JPH09188694A - Peptide, angiotensinase inhibiting composition and their production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new peptide obtained by the culture of Lactococcus lactis, Lactococcus confusus, etc., having a specific amino acid sequence, exhibiting angiotensin I converting enzyme inhibiting action and useful for the prevention and treatment of hypertension, etc. SOLUTION: This new peptide having angiotensin I converting enzyme inhibiting action is expressed by the formula I and the formula II. It inhibits angiotensin I converting enzyme having an action to convert angiotensin I into angiotensin II which is a strong hypertensive peptide and is effective e.g. for the prevention and treatment of hypertension. The peptide can be produced by dissolving skim milk in water, sterilizing at 115 deg.C for 20min, inoculating at least one kind of microorganism selected from Lactococcus lactis subsp. lactic (FERM P-13647) and Lactococcus confusus (FERM P-13646), fermenting at 30 deg.C for 24hr and collecting the produced peptide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide, more specifically to a peptide useful as a composition for inhibiting angiotensin I converting enzyme, its use and a method for producing the same.

[0002]

2. Description of the Related Art Angiotensin I converting enzyme (hereinafter referred to as "A
(Abbreviated as "CE") is known as a key enzyme of blood pressure control system. This enzyme has an action of converting angiotensin I into angiotensin II, which is a strong pressor peptide, and also has an action of inactivating bradykinin, which is a hypotensive peptide, thereby increasing blood pressure. Therefore, a substance that inhibits ACE is considered to exert an effect of suppressing an increase in blood pressure.

[0003] However, as the above-mentioned inhibitors that are already in practical use as pharmaceuticals, synthetic inhibitors such as captopril are known.

On the other hand, in the rapidly advancing aging society, prevention of cardiovascular diseases is urgently needed in this field, and hypertension triggers it. Therefore, the above-mentioned ACE for preventing or treating hypertension is required. Research and development of inhibitors is required. In particular, the hypertension is one of the diseases with the highest morbidity for Japanese who have a high salt intake, and the development of substances for its prevention or treatment has been earnestly desired in the art.

[0005]

Therefore, the present invention is to develop an ACE inhibitor for the prevention or treatment of hypertension, which is highly desired in the art, and in particular, it is highly safe and is ingested as a daily food. The aim is to develop said ACE inhibitor which can also be

[0006] From the above-mentioned purpose, the present inventors have conducted extensive studies on various food resources using this as a material, and as a result, in a specific enzymatic degradation product of a certain protein, an excellent ACE meeting the above-mentioned purpose. It was confirmed that there is a novel peptide having inhibitory activity, and the present invention has been completed here.

[0007]

That is, according to the present invention,
An angiotensin I-converting enzyme inhibitory composition containing as an active ingredient at least one selected from the peptide having the amino acid sequence represented by SEQ ID NO: 1, the peptide having the amino acid sequence represented by SEQ ID NO: 2, and other peptides. Also provided is a method for producing a peptide, in which a protein raw material containing a homologous peptide as a constituent is fermented with at least one of Lactococcus lactis and Lactococcus confusus to collect a target peptide.

Since the peptide of the present invention has an excellent ACE inhibitory action and is highly safe, it is used as a health food or a functional food for the purpose of preventing hypertension or a material thereof, and a drug such as an antihypertensive agent. Is useful as

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The novel peptide according to the present invention has a conventional chemical structure in which each amino acid is sequentially subjected to a peptide bond reaction based on its amino acid sequence, or after a plurality of suitable fragments are synthesized, they are combined with each other. Of course, it can be produced by a synthetic method (including both a liquid phase method and a solid phase method), but more advantageously, it can be produced by fermenting a protein raw material containing this as a constituent component with a specific lactic acid bacterium. .

In the method utilizing the enzyme treatment, a protein such as casein can be typically exemplified as a raw material, but it is not limited thereto as long as it contains the peptide of the present invention as a constituent.

The enzyme to act on the protein raw material is Lactobacillus.
confusus ) and Lactococ
us lactis ). Each lactic acid bacterium is, for example, FERM (Institute of Industrial Science and Technology, Institute of Biotechnology, Tsukuba City, Ibaraki Prefecture), JCM (RIKEN Microorganism Preservation Facility, Wako City, Saitama Prefecture), IFO (Fermentation Research Institute, Osaka City). ), ATCC (American Type Culture Collection, Maryland, USA) and the like, and commercially available products such as these may be used as they are, or may be separately screened and purified before use. Examples of typical enzymes include FER in Lactococcus lactis.
M P-13646 and Lactococcus confusus can be exemplified by FERM P-13647.

More specifically, the above method uses, as a substrate solution, whole milk of animal milk such as cow, sheep, goat, etc. or skim milk or casein as a substrate solution, as a protein raw material containing a desired peptide. To this, pH is adjusted to 5.5 to 7.5 by adding NaOH, HCl or the like as a pH adjusting agent, and this solution is preferably inoculated with lactic acid bacteria at 5 × 10 ^{5 to} 5 × 10 ⁷ cells / ml. However, it is preferable to culture the cells under a temperature condition of 20 to 40 ° C. for about 10 to 120 hours.

The culture thus obtained is rich in the peptide of the present invention and has an excellent ACE inhibitory activity, so that it can be used as it is as an ACE inhibitory composition. Further, the peptide of the present invention is obtained by centrifugation, extraction, concentration, dryness, ultrafiltration, ultrafiltration, or
It can be isolated and purified by performing salting out, ethanol precipitation, various chromatographic operations (ion exchange, gel filtration, reverse phase, affinity, etc.), and the purified form is put to practical use as an ACE inhibitory composition. You can also

The peptide of the present invention obtained as described above is a novel peptide having an amino acid sequence represented by SEQ ID NO: 1 and: 2, and has a unique ACE inhibitory activity based on the amino acid sequence. ing.

The ACE inhibitory activity can be determined by a conventional method such as the method of Cushman et al. [Cushman and Cheung, Biochemi.
cal Pharmacology, 20 , 1637 (1971)]. That is, 100 mM HEPES buffer (pH 8.
3), 200 μl of a reaction solution containing 300 mM NaCl, 0.25 mU ACE and an appropriate amount of ACE inhibitor,
Pre-incubate at 37 ℃ and add 5m as substrate
Bz-Gly-His-Leu of M was dissolved in 100 mM HEPES buffer (pH 8.3) containing 300 mM NaCl to start the reaction by adding 300 μl of the substrate solution, and after the reaction at 37 ° C. for 30 minutes, the reaction was performed. 0.1N N in the thing
The reaction was stopped by adding 2 ml of aOH, and then 0.1 ml of 0.2% o-phthaldialdehyde dissolved in methanol was added, and the mixture was allowed to stand at room temperature for 15 minutes. After that, 0.4 ml of 1.5 M phosphoric acid was added, and the fluorescence intensity of the obtained solution was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 480.
nm using a fluorimeter, and
The CE inhibition rate is calculated.

ACE inhibition rate (%) = [1- (S-Bs)
/ (C-Bc)] × 100 S: Fluorescence intensity when SACE and ACE inhibitor sample are added C: Fluorescence intensity when ACE is added and ACE inhibitor sample is not added Bc: ACE and ACE inhibitor sample Fluorescence intensity without addition of Bs: Fluorescence intensity without addition of ACE and addition of ACE inhibitor

The inhibition strength showing 50% inhibition calculated above is defined as 1 unit (U). The degree of inhibition is I
It is expressed using C ₅₀ , and the IC ₅₀ value indicates the concentration of the sample required for 50% inhibition in the above measurement method.

The present invention also provides an ACE inhibitory composition in the form of a drug or food containing the above peptide as an active ingredient. The ACE inhibitory composition in the form of a drug usually uses a suitable pharmaceutical carrier. Then, it is prepared into a suitable pharmaceutical preparation composition according to a conventional method and put into practical use.

As the pharmaceutical carrier, a filler, a bulking agent, a binder, a moistening agent, which is usually used, is selected depending on the use form of the pharmaceutical.
Examples of the diluents or excipients include disintegrants, surfactants, lubricants and the like, and these are appropriately selected and used according to the dosage unit form of the resulting preparation.

The dosage unit form of the pharmaceutical preparation of the present invention is:
Various forms can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, solutions, suspensions, emulsions,
Granules, capsules, suppositories, injections (solutions, suspensions, etc.)
And the like.

In the case of molding in the form of tablets, as the above-mentioned carrier for the formulation, for example, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc.,
Binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, sodium carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried Starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate and other disintegrants, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, surfactants such as stearic acid monoglyceride, sucrose, stearin, cocoa butter, Disintegration inhibitors such as hydrogenated oil, quaternary ammonium bases, absorption enhancers such as sodium lauryl sulfate, glycerin, starch, etc. Moisturizers, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, stearates, boric acid powder, a lubricant such as polyethylene glycol can be used.

Further, the tablets may be tablets coated with a usual coating, if necessary, for example, sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double-layered tablets, multi-layered tablets.

In the case of molding in the form of pills, pharmaceutical carriers such as glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

In the case of molding in the form of suppositories, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like can be used as a pharmaceutical carrier. Capsules are prepared by mixing the active ingredient compound of the present invention with the various pharmaceutical carriers exemplified above and filling the mixture into hard gelatin capsules, soft capsules and the like according to a conventional method.

When the drug of the present invention is prepared as an injection such as a solution, emulsion or suspension, it is preferable that the drug is sterilized and isotonic with blood. As the diluent, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. can be used. In this case, a sufficient amount of salt, glucose or glycerin to adjust the isotonic solution may be contained in the drug of the present invention, and ordinary solubilizers, buffers, soothing agents and the like are added. May be.

Further, the drug of the present invention may contain a colorant, a preservative, a flavor, a flavoring agent, a sweetening agent and the like and other pharmaceuticals, if necessary.

The amount of the active ingredient compound to be contained in the drug of the present invention is not particularly limited and is appropriately selected from a wide range, but it is usually contained in a pharmaceutical preparation in an amount of about 0.1 to 100% by weight. Good to do.

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, patient's age, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are intravenously administered alone or in a mixture with a normal replenishing solution such as glucose and amino acid. Independently, it is intramuscularly, intradermally, subcutaneously or intraperitoneally administered, and the suppository is intrarectally administered.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on the usage, age of the patient, sex and other conditions, degree of disease and the like, but usually the amount of the compound of the present invention as an active ingredient is 1.
The amount is preferably about 0.5 to 20 mg per kg body weight per day, and the preparation can be administered in 1 to 4 divided doses per day.

When the ACE-inhibiting substance of the present invention is put into practical use in the form of food, it may be shaped into various suitable forms of food such as solid preparations and liquid preparations by a general method using suitable edible materials. It is also possible to add an appropriate amount thereof to ordinary food materials or processed foods. The intake amount is not particularly limited, and can be appropriately determined by those skilled in the art using the dose in the above-mentioned pharmaceutical form as a guide.

The ACE-inhibiting composition of the present invention in the form of pharmaceuticals and foods can exert an effect of lowering blood pressure, suppressing inactivation of bradykinin, etc. due to its ACE-inhibiting action, so that essential hypertension, renal hypertension, adrenal hypertension, etc. It is effective in preventing or treating various types of hypertension. The composition of the present invention can also be used as a blood pressure-lowering agent used for diagnosing diseases and the progress of pathological conditions, an increase in the threshold of angina attack, a reduction in myocardial infarction, a congestive heart failure improving agent, and the like.

[0032]

The present invention will now be described in further detail with reference to examples.

[0033]

Example 1 (1) Production of Fermented Milk Containing Peptide of the Present Invention 5 g of skim milk powder was dissolved in 50 ml of water, and 115 ° C., 20
Two sterilized pieces were prepared. After cooling them to room temperature, one of them will contain Lactococcus lactis
Sub Species Ractis FERM P-136
Forty-seven platinum loops were inoculated. In addition, another platinum loop was inoculated with Lactobacillus confusus FERM P-13646.

Each of them was incubated at 30 ° C. for 24 hours,
It was a starter.

Next, 200 g of skim milk powder was dissolved in 2000 g of water, sterilized to a temperature of 90 ° C., and 20 ml of each of the starters was inoculated (5 × 10 ⁶ / ml).

After culturing at 30 ° C. for 60 hours, about 2 k of fermented milk
g was obtained.

The main fermented milk had excellent ACE as shown in Table 1.
It exhibited inhibitory activity.

Commercially available sour milk drinks (trade name: Calpis,
Although fermented with lactic acid bacteria such as Milton, chorus, Lactobacillus helveticus, etc., and added with sugar, flavor, etc.) also showed excellent ACE inhibitory activity, but this is usually 5-6 times when drinking. Diluted to.

[0039]

[Table 1]

In Table 1, commercially available yogurts 1-4 (manufactured by different manufacturers), kefir (traditional fermented milk of the Caucasus region, several kinds of lactic acid bacteria, and one fermented with yeast, usually about 1% Of the same A) obtained for unfermented milk (raw skim milk)
CE inhibitory activity is also shown.

(2) Purification of the peptide of the present invention The fermented milk obtained in the above (1) was centrifuged for 10 minutes at a centrifugal force of 3000 × g, and 4 times amount of ethanol was added to the obtained supernatant, resulting precipitate. Was centrifuged at 10,000 xg for 10 minutes. The obtained supernatant fraction was concentrated under reduced pressure to 1/25 volume,
0.5 ml of this concentrate was subjected to HPLC using an ODS column under the following conditions.

First chromatographic conditions by ODS column: Column: Cosmosil 5C18 (8 × 250 mm, manufactured by Nacalai Tesque) Detection: UV220 nm Flow rate: 2 ml / min Elution solvent: Gradient elution B / (B + A) × 100%: 0% → 0% (0 → 10 minutes)
→ 50% (10 → 40 minutes) Solution A: 0.1 wt% TFA (trifluoroacetic acid) aqueous solution Solution B: 0.1 wt% TFA (trifluoroacetic acid-containing acetonitrile)

The elution results are shown in FIG.

In the figure, the upper part shows elution results (horizontal axis: retention time (min), vertical axis: absorbance (Absobance at 220 n).
m), the lower row shows the ACE inhibitory activity (horizontal axis: fraction No. (Fraction No.)) and vertical axis: ACE inhibition rate (ACE inhibition (%)) of each elution fraction.

The eluate was fractionated into 8 fractions (indicated as Fr. 1 to 8 in the figure), and the seventh fraction showing the highest ACE inhibitory activity.
The fraction was dried under reduced pressure, dissolved in 0.25 ml of water, and re-chromatographed under the following conditions.

Second chromatography condition by ODS column: Column: Cosmocil 5C18 (8 × 250 mm, manufactured by Nacalai Tesque) Detection: UV220 nm Flow rate: 2 ml / min Elution solvent: Gradient elution B / (B + A) × 100%: 25% → 25% (0 → 5
Min) → 50% (5 → 35 min) A liquid: 0.1 wt% TFA (trifluoroacetic acid) aqueous solution B liquid: 0.1 wt% TFA (trifluoroacetic acid-containing acetonitrile)

The results are shown in FIG.

In the figure, the upper part shows the elution results (horizontal axis: retention time (min), vertical axis: absorbance (Absobance at 220).
nm) and acetonitrile (Acetonitorile)%), and the lower row shows the ACE inhibitory activity (horizontal axis: fraction No. (Fraction No.), vertical axis: ACE inhibitory rate (ACE inhi).
bition (%)) is shown.

The eluate was fractionated into 13 fractions, the 10th and 11th fractions having high activity were collected, and further subjected to chromatography under the following conditions using Asahi Pack GS-320HQ.

Column: Asahi Pack GS-320HQ
(Manufactured by Showa Denko) Detection: UV220nm Flow rate: 1.0 ml / min Elution solvent: 50 mM ammonium acetate containing 20% acetonitrile

The results are shown in FIG.

In the figure, the left column (I) shows the result of using the tenth fraction, and the right column (II) shows the same result of the eleventh fraction. The upper column of each column shows the elution results (horizontal axis: retention time (min), vertical axis: absorbance (Absobance at 220).
nm)), and the lower part shows the ACE inhibitory activity (horizontal axis: retention time (min), vertical axis: ACE inhibition rate (ACE inhibition (%)) of the corresponding elution fraction.

Each active fraction eluted as the main peak was collected and desalted on the ODS column (under the same conditions as the second chromatography). The active ingredients thus obtained are designated peptides I and II, respectively.

(3) Identification of peptide of the present invention and activity measurement The amino acid sequence of the peptide isolated in (2) above was determined using an amino acid sequencer (PQS-1 type manufactured by Shimadzu Corp.), and a peptide conforming to the obtained sequence was determined. It was confirmed that they were the same by chemically synthesizing separately and comparing the behavior, activity, etc. using an ODS column.

The results are shown in Table 2.

[0056]

[Table 2]

From the table, it can be seen that the peptides I and II of the present invention have a novel amino acid sequence consisting of 6 and 12 amino acids shown in SEQ ID NOS: 2 and 1, respectively, and have strong AC.
It was confirmed to have E inhibitory activity.

[0058]

Example 2 Lactococcus prepared in the same manner as in Example 1 by sterilizing 1000 ml of milk at 115 ° C. for 20 minutes
Ractis Subspecies Ractis FERM
P-13647 and Lactobacillus confusus F
10 ml of each starter of ERM P-13646 was inoculated and cultured at 35 ° C. for 72 hours.

The culture was centrifuged at 3000 xg for 10 minutes,
The precipitate was removed. The obtained inhibitory substance solution was 44 μl
An IC ₅₀ value of / ml is shown.

Further, this was freeze-dried to obtain an inhibitor composition powder.

[0061]

[Sequence list]

SEQ ID NO: 1 Sequence length: 11 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 7 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

[Brief description of the drawings]

FIG. 1 is a graph showing the elution result in the first chromatography with an ODS column when purifying the peptide of the present invention according to (2) of Example 1.

FIG. 2 is a graph showing the elution result in the second chromatography with an ODS column when purifying the peptide of the present invention according to (2) of Example 1.

FIG. 3 is a graph showing an elution result in chromatography by Asahi Pack GS-320HQ when purifying the peptide of the present invention according to (2) of Example 1.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:01)

Claims (4)

[Claims] 1. A peptide having an amino acid sequence represented by SEQ ID NO: 1. 2. A peptide having an amino acid sequence represented by SEQ ID NO: 2. 3. Angiotensin I conversion comprising as an active ingredient at least one selected from the peptide having the amino acid sequence represented by SEQ ID NO: 1 and the peptide having the amino acid sequence represented by SEQ ID NO: 2. Enzyme inhibiting composition. 4. A Lactococcus lactis and Lactococcus confusus a protein raw material containing a peptide having the amino acid sequence represented by SEQ ID NO: 1 and / or a peptide having the amino acid sequence represented by SEQ ID NO: 2 as a constituent component. And a peptide having the amino acid sequence represented by SEQ ID NO: 1 and / or the peptide having the amino acid sequence represented by SEQ ID NO: 2 is collected. .