WO2006067856A1 - Functional food or pharmaceutical and process for producing the same - Google Patents

Functional food or pharmaceutical and process for producing the same Download PDF

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Publication number
WO2006067856A1
WO2006067856A1 PCT/JP2004/019367 JP2004019367W WO2006067856A1 WO 2006067856 A1 WO2006067856 A1 WO 2006067856A1 JP 2004019367 W JP2004019367 W JP 2004019367W WO 2006067856 A1 WO2006067856 A1 WO 2006067856A1
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WIPO (PCT)
Prior art keywords
phe lys
lys
action
thr phe
gly
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PCT/JP2004/019367
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French (fr)
Japanese (ja)
Inventor
Yoko Takenaka
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Yoko Takenaka
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Priority to PCT/JP2004/019367 priority Critical patent/WO2006067856A1/en
Publication of WO2006067856A1 publication Critical patent/WO2006067856A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L21/00—Marmalades, jams, jellies or the like; Products from apiculture; Preparation or treatment thereof
    • A23L21/20—Products from apiculture, e.g. royal jelly or pollen; Substitutes therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a functional food or medicament exhibiting at least one of an insulin-like action, an antihypertensive action and an anti-acidic action, and a method for producing the same.
  • Royal jelly (hereinafter abbreviated as RJ) is generally ingested as a nourishing tonic or as a food having functions such as growth promotion, aging prevention, and rejuvenation.
  • RJ Royal jelly
  • 10-hydroxydecenoic acid and the like have been known so far (Japanese Patent Laid-Open No. 967252).
  • an organic solvent extract of RJ has an anti-oxidative action (Japanese Patent Laid-Open No. 2002-173413), and in order to further improve the anti-oxidative action. It is also known to thermally decompose proteins in RJ (Japanese Patent Laid-Open No. 2001-112715).
  • An object of the present invention is to provide a functional food or a medicine that has at least one of insulin-like action, antihypertensive action and anti-acidic action and can be safely ingested. Another purpose of is at least one of insulin-like action, antihypertensive action and anti-acidic action It is an object of the present invention to provide a method for producing a functional food or a medicine capable of easily producing a functional food or a medicine containing an active ingredient having two actions.
  • a degradation product of royal jelly (RJ) protein including Thr Phe Lys ⁇ Leu Gly Lys ⁇ Gly Gly Cys Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys is included as an effective component.
  • RJ royal jelly
  • a functional food or medicament exhibiting at least one of insulin-like action, antihypertensive action and antioxidant action.
  • the step of enzymatically decomposing RJ protein under the conditions that a degradation product containing Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys is obtained.
  • a method for producing the functional food or medicine is provided.
  • Use of degradation products of RJ proteins including Ser Phe Lys and Asp Gly Val Thr Phe Lys is provided.
  • the functional food or medicament of the present invention contains a specific degradation product of RJ protein as an active ingredient, it has at least one of insulin-like action, antioxidant action and antihypertensive action, and can be safely ingested. can do.
  • the production method of the present invention includes a step of enzymatic degradation of RJ protein under specific conditions, a functional food or pharmaceutical having at least one of insulin-like action, antioxidant action and antihypertensive action can be easily produced. can do.
  • Fig. 1 is a graph showing the measurement results of antioxidative activity in Example 1-14.
  • FIG. 2 is a graph showing the measurement results of ACE inhibitory activity in Examples 1 to 4.
  • FIG. 3 is a graph showing the measurement results of lipolysis inhibiting ability in Example 5.
  • FIG. 4 is a graph showing the measurement results of fat synthesis promoting ability in Example 5.
  • FIG. 5 is a graph showing the results of separation of RJ protein degradation products by gel chromatography in Example 5.
  • FIG. 6 is a graph showing the results of separation by HPLC in the first stage in Example 5.
  • FIG. 7 is a graph showing the results of separation by HPLC in the second stage in Example 5.
  • the functional food or medicament of the present invention contains a specific degradation product of RJ protein as an active ingredient, and has an antihypertensive action and an anti-oxidative action expected by an insulin-like action, angiotensin converting enzyme (ACE) inhibitory activity. It exhibits at least one action, preferably two of these actions, particularly preferably all these actions.
  • ACE angiotensin converting enzyme
  • the insulin-like action can be expected to lower blood glucose levels, and the ACE inhibitory activity can be expected to develop a hypotensive action.
  • the antioxidative action can be expected to produce various effects based on the anti-oxidative properties both in the food or medicine and in the living body.
  • the specific degradation product of the RJ protein is Thr Phe Lys, Leu Gly Lys, Gly Gly Cys.
  • a degradation product containing Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys is preferred.
  • a degradation product as an active ingredient, all of the insulin-like action, antihypertensive action and anti-acidic action can be imparted to the functional food or medicament of the present invention.
  • the specific degradation product of the RJ protein is a Thr Phe obtained when the RJ protein is degraded so that at least one of the insulin-like action, antihypertensive action and anti-acidic action is obtained.
  • At least 1 fraction containing Lys peptide, fraction containing Leu Gly Lys peptide, fraction containing Gly Gly Cys Glu Lys peptide, fraction containing Ser Phe Lys peptide and fraction containing Asp Gly Val Thr Phe Lys peptide One fraction or a mixture of two or more of these fractions can be used.
  • the active ingredient is, if necessary, a supernatant produced when RJ protein is prepared from RJ in the production method of the present invention, which will be described later, or a component that works on the supernatant. Including, may be,.
  • the component of the RJ protein as the active ingredient is usually 0.01 to 100% by weight, based on the total amount of the functional food or medicine, and is usually 0.01 to 30%, especially when it is a functional food. Weight% is appropriate. Further, the content ratio of the supernatant or the components related to the supernatant is usually 30% by weight or less, or 0.01 to 30% by weight as dry matter based on the total amount of the functional food or medicine. .
  • the form of the functional food of the present invention is not particularly limited.
  • foods such as tablets, powders, granules, seasonings, edible oils, confectionery, breads, rice cakes, pasta, soup-like or It can be in the form of jelly-like food, soy milk such as soybean fermented soy milk, and beverages such as sports drinks.
  • soy milk such as soybean fermented soy milk
  • beverages such as sports drinks.
  • it can also be set as the form suitable for ingestion of animals other than humans, such as pet food, feed, and a feed.
  • the form of the medicament of the present invention is not particularly limited, and for example, it can be in the form of tablets, powders, granules, solutions, force capsules, syrups and the like.
  • the functional food or medicament of the present invention can be applied not only to humans but also to non-human animals such as mammals.
  • the functional food or medicament of the present invention can be ingested by oral administration, and the dose can be appropriately selected according to the age, sex, physique, form, etc. of the ingestor or patient.
  • a functional food or a medicine containing a fraction containing the above peptide can be ingested so that the total amount of the above peptide is usually 300-1500 mgZ 50 kg per day.
  • the RJ protein comprises a digest containing Thr Phe Lys, Leu Gly Lys ⁇ Gly Gly Cys Glu Lys ⁇ Ser Phe Lys, and Asp Gly Val Thr Phe Lys. A step of enzymatic degradation under the conditions obtained.
  • the enzyme decomposing step can be carried out by subjecting RJ in the form of raw, frozen, dried, etc. to the enzyme reaction as it is, preferably subjecting the protein obtained by separating the RJ force to the enzyme reaction. Can be performed.
  • the RJ may be a normal RJ produced by honeybee, and may be a known raw RJ or dry RJ.
  • the raw RJ include RJ having a water content of 63-68% by weight, crude protein 11-14.5% by weight, and 10-hydroxydecenoic acid 1.6% by weight or more.
  • Dry RJ Examples thereof include RJ having a water content of 5% by weight or less, a crude protein of 30-41% by weight, and 10-hydroxydecenoic acid of 3.85% by weight or more.
  • Examples of the method for separating the protein from the RJ catalyst include a method in which RJ is used as an aqueous solution, the protein therein is precipitated by ethanol precipitation or the like, and the precipitate is recovered.
  • RJ is dissolved in water at a ratio of 5 to 20% by weight to form an RJ aqueous solution, the pH is adjusted to 6.5-5. It can be carried out by a precipitation method.
  • the ethanol can be added at a ratio of 50-80% by weight as the amount of ethanol in the whole solution after addition.
  • Examples of the enzyme used for the enzymatic degradation include a protease derived from Aspergillus oryzae, a protease derived from Bacillus subtillis, and a Bacillus stearothermophilus derived from Bacillus stearothermophilus. Other proteases, and enzymes selected from the group consisting of these mixtures can also be used. As these enzymes, commercially available products can be used. For example, registered trademarks Proteaase 8, Proteaase 8, Protease S, Protease M, etc. commercially available from Amano Enzym Co., Ltd. can be used. These enzymes are particularly preferably enzymes capable of degrading avidin and Z or glycoprotein having a molecular weight of 5500.
  • Enzymatic degradation conditions for obtaining a degradation product containing the specific peptide can be appropriately selected according to the optimum pH and temperature when the enzyme reacts with the substrate.
  • the enzyme weight ratio is 100-500, and the reaction time is 1 to 20 hours.
  • the reaction temperature is, for example, a force that can be set to 50 to 60 ° C.
  • the optimal temperature is high, such as the above-mentioned Proteaase® and Protease S.
  • the reaction is performed at a higher temperature. Can be performed.
  • the degradation product obtained by the enzymatic degradation is subjected to a step of inactivating the enzyme and a step of filtering, if necessary, and the pH is adjusted as necessary, and further an excipient or the like is added.
  • the functional food or medicinal product of the present invention can be obtained by formulating it with an agent or blending it into various food materials.
  • the step of inactivating the enzyme can be performed by heating the suspension after completion of the enzyme reaction at a temperature of about 60-100 ° C. for about 5-20 minutes.
  • the filtering step This can be performed, for example, by filtering out molecules having a molecular weight of 2000 or less by ultrafiltration or the like.
  • the pH adjustment can be performed, for example, by adding a citrate aqueous solution.
  • a functional food can be produced by mixing the RJ aqueous solution protein after separating the protein or the component applied to the supernatant into the degradation product. .
  • both the effective component of the degradation product of RJ protein and the effective component in the supernatant such as the ethanol-soluble fraction are effectively used.
  • Functional food that can be ingested can be produced.
  • Raw RJ was dissolved in water at a rate of 10% by weight and the pH was adjusted between 6.5 and 7.5. To this, ethanol was added as appropriate until the protein in RJ precipitated at a rate in the range of a final concentration of 50-80 wt%. The supernatant and precipitated protein were then separated by centrifugation and filtration, and the supernatant was stored, while the precipitated protein was suspended in water at a rate of 10 wZv%.
  • protease A (derived from Aspergillus oryzae, optimum pH 6-8, optimum temperature 50 ° C) (Example 1)
  • protease N derived from Bacillus subtilis, optimum pH 6-8, optimum
  • Optimum temperature 55 ° C) (Example 2)
  • Protease S (derived from Bacillus stearothermophilus, Optimal pH 7-9, Optimal temperature 70 ° C) (Example 3) or Protease M (Aspergillus olisee) 1
  • optimal pH 3-6, optimal temperature 50 ° C) (Example 4) both manufactured by Amano Enzym Co., Ltd., registered trademark) were added at a ratio of 1Z100 by weight to the substrate, and each enzyme was added. It was carried out enzymatic reactions in optimal P H and optimum temperature.
  • the absorbance at 280 nm of each obtained fraction was measured.
  • the relative antioxidant activity, ACE inhibitory activity, and fat synthesis promoting ability of each fraction were measured in the same manner as described later, and a plurality of fractions of each peak were separated, separated by HPLC, and contained.
  • the sequence of the peptide was measured by the product name “Procise 494cLC protein sequencer” (manufactured by Biosystems of PERKIN ELMER).
  • any of the suspensions of Examples 1 to 4 contained Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys, and Asp Gly Val Thr Phe Lys peptides. understood.
  • Example 1 1 4 After the start of each enzyme reaction, 1, 2, 3, 4 or 20 hours later, an aqueous suspension of RJ proteolysate was used as a sample for measuring ACE inhibitory activity. (Measured according to Biochem. Pharm. 20, 1637 (1971).
  • Example 2 (1) In the same manner as in Example 2, an aqueous suspension containing a degradation product of RJ protein was obtained. However, the enzyme reaction time was 3 hours. The resulting suspension is lyophilized to give a dry product, which is further dissolved in KRB (Krebs-Ringer bicarbonate) buffer (pH 7.4) without bovine serum albumin to decompose various RJ proteins. A sample solution having a physical concentration was obtained.
  • KRB Kerat-Ringer bicarbonate
  • Adipocytes were prepared from rodent adipose tissue of Wister male rats by the method of Rodbell (Rodbell, M .: J. Biol. Chem. 239,375 (1964), containing 2.5% albumin.
  • adipocyte suspension 40 1 obtained in the same manner as 2 and 5% bovine serum albumin 3 mM 1 4 C-glucose containing (0. 25 Ci) Krebs- Ringer buffer (pH 7. 4) 160 1 and then ⁇ insulin 25 1 (final concentration InM), 37 ° C under 5% CO
  • sample solutions 251 having various RJ protein degradation product concentrations obtained in the same manner as in (1) above are collected and incubated for 1 hour at 37 ° C in 5% CO.
  • the reaction was stopped with 2.4 ml of alcohol, 0.6 ml of heptane and 0.25 ml of water. After centrifugation, the supernatant was taken, and the radiation dose (cpm) of tridalylide 14C was measured with a liquid scintillator. The results are shown in Fig. 4. As a control, radiation dose was measured in the same manner as above except that ⁇ insulin was used instead of the sample solution. The results are also shown in Fig. 4.
  • the suspension obtained in (1) was centrifuged to obtain a sample solution.
  • This sample solution was separated by gel chromatography according to the following conditions.
  • the No. 28-35 peak fraction shown in Fig. 5 was collected and separated by HPLC according to the following conditions.
  • Figure 6 shows the separation results.
  • the relative antioxidant activity of each fraction was measured in the same manner as described above.
  • the results are also shown in FIG.
  • Fractions containing peaks F1-F5 shown in FIG. 6 were fractionated, and further separated by HPLC under the same conditions as above except that the concentration gradient was changed appropriately as shown in FIG.
  • the relative antioxidant activity of each separated fraction was measured. The results are shown in FIG.
  • Peaks P1—P5 with high anti-acid activity against absorbance shown in FIG. 7 were fractionated, and the peptide sequences contained therein were identified by the product name “Procise 494cLC protein sequenced (PERKIN ELMER Biosystems The PI-P5 peaks were found to contain Thr Phe Lys, Leu uly Lys, uly Gly and ys ulu Lys, ber Phe Lys, or Asp Gly Val Thr Phe Lys, respectively.
  • the ACE inhibition rate was measured using the obtained peak P1—P5 as a sample in the same manner as in Example 1-14. The promotion ability was measured and the results are shown in Table 1. [table 1]
  • Example 2 In the same manner as in Example 2, the aqueous RJ solution was subjected to ethanol precipitation to separate the supernatant and the precipitated protein. The precipitated protein was subjected to an enzyme reaction for 3 hours to obtain an aqueous suspension of RJ protein degradation product. 6: 4 A mixture of the dried supernatant and the dried protein suspension in an equal amount (weight ratio) and an excipient such as lactose-starch-pullanka — Blended at a ratio of 8: 2 (weight ratio) and filled into capsules to obtain a functional food.
  • an excipient such as lactose-starch-pullanka — Blended at a ratio of 8: 2 (weight ratio) and filled into capsules to obtain a functional food.

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Abstract

A functional food or pharmaceutical that exhibits at least one activity selected from among insulin-like activity, hypotensive activity and antioxidant activity and can be safely uptaken; and a process for producing the same in a simple and easy manner. The functional food or pharmaceutical comprises as an active ingredient a decomposition product of royal jelly protein containing Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys. Further, there is provided a production process comprising the step of conducting zymolysis under such conditions that the above peptide-containing decomposition product can be obtained.

Description

機能性食品又は医薬及びその製造方法  Functional food or medicine and method for producing the same
技術分野  Technical field
[0001] 本発明は、インスリン様作用、降圧作用及び抗酸ィ匕作用の少なくとも 1つの作用を 示す機能性食品又は医薬及びその製造方法に関する。  [0001] The present invention relates to a functional food or medicament exhibiting at least one of an insulin-like action, an antihypertensive action and an anti-acidic action, and a method for producing the same.
背景技術  Background art
[0002] ローヤルゼリー (以下 RJと略す)は、一般に滋養強壮剤として、又は発育促進、老化 防止、若返り等の機能を有する食品として摂取されている。 RJ中に含まれる生理活 性物質としては、 10—ヒドロキシデセン酸等がこれまでに知られている (特開平 9 672 52号公報)。また、化粧品添加物の分野において、 RJの有機溶媒抽出物は抗酸ィ匕 作用を有することが知られており (特開 2002-173413号公報)、更に抗酸ィ匕作用を 向上させるために RJ中のタンパク質を熱分解することも知られて ヽる (特開 2001-11 2715号公報)。  [0002] Royal jelly (hereinafter abbreviated as RJ) is generally ingested as a nourishing tonic or as a food having functions such as growth promotion, aging prevention, and rejuvenation. As a physiologically active substance contained in RJ, 10-hydroxydecenoic acid and the like have been known so far (Japanese Patent Laid-Open No. 967252). Further, in the field of cosmetic additives, it is known that an organic solvent extract of RJ has an anti-oxidative action (Japanese Patent Laid-Open No. 2002-173413), and in order to further improve the anti-oxidative action. It is also known to thermally decompose proteins in RJ (Japanese Patent Laid-Open No. 2001-112715).
ところで、近年、抗酸化作用を有する物質が、生体内等において血圧降下、活性 酸素消去、過酸化物生成抑制、コレステロール上昇抑制及び脂肪代謝促進等の様 々な好ましい効果を発現することが明らかになってきている。このような作用を有する 物質は、医薬として、また食品に添加して摂取することが好ましいことも知られている 。また、生活習慣病の一つとして多く発生する高血圧を、有効に制御するニーズが近 年特に大きくなつている。更に、血糖値の低下を抑制、改善するための、生体内でィ ンスリン様作用を発揮する物質についてのニーズもある。そして、これらの効果が安 全に得られる医薬及び機能性食品が求められている。  By the way, in recent years, it has been clarified that substances having an antioxidative action exhibit various favorable effects such as lowering blood pressure, eliminating active oxygen, inhibiting peroxide formation, inhibiting cholesterol elevation and promoting fat metabolism in vivo. It has become to. It is also known that a substance having such an action is preferably taken as a medicine or added to food. In recent years, the need to effectively control high blood pressure, which frequently occurs as a lifestyle-related disease, has become particularly large. Furthermore, there is a need for a substance that exerts an insulin-like action in vivo in order to suppress and improve the decrease in blood glucose level. There is a need for pharmaceuticals and functional foods that can safely achieve these effects.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0003] 本発明の目的は、インスリン様作用、降圧作用及び抗酸ィ匕作用の少なくとも 1つの 作用を有し、安全に摂取することができる機能性食品又は医薬を提供することにある 本発明の別の目的は、インスリン様作用、降圧作用及び抗酸ィ匕作用の少なくとも 1 つの作用を有する有効成分を含む機能性食品又は医薬を簡便に製造することがで きる機能性食品又は医薬の製造方法を提供することにある。 [0003] An object of the present invention is to provide a functional food or a medicine that has at least one of insulin-like action, antihypertensive action and anti-acidic action and can be safely ingested. Another purpose of is at least one of insulin-like action, antihypertensive action and anti-acidic action It is an object of the present invention to provide a method for producing a functional food or a medicine capable of easily producing a functional food or a medicine containing an active ingredient having two actions.
課題を解決するための手段  Means for solving the problem
[0004] 本発明によれば、 Thr Phe Lysゝ Leu Gly Lysゝ Gly Gly Cys Glu Lys、 Ser Phe Lys及 び Asp Gly Val Thr Phe Lysを含むローヤルゼリー (RJ)タンパク質の分解物を有効成 分として含み、インスリン様作用、降圧作用及び抗酸化作用の少なくとも 1つの作用 を示す機能性食品又は医薬が提供される。 [0004] According to the present invention, a degradation product of royal jelly (RJ) protein including Thr Phe Lys ゝ Leu Gly Lys ゝ Gly Gly Cys Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys is included as an effective component. There is provided a functional food or medicament exhibiting at least one of insulin-like action, antihypertensive action and antioxidant action.
また、本発明によれば、 RJタンパク質を、 Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys及び Asp Gly Val Thr Phe Lysを含む分解物が得られる条件で 酵素分解する工程を含む、前記機能性食品又は医薬の製造方法が提供される。 更に、本発明によれば、インスリン様作用、降圧作用及び抗酸化作用の少なくとも 1 つの作用を示す機能性食品又は医薬を製造するための、 Thr Phe Lys、 Leu Gly Lys 、 Gly Gly Cys Glu Lys, Ser Phe Lys及び Asp Gly Val Thr Phe Lysを含む RJタンパク 質の分解物の使用が提供される。  Further, according to the present invention, the step of enzymatically decomposing RJ protein under the conditions that a degradation product containing Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys is obtained. A method for producing the functional food or medicine is provided. Furthermore, according to the present invention, Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, for producing a functional food or a medicine exhibiting at least one of insulin-like action, antihypertensive action and antioxidant action, Use of degradation products of RJ proteins including Ser Phe Lys and Asp Gly Val Thr Phe Lys is provided.
発明の効果  The invention's effect
[0005] 本発明の機能性食品又は医薬は、 RJタンパク質の特定の分解物を有効成分として 含むので、インスリン様作用、抗酸化作用及び降圧作用の少なくとも 1つの作用を有 し、しかも安全に摂取することができる。また、本発明の製造方法では、 RJタンパク質 を特定条件による酵素分解する工程を含むので、インスリン様作用、抗酸化作用及 び降圧作用の少なくとも 1つの作用を有する機能性食品又は医薬を簡便に製造する ことができる。  [0005] Since the functional food or medicament of the present invention contains a specific degradation product of RJ protein as an active ingredient, it has at least one of insulin-like action, antioxidant action and antihypertensive action, and can be safely ingested. can do. In addition, since the production method of the present invention includes a step of enzymatic degradation of RJ protein under specific conditions, a functional food or pharmaceutical having at least one of insulin-like action, antioxidant action and antihypertensive action can be easily produced. can do.
図面の簡単な説明  Brief Description of Drawings
[0006] [図 1]実施例 1一 4における抗酸ィ匕活性の測定結果を示すグラフである。 [0006] Fig. 1 is a graph showing the measurement results of antioxidative activity in Example 1-14.
[図 2]実施例 1一 4における ACE阻害活性の測定結果を示すグラフである。  FIG. 2 is a graph showing the measurement results of ACE inhibitory activity in Examples 1 to 4.
[図 3]実施例 5における脂肪分解阻害能の測定結果を示すグラフである。  FIG. 3 is a graph showing the measurement results of lipolysis inhibiting ability in Example 5.
[図 4]実施例 5における脂肪合成促進能の測定結果を示すグラフである。  FIG. 4 is a graph showing the measurement results of fat synthesis promoting ability in Example 5.
[図 5]実施例 5におけるゲルクロマトグラフィーによる RJタンパク質分解物の分離結果 を示すグラフである。 [図 6]実施例 5における第 1段階の HPLCによる分離結果を示すグラフである。 FIG. 5 is a graph showing the results of separation of RJ protein degradation products by gel chromatography in Example 5. FIG. 6 is a graph showing the results of separation by HPLC in the first stage in Example 5.
[図 7]実施例 5における第 2段階の HPLCによる分離結果を示すグラフである。  FIG. 7 is a graph showing the results of separation by HPLC in the second stage in Example 5.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0007] 以下、本発明を更に詳細に説明する。 [0007] Hereinafter, the present invention will be described in more detail.
本発明の機能性食品又は医薬は、 RJタンパク質の特定の分解物を有効成分として 含み、インスリン様作用、アンジォテンシン変換酵素 (ACE)阻害活性により期待され る降圧作用及び抗酸ィ匕作用の少なくとも 1つの作用、好ましくはこれらのうちの 2つの 作用、特に好ましくはこれら全ての作用を示す。  The functional food or medicament of the present invention contains a specific degradation product of RJ protein as an active ingredient, and has an antihypertensive action and an anti-oxidative action expected by an insulin-like action, angiotensin converting enzyme (ACE) inhibitory activity. It exhibits at least one action, preferably two of these actions, particularly preferably all these actions.
本発明の機能性食品又は医薬をヒト等が摂食した場合には、前記インスリン様作用 により、血糖値を低下させる効果が期待でき、前記 ACE阻害活性により、降圧作用 の発現が期待でき、また、前記抗酸化作用により、その食品又は医薬中、並びに生 体内の両方において、抗酸ィ匕に基づく種々の効能発現が期待できる。  When a human or the like ingests the functional food or medicine of the present invention, the insulin-like action can be expected to lower blood glucose levels, and the ACE inhibitory activity can be expected to develop a hypotensive action. The antioxidative action can be expected to produce various effects based on the anti-oxidative properties both in the food or medicine and in the living body.
[0008] 前記 RJタンパク質の特定の分解物は、 Thr Phe Lys, Leu Gly Lys, Gly Gly Cys[0008] The specific degradation product of the RJ protein is Thr Phe Lys, Leu Gly Lys, Gly Gly Cys.
Glu Lys, Ser Phe Lys及び Asp Gly Val Thr Phe Lysを含む分解物であることが好まし い。このような分解物を有効成分として含むことにより、前記インスリン様作用、降圧作 用及び抗酸ィ匕作用の全ての作用を本発明の機能性食品又は医薬に付与することが できる。 A degradation product containing Glu Lys, Ser Phe Lys and Asp Gly Val Thr Phe Lys is preferred. By including such a degradation product as an active ingredient, all of the insulin-like action, antihypertensive action and anti-acidic action can be imparted to the functional food or medicament of the present invention.
本発明においては、前記 RJタンパク質の特定の分解物を、前記インスリン様作用、 降圧作用及び抗酸ィ匕作用の少なくとも 1つが得られるように、 RJタンパク質を分解し た際に得られる、 Thr Phe Lysペプチドを含むフラクション、 Leu Gly Lysペプチドを含 むフラクション、 Gly Gly Cys Glu Lysペプチドを含むフラクション、 Ser Phe Lysぺプチ ドを含むフラクション及び Asp Gly Val Thr Phe Lysペプチドを含むフラクションの少な くとも 1つのフラクション又はこれら 2以上のフラクションを組合せた混合物とすることも 可能である。  In the present invention, the specific degradation product of the RJ protein is a Thr Phe obtained when the RJ protein is degraded so that at least one of the insulin-like action, antihypertensive action and anti-acidic action is obtained. At least 1 fraction containing Lys peptide, fraction containing Leu Gly Lys peptide, fraction containing Gly Gly Cys Glu Lys peptide, fraction containing Ser Phe Lys peptide and fraction containing Asp Gly Val Thr Phe Lys peptide One fraction or a mixture of two or more of these fractions can be used.
[0009] 本発明の機能性食品又は医薬においては、前記有効成分が、必要により後述する 本発明の製造方法において RJから RJタンパク質を調製した際に生成する上清又は 該上清に力かる成分を含んで 、ても良 、。  [0009] In the functional food or medicament of the present invention, the active ingredient is, if necessary, a supernatant produced when RJ protein is prepared from RJ in the production method of the present invention, which will be described later, or a component that works on the supernatant. Including, may be,.
[0010] 本発明の機能性食品又は医薬において、前記有効成分である RJタンパク質の分 解物の含有割合は、該分解物の乾物量として、機能性食品又は医薬の全量に対し て、通常 0. 01— 100重量％、特に機能性食品とする場合には通常 0. 01— 30重量 %が適当である。また、前記上清又は該上清にかかる成分の含有割合は、機能性食 品又は医薬の全量に対して、乾物量として、通常 30重量％以下、若しくは 0. 01— 3 0重量％である。 [0010] In the functional food or medicament of the present invention, the component of the RJ protein as the active ingredient The content of the product is usually 0.01 to 100% by weight, based on the total amount of the functional food or medicine, and is usually 0.01 to 30%, especially when it is a functional food. Weight% is appropriate. Further, the content ratio of the supernatant or the components related to the supernatant is usually 30% by weight or less, or 0.01 to 30% by weight as dry matter based on the total amount of the functional food or medicine. .
[0011] 本発明の機能性食品の形態は特に限定されず、例えば、錠剤、散剤、顆粒剤、調 味料、食用油、菓子類、パン類、麵、パスタ類等の食品、スープ状又はゼリー状の食 品、大豆発酵豆乳等の豆乳、スポーツ飲料等の飲料の形態とすることができる。また 、ペットフード、飼料、餌料等の、ヒト以外の動物の摂取に適した形態とすることもでき る。  [0011] The form of the functional food of the present invention is not particularly limited. For example, foods such as tablets, powders, granules, seasonings, edible oils, confectionery, breads, rice cakes, pasta, soup-like or It can be in the form of jelly-like food, soy milk such as soybean fermented soy milk, and beverages such as sports drinks. Moreover, it can also be set as the form suitable for ingestion of animals other than humans, such as pet food, feed, and a feed.
本発明の医薬の形態は、特に限定されず、例えば、錠剤、散剤、顆粒剤、液剤、力 プセル、シロップ等の形態とすることができる。  The form of the medicament of the present invention is not particularly limited, and for example, it can be in the form of tablets, powders, granules, solutions, force capsules, syrups and the like.
本発明の機能食品又は医薬は、ヒトのみならず、哺乳類等のヒト以外の動物をも投 与対象とすることができる。  The functional food or medicament of the present invention can be applied not only to humans but also to non-human animals such as mammals.
[0012] 本発明の機能性食品又は医薬は、経口投与により摂取することができ、該投与量 は、摂取者や患者の年齢、性別、体格、様態等に応じて適宜選択できる。例えば、前 記ペプチドの合計量として、通常 1日あたり 300— 1500mgZヒト 50kgとなるよう〖こ前 記ペプチドを含むフラクションを含有した機能性食品又は医薬を摂取することができ る。 [0012] The functional food or medicament of the present invention can be ingested by oral administration, and the dose can be appropriately selected according to the age, sex, physique, form, etc. of the ingestor or patient. For example, a functional food or a medicine containing a fraction containing the above peptide can be ingested so that the total amount of the above peptide is usually 300-1500 mgZ 50 kg per day.
[0013] 本発明の機能性食品又は医薬の製造方法は、 RJタンパク質を、 Thr Phe Lys、 Leu Gly Lysゝ Gly Gly Cys Glu Lysゝ Ser Phe Lys及び Asp Gly Val Thr Phe Lysを含む分 解物が得られる条件で酵素分解する工程を含む。  [0013] In the method for producing a functional food or medicament of the present invention, the RJ protein comprises a digest containing Thr Phe Lys, Leu Gly Lys ゝ Gly Gly Cys Glu Lys ゝ Ser Phe Lys, and Asp Gly Val Thr Phe Lys. A step of enzymatic degradation under the conditions obtained.
前記酵素分解する工程は、生、冷凍物、乾燥物等の形態の RJを、そのまま酵素反 応に供することにより行なうこともできる力 好ましくは RJ力も分離して得たタンパク質 を酵素反応に供することにより行なうことができる。  The enzyme decomposing step can be carried out by subjecting RJ in the form of raw, frozen, dried, etc. to the enzyme reaction as it is, preferably subjecting the protein obtained by separating the RJ force to the enzyme reaction. Can be performed.
[0014] 前記 RJは、セィヨウミツバチが産生する通常の RJが使用でき、公知の生 RJ又は乾 燥 RJが挙げられる。生 RJとしては、例えば、水分 63— 68重量％、粗タンパク質 11一 14. 5重量％、 10—ヒドロキシデセン酸 1. 6重量％以上の RJが挙げられる。乾燥 RJと しては、例えば、水分量 5重量％以下、粗タンパク質 30— 41重量％、 10—ヒドロキシ デセン酸 3. 85重量％以上の RJが挙げられる。 [0014] The RJ may be a normal RJ produced by honeybee, and may be a known raw RJ or dry RJ. Examples of the raw RJ include RJ having a water content of 63-68% by weight, crude protein 11-14.5% by weight, and 10-hydroxydecenoic acid 1.6% by weight or more. Dry RJ Examples thereof include RJ having a water content of 5% by weight or less, a crude protein of 30-41% by weight, and 10-hydroxydecenoic acid of 3.85% by weight or more.
[0015] 前記 RJカゝらタンパク質を分離する方法としては、例えば、 RJを水溶液とし、エタノー ル沈殿等によりその中のタンパク質を沈殿させ、該沈殿を回収する方法が挙げられる 。該エタノール沈殿は、例えば、 RJを水に 5— 20重量％の割合で溶解して RJ水溶液 とし、 pHを 6. 5-7. 5に調整し、これにエタノールを適宜添カ卩しタンパク質を沈殿さ せる方法により行なうことができる。エタノールの添加割合は、添加後の溶液全体に 対するエタノール量として、 50— 80重量％の割合で添加することができる。  [0015] Examples of the method for separating the protein from the RJ catalyst include a method in which RJ is used as an aqueous solution, the protein therein is precipitated by ethanol precipitation or the like, and the precipitate is recovered. In the ethanol precipitation, for example, RJ is dissolved in water at a ratio of 5 to 20% by weight to form an RJ aqueous solution, the pH is adjusted to 6.5-5. It can be carried out by a precipitation method. The ethanol can be added at a ratio of 50-80% by weight as the amount of ethanol in the whole solution after addition.
[0016] 前記酵素分解に用いる酵素としては、例えば、ァスペルギルス 'ォリゼ (Aspergillus oryzae)由来のプロテアーゼ、バチルス'ズブチリス (Bacillus subtillis)由来のプロテア ーゼ、バチルス'ステアロサーモフィラス (Bacillus stearothermophilus)由来のプロテア ーゼ、及びこれらの混合物力もなる群より選択される酵素を用いることができる。これ ら酵素としては、市販品が使用でき、例えば、天野ェンザィム株式会社等より市販さ れる登録商標プロテア一ゼ八、プロテア一ゼ?^、プロテアーゼ S又はプロテアーゼ M 等を用いることができる。また、これら酵素は、特に、アビシン及び Z又は分子量 550 00の糖タンパク質を分解しうる酵素であることが好ましい。  [0016] Examples of the enzyme used for the enzymatic degradation include a protease derived from Aspergillus oryzae, a protease derived from Bacillus subtillis, and a Bacillus stearothermophilus derived from Bacillus stearothermophilus. Other proteases, and enzymes selected from the group consisting of these mixtures can also be used. As these enzymes, commercially available products can be used. For example, registered trademarks Proteaase 8, Proteaase 8, Protease S, Protease M, etc. commercially available from Amano Enzym Co., Ltd. can be used. These enzymes are particularly preferably enzymes capable of degrading avidin and Z or glycoprotein having a molecular weight of 5500.
[0017] 前記特定のペプチドを含む分解物を得るための酵素分解の条件は、上記酵素が 基質と反応する際の至適 pH及び温度に応じて適宜選択でき、例えば、水溶液中で 、基質 Z酵素の重量比を 100— 500とし、反応時間 1一 20時間で行なうことができる 。反応温度は、例えば、 50— 60°Cとすることができる力 前記プロテア一ゼ?^、プロ テアーゼ Sのように至適温度の高 、酵素を用いる場合は、これよりも高 、温度で反応 を行なうことができる。  [0017] Enzymatic degradation conditions for obtaining a degradation product containing the specific peptide can be appropriately selected according to the optimum pH and temperature when the enzyme reacts with the substrate. The enzyme weight ratio is 100-500, and the reaction time is 1 to 20 hours. The reaction temperature is, for example, a force that can be set to 50 to 60 ° C. The optimal temperature is high, such as the above-mentioned Proteaase® and Protease S. When using an enzyme, the reaction is performed at a higher temperature. Can be performed.
[0018] 前記酵素分解により得られる分解物は、必要に応じて、酵素を失活させる工程及び ろ過する工程に供し、更に必要に応じて pHを調整したり、更には賦形剤等の添加剤 により製剤化するか、各種食品材料に配合する等して、本発明の機能性食品又は医 薬とすることができる。  [0018] The degradation product obtained by the enzymatic degradation is subjected to a step of inactivating the enzyme and a step of filtering, if necessary, and the pH is adjusted as necessary, and further an excipient or the like is added. The functional food or medicinal product of the present invention can be obtained by formulating it with an agent or blending it into various food materials.
前記酵素を失活させる工程は、酵素反応終了後の懸濁液を、 60— 100°C程度の 温度で 5— 20分間程度加熱することにより行うことができる。また、前記ろ過する工程 は、例えば、限外ろ過等により、分子量 2000以下の分子をろ取することにより行うこと ができる。前記 pH調節は、例えば、クェン酸水溶液を添加する等して行なうことがで きる。 The step of inactivating the enzyme can be performed by heating the suspension after completion of the enzyme reaction at a temperature of about 60-100 ° C. for about 5-20 minutes. Also, the filtering step This can be performed, for example, by filtering out molecules having a molecular weight of 2000 or less by ultrafiltration or the like. The pH adjustment can be performed, for example, by adding a citrate aqueous solution.
[0019] 本発明の製造方法では、前記 RJの水溶液力 タンパク質を分離した後の上清又は 該上清にかかる成分を、前記タンパク質を分解物に混合して機能性食品を製造する こともできる。このように、上清又は該上清に力かる成分を更に含むことにより、 RJタン パク質の分解物の有効成分と、エタノール可溶画分等の上清中の有効成分とを両方 有効に摂取しうる機能性食品を製造することができる。  [0019] In the production method of the present invention, a functional food can be produced by mixing the RJ aqueous solution protein after separating the protein or the component applied to the supernatant into the degradation product. . As described above, by further including the supernatant or a component that works on the supernatant, both the effective component of the degradation product of RJ protein and the effective component in the supernatant such as the ethanol-soluble fraction are effectively used. Functional food that can be ingested can be produced.
実施例  Example
[0020] 以下、本発明を、実施例を参照してより詳細に説明するが、本発明はこれらに限定 されない。  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
実施例 1一 4  Example 1-1 4
生 RJを水に 10重量％の割合で溶解し、 pHを 6. 5-7. 5の間に調整した。これに、 エタノールを、最終濃度が 50— 80重量％となる範囲の割合で、 RJ中のタンパク質が 沈殿するまで適宜添加した。次いで、遠心分離及びろ過により、上清と沈殿タンパク 質とを分離し、上清は保存し、一方沈殿タンパク質は水に 10wZv%の割合で懸濁さ せた。  Raw RJ was dissolved in water at a rate of 10% by weight and the pH was adjusted between 6.5 and 7.5. To this, ethanol was added as appropriate until the protein in RJ precipitated at a rate in the range of a final concentration of 50-80 wt%. The supernatant and precipitated protein were then separated by centrifugation and filtration, and the supernatant was stored, while the precipitated protein was suspended in water at a rate of 10 wZv%.
該懸濁液に 20%NaOH及び 30%NaCOを添カ卩して、下記各酵素の至適 pHに懸  Add 20% NaOH and 30% NaCO to the suspension to ensure optimum pH of each enzyme below.
3  Three
濁液 pHを調整した後、プロテアーゼ A (ァスペルギルス'オリゼー由来、至適 pH6— 8 ,至適温度 50°C)(実施例 1)、プロテアーゼ N (バチルス ·ズブチリス由来、至適 pH6— 8,至適温度 55°C)(実施例 2)、プロテアーゼ S (バチルス'ステアロサーモフィラス由来 、至適 pH7— 9,至適温度 70°C)(実施例 3)又はプロテアーゼ M (ァスペルギルス 'ォ リゼ一由来、至適 pH3— 6,至適温度 50°C)(実施例 4)(いずれも天野ェンザィム株式 会社製、登録商標)を、基質に対する重量比で 1Z100の割合で添加し、各酵素の至 適 PH及び至適温度で酵素反応を行なった。 After adjusting the pH of the suspension, protease A (derived from Aspergillus oryzae, optimum pH 6-8, optimum temperature 50 ° C) (Example 1), protease N (derived from Bacillus subtilis, optimum pH 6-8, optimum) (Optimum temperature 55 ° C) (Example 2), Protease S (derived from Bacillus stearothermophilus, Optimal pH 7-9, Optimal temperature 70 ° C) (Example 3) or Protease M (Aspergillus olisee) 1), optimal pH 3-6, optimal temperature 50 ° C) (Example 4) (both manufactured by Amano Enzym Co., Ltd., registered trademark) were added at a ratio of 1Z100 by weight to the substrate, and each enzyme was added. It was carried out enzymatic reactions in optimal P H and optimum temperature.
酵素反応開始後、 1、 2、 3、 4又は 20時間後に、 10%クェン酸を添加して pHを 5— 6に調整し、 80°Cで 10分間加熱して酵素を失活させ、遠心分離及び膜ろ過により不 純物及び不溶成分を除去し、 RJタンパク質の酵素分解物の水懸濁液を得た。 [0021] 得られた各懸濁液を遠心分離し、下記条件に従い、ゲルクロマトグラフィー法により 分離した。 1, 2, 3, 4 or 20 hours after the start of the enzyme reaction, add 10% citrate to adjust the pH to 5-6, heat at 80 ° C for 10 minutes to inactivate the enzyme, and centrifuge Impurities and insoluble components were removed by separation and membrane filtration to obtain an aqueous suspension of the enzymatic degradation product of RJ protein. [0021] The obtained suspensions were centrifuged and separated by gel chromatography according to the following conditions.
(分離条件）  (Separation conditions)
カラム： Sephadex G-25(アマシャム'バイオサイエンス社製)、 1. 6 X 75cm、溶出液： 5 OmM酢酸、流量： 20mlZ時間  Column: Sephadex G-25 (Amersham Biosciences), 1.6 X 75 cm, eluent: 5 OmM acetic acid, flow rate: 20 mlZ time
得られた各フラクションにおける 280nmにおける吸光度を測定した。また、各フラク シヨンの相対的な抗酸化活性、 ACE阻害活性及び脂肪合成促進能を、後述する方 法と同様に測定し、各ピークの複数のフラクションを分取し、 HPLCにより分離し、含 まれるペプチドの配列を、製品名「Procise 494cLC protein sequencer] (PERKIN ELMER社の Biosystems社製)により測定した。  The absorbance at 280 nm of each obtained fraction was measured. In addition, the relative antioxidant activity, ACE inhibitory activity, and fat synthesis promoting ability of each fraction were measured in the same manner as described later, and a plurality of fractions of each peak were separated, separated by HPLC, and contained. The sequence of the peptide was measured by the product name “Procise 494cLC protein sequencer” (manufactured by Biosystems of PERKIN ELMER).
その結果、実施例 1一 4のいずれの懸濁液においても、 Thr Phe Lys、 Leu Gly Lys 、 Gly Gly Cys Glu Lys、 Ser Phe Lys及び Asp Gly Val Thr Phe Lysのペプチドが含ま れていることが判った。  As a result, any of the suspensions of Examples 1 to 4 contained Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys, and Asp Gly Val Thr Phe Lys peptides. understood.
[0022] 次に、得られた各懸濁液につ!ヽて、抗酸化活性及び ACE阻害活性を、下記方法 に従い測定した。結果を図 1及び図 2に示す。  Next, each suspension obtained was measured for antioxidant activity and ACE inhibitory activity according to the following methods. The results are shown in Figs.
(ロダン鉄法による抗酸ィ匕活性の測定）  (Measurement of anti-acid activity by the rodan iron method)
リノール酸 1. 3mlにエタノール 100mlをカ卩え、リノール酸を完全に溶解した後、 50 mMリン酸緩衝液 (pH7. 0)100mlを加え、攪拌し、これをリノール酸溶液とした。この 溶液 20mlを 50ml容褐色サンプル瓶に採り、実施例 1一 4それぞれの酵素反応開始 後、 1、 2、 3、 4又は 20時間後の RJタンパク質分解物の水懸濁液 200 1及び蒸留水 4. 8mlをカロえ、全量を 25mlとして抗酸化試験に供する試料液とした。  100 ml of ethanol was added to 1.3 ml of linoleic acid to completely dissolve linoleic acid, and then 100 ml of 50 mM phosphate buffer (pH 7.0) was added and stirred to obtain a linoleic acid solution. 20 ml of this solution is put into a 50 ml brown sample bottle, and each of Examples 1 and 4 after the start of each enzyme reaction, 1, 2, 3, 4 or 20 hours later, RJ proteolysate water suspension 200 1 and distilled water 4. Calored 8 ml and made a total of 25 ml to prepare the sample solution for the antioxidant test.
次に、 20ml容試験管に 75%エタノールを 4. 7ml、試料液 0. 1ml及び 30%NH S  Next, in a 20 ml test tube, 4.7 ml of 75% ethanol, 0.1 ml of sample solution and 30% NH 3 S
4 Four
CN溶液 0. 1mlを採り攪拌した。これに 0. 02M FeCl溶液 (0. 02M FeCl /3. 5 0.1 ml of CN solution was taken and stirred. Add 0.02M FeCl solution (0.02M FeCl /3.5
2 2 twenty two
%HC1)0. 1mlを添加し、再度攪拌した。その後、 30分間放置し、 500nmで吸光度 を計測することにより、リノール酸の酸化度を評価し、抗酸ィ匕活性を測定した。 % HC1) 0.1 ml was added and stirred again. Thereafter, the mixture was allowed to stand for 30 minutes, and the absorbance at 500 nm was measured to evaluate the degree of oxidation of linoleic acid, and the antioxidant activity was measured.
[0023] (ACE阻害活性の測定） [0023] (Measurement of ACE inhibitory activity)
実施例 1一 4それぞれの酵素反応開始後、 1、 2、 3、 4又は 20時間後の RJタンパク 質分解物の水懸濁液を ACE阻害活性測定の試料として用い、 H.S.Cheungらの方法 (Biochem.Pharm. 20, 1637(1971》に従って測定した。 Example 1 1 4 After the start of each enzyme reaction, 1, 2, 3, 4 or 20 hours later, an aqueous suspension of RJ proteolysate was used as a sample for measuring ACE inhibitory activity. (Measured according to Biochem. Pharm. 20, 1637 (1971).
[0024] 実施例 5 [0024] Example 5
(脂肪分解阻害能の測定）  (Measurement of lipolysis inhibition ability)
(1)実施例 2と同様に、 RJタンパク質の分解物を含む水懸濁液を得た。但し、酵素反 応時間を 3時間とした。得られた懸濁液を凍結乾燥処理にて乾燥物とし、更に牛血 清アルブミンを含まな 、KRB(Krebs- Ringer bicarbonate)緩衝液 (pH 7. 4)に溶解し、 様々な RJタンパク質の分解物濃度を有する検体溶液を得た。  (1) In the same manner as in Example 2, an aqueous suspension containing a degradation product of RJ protein was obtained. However, the enzyme reaction time was 3 hours. The resulting suspension is lyophilized to give a dry product, which is further dissolved in KRB (Krebs-Ringer bicarbonate) buffer (pH 7.4) without bovine serum albumin to decompose various RJ proteins. A sample solution having a physical concentration was obtained.
(2) Wister系雄性ラットの副睾丸脂肪組織から、 Rodbellの方法 (Rodbell, M.:J.Biol. Chem.239,375(1964》で脂肪細胞を調製し、 2. 5%アルブミンを含む  (2) Adipocytes were prepared from rodent adipose tissue of Wister male rats by the method of Rodbell (Rodbell, M .: J. Biol. Chem. 239,375 (1964), containing 2.5% albumin.
KRB(Krebs-Ringer bicarbonate)緩衝液 (pH 7. 4)中に 106個/ mlの細胞が含まれる 脂肪細胞懸濁液を得た。 KRB (Krebs-Ringer bicarbonate) buffer (pH 7. 4) to give the adipocyte suspension containing the 10 6 cells / ml in.
(3)牛血清アルブミン 2. 5%を含む KRB(Krebs- Ringer bicarbonate)緩衝液 (pH 7. 4) 200 μ 1に、上記 (1)で得た検体溶液 25 μ 1、ェピネフリン 5 μ g/ml溶液 25 μ 1(最終 濃度 0. 42 gZml)及び上記 (2)で得た脂肪細胞懸濁液 50 1をカ卩え、 5%CO下で  (3) KRB (Krebs-Ringer bicarbonate) buffer solution (pH 7.4) containing 2.5% bovine serum albumin (pH 7.4) 200 μ1, the sample solution 25 μ1 obtained in (1) above, epinephrine 5 μg / Prepare 25 μ1 of the ml solution (final concentration 0.42 gZml) and 50 1 of the adipocyte suspension obtained in (2) above, under 5% CO.
2 2
37°Cで 1時間インキュベートし、遊離する脂肪酸を、 NEFA-テスト (商品名、和光純薬 社製)を用いて Duncombeの変法 (J.Boberg, L.A. Carlson,: Clin. chim.Acta 10, 420(1964))により定量した。一方、検体溶液の代わりに水を加えた他は上記と同様に 操作して対照とした。各検体について測定した遊離脂肪酸量の、対照の遊離脂肪酸 量に対する割合を、脂肪分解率として求めた。結果を図 3に示す。 Incubate at 37 ° C for 1 hour, and release the fatty acids using the NEFA-test (trade name, manufactured by Wako Pure Chemical Industries, Ltd.), a modified Duncombe method (J. Boberg, LA Carlson ,: Clin. Chim. Acta 10, 420 (1964)). On the other hand, except that water was added instead of the sample solution, the same operation as above was performed as a control. The ratio of the amount of free fatty acid measured for each specimen to the amount of free fatty acid in the control was determined as the lipolysis rate. The results are shown in Figure 3.
[0025] (脂肪合成促進能の測定） [0025] (Measurement of ability to promote fat synthesis)
上記 (2)と同様にして得た脂肪細胞懸濁液 40 1を 2. 5%牛血清アルブミンと 3mM 14C-グルコース (0. 25 Ci)を含む Krebs- Ringer緩衝液 (pH7. 4)160 1にカロえ、更 に ΙΟηΜインスリン 25 1 (最終濃度 InM)をカ卩え、 5%CO下で 37°C (2) and the adipocyte suspension 40 1 obtained in the same manner as 2 and 5% bovine serum albumin 3 mM 1 4 C-glucose containing (0. 25 Ci) Krebs- Ringer buffer (pH 7. 4) 160 1 and then ΜηΜ insulin 25 1 (final concentration InM), 37 ° C under 5% CO
2 、 20分間インキ ュペートした。次に、上記の (1)と同様にして得た様々な RJタンパク質の分解物濃度を 有する検体溶液 25 1をカ卩え、 5%CO下 37°Cで 1時間インキュベートし、イソプロピ  2. Ink for 20 minutes. Next, sample solutions 251 having various RJ protein degradation product concentrations obtained in the same manner as in (1) above are collected and incubated for 1 hour at 37 ° C in 5% CO.
2  2
ルアルコール 2. 4ml、ヘプタン 0. 6ml及び水 0. 25mlをカ卩ぇ反応を停止させた。遠 心分離後上清を取り、液体シンチレ一ターでトリダリセライド 14 Cによる放射線量 (cpm)を測定した。結果を図 4に示す。 また、対照として、検体溶液の代わりに ΙΟηΜインスリンを用いた他は上記と同様に 操作し、放射線量を測定した。結果を併せて図 4に示す。 The reaction was stopped with 2.4 ml of alcohol, 0.6 ml of heptane and 0.25 ml of water. After centrifugation, the supernatant was taken, and the radiation dose (cpm) of tridalylide 14C was measured with a liquid scintillator. The results are shown in Fig. 4. As a control, radiation dose was measured in the same manner as above except that ΙΟηΜ insulin was used instead of the sample solution. The results are also shown in Fig. 4.
[0026] 上記 (1)で得られた懸濁液を遠心分離し、試料溶液を得た。この試料溶液を下記の 条件に従い、ゲルクロマトグラフィー法により分離した。 [0026] The suspension obtained in (1) was centrifuged to obtain a sample solution. This sample solution was separated by gel chromatography according to the following conditions.
(分離条件）  (Separation conditions)
カラム： Sephadex G-25(アマシャム'バイオサイエンス社製)、 1. 6 X 75cm、溶出液： 5 OmM酢酸、流量： 20mlZ時間  Column: Sephadex G-25 (Amersham Biosciences), 1.6 X 75 cm, eluent: 5 OmM acetic acid, flow rate: 20 mlZ time
各フラクションにおける 280nmにおける吸光度を測定した。また、各フラクションの 相対的な抗酸化活性、 ACE阻害活性及び脂肪合成促進能を、上記と同様に測定し た。結果を図 5に示す。  The absorbance at 280 nm in each fraction was measured. In addition, the relative antioxidant activity, ACE inhibitory activity and fat synthesis promoting ability of each fraction were measured in the same manner as described above. The results are shown in FIG.
図 5中に示される No. 28— 35のピークのフラクションを分取し、下記条件に従い H PLCにより分離した。  The No. 28-35 peak fraction shown in Fig. 5 was collected and separated by HPLC according to the following conditions.
[0027] (分離条件） [0027] (Separation conditions)
カラム： TSK- gel ODS- 120T (東ソ一社製)、カラム温度： 35°C、溶出液： 0. 1%TFA ァセトニトリル 0%(0分)から 50%(60分)の直線濃度勾配、流量： 1. OmlZ分、検出： 230nmにおける吸光度  Column: TSK-gel ODS-120T (manufactured by Tosohichi Co., Ltd.), column temperature: 35 ° C, eluent: 0.1% TFA acetonitrile 0% (0 min) to 50% (60 min) linear concentration gradient, Flow rate: 1. OmlZ min, detection: absorbance at 230nm
分離結果を図 6に示す。また、各フラクションの相対的な抗酸ィ匕活性を、上記と同様 に測定した。結果を図 6に併せて示す。図 6中に示すピーク F1— F5を含むフラクショ ンをそれぞれ分取し、さらに、濃度勾配を図 7中に示するように適宜変更した以外は 上記と同一の条件の HPLCで分離した。分離した各フラクションの相対抗酸ィ匕活性 を測定した。結果を図 7に示す。  Figure 6 shows the separation results. In addition, the relative antioxidant activity of each fraction was measured in the same manner as described above. The results are also shown in FIG. Fractions containing peaks F1-F5 shown in FIG. 6 were fractionated, and further separated by HPLC under the same conditions as above except that the concentration gradient was changed appropriately as shown in FIG. The relative antioxidant activity of each separated fraction was measured. The results are shown in FIG.
[0028] 図 7に示す、吸光度に対して抗酸ィ匕活性が高力つたピーク P1— P5を分取し、含ま れるペプチドの配列を、製品名「Procise 494cLC protein sequenced (PERKIN ELMER社の Biosystems社製)により決定したところ、 PI— P5のピークにはそれぞれ、 Thr Phe Lys、 Leu uly Lys、 uly Glyし ys ulu Lys、 ber Phe Lys又は Asp Gly Val Thr Phe Lysを含んでいることが判った。また、得られたピーク P1— P5を試料として、実施 例 1一 4と同様に ACE阻害率を測定した。更に、ピーク P1— P5を検体溶液として、 上記と同様に ACE阻害率及び脂肪合成促進能を測定した。結果を表 1に示す。 [表 1]
Figure imgf000012_0001
[0028] Peaks P1—P5 with high anti-acid activity against absorbance shown in FIG. 7 were fractionated, and the peptide sequences contained therein were identified by the product name “Procise 494cLC protein sequenced (PERKIN ELMER Biosystems The PI-P5 peaks were found to contain Thr Phe Lys, Leu uly Lys, uly Gly and ys ulu Lys, ber Phe Lys, or Asp Gly Val Thr Phe Lys, respectively. In addition, the ACE inhibition rate was measured using the obtained peak P1—P5 as a sample in the same manner as in Example 1-14. The promotion ability was measured and the results are shown in Table 1. [table 1]
Figure imgf000012_0001
処方例 1  Formulation example 1
実施例 2と同様に、 RJ水溶液をエタノール沈殿に供して上清と沈殿タンパク質とに 分けた。沈殿タンパク質を 3時間の酵素反応に供し、 RJタンパク質の分解物水懸濁 液を得た。上記上清を乾燥させたもの及びタンパク質分解物水懸濁液を乾燥させた ものを等量 (重量比)で混合した混合物と、乳糖-デンプン-プルランカゝらなる賦形剤と を 6:4— 8:2(重量比)の割合で配合し、カプセルに充填し、機能性食品を得た。  In the same manner as in Example 2, the aqueous RJ solution was subjected to ethanol precipitation to separate the supernatant and the precipitated protein. The precipitated protein was subjected to an enzyme reaction for 3 hours to obtain an aqueous suspension of RJ protein degradation product. 6: 4 A mixture of the dried supernatant and the dried protein suspension in an equal amount (weight ratio) and an excipient such as lactose-starch-pullanka — Blended at a ratio of 8: 2 (weight ratio) and filled into capsules to obtain a functional food.

Claims

請求の範囲 The scope of the claims
[1] Thr Phe Lys、 Leu Gly Lys、 Gly Gly Cys Glu Lys、 Ser Phe Lys及び Asp Gly Val [1] Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys and Asp Gly Val
Thr Phe Lysを含むローヤルゼリータンパク質分解物を有効成分として含み、インスリ ン様作用、アンジォテンシン変換酵素阻害作用及び抗酸ィ匕作用の少なくとも 1つの 作用を示す機能性食品又は医薬。 A functional food or medicine comprising a royal jelly protein degradation product containing Thr Phe Lys as an active ingredient and exhibiting at least one of insulin-like action, angiotensin converting enzyme inhibitory action and anti-acidic action.
[2] ローヤルゼリー水溶液力もタンパク質を除 、た上清又は該上清成分を更に含む請 求項 1の機能性食品又は医薬。  [2] The functional food or medicament according to claim 1, further comprising a supernatant or a component of the supernatant after removing protein from the aqueous solution of royal jelly.
[3] ローヤルゼリータンパク質を、 Thr Phe Lysゝ Leu Gly Lys, Gly Gly Cys Glu Lys, Ser  [3] Royal jelly protein, Thr Phe Lys Ly Leu Gly Lys, Gly Gly Cys Glu Lys, Ser
Phe Lys及び Asp Gly Val Thr Phe Lysを含む分解物が得られる条件で、酵素分解す る工程を含む請求項 1の機能性食品又は医薬の製造方法。  2. The method for producing a functional food or medicine according to claim 1, comprising a step of enzymatic degradation under the condition that a degradation product containing Phe Lys and Asp Gly Val Thr Phe Lys is obtained.
[4] 前記酵素分解する工程に用いる酵素力 ァスペルギルス 'ォリゼ (Aspergillus  [4] Enzymatic power used in the enzymatic decomposition step Aspergillus
oryzae)由来のプロテアーゼ、バチルス'ズブチリス (Bacillus subtillis)由来のプロテア ーゼ、バチルス'ステアロサーモフィラス (Bacillus stearothermophilus)由来のプロテア ーゼ、及びこれらの混合物からなる群より選択される請求項 3の製造方法。  oryzae), a protease derived from Bacillus subtillis, a protease derived from Bacillus stearothermophilus, and a mixture thereof. Manufacturing method.
[5] 前記酵素が、アビシンを分解しうる酵素である請求項 4の製造方法。  [5] The production method of claim 4, wherein the enzyme is an enzyme capable of degrading avidin.
[6] インスリン様作用、降圧作用及び抗酸化作用の少なくとも 1つの作用を示す機能性 食品又は医薬を製造するための、 Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys及び Asp Gly Val Thr Phe Lysを含むロイヤルゼリータンパク質の分解物 の使用。  [6] Thr Phe Lys, Leu Gly Lys, Gly Gly Cys Glu Lys, Ser Phe Lys, and Asp for the production of a food or medicine having at least one of insulin-like action, antihypertensive action and antioxidant action Use of royal jelly protein degradation products including Gly Val Thr Phe Lys.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009274960A (en) * 2008-05-12 2009-11-26 Api Co Ltd Royal jelly peptide, method for producing the same, inhibitor for blood sugar elevation and antioxidant

Citations (2)

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Publication number Priority date Publication date Assignee Title
JPH03151838A (en) * 1989-11-09 1991-06-28 Gifu Youhou Kk Production of solubilized royal jelly and royal jelly drink
JP2003334003A (en) * 2002-05-15 2003-11-25 Nippon Yoho Kk Method for producing transparent royal jelly solution

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Publication number Priority date Publication date Assignee Title
JPH03151838A (en) * 1989-11-09 1991-06-28 Gifu Youhou Kk Production of solubilized royal jelly and royal jelly drink
JP2003334003A (en) * 2002-05-15 2003-11-25 Nippon Yoho Kk Method for producing transparent royal jelly solution

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Title
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Publication number Priority date Publication date Assignee Title
JP2009274960A (en) * 2008-05-12 2009-11-26 Api Co Ltd Royal jelly peptide, method for producing the same, inhibitor for blood sugar elevation and antioxidant

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