JPH0223885A - Production of peptide - Google Patents

Abstract

PURPOSE:To perform mass-production of an angiotensinase-inhibiting peptide effective in suppressing the blood pressure by decomposing casein with trypsin, completely inactivating the trypsin by the heat-treatment under pressure and separating the objective peptide from the supernatant liquid. CONSTITUTION:Casein is decomposed with trypsin and is subjected to the treatment at high temperature under pressure to completely inactivate the trypsin. After separating and removing the precipitate from the decomposition liquid, the objective angiotensinase-inhibiting peptide is separeted from the supernatant liquid. The decomposition of casein with trypsin is carried out under normal decomposition condition and the complete inactivation of trypsin is preferably performed at 105 deg.C (about 1.2atm) for >=15min or at 130 deg.C (about 2.7atm) for >=3min. The angiotensinase-inhibiting peptide can be produced in the form of white powder by the spray-drying or freeze-drying of the supernatant liquid.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention completely deactivates trypsin by subjecting a tryponic solution of casein to a heat treatment under high temperature and pressure, and then separating and removing the resulting precipitate. The present invention relates to a method for obtaining an angiotensin converting enzyme inhibitory peptide, which comprises collecting a supernatant.

[Conventional technology]

It is well known that the renin-angiotensin system is deeply involved in the onset of hypertension. This renin-angiotene system is an angiotensin convertase (Angiotensin Convertin enzyme) that is involved in blood pressure regulation.
g Enzyme (hereinafter abbreviated as ACE) exists, and this enzyme is involved in increasing blood pressure by converting angiotensin ■ to angiotensin ■, which has a strong vascular wall smooth muscle contraction effect. Therefore, it is possible to prevent an increase in blood pressure by suppressing this enzyme activity.

Various substances have already been discovered as activity inhibitors of angiotensin converting enzyme, such as D-2-methyl-3-mercaptopropanoyl-
Due to its high inhibitory activity, proline (-ship name: cabtopril) is currently in practical use as an oral antihypertensive agent. In addition, as natural products or inhibitors derived from natural products, snake venom peptides and their analogs, peptides obtained by tryptic decomposition of Ushiyama casein, and the like are known.

Among these, natural products or inhibitors derived from natural products are expected to be safer antihypertensive agents, whereas synthetic products still have problems in terms of toxicity and side effects. However, casein-derived inhibitory peptides are being considered for practical use as antihypertensive agents, as they are expected to be advantageous in terms of safety and efficacy, as well as safety and efficacy. (
JP-A-58-109425).

Examples of casein-derived ACE-inhibiting peptides include CE, which has the amino acid sequence shown below. CEI
? and CE 16 etc.

CEI+1: Phe-Phe-Val-Ala-Pr
o-Phe-Pro-Glu-Va1-Phs-Gly-Lys CEI, :Ala-Val-Pro-Tyr-Pro-Gln-Arg CEl, :Thr-Thr-Met-Pr.

Leu-Trp In addition to safety, these natural product-derived ACE inhibitors are expected to be mass-produced relatively easily and at low cost; however, due to the complexity of the purification method, , has not yet been put into practical use.

[Problem to be solved by the invention]

Conventionally known methods for removing unreacted casein from the tryptic casein solution include heat denaturation precipitation, isoelectric precipitation,
Although organic solvent precipitation, acid denaturation precipitation, and other methods were possible, trypsin activity added for decomposition remained in either method, making it difficult to completely remove trypsin. Further, when a step of adding an acid such as isoelectric point precipitation is performed, a subsequent desalting operation is required, which is industrially complicated. Therefore, there was a desire for an industrially simple method for obtaining the target peptide, which completely deactivates the enzymatic activity of trypsin and does not require desalting.

In addition, the sterilization process after casein decomposition was also an issue.

That is, the present invention focuses on the above-mentioned casein-derived inhibitory peptide, and aims to provide a method for obtaining it easily, quickly, in large quantities, completely inactivating trypsin, and having high bactericidal properties.

[Means to solve the problem]

The present invention is characterized in that casein is decomposed with trypsin, the decomposed solution is subjected to high temperature pressure and heat treatment to completely inactivate the trypsin, the resulting precipitate is separated and removed, and the supernatant is collected. This is a method for obtaining an angiotensin converting enzyme inhibiting peptide.

The method of the present invention will be explained below.

Casein, which is a raw material used in this production, is best casein such as various acid caseins, sodium caseinate, calcium casein, etc., but unpurified casein such as milk, skim milk, etc. can also be used as a raw material.0 Enzymes used in the present invention must be trypsin, and for enzymes such as pancreatin that contain trypsin,
This is inappropriate because the effect of obtaining peptides such as CEI+s is drastically reduced.

Any known method may be used to decompose casein with trypsin.
．． Add 0.1 to 5 parts by weight of trypsin per substrate under conditions of
A casein decomposition solution can be obtained by reacting for minutes to 18 hours.

The conditions for the high temperature, pressure and heat treatment of this decomposition liquid are r1
05℃ (approximately 1.2 atm) for more than 15 minutes to 130℃
(about 2.7 atm) for 3 minutes or more is preferable.For example, conditions such as 110°C for 10 minutes or more and 120°C for 5 minutes or more are applicable.

By performing such high-temperature, pressure, and heat treatment, trypsin is completely inactivated and precipitated together with denatured and unreacted casein. This precipitate is separated and removed by a method such as filtration or centrifugation. The sterilized supernatant is dried using a method such as spray drying or freeze drying, and a white powder of AC is obtained.
An E inhibitory peptide was obtained.

The ACE inhibitory peptides obtained by the method of the present invention include the above-mentioned CEI, CE +7. and CE 1. .

It is a crude peptide mainly composed of When we investigated this drug, we found that, unexpectedly, oral administration showed a hypotensive effect that could be expected to be effective in preventive blood pressure control.Coupled with its low toxicity and high safety, we found that prevention,
It has been found that it is possible to provide oral food that is useful as a health food for alleviating hypertension tendencies.

The above-mentioned casein-derived ACE inhibitory peptides are usually isolated and obtained in the form of a powder, and then used as is, or more preferably, with an appropriate non-toxic carrier for oral administration (ingestion) in an appropriate form. It is provided as a composition for oral consumption.

Examples of compositions include ACE-inhibiting peptides, together with pharmaceutically acceptable carriers (excipients, lubricants, binders, colorants, colorants, flavoring agents, etc.), in a pharmaceutical composition for oral administration. Preparation forms include, for example, tablets (tJ! coated tablets, effervescent tablets, film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, and the like.

It may also be in the form of solid or liquid foods or luxury items, such as confectionery, powdered tea, alcoholic beverages, sports drinks, and the like.

As a method for manufacturing these oral foods, conventional methods for manufacturing formulations or foods can be used.

The dose is generally 5 mg to 10 g/kg per day for adult males.
The appropriate amount is selected from this range depending on the purpose of feeding.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Furthermore, the activity (IDs) of the obtained ACE inhibitory peptides
was measured by the following method.

(Measurement of ACE inhibitory activity of ACE inhibitory peptide) i) A
Preparation of CE liquid 5g rabbit lang acetone powder (manufactured by Sigma)
was dissolved in 50ml of 0.1M borate buffer (pH 8.3) and centrifuged at 40,000Xg for 40 minutes.The synovial fluid was further diluted 10 times with the above buffer, and A.
A CE solution was obtained.

ii) Measurement of activity 0.03 ml of the sample was placed in a test tube, and 250 μl of hybridyl-histidyl-leucine (manufactured by Sigma, final concentration 5 mM) was added as a substrate.

(containing 1300 mM NaC) was added and incubated at 37°C for 1
After incubating for 0 minutes, add 0.1 ml of the above enzyme solution,
The reaction was carried out at 7°C for 30 minutes. Then IN hydrochloric acid 0,25
After terminating the reaction by adding 1.5 ml of ethyl acetate, the mixture was stirred vigorously for 15 seconds. after that,
3. Centrifuge at 50 rpm for 15 minutes to remove ethyl acetate layer 1.
ml was taken. The ethyl acetate layer was heated at 120° C. for 30 minutes to remove the solvent. After removing the solvent, add 1 ml of distilled water.
was added, and the absorption of the extracted hypobrilic acid (absorbance at 228 nm) was measured, which was taken as the enzyme activity.

The inhibition rate was calculated using the following formula.

Inhibition rate = (A-B)/AX 100% A: Absorbance at 228 nm without inhibitor B: Absorbance at 228 nm when inhibitor is added And inhibition in enzyme reaction solution when inhibition rate is 50% IDs the concentration of the agent
. shall be.

Example-1 Bovine-derived casein (manufactured by BESNIER, food grade acid casein) 2.0 in 501 Jarfermenter (manufactured by Mitsuwa)
Kg is suspended in 201 ml of water and the pH is adjusted to 7.5 with 1ONK OI+. Incubate at 37℃ and add trypsin (manufactured by Novo Industries, PTN3.0S EC3)
, 4, 21, 4 derived from pig pancreas) was added, and digestion was performed for 4 hours while stirring.

This is heated under high temperature pressure in a jar fermenter (12
1°C, 10 minutes), and the resulting precipitate was heated to, OOOrpm,
The supernatant was obtained by continuous centrifugation at 4°C. When the residual trypsin activity of the obtained supernatant was measured by the method shown later, the residual rate was 0%. This supernatant was dried to obtain 1.4 kg of peptide-containing material.

Residual trypsin activity was measured and calculated by the following method.

(Residual trypsin activity measurement method) Synthetic aTt benzoyl-arginine-4-nitroanilide (N'-benzoyl-L-argin
ine-4nitroanilide: Bz
The residual rate of trypsin activity was calculated by measuring the amount of color development of free 4-nitroanilide by enzymatic hydrolysis of -Arg-ANA).

i) Substrate Bz-Arg-4NA containing 5mM calcium chloride
．． It was dissolved in 15 M Tris-HCl buffer (pH 7,8) to give a solution with a concentration of 0.8 mM.

1i)) Lipsin activity measurement Add 0.1 mf of the sample (digested fluid supernatant after heat treatment etc.) to 0.9 ml of the substrate from i) heated to 37°C.
(diluted as necessary) and reacted at 37°C for 1 hour. Add 9.2ml of 50% acetic acid to this! was added to stop the reaction. This was centrifuged, and the absorption of free 4-nitroanilide (absorbance at 405 nm) in the supernatant was measured.

Furthermore, similar measurements were carried out for the above samples, ``digestive fluid before the treatment process'' and ``water and tochino'', and the residual trypsin activity was calculated using the following formula.

Survival rate-(X-B)/(x*-B)X 100%X:
Absorbance X when the digestive fluid supernatant after heat treatment etc. is used as the sample: Absorbance B when the digestive fluid before the treatment process is used as the sample: Absorbance when water is used as the sample Example - 2-3 High temperature pressure heat treatment conditions: r110℃, 10 minutes (Example 2)
” and 1105°C, 15 minutes (Example 3) Example as J-
1811! A supernatant containing caseinolytic peptides was obtained by the method described above. When the residual trypsin activity of this supernatant was measured by the above method, the residual rate was 0% in all cases.

Comparative Example-1 Instead of high-temperature pressure heat treatment, heat treatment using a boiling R water bath (
A supernatant containing caseinolytic peptides was obtained by the method described in Example 1. When the residual trypsin activity of this supernatant was measured by the above method, the residual rate was 10.2%.

From the above, it can be seen that residual trypsin is completely inactivated by the high-temperature, pressure, and heat treatment.

The peptides obtained in Examples 1 to 3 have an ACE inhibitory effect, have an ID5o of 0.23 mg7ml, and have an ACE inhibitory effect.
Among the peptides with E inhibitory effect, CE I +□ was measured by liquid chromatography. ! It contained 1.3% per amount.

Next, the antihypertensive effect of the peptide obtained in Example-1 was tested.

Test Example = 1 Antihypertensive effect when the peptide obtained in Example 1 was administered orally in a single dose to rats (11 Test method) 10-week-old spontaneously hyperbaric rats (Charles River Japan) were treated at a temperature of 23 ± 2°C. , housed in an animal room with a humidity of 50±1O%, and provided with water and feed (manufactured by Oriental Yeast Co., Ltd., MF
) were fed ad libitum. After preliminarily breeding the rats for one week, healthy rats with a systolic blood pressure of about 180 mmHg or more were used for the test.

That is, the peptide obtained in Example 1 was orally administered at 3.0 g per rat body weight, and blood pressure and heart rate were measured over time to examine the antihypertensive effect. Measurements were made 8 times in total: before administration and 0.5, 1, 2, 3, 4, 6, and 8 degrees 24 hours after administration.

Measurements were performed three times for each animal using a non-invasive non-warming tail artery blood pressure/heart rate measuring device (manufactured by Riken Kaihatsu Co., Ltd.).

The results are shown as the average value of 7 animals in the − group.

(2) Test results Figure 1 shows the blood pressure measurement results.

As is clear from FIG. 1, A obtained by the method of the present invention
It can be seen that the CE inhibitory peptide has a remarkable hypotensive effect upon oral administration. On the other hand, heart rate was not affected at all and remained constant.

Oral food will be shown below as an application example of the peptide obtained by the method of the present invention.

Application example - 1 Yogurt content Weight % Milk 67.0% Whole milk 4.0% Skim milk powder 5.0% Granule 1! 7.0% water
12.0% Peptide of Example-1 5.0% Produced by normal manufacturing method. (10.0 g/cup) [Effects of the Invention] Since the present invention is configured as described above, it produces the effects described below.

The method for obtaining peptides of the present invention enables: ■ Simple, rapid, and large quantities; ■ No residual enzymatic activity; and ■ Few contaminating salts.
■ACE inhibitory peptides can be obtained in a sterilized state.

Furthermore, as shown in the test examples above, the ACE-inhibiting peptide obtained according to the present invention exhibits a favorable hypotensive effect when administered orally. Therefore, it is of great significance that this method has made it possible to put into practical use an oral preparation derived from natural products that is highly safe and has an effective antihypertensive effect.

In addition, in this acquisition method, CEI+t is obtained by subsequent purification and separation.
, CEI , and CBI b peptides, it is also useful as a primary purification method for obtaining single peptides.

[Brief explanation of the drawing]

FIG. 1 is a graph showing changes over time in a test group in which a single oral administration of the peptide obtained by the method of the present invention was administered to spontaneously hypertensive rats. 1゜2゜3゜Procedural amendment (voluntary) April 17, 1999 Indication of the case 1986 Patent application No. 173393 Name of the invention Person who amends the method for obtaining peptide Relationship with the case Patent applicant address Tokyo 17-4, Sumida 5-chome, Sumida-ku, Tokyo Details of the amendment to the "Detailed Description of the Invention" section of the specification subject to the amendment (1) In the 20th line of page 1 of the specification, the term "angioten" was replaced with "angiotensin 1". (2) In the 4th line of page 13 of the specification, insert 1 for r peptide only before j per r weight. Gokanbo Co., Ltd. Patent Department Phone (06) 921-1251

Claims (1)

[Claims] An angiotensin converting enzyme inhibiting peptide characterized by decomposing casein with trypsin, completely inactivating trypsin by subjecting the decomposed solution to high temperature, pressure and heat treatment, separating and removing the resulting precipitate, and collecting the supernatant. How to obtain.