSA solution was discarded.
ヒト血清は、1%BSAを含むPBSで1/2、1/5、1/10と段階希釈を行い、ウェルに50μl添加した後、2時間室温でインキュベートした。ウェル内の抗血清を捨て、0.05% Tween20−PBS溶液でウェルを3回洗浄した。HRP標識抗ヒトIgE(0.5mg/ml)を1%BSAを含むPBSにて500倍に希釈したものを50μl添加した。ウェル中の抗体液を捨て、0.05% Tween20−PBS溶液でウェルを3回洗浄した。検出は、Protein Detector ELISA Kitを用い、マイクロプレートリーダーで405nmにおける吸光度を測定した。なお、ブランクとしてヒト血清のかわりに1%BSAを用いた。結果を表8に示す。 Human serum was serially diluted 1/2, 1/5, 1/10 with PBS containing 1% BSA, and 50 μl was added to the wells, followed by incubation at room temperature for 2 hours. The antiserum in the wells was discarded and the wells were washed 3 times with 0.05% Tween20-PBS solution. 50 μl of HRP-labeled anti-human IgE (0.5 mg / ml) diluted 500-fold with PBS containing 1% BSA was added. The antibody solution in the well was discarded, and the well was washed 3 times with 0.05% Tween20-PBS solution. Detection was performed using a Protein Detector ELISA Kit, and the absorbance at 405 nm was measured with a microplate reader. In addition, 1% BSA was used instead of human serum as a blank. The results are shown in Table 8.
<反転腸管試験によるアミノ酸吸収能比較試験>
一晩絶食させたモルモットを放血にて屠殺し、腸管を取り出した。この腸管を反転し、腸上部、腸中部、腸下部と三等分に切断した。反転腸管の一端を糸で縛り、その中に0.3%グルコース含有0.9%NaCl溶液を2ml注入し、もう一方の端を縛って反転嚢とした。この反転嚢を5%の凍結乾燥RJ、試験例2,3の等量混合物、又は試験例11,12の等量混合物溶液が30ml入ったフラスコにそれぞれ入れ、10分間酸素通気した。その後37℃、1時間インキュベートして腸管内液を取り出しアミノ酸吸収能試験用のサンプルとした。
<Amino acid absorption ability comparison test by inversion intestinal tract test>
Guinea pigs fasted overnight were sacrificed by exsanguination and the intestine was removed. The intestinal tract was inverted and cut into three equal parts: the upper intestine, the middle intestine, and the lower intestine. One end of the inverted intestinal tract was tied with a thread, and 2 ml of 0.9% NaCl solution containing 0.3% glucose was injected into it, and the other end was tied to form an inverted sac. The inverted sac was placed in a flask containing 30 ml of a 5% freeze-dried RJ, an equal amount mixture of Test Examples 2 and 3, or an equal amount mixture solution of Test Examples 11 and 12, and aerated with oxygen for 10 minutes. Thereafter, the mixture was incubated at 37 ° C. for 1 hour, and the intestinal fluid was taken out and used as a sample for amino acid absorption ability test.
これらのサンプルを用いてFolin&Ciocalteu法によるアミノ酸量の測定を行った。即ち、各サンプル又はL−チロシン標準溶液0.5mlをそれぞれ遠心管に入れ、その中に20%トリクロロ酢酸を0.5ml加えた。よく混合して遠心した後、上清0.1mlを採取して試験管内に入れた。ここに2%Na2CO3/0.1規定NaOHを5ml加え、さらにフェノール試薬0.5mlを加えて混合し、30分間静置した。660nmにおける吸光度を測定し、L−チロシンにて検量線を作成、各サンプルの吸光度からアミノ酸吸収量(L−チロシン量として)を算出した。なお、腸管の上部・中部・下部でのアミノ酸の吸収率に差が認められなかったことから、それらの平均値を取り(n=3)L−チロシン含量を算出した。L−チロシンについてはあらかじめ検量線を作成し、直線性を確認した。結果を表9に示す。 Using these samples, the amount of amino acids was measured by the Folin & Ciocalteu method. That is, each sample or 0.5 ml of L-tyrosine standard solution was placed in a centrifuge tube, and 0.5 ml of 20% trichloroacetic acid was added thereto. After thorough mixing and centrifugation, 0.1 ml of the supernatant was collected and placed in a test tube. To this, 5 ml of 2% Na 2 CO 3 /0.1N NaOH was added, 0.5 ml of phenol reagent was further added and mixed, and the mixture was allowed to stand for 30 minutes. Absorbance at 660 nm was measured, a calibration curve was prepared with L-tyrosine, and the amount of amino acid absorption (as L-tyrosine amount) was calculated from the absorbance of each sample. Since there was no difference in the absorption rate of amino acids in the upper, middle and lower parts of the intestinal tract, the average value thereof was taken (n = 3) and the L-tyrosine content was calculated. For L-tyrosine, a calibration curve was prepared in advance to confirm linearity. The results are shown in Table 9.
<酵素処理RJの分子量分析>
試験例2,3の等量混合物、又は試験例11,12の等量混合物について、ゲル濾過カラムを用いたHPLCにて分子量の測定を行った。HPLC条件は、カラム;Superdex-peptide 10/300 GL (1.0cm×30cm)、移動相;30%CH3CN、0.1%TFA/H2O、ポンプ;TOYOSODA CCPM、流速;0.3ml/分、検出波長;220nmである。試験例2,3の等量混合物の結果を図1(b)、試験例11,12の等量混合物の結果を図1(c)に示す。なお、図1(a)は分子量マーカーを示し、左側のピークから順に分子量12.5kDa(retention time Y = 26.958)、分子量6.5kDa(retention time Y = 31.242)、分子量1274Da(retention time Y = 38.993)、分子量393Da(retention time Y = 49.5)、分子量132Da(retention time Y = 56.435)となっている。なお、図1(a)に示されるゲル濾過の結果を片対数プロットした検量線より、Y=87.495−6.4744Ln(X)、R2=0.990、Y;retention time、X;分子量であった。
<Molecular weight analysis of enzyme-treated RJ>
About the equal amount mixture of Test Examples 2 and 3, or the equal amount mixture of Test Examples 11 and 12, the molecular weight was measured by HPLC using a gel filtration column. HPLC conditions were as follows: column; Superdex-
図1(b)に示されるクロマトグラムでは retention time Y = 33.008のピーク(分子量4523Da)及び retention time Y = 42.09のピーク(分子量1111Da)が確認されており、図1(c)に示されるクロマトグラムでは前記分子量4523Daのピーク及びペプチド性成分が消失し、分子量1111Daのピークが残っていた。さらに、試験例11,12はいずれも、例えば上記表4に示されるような予備的な試験において、SDS−PAGE(クマシー染色及び銀染色)にて分子量4kDa以上の蛋白質のバンドを含め、ほとんどの蛋白質バンドが検出されなかったことが確認されている。従って、試験例11,12では分子量4.5kDaを超えるペプチド性成分が存在せず、分子量1.2kDa以上のピークが存在しないことが確認された。 In the chromatogram shown in FIG. 1 (b), a peak at retention time Y = 33.008 (molecular weight 4523Da) and a peak at retention time Y = 42.09 (molecular weight 1111Da) were confirmed, and the chromatogram shown in FIG. 1 (c). Then, the peak with the molecular weight of 4523 Da and the peptide component disappeared, and the peak with the molecular weight of 1111 Da remained. Furthermore, all of Test Examples 11 and 12, including a protein band having a molecular weight of 4 kDa or more by SDS-PAGE (Coomassie staining and silver staining) in a preliminary test as shown in Table 4 above, for example, It has been confirmed that no protein band was detected. Therefore, it was confirmed in Test Examples 11 and 12 that there was no peptide component having a molecular weight of 4.5 kDa and no peak having a molecular weight of 1.2 kDa or more.
<抗疲労効果の検討>
凍結乾燥RJ粉末及び試験例11,12の等量混合物について抗疲労効果を確認するために、マウスに強制水泳運動負荷を与えた状態での水泳運動の持続時間を測定した。5週齢のddy系雄性マウス(体重28g前後)を日本エスエルシー社から購入し、試験時間以外は餌と水を自由に摂取させた。次に、各サンプルを下記表10に示される用量でマウスに1日1回3日間経口投与し、4日目に水槽(直径19cm、深さ27cm、水温25℃)内で5分間強制水泳(第1回目)を試行させた。その直後に各サンプルを下記表10に示される用量でマウスに経口投与し、60分経過後に第2回目の強制水泳を5分間試行させた。第2回目の強制水泳時にマウスが水中で不動であった時間を測定し、その累計不動時間を抗疲労効果の指標とした。なお、各サンプルはそれぞれ蒸留水に懸濁し、対照群には蒸留水のみを経口投与させた。結果を表10に示す。
<Examination of anti-fatigue effect>
In order to confirm the anti-fatigue effect of the lyophilized RJ powder and the equivalent mixture of Test Examples 11 and 12, the duration of the swimming exercise was measured in a state where a forced swimming exercise load was applied to the mouse. Five-week-old ddy male mice (body weight of about 28 g) were purchased from Japan SLC, and were allowed to freely ingest food and water except for the test time. Next, each sample was orally administered to mice once a day for 3 days at the doses shown in Table 10 below, and forced swimming for 5 minutes in a water bath (diameter 19 cm, depth 27 cm, water temperature 25 ° C.) on the 4th day ( The first time) was tried. Immediately thereafter, each sample was orally administered to mice at the doses shown in Table 10 below, and after 60 minutes, the second forced swimming was tried for 5 minutes. The time during which the mouse was immobile in the water during the second forced swimming was measured, and the cumulative immobility time was used as an index of the anti-fatigue effect. Each sample was suspended in distilled water, and only distilled water was orally administered to the control group. The results are shown in Table 10.
Claims (1)
pHが6.5〜8.0に調整されたローヤルゼリー希釈液に蛋白質分解酵素としてエンド型中性プロテアーゼ及び糖分解酵素としてβ−マンノシダーゼを同時に添加して行う第1分解工程と、該第1分解工程後のローヤルゼリー希釈液のpHを6.5〜8.0に調整するpH調整工程と、該pH調整工程後のローヤルゼリー希釈液に両酵素処理を行う第2分解工程とを含むことを特徴とする低アレルゲン化ローヤルゼリーの製造方法。a first decomposition step in which endo-type neutral protease as a proteolytic enzyme and β-mannosidase as a glycolytic enzyme are simultaneously added to a royal jelly diluent adjusted to pH 6.5 to 8.0, and the first decomposition A pH adjusting step for adjusting the pH of the royal jelly diluted solution after the step to 6.5 to 8.0, and a second decomposition step for performing both enzyme treatments on the royal jelly diluted solution after the pH adjusting step, To produce a low allergenized royal jelly.
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JP2008056645A (en) * | 2006-09-04 | 2008-03-13 | Yumedeika Kk | Anti-oxidant peptide obtained by reaction of protein in enzymatically treated royal jelly with polypeptide and method for producing the same |
JP2008208088A (en) * | 2007-02-27 | 2008-09-11 | Ehime Univ | Composition for treatment of stomatitis |
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JP5027001B2 (en) * | 2007-07-05 | 2012-09-19 | アピ株式会社 | Enzyme-treated royal jelly and skin fibroblast growth promoter containing the same |
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KR101617320B1 (en) * | 2013-12-03 | 2016-05-02 | 주식회사 삼양사 | Royal jelly beverage composition having improved dispersability in water and method for preparing the same |
AU2014369278A1 (en) * | 2013-12-19 | 2016-06-16 | Nestec Sa | Compositions and methods for reducing cat allergens in the environment |
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KR101900258B1 (en) * | 2016-08-16 | 2018-09-19 | (주) 바이텍 | Preparation method of enzyme treated royal jelly and Enzyme treated royal jelly |
US11998031B2 (en) | 2018-03-28 | 2024-06-04 | Yamada Bee Company | Stabilization of enzyme treated royal jelly |
CN115105459B (en) * | 2022-07-05 | 2024-04-12 | 广东先强药业有限公司 | Royal jelly extract and extraction method and application thereof |
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