JP2013241470A - Peptide-containing composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel ACE (Angiotensin Converting Enzyme) inhibitory peptide which effectively inhibits ACE with a small amount of ingestion without a side effect and can be easily orally ingested by a hypertensive person in an ordinary life, and to provide a composition containing the peptide.SOLUTION: A composition is provided, which contains a peptide Tyr-Thr having an angiotensin converting enzyme inhibitory activity, and its salt.

Description

  The present invention relates to novel peptides that exhibit the function of lowering blood pressure by inhibiting angiotensin converting enzyme, compositions containing these peptides, and methods for producing the same.

  Angiotensin-converting enzyme (hereinafter abbreviated as ACE) is a kind of carboxypeptidase present in an animal body, and cleaves one of the peptide bonds of angiotensin I to produce angiotensin II. Angiotensin II is a peptide hormone having a strong pressor action and causes an increase in blood pressure through vasoconstriction and aldosterone secretion promotion. Therefore, if ACE activity is inhibited, the production of angiotensin II can be suppressed, and hypertension can be treated. So far, pharmaceuticals such as captopril have been put into practical use and widely used as ACE inhibitors. However, since these drugs have a strong action, there is a concern about side effects, and many ACE inhibitors require attention to side effects such as dry cough.

  On the other hand, since angiotensin I, which is a substrate for ACE, is a peptide, research has been conducted to competitively inhibit ACE with a natural product-derived peptide and to provide an antihypertensive agent having no side effects (for example, patent documents). 1-5). These peptides are expected to be highly safe because of their mild action, but in order to ingest an effective amount, a relatively large amount of the peptide or a composition containing the peptide must be ingested. There's a problem. Furthermore, many of these peptides have high overall hydrophobicity, and such peptides are generally said to have a strong bitter taste (see, for example, Patent Document 6 and Non-Patent Documents 1 and 2). There is also a problem that it is difficult to take orally.

JP-A-4-091097 Japanese Patent Laid-Open No. 5-262790 JP-A-6-040944 JP 7-188282 A JP 10-175997 A JP 2006-75064 A

Wenyi Wang et al. Comprehensive Reviews in Food Science and Food Safety, 2005, (4), p. 63-78. Written by Masao Fujimaki et al., “Revised New Food Chemistry”, Asakura Shoten, March 1976, p. 117-118

  The problems to be solved by the present invention are novel ACE-inhibiting peptides that effectively inhibit ACE with a small amount of intake, can be easily taken orally in daily life by hypertensives, and these peptides. And a method for producing the same.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found a peptide having a chemical structure (amino acid sequence) that has not been known to have an ACE inhibitory action so far and that has an extremely strong action. The headline, the present invention has been reached.

That is, the present invention
1) A composition comprising a peptide Tyr-Thr having angiotensin converting enzyme inhibitory activity and a salt thereof,
2) The composition according to 1) above, which has an action of alleviating symptoms of hypertension,
3) The method for producing the composition as described in 1) above, wherein the soybean is mixed and stirred together with the koji mold culture at 25 ° C or higher.
Is to provide.

  According to the present invention, novel ACE-inhibiting peptides that effectively inhibit ACE with a small amount of intake, have no worry of side effects, and can be easily taken orally in daily life, and compositions containing these peptides, and The manufacturing method was provided.

It is a figure which shows the UV peak at the time of isolating Tyr-Thr from the composition 1. FIG. It is a figure which shows the change of the blood-pressure value at the time of administering the composition 1 to a hypertensive rat for a long term.

Hereinafter, the present invention will be described in detail.
The peptide Tyr-Thr of the present invention can be produced, for example, by a chemical synthesis method. As a well-known method, for example, the amino group of the amino terminal amino acid is protected with a benzyloxycarbonyl group, the carboxyl group is activated with a p-nitrophenyl ester group, and condensed with the carboxyl terminal amino acid in the presence of triethylamine. After that, a liquid phase method in which the protective group is removed by catalytic reduction or trifluoroacetic acid, and an amino acid at the carboxyl terminal side is bound to a polymeric solid support to protect the amino group and activate the carboxyl group There is a solid phase method in which the amino acid side chain protecting group is removed by sequentially cleaving from the solid phase support using trifluoroacetic acid, hydrogen fluoride, etc. be able to. The same effect can be expected not only with a peptide alone but also with a salt with a physiologically acceptable ion.

  The peptide of the present invention can also be produced by separating and purifying the protein containing the amino acid sequence from a composition obtained by hydrolyzing the protein with an appropriate protease agent using column chromatography or the like. In this case, the desired effect can be obtained even when ingested in the form of a composition containing the peptide. Compared with the above-mentioned chemical synthesis method, mass production is easy and production cost is low, which is more preferable.

  As a protein source for obtaining the peptide of the present invention by protein hydrolysis, any protein source may be used as long as the object of the present invention can be achieved, but a leguminous plant is preferably used. More preferably, soybean is preferably used because it has a large amount of cultivation, a low price, a high protein content, a rich dietary experience, and a good taste of the composition after decomposition. Examples of soybeans include yellow soybeans, red soybeans, and black soybeans, with yellow soybeans being particularly preferable. As an aspect of soybean, whole soybean (full fat soybean), defatted soybean, refined soybean protein and the like can be used after being subjected to protein denaturation treatment as appropriate or after being crushed and pulverized. As the protein denaturation treatment, steaming under pressure which is widely performed in industry is preferable.

  As the protease agent in the case of obtaining the peptide of the present invention by hydrolysis of protein, any one can be used as long as the object of the present invention can be achieved, but the protease activity is high, the price is low, the food experience is abundant, From the viewpoint that safety is guaranteed and the taste of the composition after decomposition is good, it is preferable to use a koji mold culture. As the koji mold, Aspergillus oryzae and / or Aspergillus soya are particularly suitable. These microorganisms have not only been used for proteolysis in soy sauce and miso brewing since ancient times, but are also listed on the list of GRAS (Generally Recognized As Safe) by the US Food and Drug Administration (FDA) and recognized for safety. Yes. Aspergillus oryzae culture is obtained by using soybean, wheat, rice or the like as a culture medium and ingesting and culturing aspergillus. Depending on the culture method, it is classified into a liquid anther culture and a solid anther culture, and any of them can be used in the present invention. For example, the liquid koji culture can be obtained by inoculating koji mold on a liquid medium containing 1 to 5% of soybean, wheat, wheat bran, etc., and culturing at 25 to 40 ° C. for 24 to 120 hours. The solid koji culture can be obtained by inoculating koji mold on a solid medium containing soybeans, wheat and the like and culturing at 25 to 40 ° C. for 24 to 120 hours.

  As the conditions for the hydrolysis reaction, the koji mold culture has a final concentration of 10 to 70%, preferably 30 to 50%, the soybean has a final concentration of 5 to 40%, preferably 15 to 30%, and the salt has a final concentration of 0 to 0. 25%, preferably 6-18%, and further mixed with water at a final concentration of 0-85%, and prepared so that the final concentration of soybean and salt is in the above range, then 25-60 ° C, preferably 30-55 The reaction is carried out at a stirring speed of 10 to 150 rpm for 12 to 240 hours, preferably 24 to 168 hours. In order to improve the flavor appropriately, it is also possible to add yeasts such as Tychosaccharomyces and ferment them at the same time.

  Although the composition prepared by the above-described production method contains the peptide of the present invention, it is remarkably different from the conventionally known ACE-inhibiting peptide-containing composition, hardly having a bitter taste, and being mellow and rich. It had deliciousness and richness, and had a mild and excellent fragrance, and was deliciously ingested as a food.

  Examples of the method for separating and purifying the peptide of the present invention from the composition include ultrafiltration, dialysis, various chromatography and the like. These are generally widely used methods. In particular, cation exchange chromatography and reverse phase chromatography are effective from the viewpoint of sample throughput and purification efficiency.

  The peptide of the present invention thus obtained can be used as an ACE inhibitor, that is, an antihypertensive / lowering agent, by oral or parenteral administration. According to a conventional method, in the case of oral administration, it can be in the form of tablets, granules, powders, capsules, etc., and in the case of parenteral administration, an injection preparation, a transdermal agent, a suppository, etc. can do.

  Moreover, it can use for the food-drinks (for example, health food, health-oriented food, functional food, food for specified health, etc.) aiming at prevention and / or treatment of hypertension. The peptide of the present invention can be produced by adding it to various foods and drinks. However, as described above, the composition containing the peptide is easy to add to various foods and drinks because of its good flavor. It is. Furthermore, it is easy to take the composition itself. Examples of the food and drink obtained in this way include soy sauce, processed soy sauce, powdered soy sauce, miso, soup, soup, soup stock, soy milk, fermented milk, soft drink, beverage concentrate concentrate and powder for adjustment, alcoholic beverages, Examples include fat and oil-containing foods, noodles, processed fishery products, processed meat products, semi-solid foods, pasty foods, and solid foods.

The dose of the peptide of the present invention varies depending on the purpose or means of prevention or treatment, but is preferably in the range of 0.01 mg to 100 mg per day as a peptide. In addition, when ingested as various foods and drinks, preferably less than or equal to 1mg / ml as a whole ACE inhibitory activity represented by the _{IC 50} of.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the technical scope of the present invention is not limited by these descriptions.

<Production of peptides by chemical synthesis>
The production of Tyr-Thr will be exemplified below. Reagents other than those specifically mentioned were manufactured by Wako Pure Chemical Industries. First, 120 mg of L-threonine hydrochloride was dissolved in 2 ml of dimethylformamide, 150 μl of triethylamine and 300 mg of Z-Tyr-Onp (made by Kokusan Kagaku) were added, and the mixture was stirred at room temperature for 24 hours. Next, 20 ml of 1% aqueous ammonia was added, and the resulting precipitate was left to stand at −10 ° C. for 1 hour, and the resulting precipitate was collected by filtration and washed with cold water. This precipitate was dissolved in 20 ml of methanol, 20 mg of palladium activated carbon was added, nitrogen gas was sealed, and a catalytic reduction reaction was performed. The reaction was carried out for 3 days by contacting with hydrogen gas while shaking at room temperature and atmospheric pressure. After the reaction, insoluble matter was removed by filtration, methanol was distilled off, and the residue was dissolved in distilled water. Subsequently, preparative purification was performed using an ODS column (Cosmosil 5C18-AR 20 × 250 mm, manufactured by Nacalai Tesque) connected to HPLC (LC-8A, manufactured by Shimadzu Corporation). The eluent A was ultrapure water + 0.1% trifluoroacetic acid (TFA), the eluent B was acetonitrile + 0.1% TFA, and gradient elution was performed according to a conventional method. Each fraction was developed by thin layer chromatography (TLC), and 55 mg of Tyr-Thr was obtained from the fraction in which spots appeared with the ninhydrin reagent. This chemical structure is a protein sequencer (Procise 492 manufactured by Applied Biosystems), NMR (AVANCE 500 manufactured by Bruker), and LC-MS (1100 manufactured by Agilent Technologies, QSTAR Elite manufactured by Applied Biosystems, and the column is Develosil RPAQUEOUS- manufactured by Nomura Chemical. AR) was used according to a conventional method.

  The ACE inhibitory activity of the peptide obtained by the synthesis method was measured. As a measuring method, the method of Cushman et al., Which is widely used, was partially modified. That is, 140 μl of sample solution and 10 μl of enzyme solution (0.3 U / ml ACE (rabbit) were added to 100 μl of substrate solution (12.5 mM Hippuryl-His-Leu (manufactured by Sigma), 100 mM borate buffer pH 8.3, 1 M NaCl). Lung origin, Sigma) and 50 mM borate buffer pH 8.3) were added and reacted at 37 ° C. for 30 minutes. Next, 250 μl of 1N hydrochloric acid was added to stop the reaction, and 1.5 ml of ethyl acetate was added and stirred. After centrifugation, 1 ml of the ethyl acetate layer was concentrated to dryness and dissolved in 1 ml of ultrapure water. Then, the absorbance at 228 nm was measured. The ACE inhibition rate is expressed by the following formula.

ACE inhibition rate (%) = {1− (ODs−ODsb) / (ODc−ODcb)} × 100
ODs is the absorbance when the enzyme solution is added to the sample as described above, ODsb is the absorbance when ultrapure water is added instead of the enzyme solution, and ODc is ultrapure water instead of the sample solution. ODcb is the absorbance when ultrapure water is added instead of the enzyme solution and the sample solution. In addition, the concentration of a sample that gives an enzyme activity inhibition rate of 50% to this reaction system is defined as an IC ₅₀ value.

  Table 1 shows the ACE inhibitory activity of the peptides of the present invention. As a comparative example, an ACE inhibitory peptide obtained by decomposing soybean with protease is also shown. Since the peptide of the present invention has sufficiently strong activity as compared with known peptides, it is considered industrially useful.

<Manufacture of peptides by a method of decomposing soybeans in koji mold culture>
A gonococcal aspergillus oryzae spore was added to a 20 L liquid medium containing defatted soybean 2.0% and soy sauce oil 0.4%, and cultured at 30 ° C. for 72 hours to obtain a koji mold culture. Next, 8.22 kg of defatted soybeans, 15.1 L of koji mold culture, 13.1 L of water, 3.1 kg of salt, and yeast Tigosaccharomyces rouxii were mixed at a rate of 1.5 × 10 ⁶ pieces / ml, 39 ° C., The moromi was obtained by stirring at 100 rpm for 48 hours and further at 45 ° C. and 100 rpm for 72 hours. Next, an insoluble solid was removed from the moromi using a filter press (PR66 / 40 manufactured by Iwata Sangyo) to obtain a clarified liquid. Furthermore, the enzyme was inactivated and sterilized by heating at 117 ° C. for 5 seconds using an HS sterilizer (manufactured by Nisaka Seisakusho). After leaving at 40 ° C. for 3 days, only the supernatant containing no sputum component was removed. 20 L was collected (Composition 1).

  5 L of Composition 1 was desalted to about 0% of sodium chloride by electrodialysis and loaded onto an 18 L ODS column (SP-120-40 / 60-ODS-B manufactured by Daiso). Stepwise elution of 72L of distilled water, 18L of 30% ethanol, and 27L of 99% ethanol, two fractions of water elution fraction (ODS-A, ODS-B in order of elution) and one fraction of ethanol elution fraction Each (ODS-C) was concentrated with an evaporator and freeze-dried.

  Next, 160 g of the ODS-B fraction was loaded onto an ODS column (SP-120-40 / 60-ODS-B manufactured by Daiso) having a column volume of 9 L. The eluent A was distilled water + 0.1% TFA, and the eluent B was acetonitrile + 0.1% TFA. The gradient conditions were (A100%: 200 minutes) → (gradient to B100%: 960 minutes), and fractionation was performed at a flow rate of 45 ml / min and a fraction size of 1200 ml. 100 μl was sampled from each fraction, dried by centrifugation, dissolved in 500 μl of water, and measured for ACE inhibitory activity. As a result, strong activity was observed in the fraction of ODS # 24. This fraction was concentrated with an evaporator and freeze-dried.

  3 g of the ODS # 24 fraction was loaded onto a 300 ml column cation exchange column (Toyopearl SP550C, Type H manufactured by Tosoh Corporation). The eluent A is 0.2% formic acid, the eluent B is 1% ammonium formate pH 6.0, the eluent C is 1% aqueous ammonia, and the gradient conditions are (A 100%: 20 minutes) → (B gradient to B 100%: 80 minutes) → (gradient to C 100%: 30 minutes) → (C 100%: 10 minutes), and fractionation was performed at a flow rate of 20 ml / min and a fraction size of 80 ml. ACE inhibitory activity was measured in the same manner as described above, and a fraction of SP # 18 having strong activity was obtained. This fraction was concentrated by an evaporator.

  The entire fraction of SP # 18 was loaded onto an ODS column (Cosmosil 5C18-AR 20 × 250 mm manufactured by Nacalai Tesque). The eluent A is ultrapure water + 0.1% TFA, the eluent B is 70% acetonitrile + 0.1% TFA, and the gradient condition is (A 100%: 10 minutes) → (B 30% gradient: 60 minutes) → ( B was gradient to 100%: 30 minutes), and fractionation was performed at a flow rate of 5 ml / min and a fraction size of 8 ml. The ACE inhibitory activity was measured in the same manner as described above to obtain a CS # 6 fraction with strong activity. This fraction was concentrated by an evaporator.

  The entire fraction of CS # 6 was loaded onto a C30 column (Develosil RPAQUEOUS-AR 20 × 250 mm manufactured by Nomura Chemical). The eluent A is ultrapure water + 0.1% TFA, the eluent B is 40% acetonitrile + 0.1% TFA, and the gradient conditions are (A 100%: 10 minutes) → (B 30% gradient: 60 minutes) → ( B: gradient to 100%: 30 minutes), fractionation at a flow rate of 5 ml / min and a fraction size of 6 ml. The chromatogram is shown in FIG. When the ACE inhibitory activity was measured, the activity was found only at the peak indicated by the arrow. This fraction was concentrated with an evaporator, and the structure was analyzed with a protein sequencer, NMR, and LC-MS. As a result, it was Tyr-Thr.

  The peptide of the present invention contained in Composition 1 was quantified using the LC-MS. First, a calibration curve was prepared using a peptide obtained by chemical synthesis as a standard. Subsequently, the composition 1 was desalted by electrodialysis, analyzed by appropriate dilution, and the content was calculated from the amount of ions detected derived from each peptide. The results are shown in Table 2.

  Similarly, Table 3 shows the content of the peptide of the present invention contained in the composition obtained by the production method under different conditions. As a comparative example, wheat protease was used instead of soybean, and a food protease agent (alkalase 2.4L-FG manufactured by Novozymes) was used instead of the koji mold culture. From the results of Table 3, it can be seen that soybean is excellent as a raw material, a koji mold culture is excellent as an enzyme agent, and a reaction temperature of 25 ° C. or higher is excellent. That is, the peptide of the present invention can be produced particularly efficiently by mixing and stirring soybean with a koji mold culture at 25 ° C. or higher.

<Verification of antihypertensive effect by long-term administration of rat diet>
Seven 5-week-old male salt-sensitive hypertensive rats (Dahl-S) were divided into 5 groups as follows, and a 30-day mixed feed administration test was conducted. The salt concentration of the sample was analyzed by flame analysis, and was adjusted by adding salt so that the salt concentration in the feed was the same between the groups. During the test period, the food was freely consumed, but the amount of food consumed was almost the same between the groups.
Group 1 (control) (▲ in the figure): A feed in which low-salt soy sauce (salt concentration: 7% w / v) was mixed with 25% v / w of the feed (salt concentration in feed: 3% w / w).
Group 2 (Composition 1) (● in the figure): Feed obtained by mixing 20% v / v of the composition 1 with low-salt soy sauce and mixing 25% v / w of the feed (salt concentration in feed: 3% w / W, composition 1 concentration 1.5% w / w).
Group 3 (fraction obtained by removing peptide from composition 1) (◯ in the figure): ODS-A fraction (fraction obtained by fractionating composition 1 using an ODS column) in reduced salt soy sauce ) And 25% v / w of the feed (salt concentration in feed 3% w / w, ODS-A fraction concentration 1.3% w / w).
Group 4 (peptide fraction of composition 1) (□ in the figure): Reduced salt soy sauce with ODS-B fraction and ODS-C fraction (composition 1 was fractionated with an ODS column, containing a large amount of peptide Fraction) is mixed according to the weight ratio, and 25% v / w of the feed is mixed (salt concentration in feed is 3% w / w, ODS-B / ODS-C fraction total concentration is 0.2% w) / W).
Group 5 (hydrochloric acid-degraded peptide fraction) (■ in the figure): ODS-B fraction and ODS-C fraction were hydrolyzed with hydrochloric acid according to a conventional method to completely decompose the peptide. Mixed feed (feed salt concentration 3% w / w, ODS-B / ODS-C fraction total concentration 0.2% w / w).
Systolic blood pressure was measured using a non-invasive blood pressure monitor MK-2000 every week from the start of the test. These results are shown in FIG. In addition, statistical processing was performed by Tukey's multiple comparison test using the systolic blood pressure value on the 30th day after administration.

  From the results of FIG. 2, Composition 1 containing the peptide of the present invention significantly suppressed an increase in blood pressure. Furthermore, while the antihypertensive action was observed in the fraction containing peptide, the antihypertensive action of composition 1 was derived from the peptide because the antihypertensive action was lost by decomposing the peptide. It was.

  The novel angiotensin converting enzyme-inhibiting peptide obtained in the present invention effectively inhibits ACE when ingested in a small amount and has no worry of side effects, and can be easily orally taken by hypertensives in daily life. In addition, by proposing a method for producing a composition containing the peptide, excellent in flavor, high in safety, and easy to ingest as a food, it can greatly contribute to the improvement of the quality of life of hypertensives. Become.

Claims (3)

A composition comprising a peptide Tyr-Thr having an angiotensin converting enzyme inhibitory activity and a salt thereof. The composition according to claim 1, which has an action of alleviating symptoms of hypertension. The method for producing a composition according to claim 1, wherein the soybean is mixed and stirred together with the koji mold culture at 25 ° C or higher.

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