JP3048594B2 - Peptide composition - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a peptide composition having an angiotensin converting enzyme inhibitory ability.

[Prior Art] Various factors are considered to be involved in the onset and maintenance of hypertension. Among them, an enzyme system generally called a renin-angiotensin system plays an important role in maintaining blood pressure. Angiotensin I converting enzyme (hereinafter abbreviated as ACE) plays a central role in the renin-angiotensin system, and converts angiotensin I having no physiological activity into angiotensin II having a strong vascular wall smooth muscle contraction action. It is involved in raising blood pressure. Therefore, it is possible to prevent an increase in blood pressure (lower blood pressure) by suppressing this enzyme activity.

Various substances have already been found as ACE activity inhibitors. For example, as a natural product, a peptide derived from snake venom, a peptide in a tryptic digest of milk casein, a peptide derived from fish meat, and the like are known.

Regarding the peptide derived from fish meat, see Journal of the Japan Nutrition and Food Society, Vol. 42, No. 1, pages 47-54 (1989).
1,000 to 2,000 peptide compositions are disclosed in JP-A-64-9012.
No. 8 discloses that peptide fractions having a molecular weight of 500 to 5,000 each have an ability to inhibit ACE activity.

Further, as a synthetic product, D-2-methyl-3-mercaptopropynol-L-proline [generic name captopril] has been put to practical use as an oral antihypertensive due to its high inhibitory activity. All practical hypotensive drugs are synthetic.

[Problems to be Solved by the Invention] In recent years, it has been known that various substances having excellent physiological functions in marine fish and shellfish, such as certain peptides, polyunsaturated fatty acids, and taurine, are present due to the advancement of learning, Its use is being reviewed for health promotion. Also, 70% of sardines and other high catch fish are used for feed and fertilizer,
Its effective use is desired.

All of the natural ACE inhibitory peptides reported to date are relatively long, about 10 peptides. However, when these peptides are actually taken orally, there is a high probability that they will be degraded in the digestive system, and the probability that they will be absorbed as they are in the intestine is low, and there is often no decrease in blood pressure. Also, recently, rather than the protein source being digested and absorbed by amino acids, the
The finding that the molecule is absorbed in a digested form up to the peptide to which the molecule is bound, and the fact that absorption in the form of this peptide is absorbed more efficiently than an amino acid mixture of the same composition becomes influential, Enteral and oral nutritional supplements using peptides as a protein source have attracted attention as clinical applications [Chiba Medical, Vol. 61, pp. 389-396 (1985)].

An object of the present invention is to solve these problems and use an inexpensive natural-derived AC with excellent absorbability, which makes effective use of fish resources.
An object of the present invention is to provide a peptide composition having E inhibitory ability.

[Means for Solving the Problems] To summarize the present invention, the present invention provides a fish protein,
The present invention relates to a peptide composition having an angiotensin converting enzyme inhibitory ability and having an average number of amino acid residues of less than 3, which is obtained by hydrolysis by adding an enzyme.

The present inventors have conducted intensive studies to solve the above problems, and as a result, by hydrolyzing fish proteins, a target peptide composition can be produced, and further, this peptide composition can be used. The present inventors have found that it can be purified in a large amount and with high purity, and completed the present invention.

The peptide composition of the present invention can be obtained by the following method. That is, a peptide composition is produced by hydrolyzing fish (including self-digestion treatment), and then separating and purifying the peptide composition by ion-exchange resin treatment or the like to obtain the desired peptide composition.

The fish in the present invention is not particularly limited, and sardines, mackerel, shrimp, octopus, and the like can be appropriately used. The hydrolysis in the present invention may be any of acid decomposition, alkali decomposition and enzymatic decomposition, but enzymatic decomposition is preferred. Furthermore, the enzyme used in the present invention may be any enzyme as long as it is a proteolytic enzyme, and can be used as needed as described below.

As shown in Table 1, the composition of the peptide varies depending on the type of proteolytic enzyme used and the hydrolysis reaction conditions such as temperature, time, pH, and concentration. When using enzymes with high exopeptidase activity, the degradation rate is low,
Although the average number of amino acid residues is also reduced and a desired peptide composition having an average number of amino acid residues of less than 3 is obtained, the production of free amino acids also increases, which is accompanied by a decrease in ACE inhibitory activity. In addition, when an enzyme having high substrate specificity such as trypsin is used, the production of free amino acids can be suppressed, but the degree of degradation decreases.

Also, if necessary, by adding the peptide composition and various amino acids, other fats, polyols, glucose, oligosaccharides, nutrients such as minerals and vitamins alone or in combination, foods for preventing or treating hypertension, oral Ingestion can be manufactured.

The peptide composition of the present invention is derived from natural food products and has extremely low toxicity.

The method for analyzing a product in the present invention is as follows.

(1) Peptide amount The total nitrogen was measured by the Kjeldahl method, and a value obtained by multiplying the total nitrogen by 6.25 was used.

(2) Average number of amino acid residues (l) The average number of amino acid residues (l) was determined by the following equation.

The hydrolysis was performed with 6N hydrochloric acid at 10 ° C. for 24 hours, and the quantification of amino groups was performed by the TNBS method.

(3) Amino acid composition The sample was hydrolyzed with 6N hydrochloric acid at 110 ° C. for 24 hours, and then measured by an automatic amino acid analysis method.

(4) Quantification of Free Amino Acid A sample was measured by automatic amino acid analysis in the same manner as in the above (2) without hydrolyzing hydrochloric acid, and the total amount of individual amino acids was defined as the amount of free amino acids.

(5) Measurement of ACE inhibitory activity A sample was placed in a test tube at 50 µl, 100 µl of ACE (Sigma, 2.5 mM) solution was added thereto, and the mixture was incubated at 37 ° C for 5 minutes, and then 100 µl of Bz-Gly- L-His-L-Leu
(Manufactured by Peptide Research Laboratories, containing a final concentration of 5 mM and NaCl of 400 mM) and reacted at 37 ° C. for 60 minutes. Thereafter, 0.25 ml of 0.5N hydrochloric acid was added to stop the reaction, and 1.5 ml of ethyl acetate was added, followed by vigorous stirring for 15 seconds. Then 3000
After centrifugation at rpm for 10 minutes, the ethyl acetate layer was heated at 140 ° C. for 20 minutes to remove the solvent. After removing the solvent, the solution was dissolved in 3 ml of a 1M aqueous solution of NaCl, and the absorption (228 nm absorbance) of the extracted hippuric acid was measured.

Inhibition rate = (AB) / A × 100 (%) A; Absorbance at 228 nm when no inhibitor is contained B; Absorbance at 228 nm when inhibitor is added And the inhibitory concentration at 50% inhibition rate ID ₅₀ .

(6) Average molecular weight () = · l-18 (1-1); average molecular weight of amino acid determined from amino acid composition l; average number of amino acid residues [Example] Hereinafter, the present invention will be more specifically described with reference to examples. Although described, the present invention is not limited to these examples.

Example 1 To 500 g of sardine meat surimi, 500 ml of water was added, 2 g of denazyme AP was added, and enzymatic degradation was carried out at 40 ° C for 5 hours. After the reaction was completed, the enzyme was inactivated by boiling at 95 ° C. for 15 minutes, cooled, centrifuged and filtered to obtain 500 ml of a filtrate. Then, 5 g of activated carbon powder (Carboraffin manufactured by Takeda Pharmaceutical Co., Ltd.) was added, and the mixture was stirred and filtered to obtain 480 ml of a filtrate. This filtrate was applied to a column (2.5φ ×) packed with Dowex 50W (H ⁺ ).
20 cm). Then, it was washed with 500 ml of deionized water and eluted with 300 ml of 2N NH ₄ OH. The eluate was concentrated under reduced pressure to remove ammonia, and then lyophilized to obtain peptide powder I (12 g). The ID ₅₀ value of this powder is 1.1 mg / m
l, average number of amino acid residues 1.8, free amino acid 35.4% (W /
W), the average molecular weight was 226.

Example 2 Water 1 was added to 100 g of sardine meat surimi and a denateam
2 g of AP was added and enzymatic degradation was performed at 40 ° C. for 5 hours. Then
After boiling at 95 ° C. for 15 minutes to inactivate the enzyme, the mixture was cooled and centrifuged and filtered to obtain 1050 ml of three liquids. This filtration was injected into a column (2.5φ × 20 cm) packed with Dowex 50W (H ⁺ ). Then, wash with 500 ml of deionized water and 2N NH ₄ OH
Elution was at 300 ml. The eluate was concentrated under reduced pressure to remove ammonia, and then lyophilized to obtain peptide powder (12 g).
I got The analysis values are shown in Tables 2 and 3 below.

Example 3 Dextrin 11.5 g, vitamin mix 1 mg, skim milk powder 0.7 g, whole grain powder 8 g, sugar ester 0.1 g, rice oil 1.0 g, lecithin 0.1 g, emulsifier 0.05 in 3 g of peptide powder of Example 2
g, baking soda 0.1 g and some flavoring agents were added, and water was added to make 100 ml to obtain an energy drink.

Example 4 0.2 g of hawthorn extract, 0.01 g of vitamin mix, 0.5 g of honey, 0.01 g of vitamin C were added to 3 g of the peptide powder of Example 2.
g, citric acid 0.3 g, fructose 8.8 g, and flavor were added, water was added to make 100 ml, and carbon dioxide was added to obtain a superb soft drink.

〔The invention's effect〕

According to the present invention, by using a hydrolyzate of fish protein, it is possible to obtain a highly safe and highly safe peptide composition for preventing or treating hypertension. Furthermore, if sardines and other multi-catch fish are used as raw materials,
Effective utilization of resources is provided, and an inexpensive and useful peptide composition is provided.

Continuing on the front page (72) Inventor Toshinori Nagaoka 3-4-1, Seta, Otsu City, Shiga Prefecture Inside Takara Shuzo Co., Ltd. Central Research Laboratory (72) Inventor Katsuhiro Kishijima 779-2 Noda, Hirano-cho, Ozu City, Ehime Prefecture (72) Inventor Sawabe Satoshi 4-5-9 Sendagaya, Shibuya-ku, Tokyo (56) References JP-A-62-169732 (JP, A) JP-A-63-54326 (JP, A) JP-A-59-44324 (JP) , A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 38/55 A61K 38/00 A61P 9/12 A61P 43/00 A61K 35/60 CA (STN) MEDLINE (STN)

Claims (1)

(57) [Claims] 1. A peptide composition having an angiotensin converting enzyme inhibitory activity and having an average number of amino acid residues of less than 3, which is obtained by hydrolyzing fish protein by adding oxygen.