JP2820868B2 - Angiotensin converting enzyme inhibitory protein degradation product - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an angiotensin converting enzyme (hereinafter referred to as "angiotensin converting enzyme") which is prepared by allowing a protease to act on a general-purpose food, sesame, and is useful as a hypotensive agent or a food for preventing hypertension. , "AC
E)).

[0002]

2. Description of the Related Art The number of patients with hypertension, which is considered to be a typical adult disease, has been increasing year by year, and its cause has been investigated and effective countermeasures have been studied. It is well known that a hypertensive enzyme system called a renin-angiotensin system and a hypotensive enzyme system called a kallikrein kinin system play an important role in the onset of hypertension.

[0003] According to this, in the enzyme system, angiotensin converting enzyme converts angiotensin I to angiotensin II, a potent vasopressor peptide, and has the effect of inactivating bradykinin, a hypotensive peptide, thereby increasing blood pressure. Involve deeply.

[0004] Therefore, inhibiting ACE activity is closely correlated with the effect of suppressing blood pressure elevation. Based on this recognition, many substances having ACE inhibitory activity have been searched for and reported. .

[0005] That is, many components having ACE inhibitory activity have been studied as food components, for example, collagenase digested gelatin (JP-A-52-148631),
Trypsin hydrolyzate of casein derived from cattle [JP-A-57-15435]
, JP-B-60-23086, JP-B-60-23087], thermolysin decomposition product of zein (γ-zein)
No. 36127], a pepsin decomposition product of sardine muscle [Japanese Unexamined Patent Publication No.
11097], or an ACE inhibitory substance / inhibitor contained in a digested lysate of bonito thermolysin [Japanese Patent Application Laid-Open No. 4-144696].

[0006] Sesame (Sesamun indicum L.), which is the starting material of the proteolysate of the present invention, has been regarded as a "secret agent for longevity" in China and Japan since ancient times. It was not clear about. Recently, it has been revealed that sesaminol, which contributes to the oxidative stability of sesame oil, has a function of suppressing peroxidation in vivo ("Food Function", supervised by Masao Fujimaki, Academic Society) The Publishing Center, p. 227) has yet to fully elucidate the effects and effects of sesame on living organisms.

[0007]

In order to provide an ACE inhibitor in an industrially large amount in the form of food, it is necessary to provide not only the physiological action and safety of the ACE inhibitor but also the production thereof. -Although it is necessary to sufficiently take into account the cost required for distribution, and also the functionality and taste of consumers, the above-mentioned conventional ACE inhibitors did not sufficiently satisfy these conditions. Therefore, there is a current need in the art for new and useful ACE inhibitors that can withstand industrial production.

[0008]

Means for Solving the Problems In view of the above problems, the present inventors have conducted intensive studies, and as a result, a protein decomposed product prepared by decomposing a sesame protein by the action of a protease is obtained.
They found that they exhibited ACE inhibitory activity, and based on this, completed the present invention.

[0009] The AC contained in these proteolysates is
By selecting a peptide that is particularly effective for inhibiting E activity, a more potent ACE inhibitor can of course be prepared.

Hereinafter, the present invention will be described in detail.

The sesame used in the present invention includes raw sesame, roasted sesame, defatted sesame produced in the sesame oil production process, and the like.
Although various types of sesame can be used, it is preferable to use defatted sesame as a raw material from the viewpoint of effective utilization of unused resources.

It is also possible to pulverize these sesame seeds and directly subject the pulverized substances to an enzymatic reaction. However, in order to efficiently obtain a protein decomposition product, sesame protein is extracted from sesame as a raw material, It is preferable to carry out an enzymatic reaction using the extracted sesame protein. As a method for extracting sesame protein, for example, Journal of Food Science, Vol. 47, pp. 457-460
(1982), i.e., defatting the raw sesame with hexane, dissolving and extracting the protein from the obtained defatted sesame with an alkaline solution, and then obtaining the protein as a precipitate by isoelectric point precipitation or the like. The present invention can be applied to the production process of the protein hydrolyzate of the present invention.

As the enzyme usable in the present invention, various proteases can be used. However, considering safety, commercially available proteases for foods, for example,
Alcalase [registered trademark: manufactured by Novo Industry Japan Co., Ltd.], samoase [brand name: manufactured by Daiwa Kasei Co., Ltd.], Proleather [registered trademark: manufactured by Amano Pharmaceutical Co., Ltd.], Protin FA [brand name: manufactured by Daiwa Kasei Co., Ltd.] ] Can be used.

[0014] The reaction conditions of the enzyme reaction are basically set in consideration of the optimum reaction temperature and the optimum reaction pH of the proteolytic enzyme to be used. As for the reaction time, in consideration of stably obtaining a protein hydrolyzate having a good ACE inhibitory activity, it is preferable to carry out the enzymatic reaction under the condition that a peptide bond decomposition rate of 3% or more is obtained, Further, considering the recovery rate of the proteolysate, it is more preferable that the conditions are such that a peptide bond decomposition rate of 15% or more can be obtained.

Further, in order to avoid phenomena such as contamination of various bacteria, it is preferable to appropriately adjust the reaction conditions by adjusting the reaction temperature to 50 ° C. or more.

In order to separate the target proteolysate from the enzyme reaction solution, as a method for removing undecomposed sesame protein and used proteolytic enzymes, heat treatment or isoelectric point precipitation treatment is used. A method such as filtration or ultrafiltration can be applied later.
A proteolysate solution can be obtained. Furthermore, if the obtained protein hydrolyzate solution is dried by a method such as spray drying or freeze drying, a powdery protein hydrolyzate can be obtained.

[0017]

Embodiments of the present invention will be described below in detail.

The AC of the decomposition product obtained in this embodiment
The method for measuring the E inhibitory activity (IC ₅₀ ) and the protease activity is as follows.

1. ACE Inhibitory Activity Measurement Method As an enzyme, a conventional method (for example, J. of Bioc.
hemistry, 90, p.1304 (1981)) and an angiotensin-converting enzyme, and as a substrate, hypryl-L-histidyl-L-leucine.

The above-mentioned angiotensin converting enzyme of 4.8 mU (1 U is an enzyme titer producing 1 μmole of hypoprilic acid per minute), 2 mmole of hypopril-L-histidyl-L-leucine, an appropriate amount of the peptide of the present invention, and mmole NaCl
Was added to 0.4 ml of a phosphate buffer (pH 8.3), and at 37 ° C, 3
The reaction was performed for 0 minutes.

After the reaction was stopped by adding 0.1 ml of 2N hydrochloric acid, 1 ml of ethyl acetate was added thereto, followed by vigorous stirring for 15 seconds. Next, centrifuge at 3,000 rpm for 10 minutes,
0.5 ml of the ethyl acetate layer was collected. This ethyl acetate layer
After removing the solvent at 120 ° C., 1 ml of distilled water was added, and the absorbance at 228 nm of the re-dissolved hypoprilic acid was measured.

The ACE inhibition rate was calculated according to the following formula 1 and expressed as the concentration of the inhibited protein hydrolyzate at an inhibition rate of 50% (IC ₅₀ ).

[0023]

(Equation 1)

2. Protease activity measurement method (casein
Digestion method) A 0.6% milk casein solution was used as a substrate.

First, 0.2 ml of the enzyme solution was added to 1.0 ml of the substrate solution and reacted for 10 minutes under the reaction conditions shown in Table 1 below. Then, after terminating the reaction with a trichloroacetic acid solution, the amount of the acid-soluble decomposed product is determined by the Folin method (see J. o.
f Biological Chemistry, 73, p.627 (1972)). The amount of the enzyme that causes an increase in the color substance in the Folin TS solution equivalent to 1 μg of tyrosine per minute is defined as 1 enzyme unit.
(U) indicates the protease activity.

Example 1: A method using various proteases
ACE Inhibitory Activity of Decomposed Protein After roasting commercially available roasted sesame with hexane, place the obtained defatted sesame in 0.05N sodium hydroxide solution at 55 ° C.
For 45 minutes to dissolve and extract the sesame protein. Next, after removing the residue by centrifugation (8,000 × g, 15 minutes), the pH was adjusted to 4.5 with 2N hydrochloric acid, and the protein was obtained by isoelectric focusing.

1 g of the sesame protein obtained by the above method was
Suspended in 100 ml, and under the reaction conditions shown in Table 1 below, 5,000 U of various proteases (measured by casein digestion method)
For 5 hours to decompose the protein. After heating the enzyme reaction solution in a boiling water bath for 10 minutes, undegraded sesame protein and protease which precipitated by the heat treatment were removed by centrifugation (8,000 × g, 15 minutes). ACE inhibitory activity was measured. The protein content was measured by the Kehldahl method, and the measurement results are shown in Table 1 below.

[0028]

[Table 1]

From the results shown in Table 1, it can be seen that the sesame protein hydrolyzate obtained by the action of various proteases exhibits an excellent ACE inhibitory activity as compared with the undegraded sesame protein to which no protease is added. confirmed.

Example 2 Roasted Roasted by Alcalase Decomposition
From 40 g of roasted sesame seeds, which is a commercial product of decomposed protein, 100 ml of water was added to 5 g of sesame protein obtained in the same manner as in Example 1, and the pH was adjusted to 7.0 with sodium hydroxide.
0 U (measured by casein digestion method)
For 5 hours. Next, adjust the pH to 4.5 with 2N HCl.
After heating to 80 ° C for 10 minutes to inactivate the enzyme,
The supernatant was collected by centrifugation (8,000 × g, 15 minutes).

The supernatant is freeze-dried and the freeze-dried product 3.8 g
I got

When the ACE inhibitory activity of the obtained protein hydrolyzate was measured, it was IC ₅₀ = 0.10 mg / ml.

Example 3 Roasted Sesame Seed by Decomposition of Samoases
From 40 g of roasted sesame protein, a commercially available protein digest, 5 ml of sesame protein obtained in the same manner as in Example 1 was added to 100 ml of water, and the pH was adjusted to 7.0 with sodium hydroxide.
(Measured by casein digestion method)
Time acted. Next, after adjusting the pH to 4.5 with 2N HCl, the enzyme was inactivated by heating at 80 ° C. for 10 minutes, and the supernatant was collected by centrifugation (8,000 × g, 15 minutes).

The supernatant was freeze-dried and the freeze-dried product 3.7 g
I got

When the ACE inhibitory activity of the obtained protein hydrolyzate was measured, the IC ₅₀ was 0.09 mg / ml.

Example 4: Raw sesame protein by samoase degradation
From 40 g of white digested raw sesame, 100 ml of water was added to 5 g of sesame protein obtained in the same manner as in Example 1, and sodium hydroxide was added.
After adjusting H to 7.0, 30,000 U (measured by the casein digestion method) of samoase was added, and the mixture was allowed to act at 65 ° C for 5 hours.

Next, the pH was adjusted to 4.5 with 2N HCl, and the mixture was heated at 80 ° C. for 10 minutes to inactivate the enzyme.
(8,000 × g, 15 minutes) to separate the supernatant.

The supernatant was freeze-dried, and the freeze-dried product 3.3 g
I got

The ACE inhibitory activity of the obtained protein hydrolyzate was measured and found to be IC ₅₀ = 0.11 mg / ml.

Example 5: Defatted sesame by digestion with samoase
15g of defatted sesame produced as a by-product in the proteolytic sesame oil production process
In a 0.05 N sodium hydroxide solution, the mixture was stirred at 55 ° C. for 45 minutes to dissolve and extract sesame protein.

After removing the residue by centrifugation (8,000 × g, 15 minutes), the pH was adjusted to 4.5 with 2N HCl, and 5 g of sesame protein was recovered by isoelectric focusing. To 5 g of this sesame protein, add 100 ml of water, and add
After H was adjusted to 7.0, 30,000 U of samoase (measured by the casein digestion method) was added, and the mixture was allowed to act at 65 ° C. for 5 hours.

Next, after adjusting the pH to 4.5 with 2N HCl, the enzyme was inactivated by heating at 80 ° C. for 10 minutes, followed by centrifugation.
(8,000 × g, 15 minutes) to separate the supernatant.

The supernatant was freeze-dried and the freeze-dried product 3.2 g
I got

The ACE inhibitory activity of the obtained protein hydrolyzate was measured and found to be IC ₅₀ = 0.10 mg / ml.

Example 6: ACE inhibitory activity by proteolysis rate
Influence on properties and recovery rate From commercially available roasted sesame seeds, 500 ml of sesame protein prepared in the same manner as in Example 1 was added to 50 ml of water, the pH was adjusted to 7.0 with sodium hydroxide, and 5,000 U (casein digestion method). (Measured value according to) was added, and the mixture was allowed to act at 65 ° C.

Next, the pH was adjusted to 4.5 with 2N HCl to remove undegraded sesame protein, and the mixture was heated at 80 ° C. for 10 minutes to inactivate the enzyme, followed by centrifugation (8,000 × g, (15 minutes).

The supernatant was freeze-dried to obtain a sesame protein degradation product.

Proteolysis rate depending on the reaction time, AC
The E inhibitory activity (IC ₅₀ ) and the recovery of the obtained sesame protein hydrolyzate were measured, and the results are shown in Table 2 below. The decomposition rate in Table 2 is shown as the content (%) of amino nitrogen generated by decomposition with respect to the total nitrogen.

[0049]

[Table 2]

From the results shown in Table 2 above, it was found that AC which was degraded at a decomposition rate of 3% or more was superior to undegraded sesame protein.
From the viewpoint of obtaining a protein hydrolyzate having E-inhibitory activity and the sesame protein recovery rate (recovery efficiency), the degradation rate was 15%.
% Has been found to be preferable.

Test Example 1 Eighteen-year-old 18 spontaneously hypertensive rats (Charles River Japan) were subjected to SPF at 23.5 ± 2.0 ° C. and 55 ± 10% humidity.
Water and feed CFR-1 (trade name:
Oriental Yeast Co., Ltd.) was fed ad libitum, acclimated for 2 weeks, and rats whose systolic blood pressure increased to 180-200 mmHg were subjected to the test.

To a hypertensive rat fasted overnight, the sesame protein hydrolyzate prepared according to the method of Example 2 was dissolved in water and administered by gavage at a rate of 500 mg / kg of rat body weight.
The same amount of water was forcibly administered orally as a control group.

After administration, a non-invasive blood pressure measurement device (“MK-10
0 ": Muromachi Kikai Co., Ltd.), the change in systolic blood pressure was measured over time by the Tail-Cuff method, and the results are shown in Table 3 below.

[0054]

[Table 3]

As is apparent from Table 3 above, A of the present invention
Sesame protein hydrolyzate having CE inhibitory activity is
After 6 hours, a significant blood pressure lowering effect was exhibited.

Test Example 2 Ten 8-week-old spontaneously hypertensive rats (Charles River Japan) were cultured at 23.5 ± 2.0 ° C. and 55 ± 10% humidity.
Rats housed in a PF room (sterile room) were allowed free access to water and feed CFR-1 (trade name: Oriental Yeast Co., Ltd.), bred for 2 weeks, and raised in systolic blood pressure to 180-200 mmHg. did.

The sesame protein hydrolyzate prepared according to the method of Example 3 was dissolved in water, and gavaged orally at a ratio of 500 mg / kg rat weight to hypertensive rats fasted overnight.
The same amount of water was forcibly administered orally as a control group.

After administration, a non-invasive blood pressure measuring device (“MK-10
0 ": Muromachi Kikai Co., Ltd.), the change in systolic blood pressure was measured over time by the Tail-Cuff method, and the results are shown in Table 4 below.

[0059]

[Table 4]

As is apparent from Table 4 above, A of the present invention
Sesame protein hydrolyzate having CE inhibitory activity is
After 4 hours, a significant blood pressure lowering effect was exhibited.

[0061]

That is, since the protein hydrolyzate of the present invention is produced from sesame, which is widely spread and taken daily, there is no problem in terms of safety when used as a food ingredient. Since raw materials (sesame) can be procured inexpensively and at low cost, it is possible to reduce production costs.

Further, the protein hydrolyzate of the present invention has a sufficient physiological effect (ACE inhibitory activity, blood pressure) in the form of the protein hydrolyzate without separating and purifying the active ingredient from the protein hydrolyzate. Lowering effect), which simplifies the manufacturing process (omission of the refining process) and reduces bitterness.
It can be provided as a food material that has a slightly sesame flavor and is also organoleptically excellent.

Further, the protein hydrolyzate of the present invention can be easily processed into a powder form by using a method such as spray drying or freeze drying, and has high heat resistance. It can be applied to a wide range of applications such as sports drinks, beverages such as powdered tea, and the like.

Continuation of front page (72) Inventor Mitsumune Takatsu 4-1-1, Nishinakajima, Yodogawa-ku, Osaka-shi, Osaka Nissin Foods Co., Ltd. (56) References JP-A-3-266957 (JP, A) JP-A Sho 63-22166 (JP, A) JP-A-2-167049 (JP, A) JP-A-2-240028 (JP, A) JP-A-4-2999991 (JP, A) JP-A-4-349893 (JP, A A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 35/78 A61K 38/00-38/58 A23L 1/227 CA (STN)

Claims (6)

(57) [Claims] (1) treating sesame with a protease;
Angiotensin modification consisting of proteolysates obtained from
Enzyme inhibitors . Wherein said protein degradation product is, angiotensin converting enzyme inhibitor according to claim 1 comprising a peptide having the angiotensin converting enzyme inhibitory activity. 3. The angiotensin according to claim 1, wherein the sesame is one or more sesames selected from the group consisting of raw sesame, roasted sesame, and sesame after oiling.
Conversion enzyme inhibitors . 4. A method for treating sesame with a protease.
The angiotensin converting enzyme inhibitory activity
A food material consisting of degraded protein. 5. The method according to claim 1, wherein the proteolytic product is an angiotensin-modified protein.
5. The method according to claim 4, which comprises a peptide having an enzyme inhibiting activity.
Food material. 6. The sesame is raw sesame, roasted sesame and squeezed sesame.
One or more selected from the group consisting of sesame after oil
The food material according to claim 4 or 5, which is sesame.