JP3997114B2 - Arginine-containing peptide having cholecystokinin secretion promoting activity and food containing the same - Google Patents

Description

【図面の簡単な説明】
【図１】 アルギニン含有ペプチドのコレシストキニン分泌促進活性
請求項１から７のペプチドを含む７種のアルギニン含有合成ペプチドを単離ラット小腸粘膜細胞と反応させた際のCCK分泌活性を表した図である。対照区（ペプチドを加えなかった実験区）でのCCK分泌量を100%として表示した。（実施例１）
【符号の説明】
ペプチド１：配列表の配列番号６記載のアミノ酸配列からなるペプチド
ペプチド２：配列表の配列番号７記載のアミノ酸配列からなるペプチド
ペプチド３：配列表の配列番号８記載のアミノ酸配列からなるペプチド
ペプチド４：配列表の配列番号９記載のアミノ酸配列からなるペプチド
ペプチド５：配列表の配列番号１０記載のアミノ酸配列からなるペプチド
ペプチド６：配列表の配列番号１１記載のアミノ酸配列からなるペプチド
ペプチド７：配列表の配列番号１２記載のアミノ酸配列からなるペプチド
【図２】 アルギニン含有ペプチドの小腸粘膜への特異的結合能
請求項１から７のペプチドを含む８種のアルギニン含有合成ペプチドとラット小腸粘膜細胞膜可溶化成分との結合量を表した図である。小腸粘膜可溶化成分との結合は分子間相互作用解析装置(BIACORE 3000)を用いて測定した。（実施例１）
【符号の説明】
- -△- -（破線）：配列表の配列番号６記載のアミノ酸配列からなるペプチド
- -□- -（破線）：配列表の配列番号７記載のアミノ酸配列からなるペプチド
- -○- -（破線）：配列表の配列番号８記載のアミノ酸配列からなるペプチド
−◇−（実線）：配列表の配列番号９記載のアミノ酸配列からなるペプチド
−△−（実線）：配列表の配列番号１０記載のアミノ酸配列からなるペプチド
−□−（実線）：配列表の配列番号１１記載のアミノ酸配列からなるペプチド
−○−（実線）：配列表の配列番号１２記載のアミノ酸配列からなるペプチド
- -×- -（破線）：配列表の配列番号１３記載のアミノ酸配列からなるペプチド
【図３】 大豆β−コングリシニンおよびそのペプシン分解物の小腸粘膜への特異的結合
β-コングリシニン及びそのペプシン分解物とラット小腸粘膜細胞膜可溶化成分との結合量を表した図である。小腸粘膜可溶化成分との結合は分子間相互作用解析装置(BIACORE 3000)を用いて測定した。（実施例２）
【符号の説明】
−○−：大豆β-コングリシニン
−●−：大豆β-コングリシニンのペプシン分解物
【図４】 大豆β−コングリシニンおよびそのペプシン分解物のコレシストキニン分泌促進活性
β-コングリシニン及びそのペプシン分解物を単離ラット小腸粘膜細胞と反応させた際のCCK分泌率を表した図である。（実施例２）
【符号の説明】
＊：コントロール群に対し危険率５％で有意差有り
【図５】 ラット血中におけるコレシストキニン濃度
ラット小腸にβ-コングリシニンペプシン分解物溶液を投与して45分後の門脈血中CCK濃度を表した図である。（実施例２）
【符号の説明】
＊：コントロール群に対し危険率５％で有意差有り
【図６】 摂食量抑制効果
β-コングリシニンペプシン分解物溶液を小腸に投与したラットの一時間の摂食量を表した図である。（実施例２）
【符号の説明】
＊：コントロール群に対し危険率５％で有意差有り
【図７】 大豆βコングリシニンのアルギニン含有ペプチド部分の小腸粘膜との特異的結合活性
大豆タンパク質ペプシン分解物、大豆β-コングリシニンのペプシン分解物、および請求項８から１２のペプチドを含む６種のアルギニン含有ペプチドとラット小腸細胞膜可溶化成分との結合を表した図である。小腸粘膜可溶化成分との結合は分子間相互作用解析装置(BIACORE 3000)を用いて測定した。なお、各ペプチド溶液は100 micro g/mlの濃度で使用した。（実施例３）
【符号の説明】
ペプチドＡ：大豆タンパク質ペプシン分解物
ペプチドＢ：大豆β-コングリシニンのペプシン分解物
ペプチドＣ：配列表の配列番号１記載のアミノ酸配列からなるペプチド
ペプチドＤ：配列表の配列番号２記載のアミノ酸配列からなるペプチド
ペプチドＥ：配列表の配列番号３記載のアミノ酸配列からなるペプチド
ペプチドＦ：配列表の配列番号１４記載のアミノ酸配列からなるペプチド
ペプチドＧ：配列表の配列番号４記載のアミノ酸配列からなるペプチド
ペプチドＨ：配列表の配列番号５記載のアミノ酸配列からなるペプチド [0001]
[Technical field to which the invention belongs]
  The present invention relates to a food material that exerts an appetite suppressing action by promoting the secretion of cholecystokinin, which is a gastrointestinal hormone.
[0002]
[Prior art]
  Cholecystokinin (hereinafter referred to as CCK) is a gastrointestinal hormone secreted from duodenal mucosal cells, secreted with fat intake, and promotes gallbladder contraction and pancreatic enzyme secretion. It is a straight-chain polypeptide and a hormone consisting of 33 amino acid residues, and the C-terminal octapeptide is particularly active.
[0003]
  It is known that the physiological action of CCK has the effect of suppressing gastric emptying of foods eaten, or the ability to promote pancreatic enzyme secretion, and it also gives a feeling of satiety to suppress eating behavior. (Weller, A. et al., Science, 247, 1589-1591, 1990, and Woltman, T and Reidelberger, R. American Journal of Physiology, 276, R1701-R1709, 1999). It is also known to stimulate the secretion of insulin, a hormone that regulates blood sugar levels (Rossetti, L. et al., Diabetes, 36, 1212-1215, 1987, and Ruchakoff, RJ et al., Journal of Clinical Endocrinological Metabolism, 65, 395-401, 1987). There are examples of using these actions to prevent and treat lifestyle-related diseases such as obesity, diabetes, and pancreatitis. Conventionally, there was only a method of administering CCK from outside the body into the blood vessel. For that purpose, when CCK was administered by injection or the like, a large amount of CCK exceeding the physiological level was required. In addition, when daily administration is required for disease treatment, there is a disadvantage that the cost becomes large in addition to the complexity of daily administration.
[0004]
  In contrast to the method of administering CCK from outside the body, there is also a means of utilizing endogenous CCK secreted from CCK producing cells in the small intestinal mucosa by dietary protein (peptide), amino acid or fatty acid. A method that uses food is less expensive than medicine. However,fatty acid,In other words, since lipid intake itself is a risk factor for lifestyle-related diseases, it was inappropriate to use it as a stimulating factor for endogenous CCK secretion. Appropriate with CCK secretion promoting action to make an appetite-suppressing foodFood materialdevelopment ofButIt was sought after.
[0005]
  Regarding the endogenous CCK secretion action by proteins and their degradation peptides, the existence of a mechanism that promotes CCK secretion by directly acting on proteins and peptides themselves in the small intestinal cells has recently been proposed (Ritter, R.C., et al.,Neuropeptides 33, 387-399, 1999). At present, the mechanism of intracellular signal transduction in CCK secretion by direct action of food-based proteins is gradually becoming clear by research using cultured cells. However, few approaches have been taken from the side of orally ingested proteins that act on cells, especially the amino acid sequences that make up proteins, and food-derived functional peptides that have CCK secretion activity via small intestinal cell stimulation. Development was delayed.
[0006]
[Problems to be solved by the invention]
  The problem to be solved is to promote the secretion of endogenous CCK secreted from the small intestinal mucosa and have an appetite suppressing effectFood materialIs safe and cheap to developFood materialIt must be.
[0007]
[Means for Solving the Problems]
  The main feature of the present invention is to use a specific amino acid sequence, that is, an arginine-containing peptide in which arginine is bound at a specific position, in order to promote CCK secretion and thereby exert an appetite suppression function. Furthermore, the present invention is characterized in that an arginine-containing peptide having a specific amino acid sequence is also found in a conventional food material, and is made into a food material having an appetite suppressing action by a specific processing method.
  That is, the present invention provides [1] a specific oligopeptide selected from the following:
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in the sequence listing wherein arginine is located at the 3rd and 5th positions from the N-terminus of the sequence of 7 consecutive amino acids;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing wherein arginine is located at the 2nd and 5th positions from the N-terminus of the sequence of 7 consecutive amino acids;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing wherein arginine is located at the 2nd and 6th positions from the N-terminus of the sequence of 7 consecutive amino acids;
Sequence listing wherein arginine is located at the 2nd, 4th and 6th positions from the N-terminus of the sequence of 7 consecutive amino acidsArrangement ofAn arginine-containing peptide comprising the amino acid sequence of column number 13;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 5 in the Sequence Listing;
An arginine-containing peptide comprising the amino acid sequence set forth in SEQ ID NO: 14 in the Sequence Listing;
SEQ ID NOs: 1 to 5 in the sequence listing, 9-11, 13 andAny one amino acid sequence selected from 14ColumnAn appetite-suppressing peptide having cholecystokinin secretion promoting activity or binding ability to a small intestinal brush border membrane soluble component, [2〕BigThe peptide obtained from the amino acid sequence of bean β-conglycinin, [3An appetite-suppressing food material containing one or more of such peptides,4The food material which is a pepsin degradation product of soybean β-conglycinin, and [5An food for suppressing appetite containing such a food material is provided.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
  Research into factors that promote CCK secretion from small intestinal mucosal cells, and to promote CCK secretion triggered by the specific binding of arginine-containing peptides with specific amino acid sequences to intestinal cells. discovered. It was clarified that these arginine-containing peptides have an appetite suppressing effect by promoting CCK secretion. Furthermore, it was clarified that arginine-containing peptides having these specific amino acid sequences exist in conventional food materials. Using these discoveries, we can develop food ingredients that have an appetite-suppressing effect.,Realized by processing conventional food ingredients.
  Based on the inventor's series of research results, ingested proteins and peptides themselves directly bind to the digestive tract and promote CCK secretion.DoI found the existence of the mechanism. There has been no report in the past that a digestive tract directly recognizes a protein or peptide to express some physiological phenomenon. This is a new possibility of proteins and peptides from the viewpoint of the bioregulatory function that is the tertiary mechanism of food.
[0009]
[Example 1]
  In order to examine the CCK secretion promoting activity of various peptides,Sequence number 6-12 description of a sequence tableThe amount of CCK secreted from the small intestinal cells was measured by reacting seven peptides corresponding to the amino acid sequence with isolated rat small intestinal mucosal cells.
[0010]
  Arginine-containing peptides used in the experiment are the following seven types of peptides. All were synthesized into each sequence, and there were no peptides other than this sequence in each experimental group.
Peptide 1,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing, (GGGRGGG)
Peptide 2,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing, (GGGGGGR)
Peptide 3,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing, (GGGRRGG)
Peptide 4,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in the Sequence Listing, (GGRGRGG)
Peptide 5,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing, (GRGGRGG)
Peptide 6,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 in the Sequence Listing, (GRGGGRG)
Peptide 7,A peptide comprising the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing, (GRGRGRG)
[0011]
  Isolation and preparation of small intestinal mucosal cells: All experiments used Sprague-Dawley male rats with an initial weight of about 250 g. A 25% casein diet was fed as a feed, and the animals were bred with a 12-hour light-dark cycle. Rats fasted overnight were sacrificed, and the small intestine of 20 cm was removed from the common bile duct opening of the duodenum and washed with saline in the lumen. Invert the small intestine and add 10 ml Krebs-Henseleit bicarbonate (KHB) solution containing 10 mM EDTA (37℃, PH 7.4, oxygenated) 37℃Incubated for 5 minutes with shaking. After the incubation was completed, the small intestinal mucosa was peeled off using a plastic stick, mixed with the KHB solution and stirred well, and then centrifuged at 500 rpm for 3 minutes. The supernatant was removed, and the precipitate was washed with 5 ml of KHB solution (containing EDTA, 37℃, PH 7.4, oxygenated), and use a Pipetman tip to disperse the cells.℃Incubated for 5 minutes with shaking, and after the reaction, centrifuged at 500 rpm for 3 minutes. This operation was performed twice to prepare isolated small intestinal mucosal cells. Combine the small intestinal mucosa cells of three rats into one, and add 200 times the amount of HEPES buffer (37℃, PH 7.4, oxygen aerated). 1 hour 37℃After equilibrating the cells by incubating with shaking, the buffer was replaced with fresh.
[0012]
  Method of reaction between test sample and small intestinal mucosal cellsPurified water (Milli-Q water)1 ml of the solution dissolved in each was put into a plastic vial and freeze-dried to remove water. Add 1 ml of small intestinal mucosal cell suspension and add 37℃Incubated for 30 minutes with shaking. After completion of the incubation, the contents were transferred to a small centrifuge tube and centrifuged at 10,000 rpm for 7 seconds, and 0.7 ml of the supernatant was collected. 0.5 ml of the solution was passed through a Sep-pak C18 cartridge, and CCK was extracted with 50% acetonitrile. The recovered CCK was lyophilized and quantified by a bioassay method using pancreatic acinar cells.
[0013]
  As a result of the CCK secretion promotion test using an oligopeptide, CCK secretion was promoted in any peptide-added group as compared to the control group (experiment group in which no peptide was added) (FIG. 1). In particular, CCK secretion was strongly promoted in peptides 4, 5, 6, and 7.
[0014]
  One important finding in the inventors' research is the promotion of CCK secretionDoIt was discovered that the substance specifically binds to the small intestinal brush border membrane solubilizing component.
  Binding between various peptides and small intestinal brush border membrane solubilized components was measured using an intermolecular interaction analyzer (BIACORE3000) (Hira, T. et al., Bioscience Biotechnology Biochemistry, 65, 1007-1015, 2001) . Cell membrane solubilized components recovered from rat small intestinal mucosal cells were immobilized on a sensor chip of an intermolecular interaction analyzer, and various peptides were flowed there to measure the amount of binding to cell membrane components. In all experiments, male Sprague-Dawley rats with an initial weight of about 250 g were used. A 25% casein diet was fed as a feed, and the animals were bred with a 12-hour light-dark cycle. Brush border membrane vesicles obtained by applying calcium chloride and KSCN treatment and ultracentrifugation to mucous membrane of rat jejunum were added to Triton X-100 to a concentration of 0.1% and stirred well.℃After shaking for 1 hour, the supernatant obtained by ultracentrifugation was used as a small intestine brush border membrane solubilizing component. Triton X-100 was removed from the solubilized components using Exratcti Gel D affinity Pak column, and then diluted with 10 mM acetate buffer (pH 4.0) was immobilized on sensor chip CM5. In the binding test, various peptides were dissolved in HEPES buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, pH 7.4) at various concentrations (0, 50, 100, 200, 500 microg / ml) It was injected into the intermolecular interaction analyzer (BIACORE3000).
[0015]
  The results of the binding test between various arginine-containing peptides and the small intestinal brush border membrane solubilizing component are shown in FIG. In the figure, four types of arginine-containing peptides (peptide 4:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in the sequence listing (GGRGRGG)Peptide 5:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing (GRGGRGG)Peptide 6:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 in the sequence listing (GRGGGRG)And peptide 7:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 12 in the sequence listing (GRGRGRG)) Was observed to bind to the small intestinal membrane component. In contrast, peptide 8 in which the glycine part of peptide 7 is replaced with proline.(Peptide consisting of the amino acid sequence described in SEQ ID NO: 13 in the Sequence Listing)It was shown that the binding activity was present although the binding amount was low.
[0016]
  These results indicate that a peptide having a structure with multiple arginines and sandwiching another amino acid between them has the ability to directly bind to the small intestinal cell membrane, and its direct action stimulates CCK secretion. It has been suggested.
  In addition, these results indicate that the strength of CCK secretion promoting activity correlates with the activity of the peptide binding to specific receptors present on the surface of rat small intestinal mucosal cells. It was also found that this binding activity can be measured strictly using an intermolecular interaction analyzer.
[0017]
[Example 2]
  Among the naturally occurring food peptidesSequence number 6-12 description of a sequence tableAs for soybean β-conglycinin having a structure similar to the arginine-containing peptide in the molecule, (1) binding activity with a solubilized component of the small intestine brush border membrane, (2) CCK secretion promoting activity, and (3) feeding The inhibitory effect was investigated.
In all experiments, male Sprague-Dawley rats with an initial weight of about 250 g were used. A 25% casein diet was fed as a feed and reared in a 12-hour light-dark cycle.
[0018]
  Isolation of β-conglycinin and preparation method of pepsin degradation product: The defatted soybean powder was mixed with 15 volumes of Tris-HCl buffer (pH 8.0) and stirred at room temperature for 1 hour. After gauze filtration, centrifuge (8,000 rpm, 20 minutes, 4℃And the supernatant was collected. After adjusting the supernatant to pH 6.0, leave it overnight in a cold room and centrifuge again (8,000 rpm, 20 minutes, 4℃And the supernatant was collected. Subsequently, the supernatant is adjusted to pH 4.8 and centrifuged (8,000 rpm, 30 minutes, 4℃And the precipitate was recovered. The obtained precipitate was dissolved in a standard buffer (35 mM Potassium phosphate, 0.4 M NaCl, 10 mM 2-mercaptoethanol, pH 7.6) so as to have a concentration of 3%, and left overnight in a low temperature room. To this, ammonium sulfate was added to a concentration of 75%, and ammonium sulfate was further added to the solubilized component to a concentration of 90%. The obtained precipitate was dissolved in Milli-Q water, dialyzed against running water, and then freeze-dried to obtain β-conglycinin. This β-conglycinin 2g was mixed with 100 times the amount of phosphoric acid solution, adjusted to pH 1.85, 37℃Kept warm. Add 1 ml of pepsin solution (20 mg / ml),℃And incubated for 10 minutes with shaking. After completion of the reaction, the solution was boiled to inactivate pepsin. After cooling the solution, centrifuge (3,000 rpm, 20 minutes, 4℃And the supernatant was collected. After neutralizing the supernatant with calcium hydroxide, centrifugation (3,000 rpm, 20 minutes, 4℃The salt was removed, and the β-conglycinin / pepsin degradation product was recovered by lyophilization.
[0019]
  Binding test method for β-conglycinin pepsin degradation product and small intestinal brush border membrane solubilization component: Binding between β-conglycinin pepsin degradation product and small intestinal brush border membrane solubilization component is an intermolecular interaction analyzer (BIACORE 3000) It measured using. Brush border membrane vesicles obtained by applying calcium chloride and KSCN treatment and ultracentrifugation to mucous membrane of rat jejunum were added to Triton X-100 to a concentration of 0.1% and stirred well.℃After shaking for 1 hour, the supernatant obtained by ultracentrifugation was used as a small intestine brush border membrane solubilizing component. Triton X-100 was removed from the solubilized components using Exratcti Gel D affinity Pak column, and then diluted with 10 mM acetate buffer (pH 4.0) was immobilized on sensor chip CM5. The pepsin degradation product of β-conglycinin is dissolved in HEPES buffer (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, pH 7.4) at a concentration of (0, 50, 100, 250, 500 micro g / ml), respectively. Was injected into the intermolecular interaction analyzer (BIACORE3000). Β-conglycinin that was not subjected to pepsin degradation was also used as the analyte.
[0020]
  The result of the binding test between the pepsin degradation product of β-conglycinin and the small intestinal brush border membrane solubilizing component is shown in FIG. Both undegraded β-conglycinin and pepsin degradation products of β-conglycinin showed a dose-dependent binding to small intestinal membrane components. However, β-conglycinin pepsin degradation product is 3 to 4 times higher than undegraded β-conglycinin, indicating that pepsin degradation increases the binding ability of β-conglycinin to the small intestinal cell membrane. It was.
[0021]
  Method for promoting CCK secretion in vitro by pepsin degradation product of β-conglycinin: (1) Isolation and preparation of small intestinal mucosal cells: After killing overnight fasted rats, 20 cm small intestine starting from the common bile duct opening of the duodenum Was removed and washed with physiological saline in the lumen. Invert the small intestine and add 10 ml Krebs-Henseleit bicarbonate (KHB) solution containing 10 mM EDTA (37℃, PH 7.4, oxygen aerated) 37℃Incubated for 5 minutes with shaking. After completion of the reaction, the small intestinal mucosa was peeled off using a plastic stick, mixed with the KHB solution, stirred well, and then centrifuged at 500 rpm for 3 minutes. The supernatant was removed, and the precipitate was washed with 5 ml of KHB solution (containing EDTA, 37℃, PH 7.4, oxygen-vented) and disperse the cells using a Pipetman tip.℃And incubated for 5 minutes with shaking, and after completion, the mixture was centrifuged at 500 rpm for 3 minutes. This operation was performed twice to prepare isolated small intestinal mucosal cells. Combine the small intestinal mucosa cells of three rats into one, and add 200 times the amount of HEPES buffer (37℃, PH 7.4, oxygen aerated). 1 hour 37℃After equilibrating the cells by incubating with shaking, the buffer was replaced with fresh.
[0022]
  (2) Method of reaction between test sample and small intestinal mucosa cells: 1 ml each of undegraded β-conglycinin and β-conglycinin pepsin degradation product dissolved in milli-Q water (200 or 500 microg / ml) It was placed in a plastic vial and freeze-dried to remove water. Add 1 ml of small intestinal mucosal cell suspension and add 37℃Incubated for 30 minutes with shaking. After completion, the contents were transferred to a small centrifuge tube and centrifuged at 10,000 rpm for 7 seconds, and 0.7 ml of supernatant was collected. 0.5 ml of the solution was passed through a Sep-pak C18 cartridge, and CCK was extracted with 50% acetonitrile. The recovered CCK was quantified by a bioassay method using pancreatic acinar cells after lyophilization.
[0023]
  The results of an in vitro CCK secretion enhancement test using a pepsin degradation product of β-conglycinin are shown in FIG. Although undegraded β-conglycinin showed some CCK secretion in the reaction with small intestinal cells, no significant difference was observed compared with the amount of CCK secretion in the control group. On the other hand, in the pepsin degradation product of β-conglycinin, a significantly larger amount of CCK secretion was observed compared with the amount of CCK secretion in the control. It was proved that the pepsin degradation product of soybean β-conglycinin has CCK secretion promoting activity.
  Furthermore, when the amount of food intake of rats administered with the synthetic trypsin inhibitor camostat (0.25 microg / ml) was measured, the amount of CCK secreted by undegraded β-conglycinin was further decreased, It was not recognized at all. On the other hand, the CCK secretion activity in the pepsin degradation product of soybean β-conglycinin was the same as that in the absence of camostat, and showed strong secretion promoting activity.
[0024]
  A pepsin degradation product of β-conglycinin, which showed strong CCK secretion-promoting activity in in vitro experiments, showed an increase in blood CCK concentration even in an in vivo model using experimental animals, which is one of the physiological functions of CCK. Demonstrate the effect of suppressing food.
[0025]
  Test method for inhibition of eating by β-conglycinin pepsin degradation product: (1) Surgical preparation of rats: SD male rats were selected as experimental animals, and catheters were placed in the duodenum before the experiment. Rats fasted overnight were anesthetized, and a catheter was inserted into the duodenum, 1 cm below the pylorus. The inserted catheter was fixed to the abdominal wall, then passed through under the skin, then taken out from the base of the back neck and protected by a vinyl chloride protector. Rats were used for experiments after a one week recovery period.
  (2) Feeding suppression test method: Rats fasted overnight were administered 2.5 ml of a 2 mg / ml β-conglycinin pepsin degradation product aqueous solution into the duodenum using a syringe pump at a flow rate of 0.5 ml / min. Rats were allowed to freely take a 25% casein diet for 1 hour from 15 minutes after the administration, and the weight of the fed food was measured. On the last day of the experiment, portal vein blood was collected under ether anesthesia for 45 minutes after administration of an aqueous solution of β-conglycinin pepsin degradation product. The CCK concentration in plasma was quantified by a bioassay method using pancreatic acinar cells.
[0026]
  A β-conglycinin / pepsin degradation product aqueous solution was administered, and after 45 minutes, the rat was laparotomized, and portal blood was collected and centrifuged immediately to collect plasma. As a result, a significant increase in blood CCK concentration was observed in the group administered with the pepsin degradation product of β-conglycinin compared with the control group (FIG. 5).
[0027]
  Changes in food intake were also significant. The aqueous solution of β-conglycinin / pepsin degradation product was fed to the duodenum at a flow rate of 0.5 ml 15 minutes after feeding and fed for 1 hour, and then the food was collected and the amount of food intake was measured. As a result, a significant decrease in food intake was observed in the β-conglycinin pepsin degradation product administration group compared to the solvent administration group (FIG. 6). In addition, it was also found that duodenal administration of a β-conglycinin pepsin degradation product causes feeding suppression even as early as 30 minutes.
[0028]
[Example 3]
  The following experiment proved that the arginine localization site in the amino acid sequence of soybean β-conglycinin showed extremely high binding activity to the rat small intestinal cell membrane and also had a strong feeding-suppressing effect. The experimental method is the same as in Example 2.
[0029]
  From the amino acid sequence of β-conglycinin, peptides in which arginine was localized were synthesized, and their binding to the rat small intestinal cell membrane solubilizing component and feeding inhibition were evaluated. The peptides subjected to the experiment are as follows. Peptide A is soybean peptone, and peptide B is a pepsin degradation product of soybean β-conglycinin. Peptide C is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing., Β-It is the 51st to 63rd sequence of the β subunit of conglycinin. Peptide D is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 in the Sequence Listing., Β-It is the 53rd to 63rd sequence of the β subunit of conglycinin.
  Peptide E is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing., Β-It is the 51st-59th arrangement | sequence of the beta subunit of conglycinin. Peptide F is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing,Arginine is located at the 2nd and 6th positions from the N-terminus of the sequence of 7 consecutive amino acids.An array of the smallest unit that satisfies the condition. Peptide G is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 4 in the Sequence Listing., ΒThe sequence of the α subunit of conglycinin. Peptide H is, ArrangementA peptide consisting of the amino acid sequence set forth in SEQ ID NO: 5, ΒThe sequence of the α-prime subunit of conglycinin. Each peptide solution was used at a concentration of 100 microg / ml.
[0030]
Results of measurement of binding to solubilized small intestinal mucosa using an intermolecular interaction analyzer (BIACORE 3000), Β-A peptide having the sequence 51-63 of the β subunit of conglycinin (refer to the amino acid sequence described in SEQ ID NO: 1 in the sequence listing) is about 3.5 times the β-conglycinin pepsin degradation product, soy protein pepsin degradation product It showed a large binding activity of about 15 times (FIG. 7). The binding activity isArginine is located at the 2nd and 6th positions from the N-terminus of the sequence of 7 consecutive amino acids.A peptide comprising the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing of the smallest unit that satisfies the conditions(Ile-Arg-Leu-Leu-Gln-Arg-Phe)However, it is similar to β-conglycinin pepsin degradation product,Arginine is located at the 3rd and 5th positions from the N-terminus of the sequence of 7 consecutive amino acids, and 7 consecutive arginines are located at the 2nd and 5th position from the N-terminus of the sequence of 7 consecutive amino acids Arginine is located at the second and sixth positions from the N-terminus of the amino acid sequence ofIt has been found that the amount of binding to the cell membrane component increases as the number of portions satisfying the condition increases.
[0031]
Of the amino acid sequence described in SEQ ID NO: 1 in the sequence listingAmino acid sequences similar to peptides are also present in β-conglycinin α subunit (see amino acid sequence described in SEQ ID NO: 4 in the sequence listing) and α prime subunit (see amino acid sequence described in SEQ ID NO: 5 in the sequence listing). Exists. The ability to bind to these cell membrane components was also examined. As a result, it was found that the binding amount was higher than that of the β-conglycinin pepsin degradation product, and the CCK secretion promoting effect could be expected. However, the binding activity per unit weight isOf the amino acid sequence described in SEQ ID NO: 1 in the sequence listingIt was about half compared with the peptide (FIG. 7).
[0032]
  These peptides were examined for feeding suppression effects. Food consumption was significantly reduced by duodenal administration of the peptide solution.
[0033]
【The invention's effect】
  When the oligopeptide of the present invention is ingested and reaches the small intestine, the secretion of cholecystokinin (CCK) is remarkably promoted. As a result, CCK acts on the brain center and causes an antifeedant effect. At the same time, acting on the stomach to suppress gastric excretionConductGives a feeling of fullness. By taking advantage of this property of the present invention, such as overeating preventive foods, eating appetitesuppressFood andFood materialCan be a food that prevents obesity and bulimia.
[0034]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 Cholecystokinin secretion promoting activity of arginine-containing peptides
  It is a figure showing the CCK secretion activity at the time of making seven types of arginine containing synthetic peptides containing the peptide of Claims 1-7 react with an isolated rat small intestinal mucosa cell. The amount of CCK secretion in the control group (experiment group in which no peptide was added) was displayed as 100%. Example 1
[Explanation of symbols]
Peptide 1:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing
Peptide 2:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing
Peptide 3:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing
Peptide 4:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in the Sequence Listing
Peptide 5:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing
Peptide 6:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 in the Sequence Listing
Peptide 7:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing
[Figure 2] Specific binding ability of arginine-containing peptides to the small intestinal mucosa
  It is a figure showing the binding amount of eight kinds of arginine containing synthetic peptides containing the peptide of Claims 1-7, and a rat small intestine mucosa cell membrane solubilization component. The binding to the small intestinal mucosa solubilizing component was measured using an intermolecular interaction analyzer (BIACORE 3000). Example 1
[Explanation of symbols]
--△--(Dashed line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing
--□--(Dashed line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 7 in the sequence listing
--○--(Dashed line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 8 in the sequence listing
-◇-(solid line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 9 in the Sequence Listing
-△-(solid line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 10 in the sequence listing
-□-(solid line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 11 in the Sequence Listing
-○-(solid line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing
--×--(dashed line):A peptide comprising the amino acid sequence set forth in SEQ ID NO: 13 in the sequence listing
FIG. 3 Specific binding of soybean β-conglycinin and its pepsin degradation product to the small intestinal mucosa.
  It is a figure showing the binding amount of β-conglycinin and its pepsin degradation product and rat small intestinal mucosa cell membrane solubilizing component. The binding to the small intestinal mucosa solubilizing component was measured using an intermolecular interaction analyzer (BIACORE 3000). (Example 2)
[Explanation of symbols]
-○-: Soybean β-conglycinin
-●-: Pepsin degradation product of soybean β-conglycinin
FIG. 4 Cholecystokinin secretion promoting activity of soybean β-conglycinin and its pepsin degradation product
  It is a figure showing the CCK secretion rate at the time of making beta-conglycinin and its pepsin degradation product react with an isolated rat small intestinal mucosa cell. (Example 2)
[Explanation of symbols]
*: Significant difference at 5% risk with respect to control group
FIG. 5: Cholecystokinin concentration in rat blood
  It is a figure showing the CCK density | concentration in portal vein blood 45 minutes after administering the beta-conglycinin pepsin degradation product solution to a rat small intestine. (Example 2)
[Explanation of symbols]
*: Significant difference at 5% risk with respect to control group
[Fig. 6] Food intake suppression effect
  It is a figure showing the food intake per hour of the rat which administered the beta-conglycinin pepsin degradation product solution to the small intestine. (Example 2)
[Explanation of symbols]
*: Significant difference at 5% risk with respect to control group
FIG. 7 Specific binding activity of arginine-containing peptide part of soybean β-conglycinin with small intestinal mucosa.
  It is a figure showing the coupling | bonding of the soy protein pepsin degradation product, the pepsin degradation product of soybean beta-conglycinin, and six types of arginine containing peptides containing the peptide of Claims 8-12, and a rat small intestine cell membrane solubilization component. The binding to the small intestinal mucosa solubilized component was measured using an intermolecular interaction analyzer (BIACORE 3000). Each peptide solution was used at a concentration of 100 microg / ml. (Example 3)
[Explanation of symbols]
Peptide A: Soy protein pepsin degradation product
Peptide B: Soybean β-conglycinin degradation product of pepsin
Peptide C:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing
Peptide D:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing
Peptide E:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 3 in the sequence listing
Peptide F:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 14 in the sequence listing
Peptide G:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 4 in the sequence listing
Peptide H:A peptide comprising the amino acid sequence set forth in SEQ ID NO: 5 in the sequence listing

Claims (5)

SEQ ID NO: 1-5 of the Sequence Listing, appetite capable of binding to any one of the amino acid sequence or Rannahli, cholecystokinin secretion promoting activity or small intestine brush Enmaku soluble component selected from 9～11,13 and 14 Inhibitory peptide. Amino acid sequences are obtained from soybean β- conglycinin amino acid sequence of claim 1, wherein the peptide. An appetite-suppressing food material containing one or more of the peptides according to claim 1 or 2 . The food material according to claim 3 , which is a pepsin degradation product of soybean β-conglycinin. An appetite-suppressing food containing the food material according to claim 4 .

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