阅读说明：本技术 抗氧化肽、含有该抗氧化肽的大豆蛋白水解物 (Antioxidant peptide and soybean protein hydrolysate containing the same ) 是由 本山贵康 于 2020-06-10 设计创作，主要内容包括：提供能用于食品或药品的、显示高抗氧化活性的抗氧化肽、含有该抗氧化肽的大豆蛋白水解物。所述抗氧化肽序列为显示高ORAC值的抗氧化肽序列KYQ、显示高DPPH值的抗氧化肽序列ERQ、显示高ORAC值的抗氧化肽序列KYR、显示高DPPH值的抗氧化肽序列ERL。(The antioxidant peptide sequence is an antioxidant peptide sequence KYQ with high ORAC value, an antioxidant peptide sequence ERQ with high DPPH value, an antioxidant peptide sequence KYR with high ORAC value and an antioxidant peptide sequence ER L with high DPPH value.)

1. An antioxidant peptide has an amino acid sequence of KYQ.

2. A soy protein hydrolysate comprising the antioxidant peptide of claim 1.

3. The soy protein hydrolysate of claim 2 further comprising an antioxidant peptide having the amino acid sequence ERQ.

4. An antioxidant peptide has an amino acid sequence of ER L.

5. A soy protein hydrolysate comprising the antioxidant peptide of claim 4.

6. The soy protein hydrolysate of claim 5 further comprising an antioxidant peptide having the amino acid sequence KYR.

7. A food product comprising the antioxidant peptide of claim 1 or 4 or the soybean protein hydrolysate of any one of claims 2, 3, 5 and 6.

8. An antioxidant pharmaceutical product comprising the antioxidant peptide according to claim 1 or 4 or the soybean protein hydrolysate according to any one of claims 2, 3, 5 and 6.

9. Use of the antioxidant peptide of claim 1 or 4, or use of the soy protein hydrolysate of any one of claims 2, 3, 5, or 6 in the manufacture of an antioxidant.

10. Use of the antioxidant peptide of claim 1 or 4 or the soybean protein hydrolysate of any one of claims 2, 3, 5 and 6 for the manufacture of an antioxidant pharmaceutical product.

Technical Field

The present invention relates to an antioxidant peptide, a soybean protein hydrolysate containing the antioxidant peptide, a food or a pharmaceutical product containing the antioxidant peptide or the soybean protein hydrolysate, and use of the antioxidant peptide or the soybean protein hydrolysate for the production of an antioxidant.

Background

Aerobic organisms including humans take in oxygen through respiration for survival, and a part of the oxygen taken in the body is converted into active oxygen species such as peroxide radicals, hydrogen peroxide, hydroxyl radicals and the like during energy metabolism. Such active oxygen species are originally involved in organism defense and the like represented by a mechanism of removing a pathogen from macrophages when infected with bacteria or viruses, and play an important role in maintaining health. However, it is considered that environmental factors such as air pollution and ultraviolet rays, living habits such as smoking, stress, and the like destroy the balance in the living body, and that excessive reactive oxygen species react with proteins, lipids, DNA, and the like in the living body to cause protein denaturation, lipid peroxide production, genetic damage, and the like, thereby causing living habit disorders and aging. Therefore, it is gradually recognized that the intake of antioxidant active ingredients from the diet is very important for maintaining health in addition to the mechanism possessed by the organism, and it is recommended to actively take antioxidant substances as the 7 th nutrient.

Soybeans are reported to have various physiological functions, and there are many reports on antioxidant activity. For example, soy isoflavones have been identified as having as high antioxidant activity as other flavonoids. In addition, regarding soybean protein, it has been reported that a hydrolysate thereof has antioxidant activity, and further, a peptide sequence showing antioxidant activity has been identified.

Among them, patent document 1 discloses: provided is a soybean protein hydrolysis method which hydrolyzes soybean protein using an enzyme having both endo-and exo-peptidase activities, thereby increasing the soluble fraction, avoiding the bitter fraction, and enhancing or improving the antioxidant ability. However, the soluble soybean protein described in patent document 1 contains peptides of about 3kDa to 30kDa in part, and does not substantially contain a low molecular weight soybean protein. As described in non-patent document 1, in soybean protein, dipeptide and tripeptide are best absorbed in the intestinal tract, and larger peptides are generally difficult to be directly absorbed. Therefore, the peptide described in patent document 1 is too large to be absorbed and is not considered to be practical because it does not exhibit a significant action in vivo. Furthermore, the peptide described in patent document 2 is also considered to be impractical because it has a size of 500 to 5000, and is too large as compared with a dipeptide and a tripeptide.

Further, various methods for measuring the oxidation resistance have been developed for measuring a reaction product obtained by a radical or the like, but even if the same antioxidant substance is used, the measured values differ depending on the measurement method, and therefore, the measured values obtained by different measurement methods cannot be compared with each other. As a representative method for measuring antioxidant activity, the ORAC method is a method for measuring a function of an antioxidant substance to inhibit substrate oxidation by supplying a hydrogen atom to a radical, and the DPPH method is a method for measuring a function of an antioxidant substance to inhibit substrate oxidation by supplying an electron to a radical or an oxide.

Although various peptides contained in soybean protein hydrolysates have been evaluated for their antioxidant function by various methods, no sequence of dipeptide or tripeptide has been reported for each measurement method, and no soybean protein hydrolysate has been reported for each measurement method such as ORAC method and DPPH method.

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the above-mentioned current state of the art, and an object of the present invention is to provide an antioxidant peptide which can be used in foods and medicines and exhibits high antioxidant activity, and a soybean protein hydrolysate which exhibits high antioxidant activity in various measurement methods such as the ORAC method and the DPPH method.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a tripeptide having a specific amino acid sequence in a soybean protein hydrolysate obtained by decomposing soybean protein with an appropriate proteolytic enzyme exhibits high antioxidant activity in the ORAC method or DPPH method, thereby completing the present invention.

Specifically, the present invention provides the following technical solutions.

[1] An antioxidant peptide has an amino acid sequence of KYQ.

[2] An antioxidant peptide has an amino acid sequence of ERQ.

[3] A soybean protein hydrolysate which comprises the antioxidant peptide of [1] and/or the antioxidant peptide of [2 ].

[4] An antioxidant peptide has an amino acid sequence of KYR.

[5] An antioxidant peptide has an amino acid sequence of ER L.

[6] A soybean protein hydrolysate comprising the antioxidant peptide of [4] and/or the antioxidant peptide of [5 ].

[7] A food product comprising the antioxidant peptide according to any one of the above [1], [2], [4] and [5], or the soybean protein hydrolysate according to the above [3] or [6 ].

[8] An antioxidant pharmaceutical product comprising the antioxidant peptide according to any one of the above [1], [2], [4] and [5], or the soybean protein hydrolysate according to the above [3] or [6 ].

[9] Use of the antioxidant peptide of any one of [1], [2], [4] and [5], or the soybean protein hydrolysate of [3] or [6] for the production of an antioxidant.

[10] Use of the antioxidant peptide of any one of the above [1], [2], [4] and [5], or the soybean protein hydrolysate of [3] or [6] for the manufacture of an antioxidant drug.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, an antioxidant peptide having a sufficiently low molecular weight and being easily absorbed and exhibiting high antioxidant activity, and a soybean protein hydrolysate containing the antioxidant peptide can be provided, and an antioxidant exhibiting high antioxidant activity can be provided based on the antioxidant peptide.

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and various modifications can be made within the scope of the gist of the present invention.

(antioxidative peptide)

The antioxidant peptide disclosed by the invention has high antioxidant activity in an ORAC method or a DPPH method, and has the amino acid sequences of KYQ, ERQ, KYR and ER L, wherein the antioxidant peptide sequence KYQ shows a high ORAC value, the antioxidant peptide sequence ERQ shows a high DPPH value, the antioxidant peptide sequence KYR shows a high ORAC value, and the antioxidant peptide sequence ER L shows a high DPPH value.

Wherein the amino acid sequence KYQ represents lysine-tyrosine-glutamine, the amino acid sequence ERQ may be represented by L ys-Tyr-gln, the amino acid sequence KYR may be represented by glutamic acid-arginine-glutamine, the amino acid sequence Glu-Arg-gln, the amino acid sequence KYR may be represented by lysine-tyrosine-arginine, the amino acid sequence ER L may be represented by L ys-Tyr-Arg, the amino acid sequence ER L may be represented by glutamic acid-arginine-leucine, and the amino acid sequence Glu-Arg-L eu.

The method for producing the antioxidant peptide of the present invention is not particularly limited, and for example, a chemical synthesis method or a biological synthesis method generally used for producing a polypeptide can be used. Examples of the chemical synthesis method include a Boc synthesis method and an Fmoc synthesis method. Among these, the Fmoc synthesis method is preferred from the viewpoint of avoiding side reactions caused by acids because the final deprotection reaction conditions are mild.

Examples of the biosynthesis method include a fermentation method, an enzymatic hydrolysis method, and a genetic engineering method. As a typical example, a soybean protein hydrolysate containing the antioxidant peptide of the present invention can be prepared by decomposing a soybean protein material described later by an enzymatic hydrolysis method. The antioxidant peptide of the present invention may be used as an antioxidant after being separated from a soybean protein hydrolysate containing the antioxidant peptide, or may be used as an antioxidant in a form contained in a soybean protein hydrolysate.

(antioxidation: ORAC)

The antioxidant peptide of the present invention shows high antioxidant activity. The antioxidant activity of the antioxidant peptide can be measured by the method described in non-patent document 2, using the value of "ORAC" (Oxygen Radical absorbance capacity) measured in the unit of. mu. mol TE/mmol. In the unit, "TE" is an abbreviation of "Trolox equivalent," and "Trolox" is a registered trademark of the standard substance "6-hydroxy-2, 5,7, 8-tetramethyl chroman-2-carboxylic acid". The higher the ORAC value, the higher the oxygen radical absorption ability, i.e., the antioxidant activity.

(antioxidation: DPPH)

The antioxidant peptide of the present invention shows high antioxidant activity. The antioxidant activity of the antioxidant peptide can be measured by the method described in non-patent document 3, and the value (unit: μmol TE/mmol) of "DPPH" (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging activity can be used as an index. In the unit, "TE" is an abbreviation of "Trolox equivalent," and "Trolox" is a registered trademark of the standard substance "6-hydroxy-2, 5,7, 8-tetramethyl chroman-2-carboxylic acid". The higher the DPPH radical scavenging ability value (DPPH value), the higher the radical scavenging ability, i.e., antioxidant activity.

(Soybean protein Material)

The soybean protein hydrolysate of the present invention is obtained by hydrolyzing a soybean protein material with a protease. Here, as an example of the soybean protein material, there can be mentioned: whole soybean milk and defatted soybean milk are obtained by extracting protein components from whole soybean, defatted soybean, etc. with water, and removing bean dregs. As another example of the soybean protein material, there can be mentioned: and a separated soybean protein obtained by concentrating the protein from the soybean milk by treatment with an ultrafiltration membrane or isoelectric precipitation using an acid. As another example of the soybean protein material, a concentrated protein containing fiber and a decomposed product of soybean powder can be cited. These soy protein materials may also be sterilized/dried materials. The finally obtained soybean protein material preferably contains 80 wt% or more of protein based on the dry weight, and for example, preferably isolated soybean protein or the like.

Isolated soy protein is generally prepared as follows. That is, water is added to defatted soybeans, extraction is performed near neutrality, and soybean milk is obtained by separating soybean dregs. Subsequently, the pH of the soybean milk was adjusted to about 4.5, and the isoelectric point precipitate was collected. Adding water and an alkaline agent to the precipitate to obtain an aqueous solution having a solid content concentration of 5 to 15 wt%, a pH of 5.7 to 8.0, and preferably a pH of about 6.8 to 7.5. As the alkali agent, sodium hydroxide, potassium hydroxide, or the like can be used. The isolated soy protein thus obtained may be used as it is in the following steps, or may be used without sterilization or after sterilization by drying and then redissolving. However, the isolated soybean protein is not limited to the above-mentioned production method, and may be produced by various modifications of the production method.

(Soybean protein hydrolysate)

In the present invention, the soy protein hydrolysate comprises at least one selected from the group consisting of an antioxidant peptide sequence KYQ and an antioxidant peptide sequence KYR exhibiting a high ORAC value, and/or at least one selected from the group consisting of an antioxidant peptide sequence ERQ and an antioxidant peptide sequence ER L exhibiting a high DPPH value, from the viewpoint of overall exhibiting higher antioxidant activity, it is preferable that the soy protein hydrolysate of the present invention comprises at least one selected from the group consisting of an antioxidant peptide sequence KYQ and an antioxidant peptide sequence KYR exhibiting a high ORAC value, and at least one selected from the group consisting of an antioxidant peptide sequence ERQ and an antioxidant peptide sequence ER L exhibiting a high DPPH value.

The soybean protein hydrolysate of the present invention may contain the antioxidant peptide sequence, and the content of each antioxidant peptide is not particularly limited. For example, each may be 3% by weight or more, preferably 5% by weight or more, more preferably 8% by weight or more, and still more preferably 10% by weight or more.

The content ratio of at least one selected from the antioxidant peptide sequence KYQ and the antioxidant peptide sequence KYR exhibiting a high ORAC value and at least one selected from the antioxidant peptide sequence ERQ and the antioxidant peptide sequence ER L exhibiting a high DPPH value in the soybean protein hydrolysate of the present invention is not particularly limited, and may be, for example, 1:9 to 9:1, preferably 2:8 to 8:2, more preferably 3:7 to 7:3, and further preferably 4:6 to 6:4 in terms of weight ratio.

The soybean protein hydrolysate of the present invention may contain tripeptides that do not satisfy the sequence of the antioxidant peptide, in addition to the antioxidant peptide. The other tripeptides are not particularly limited, and may be those exhibiting a high ORAC value or those exhibiting a high DPPH value.

The tripeptide having a high ORAC value may be, for example, a tripeptide sequence having an ORAC value of 350. mu. mol TE/mmol or more, preferably 450. mu. mol TE/mmol or more, and more preferably 500. mu. mol TE/mmol or more, the upper limit of the ORAC value of the tripeptide is not particularly limited, but is usually lower than the ORAC values of the antioxidative peptide sequence KYQ and the antioxidative peptide sequence KYR, and may be, for example, 1000. mu. mol TE/mmol or less.

The tripeptides exhibiting a high DPPH value may be, for example, tripeptide sequences having a DPPH value of preferably 100. mu. mol TE/mmol or more, more preferably 200. mu. mol TE/mmol or more, and still more preferably 300. mu. mol TE/mmol or more, the upper limit of the DPPH value of the tripeptides is not particularly limited, and is usually lower than the DPPH values of the antioxidative peptide sequence ERQ and the antioxidative peptide sequence ER L, and may be, for example, 420. mu. mol TE/mmol or less.

The soybean protein hydrolysate of the present invention is typically a peptide mixture obtained by treating the above-described soybean protein material with a protease. The protein hydrolysate is preferably decomposed to a higher degree, and particularly preferably, the ratio of a dipeptide and a tripeptide (hereinafter, sometimes collectively referred to as "dipeptide/tripeptide") to the total amount of peptides and free amino acids in the hydrolysate is high. Specifically, for example, from the viewpoint of being easily absorbed in a living body and effectively exhibiting antioxidant activity, the total content of the dipeptide/tripeptide is preferably 50% by weight or more, more preferably 60% by weight or more, and particularly preferably 70% by weight or more, based on the total amount of the peptide and the free amino acid.

The dipeptide/tripeptide content was calculated as follows: the ratio of the di/tri peptide and free amino acid fractions in the protein hydrolysate was determined by peptide gel filtration chromatography, and the free amino acid content in the protein hydrolysate calculated by amino acid analysis was subtracted.

In addition, from the viewpoint of easy absorption in the living body and exerting high antioxidant activity, the soybean protein hydrolysate of the present invention preferably has a reduced ratio of peptides other than di-and tripeptides to free amino acids as much as possible. That is, the content of free amino acids in the protein hydrolysate is preferably 12% by weight or less, more preferably 5% by weight or less, and still more preferably 3% by weight or less, based on the total amount of peptides and free amino acids. In addition, since the peptide body in the protein hydrolysate is preferably a lower molecular peptide, the fraction ratio of the dipeptide/tripeptide in the protein hydrolysate is preferably 40 wt% or less, more preferably 38 wt% or less, and further preferably 35 wt% or less with respect to the total amount of the peptide and the free amino acid.

(protease)

The protease used for obtaining the soybean protein hydrolysate of the present invention may be of any animal, plant or microbial origin, and may be suitably selected from proteases classified into "metalloprotease", "acid protease", "thiol protease" and "serine protease" in the classification of proteases, and preferably from proteases classified into "metalloprotease", "thiol protease" and "serine protease". In particular, a decomposition method in which enzymes belonging to 2 or more or 3 or more different classes are allowed to act sequentially or simultaneously is particularly preferable because the ratio of peptides having a relatively low molecular weight, such as dipeptides and tripeptides, can be increased.

The protease is classified into a classification method according to the kind of amino acid in the active center, which is generally used in the field of enzymology. Representative examples of the "metalloprotease" include neutral protease derived from Bacillus (Bacillus), neutral protease derived from Streptomyces (Streptomyces), neutral protease derived from Aspergillus (Aspergillus), and "Thermoase", and examples of the "acid protease" include pepsin, acid protease derived from Aspergillus (Aspergillus), and "submizyme FP", examples of the "thiol protease" include bromelain, papain, and examples of the "serine protease" include trypsin, chymotrypsin, subtilisin, alkaline protease derived from Streptomyces (Streptomyces), "Alcalase", and "Bioprase". Even enzymes other than these enzymes can be classified according to the action pH or reactivity with inhibitors. Since enzymes having different active centers have greatly different sites of action on substrates, enzymatic decomposition products can be efficiently obtained with reduced "cleavage residues". Further, by using enzymes of different origins (organisms of origin) in combination, an enzymatic decomposition product can be produced more efficiently. Even if the classification is the same, the site of action on the substrate protein differs depending on the source, and as a result, the ratio of the dipeptide or tripeptide can be increased. These proteases are preferably those having a small exonuclease activity.

The reaction pH or reaction temperature of the protease treatment may be set according to the characteristics of the protease used, and usually, the reaction pH may be set to a value near the optimum pH and the reaction temperature may be set to a value near the optimum temperature. The reaction temperature is generally 20 to 80 ℃ and preferably 40 to 60 ℃. After the reaction, the reaction mixture is heated to a temperature (about 60 to 170 ℃) sufficient to inactivate the enzyme, thereby inactivating the residual enzyme activity.

The reaction solution after the protease treatment may be used as it is or after concentration, but is usually used in the form of a dry powder by sterilizing, spray drying, freeze drying, or the like. The sterilization is preferably performed by heat sterilization, and the heating temperature is preferably 110 to 170 ℃, and more preferably 130 to 170 ℃. The heating time is preferably 3 to 20 seconds. The reaction solution may be adjusted to an arbitrary pH. Insoluble matter (precipitate, suspended matter) generated during the protease treatment or pH adjustment can be removed by centrifugation, filtration, or the like. Removal of insoluble matter is preferred because it can improve the antioxidant activity of the soybean protein hydrolysate. And further purifying with activated carbon and adsorbent resin.

(use as antioxidant)

The antioxidant peptide and the soybean protein hydrolysate containing the same of the present invention can be used alone as an antioxidant, but are preferably used in combination with 1 or more other substances exhibiting antioxidant activity or extracts containing these substances, because they synergistically enhance the antioxidant activity.

Examples of the other antioxidant substance include vitamins, polyphenols, carotenoids, saponins, garlicin, and the like, examples of the vitamins include vitamin C, vitamin a, vitamin E, and the like, examples of the polyphenols include flavonoids such as isoflavone, quercetin, myricetin, kaempferol, hesperidin, naringin, anthocyanin, catechin, chrysin, apigenin, luteolin, sesamin, sesaminol, asanol, asaricin, sesamol, sesamolin, and the like, and examples of the carotenoids include carotenoids (α -, β -, γ -, -, -lycopene, and the like, or xanthophyll, astaxanthin, zeaxanthin, canthaxanthin, phytoxanthin, lutein, phytoxanthin, theophyllin, and the like, and examples of the carotenoid extract may include a phytolaccin extract, a capsanthin which these saponins and the aforementioned saponins are used in place of the antioxidant substance, and the extract of rubus berries, and the extract may be used in combination with the aforementioned extracts.

(utilization in food)

The antioxidant peptide of the present invention and the soybean protein hydrolysate containing the same can be used in various foods in the form of an antioxidant containing the same as an active ingredient. For example, it can be added to a food for the purpose of preventing oxidation of the food itself. In addition, by adding the antioxidant peptide of the present invention or a soybean protein hydrolysate containing the same, it is possible to obtain a beverage, a tablet, a food block (a processed food in a block form), a sauce for salad, a meat product, a snack, a dessert, a snack, a nutritional supplement, and the like, which exhibit an antioxidant ability as a physiological function to an ingester.

(utilization in medicine)

The antioxidant peptide of the present invention and the soybean protein hydrolysate containing the same can also be used as a pharmaceutical, typically, an antioxidant pharmaceutical. Examples of diseases caused by oxidation of a living body include arteriosclerosis, myocardial infarction, cancer, diabetes, alzheimer disease, pollinosis, and the like. The antioxidant peptide of the present invention and the soybean protein hydrolysate containing the same can be used as a medicine for preventing and treating these diseases. When the drug is supplied in the form of a pharmaceutical product, it can be used in various forms such as liquid, powder, tablet, capsule, etc.