WO2006132273A1 - Composition comprising peptide having suppressive effect on food intake - Google Patents

Composition comprising peptide having suppressive effect on food intake Download PDF

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Publication number
WO2006132273A1
WO2006132273A1 PCT/JP2006/311426 JP2006311426W WO2006132273A1 WO 2006132273 A1 WO2006132273 A1 WO 2006132273A1 JP 2006311426 W JP2006311426 W JP 2006311426W WO 2006132273 A1 WO2006132273 A1 WO 2006132273A1
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WIPO (PCT)
Prior art keywords
peptide
conglycinin
composition
activity
enzyme
Prior art date
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PCT/JP2006/311426
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French (fr)
Japanese (ja)
Inventor
Toru Hira
Motohiko Hirotsuka
Toshihiro Nakamori
Hiroshi Hara
Kozo Asano
Hitoshi Chiba
Fusao Tomita
Original Assignee
A-Hitbio Inc.
National University Corporation Hokkaido University
Fuji Oil Co., Ltd.
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Publication date
Application filed by A-Hitbio Inc., National University Corporation Hokkaido University, Fuji Oil Co., Ltd. filed Critical A-Hitbio Inc.
Priority to JP2007520140A priority Critical patent/JPWO2006132273A1/en
Publication of WO2006132273A1 publication Critical patent/WO2006132273A1/en

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • 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
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
    • 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
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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 peptide that suppresses food intake and reduces hunger when taken orally, and various compositions containing the peptide.
  • the amphetamine class of mazindol has been put to practical use as a drug that suppresses appetite for the treatment of obesity.
  • due to the direct effects on the center and the habitual nature associated with this drug its use is limited only to highly obese patients, and a safer method is desired.
  • Non-Patent Documents 1 to 4 It is known that various hormones (gastrointestinal hormones) secreted from endocrine cells of the gastrointestinal tract have a function of suppressing appetite. Since these peptide hormones are easily degraded in the digestive tract lumen when administered orally, it is difficult to absorb them and exert their effects, and they must be administered in blood. Yes. In that case, there are high costs involved in peptide synthesis, complicated administration methods, and problems.
  • Cholecystokinin (hereinafter sometimes abbreviated as “CCK”) has been found to have an effect of suppressing appetite ahead of other gonadotropins (GLP-1, PYY) and the like. It is a gastrointestinal hormone and is caused by dietary lipids, proteins, amino acids, etc. Secretion is stimulated (Non-patent Document 2). However, in order to limit calorie intake, energy efficiency is high!
  • Patent Document 1 JP 2004-10569 A
  • Non-Patent Document 1 Woods SC, Am J Physiol Gastrointest Liver Physiol, 286 (1): G7-13, 2004
  • Non-Patent Document 2 Moran TH, et al, Am J Physiol Gastrointest Liver Physiol, 286 (2): G 183-8, 2004
  • Non-Patent Document 3 Moran TH, et al, Am J Physiol Gastrointest Liver Physiol, 286 (5): G 693-7, 2004
  • Non-Patent Document 4 Tso P, et al, Am J Physiol Gastrointest Liver Physiol, 286 (6): G885—90, 2004
  • Non-Patent Document 5 Nishi T, et al, J Nutr, 133 (2): 352-7, 2003
  • Non-Patent Document 6 Nishi T, et al, J Nutr, 133 (8): 2537-42, 2003
  • the present invention is a peptide having an anti-feeding action that can be orally administered or ingested and can also be applied to humans, in particular, a peptide that is low-cost and practical, less bitter and safe and easy to ingest. And an antifeedant, food, pharmaceutical composition, feed and the like containing the same.
  • the present inventor prepares a peptide composition by degrading soybean ⁇ -conglycinin, which is a food-derived protein, with various food-adding enzymes. By selecting a peptide composition with high CCK release activity, and further reducing the feeling of hunger and satiety due to actual ingestion in humans. By observing the triggering, the present invention was completed.
  • the present invention is a.
  • a peptide obtained by degrading ⁇ -conglycinin with an enzyme having an enzyme activity equivalent to that of bromelain or promelain, and contains a peptide having a cholecystocone secretion promoting activity or an antifeedant activity A composition to make;
  • composition according to (1) or (2), wherein the enzyme is bromelain
  • a cholecystokinin secretion promoter comprising the composition according to any one of (1) to (3) above;
  • a pharmaceutical composition comprising the antifeedant or cholecystokinin secretion promoter according to (4) or (5) above;
  • a peptide having cholecystokinin secretion-promoting activity or feeding-suppressing activity comprising a step of decomposing ⁇ -conglycinin by an enzyme that preferentially cleaves a peptide bond on the carboxyl group side of an arginine residue.
  • composition of the present invention an antifeedant, a cholecystocun secretion promoter, and a pharmaceutical composition, food, and feed containing these (hereinafter referred to as "the composition of the present invention") )
  • the composition of the present invention has the activity of stimulating the secretion of cholecystokinin, which has an appetite-suppressing effect, so that hunger can be reduced by taking it orally.
  • the composition of the present invention is obtained by treating a safe food material with an enzyme for food processing, so that the safety is high and the cost is low.
  • composition of the present invention can be taken not only in a solid form such as a capsule but also in a beverage or the like.
  • the effect of the composition of the present invention is exhibited about 15 minutes after ingestion, the user feels full before taking the peptide composition of the present invention about 15 minutes before eating. This can prevent excessive caloric intake thereafter.
  • a synergistic effect with the feeling of fullness caused by the actual meal can be expected.
  • FIG. 1 is a graph showing the amount of CCK released from gastrointestinal endocrine cell line STC-1 by various ⁇ -conglycinin degradation products. The value indicates the concentration of CCK released into the culture supernatant during 60 minutes.
  • FIG. 2 is a graph showing food intake from 30 minutes to 90 minutes after administration of a j8-conglycinin degradation product solution into the rat stomach.
  • the values are the average of 12 animals and the standard error. * Indicates that there is a significant difference (P ⁇ 0. 05) compared to the water-administered group.
  • FIG. 3 shows the total of 2 days after administration of
  • the numbers represent the mean (24-30) change from the pre-tasting value for each subject scored by the VAS method and the standard error.
  • ⁇ Hainu Mouth 3g (25 people) Mouth: High-Ute 1.5 g + ⁇ —Conglycine bromelain degradation product 1.5 g (24 people)
  • FIG. 5 is a graph showing changes in satiety after ingestion of ⁇ -conglycinin bromelain degradation product or soybean peptide solution.
  • the numerical value represents the average value (24-30 people) and standard error of changes in value before each tasting of each subject scored by the VAS method.
  • ⁇ High Newt 3g (25 people) Mouth: High-Ute 1.5g + ⁇ —Conglycine Bromelain Degradation Product 1.5g (24 people)
  • Plots with different alphabets indicate that they are significantly different from each other at the same time.
  • FIG. 6 is a graph showing changes in the allowable dietary intake after ingestion of ⁇ -conglycinin bromelain degradation product or soybean peptide solution.
  • the numbers represent the mean (24-30) change from the pre-tasting value for each subject scored by the VAS method and the standard error.
  • ⁇ High-yuteu 3g (25 people) Mouth: High-yute 1.5g + j8—Conglycine bromelain degradation product 1.5g (24 people)
  • Plots with different alphabets indicate that there is a significant difference from each other at the same time.
  • FIG. 7 is a diagram showing the CCK releasing activity of the active peptide fraction in ⁇ -conglycinin degradation product. Indicates that there is a significant difference between groups with different alphabets ( ⁇ ⁇ 0. 05).
  • the peptide of the present invention is obtained by degrading j8-conglycinin.
  • j8 Conglycinin is a major component of soy protein.
  • ⁇ -conglycinin except for soy allergies, is usually already eaten and has been confirmed to be safe by ingesting 5 grams a day for 3 months in humans. (Kanbara No. 2 ⁇ 16 ⁇ 17, Kanbara et al.) Has been confirmed to be safe.
  • ⁇ -conglycinin used in the present invention may be derived from soybeans or may be derived from natural raw materials such as other beans. It may be a thing. From the viewpoint of cost and the like, j8-conglycinin derived from soybean is preferable.
  • the enzyme used for the degradation of ⁇ -conglycinin is not particularly limited as long as it is an enzyme that preferentially cleaves the peptide bond on the carboxyl group side of the arginine residue in the polypeptide, such as bromelain.
  • an enzyme that preferentially cleaves the peptide bond on the carboxyl group side of the arginine residue in the polypeptide such as bromelain.
  • a peptide composition having little or no bitterness due to the presence of an aromatic amino acid at the end of the peptide can be obtained.
  • enzymes that preferentially cleave the peptide bond on the carboxyl side of the arginine residue in the polypeptide include thrombin, endoproteinase Arg-C, and cathebsin B1. From the viewpoints of safety, availability, cost, etc., bromelain is preferable.
  • Bromelain is a cystine protease derived from a plant belonging to the pineapple family (Bromeliaceae), and is mainly contained in pineapple rhizomes and fruits.
  • stem bromelain EC.3.4.22.4, EC.3.4.22.32
  • funolate bromelain EC.3.4.22.5, EC.3.4.22.33
  • bromelain derived from pineapple stem EC.3.4.22.4
  • Bromelain is an enzyme that has wide substrate recognition in proteolysis, but has high resolution against synthetic substrates including arginine. It is used for meat processing for food processing, and for inflammation for medical use. It is also used for improvement and has many advantages such as being easy to obtain and highly safe.
  • the degradation of ⁇ -conglycinin can be performed, for example, as follows.
  • ⁇ -Conglycinin is suspended in ion-exchanged water at a weight concentration of 10%, and ⁇ is adjusted to 6.0 with sodium hydroxide aqueous solution. Add bromelain to the substrate concentration of 0.1%, incubate at 50 ° C for 1 hour, and raise to 100 ° C to stop the enzyme reaction. The insoluble fraction is removed by centrifugation and filtration, and the resulting peptide solution is lyophilized to obtain a ⁇ -conglycinin bromelain degradation product. Any concentration of ⁇ -conglycinin can be used as long as it can be suspended (maximum 10%).
  • the concentration with respect to the substrate is 0.01% to 10%, 0.05 to 5% is preferred, and 0.05 to 1% is particularly preferred.
  • the optimum reaction temperature is 45 ° C to 60 ° C, with 50 ° C being optimal.
  • the reaction time is a force depending on the enzyme concentration'reaction temperature and the like. Generally, about 15 minutes to 20 hours is appropriate, and 30 minutes to 5 hours is preferable.
  • pH 4. 4. 0 to 7.0 force is preferred, 4. 0 to 6.5 force is particularly preferred, in vivo and in vitro tests Therefore, 6.0 was judged to be optimal.
  • the active peptide can be concentrated by treating the degradation product of ⁇ -conglycinin with a cation exchange resin under appropriate conditions.
  • the cation exchange resin used is preferably a weakly acidic cation exchange resin such as acrylic acid or methacrylic acid.
  • “Diaion” (registered trademark) WK series of Mitsubishi Igaku Co., Ltd., or commercially available products equivalent to them can be used.
  • the active peptide is recovered in the fraction adsorbed on the cation exchange resin, it is thought that it contains a large amount of basic amino acid such as arginine. Elution from the cation exchange resin is carried out using 0.7 to 0.9%, preferably 0.8% ammonia.
  • peptide is used as an encompassing term if it is composed of two or more amino acid residues and is smaller than the original protein or polypeptide.
  • the peptide is preferably a hydrolyzate having a molecular weight of about 200 to 20,000 (2 to 200 amino acid residues).
  • composition of the present invention means that it contains at least one component (may be another peptide! /) In addition to one kind of peptide.
  • cholecystocun secretion promoting activity can be examined as follows (CCK secretion test).
  • a mouse duodenum-derived cell line STC-1 widely used as a model of CCK-producing cells is used.
  • STC-1 cells cultured in 24-well plates were reacted in ⁇ -conglycinin hydrolyzate solution (lmg / ml) for 60 minutes, and the amount of CCK released into the supernatant was determined using Enzyme Immuno Assay (EIA). taking measurement.
  • EIA Enzyme Immuno Assay
  • 13-conglycinin hydrolyzate or a buffer not supplemented with its peptide (10 mM Hepes, pH 7.4) is used as a control.
  • cholecystocun secretion promoting activity means that cholecystocun secretion is significantly promoted as compared to the control when examined by the test method described above.
  • “having anti-feeding activity” means that, when examined by the above test method, it significantly inhibits feeding compared to the control.
  • composition and the like of the present invention can be administered orally orally.
  • the administration or ingestion amount can be 2 to 5 g, preferably 3 to 5 g, as the above-mentioned j8-conglycinin degradation product. Such intake is sufficiently safe.
  • the peptide-containing composition of the present invention can be used as an antifeedant or a cholecystoki secretion promoter containing the same.
  • the antifeedant or cholecystokinin secretion promoter of the present invention can be combined with components such as a pharmaceutically acceptable general carrier or excipient to form a pharmaceutical composition.
  • a pharmaceutically acceptable general carrier or excipient such as a pharmaceutically acceptable general carrier or excipient.
  • the term “medicine” is used as a concept encompassing not only those applied to humans but also those applied to animals (veterinary medicine).
  • the feeding inhibitor and the cholecystocun secretion promoter are effective for animals, they are combined with known general nutritional components. Or a feed containing the antifeedant or the secretecone secretion promoter of the present invention (pet food, livestock feed, etc.) in combination with other active ingredients.
  • Soybean j8 -.. The conglycinin was suspended in deionized water, pH was adjusted to 4 0-9 0 at Mizusani ⁇ sodium solution, which was used as a decomposition start P H. No buffer solution is used to eliminate the need for desalting.
  • Abbreviated names (labels) from A-1 to 14 were provided for the degradation products with different enzyme names and degradation pH.
  • the upper small intestinal brush border membrane soluble component of 3 & ⁇ -0 & ⁇ male rats aged 8-10 weeks was fixed on the sensor chip CM5 of the biomolecular interaction analyzer BIACORE3000.
  • Each type of conglycinin degradation product 500 / zg / ml was added as an analyte, and the binding amount (Resonance Unit: RU) to the small intestinal brush border membrane component on the sensor chip was measured.
  • Example 1 Specifically, using the mouse duodenum-derived cell line STC-1 widely used as a model for CCK-producing cells, various hydrolysates selected based on the binding test results of Example 1 (prepared in the same manner as in Example 1). The CCK release activity of the digestive tract endocrine cell strength of soybean ⁇ 8—conglycinin degradation product underlined in Table 1 was measured. STC-1 cells cultured in 24-well plates are reacted in the hydrolyzate solution (lmg / ml) for 60 minutes, and the amount of CCK released in the supernatant is determined using the Enzyme Immuno Assay (EIA) kit (Cholecystokinin Octapeptide EIA kit, Phenix Pharmaceuticals, EK-069-04).
  • EIA Enzyme Immuno Assay
  • the results are shown in Figure 1.
  • the vertical axis shows the concentration of CCK in the STC-1 cell supernatant 60 minutes after sample addition.
  • 9 hydrolysates excluding protease S hydrolyzate (“E-1” in Figure 1)
  • the product showed higher CCK release activity than the negative control without the hydrolyzate (“Blk” in FIG. 2).
  • pepsin decomposition product was used as a positive control in (in FIG. 2, "P mark ton e") the same extent showed the strongest ⁇ CCK releasing activity of 3-4 times that of the positive control It was decided to examine the feeding suppression effect of the thermolysin degradation product (“C-1” in FIG. 2) and bromelain degradation product (“G-1” in FIG. 1) in the rat feeding test.
  • a soy-conglycone bromelain degradation product 1.5 g or 3 g dissolved in 100 ml of low-calorie sports drinks sold on the market (“Pocari Sweat Stevia", Otsuka Pharmaceutical Co., Ltd., l lkcal / 100 ml) was used.
  • a commercially available soy peptide (“Hyute”, Fuji Oil Co., Ltd.) prepared at the same concentration was used.
  • the following three test samples (100 ml each) were prepared and tested by the double blind method. Each calorie intake is 23 kcal (protein 12 kcal, beverage l lkcal).
  • Group H High-Ute 3.
  • Group HB High-Ute 1.5 g + j8—Conglycine bromelain degradation product 1.5 g •
  • Group B j8—Conglycine bromelain degradation product 3.
  • Subjects were healthy men and women aged 21-35 years (19 men and 16 women). These subjects were fasted for 3 hours after lunch, had 100 ml of any of the above test samples taken, immediately before taking the test sample (set to 0 minutes), and every 15 minutes after ingestion (15, 30, 45 minutes later), appetite indicators (hunger, satiety, acceptable intake) were recorded on the Visual Analogue Scale (VAS). With the VAS method, the left end of the 100mm horizontal line is 0 (nothing at all) and the right end is 100 (very much), and each item is marked at any position with the degree of! Is the method.
  • VAS Visual Analogue Scale
  • the food intake group (Powder group) was always higher than the other two sample groups, and the mixed soy peptide and j8-conglycine bromelain degradation product group (HB group) was intermediate between the other two samples. Although there was a tendency to show a value, no significant change was seen.
  • Hungry sensation and tolerable intake remained lower than the initial values for 45 minutes in the ⁇ -conglycone bromelain degradation product intake group (group IV), but in the other two sample groups than before administration. But their degree increased.
  • the feeling of satiety remained higher in the ⁇ -conglycone bromelain-digested group (group IV) than the initial value for 45 minutes, and decreased in the other two groups compared to before administration. did.
  • the active peptide in the bromelain degradation product of ⁇ -conglycinin was concentrated by fractionation by batch method using cation exchange resin.
  • Peptide solution in weak cation exchange resin (Mitsubishi Chemical Diaion WK100), which was prepared in Example 1 from the bromelain degradation product of j8-conglycinin, conditioned to H + form with 1N hydrochloric acid tower and washed with demineralized water And stirred at 4 ° C. Thereafter, the non-adsorbed fraction was collected with demineralized water, and the adsorbed fraction was collected by eluting with 0.8 M and 1. OM ammonia. As for the ammonia elution fraction, the ammonia was removed by a rotary evaporator, and then freeze-dried powder was obtained. The non-adsorbed fraction was lyophilized without being concentrated.
  • weak cation exchange resin Mitsubishi Chemical Diaion WK100
  • the collected fraction was 59% in terms of dry matter weight ratio in the non-adsorbed fraction and 41% in the adsorbed fraction (0.8 M ammonia: 16%, 1. OM ammonia: 25%).
  • the CCK releasing activity of each of the above fractions was measured using a mouse small intestine-derived CCK producing cell line STC-1.
  • STC-1 cells cultured in a 48-well plate are reacted in a buffer containing the above peptide fractions for 60 minutes, and the amount of CCK released in the supernatant is used with a commercially available Enzyme 'Imnoassay' kit (Phoenix pharmaceutical). I made a mistake.
  • the bromelain degradation product of ⁇ -conglycinin prepared in Example 7 was fractionated with ion-exchange resin.
  • the 8% ammonia-eluted fraction was dissolved in demineralized water, and the residual ammonia concentration was measured using a kit (Ammonia Test Measured using Yakuhin Kogyo).

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Abstract

Discloses are: a peptide having a suppressive effect on food intake which can be administered or taken orally and is applicable to a human body, particularly the peptide which is inexpensive and practically useful, has less bitter taste, and is safe and easy to be taken into a body; and a food-intake suppresser, food, pharmaceutical composition, feeding stuff or the like comprising the peptide. A composition comprising a peptide which can be produced by digesting β-conglycinin with an enzyme capable of predominantly cleaving a peptide bond on the carboxyl side in an arginine residue and which has a promoting activity on the secretion of cholecystokinin or a suppressive activity on food intake.

Description

明 細 書  Specification
摂食抑制作用を有するペプチドを含有する組成物  Composition containing peptide having antifeedant action
技術分野  Technical field
[0001] 本発明は、経口摂取することにより、摂食を抑制し、空腹感を低減させるペプチドお よびそれを含有する各種組成物等に関する。  [0001] The present invention relates to a peptide that suppresses food intake and reduces hunger when taken orally, and various compositions containing the peptide.
背景技術  Background art
[0002] 糖尿病、高脂血症、高血圧などの生活習慣病予防は、現代の先進国社会における 重要な課題であり、これらの大きな原因である肥満を予防または改善することが望ま れている。規則正しい食事摂取および摂取カロリーの制限こそが肥満を予防、改善 する最善策と考えられるが、食物がありふれた現代社会において、その実行は困難 である場合が少なくない。  [0002] Prevention of lifestyle-related diseases such as diabetes, hyperlipidemia, and hypertension is an important issue in the society of modern developed countries, and it is desired to prevent or improve obesity, which is a major cause of these diseases. Regular dietary intake and caloric restriction are considered the best way to prevent and ameliorate obesity, but in modern societies where food is common, doing so is often difficult.
[0003] 肥満治療のために食欲を抑制する薬剤として、アンフェタミン類のマジンドールが 実用化されている。しかし、この薬剤に付随する中枢への直接作用や習慣性などの 危険性から、高度肥満患者のみにその使用は限られており、より安全な手法が望ま れている。  [0003] The amphetamine class of mazindol has been put to practical use as a drug that suppresses appetite for the treatment of obesity. However, due to the direct effects on the center and the habitual nature associated with this drug, its use is limited only to highly obese patients, and a safer method is desired.
[0004] 消化管の内分泌細胞より分泌される種々のホルモン (消化管ホルモン）が食欲を抑 制する機能を有することが知られている（非特許文献 1〜4)。これらのペプチドホル モンは、経口投与の場合、消化管管腔内において容易に分解されるので、経口投与 によって吸収させてその効果を発揮させることは困難であり、血中投与せざるを得な い。その場合、ペプチド合成に力かる高いコストと投与法の煩雑さといつた課題が存 在する。  [0004] It is known that various hormones (gastrointestinal hormones) secreted from endocrine cells of the gastrointestinal tract have a function of suppressing appetite (Non-Patent Documents 1 to 4). Since these peptide hormones are easily degraded in the digestive tract lumen when administered orally, it is difficult to absorb them and exert their effects, and they must be administered in blood. Yes. In that case, there are high costs involved in peptide synthesis, complicated administration methods, and problems.
[0005] しかし、これらの消化管ホルモンは、食事摂取が強 、分泌刺激となって分泌される ことから、これらのホルモンの分泌を効果的に刺激できれば、食品成分の経口投与と いう安全な手法により食欲を抑制することが可能となる。  [0005] However, since these gastrointestinal hormones are secreted and stimulated to be secreted, they are secreted, so if these hormones can be effectively stimulated, a safe method of oral administration of food ingredients is possible. Can suppress appetite.
[0006] コレシストキニン（Cholecystokinin、以下「CCK」と略称することがある）は、他の消 化管ホルモン (GLP—1、PYY)などに先駆けて食欲を抑制する作用を有することが 見いだされた消化管ホルモンであり、食事中の脂質、タンパク質、アミノ酸などにより 分泌が刺激される (非特許文献 2)。しかし、摂取カロリーの制限を目指す上で、エネ ルギー効率の高!、脂質を用いるのは不適である。 [0006] Cholecystokinin (hereinafter sometimes abbreviated as “CCK”) has been found to have an effect of suppressing appetite ahead of other gonadotropins (GLP-1, PYY) and the like. It is a gastrointestinal hormone and is caused by dietary lipids, proteins, amino acids, etc. Secretion is stimulated (Non-patent Document 2). However, in order to limit calorie intake, energy efficiency is high!
[0007] 動物試験において、大豆 j8—コングリシニン由来のペプチド（ペプシン分解物およ び合成ペプチド）により CCK放出を刺激することができることは報告されている（特許 文献 非特許文献 5〜6)。しかし、ペプシンで分解した場合はその基質特異性によ りペプチドが末端に芳香族アミノ酸を含むため、苦みが強いという問題がある。一方、 合成ペプチドは高コストである。したがって、ヒトに適用できる、低コストで実用的な、 苦みが少なく摂取しやす 、ペプチド糸且成物の開発が望まれて 、る。 In animal studies, it has been reported that CCK release can be stimulated by soybean j8-conglycinin-derived peptides (pepsin degradation products and synthetic peptides) (Patent Documents Non-Patent Documents 5 to 6). However, when pepsin decomposes, there is a problem that the bitterness is strong because the peptide contains an aromatic amino acid at the terminal due to its substrate specificity. On the other hand, synthetic peptides are expensive. Therefore, there is a demand for the development of a low-cost, practical, low-bitter, easy-to-take peptide yarn and composition that can be applied to humans.
特許文献 1 :特開 2004— 10569  Patent Document 1: JP 2004-10569 A
非特許文献 1 : Woods SC, Am J Physiol Gastrointest Liver Physiol, 286(1):G7- 13, 2004  Non-Patent Document 1: Woods SC, Am J Physiol Gastrointest Liver Physiol, 286 (1): G7-13, 2004
非特許文献 2 : Moran TH, et al, Am J Physiol Gastrointest Liver Physiol, 286(2):G 183-8, 2004  Non-Patent Document 2: Moran TH, et al, Am J Physiol Gastrointest Liver Physiol, 286 (2): G 183-8, 2004
非特許文献 3 : Moran TH, et al, Am J Physiol Gastrointest Liver Physiol, 286(5):G 693-7, 2004  Non-Patent Document 3: Moran TH, et al, Am J Physiol Gastrointest Liver Physiol, 286 (5): G 693-7, 2004
非特許文献 4 : Tso P, et al, Am J Physiol Gastrointest Liver Physiol, 286(6):G885— 90, 2004  Non-Patent Document 4: Tso P, et al, Am J Physiol Gastrointest Liver Physiol, 286 (6): G885—90, 2004
非特許文献 5 : Nishi T, et al, J Nutr, 133(2):352-7, 2003  Non-Patent Document 5: Nishi T, et al, J Nutr, 133 (2): 352-7, 2003
非特許文献 6 : Nishi T, et al, J Nutr, 133(8):2537-42, 2003  Non-Patent Document 6: Nishi T, et al, J Nutr, 133 (8): 2537-42, 2003
発明の開示  Disclosure of the invention
[0008] 本発明は、経口的に投与または摂取可能であり、ヒトにも適用できる、摂食抑制作 用を有するペプチド、特に、低コストで実用的な、苦みが少なく安全で摂取しやすい ペプチド、およびこれを含有する摂食抑制剤、食品、医薬組成物、飼料等を提供す ることを目的とする。  [0008] The present invention is a peptide having an anti-feeding action that can be orally administered or ingested and can also be applied to humans, in particular, a peptide that is low-cost and practical, less bitter and safe and easy to ingest. And an antifeedant, food, pharmaceutical composition, feed and the like containing the same.
[0009] 本発明者は、食品由来のタンパク質である大豆 β コングリシニンを種々の食品加 ェ用酵素で分解してペプチド組成物を調製し、分子レベルカゝら細胞レベル、個体レ ベルと 、う順序でスクリーニングすることにより、 CCK放出活性の高 、ペプチド組成 物を選択し、さらに、ヒトにおいて、実際に経口摂取による空腹感の低減、満腹感の 惹起を観察することにより、本発明を完成した。 [0009] The present inventor prepares a peptide composition by degrading soybean β-conglycinin, which is a food-derived protein, with various food-adding enzymes. By selecting a peptide composition with high CCK release activity, and further reducing the feeling of hunger and satiety due to actual ingestion in humans. By observing the triggering, the present invention was completed.
本発明は、  The present invention
(1) β コングリシニンを、アルギニン残基のカルボキシル基側のペプチド結合を 優先的に切断する酵素によって分解して得られるペプチドであって、コレシストキュン 分泌促進活性または摂食抑制活性を有するペプチドを含有する組成物；  (1) A peptide obtained by degrading β-conglycinin with an enzyme that preferentially cleaves the peptide bond on the carboxyl group side of an arginine residue, and having a cholecystocone secretion-promoting activity or an anti-feeding activity Containing composition;
(2) β コングリシニンを、ブロメラインまたはプロメラインと同等の酵素活性を有す る酵素によって分解して得られるペプチドであって、コレシストキュン分泌促進活性ま たは摂食抑制活性を有するペプチドを含有する組成物；  (2) A peptide obtained by degrading β-conglycinin with an enzyme having an enzyme activity equivalent to that of bromelain or promelain, and contains a peptide having a cholecystocone secretion promoting activity or an antifeedant activity A composition to make;
(3) 前記酵素が、ブロメラインである、前記（1)または（2)記載の組成物；  (3) The composition according to (1) or (2), wherein the enzyme is bromelain;
(4) 前記（1)〜（3)の 、ずれか 1項記載の組成物を含有する摂食抑制剤； (4) The antifeedant containing the composition according to any one of (1) to (3) above;
(5) 前記（1)〜（3)の ヽずれか 1項記載の組成物を含有するコレシストキニン分泌 促進剤； (5) A cholecystokinin secretion promoter comprising the composition according to any one of (1) to (3) above;
(6) 前記 (4)または（5)記載の摂食抑制剤またはコレシストキニン分泌促進剤を含 有する医薬組成物；  (6) A pharmaceutical composition comprising the antifeedant or cholecystokinin secretion promoter according to (4) or (5) above;
(7) 前記 (4)または（5)記載の摂食抑制剤またはコレシストキニン分泌促進剤を含 有する食品；および  (7) A food containing the antifeedant or cholecystokinin secretion promoter according to (4) or (5) above; and
(8) 前記 (4)または（5)記載の摂食抑制剤またはコレシストキニン分泌促進剤を含 有する飼料、  (8) A feed containing the antifeedant or cholecystokinin secretion promoter according to (4) or (5),
(9) β コングリシニンを、アルギニン残基のカルボキシル基側のペプチド結合を 優先的に切断する酵素によって分解する工程を含むことを特徴とする、コレシストキ ニン分泌促進活性または摂食抑制活性を有するペプチド又はこのペプチドを含有す る組成物の製造方法、  (9) A peptide having cholecystokinin secretion-promoting activity or feeding-suppressing activity, comprising a step of decomposing β-conglycinin by an enzyme that preferentially cleaves a peptide bond on the carboxyl group side of an arginine residue. A method for producing a composition containing the peptide,
(10) β コングリシニンを、アルギニン残基のカルボキシル基側のペプチド結合を 優先的に切断する酵素によって分解する工程、および得られた分解物力ゝら陽イオン 交換榭脂吸着画分を回収する工程、を含むことを特徴とする、コレシストキニン分泌 促進活性または摂食抑制活性を有するペプチド又はこのペプチドを含有する組成物 の製造方法、  (10) a step of decomposing β-conglycinin by an enzyme that preferentially cleaves a peptide bond on the carboxyl group side of an arginine residue, and a step of recovering a cation-exchanged resin-absorbing fraction from the resulting degradation product force, A method for producing a peptide having cholecystokinin secretion-promoting activity or feeding-suppressing activity or a composition containing this peptide,
を提供する。 [0011] 本発明の組成物、摂食抑制剤、コレシストキュン分泌促進剤およびこれらを含有す る医薬組成物、食品、飼料 (以下、「(本発明の)組成物等」と総称することがある）は、 食欲抑制作用を有するコレシストキニンの分泌を刺激する活性を有するため、経口 摂取することにより、空腹感を低減させることができる。また、本発明の組成物等は、 安全な食品素材を食品加工用の酵素で処理することにより得られるので、安全性が 高ぐ低コストである。 I will provide a. [0011] The composition of the present invention, an antifeedant, a cholecystocun secretion promoter, and a pharmaceutical composition, food, and feed containing these (hereinafter referred to as "the composition of the present invention") ) Has the activity of stimulating the secretion of cholecystokinin, which has an appetite-suppressing effect, so that hunger can be reduced by taking it orally. In addition, the composition of the present invention is obtained by treating a safe food material with an enzyme for food processing, so that the safety is high and the cost is low.
本発明の組成物は、その高い溶解性により、カプセル等の固体形態による摂取の みならず、飲料等に溶解して摂取することも可能である。  Due to its high solubility, the composition of the present invention can be taken not only in a solid form such as a capsule but also in a beverage or the like.
[0012] さらに、本発明の組成物等は、摂取後約 15分でその効果が発揮されることから、食 事の 15分ほど前に本発明のペプチド組成物を摂取して予め満腹感を惹起すること により、その後の過剰なカロリー摂取を予防することができる。また、食事開始と同時 に摂取する場合には、実際の食事によって惹起される満腹感との相乗効果が期待で きる。  [0012] Further, since the effect of the composition of the present invention is exhibited about 15 minutes after ingestion, the user feels full before taking the peptide composition of the present invention about 15 minutes before eating. This can prevent excessive caloric intake thereafter. In addition, when taken at the same time as the start of a meal, a synergistic effect with the feeling of fullness caused by the actual meal can be expected.
図面の簡単な説明  Brief Description of Drawings
[0013] [図 1]は、各種 β —コングリシニン分解物による消化管内分泌細胞株 STC— 1からの CCK放出量を示す図である。数値は 60分間に培養上清中に放出された CCKの濃 度を示す。  [0013] FIG. 1 is a graph showing the amount of CCK released from gastrointestinal endocrine cell line STC-1 by various β-conglycinin degradation products. The value indicates the concentration of CCK released into the culture supernatant during 60 minutes.
[図 2]は、ラット胃内に j8—コングリシニン分解物溶液を投与して、 30分後から 90分 後までの摂食量を示す図である。数値は 12匹の平均値と標準誤差である。 *は、水 投与群に比べて有意差 (P < 0. 05)があることを示す。  FIG. 2 is a graph showing food intake from 30 minutes to 90 minutes after administration of a j8-conglycinin degradation product solution into the rat stomach. The values are the average of 12 animals and the standard error. * Indicates that there is a significant difference (P <0. 05) compared to the water-administered group.
[図 3]は、雄雌ラットの胃内に過剰量 (雄： 10g/kg体重、雌： 12g/kg体重）の |8—コン グリシ-ンブロメライン分解物溶液を投与して 2日間の積算摂食量を示す図である。 数値は各群 6匹のラットにおける、各時点までに摂取した食事量の積算値の平均値 を示す。 口：雄ラット、水投与 國：雄ラット、 β —コングリシ-ンブロメライン分解物 投与 〇：雌ラット、水投与 參:雌ラット、 |8—コングリシニンブロメライン分解物投与 [図 4]は、 β コングリシニンブロメライン分解物または市販の大豆ペプチド溶液摂取 後の、空腹感の変化を示す図である。数値は、 VAS法によりスコアリングした各被検 者の試飲前の値からの変化の平均値 (24〜30人）と標準誤差を表す。 〇：ハイニュ ート 3g (25人） 口：ハイ-ユート 1. 5g+ β —コングリシ-ンブロメライン分解物 1. 5g (24人） 參： j8—コングリシ-ンブロメライン分解物 3g (30人） * :摂取前に比べて 有意な差があることを示す。 (P< 0. 05) [Fig. 3] shows the total of 2 days after administration of | 8-conglycine bromelain degradation product solution (male: 10 g / kg body weight, female: 12 g / kg body weight) in the stomach of male and female rats. It is a figure which shows the amount of food intake. The numerical value shows the average value of the cumulative amount of food consumed up to each time point in 6 rats in each group. Mouth: Male rat, water administration Country: Male rat, β-conglycin bromelain degradation product Administration ○: Female rat, water administration 參: Female rat, | It is a figure which shows the change of a feeling of hunger after ingesting degradation product or a commercially available soybean peptide solution. The numbers represent the mean (24-30) change from the pre-tasting value for each subject scored by the VAS method and the standard error. ◯: Hainu Mouth 3g (25 people) Mouth: High-Ute 1.5 g + β —Conglycine bromelain degradation product 1.5 g (24 people) 參: j8—Conglycine bromelain degradation product 3 g (30 people) *: Before consumption It shows that there is a significant difference. (P <0. 05)
[図 5]は、 β コングリシニンブロメライン分解物または大豆ペプチド溶液摂取後の、 満腹感の変化を示す図である。数値は、 VAS法によりスコアリングした各被検者の試 飲前の値力もの変化の平均値 (24〜30人）と標準誤差を表す。 〇：ハイニュート 3g (25人） 口：ハイ-ユート 1. 5g+ β —コングリシ-ンブロメライン分解物 1. 5g (24人 ) 參：j8—コングリシ-ンブロメライン分解物 3g (30人） * :摂取前に比べて有意な 差があることを示す。 （Pく 0. 05) 異なるアルファベットの付いたプロットは、同じ時 間において、互いに有意な差があることを示す。（P< 0. 05)  FIG. 5 is a graph showing changes in satiety after ingestion of β-conglycinin bromelain degradation product or soybean peptide solution. The numerical value represents the average value (24-30 people) and standard error of changes in value before each tasting of each subject scored by the VAS method. ○: High Newt 3g (25 people) Mouth: High-Ute 1.5g + β —Conglycine Bromelain Degradation Product 1.5g (24 people) 參: j8—Conglycine Bromelain Degradation Product 3g (30 people) *: Ingestion It shows that there is a significant difference compared to before. (P 0. 05) Plots with different alphabets indicate that they are significantly different from each other at the same time. (P <0. 05)
[図 6]は、 β コングリシニンブロメライン分解物または大豆ペプチド溶液摂取後の、 許容食事摂取量の変化を示す図である。数値は、 VAS法によりスコアリングした各被 検者の試飲前の値からの変化の平均値 (24〜30人）と標準誤差を表す。 〇：ハイ 二ユート 3g (25人） 口：ハイ-ユート 1. 5g+ j8—コングリシ-ンブロメライン分解物 1 . 5g (24人） 參： j8—コングリシ-ンブロメライン分解物 3g (30人） * :摂取前に比 ベて有意な差があることを示す。 (P< 0. 05) 異なるアルファベットの付いたプロット は、同じ時間において、互いに有意な差があることを示す。 (P< 0. 05) FIG. 6 is a graph showing changes in the allowable dietary intake after ingestion of β-conglycinin bromelain degradation product or soybean peptide solution. The numbers represent the mean (24-30) change from the pre-tasting value for each subject scored by the VAS method and the standard error. ○: High-yuteu 3g (25 people) Mouth: High-yute 1.5g + j8—Conglycine bromelain degradation product 1.5g (24 people) 參: j8—Conglycine bromelain degradation product 3g (30 people) *: It shows that there is a significant difference before ingestion. (P <0. 05) Plots with different alphabets indicate that there is a significant difference from each other at the same time. (P <0. 05)
[図 7]は、 β コングリシニン分解物中の活性ペプチド分画物の CCK放出活性を示 す図である。異なるアルファベットの付いた群間には有意差があることを示す (Ρ < 0. 05)。 FIG. 7 is a diagram showing the CCK releasing activity of the active peptide fraction in β-conglycinin degradation product. Indicates that there is a significant difference between groups with different alphabets (Ρ <0. 05).
本発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
本発明のペプチドは、 j8—コングリシニンを分解することによって得られる。 j8 —コ ングリシニンは、大豆タンパクの主要な成分である。 β コングリシニンは、大豆ァレ ルギー患者を除いては、通常、既に食経験があり、ヒトにおいて一日 5グラムを 3ヶ月 間連続摂取させて安全性を確認した報告（日本農芸化学会 2004年度大会、 口演番 号 2Α16ρ17、神原ら）があることから、安全性は充分確認されている。本発明におい て使用される β コングリシニンは、大豆由来のものであってもよぐ他の豆類のよう な天然原料由来のものであってもよぐまた、遺伝子組換え法等によって製造された ものであってもよい。コスト等の観点から、大豆由来の j8 —コングリシニンが好ましい。 The peptide of the present invention is obtained by degrading j8-conglycinin. j8 —Conglycinin is a major component of soy protein. β-conglycinin, except for soy allergies, is usually already eaten and has been confirmed to be safe by ingesting 5 grams a day for 3 months in humans. (Kanbara No. 2 口 16ρ17, Kanbara et al.) Has been confirmed to be safe. Β-conglycinin used in the present invention may be derived from soybeans or may be derived from natural raw materials such as other beans. It may be a thing. From the viewpoint of cost and the like, j8-conglycinin derived from soybean is preferable.
[0015] β コングリシニンの分解に使用される酵素としては、ブロメラインのような、ポリべ プチド中のアルギニン残基のカルボキシル基側のペプチド結合を優先的に切断する 酵素であれば、特に制限されない。このような酵素を用いることにより、ペプチド末端 に芳香族アミノ酸が存在することによる苦味を、ほとんど、またはまったく有さないぺ プチド組成物を得ることができる。ポリペプチド中のアルギニン残基のカルボキシル 基側のペプチド結合を優先的に切断する酵素としては、トロンビン、エンドプロティナ ーゼ Arg— C、カテブシン B1などが挙げられる。安全性、入手の容易性およびコスト 等の観点などから、好ましくはブロメラインである。 [0015] The enzyme used for the degradation of β-conglycinin is not particularly limited as long as it is an enzyme that preferentially cleaves the peptide bond on the carboxyl group side of the arginine residue in the polypeptide, such as bromelain. By using such an enzyme, a peptide composition having little or no bitterness due to the presence of an aromatic amino acid at the end of the peptide can be obtained. Examples of enzymes that preferentially cleave the peptide bond on the carboxyl side of the arginine residue in the polypeptide include thrombin, endoproteinase Arg-C, and cathebsin B1. From the viewpoints of safety, availability, cost, etc., bromelain is preferable.
[0016] ブロメラインは、パイナップル科 (Bromeliaceae)に属する植物由来のシスティンプロ テアーゼであり、主にパイナップルの根茎および果実に含有される。ステムブロメライ ン（EC.3.4.22.4、 EC.3.4.22.32)およびフノレートブロメライン (EC.3.4.22.5, EC.3.4.22. 33)があり、本発明においてはいずれを用いてもよい。好ましくは、パイナップル茎由 来のブロメライン（EC.3.4.22.4)である。ブロメラインは、タンパク質分解において広い 基質認識性を持つが、アルギニンを含む合成基質に対し高!ヽ分解能を持つ酵素で あり、食品加工用としては肉の軟ィ匕に用いられ、医療用として炎症の改善にも用いら れており、入手が容易であるうえ、安全性が高いなど、多くの利点を有する。 [0016] Bromelain is a cystine protease derived from a plant belonging to the pineapple family (Bromeliaceae), and is mainly contained in pineapple rhizomes and fruits. There are stem bromelain (EC.3.4.22.4, EC.3.4.22.32) and funolate bromelain (EC.3.4.22.5, EC.3.4.22.33), any of which may be used in the present invention. Preferred is bromelain derived from pineapple stem (EC.3.4.22.4). Bromelain is an enzyme that has wide substrate recognition in proteolysis, but has high resolution against synthetic substrates including arginine. It is used for meat processing for food processing, and for inflammation for medical use. It is also used for improvement and has many advantages such as being easy to obtain and highly safe.
[0017] β —コングリシニンの分解は、例えば以下のようにして行うことができる。 [0017] The degradation of β-conglycinin can be performed, for example, as follows.
β —コングリシニンを重量濃度 10%にてイオン交換水に懸濁し、水酸ィ匕ナトリウム 水溶液にて ρΗを 6. 0に調整する。対基質濃度 0. 1%のブロメラインを添加して、 50 °Cにて 1時間保温し、 100°Cに上昇させて酵素反応を停止する。遠心分離、ろ過によ り、不溶性画分を除去し、得られたペプチド溶液を凍結乾燥して、 β コングリシニン ブロメライン分解物とする。 β —コングリシニンの溶解濃度は、懸濁できる範囲（最大 10%)であれば、どの濃度を用いてもよい。一般的には、対基質濃度 0. 01%〜10 %であり、 0. 05〜5%が好ましぐ 0. 05〜1%が特に好ましい。至適反応温度は 45 °C〜60°Cであり、 50°Cが最適である。反応時間は、酵素の濃度'反応温度等にもよ る力 一般に 15分〜 20時間程度が適当であり、 30分〜 5時間が好ましい。 pHにつ ヽて ίま、 4. 0〜7. 0力好ましく、 4. 0〜6. 5力特に好ましく、 in vivo, in vitroの試験 により、 6. 0が最適と判断された。 β-Conglycinin is suspended in ion-exchanged water at a weight concentration of 10%, and ρΗ is adjusted to 6.0 with sodium hydroxide aqueous solution. Add bromelain to the substrate concentration of 0.1%, incubate at 50 ° C for 1 hour, and raise to 100 ° C to stop the enzyme reaction. The insoluble fraction is removed by centrifugation and filtration, and the resulting peptide solution is lyophilized to obtain a β-conglycinin bromelain degradation product. Any concentration of β-conglycinin can be used as long as it can be suspended (maximum 10%). In general, the concentration with respect to the substrate is 0.01% to 10%, 0.05 to 5% is preferred, and 0.05 to 1% is particularly preferred. The optimum reaction temperature is 45 ° C to 60 ° C, with 50 ° C being optimal. The reaction time is a force depending on the enzyme concentration'reaction temperature and the like. Generally, about 15 minutes to 20 hours is appropriate, and 30 minutes to 5 hours is preferable. For pH, 4. 4. 0 to 7.0 force is preferred, 4. 0 to 6.5 force is particularly preferred, in vivo and in vitro tests Therefore, 6.0 was judged to be optimal.
β コングリシニンの分解物を、適当な条件下で陽イオン交換榭脂によって処理す ることにより、活性ペプチドを濃縮することができる。使用する陽イオン交換榭脂として は、アクリル酸系又はメタクリル酸系等の、弱酸性陽イオン交換樹脂が好ましい。具 体的には、例えば三菱ィ匕学 (株）の「ダイヤイオン」（登録商標）の WKシリーズのもの 、又はそれらと同等の市販品を使用することができる。  The active peptide can be concentrated by treating the degradation product of β-conglycinin with a cation exchange resin under appropriate conditions. The cation exchange resin used is preferably a weakly acidic cation exchange resin such as acrylic acid or methacrylic acid. Specifically, for example, “Diaion” (registered trademark) WK series of Mitsubishi Igaku Co., Ltd., or commercially available products equivalent to them can be used.
活性ペプチドは、陽イオン交換樹脂に吸着する画分に回収されるため、アルギニン のような塩基性アミノ酸を多く含むと考えられる。陽イオン交換榭脂からの溶出は、 0. 7〜0. 9Μ、好ましくは 0. 8Μアンモニアを用いて行なう。  Since the active peptide is recovered in the fraction adsorbed on the cation exchange resin, it is thought that it contains a large amount of basic amino acid such as arginine. Elution from the cation exchange resin is carried out using 0.7 to 0.9%, preferably 0.8% ammonia.
[0018] 本発明に関して、「ペプチド」は、 2以上のアミノ酸残基からなり、もとのタンパク質ま たはポリペプチドよりも小さいものであれば、すべて包含する用語として使用される。 ここで 、うペプチドは、好ましくは分子量 200〜20,000 (2〜200アミノ酸残基）程度 の加水分解物である。また、本発明の「組成物」は、 1種のペプチドのほかに、少なく とも 1つの成分 (別のペプチドであってもよ!/、）を含むものであることを意味する。  [0018] In the context of the present invention, "peptide" is used as an encompassing term if it is composed of two or more amino acid residues and is smaller than the original protein or polypeptide. Here, the peptide is preferably a hydrolyzate having a molecular weight of about 200 to 20,000 (2 to 200 amino acid residues). Further, the “composition” of the present invention means that it contains at least one component (may be another peptide! /) In addition to one kind of peptide.
[0019] コレシストキュン分泌促進活性の有無または程度は、以下のようにして調べることが できる（CCK分泌試験)。  [0019] The presence or absence of cholecystocun secretion promoting activity can be examined as follows (CCK secretion test).
[0020] CCK産生細胞のモデルとして広く用いられるマウス十二指腸由来の細胞株 STC — 1を用いる。 24— wellプレートに培養した STC—1細胞を、 β —コングリシニン加水 分解物溶液（lmg/ml)中で 60分間反応させ、上清中に放出された CCK量を Enzyme Immuno Assay (EIA)にて測定する。対照としては、 13 —コングリシニン加水分解物 またはそのペプチドを添カ卩していないバッファー（10mM Hepes、 pH7. 4)を用い る。  [0020] A mouse duodenum-derived cell line STC-1 widely used as a model of CCK-producing cells is used. STC-1 cells cultured in 24-well plates were reacted in β-conglycinin hydrolyzate solution (lmg / ml) for 60 minutes, and the amount of CCK released into the supernatant was determined using Enzyme Immuno Assay (EIA). taking measurement. As a control, 13-conglycinin hydrolyzate or a buffer not supplemented with its peptide (10 mM Hepes, pH 7.4) is used.
[0021] 本発明に関して、「コレシストキュン分泌促進活性を有する」とは、上記の試験方法 によって調べた場合に、対照と比較して有意にコレシストキュンの分泌を促進するこ とを意味する。  [0021] In the context of the present invention, "having cholecystocun secretion promoting activity" means that cholecystocun secretion is significantly promoted as compared to the control when examined by the test method described above. .
[0022] 摂食抑制活性の有無または程度は、以下のようにして調べることができる。  [0022] The presence or absence or degree of antifeedant activity can be examined as follows.
[0023] 8週齢の Sprague-Dawley系雄ラットを、精製飼料にて予備飼育後、一夜絶食させ、 フィーディングチューブにより j8—コングリシニン加水分解物溶液（50mg/ml、 1ml)を 胃内投与する。対照群には、水 lmlを投与する。投与 30分後に精製飼料を再給餌し[0023] 8-week-old Sprague-Dawley male rats were preliminarily raised on purified feed, fasted overnight, and j8-conglycinin hydrolyzate solution (50 mg / ml, 1 ml) was fed into the feeding tube. Administer intragastric. The control group receives 1 ml of water. 30 minutes after administration, re-feed the purified feed
、 60分間の摂食量を測定する。 Measure food intake for 60 minutes.
[0024] 本発明に関して、「摂食抑制活性を有する」とは、上記の試験方法によって調べた 場合に、対照と比較して有意に摂食を抑制することを意味する。 [0024] In the context of the present invention, "having anti-feeding activity" means that, when examined by the above test method, it significantly inhibits feeding compared to the control.
[0025] 本発明の組成物等は、経口的に投与したり、摂取させることができる。一般的には、 投与または摂取量としては、上記のような j8—コングリシニン分解物にして 2g〜5gで あることができ、好ましくは 3g〜5gに相当する量である。このような摂取量は、充分安 全である。 [0025] The composition and the like of the present invention can be administered orally orally. In general, the administration or ingestion amount can be 2 to 5 g, preferably 3 to 5 g, as the above-mentioned j8-conglycinin degradation product. Such intake is sufficiently safe.
[0026] 本発明のペプチド含有組成物は、これを含有する摂食抑制剤またはコレシストキ- ン分泌促進剤として使用することができる。  [0026] The peptide-containing composition of the present invention can be used as an antifeedant or a cholecystoki secretion promoter containing the same.
[0027] さらに、本発明の摂食抑制剤またはコレシストキニン分泌促進剤は、医薬的に許容 されうる一般的な担体または賦形剤などの成分と一緒にして医薬組成物とすることが できる。なお、本発明に関して、「医薬」という用語は、人に適用するものだけでなぐ 動物に適用するもの (獣医薬)をも包含する概念として使用される。  [0027] Furthermore, the antifeedant or cholecystokinin secretion promoter of the present invention can be combined with components such as a pharmaceutically acceptable general carrier or excipient to form a pharmaceutical composition. . In the context of the present invention, the term “medicine” is used as a concept encompassing not only those applied to humans but also those applied to animals (veterinary medicine).
[0028] また、本発明の摂食抑制剤またはコレシストキュン分泌促進剤を食品に添加するこ とにより、摂食抑制作用またはコレシストキニン分泌促進作用を有する各種の食品（ 飲料であってもよ 、）とすることができる。  [0028] Furthermore, by adding the food intake inhibitor or cholecystocone secretion promoter of the present invention to foods, various foods (even beverages having a food intake suppressive action or cholecystokinin secretion promoting action) can be obtained. Yo)).
[0029] 本発明のペプチド含有組成物、および摂食抑制剤およびコレシストキュン分泌促 進剤の効果は、動物に対しても有効であるので、公知の一般的な栄養成分と組み合 わせて、またはさらに他の有効成分と組み合わせて、本発明の摂食抑制剤またはコ レシストキュン分泌促進剤を含有する飼料 (ペットフード、家畜用飼料など）とすること ちでさる。  [0029] Since the effects of the peptide-containing composition of the present invention, the feeding inhibitor and the cholecystocun secretion promoter are effective for animals, they are combined with known general nutritional components. Or a feed containing the antifeedant or the secretecone secretion promoter of the present invention (pet food, livestock feed, etc.) in combination with other active ingredients.
[0030] 次いで、実施例により本発明をさらに詳細に説明するが、本発明はこれらの実施例 に限定されるものではない。  Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
[0031] l [0031] l
大豆 j8 —コングリシニンをイオン交換水に懸濁し、水酸ィ匕ナトリウム溶液にて pHを 4 . 0〜9. 0に調整し、これを分解スタート PHとした。脱塩処理を不要とするために、緩 衝液は用いていない。対基質濃度 0. 1%〜0. 5%の表 1に示す各種食品加工用プ 口テアーゼ（「酵素」）を添加して、 60分間加水分解した。酵素名、分解 pHの異なる 分解物に、 A— 1〜1 4まで略称 (ラベル)を設けた。 Soybean j8 -.. The conglycinin was suspended in deionized water, pH was adjusted to 4 0-9 0 at Mizusani匕sodium solution, which was used as a decomposition start P H. No buffer solution is used to eliminate the need for desalting. Various food processing formulations shown in Table 1 with substrate concentrations from 0.1% to 0.5% Oral thease (“enzyme”) was added and hydrolyzed for 60 minutes. Abbreviated names (labels) from A-1 to 14 were provided for the degradation products with different enzyme names and degradation pH.
得られた分解物を用いて、特開 2004— 10569 (特許文献 1)に記載された方法に したがって、ラット小腸粘膜との結合活性を調べた。  Using the resulting degradation product, the binding activity to the rat small intestinal mucosa was examined according to the method described in JP-A-2004-10569 (Patent Document 1).
具体的には、 8〜10週齢の3 &§ -0& ^系雄ラットの上部小腸刷子縁膜可溶ィ匕 成分（Hira T, et al., Biosci Biotechnol Biochem, 65(5):1007- 15, 2001参照）を、生体 分子間相互作用解析装置 BIACORE3000のセンサーチップ CM5に固定ィ匕した。各 種 —コングリシニン分解物（500 /z g/ml)をアナライトとして添カ卩し、センサーチップ 上の小腸刷子縁膜成分への結合量 (Resonance Unit :RU)を測定した。 Specifically, the upper small intestinal brush border membrane soluble component of 3 & § -0 & ^ male rats aged 8-10 weeks (Hira T, et al., Biosci Biotechnol Biochem, 65 (5): 1007- 15, 2001) was fixed on the sensor chip CM5 of the biomolecular interaction analyzer BIACORE3000. Each type of conglycinin degradation product (500 / zg / ml) was added as an analyte, and the binding amount (Resonance Unit: RU) to the small intestinal brush border membrane component on the sensor chip was measured.
結果を表 1 (「結合量」）に示す。数値は 3回の測定の平均値を示す。  The results are shown in Table 1 (“Binding amount”). The numerical value shows the average value of three measurements.
[表 1] [table 1]
J丁 ¾l v Λ J ¾l v Λ
71二— *¾* (¾)Hヽ , 71 2— * ¾ * (¾) H ヽ,
Figure imgf000011_0001
表 1に示すように、各酵素において、 pH6にて分解した産物に高い結合活性 (表 1 中、下線を付して表す）が見られた。この結果は、これらの加水分解物と小腸刷子縁 膜との間に強い相互作用があることを示しており、これらの加水分解物中に消化管内 分泌細胞からの消化管ホルモンの分泌を強く刺激できるものが存在することを示唆し ている。
Figure imgf000011_0001
As shown in Table 1, a high binding activity (represented by underlining in Table 1) was observed for the products degraded at pH 6 in each enzyme. This result indicates that there is a strong interaction between these hydrolysates and the small intestinal brush border membrane, and in these hydrolysates the gastrointestinal tract This suggests that there are those that can strongly stimulate the secretion of gastrointestinal hormones from secretory cells.
[0034] 例 2  [0034] Example 2
消化管内分泌細胞力 の消化管ホルモンコレシストキュン (CCK)の放出活性によ る in vitroでのスクリーニングを行った。  In vitro screening of the gastrointestinal endocrine cell strength by the release activity of the gastrointestinal hormone cholecystocun (CCK) was performed.
具体的には、 CCK産生細胞のモデルとして広く用いられるマウス十二指腸由来の 細胞株 STC— 1を用いて、例 1の結合試験結果に基づいて選抜した各種加水分解 物 (例 1と同様に調製した大豆 ι8 —コングリシニン分解物のうち、表 1中、下線を付し たもの）の消化管内分泌細胞力もの CCK放出活性を測定した。 24— wellプレートに 培養した STC— 1細胞を上記加水分解物溶液（lmg/ml)中で 60分間反応させ、上 清中に放出された CCK量を Enzyme Immuno Assay (EIA)キット（Cholecystokinin O ctapeptide EIA kit, Phenix Pharmaceuticals, EK— 069— 04)にて孭 U定した。  Specifically, using the mouse duodenum-derived cell line STC-1 widely used as a model for CCK-producing cells, various hydrolysates selected based on the binding test results of Example 1 (prepared in the same manner as in Example 1). The CCK release activity of the digestive tract endocrine cell strength of soybean ι8—conglycinin degradation product underlined in Table 1 was measured. STC-1 cells cultured in 24-well plates are reacted in the hydrolyzate solution (lmg / ml) for 60 minutes, and the amount of CCK released in the supernatant is determined using the Enzyme Immuno Assay (EIA) kit (Cholecystokinin Octapeptide EIA kit, Phenix Pharmaceuticals, EK-069-04).
結果を図 1に示す。縦軸は、サンプル添加 60分間後の STC— 1細胞上清中の CC K濃度を示す。検討した 10種類 (表 1中、下線を付したもの 8種ならびに陽性および 陰性対照）の加水分解物のうち、プロテアーゼ S分解物（図 1中「E— 1」）を除く 9種の 加水分解物が、加水分解物無添加の陰性対照（図 2中「Blk」）よりも高い CCK放出 活性を示した。  The results are shown in Figure 1. The vertical axis shows the concentration of CCK in the STC-1 cell supernatant 60 minutes after sample addition. Of the 10 hydrolysates examined (8 underlined in Table 1 and the positive and negative controls), 9 hydrolysates excluding protease S hydrolyzate (“E-1” in Figure 1) The product showed higher CCK release activity than the negative control without the hydrolyzate (“Blk” in FIG. 2).
これらの結果より、陽性対照として用いたペプシン分解物（図 2中「P印 tone」）と同程 度で、この陽性対照に比べて 3〜4倍の最も強 ヽ CCK放出活性を示したサーモライ シン分解物（図 2中「C— 1」）およびブロメライン分解物（図 1中「G— 1」）の摂食抑制 効果をラット摂食試験により検討することとした。 These results, pepsin decomposition product was used as a positive control in (in FIG. 2, "P mark ton e") the same extent showed the strongestヽCCK releasing activity of 3-4 times that of the positive control It was decided to examine the feeding suppression effect of the thermolysin degradation product (“C-1” in FIG. 2) and bromelain degradation product (“G-1” in FIG. 1) in the rat feeding test.
[0035] 例 3 [0035] Example 3
8週齢の Sprague-Dawley系雄ラットを、精製飼料にて予備飼育後、一夜絶食させた 。これらのラットに、フィーディングチューブにより上記 2種のいずれかの加水分解物 溶液（50mg/ml、 1ml)を胃内投与した（1群 12匹)。対照群には、水 lmlを投与した。 投与 30分後に精製飼料を再給餌し、その後 60分間の摂食量を測定した。  8-week-old Sprague-Dawley male rats were preliminarily raised on purified feed and fasted overnight. These rats were intragastrically administered with one of the above two hydrolyzate solutions (50 mg / ml, 1 ml) via a feeding tube (12 rats per group). The control group received 1 ml of water. 30 minutes after the administration, the purified feed was re-fed, and the food intake for 60 minutes was measured.
結果を図 2に示す。数値は 12匹の平均値および標準誤差であり、「*」は水投与群 に比べて有意差 (P< 0. 05)があることを示す。ブロメライン分解物投与により、有意 な摂食量の低下が観察された（図 2)。サーモライシン分解物では、摂食量の増大傾 向が見られたが、統計的に有意な変化ではな力つた。同濃度のペプチド投与により 作用に差があるということから、ブロメライン分解物による摂食量の低下は、摂取カロリ 一に依存するものではなぐブロメライン分解物特異的作用と考えられる。 The result is shown in figure 2. The values are the mean and standard error of 12 animals, and “*” indicates that there is a significant difference (P <0. 05) compared to the water-treated group. Significant by bromelain degradation product administration A significant decrease in food intake was observed (Figure 2). The thermolysin degradation product showed an increasing trend in food intake, but it was not a statistically significant change. Since there is a difference in action due to the administration of peptides at the same concentration, the decrease in food intake due to bromelain degradation products is considered to be a bromelain degradation product-specific action that does not depend on the caloric intake.
[0036] 例 4 [0036] Example 4
上記の j8—コングリシ-ンブロメライン分解物をヒト試験に用いるにあたり、急性の 毒性試験として、雄雌両ラッ HSprague- Dawley系、 15週齢、体重：雄 400g、雌 250g )に、過剰量 (雄： 10g/kg体重、雌： 12g/kg体重）の β コングリシ-ンブロメライン分 解物を胃内投与し、その後の 2日間の摂食量を測定した。対照群には、等量の水 (雄 ： 20ml/kg体重、雌： 24ml/kg体重）を胃内投与した。  When using the above-mentioned degradation product of j8-conglycone bromelain for human studies, as an acute toxicity test, both males and females HSprague-Dawley, 15 weeks old, body weight: 400 g male, 250 g female, : 10 g / kg body weight, female: 12 g / kg body weight) β-conglycin bromelain digest was administered intragastrically, and the food intake was measured for the following 2 days. In the control group, an equal amount of water (male: 20 ml / kg body weight, female: 24 ml / kg body weight) was administered intragastrically.
結果を図 3に示す。雌雄ラットともに、投与後に嘔吐や下痢などの症状はなぐ水、 β コングリシニン分解物投与群ともに体重も正常に増加した。摂食量に関しては（ 図 3)、雌雄ラットともに コングリシニン分解物投与群が、水投与群に比べて投与 後 6〜9時間後は低値を示した。その後両群とも同様の摂食量を示し、初期の摂食量 の差が投与後 2日目まで維持された。  The results are shown in Figure 3. In both male and female rats, the body weight increased normally in both the water and β-conglycinin hydrolyzate groups after vomiting and diarrhea. Concerning food intake (Figure 3), the conglycinin degradation product-treated group in both male and female rats showed a lower value 6 to 9 hours after administration compared to the water-treated group. Thereafter, both groups showed similar food intake, and the difference in the initial food intake was maintained until the second day after administration.
[0037] 5 [0037] 5
上記の in vitro, in vivoの試験結果より、 j8—コングリシニンのブロメライン分解物が 、消化管力もの CCK放出を活性ィ匕して、飽食感をもたらすこと、およびラットでの安 全性が確認されたことより、この物質を用いたヒト試験を実施した。ヒト試験実施に際 し、北海道大学医学研究科 ·医学部において医の倫理委員会臨床研究審査専門委 員会の承認を得た。  From the above in vitro and in vivo test results, it was confirmed that bromelain degradation product of j8-conglycinin activated gastrointestinal CCK release, resulting in satiety, and safety in rats. Therefore, a human test using this substance was conducted. In conducting human tests, we received approval from the Clinical Ethics Committee Clinical Research Review Committee at the Graduate School of Medicine and Faculty of Medicine, Hokkaido University.
試験物質として、大豆 —コングリシ-ンブロメライン分解物 1. 5gまたは 3gを巿販 の低カロリースポーツ飲料 100ml (「ポカリスエットステビア」、大塚製薬 (株）、 l lkcal /100ml)に溶解したものを使用した。対照物質としては、市販の大豆ペプチド（「ハ ィ-ユート」、不二製油 (株)）を同濃度にて調製したものを使用した。下記 3つの試験 試料 (それぞれ 100ml)を調製し、二重盲検法により試験を実施した。摂取カロリーは 、いずれも 23kcal (タンパク質 12kcal、飲料 l lkcal)である。  As a test substance, a soy-conglycone bromelain degradation product 1.5 g or 3 g dissolved in 100 ml of low-calorie sports drinks sold on the market ("Pocari Sweat Stevia", Otsuka Pharmaceutical Co., Ltd., l lkcal / 100 ml) was used. . As a control substance, a commercially available soy peptide (“Hyute”, Fuji Oil Co., Ltd.) prepared at the same concentration was used. The following three test samples (100 ml each) were prepared and tested by the double blind method. Each calorie intake is 23 kcal (protein 12 kcal, beverage l lkcal).
•H群：ハイ-ユート 3. Og •HB群：ハイ-ユート 1. 5g+ j8—コングリシ-ンブロメライン分解物 1. 5g •B群： j8—コングリシ-ンブロメライン分解物 3. Og • Group H: High-Ute 3. Og • Group HB: High-Ute 1.5 g + j8—Conglycine bromelain degradation product 1.5 g • Group B: j8—Conglycine bromelain degradation product 3. Og
被験者は、 21〜35歳の健康な男女 (男性 19名、女性 16名）であった。これらの被 験者を昼食摂取後 3時間絶食とし、上記の 、ずれかの試験試料 100mlを飲んでもら い、試験試料の摂取直前 (0分とした)および摂取後 15分おき（15、 30、 45分後）に 、食欲に関する指標 (空腹感、満腹感、許容摂取量)を視覚的アナログ尺度 (Visual Analogue Scale:VAS)により記録してもらった。 VAS法とは、 100mmの横線の左端を 0 (全く無 、）、右端を 100 (とてもある）とし、それぞれの項目につ 、ての度合!/、を、任 意の位置にマーキングしてもらう方法である。  Subjects were healthy men and women aged 21-35 years (19 men and 16 women). These subjects were fasted for 3 hours after lunch, had 100 ml of any of the above test samples taken, immediately before taking the test sample (set to 0 minutes), and every 15 minutes after ingestion (15, 30, 45 minutes later), appetite indicators (hunger, satiety, acceptable intake) were recorded on the Visual Analogue Scale (VAS). With the VAS method, the left end of the 100mm horizontal line is 0 (nothing at all) and the right end is 100 (very much), and each item is marked at any position with the degree of! Is the method.
結果を図 4〜6に示す。空腹感 (図 4)、満腹感 (図 5)、許容摂取量 (図 6)のいずれ の指標においても、試験試料間ならびに、各時間において 2元配置分散分析による 統計的有意差 (P< 0. 05)が検出された。 |8—コングリシニンブロメライン分解物摂 取 (B群）により摂取 15分後において、統計的に有意な空腹感の低下と、摂取 15分、 30分後の満腹感の有意な増加が見られた。空腹感、許容食事量においては、 β コングリシ-ンブロメライン分解物摂取群 (Β群は、他の 2試料の群よりも常に低値を 示し、満腹感においては逆に j8—コングリシ-ンブロメライン分解物摂取群 (Β群）は 他の 2試料の群よりも常に高値を示した。巿販大豆ペプチドと j8—コングリシ-ンブロ メライン分解物の混合物の群 (HB群）は他の 2試料の中間値を示す傾向にあつたが 、有意な変化は見られな力つた。  The results are shown in Figs. Statistically significant differences (P <0) between two test samples and at each time for each index of hunger (Fig. 4), satiety (Fig. 5), and allowable intake (Fig. 6). 05) was detected. | 8—By taking conglycinin bromelain degradation product (Group B), a statistically significant decrease in hunger was observed 15 minutes after ingestion and a significant increase in satiety at 15 and 30 minutes after ingestion. The β-conglycone bromelain digested group in the sense of hunger and tolerable diet (group IV always showed a lower value than the other two samples, and conversely in the sense of satiety j8-conglycine bromelain degradation. The food intake group (Powder group) was always higher than the other two sample groups, and the mixed soy peptide and j8-conglycine bromelain degradation product group (HB group) was intermediate between the other two samples. Although there was a tendency to show a value, no significant change was seen.
空腹感、許容摂取量は、 β コングリシ-ンブロメライン分解物摂取群 (Β群）にお いては、 45分間は初期値よりも低い値を維持したが、他の 2試料の群では投与前より もそれらの度合いは増加した。満腹感については、 β コングリシ-ンブロメライン分 解物摂取群 (Β群）においては、 45分間は初期値よりも高値を維持し、他の 2試料の 群では、投与前よりもその度合いが低下した。  Hungry sensation and tolerable intake remained lower than the initial values for 45 minutes in the β-conglycone bromelain degradation product intake group (group IV), but in the other two sample groups than before administration. But their degree increased. The feeling of satiety remained higher in the β-conglycone bromelain-digested group (group IV) than the initial value for 45 minutes, and decreased in the other two groups compared to before administration. did.
以上の結果より、大豆由来のタンパク質 β コングリシニンを食品加工用酵素ブロ メラインで分解したペプチドを含む組成物を 3g摂取することで、ヒトにお ヽて空腹感 ならびに許容食事摂取量を低減させること、満腹感を与えること、その効果が少なくと も 45分間持続すること、が明らかとなった。 [0039] この効果は、どの投与物質も等カロリーであることから、摂取したカロリーに依存する ものではなく、 β コングリシ-ンをブロメラインで分解することで生じるものであり、こ れにはペプチド中の特異構造が上述の CCK放出刺激として、消化管で認識されるこ とによるものと考えられる。 Based on the above results, ingesting 3 g of a composition containing a peptide obtained by degrading soybean-derived protein β-conglycinin with the enzyme bromelain for food processing reduces hunger and acceptable food intake in humans. It became clear that it gives a feeling of fullness and that the effect lasts for at least 45 minutes. [0039] This effect is not dependent on the calorie intake, since every administered substance is isocaloric, but it is caused by the degradation of β-conglycone with bromelain, which is caused in peptides. This unique structure is considered to be recognized in the digestive tract as the above-mentioned CCK release stimulus.
[0040] 6  [0040] 6
β コングリシニンのブロメライン分解物中の活性ペプチドを、陽イオン交換榭脂を 用いたバッチ法による分画によって濃縮した。  The active peptide in the bromelain degradation product of β-conglycinin was concentrated by fractionation by batch method using cation exchange resin.
例 1で作製した j8 —コングリシニンのブロメライン分解物を、 1N塩酸の通塔によって H+型にコンディショニングした後、脱塩水にて洗浄した弱陽イオン交換榭脂（三菱化 学ダイヤイオン WK100)にペプチド溶液を添加し、 4°Cにて攪拌した。その後、脱塩 水にて非吸着画分を回収し、吸着画分を 0. 8Mおよび 1. OMアンモニアにて溶出し て回収した。アンモニア溶出画分については、ロータリーエバポレーターによりアンモ ニァを除去後、凍結乾燥粉末を得た。非吸着画分については、濃縮せずに凍結乾 燥した。  Peptide solution in weak cation exchange resin (Mitsubishi Chemical Diaion WK100), which was prepared in Example 1 from the bromelain degradation product of j8-conglycinin, conditioned to H + form with 1N hydrochloric acid tower and washed with demineralized water And stirred at 4 ° C. Thereafter, the non-adsorbed fraction was collected with demineralized water, and the adsorbed fraction was collected by eluting with 0.8 M and 1. OM ammonia. As for the ammonia elution fraction, the ammonia was removed by a rotary evaporator, and then freeze-dried powder was obtained. The non-adsorbed fraction was lyophilized without being concentrated.
回収された画分は、非吸着画分に乾物重量比として 59%、吸着画分に 41% (0. 8 Mアンモニア： 16%、 1. OMアンモニア： 25%)の割合であった。  The collected fraction was 59% in terms of dry matter weight ratio in the non-adsorbed fraction and 41% in the adsorbed fraction (0.8 M ammonia: 16%, 1. OM ammonia: 25%).
[0041] 例 7 [0041] Example 7
上記の各画分の CCK放出活性を、マウス小腸由来の CCK産生細胞株 STC— 1を 用いて測定した。  The CCK releasing activity of each of the above fractions was measured using a mouse small intestine-derived CCK producing cell line STC-1.
48 wellプレートに培養した STC— 1細胞を上記ペプチド画分を含む緩衝液中で 60分間反応させ、上清中に放出された CCK量を市販のェンザィム 'ィムノアッセィ' キット (Phoenix pharmaceutical)を用 ヽて U疋した。  STC-1 cells cultured in a 48-well plate are reacted in a buffer containing the above peptide fractions for 60 minutes, and the amount of CCK released in the supernatant is used with a commercially available Enzyme 'Imnoassay' kit (Phoenix pharmaceutical). I made a mistake.
結果を図 7に示す。  The results are shown in FIG.
[0042] イオン交換榭脂によって分画する前の j8—コングリシニンのブロメライン分解物は、 濃度 5mg/ml (乾重量/ ml緩衝液)で有意な CCK分泌を示した。  [0042] The bromelain degradation product of j8-conglycinin before fractionation with ion-exchanged rosin showed significant CCK secretion at a concentration of 5 mg / ml (dry weight / ml buffer).
陽イオン交換樹脂に吸着させ、分画したペプチド画分の活性を測定するため、それ ぞれの画分の乾物 5mgを lml緩衝液に溶解して反応に供したところ、 0. 8Mアンモ- ァ溶出画分が最も強く CCK分泌を促進した。 0. 8Mアンモニア溶出画分は、乾物 0 . 8mg/ml緩衝液という低濃度で、イオン交換榭脂処理に供する前の 5mg/mlの 13 コングリシニン分解物と同程度の CCK分泌を引き起こした。即ち、乾重量あたり 6. 2 5倍 (5ZO. 8倍）に活性が濃縮されたことになる。 In order to measure the activity of peptide fractions that were adsorbed on a cation exchange resin and fractionated, 5 mg of the dry matter of each fraction was dissolved in lml buffer and subjected to the reaction. The elution fraction was the strongest and promoted CCK secretion. 0. 8M ammonia elution fraction is dry matter 0 The low concentration of 8 mg / ml buffer caused CCK secretion to the same extent as 5 mg / ml 13 conglycinin degradation products before being subjected to ion-exchange resin treatment. In other words, the activity was concentrated to 6.25 times (5ZO.8 times) per dry weight.
各画分の乾物あたりの濃度を 5mg/mlにそろえた場合も 0. 8Mアンモニア溶出画分 が最も強ぐ分画前の 3倍以上の CCK分泌を引き起こした。  Even when the concentration per dry matter of each fraction was adjusted to 5 mg / ml, the 0.8M ammonia elution fraction caused the CCK secretion more than three times before the strongest fraction.
この結果より、 β コングリシニンペプチドの陽イオン交換榭脂吸着画分に強力な CCK分泌活性があることが明らかとなり、陽イオン交換榭脂を用いた本手法により、 CCK放出活性ペプチドを濃縮することが可能となった。  From this result, it was found that the cation-exchanged fat-absorbing fraction of β-conglycinin peptide has strong CCK secretion activity, and this method using cation-exchanged fat can concentrate CCK-releasing peptide. It has become possible.
[0043] 例 8 [0043] Example 8
例 7で作製した β コングリシニンのブロメライン分解物をイオン交換榭脂にて分画 した 0. 8Μアンモニア溶出画分を脱塩水に溶解し、残存アンモニア濃度を測定キット (アンモニアテストヮコ一、和光純薬工業)を用いて測定した。  The bromelain degradation product of β-conglycinin prepared in Example 7 was fractionated with ion-exchange resin. The 8% ammonia-eluted fraction was dissolved in demineralized water, and the residual ammonia concentration was measured using a kit (Ammonia Test Measured using Yakuhin Kogyo).
0. 8Μアンモニア溶出画分で 6. 06 μ g/mg, 1. OMアンモニア溶出画分で 3. 94 μ g/mgであった。納豆のアンモニア濃度は約 8 g/ml (道南平塚食品）であることか ら、このペプチド画分のアンモニア毒性は無!、と!/、える。  It was 6.06 μg / mg for the 8% ammonia elution fraction, and 3.94 μg / mg for the OM ammonia elution fraction. Since the natto ammonia concentration is about 8 g / ml (Donan Hiratsuka Foods), there is no ammonia toxicity in this peptide fraction!
[0044] この出願は、平成 17年 6月 8日出願の日本特許出願、特願 2005— 167862に基 づくものであり、特願 2005— 167862の明細書及び特許請求の範囲に記載された 内容は、すべてこの出願明細書に包含される。 [0044] This application is based on Japanese Patent Application No. 2005-167862, filed on June 8, 2005, and is described in the description and claims of Japanese Patent Application 2005-167862. Are all encompassed in this application.

Claims

請求の範囲 The scope of the claims
[1] β コングリシニンを、アルギニン残基のカルボキシル基側のペプチド結合を優先 的に切断する酵素によって分解して得られるペプチドであって、コレシストキニン分泌 促進活性または摂食抑制活性を有するペプチドを含有する組成物。  [1] A peptide obtained by degrading β-conglycinin with an enzyme that preferentially cleaves the peptide bond on the carboxyl group side of an arginine residue, and having a cholecystokinin secretion-promoting activity or a feeding-suppressing activity Containing composition.
[2] β —コングリシニンを、ブロメラインまたはプロメラインと同等の酵素活性を有する酵 素によって分解して得られるペプチドであって、コレシストキュン分泌促進活性または 摂食抑制活性を有するペプチドを含有する組成物。  [2] A composition obtained by degrading β-conglycinin with an enzyme having an enzyme activity equivalent to that of bromelain or promelain, and containing a peptide having a cholecystocone secretion-promoting activity or an eating-suppressing activity object.
[3] 前記酵素が、ブロメラインである、請求の範囲 1または 2記載の組成物。 [3] The composition according to claim 1 or 2, wherein the enzyme is bromelain.
[4] 請求の範囲 1〜3の 、ずれか 1項記載の組成物を含有する摂食抑制剤。 [4] An antifeedant comprising the composition according to any one of claims 1 to 3.
[5] 請求の範囲 1〜3の ヽずれか 1項記載の組成物を含有するコレシストキュン分泌促 進剤。 [5] A cholecystocun secretion promoter containing the composition according to any one of claims 1 to 3.
[6] 請求の範囲 4または 5記載の摂食抑制剤またはコレシストキニン分泌促進剤を含有 する医薬組成物。  [6] A pharmaceutical composition comprising the feeding inhibitor or cholecystokinin secretion promoter according to claim 4 or 5.
[7] 請求の範囲 4または 5記載の摂食抑制剤またはコレシストキニン分泌促進剤を含有 する食品。  [7] A food containing the feeding inhibitor or cholecystokinin secretion promoter according to claim 4 or 5.
[8] 請求の範囲 4または 5記載の摂食抑制剤またはコレシストキニン分泌促進剤を含有 する飼料。  [8] A feed containing the antifeedant or cholecystokinin secretion promoter according to claim 4 or 5.
[9] β —コングリシニンを、アルギニン残基のカルボキシル基側のペプチド結合を優先 的に切断する酵素によって分解する工程を含むことを特徴とする、コレシストキニン分 泌促進活性または摂食抑制活性を有するペプチド又はこのペプチドを含有する組成 物の製造方法。  [9] β-conglycinin has a cholecystokinin secretion-promoting activity or an anti-feeding activity characterized by including a step of degrading conglycinin by an enzyme that preferentially cleaves the peptide bond on the carboxyl side of the arginine residue. A method for producing a peptide having or a composition containing the peptide.
[10] β コングリシニンを、アルギニン残基のカルボキシル基側のペプチド結合を優先 的に切断する酵素によって分解する工程、および得られた分解物力ゝら陽イオン交換 榭脂吸着画分を回収する工程、を含むことを特徴とする、コレシストキュン分泌促進 活性または摂食抑制活性を有するペプチド又はこのペプチドを含有する組成物の製 造方法。  [10] A step of decomposing β-conglycinin by an enzyme that preferentially cleaves a peptide bond on the carboxyl group side of an arginine residue, and a step of recovering a cation exchange saccharose-absorbing fraction from the resulting degradation product force, A method for producing a peptide having cholecystocun secretion promoting activity or feeding inhibition activity or a composition containing this peptide, which comprises
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WO2012137825A1 (en) 2011-04-04 2012-10-11 味の素株式会社 Manufacturing method for food with enhanced taste and method for enhancing taste of food
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JP2019530730A (en) * 2016-09-07 2019-10-24 国立大学法人京都大学 Peptides and peptide conjugates for treating psychiatric disorders
JP2020079266A (en) * 2016-09-07 2020-05-28 国立大学法人京都大学 Peptides and peptide conjugates for treating mental disorders
JP7061764B2 (en) 2016-09-07 2022-05-02 国立大学法人京都大学 Peptides and Peptide Conjugates for the Treatment of Mental Illness
JP2022091961A (en) * 2016-09-07 2022-06-21 国立大学法人京都大学 Peptides and Peptide Conjugates for Treating Mental Illness

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