JP2008156307A - Angiotensin converting enzyme inhibitor, angiotensin converting enzyme inhibiting composition and food and drink containing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new and useful angiotensin converting enzyme inhibitor suitable for industrial production. <P>SOLUTION: The angiotensin converting enzyme inhibitor is composed of a peptide having either one of the following structure formulas (1) to (9); (1) Ser-Glu-Val-Tyr, (2) Ala-Val-Ser-Lys-Val-Tyr, (3) Asp-Leu-Tyr-Thr-Lys-Pro, (4) Thr-Glu-Lys-Gly-Val, (5) Ser-Ile-Val-Pro, (6) Ala-Met-Tyr, (7) Asn-Val-Pro-Leu, (8) Asp-Leu-Thr and (9) Ala-Ile-Phe. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a yeast extract, an angiotensin converting enzyme inhibitor comprising a novel peptide contained in the yeast extract, an angiotensin converting enzyme inhibitor composition containing the peptide, and a food or drink containing them.

  Hypertension, which is one of lifestyle-related diseases, has become a problem as many disease factors. Various drugs such as angiotensin converting enzyme inhibitors, β blockers, diuretics and the like have been developed as drugs that lower blood pressure. Typical drugs for angiotensin converting enzyme inhibitors include captopril and enalapril, all of which are synthetic products. It is desirable to obtain the same effect from food with high safety. From this point of view, many patent applications have been filed for angiotensin converting enzyme inhibitors and inhibitory compositions containing natural food-derived protein degradation products and peptides as active ingredients.

For example, Patent Documents 1 to 3 (microbe culture such as lactic acid bacteria) Patent Documents 4 to 7 (milk proteins such as casein), Patent Documents 8 to 17 (fish meat such as sardines), Patent Documents 18 to 19 (derived from grains such as wheat), Patent Document 20
(Egg white), patent document 21 (garlic), patent document 22-23 (mammalian blood component), patent document 24-27 (soybean), patent document 28-32 (algae such as seaweed), patent document 33-35 (royal jelly) Patent Documents 36 to 37 (fish bones) disclose angiotensin converting enzyme inhibitory peptides derived from natural products.

Japanese Patent Laid-Open No. 03-251543 Japanese Patent Laid-Open No. 06-40944 JP 2000-229996 A Japanese Patent Laid-Open No. 04-82898 JP-T-2003-513636 Japanese translation of PCT publication No. 2005-530851 WO2003 / 044044 pamphlet Japanese Patent Laid-Open No. 04-95099 Japanese Patent Laid-Open No. 06-166697 Japanese Patent Laid-Open No. 06-340692 JP 05-262790 A JP-A-11-343297 JP 2001-106699 A JP 2001-261698 A Japanese Patent Laid-Open No. 2003-24012 WO2003 / 055901 pamphlet JP 2006-56805 A Japanese Patent Laid-Open No. 04-164094 Japanese Patent Laid-Open No. 06-16568 Japanese Patent Laid-Open No. 04-20200 Japanese Patent Laid-Open No. 06-256387 JP 07-188283 A JP 07-289283 A JP 07-188282 A Japanese Patent Laid-Open No. 08-225593 Japanese Patent Laid-Open No. 08-269087 WO2002 / 055546 pamphlet JP 09-59296 A JP 2001-64299 A JP 2003-267994 A JP 2003-267993 A JP 2003-128694 A JP 2001-261168 A JP 2002-338594 A JP 2006-28065 A JP 2005-154326 A JP 2005-206469 A

In addition, it was reported that the yeast extract and Gly-Phe and Ala-Phe isolated from the extract have an angiotensin converting enzyme inhibitory action in administration tests to spontaneously hypertensive rats. ing.
Osamu Kanauchi et al. “A Yeast Extract High in Bioactive Peptides has a Blood-Pressure Lowering Effect in Hypertensive Model”, Current Medicinal Chemistry, 2005, 30, 308.

  In order to provide an angiotensin-converting enzyme inhibitor in a large amount industrially in the form of food, it is possible to provide the physiological action and safety of angiotensin-converting enzyme inhibitor, as well as the cost required for production and distribution. Furthermore, the above-mentioned conventional angiotensin converting enzyme inhibitor does not sufficiently satisfy these conditions, although it is necessary to take into account the consumer's functionality and taste. Accordingly, the present situation is that a new and useful angiotensin converting enzyme inhibitor capable of withstanding industrial production has been desired.

  As a result of intensive studies in view of the above-mentioned problems, the present inventors have shown that a proteolytic product prepared by degrading yeast protein by acting a proteolytic enzyme, particularly a neutral proteolytic enzyme, has angiotensin converting enzyme inhibitory activity. Furthermore, the present invention was completed based on the fact that several kinds of novel peptides were found in the degradation product.

  That is, the present invention relates to the following (1) to (6).

(1) An angiotensin converting enzyme inhibitor comprising a peptide having any one of the following structural formulas (1) to (9).

(1) Ser-Glu-Val-Tyr
(2) Ala-Val-Ser-Lys-Val-Tyr
(3) Asp-Leu-Tyr-Thr-Lys-Pro
(4) Thr-Glu-Lys-Gly-Val
(5) Ser-Ile-Val-Pro
(6) Ala-Met-Tyr
(7) Asn-Val-Pro-Leu
(8) Asp-Leu-Thr
(9) Ala-Ile-Phe

(2) An angiotensin converting enzyme inhibitory composition characterized by containing 2 or more types of peptides which have structural formula shown by following (1)-(9).

(1) Ser-Glu-Val-Tyr
(2) Ala-Val-Ser-Lys-Val-Tyr
(3) Asp-Leu-Tyr-Thr-Lys-Pro
(4) Thr-Glu-Lys-Gly-Val
(5) Ser-Ile-Val-Pro
(6) Ala-Met-Tyr
(7) Asn-Val-Pro-Leu
(8) Asp-Leu-Thr
(9) Ala-Ile-Phe

(3) Food / beverage products containing the angiotensin converting enzyme inhibition composition as described in (2) term.
(4) An angiotensin converting enzyme inhibitory composition comprising a proteolytic product obtained by treating yeast with a neutral proteolytic enzyme.
(5) The angiotensin converting enzyme inhibitory composition according to (4), wherein the yeast is one or more yeasts selected from the group consisting of brewing yeast, baker's yeast and torula yeast.
(6) Food / beverage products containing the angiotensin converting enzyme inhibition composition as described in the (4) term or (5) term.

  That is, the proteolysate of the present invention is produced from yeast that is generally spread and consumed daily, so there is no problem in terms of safety when used as a food ingredient, and it occurs after brewing. When yeast is used as a raw material, it can be procured at a particularly low cost, so that the production cost can be suppressed.

  In addition, the proteolytic product of the present invention has sufficient physiological action (angiotensin converting enzyme inhibitory activity, blood pressure lowering effect) in the form of the proteolytic product without particularly separating and purifying the active ingredient from the proteolytic product. Therefore, the manufacturing process can be simplified, and in addition, it is originally used as a seasoning as a yeast extract and can be provided as a food material that is also excellent in terms of sensuality.

  Furthermore, the proteolysate of the present invention can be easily processed into a powder by using a method such as spray drying or freeze drying, and its heat resistance is high, so it can be applied to foods that require heating and sterilization processes. Is possible. Furthermore, the present invention has various excellent effects such as application to a wide range of uses such as sports drinks and soups.

  Hereinafter, the present invention will be described in detail.

As the yeast used in the present invention, various yeasts such as brewing yeast, baker's yeast and torula yeast can be used, but industrially, from the viewpoint of effective utilization of by-products, brewing yeast, that is, yeast generated after brewing is used as a raw material. Is preferred.
In addition, these yeasts can be pulverized and the pulverized product can be directly subjected to an enzyme reaction. However, in order to obtain a proteolysate efficiently, it is preferable to subject the yeast as a raw material to the enzyme reaction as it is.

  As the enzyme that can be used in the present invention, various proteolytic enzymes can be used. However, in consideration of safety, commercially available food proteolytic enzymes such as Neutase [registered trademark: manufactured by Novo Industry Japan Co., Ltd.] ], Protin P [trade name: manufactured by Daiwa Kasei Co., Ltd.], protease A [registered trademark: manufactured by Amano Enzyme Co., Ltd.], YL-15 [registered trademark: manufactured by Amano Enzyme Co., Ltd.], YL-NL [registered trademark: Amano] Enzyme Co., Ltd.], Sumiteam FP [trade name: Manufactured by Shin Nippon Chemical Industry Co., Ltd.] and the like can be used.

  In addition, the reaction conditions for the enzyme reaction are basically set in consideration of the optimum reaction temperature and optimum reaction pH of the proteolytic enzyme to be used. More preferably, the conditions are such that a proteolysate having good angiotensin converting enzyme inhibitory activity can be stably obtained.

  Furthermore, it is preferable to appropriately adjust the reaction conditions by adjusting the reaction temperature to 50 ° C. or higher in order to avoid phenomena such as contamination with bacteria.

  In addition, as a method for removing undegraded yeast residues and used proteolytic enzymes in order to separate the target proteolysate from the enzyme reaction solution, it is filtered after heat treatment or isoelectric precipitation. A method or a method such as ultrafiltration can be applied, whereby a protein degradation product solution can be obtained. Furthermore, if the obtained protein degradation product solution is dried by a method such as spray drying or freeze drying, a powdered protein degradation product can be obtained.

  The angiotensin converting enzyme inhibitor of the present invention is characterized by comprising a peptide having any one of the following structural formulas (1) to (9).

(1) Ser-Glu-Val-Tyr
(2) Ala-Val-Ser-Lys-Val-Tyr
(3) Asp-Leu-Tyr-Thr-Lys-Pro
(4) Thr-Glu-Lys-Gly-Val
(5) Ser-Ile-Val-Pro
(6) Ala-Met-Tyr
(7) Asn-Val-Pro-Leu
(8) Asp-Leu-Thr
(9) Ala-Ile-Phe

  In order to isolate an angiotensin converting enzyme inhibitor represented by the structural formula of any one of (1) to (9) above from a proteolytic product obtained by treating yeast with a neutral proteolytic enzyme, a yeast suspension is used. The solution obtained by treating the protein with a neutral proteolytic enzyme is subjected to microfiltration, followed by chromatography using Dowex 50W (H +), Sephadex G-25, etc., and the active peptide fraction from the eluate. Get. Next, this peptide fraction is subjected to reverse phase HPLC treatment to obtain peptide fragments (1) to (9) having higher activity.

  The angiotensin converting enzyme inhibitory composition of the present invention is characterized by containing two or more peptides having the structural formulas represented by the following (1) to (9).

(1) Ser-Glu-Val-Tyr
(2) Ala-Val-Ser-Lys-Val-Tyr
(3) Asp-Leu-Tyr-Thr-Lys-Pro
(4) Thr-Glu-Lys-Gly-Val
(5) Ser-Ile-Val-Pro
(6) Ala-Met-Tyr
(7) Asn-Val-Pro-Leu
(8) Asp-Leu-Thr
(9) Ala-Ile-Phe

The food / beverage products of this invention contain the said angiotensin converting enzyme inhibitory composition, and as food / beverage products, drinks, such as a soft drink, a carbonated drink, a nutritive drink, a fruit drink, a lactic acid drink (Concentrated undiluted | stock solution and preparation of these drinks) Ice cream, ice sherbet, shaved ice, etc .; noodles such as buckwheat, udon, harusame, gyoza skin, shumai skin, Chinese noodles, instant noodles; rice cakes, candy, gum, chocolate, tablets , Snacks, biscuits, jelly, jam, cream, baked confectionery, etc .; fish and fishery processed foods such as kamaboko, chikuwa, ham, sausage; dairy products such as processed milk, fermented milk; margarine, mayonnaise, shortening, whipped Fat and oil processed foods such as cream and dressing; Seasonings such as sauces and sauces; Soups, stews, salads, salmon , Pickles; Other health food various forms, dietary supplements, food for specified health use; tablets, capsules, drinks, pharmaceuticals troches and the like, quasi-drugs and the like. These can be added together with auxiliary materials and additives usually used in the production.
Examples of such raw materials and additives include glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, lactose, mannitol, dextrin, citric acid, sodium citrate, tartaric acid, malic acid, succinic acid. Acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, sucrose fatty acid ester, arabic gum, carrageenan, casein, gelatin, pectin, agar, Examples include vitamin C, vitamin B group, vitamin E, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, surfactant, pigment, fragrance, and preservative.

  Examples Hereinafter, examples of the present invention will be described in detail.

After brewing angiotensin-converting enzyme inhibitory activity of yeast enzyme degradation product, brewer's yeast (15% solid content) 1,000 g was treated with protease YL-15 0.15 g, heated at 50 ° C. for 7 hours, and then soluble by centrifugation. The fraction was dried to obtain 65 g of a yeast enzyme degradation product. The angiotensin converting enzyme inhibitory activity of the obtained yeast enzyme degradation product was measured. The angiotensin converting enzyme inhibitory activity was measured using a modified Lieberman method using Hippuryl-His-Leu (hereinafter referred to as HHL) as a substrate.

  Angiotensin-converting enzyme inhibitory activity of yeast enzyme degradation product IC50 = 0.515 mg / ml

Angiotensin-converting enzyme inhibitory activity of yeast enzyme-degraded product GPC fraction The yeast enzyme-degraded product obtained in Example 1 above was suspended in water again to 10% w / v, and the molecule was analyzed by the following GPC-HPLC method. Four fractions were collected for each size. The angiotensin converting enzyme inhibitory activity of the 4 fractions thus obtained was measured. The measurement results are shown in Table 1.

Column; Amersham Biosciences Superdex Peptide HR10 / 30 (1.0x30cm)
Eluent: 30% acetonitrile / 0.1% trifluoroacetic acid Flow rate: 0.3 ml / min
Detection wavelength: 220 nm
Column temperature: 35 ° C
Chromatogram diagram; see Figure 1

Angiotensin-converting enzyme inhibitory activity of peptides contained in yeast enzyme degradation product GPC-treated fraction 3 The above-mentioned fraction 3 was dissolved in 5% acetonitrile to 1% w / v, and peptide fragments were separated under the following HPLC conditions. . The peptide fragments thus obtained were measured for angiotensin converting enzyme inhibitory activity. The amino acid sequence was determined for each of the peaks with particularly high activity, and the results shown in Table 2 were obtained.

Column: Cosmosil5C18-MS-II (Φ4.6x250mm)
Eluent: 5% -35% acetonitrile (60min)
Flow rate: 0.4ml / min
Detection wavelength: 220nm
Column temperature: 35 ° C
Chromatogram diagram; see FIGS. 2 and 3

Blood pressure lowering effect (single dose test in rats)
Twelve 16-week-old spontaneously hypertensive rats (Japan SLC) are housed in individual gauges with a temperature of 23 ± 2 ° C and humidity of 55 ± 5%, and water and feed MF food (trade name: Oriental Yeast Co., Ltd.) are free. Rats that were ingested, acclimatized for 2 weeks and whose systolic blood pressure increased to 180-200 mmHg were subjected to the test.

  1 ml of a sample (concentration: 1 g / kg body weight of rat) in which the yeast enzyme degradation product GPC fraction 3 prepared according to the method of Example 2 was dissolved in water was forcibly orally administered. As a control group, the same amount of physiological saline was forcibly administered orally.

  After administration, a non-invasive blood pressure measuring device (Softron BP-98A: manufactured by Softron) was used to measure changes in systolic blood pressure over time by the Tail-Cuff method, and the results are shown in Table 3 and FIG.

  As apparent from Table 3 above, the yeast enzyme degradation product GPC fraction 3 of the present invention showed a significant blood pressure lowering effect 6 to 9 hours after oral administration.

Blood pressure lowering effect (long-term administration test in rats)
Twelve 7-week-old spontaneously hypertensive rats (Japan SLC) are housed in individual gauges at a temperature of 23 ± 2 ° C and humidity of 55 ± 5%, and water and feed MF food (trade name: Oriental Yeast Co., Ltd.) are free. The animals were ingested and acclimatized for 1 week, and blood pressure, body weight and daily food intake were measured every week.

  A sample obtained by adding the yeast enzyme degradation product prepared according to the method of Example 1 to the MF diet so as to be 5 w / w% was freely ingested and raised from 8 weeks to 20 weeks, and blood pressure, Body weight and food intake per day were measured. As a control group, a crude protein equivalent to 5% w / w of yeast enzyme degradation product was added as 84 mg / 100 mg as milk casein, and a soluble nitrogen-free product was added as 86 mg / 100 mg as corn starch and allowed to freely ingest.

  For blood pressure measurement, after pre-incubating the rat for 10 minutes in a constant temperature bath at 40 ° C, the maximum blood pressure is measured by the Tail-Cuff method using a non-invasive blood pressure measuring device (Softron BP-98A: manufactured by Softron). did. At that time, body weight and food intake (per day) were also measured. The results are shown in Table 4 and FIG.

  As apparent from Table 4 above, the yeast enzyme degradation product of the present invention showed a significant blood pressure lowering effect from 12 weeks to 18 weeks of intake.

  Changes in body weight and food intake during the administration test are shown in FIGS. From FIG. 6 and FIG. 7, there was no significant difference between the control group and the test group.

  Functional foods containing angiotensin converting enzyme-inhibiting compositions are expected to be used in a wide range of applications, including health foods, dietary supplements, foods for specified health use, and health-oriented foods added to various beverages and foods. The

HPLC chromatogram of yeast enzyme degradation product HPLC chromatogram of peptides contained in yeast enzyme degradation product HPLC-treated fraction F3 HPLC chromatogram of peptides contained in yeast enzyme degradation product HPLC-treated fraction F3 The graph which shows the blood pressure lowering effect of yeast enzyme-degradation product HPLC processing fraction F3 (rat single administration test) Graph showing the blood pressure lowering effect of yeast enzyme degradation products (rat long-term administration test) Graph showing the effect of yeast enzyme degradation products on body weight (rat long-term administration test) Graph showing the effect of yeast enzyme degradation products on food intake (rat long-term administration test)

Claims (6)

An angiotensin converting enzyme inhibitor comprising a peptide having any one of the following structural formulas (1) to (9):
(1) Ser-Glu-Val-Tyr
(2) Ala-Val-Ser-Lys-Val-Tyr
(3) Asp-Leu-Tyr-Thr-Lys-Pro
(4) Thr-Glu-Lys-Gly-Val
(5) Ser-Ile-Val-Pro
(6) Ala-Met-Tyr
(7) Asn-Val-Pro-Leu
(8) Asp-Leu-Thr
(9) Ala-Ile-Phe An angiotensin converting enzyme inhibitory composition comprising two or more peptides having the structural formulas represented by the following (1) to (9):
(1) Ser-Glu-Val-Tyr
(2) Ala-Val-Ser-Lys-Val-Tyr
(3) Asp-Leu-Tyr-Thr-Lys-Pro
(4) Thr-Glu-Lys-Gly-Val
(5) Ser-Ile-Val-Pro
(6) Ala-Met-Tyr
(7) Asn-Val-Pro-Leu
(8) Asp-Leu-Thr
(9) Ala-Ile-Phe Food / beverage products containing the angiotensin converting enzyme inhibitory composition of Claim 2. An angiotensin converting enzyme inhibitory composition comprising a proteolytic product obtained by treating yeast with a neutral proteolytic enzyme. The angiotensin converting enzyme inhibitory composition according to claim 4, wherein the yeast is at least one yeast selected from the group consisting of brewing yeast, baker's yeast and torula yeast. Food / beverage products containing the angiotensin converting enzyme inhibition composition of Claim 4 or 5.

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