JP2003210138A - Functional food, method for producing the same, and medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a functional food and medicine both having an anti- oxidation and a hypotensive activities, capable of being taken safely and produced at a low cost, and a method for producing the functional food. <P>SOLUTION: The functional food and the medicine are characterized by containing a decomposed soybean protein mixture obtained by decomposing a heat-degenerated soybean protein with an enzyme. The method for producing the functional food is characterized by comprising a process of obtaining the degenerated soybean protein by heat-treating the soybean protein, and a process of obtaining the decomposed soybean protein mixture by decomposing the degenerated soybean protein with the enzyme. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a medicine, a functional food and a method for producing the same.

[0002]

2. Description of the Related Art It has been conventionally known that there are various useful degradation products of proteins derived from foods and the like. For example, amino acids and peptides produced as hydrolysates obtained by decomposing food proteins with proteases include those that can be used for applications in the food field such as seasonings and foaming agents, functional foods, enteral nutritional supplements, and It is known that there are those that can be used as medical supplements, cosmetic materials, and the like.

By the way, in recent years, substances having an antioxidative effect may exert various desirable effects such as blood pressure lowering, active oxygen scavenging, peroxide production suppression, cholesterol increase suppression and fat metabolism promotion in vivo. It has been clarified that it is known that it is preferable to ingest a substance having such an action as a medicine or by adding it to a food. Further, in recent years, the need for effective control of high blood pressure, which frequently occurs as one of lifestyle-related diseases, has been increasing. A pharmaceutical and a functional food that can obtain these effects safely and can be manufactured at low cost are required.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a functional food having an antioxidative effect and an antihypertensive effect, which can be safely ingested and can be manufactured at low cost.

Another object of the present invention is to provide a method for producing a functional food, which can easily produce a safe functional food having an antioxidative action and a hypotensive action.

[0006] Another object of the present invention is to provide a drug which has a blood pressure lowering effect, a peroxide production suppressing effect, or a combination thereof, which can be safely ingested and can be manufactured at a low cost. It is in.

[0007]

According to the present invention, there is provided a functional food and a medicament, which comprises a soybean protein decomposition mixture obtained by decomposing a heat-denatured soybean protein with an enzyme.

According to the present invention, the method further comprises the steps of heat-treating soybean protein to obtain modified soybean protein, and decomposing the modified soybean protein with an enzyme to obtain a soybean protein decomposition mixture. Provided is a method for producing a functional food containing the soy protein mixture.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The functional food of the present invention contains a soybean protein decomposition mixture obtained by decomposing a heat-denatured soybean protein with an enzyme.

The soybean protein is not particularly limited, and soybean protein such as defatted soybean milk, isolated soybean protein, concentrated soybean protein and defatted soybean meal extract protein can be used.

The method for denaturing the soybean protein by heating is not particularly limited, but it can be carried out by dissolving or suspending the soybean protein in a solvent such as water and heating it. The temperature of heat denaturation is 40 to 100 ° C,
It can be preferably carried out in a temperature range of 80 to 100 ° C. The heating time may be 30 to 60 minutes.

As the enzyme for decomposing the heat-denatured soybean protein used in the present invention, microbial or plant-derived acidic protease, neutral protease and alkaline protease, and mammalian-derived digestive enzyme such as pepsin and pancreatin. Examples of foods that are usually used in foods such as
arica papaya L) -derived protease, Aspergillus oryzae (Aspergillus ore)
yzae) -derived protease, Bacillus subtilis-derived protease, Aspergillus mereus (Aspergi)
bacillus s.
Aspergillus niger (Aspergillus)
s niger) and an enzyme selected from the group consisting of a mixture thereof. Particularly, a protease derived from Bacillus subtilis having an optimum pH of about 7 and an enzyme derived from Aspergillus oryzae having an optimum pH of about 7 or 3 to Protease 6 can be preferably mentioned.

The soybean protein decomposition mixture contained in the functional food of the present invention includes various products obtained by enzymatically decomposing soybean protein.
-Tyr, tripeptide Gly-Tyr-Tyr, tripeptide Ala-Asp-Phe, tripeptide Ser
Peptides selected from the group consisting of -Asp-Phe and mixtures thereof are preferably included as active ingredients.

The form of the functional food of the present invention is not particularly limited, but examples include tablets, powders, granules, seasonings,
Solid foods such as edible oil, confectionery, breads, noodles and pasta,
Soup-like and jelly-like foods, soybean milk such as soybean fermented soymilk,
It may be in the form of a drink such as a sports drink. Further, it may be in a form suitable for ingestion by animals other than humans, such as pet food, feed and feed.

The functional food of the present invention is not limited to humans,
It can also be applied to animals other than humans such as mammals.

The method for producing a functional food of the present invention comprises the steps of heat-treating soybean protein to obtain a modified soybean protein by the method described above, and a step of decomposing the modified soybean protein with an enzyme to obtain a soybean protein decomposition mixture. including.

The step of degrading the modified soybean protein with an enzyme can be carried out by adding various enzymes such as those exemplified above to the suspension of the modified soybean protein and hydrolyzing it. The reaction conditions can be appropriately selected depending on the optimum pH and temperature when the enzyme to be used reacts with the substrate, but for example, the substrate / enzyme weight ratio is 500 to
1000, reaction temperature 50 to 60 ° C., reaction time 2 to 3
Can be done in time.

The soybean protein decomposition mixture obtained in the above step may be subjected to a step of deactivating the enzyme and a step of filtering, if necessary, to obtain a functional food.

In the step of inactivating the enzyme, the suspension after the hydrolysis reaction is heated at a temperature of about 80 to 100 ° C. for 5 to 20 times.
It can be performed by heating for about a minute. Also,
The step of filtering is carried out by, for example, ultrafiltration or the like to obtain a molecular weight of 20.
It can be performed by collecting molecules of 00 or less. The soybean protein degradation mixture thus obtained is
The average peptide chain length can be 2 to 8, preferably 2 to 4, and the content ratio of free amino acids can be 20 to 30% by weight.

The medicine of the present invention contains the above-mentioned soybean protein decomposition mixture.

The form of the medicine of the present invention is not particularly limited, but examples include tablets, powders, granules, liquids, capsules,
It can be in the form of syrup or the like.

The method for producing the medicine of the present invention is not particularly limited, but for example, the soybean protein decomposition mixture obtained by the above method or the like is used as it is, or if necessary, the soybean protein decomposition mixture is obtained by the above method or the like. The drug of the present invention can be the one that has been subjected to the step of deactivating the enzyme and the step of filtering, or the one that has been further added with a material for formulation such as an excipient to form a formulation.

The drug of the present invention can be administered not only to humans but also to non-human animals such as mammals.

The method of administration of the medicine of the present invention includes, for example, oral administration. In the case of oral administration, it can be administered in the form of the above-mentioned preparation.

The dose of the drug of the present invention can be appropriately selected according to the age, sex, physique, condition of the patient, and the like, for example, as the soybean protein decomposition mixture in the state where molecules having a molecular weight of more than 2000 are removed by filtration, Usually 200 mg per day
~ 1000 mg can be used.

The functional food and drug of the present invention have both an antioxidant effect and an angiotensin converting enzyme (ACE) inhibitory effect. Regarding the antioxidant effect, when the functional food of the present invention is further added to other foods, the antioxidant effect can be expected to be expressed both in the food and in the living body. Further, due to the ACE inhibitory effect, it can be expected that a hypotensive effect is expressed when a human having hypertension eats. The IC ₅₀ value of the ACE inhibitory activity of the functional food or drug of the present invention is determined by the method of HS Cheung et al. (Biochem.Pharm. 20,
1637 (1971)), the molecular weight is 2000
The soybean protein-decomposed mixture in the state in which more than 100 molecules are removed by filtration can be 66 to 120 μg protein / ml. In addition, when the functional food and drug of the present invention are ingested, in addition to the above-mentioned effects, preferable effects such as elimination of active oxygen, suppression of cholesterol elevation, and promotion of fat metabolism can be expected.

[0027]

EFFECTS OF THE INVENTION The functional foods and medicaments of the present invention have antioxidative action and antihypertensive action, and can be taken safely.
In addition, it can be manufactured at low cost.

Further, according to the method for producing a functional food of the present invention, it is possible to easily produce a safe functional food having an antioxidant action and a blood pressure lowering action.

[0029]

EXAMPLES The present invention will be described in more detail below with reference to Examples and Comparative Examples, but the present invention is not limited to the Examples. Examples 1 to 8 50 g of the isolated soybean protein was dissolved in 1000 g of water to prepare an aqueous suspension of soybean protein. Suspension at 100 ° C
The mixture was heated at 40 ° C for about 40 minutes to denature the protein.

Then, in the suspension, protease A (optimal pH 6 to 8, optimal temperature 50 ° C.) (Example 1), protease M (optimal pH 3 to 6, optimal temperature 50 ° C.) (Example 2) ), Protease P (optimal pH 7-9, optimal temperature 45)
C) (Example 3), Protease N (optimal pH 6-8,
Optimum temperature 55 ° C (Example 4), Protease Z (optimal pH 9-12, optimal temperature 60 ° C) (Example 5), Neurase F (optimal pH 3-5, optimal temperature 45 ° C) ( Example 6), Protease S (optimal pH 7-9, optimal temperature 70)
C.) (Example 7) or papain W-40 (optimal pH 3 to
12. Optimum temperature 45 to 60 ° C.) (Example 8) (all manufactured by Amano Enzyme Inc.) Substrate / enzyme = 1000
(Weight ratio) was added, and the enzyme reaction was carried out at the optimum pH and optimum temperature of each enzyme. 1, 2, 3, after starting the enzymatic reaction
After 6 or 24 hours, the enzyme was inactivated by heating at 90 ° C. for 10 minutes to obtain an aqueous suspension of the soybean proteolysis mixture. The antioxidant activity and ACE inhibitory activity of these soybean protein degradation mixture suspensions were measured according to the following methods. The results are shown in FIGS. 1 and 2, respectively. Measurement of antioxidant activity by Rhodan iron method Add 100 ml of ethanol to 1.3 ml of linoleic acid,
After completely dissolving linoleic acid, 100 ml of 50 mM phosphate buffer solution (pH 7.0) was added and stirred to obtain a linoleic acid solution. 20 ml of this solution was placed in a 50 ml brown sample bottle, and an aqueous suspension of the soybean protein degradation mixture was filtered by 1, 2, 3, 6 or 24 hours after the initiation of the enzyme reaction of each of Examples 1 to 8 to obtain a filter. The liquid (200 μl) and distilled water (4.8 ml) were added to make the total amount 25 ml, and this was used as a sample liquid for an antioxidant test.

Next, in a 20 ml test tube, 4.7 ml of 75% ethanol, 0.1 ml of sample solution and 30% NH ₄ S were added.
0.1 ml of CN solution was taken and stirred. 0.02M for this
FeCl ₂ solution (0.02M FeCl ₂ /3.5% HC
L) 0.1 ml was added and stirred again. Then 30
By leaving it for a minute and measuring the absorbance at 500 nm,
The degree of oxidation of linoleic acid was evaluated and the antioxidant activity was measured. Measurement of ACE inhibitory activity 1, 2, 3, 6 after initiation of the enzymatic reaction of each of Examples 1 to 8
Alternatively, a filtrate obtained by filtering an aqueous suspension of a soybean protein decomposition mixture after 24 hours was used as a sample for measuring ACE inhibitory activity, and the method of HS Cheung et al. (Biochem. Pharm. 2) was used.
0, 1637 (1971)).

Further, the above water suspension is centrifuged and filtered to remove insoluble matter, and the obtained filtrate is further subjected to a pore size of 1 to 10 μm.
After passing through a filter having a larger pore size in order, the filtrate containing the substance having a molecular weight of 2000 or less is collected by ultrafiltration, further concentrated and dried to obtain a crude protein content of 93.
A 0% (by Kjeldahl method) soybean proteolysis mixture was obtained. ACE of this soy protein breakdown mixture
The IC ₅₀ value of the inhibitory activity was measured according to the method of HS Cheung et al. (Biochem. Pharm. 20, 1637 (1971)). In Examples 1 to 8, 80 μg protein / ml and 66 μg protein / ml, 110μ
g protein / ml, 85 μg protein / ml, 80
μg protein / ml, 110 μg protein / ml,
There were 115 μg protein / ml and 120 μg protein / ml. Example 9 A soybean protein suspension and an aqueous suspension of a soybean protein decomposition mixture were obtained in the same manner as in Examples 1 to 8 by using the isolated soybean protein and the protease N as the enzyme.

The aqueous suspension was centrifuged, filtered to remove insoluble matter, and the obtained filtrate was passed through a filter having a pore size of 1 to 10 μm in order from the one having a larger pore size and then subjected to ultrafiltration to obtain a molecular weight of 2000. The filtrate containing the following substances was collected, further concentrated, lyophilized, and crude protein content 93.0%
A soybean protein degradation mixture (according to the Kjeldahl method) was obtained. This soybean protein decomposition mixture was separated by gel chromatography according to the following conditions. Separation conditions: Column: Sephadex G-25 (manufactured by Amersham Bioscience), 1.6 × 90 cm Eluent: distilled water Flow rate: 0.8 ml / min Detection: Optical density separation result at 280 nm is shown in FIG. 3A. Four peaks P-1 to 1
Fractions containing P-4 were collected, and the collected eluate was used as a sample, and the antioxidant activity of each was measured by the same iron iron method as above. The results are shown in Figure 3B.

The fraction containing peak P-4 was further separated by HPLC according to the following conditions. Separation conditions: Column TSK-gel ODS-120T (manufactured by Tosoh Corporation) Column temperature: 35 ° C Eluent 0.1% TFA Acetonitrile 0% (0 minutes) →
A linear concentration gradient flow rate of 50% (60 minutes): 1.0 ml / min Detection: The result of absorbance separation at 230 nm is shown in FIG. 4A. Fractions containing some of the appearing peaks were fractionated, and the antioxidant activity was measured by the same iron iron method as above, and the ACE inhibitory activity was measured by the same method as above. The results are shown in FIGS. 4A and 4B, respectively.
Furthermore, when the sequence of the peptide contained in each peak was determined, P-4 was found to have at least Ala-Tyr and Gl as peptides having antioxidant activity and ACE inhibitory activity.
y-Tyr-Tyr, Ala-Asp-Phe and Se
It was found to contain four peptides of r-Asp-Phe. Examples 10 and 11 A soybean protein degradation mixture prepared as in Example 9 was added to soybean oil (Example 10) or lard (Example 11).
The mixture was mixed at a ratio of 5% by weight and left at 50 ° C. A sample is taken from the mixture at regular intervals after the start of standing, and the amount of peroxide contained in the sample is determined by the POV method described in Standard Oil and Fat Analysis Test Method (Japan Oil Chemistry Association) 2.4.12-71. It was measured. The results are shown in Tables 1 and 2, respectively. Comparative Examples 1 and 2 The same soybean oil (Comparative Example 1) or lard (Comparative Example 2) used in Examples 10 and 11 was left at 50 ° C.
A sample was taken from the mixture at regular intervals after the start of standing, and the amount of peroxide contained in the sample was measured by the POV method.
The results are shown in Tables 1 and 2, respectively.

[0035]

[Table 1]

Example 12 A rat diet containing modified AIN containing 25% of the soybean protein degradation mixture prepared in the same manner as in Example 9 was used for 5 rats (6
14 g per day was given to each of the animals (weekly) and they were bred for 60 days. During the rearing period, the rat was exposed to an environment in which positive ions were present at 8 × 10 ⁴ / cm ^{3 for} 4 hours per day in order to activate active oxygen in the body of the rat.

Thereafter, blood was collected from the rat, the liver and the brain were excised, and the peroxide contents in the cerebellum, brain stem, cerebrum, liver and serum were measured by the following TBA method. Results are shown in FIG. Measurement of Peroxide Content by TBA Method In the case of serum, 1.0 ml was used as a sample, and in the case of a solid sample, 1.0 ml of physiological saline was added and homogenized, and the supernatant was used as a sample. A sample is placed in a 15 ml stoppered test tube, and 1.0 ml of 35% TCA solution, 2.0 ml of 0.5% TBA solution, 0.1 ml of 1.0% BHT solution, and 0.
0.1 ml of 5% SDS solution was added, mixed, heated in a boiling water bath for 15 minutes, and immediately cooled in ice water. After cooling
Add 1.0 ml glacial acetic acid and 2.0 ml chloroform,
After vigorous stirring, centrifugation (3,000 rpm, 10 minutes) was performed, and the absorption value at a wavelength of 532 nm of the obtained supernatant was measured. Comparative Example 3 The same procedure as in Example 12 was repeated except that the soybean protein decomposition mixture was not added as the feed, and the rats were bred, and the peroxides in the cerebellum, brain stem, cerebrum, liver and serum were used. Each content was measured by the TBA method. Results are shown in FIG. Example 13 A tablet containing 100 mg of the soybean protein degradation mixture prepared in the same manner as in Example 9 was prepared. Volunteers with hypertension (maximum blood pressure 130 mmHg or more)
Twenty-four people were dosed with 4 tablets daily for 10 weeks. Blood pressure was measured daily from 3 weeks before the administration to 10 weeks after the administration. As a result, 7 mmH in 15 of 24 subjects
A drop in systolic blood pressure of g or more was observed. The average daily variation of systolic blood pressure of all subjects is shown in FIG. Examples 14-16 Defatted soy protein (Example 14), concentrated soy protein (Example 15) and isolated soy protein (Example 1)
6) 50 g of each was dissolved in 1000 g of water to prepare an aqueous suspension. The suspension was heated at 80 ° C. for 30 minutes to denature the protein.

After that, protease N (manufactured by Amano Enzyme) was added to the suspension at a ratio of substrate / enzyme = 1000 (weight ratio), and an enzyme reaction was carried out at pH 7 and 55 ° C.
Three hours after the start of the enzyme reaction, the enzyme was inactivated by heating at 90 ° C. for 10 minutes to obtain an aqueous suspension of the soybean protein decomposition mixture. With respect to these soybean protein decomposition mixture suspensions, the antioxidant activity was measured by the same method as the above-mentioned Rhodan iron method, and the ACE inhibitory activity was measured by the same method as above. The results are shown in Table 2. Comparative Examples 4 to 6 50 g of defatted soybean protein (Comparative Example 4), concentrated soybean protein (Comparative Example 5) and separated soybean protein (Comparative Example 6) were dissolved in 1000 g of water to prepare an aqueous suspension. Examples 14-16 except that the suspension was not heated
The same operation as in (1) was performed to obtain an aqueous suspension of the soybean protein decomposition mixture. With respect to these soybean protein decomposition mixture suspensions, the antioxidant activity was measured by the same method as the above-mentioned Rhodan iron method, and the ACE inhibitory activity was measured by the same method as above.
The results are shown in Table 2.

[0039]

[Table 2]

[Brief description of drawings]

FIG. 1 is a graph showing the measurement results of antioxidant activity of each soybean protein degradation mixture in Examples 1 to 8.

FIG. 2 is a graph showing the measurement results of ACE inhibitory activity of each soybean protein degradation mixture in Examples 1 to 8.

FIG. 3 is a graph showing the separation result of the soybean protein decomposition mixture by the gel chromatography method and the measurement result of the antioxidant activity of each fraction in Example 9.

FIG. 4 shows the result of separation by HPLC in Example 9.
3 is a graph showing the measurement results of antioxidant activity and ACE inhibitory activity of each fraction.

FIG. 5 is a graph showing the measurement results of the amount of peroxide in the rat body in Example 12 and Comparative Example 3.

FIG. 6 is a graph showing the measurement results of blood pressure of a subject who ingested the soybean protein decomposition mixture in Example 13.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 39/06 A61K 37/02

Claims (5)

[Claims] 1. A functional food comprising a soybean protein decomposition mixture obtained by decomposing heat-denatured soybean protein with an enzyme. 2. The soybean proteolysis mixture comprises a dipeptide Ala-Tyr and a tripeptide Gly-Tyr-.
The functional food according to claim 1, comprising a peptide selected from the group consisting of Tyr, tripeptide Ala-Asp-Phe, tripeptide Ser-Asp-Phe, and mixtures thereof. 3. The method according to claim 1, further comprising a step of heat-treating soybean protein to obtain a modified soybean protein; and a step of decomposing the modified soybean protein with an enzyme to obtain a soybean protein decomposition mixture. 2. The method for producing a functional food according to 2. 4. The step of degrading the denatured soybean protein with an enzyme is carried out by Carica papaya.
a L) -derived protease, Aspergillus oryzae-derived protease, Bacillus subtilis (Bacillus)
Aspergillus melleus, a protease derived from subtilis
(us) -derived protease, Bacillus stearotherm
The method according to claim 3, wherein the enzyme is selected from the group consisting of a protease derived from Aspergillus niger, a protease derived from Aspergillus niger, and a mixture thereof. 5. A medicine for lowering blood pressure, suppressing peroxide generation, or a combination thereof, which comprises a soybean protein decomposition mixture obtained by decomposing heat-denatured soybean protein with an enzyme.