JPH067188A - Preparation of peptide-containing food - Google Patents

Abstract

PURPOSE:To obtain a peptide mixture capable of inhibiting an angiotensin converting enzyme and having hypotensive action by using an ultrafiltration membrane and a reverse osmosis membrane. CONSTITUTION:Protein of animal, vegetable, fish or shellfish is made to react with a commercially available enzymatic preparation and/or an enzyme existing in tissue and the decomposition product is continuously filtered with an ultrafiltration membrane in the course of the reaction. Free amino acids and other low-molecular components unrelated to the inhibition of angiotensin converting enzyme and existing in the filtrate liquid are removed by filtering with a reverse osmosis membrane to obtain a peptide mixture capable of inhibiting an angiotensin converting enzyme and having high inhibition activity. The peptide mixture capable of inhibiting angiotensin converting enzyme can easily be administered by preparing a food containing the peptide mixture.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a mixture of angiotensin converting enzyme inhibitory peptides and foods using the same. Since angiotensin converting enzyme is an enzyme that produces angiotensin II, which is a blood pressure-increasing peptide in vivo, the food product produced by the present invention can be used as a functional food product having a blood pressure lowering effect, and can suppress hypertension.

[0002]

2. Description of the Related Art Hypertension is one of the diseases for which the number of patients has been increasing in recent years, and development of effective preventive and therapeutic methods is desired. It is said that various mechanisms are involved in the regulation of blood pressure in the body, and the renin-angiotensin system is known as one of them.

The renin-angiotensin system is a regulatory system that raises blood pressure. Renin, an enzyme produced in the kidney, acts on angiotesinogen in blood vessels to form angiotensin I, which is a peptide consisting of 10 amino acids.
(Asp-Arg-Val-Tyr-Ile-His-
Pro-Phe-His-Leu). By the action of angiotensin converting enzyme, angiotensin I
The dipeptide His-Leu is released from the C-terminus of the enzyme and angiotensin II (Asp-Ar) consisting of 8 amino acids is released.
g-Val-Tyr-Ile-His-Pro-Ph
e) is generated. Angiotensin II contracts vascular smooth muscle to raise blood pressure, secretes aldosterone from the adrenal cortex and reabsorbs sodium in the kidney to raise blood pressure.

The kallikrein-kinin system is known as a mechanism for lowering blood pressure, and the kinin produced here diminishes blood pressure by expanding blood vessels. However, since this quinine is decomposed by the action of angiotensin converting enzyme, the blood pressure drop is suppressed.

Thus, in both the renin-angiotensin system and the kallikrein-quinine system, angiotensin converting enzyme increases blood pressure. Therefore, attempts have been made to suppress an increase in blood pressure by inhibiting angiotensin converting enzyme. Further, it has recently been known that peptides derived from various proteins have an inhibitory action on angiotensin converting enzyme. For example, JP-A-2-240
027 is Leu-Pr derived from corn protein.
o-Pro, Val-His-Leu-Pro-Pro
Is described as an angiotensin converting enzyme inhibitory peptide, and in JP-A-3-81291, Leu-Lys-Tyr from the tissue of Antarctic krill is disclosed in Japanese Patent Publication No.
In case of 1-51564, casein derived from bovine is converted to Thr-Th.
r-Met-Pro-Leu-Trp is separated.
On the other hand, regarding a mixture containing an angiotensin converting enzyme inhibitor peptide and a method for producing the same, zein or gluten meal described in JP-A-2-240028 is enzymatically decomposed to give a molecular weight of 10
A method of obtaining a fraction of 000 or less by gel filtration or ultrafiltration, and a molecular weight of 1 derived from figs of JP-A-2-282394
Liquid containing a substance of 0000 or less, JP-A-62-283.
A method of hydrolyzing 70 fish meat proteins to fractionate the molecular weight is known.

[0006]

However, these angiotensin-converting enzyme-containing fractions contain salts, free amino acids, and other components having a smaller molecular weight than the angiotensin-converting enzyme-inhibiting peptide and no inhibitory action. Removal of these low molecular weight components is expected to increase the enzyme inhibition specific activity of the angiotensin converting enzyme inhibitor peptide-containing fraction. Therefore, there is a demand for a simple and effective method for removing these low molecular weight components. As a method for removing low molecular weight components, there is a method of performing gel filtration, but the amount of treatment of the column is small and the operation such as filling the column with gel is complicated.

[0007]

As a result of various studies to solve the above-mentioned problems, the inventors of the present invention have completed the present invention.

That is, the present invention is a method for separating and concentrating an angiotensin converting enzyme inhibitory peptide mixture efficiently and effectively.

The angiotensin-converting enzyme inhibitory peptide mixture as referred to in the present invention is obtained by degrading animal or plant or seafood proteins with an enzyme and then concentrating the angiotensin-converting enzyme inhibitory peptide by ultrafiltration membrane treatment or reverse osmosis membrane treatment. . The enzymatic degradation can be carried out by using either a commercially available proteolytic enzyme or a enzymatic degradation contained in the tissues of animals and plants, or a combination of both. After enzymatic decomposition, molecular weight cut-off is 3000 by ultrafiltration.
After filtering through a membrane of 10,000 to 10,000, the permeate is subjected to reverse osmosis membrane filtration to remove salts, free amino acids, and other low-molecular components to obtain an angiotensin converting enzyme inhibitory peptide mixture. It is also possible to increase the membrane permeability by enzymatically decomposing the permeate of the filtration membrane to reduce the molecular weight, thereby increasing the yield of the angiotensin converting enzyme-inhibiting peptide mixture or increasing the specific activity.

As the material of the present peptide, beef protein, pork protein, poultry protein, seafood protein, vegetable protein and the like can be used. As the enzyme, Alcalase (NovoIndustry), Neutrase (NovoIndustry), Samoases (Daiwa Kasei), Actinase (Kaken Pharmaceutical), Morcin (Sorijin Pharmaceutical), etc. can be used, but the present invention is limited to these enzymes. Do not mean. When using this peptide, it can be ingested alone or after being processed into food.

[0011]

[Embodiment 1] The present invention will be described in detail below with reference to an embodiment.

10 kg of minced pork meat was added to the reaction tank, and 1 part of water was added.
After adding 35 Kg and adjusting the pH to 9.0 with sodium hydroxide solution and adjusting the temperature to 55 ° C, 0.6L of Alcalase
(Novo Industry) 375 g was added and enzymatically decomposed while stirring. 15 minutes after the start of enzymatic decomposition, the enzyme reaction solution was subjected to an ultrafiltration lab module SIP-1013 (manufactured by Asahi Kasei).
Was continuously filtered. The unfiltered liquid is returned to the reaction tank for reaction, and the pH is adjusted with 4N sodium hydroxide during the reaction.
It was adjusted to 9.0. A 200-mesh filter was attached to the inlet of the module to prevent solids from entering, and the inlet pressure was 1.3 Kg / cm ² and the outlet pressure was 0.7 Kg / cm ² . Thus, 4 hours after the start of the enzyme reaction, the filtrate 50.5
Kg was obtained. 83.6Kg of the impermeable liquid was adjusted to pH 7.5 with 4N hydrochloric acid, and actinase AS (Kaken Pharmaceutical) 30g
Was added and enzymatically decomposed at 45 ° C. for 4 hours. During the decomposition, it was continuously filtered by the ultrafiltration module SIP-1013, and the filtrate 4
9.3 Kg was obtained. Then, the ultrafiltrate is combined and subjected to reverse osmosis filtration through a reverse osmosis membrane SU620 (manufactured by Toray).
A Kg impermeable fraction was obtained, which was used as an angiotensin converting enzyme inhibitor peptide mixture. This liquid contained 3.2% solids.
It contained Kg.

The measurement of angiotensin converting enzyme inhibition is as follows.
L-Hipryl histidyl leucine was used as a substrate, and the method was carried out by using an angiotensin converting enzyme derived from rabbit lung. The peptide mixture of the present invention dissolved in 280 μl of 100 mM borate buffer containing 300 mM sodium chloride having a pH of 8.3 was placed in a glass test tube and heated at 37 ° C. for 10 minutes. Rabbit lung acetone powder (manufactured by Sigma) 1 g, 100 mM borate buffer solution (pH 8.3) 30 g
Add ml, stir well and centrifuge at 30,000 G for 20 minutes, and use the supernatant as 100% angiotensin converting enzyme.
μl was added and enzyme reaction was performed at 37 ° C. for 30 minutes. The enzymatic reaction was stopped by adding 250 μl of 1N hydrochloric acid, and 1.5 ml of ethyl acetate (for ultraviolet absorption spectrum manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1
Shake for 5 seconds to extract hippuric acid generated by the enzymatic reaction,
Centrifugation was performed at 2500 rpm for 10 minutes, and 1.0 ml of an ethyl acetate layer was collected in a glass test tube. The ethyl acetate was heated in a hot dry bath at 120 ° C for 30 minutes to completely remove it, and then distilled water was added to the UV-made by Shimadzu Corporation.
The absorbance at 228 nm was measured with distilled water using 160-02 to determine the amount of hippuric acid produced by the enzymatic reaction.

The inhibitory rate of angiotensin converting enzyme is obtained when the absorbance when the amount of hippuric acid produced is measured without adding the inhibitory peptide mixture is B, and the absorbance when the inhibitory peptide of the present invention is added is S. , Inhibition rate (%) =
(B−S) ÷ B. The enzyme activity when no inhibitory peptide was added was 8 mU.

When the angiotensin converting enzyme inhibitory activity is measured under these conditions, the enzyme activity is 50% inhibited in the pre-ultrafiltration start solution 15 minutes after the start of enzymatic decomposition of minced pork.
C ₅₀ is 1000 μg / ml of solid matter, while SI is
The P1013 permeate was 630 μg / ml, and the angiotensin converting enzyme inhibitory peptide liquid of the present invention was 21 μg / ml.
It was possible to inhibit the angiotensin converting enzyme with a smaller amount.

[0016]

Example 2 The pork-derived angiotensin converting enzyme inhibitor peptide mixture prepared in Example 1 was mixed with other materials in the proportions shown in Tables 1 to 4 to prepare a semi-solid food product. Moreover, the thing which did not add each carrageenan was manufactured, and it was set as the drink. Thus, by providing the food containing the angiotensin converting enzyme inhibitory peptide, the angiotensin converting enzyme inhibiting peptide could be provided on a daily basis. Table 1 Angiotensin converting enzyme inhibitor peptide mixture 30.0% milk 43.7% banana juice 10.0% honey 15.0% vitamin mixture (VA ₁ , VB ₁ , VB ₂ , VD) Carrageenan 0.3% Table 2 Angiotensin converting enzyme inhibitor peptide mixture 30.0% Fermented milk 43.7% Apple juice 10.0% Honey 15.0% Vitamin mixture liquid (V A ₁ , V B ₁ , V B ₂ , V D 1.0% 0.2% content liquid) Carrageenan 0.3% Table 3 Angiotensin converting enzyme inhibitor peptide mixture 30.0% Fermented milk 43 .7% Konpauda - 10.0% vitamin mixture _{(V.A 1, V.B 1, V.B} 2, V.D 1. 0% 0.2% containing liquid) water 15.0% carrageenan 0. 3% Table 4 Angiotensin converting enzyme inhibition Peptide mixture 30.0% milk 43.7% pumpkin powder 10.0% Vitamin mixture _{(V.A 1, V.B 1, V.B} 2, V.D 1. 0% 0.2% containing solution) Water 15.0% Carrageenan 0.3%

[0017]

As described above, according to the present invention, the ultrafiltration membrane treatment and the reverse osmosis membrane treatment increase the angiotensin converting enzyme inhibitory activity which has an action of suppressing an increase in blood pressure, and a peptide solution can be efficiently obtained. . In addition, the food containing it can easily take the angiotensin converting enzyme inhibitory peptide.

Claims (2)

[Claims] 1. An angiotensin converting enzyme inhibitor characterized by enzymatically decomposing proteins of animals and plants or fish and shellfish, and a filtrate obtained by continuously filtering the liquid during the reaction with an ultrafiltration membrane is further filtered with a reverse osmosis membrane. Process for producing peptide mixture. 2. A food comprising the angiotensin converting enzyme inhibitory peptide mixture as set forth in claim 1.