JP6082551B2 - Insulin secretion promoter, blood glucose level increase inhibitor, and production method thereof - Google Patents
Insulin secretion promoter, blood glucose level increase inhibitor, and production method thereof Download PDFInfo
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- JP6082551B2 JP6082551B2 JP2012206466A JP2012206466A JP6082551B2 JP 6082551 B2 JP6082551 B2 JP 6082551B2 JP 2012206466 A JP2012206466 A JP 2012206466A JP 2012206466 A JP2012206466 A JP 2012206466A JP 6082551 B2 JP6082551 B2 JP 6082551B2
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- royal jelly
- protein
- blood glucose
- insulin secretion
- glucose level
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Landscapes
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Description
本発明は、インスリン分泌促進剤、血糖値上昇抑制剤およびそれらの製造方法に関する。 The present invention relates to an insulin secretion promoter, a blood sugar level increase inhibitor, and a method for producing them.
病気にかかる前に日常の食事により病気を予防することは、健康な生活を送る上で非常に重要である。従来、生活習慣病、慢性疾患などの老化に伴う現象に対応するものとして、ローヤルゼリーが配合されたサプリメントや食品などがある。 Prevention of illness by daily diet before getting ill is very important for a healthy life. Conventionally, there are supplements and foods containing royal jelly as a response to aging phenomena such as lifestyle-related diseases and chronic diseases.
ローヤルゼリーは、若い働き蜂の大顎腺および下咽頭腺から分泌される乳白色のクリーム状物質であり、全ての若い幼虫の食餌として、また、将来女王蜂になる蜂の唯一の食餌として分泌される物質である。ローヤルゼリーは、抗アレルギー作用、マウスにおけるDNA損傷抑制作用を有することが報告されており、また、その栄養学的役割や薬理学的役割についても研究が行われている。 Royal jelly is a milky white creamy substance secreted from the mammary gland and hypopharyngeal gland of young worker bees, and is secreted as the diet of all young larvae and as the sole diet of bees that will become queen bees in the future. is there. Royal jelly has been reported to have an antiallergic action and a DNA damage inhibitory action in mice, and studies on its nutritional and pharmacological roles have also been conducted.
ローヤルゼリーは、およそ66%の水分、14%のタンパク質、12%の炭水化物を含む物質である。ローヤルゼリーは、その有効成分がデセン酸であると言われており、ローヤルゼリーをドリンクに配合するときには、タンパク質が沈殿することから、タンパク質を除去した上清(上澄み液)を使用している。また、除去されたタンパク質の一部は、ローヤルゼリー由来のタンパク質として上市されている。 Royal jelly is a substance that contains approximately 66% moisture, 14% protein, and 12% carbohydrate. Royal jelly is said to have an active ingredient of decenoic acid. When royal jelly is blended in a drink, protein is precipitated, and thus a supernatant (supernatant) from which protein has been removed is used. A part of the removed protein is marketed as a protein derived from royal jelly.
既存の技術として、ローヤルゼリーをペプシン及びパンクレアチンで分解して得られる分子量500〜3,000のペプチドを含有することを特徴とする血糖値上昇抑制剤が報告されている(例えば、特許文献1参照)。 As an existing technique, a blood glucose level increase inhibitor characterized by containing a peptide having a molecular weight of 500 to 3,000 obtained by degrading royal jelly with pepsin and pancreatin has been reported (for example, see Patent Document 1). ).
また、ローヤルゼリーに含まれるタンパク質は、抗菌作用や抗アレルギー作用を有することが報告されている。ローヤルゼリーのインスリン作用については、脂肪酸であるローヤルゼリーに含まれるデセン酸がその本体として報告されている(例えば、非特許文献1または2参照)。 Moreover, it is reported that the protein contained in royal jelly has an antibacterial action and an antiallergic action. Regarding the insulin action of royal jelly, decenoic acid contained in royal jelly, which is a fatty acid, has been reported as its main body (see, for example, Non-Patent Document 1 or 2).
なお、高血糖は生活習慣病のひとつで、血糖値を高い状態で放置しておくと、糖尿病になり、血液がどろどろになる。また、神経障害、網膜症、腎症、脳梗塞、狭心症、糖尿病性壊疽などの合併症を引き起こすこともある。厚生労働省の平成20年調査によると、糖尿病の総患者数は237万人であり、平成19年の「国民健康・栄養調査の概要」によると、糖尿病予備軍は2210万人にもなる。 Hyperglycemia is a lifestyle-related disease, and if the blood sugar level is left in a high state, it becomes diabetic and blood becomes muddy. It may also cause complications such as neuropathy, retinopathy, nephropathy, cerebral infarction, angina pectoris, and diabetic gangrene. According to the 2008 survey by the Ministry of Health, Labor and Welfare, the total number of patients with diabetes is 2.37 million, and according to the “Summary of National Health and Nutrition Survey” in 2007, there are 22.1 million reserves of diabetes.
特許文献1では、酸性プロテアーゼのペプシンとアルカリ性プロテアーゼのパンクレアチンとを組み合わせて使用するため、製造工程上、酸性溶液およびアルカリ性溶液を使用する。それにより塩ができ、その処理工程を必要とするため、製造コストがかさむという課題があった。また、ローヤルゼリーをドリンクに配合したときに除去されたタンパク質は、一部は上市されているが、大部分は利用されていないという課題があった。このため、未利用のタンパク質の有効な利用方法が模索されていた。 In Patent Document 1, since acidic protease pepsin and alkaline protease pancreatin are used in combination, an acidic solution and an alkaline solution are used in the production process. As a result, salt was formed, and the processing step was required, which caused a problem that the manufacturing cost was increased. Moreover, although the protein removed when the royal jelly was mix | blended with the drink was partly marketed, there existed a subject that most was not utilized. For this reason, the effective utilization method of the unused protein was sought.
本発明は、このような課題に着目してなされたもので、製造コストを下げることができ、未利用のローヤルゼリータンパク質を利用可能な、インスリン分泌促進剤、血糖値上昇抑制剤およびそれらの製造方法を提供することを目的としている。 The present invention has been made paying attention to such a problem, and can reduce the manufacturing cost, and can utilize an unused royal jelly protein, an insulin secretion promoter, a blood sugar level increase inhibitor, and a production method thereof The purpose is to provide.
上記目的を達成するために、本発明に係るインスリン分泌促進剤は、酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせによるローヤルゼリータンパク質分解物を有効成分とすることを特徴とする。
本発明に係るインスリン分泌促進剤の製造方法は、水溶性ローヤルゼリータンパク質を酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせによりプロテアーゼ処理することを特徴とする。他の本発明に係るインスリン分泌促進剤の製造方法は、アルコール変性したローヤルゼリータンパク質を酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせによりプロテアーゼ処理することを特徴とする。
In order to achieve the above object, the insulin secretagogue according to the present invention is characterized by comprising a royal jelly proteolysate obtained by acidic protease, neutral protease or a combination of two or more thereof as an active ingredient.
The method for producing an insulin secretagogue according to the present invention is characterized by treating a water-soluble royal jelly protein with an acidic protease, a neutral protease, or a combination of two or more thereof. Another method for producing an insulin secretagogue according to the present invention is characterized in that an alcohol-modified royal jelly protein is treated with an acid protease, a neutral protease, or a combination of two or more thereof.
本発明に係る血糖値上昇抑制剤は、酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせによるローヤルゼリータンパク質分解物を有効成分とすることを特徴とする。
本発明に係る血糖値上昇抑制剤の製造方法は、水溶性ローヤルゼリータンパク質を酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせによりプロテアーゼ処理することを特徴とする。他の本発明に係る血糖値上昇抑制剤の製造方法は、アルコール変性したローヤルゼリータンパク質を酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせによりプロテアーゼ処理することを特徴とする。
The blood sugar level elevation inhibitor according to the present invention is characterized by comprising a royal jelly protein degradation product by acidic protease, neutral protease or a combination of two or more thereof as an active ingredient.
The method for producing a blood sugar level elevation inhibitor according to the present invention is characterized in that a water-soluble royal jelly protein is treated with an acid protease, a neutral protease, or a combination of two or more thereof. Another method for producing a blood sugar level increase inhibitor according to the present invention is characterized in that an alcohol-modified royal jelly protein is treated with an acid protease, a neutral protease, or a combination of two or more thereof.
本発明に係るインスリン分泌促進剤、血糖値上昇抑制剤およびそれらの製造方法において、ローヤルゼリータンパク質としては、例えば、ローヤルゼリー、ローヤルゼリーから水溶性タンパク質を抽出した水溶性ローヤルゼリータンパク質、ローヤルゼリーをアルコールで抽出したアルコール変性ローヤルゼリータンパク質を利用することができるが、これらには限らない。ローヤルゼリータンパク質分解物は、ローヤルゼリータンパク質をプロテアーゼ処理することにより製造される。プロテアーゼ処理で使用するプロテアーゼは、Rhizopus niveus、Aspergillus nigerなどの酸性プロテアーゼ、bacillus subtilis、Asperhillus oryzae、Pig pancreasなどの中性プロテアーゼを利用することができるが、これらには限らない。 In the insulin secretion promoter, the blood sugar level increase inhibitor and the production method thereof according to the present invention, as the royal jelly protein, for example, royal jelly, water-soluble royal jelly protein obtained by extracting a water-soluble protein from royal jelly, alcohol obtained by extracting royal jelly with alcohol Denatured royal jelly protein can be used, but is not limited thereto. A royal jelly protein degradation product is produced by treating a royal jelly protein with a protease. Proteases used in the protease treatment can be acidic proteases such as Rhizopus niveus and Aspergillus niger, neutral proteases such as Bacillus subtilis, Aspergillus oryzae, and Pig pancreas, but are not limited thereto.
本発明に係るインスリン分泌促進剤、血糖値上昇抑制剤およびそれらの製造方法では、プロテアーゼとして、酸性プロテアーゼ、中性プロテアーゼまたはそれらの2種以上の組み合わせを使用するため、酸性溶液およびアルカリ性溶液を使用する場合と異なり、製造工程上、塩ができず、塩の処理工程を必要としない。このため、塩の処理工程に要する製造コストを下げることができる。 In the insulin secretion promoter, the blood glucose level increase inhibitor, and the production method thereof according to the present invention, an acidic solution and an alkaline solution are used because an acidic protease, a neutral protease, or a combination of two or more thereof is used as a protease. Unlike the case, the salt is not formed in the manufacturing process, and the salt treatment process is not required. For this reason, the manufacturing cost required for the salt treatment step can be reduced.
本発明に係るインスリン分泌促進剤または血糖値上昇抑制剤の投与形態は、治療に際し最も効果的なものを使用するのが望ましく、経口投与または、例えば静脈内、腹膜内もしくは皮下投与などの非経口投与をあげることができる。これらのうち、静脈内投与または経口投与が好ましい。 The dosage form of the insulin secretagogue or blood glucose level increase inhibitor according to the present invention is preferably the most effective in the treatment, and is administered orally or parenterally such as intravenous, intraperitoneal or subcutaneous administration. Administration can be raised. Of these, intravenous administration or oral administration is preferred.
投与する剤形としては、錠剤、散剤、顆粒剤、丸剤、懸濁剤、乳剤、浸剤、カプセル剤、シロップ剤、注射剤、液剤、エリキシル剤、エキス剤、チンキ剤、流エキス剤等があげられる。経口投与に適当な、例えばシロップ剤のような液体調製物は、水、ショ糖、ソルビトール、果糖などの糖類、ポリエチレングリコール、プロピレングリコールなどのグリコール類、ごま油、オリーブ油、大豆油などの油類、p−ヒドロキシ安息香酸エステル類などの防腐剤、パラオキシ安息香酸メチル等のパラオキシ安息香酸誘導体、安息香酸ナトリウム等の保存剤、ストロベリーフレーバー、ペパーミントなどのフレーバー類などの担体を使用して製造できる。 The dosage forms to be administered include tablets, powders, granules, pills, suspensions, emulsions, soaking agents, capsules, syrups, injections, solutions, elixirs, extracts, tinctures, fluid extracts, etc. can give. Liquid preparations suitable for oral administration, such as syrups, include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, It can be produced using a carrier such as a preservative such as p-hydroxybenzoic acid esters, a paraoxybenzoic acid derivative such as methyl paraoxybenzoate, a preservative such as sodium benzoate, and a flavor such as strawberry flavor and peppermint.
また、経口投与に適当な、例えば錠剤、散剤および顆粒剤などは、乳糖、白糖、ブドウ糖、ショ糖、マンニトール、ソルビトール等の糖類、バレイショ、コムギ、トウモロコシ等の澱粉、炭酸カルシウム、硫酸カルシウム、炭酸水素ナトリウム、塩化ナトリウム等の無機物、結晶セルロース、カンゾウ末、ゲンチアナ末などの植物末、パインデックスなどの賦形剤、澱粉、寒天、ゼラチン末、結晶セルロース、カルメロースナトリウム、カルメロースカルシウム、炭酸カルシウム、炭酸水素ナトリウム、アルギン酸ナトリウムなどの崩壊剤、ステアリン酸マグネシウム、タルク、水素添加植物油、マクロゴール、シリコーン油などの滑沢剤、ポリビニルアルコール、ヒドロキシプロピルセルロース、メチルセルロース、エチルセルロース、カルメロース、ゼラチン、澱粉のり液などの結合剤、脂肪酸エステルなどの界面活性剤、グリセリンなどの可塑剤などを用いて製造できる。 Also suitable for oral administration, such as tablets, powders and granules, are sugars such as lactose, sucrose, glucose, sucrose, mannitol, sorbitol, starches such as potato, wheat, corn, calcium carbonate, calcium sulfate, carbonate Inorganic substances such as sodium hydrogen and sodium chloride, plant powders such as crystalline cellulose, licorice powder, gentian powder, excipients such as paindex, starch, agar, gelatin powder, crystalline cellulose, carmellose sodium, carmellose calcium, calcium carbonate , Disintegrating agents such as sodium bicarbonate and sodium alginate, magnesium stearate, talc, hydrogenated vegetable oil, macrogol, silicone oil and other lubricants, polyvinyl alcohol, hydroxypropylcellulose, methylcellulose, ethylcellulose, potassium Merosu, gelatin, binding agents such as starch paste solution, surfactants such as fatty acid esters, can be prepared by using a plasticizer such as glycerin.
非経口投与に適当な注射剤は、好ましくは受容者の血液と等張である活性化合物を含む滅菌水性剤からなる。例えば、注射剤の場合は、塩溶液、ブドウ糖溶液または塩水とブドウ糖溶液の混合物からなる担体などを用いて注射用の溶液を調製する。また、これら非経口剤においても、前述の防腐剤、保存剤、界面活性剤等が使用できる。 Injectables suitable for parenteral administration preferably comprise a sterile aqueous solution containing the active compound which is isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of salt water and a glucose solution. Also in these parenteral agents, the above-mentioned preservatives, preservatives, surfactants and the like can be used.
本発明に係るインスリン分泌促進剤または血糖値上昇抑制剤の投与量および投与回数は、投与形態、患者の年齢、体重、治療すべき症状の性質もしくは重篤度により異なるが、含有するローヤルゼリータンパク質分解物の量として、通常経口の場合、成人一人当り1mg〜50g、好ましくは5mg〜20gを一日一回ないし数回投与する。静脈内投与などの非経口投与の場合、成人一人当り0.1mg〜50g、好ましくは1mg〜10gを一日一回ないし数回投与する。また、動物に投与する場合、動物の年齢、種類、症状の性質もしくは重篤度により異なるが、特に制限はなく、体重1kg当たり、0.1mg〜10g、好ましくは1mg〜1gを一日一回ないし数回投与する。また、静脈内投与などの非経口投与の場合、体重1kg当たり0.01mg〜10g、好ましくは1mg〜1gを一日一回ないし数回投与する。しかしながら、これら投与量および投与回数に関しては、前述の種々の条件により変動する。 The dose and frequency of administration of the insulin secretagogue or blood glucose level increase inhibitor according to the present invention vary depending on the administration form, patient age, body weight, nature or severity of symptoms to be treated, but royal jelly proteolysis contained therein In general, in the case of oral administration, 1 mg to 50 g, preferably 5 mg to 20 g per adult is administered once or several times a day. In the case of parenteral administration such as intravenous administration, 0.1 mg to 50 g, preferably 1 mg to 10 g per adult is administered once to several times a day. In addition, when administered to an animal, it varies depending on the age, type, nature or severity of the animal, but there is no particular limitation, and 0.1 mg to 10 g, preferably 1 mg to 1 g, per kg body weight once a day. Dosage several times. In the case of parenteral administration such as intravenous administration, 0.01 mg to 10 g, preferably 1 mg to 1 g per kg body weight is administered once to several times a day. However, the dose and the number of doses vary depending on the various conditions described above.
本発明に係るインスリン分泌促進剤または血糖値上昇抑制剤は、飲食品または飼料に添加されてもよく、このような飲食品または飼料は、ローヤルゼリータンパク質分解物を飲食品または飼料の原料に添加し、一般の飲食品または飼料の製造方法で製造することができる。その飲食品または飼料は、一般の飲食品または飼料と同様、成形・造粒することができる。成形・造粒方法としては流動層造粒、攪拌造粒、押し出し造粒、転動造粒、気流造粒、圧縮成形造粒、解砕造粒、噴霧造粒、噴射造粒などの造粒方法、パンコーティング、流動層コーティング、ドライコーティング、などのコーティング方法、パフドライ、過剰水蒸気法、フォームマット方法、マイクロ波加熱方法などの膨化方法、押出造粒機やエキストルーダーなどの押出方法などがあげられる。 The insulin secretion promoter or blood sugar level increase inhibitor according to the present invention may be added to foods and drinks or feeds, and such foods and drinks or feeds add royal jelly proteolysate to the raw materials of foods and drinks or feeds. It can be produced by a general method for producing food or drink or feed. The food / drink or feed can be shaped and granulated in the same manner as general food / drink or feed. Molding / granulation methods include fluidized bed granulation, stirring granulation, extrusion granulation, rolling granulation, airflow granulation, compression molding granulation, pulverization granulation, spray granulation, spray granulation, etc. Coating methods such as pan coating, fluidized bed coating and dry coating, puff drying, excess steam method, foam mat method, microwave heating method and other extruding methods, extrusion granulators and extruders etc. It is done.
本発明に係るインスリン分泌促進剤または血糖値上昇抑制剤を飲食品または飼料に添加する場合、ローヤルゼリータンパク質分解物の飲食品または飼料中の含有量としては、含有して得られる飲食品または飼料がインスリン分泌促進作用または血糖上昇抑制作用を示す濃度であれば特に制限はないが、含有する飲食品または飼料に対して0.001〜100質量%が好ましく、0.01〜100質量%がより好ましく、0.1〜100質量%がさらに好ましい。 When the insulin secretion promoter or blood sugar level increase inhibitor according to the present invention is added to a food or drink or feed, the content of the royal jelly protein degradation product in the food or drink or feed includes: The concentration is not particularly limited as long as it has an insulin secretion promoting action or a blood glucose rise inhibiting action, but is preferably 0.001 to 100% by weight, more preferably 0.01 to 100% by weight with respect to the food or drink or feed to be contained. 0.1 to 100% by mass is more preferable.
本発明に係るインスリン分泌促進剤または血糖値上昇抑制剤を添加する具体的な飲食品としては、ジュース類、清涼飲料水、スープ類、茶類、乳酸菌飲料、発酵乳、冷菓、バター、チーズ、ヨーグルト、加工乳、脱脂粉乳等の乳製品、ハム、ソーセージ、ハンバーグ等の畜肉製品、魚肉錬り製品、だし巻き、卵豆腐等の卵製品、クッキー、ゼリー、スナック菓子、チューイングガム等の菓子類、パン類、麺類、漬け物類、薫製品、干物、佃煮、調味料等があげられる。飲食品の形態としては、例えば粉末食品、シート状食品、瓶詰め食品、缶詰食品、レトルト食品、カプセル食品、タブレット状食品、流動食品、ドリンク剤等があげられる。 Specific foods and drinks to which the insulin secretion promoter or blood sugar level increase inhibitor according to the present invention is added include juices, soft drinks, soups, teas, lactic acid bacteria beverages, fermented milk, frozen desserts, butter, cheese, Dairy products such as yogurt, processed milk, skimmed milk powder, livestock meat products such as ham, sausage and hamburger, fish smelted products, egg products such as broiled and egg tofu, cookies, jelly, snacks, chewing gum and other confectionery, bread , Noodles, pickles, salmon products, dried fish, boiled salmon, seasonings and the like. Examples of the form of food and drink include powdered foods, sheet-like foods, bottled foods, canned foods, retort foods, capsule foods, tablet-like foods, liquid foods, and drinks.
本発明に係るインスリン分泌促進剤または血糖値上昇抑制剤を添加した飲食品または飼料には、必要に応じて一般に飲食品または飼料に用いられる添加物、例えば、甘味料、着色料、保存料、増粘安定剤、酸化防止剤、発色料、漂白料、防かび剤、ガムベース、苦味料、酵素、光沢剤、酸味料、調味料、乳化剤、強化剤、製造用剤、香料、香辛料抽出物などの添加物を添加してもよい。 The food and drink or feed to which the insulin secretion promoter or blood sugar level increase inhibitor according to the present invention is added, if necessary, additives generally used in food or drink or feed, such as sweeteners, coloring agents, preservatives, Thickening stabilizers, antioxidants, coloring agents, bleaching agents, fungicides, gum bases, bittering agents, enzymes, brighteners, acidulants, seasonings, emulsifiers, fortifiers, manufacturing agents, fragrances, spice extracts, etc. These additives may be added.
本発明では、未利用のローヤルゼリータンパク質を利用して、インスリン分泌促進作用および血糖値上昇抑制作用を有する新しい健康食品素材を得ることができる。これにより、ローヤルゼリーからデセン酸などの脂溶性物質を除去した後に残るローヤルゼリータンパク質を、有効活用することができる。ローヤルゼリータンパク質分解物には、インスリン分泌促進作用、血糖上昇抑制作用のほか、糖尿病改善作用、糖尿病予防作用、成長促進作用、母乳中のインスリン増加作用なども期待できる。 In the present invention, a new health food material having an action of promoting insulin secretion and an action of suppressing an increase in blood glucose level can be obtained by utilizing an unused royal jelly protein. Thereby, the royal jelly protein remaining after removing fat-soluble substances such as decenoic acid from the royal jelly can be effectively utilized. The royal jelly protein degradation product can be expected to have an insulin secretion-promoting effect, a blood glucose rise-inhibiting effect, a diabetes-improving effect, a diabetes-preventing effect, a growth-promoting effect, and an insulin-increasing effect in breast milk.
本発明によれば、製造コストを下げることができ、未利用のローヤルゼリータンパク質を利用可能な、インスリン分泌促進剤、血糖値上昇抑制剤およびそれらの製造方法を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the manufacturing cost can be lowered | hung and the insulin secretion promoter, blood glucose level rise inhibitor, and those manufacturing methods which can utilize an unused royal jelly protein can be provided.
以下、本発明の実施の形態のインスリン分泌促進剤、血糖値上昇抑制剤およびそれらの製造方法について、実施例に基づいて説明する。なお、以下の実施例では、ローヤルゼリータンパク質およびローヤルゼリータンパク質分解物の代表的な作り方を示すが、本発明においてはこの作り方に限られない。 Hereinafter, the insulin secretion promoter, the blood sugar level increase inhibitor, and the production methods thereof according to embodiments of the present invention will be described based on examples. In the following examples, representative methods for producing royal jelly protein and royal jelly protein degradation products are shown, but the present invention is not limited to this method.
[アルコール変性したローヤルゼリータンパク質、および、アルコール変性したローヤルゼリータンパク質分解物の製造方法]
ローヤルゼリー(100g)を同量のエタノールで処理し、その残渣を60%エタノールで、室温下で2回洗浄した。この残渣はアルコール変性したローヤルゼリータンパク質であり、これを凍結乾燥した結果、その成分組成は、タンパク質(80〜81%)、炭水化物(14〜15%)および少量の脂質であった。
[Alcohol-denatured Royal Jelly Protein and Method for Producing Alcohol-Denatured Royal Jelly Protein Degradation Product]
Royal jelly (100 g) was treated with the same amount of ethanol, and the residue was washed twice with 60% ethanol at room temperature. This residue was an alcohol-denatured royal jelly protein, which was lyophilized, and as a result, its component composition was protein (80-81%), carbohydrate (14-15%), and a small amount of lipid.
得られたアルコール変性したローヤルゼリータンパク質をろ過した後、乾燥残渣(12.5g)を水(87.5g)に懸濁し、適正pHに調整した。その後、プロテアーゼ(Aspergillus niger由来プロテアーゼ0.25g)を加え、至適温度50℃に温度を維持し、5時間タンパク質分解を行った。次に、80℃の温度で30分間、酵素失活を行った。酵素反応液をセライトでろ過をした後、ろ液を凍結乾燥した。こうして、アルコール変性したローヤルゼリータンパク質分解物が得られた。なお、その収量は、8.3gであった。また、ローヤルゼリータンパク質分解物は、主要成分が酵素分解物(平均分子量879)であり、少量の脂質(0.9%)、ミネラル(3.9%)および水分(3%)を含んでいた。 After the obtained alcohol-modified royal jelly protein was filtered, the dried residue (12.5 g) was suspended in water (87.5 g) and adjusted to an appropriate pH. Thereafter, protease (0.25 g protease derived from Aspergillus niger) was added, and the temperature was maintained at an optimum temperature of 50 ° C., and proteolysis was performed for 5 hours. Next, enzyme inactivation was performed at a temperature of 80 ° C. for 30 minutes. The enzyme reaction solution was filtered through celite, and the filtrate was lyophilized. Thus, an alcohol-modified royal jelly protein degradation product was obtained. The yield was 8.3 g. The main component of the royal jelly protein degradation product was an enzyme degradation product (average molecular weight 879), and contained a small amount of lipid (0.9%), mineral (3.9%) and water (3%).
[水溶性ローヤルゼリータンパク質、および、水溶性ローヤルゼリータンパク質のタンパク質分解物の製造方法]
アルコール変性ローヤルゼリータンパク質以外の原料として、水溶性ローヤルゼリータンパク質の製造方法を、以下に示す。ローヤルゼリー(50g)に精製水(200g)を加えて、室温にて30分間攪拌した。その後、10,000rpmで30分間の遠心分離を行い、上清(1回目)を回収後、沈殿物にさらに精製水(200g)を加えて同様の操作を行い、上清(2回目)を回収した。回収した各上清を凍結乾燥し、水溶性ローヤルゼリータンパク質15.5gを得た。なお、水溶性成分の収率は87%であった。
[Method for producing water-soluble royal jelly protein and protein degradation product of water-soluble royal jelly protein]
As a raw material other than the alcohol-denatured royal jelly protein, a method for producing a water-soluble royal jelly protein is shown below. Purified water (200 g) was added to royal jelly (50 g), and the mixture was stirred at room temperature for 30 minutes. Thereafter, centrifugation is performed at 10,000 rpm for 30 minutes, and the supernatant (first time) is recovered. Then, purified water (200 g) is further added to the precipitate, and the same operation is performed to recover the supernatant (second time). did. Each collected supernatant was freeze-dried to obtain 15.5 g of a water-soluble royal jelly protein. The yield of the water-soluble component was 87%.
実施例1で示したアルコール変性したローヤルゼリータンパク質からアルコール変性したローヤルゼリータンパク質分解物を製造する方法と同様にして、水溶性ローヤルゼリータンパク質から、水溶性ローヤルゼリータンパク質のタンパク質分解物を製造した。 In the same manner as the method for producing an alcohol-denatured royal jelly protein degradation product from the alcohol-denatured royal jelly protein shown in Example 1, a protein degradation product of the water-soluble royal jelly protein was produced from the water-soluble royal jelly protein.
[ローヤルゼリータンパク質分解物のKK−Ayの強制経口投与実験]
実施例1で製造したアルコール変性したローヤルゼリータンパク質分解物の水溶液を、糖尿病マウス(日本エスエルシー株式会社製「kk−Ayマウス」)に、強制的に35日間経口投与した。また、対照群として、グアバ葉抽出物(備前化成社製)を、別の糖尿病マウスに、強制的に35日間経口投与した。これらの投与量は、350mg/kg体重で、濃度は、0.3mL/30g体重である。マウスは、室温25±1℃、湿度55%±5%、明暗周期12時間の条件下でプラスチックケージに入れて飼育し、試験開始前に1週間訓化させた。
[Robust jelly protein degradation product KK-Ay oral gavage experiment]
The aqueous solution of the alcohol-denatured royal jelly proteolysate produced in Example 1 was forcibly orally administered to diabetic mice (“KK-Ay mice” manufactured by Japan SLC Co., Ltd.) for 35 days. As a control group, guava leaf extract (manufactured by Bizen Kasei Co., Ltd.) was forcibly orally administered to another diabetic mouse for 35 days. These doses are 350 mg / kg body weight and the concentration is 0.3 mL / 30 g body weight. Mice were housed in plastic cages under conditions of room temperature 25 ± 1 ° C., humidity 55% ± 5%, light / dark cycle 12 hours, and habituated for 1 week before the start of the test.
空腹時を避けて、尾静脈から7日ごとに採血を行った。飼料(日本クレア社製「CE−2」)および水は、自由摂取とした。また、水投与群をコントロール群(対照群)とした。血中グルコース値の測定は、マウスの尾静脈血液を用いて、グルコース検出器およびグルコース試験紙で行った。データの統計解析は、一元配置分散分析および多重比較検定(Dunnett’s multiple test)により行い、有意水準は0.05とした。 Blood was collected every 7 days from the tail vein, avoiding fasting. The feed (“CE-2” manufactured by CLEA Japan) and water were freely consumed. The water administration group was used as a control group (control group). The blood glucose level was measured with a glucose detector and glucose test paper using mouse tail vein blood. Statistical analysis of data was performed by one-way analysis of variance and multiple comparison test (Dunnett's multiple test), with a significance level of 0.05.
図1に、強制投与終了後(35日間経過後)の、各群の血中グルコース値の時間変化を示す。各々の実験結果は、1群5〜8匹のマウスの平均値を示す。グラフ中の垂直線は、平均に対する標準誤差を示す。図1に示すように、ローヤルゼリータンパク質分解物投与群およびグアバ葉抽出物投与群(ポジティブコントロール)は、コントロール群に比べて、有意に血中グルコース濃度(血糖値)を抑制していることが確認された。 In FIG. 1, the time change of the blood glucose level of each group after completion | finish of forced administration (after 35-day progress) is shown. Each experimental result shows the average value of 5-8 mice per group. The vertical line in the graph indicates the standard error relative to the average. As shown in FIG. 1, it was confirmed that the royal jelly protein hydrolyzate administration group and the guava leaf extract administration group (positive control) significantly suppressed the blood glucose concentration (blood glucose level) compared to the control group. It was done.
[ローヤルゼリータンパク質分解物のKK−Ayの自由摂取実験]
実施例1で製造したアルコール変性したローヤルゼリータンパク質分解物の水溶液を、0.18w/w%(350mg/kg体重となるように調整)で粉末飼料(日本クレア社製「CE−2」)に混合し、糖尿病マウス(日本エスエルシー株式会社製「kk−Ayマウス」)に35日間自由摂取させた。また、対照群として、グアバ葉抽出物(備前化成社製)を、同じ割合で粉末飼料に混合し、別の糖尿病マウスに35日間自由摂取させた。また、コントロール群(対照群)として、何も混合しない粉末飼料を与えた。
[Experimental ingestion of royal jelly proteolysate KK-Ay]
The aqueous solution of the alcohol-denatured royal jelly proteolysate produced in Example 1 was mixed with a powdered feed (“CE-2” manufactured by CLEA Japan) at 0.18 w / w% (adjusted to 350 mg / kg body weight). Then, diabetic mice (“KK-Ay mice” manufactured by Nippon SLC Co., Ltd.) were allowed to freely ingest for 35 days. As a control group, guava leaf extract (manufactured by Bizen Kasei Co., Ltd.) was mixed with the powdered feed at the same ratio and allowed to freely take another diabetic mouse for 35 days. Moreover, the powder feed which does not mix anything was given as a control group (control group).
空腹時を避けて、尾静脈から7日ごとに採血を行った。血中インスリン値の測定は、マウスインスリンエライザキットで行った。その他の実験条件は、実施例3の強制経口投与実験と同じとした。 Blood was collected every 7 days from the tail vein, avoiding fasting. The blood insulin level was measured using a mouse insulin ELISA kit. Other experimental conditions were the same as those in the gavage oral administration experiment of Example 3.
図2に、自由摂取終了後(35日間経過後)の、各群の血中グルコース値の時間変化を示す。各々の実験結果は、1群6匹のマウスの平均値を示す。グラフ中の垂直線は、平均に対する標準誤差を示す。図2に示すように、ローヤルゼリータンパク質分解物投与群は、コントロール群に比べて有意に血中インスリン濃度を増強していることが確認された。 In FIG. 2, the time change of the blood glucose level of each group after completion | finish of free intake (after 35-day progress) is shown. Each experimental result shows the average value of 6 mice per group. The vertical line in the graph indicates the standard error relative to the average. As shown in FIG. 2, it was confirmed that the royal jelly protein hydrolyzate administration group significantly increased the blood insulin concentration compared to the control group.
図2に示すように、II型糖尿病マウスにローヤルゼリータンパク質分解物を自由摂取させたときに血中インスリン濃度が上昇したこと、および、図1に示すように、強制経口投与したときに血中グルコース濃度が抑制されたことを考え合わせると、ローヤルゼリータンパク質分解物は、血中インスリン濃度が低下した糖尿病患者への応用だけではなく、糖尿病を予防する食品素材としての応用が期待できるといえる。
As shown in FIG. 2, blood insulin concentration increased when a type II diabetic mouse was allowed to freely take a royal jelly protein degradation product, and as shown in FIG. Considering that the concentration was suppressed, it can be said that the royal jelly proteolysate can be applied not only to diabetic patients whose blood insulin concentration is lowered but also as a food material for preventing diabetes.
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