JP5015668B2 - Angiotensin converting enzyme inhibitory peptide mixture and method for producing the same - Google Patents

Description

  The present invention relates to an angiotensin converting enzyme (hereinafter referred to as “ACE”) inhibitory peptide mixture having an antihypertensive action, an ACE inhibitor and an antihypertensive agent containing the peptide mixture, and a method for producing the peptide mixture.

  Hypertension is one of the modern adult diseases that easily cause arteriosclerosis with hyperlipidemia, diabetes and obesity. The population diagnosed with hypertension is estimated to be more than 20 million in Japan, and the reserve army is said to be quite large. If hypertension is left unattended, it can cause stroke and heart disease, and also death. In spite of such a serious lifestyle-related disease, it is also known as a silent killer because it is asymptomatic and difficult to recognize.

  Several mechanisms are known to cause hypertension, and the renin-angiotensin system is one of them. The renin-angiotensin system is an important factor that regulates the balance between blood pressure, water content in the body, and electrolytes. Renin secreted from the kidney acts on angiotensinogen biosynthesized in the liver to produce angiotensin I. Next, angiotensin I is converted into angiotensin II having an effect of increasing blood pressure by the action of ACE. Therefore, if this angiotensin I is not made or the activity of ACE is inhibited, the production of angiotensin II is suppressed and the blood pressure can be lowered.

  Conventionally, in order to improve this hypertension, many research institutions have been researching and developing antihypertensives and food ingredients having blood pressure regulating functions. As natural product-derived ACE inhibitors, many peptides obtained from soybean protein, fish protein and the like are known. However, ACE inhibitors composed of natural protein-derived soy protein and fish protein contain a large amount of ingredients derived from raw materials other than active peptides, so that the effect at the time of oral ingestion is insufficient or the taste is poor and not suitable as food. Moreover, if it is going to remove the impurity derived from a raw material, a manufacturing process will become complicated, such as ultrafiltration and adsorption removal. Therefore, there is a need for a method for producing a highly active ACE-inhibiting peptide mixture from a natural product without requiring a special purification step.

Here, Patent Document 1 discloses a novel ACE inhibitor using sake lees as a raw material. Patent Document 2 discloses an oral intake for ACE inhibition using sake lees as a proteolytic enzyme degradation product of rice protein. However, these enzymatic degradation products are peptide mixtures containing a large amount of impurities, and their ACE inhibitory activity is not practically satisfactory.
Japanese Patent No. 3054462 Patent No. 3414761

  The present invention relates to a method for producing an ACE inhibitory peptide mixture capable of obtaining a highly active ACE inhibitory peptide mixture from a natural product without requiring a special purification step, and a natural product-derived ACE inhibitory peptide mixture having a high ACE inhibitory activity The main issue is to provide

  As a result of intensive studies to solve the above problems, the present inventors have made a first step in which cellulase is allowed to act on sake lees, rice shochu, heated rice, and / or koji, and a mixture obtained by the first step. According to the method including the second step of removing the liquid fraction from the second step and the third step of allowing neutral protease and / or acidic protease to act on the solid content obtained in the second step, a special purification step is required. It was found that a highly active ACE inhibitory peptide mixture was obtained from the natural product.

The present invention has been completed based on the above findings, and provides the following method for producing an ACE inhibitory peptide mixture, an ACE inhibitory peptide mixture, an ACE inhibitor, and a blood pressure lowering agent.
Item 1. The first step of allowing cellulase to act on sake lees, rice shochu, heated rice and / or koji, the second step of removing the liquid fraction from the mixture obtained by the first step, and the solid content obtained by the second step And a third step in which a neutral protease and / or an acidic protease is allowed to act on, and a method for producing an angiotensin converting enzyme-inhibiting peptide mixture.
Item 2. Item 2. The method according to Item 1, wherein in the first step, amylase is allowed to act in addition to cellulase.
Item 3. Item 3. The method according to Item 2, wherein the amylase is at least one selected from the group consisting of β-amylase, α-amylase, and / or α-glucosidase.
Item 4. Item 4. The method according to any one of Items 1 to 3, wherein the neutral protease is a neutral protease derived from Bacillus bacteria, and the acidic protease is an acidic protease derived from Aspergillus.
Item 5. Furthermore, the method in any one of claim | item 1-4 including the 4th process of removing solid content from the mixture obtained by a 3rd process.
Item 6. Item 6. An angiotensin converting enzyme inhibitory peptide mixture obtained by the method according to any one of Items 1 to 5.
Item 7. 1 part by weight of Phe-Trp and / or its salt, 4 to 12 parts by weight of Arg-Trp and / or its salt, 2 to 5 parts by weight of Ser-Trp and / or its salt, Gln-Trp and / or its An angiotensin converting enzyme inhibitory peptide mixture containing 0.1 to 0.4 parts by weight of a salt.
Item 8. Item 8. An angiotensin converting enzyme inhibitor comprising the peptide mixture obtained by the method according to any one of Items 1 to 5, or the peptide mixture according to Item 7.
Item 9. Item 8. A blood pressure lowering agent comprising the peptide mixture obtained by the method according to any one of Items 1 to 5, or the peptide mixture according to Item 7.
Item 10. An angiotensin converting enzyme inhibitor comprising at least one selected from the group consisting of the following peptides (1) to (4) and salts thereof.
(1) Phe-Trp
(2) Arg-Trp
(3) Ser-Trp
(4) Gln-Trp
Item 11. An antihypertensive agent comprising at least one selected from the group consisting of the following peptides (1) to (4) and salts thereof.
(1) Phe-Trp
(2) Arg-Trp
(3) Ser-Trp
(4) Gln-Trp
Item 12. Item 8. The food product according to (a), (b), or (c) below, comprising the peptide mixture obtained by the method according to any one of Items 1 to 5 or the peptide mixture according to Item 7.
(a) Solid or gel food containing 0.1 to 1% by weight of the peptide mixture
(b) Liquid or fluid food containing 0.1 to 1 w / v% of the above peptide mixture
(c) Food preparation containing 20 to 80% by weight of the peptide mixture. The following food (a), (b), or (c) comprising at least one peptide component selected from the group consisting of the following peptides (1) to (4) and salts thereof.
(1) Phe-Trp
(2) Arg-Trp
(3) Ser-Trp
(4) Gln-Trp
(a) Solid or gel food containing 0.1 to 1% by weight of the peptide component
(b) Liquid or fluid food containing 0.1 to 1 w / v% of the above peptide component
(c) Food preparation containing 20 to 80% by weight of the peptide component

According to the method for producing an ACE-inhibiting peptide mixture of the present invention, a peptide mixture exhibiting high ACE-inhibiting activity can be obtained using a natural food component as a raw material. The method of the present invention is a simple method including only a step of allowing an enzyme to act on a food material and a solid-liquid separation step, and does not include a special purification step. Nevertheless, according to the method of the present invention, a peptide mixture exhibiting high ACE inhibitory activity can be obtained.
More specifically, in the method of the present invention, sake lees, rice shochu, heated rice, and / or koji are used as starting materials. In sake lees and rice shochu, saccharides that are impurities are decomposed to some extent, and in heated rice, the rice becomes alpha due to heating, and the enzyme's action efficiency increases. Since there are few and many proteins are contained, the ACE inhibitory peptide contained in it can be efficiently concentrated in a subsequent process.
In the method of the present invention, cellulose is decomposed by using cellulase in the first step. By removing the decomposed cellulose in the subsequent second step, the viscosity of the raw material is reduced, the working efficiency is improved, and the action effect of an enzyme such as protease added later is increased. When α-amylase, β-amylase, and / or α-glucosidase is used in addition to cellulase in the first step, these effects are further improved.

  The method of the present invention is also characterized in that neutral protease and / or acidic protease is added in the third step. By using these proteases in combination, a peptide mixture having high ACE inhibitory activity can be efficiently obtained from the protein in the raw material.

  Since the ACE inhibitory peptide mixture obtained by the method of the present invention has a blood pressure lowering action, it can be suitably used as a blood pressure lowering agent for the prevention or treatment of hypertension. Since this peptide mixture is derived from rice, it has a good flavor and is not harmful to the human body even if it is ingested too much, and there is no worry of side effects due to long-term continuous ingestion. Therefore, this peptide mixture can be taken for a long period of time without worrying about side effects or excessive intake. Further, as described above, the ACE inhibitory peptide mixture obtained by the method of the present invention has a small amount of contaminants and high ACE inhibitory activity.

  The present invention also includes novel peptide mixtures having ACE inhibitory activity. This peptide mixture contains a dipeptide having an ACE inhibitory action, has an ACE inhibitory activity much higher than that of conventional products, and requires a small daily intake.

Hereinafter, the present invention will be described in detail.
(I) Method for Producing ACE Inhibitory Peptide Mixture The method for producing the ACE inhibitory peptide mixture of the present invention is obtained by the first step and the first step in which cellulase is allowed to act on sake lees, rice shochu, heated rice, and / or koji. A second step of removing the liquid fraction from the resulting mixture, and a third step of allowing neutral protease and / or acidic protease to act on the solid content obtained in the second step.

Raw material As a raw material used in the production method of the present invention, a protein hydrolyzate derived from rice is used. Specifically, sake lees, rice shochu, heated rice, and / or rice koji that is produced when milling is used. Among these, sake lees and rice shochu are preferred, and sake lees are more preferred in that they are low in carbohydrates, which are impurities, contain a lot of protein, and are by-products of liquor production and are easily available. The sake lees may be either sake rice brewed from steamed rice or rice koji, or liquefied rice or sake koji brewed by liquefaction preparation using rice koji.

First Step The first step is a step of allowing cellulase to act on the raw material.
As the cellulase cellulase, a known cellulase can be used without limitation. Cellulases include endoglucanases that randomly cleave cellulose chains and cellobiohydrolase that cleaves from the reducing end of cellulose to produce cellobiose. Either may be used, but cellulose can be efficiently decomposed by using both in combination.
The origin of the cellulase is not particularly limited, but a cellulase derived from Trichoderma spp. Is preferred because it easily functions with respect to the raw material of the method of the present invention.
The amount of cellulase used is preferably about 0.005 to 0.2% by weight, more preferably about 0.05 to 0.2% by weight based on the raw material (sake lees, rice shochu, heated rice, and / or koji). Preferably, about 0.05 to 0.1% by weight is even more preferred. If it is the said range, a cellulose will fully be decomposed | disassembled within a practical time, and it will solubilize in a liquid fraction. Moreover, although reaction temperature and reaction time change with the raw material to be used and the kind of cellulase, about 40-60 degreeC is preferable for reaction temperature, and about 2-20 hours are preferable for reaction time. In addition, the pH during the reaction is preferably about 3.5 to 6 where the enzyme easily functions. If it is the said range, a cellulase can fully exhibit the function, without inactivating. The reaction with cellulase does not necessarily need to be stopped, but can be stopped by heating the reaction mixture at about 80 to 90 ° C. for about 30 to 60 minutes, for example.

In the first step of amylase, amylase can be further used in order to remove carbohydrates which are contaminants. Amylase is a general term for enzymes that hydrolyze starch, and examples of amylase include α-amylase, β-amylase, glucoamylase, α-glucosidase, pullulanase, and isoamylase. Amylase can be used singly or in combination of two or more. Among these, β-amylase, α-amylase, and α-glucosidase are preferable, β-amylase is more preferable, and wheat-derived β-amylase is even more preferable.
The amount of amylase used is preferably about 0.005 to 0.2% by weight, more preferably about 0.05 to 0.1% by weight relative to the raw material (sake lees, rice shochu, heated rice, and / or koji). preferable. If it is the said range, carbohydrate will fully be solubilized within a practical time. The reaction temperature and reaction time vary depending on the raw materials and enzymes used, but the reaction temperature is preferably about 50 to 60 ° C., and the reaction time is preferably about 5 to 20 hours. The reaction with amylase is not necessarily stopped, but can be stopped by heating the reaction mixture at about 80 to 90 ° C. for about 30 to 60 minutes, for example. The pH during the reaction is preferably about 5 to 6 where the enzyme is easy to function.

Second Step The second step is a step of removing the liquid fraction from the mixture obtained in the first step. The water-soluble components are mainly removed, including carbohydrates that have been reduced in molecular weight by this process. Examples of the solid-liquid separation method include squeezing, filtration, and centrifugal separation, but it is sufficient that a certain amount of water can be removed. By this step, a solid content rich in rice-derived protein is obtained.

Third Step The third step is a step of causing protease to act on the solid content obtained in the second step. In this step, a protease is used to obtain a peptide as an active ingredient from the protein in the raw material. This process produces a peptide mixture having ACE inhibitory activity from rice-derived protein.

Protease In the method of the present invention, neutral protease and acidic protease are used alone or in combination as protease. The origin of each protease is not particularly limited. Known are proteases derived from Bacillus bacteria, Aspergillus bacteria, Rhizopus bacteria, etc. Among them, neutral proteases derived from Bacillus bacteria and acidic proteases derived from Aspergillus bacteria are preferred. The protease used in the present invention may be either an endo-type or an exo-type, but the endo-type is preferable so that the target peptide is not further fragmented. A neutral protease may be used individually by 1 type, and may be used in combination of 2 or more type. Similarly, one type of acidic protease may be used alone, or two or more types may be used in combination.
The amount of protease used is preferably about 0.1 to 1% by weight, preferably about 0.1 to 1% by weight, based on the mixture obtained after the second step for each of the neutral protease and the acidic protease, ie, the compressed product after saccharide decomposition. 0.8% by weight is more preferred, and about 0.3-0.6% by weight is even more preferred. If it is the said range, the protein in a raw material can be decomposed | disassembled to the peptide which has ACE inhibitory activity within practical time.
Although the reaction temperature and reaction time vary depending on the type of enzyme used, in the case of a neutral protease, the reaction temperature is preferably about 40 to 60 ° C., and the reaction time is preferably about 5 to 20 hours. In the case of acidic protease, the reaction temperature is preferably about 50 to 60 ° C., and the reaction time is preferably about 5 to 20 hours. The reaction with each protease may be stopped by heating the reaction mixture at, for example, about 80 to 100 ° C. for about 10 to 30 minutes. By stopping the reaction by the protease, it is avoided that the excised ACE inhibitory peptide is further decomposed to reduce the inhibitory activity. Alternatively, it is possible to prevent a decrease in the purity of the ACE-inhibiting active peptide due to excision of contaminating peptides other than the ACE-inhibiting active peptide.
The pH during the reaction is preferably about 6 to 9 for a neutral protease and about 3 to 5 for an acidic protease. If it is the said range, each protease will function easily.
Neutral protease and acidic protease may be allowed to act alone or in combination. When they are used in combination, any of them may be reacted first, but it is preferable to react a neutral protease first in that a more active peptide mixture can be obtained. Moreover, when the pH which both proteases function overlaps, you may make it react simultaneously.

Fourth Step Since the mixture obtained in the third step contains a peptide having an ACE inhibitory activity at a high concentration, it can be added to food as it is or as an active ingredient of a pharmaceutical product.
The method of the present invention may further include a fourth step of removing solid content from the mixture obtained in the third step. Thereby, solid contaminants are removed, and a liquid fraction in which the ACE inhibitory active peptide is dissolved can be obtained. The removal of the solid content can be performed by, for example, pressing, filtration, centrifugation, or the like. This liquid fraction can be added to food as it is, or used as an active ingredient of a pharmaceutical product.
Further, the liquid fraction can be concentrated or dried to be powdered. Thereby, the peptide mixture which can be preserve | saved for a long period of time and is easy to process can be obtained.

(II) ACE-inhibiting peptide mixture and use thereof The first ACE-inhibiting peptide mixture of the present invention is a peptide mixture obtained by the above steps. The second ACE inhibitory peptide mixture of the present invention contains 1 part by weight of Phe-Trp and / or a salt thereof, whereas 4 to 12 parts by weight of Arg-Trp and / or a salt thereof, Ser-Trp. And / or a salt thereof, and a peptide component mixture containing 2 to 5 parts by weight of a salt and 0.1 to 0.4 parts by weight of Gln-Trp and / or a salt thereof. Since the peptide in the second peptide mixture is a dipeptide, it is easily absorbed in the intestinal tract, and prompt action and effect can be expected after ingestion.
Since the 1st and 2nd peptide mixture of this invention has a high ACE inhibitory action and there are few impurities, it can be used conveniently as an active ingredient of an ACE inhibitor or a blood pressure lowering agent. The ACE inhibitor and the antihypertensive agent may be a pharmaceutical (a pharmaceutical composition for ACE inhibition or a pharmaceutical composition for lowering blood pressure), or a food or food additive having the respective effects.
In the case of a pharmaceutical, the first and second peptide mixtures of the present invention can be used as an active ingredient of a preventive or therapeutic agent for hypertension in mammals including humans. Moreover, since it has normalization of organ circulation and improvement of long-term prognosis (life extension effect) for congestive heart failure, it can be expected as a therapeutic agent for congestive heart failure and heart failure. The prevention in the present invention includes not only completely suppressing the onset of the disease but also suppressing the progression of the disease, and the treatment includes not only the completeness of the disease but also the improvement of the disease. A food containing the first and second peptide mixtures of the present invention is suitable as a food for specified health use or a food with a nutritional function.
Further, the present invention may be at least one selected from the group consisting of Phe-Trp, Arg-Trp, Ser-Trp, Gln-Trp and a salt thereof (hereinafter referred to as “third ACE inhibitory peptide or peptide mixture”). ACE inhibitors and antihypertensive agents are also provided. Since these peptides are dipeptides, they are easily absorbed in the intestinal tract and can be expected to have a rapid effect after ingestion.

In the case of a food having a food ACE inhibitory effect or a blood pressure lowering effect, the type of food is not particularly limited. For example, sweets such as candy, gum, cake, pie, cookies, crackers, jelly, chocolate, pudding, potato chips, sheep candy, rice crackers, buns, Chinese buns; liquors, teas, coffees, sports drinks, soft drinks Examples thereof include beverages such as water, soups and milk beverages or powdered beverages; processed rice products such as cooked rice, rice cakes and noodles. Also, so-called supplements (food preparations) such as tablets and capsules may be used.
In the case of solid or gel food, the content of the first to third peptides or peptide mixture in the food is preferably about 0.1 to 1% by weight in terms of the dry weight of the peptide, 0.25 About 0.5% by weight is more preferable. In the case of a liquid or fluid food, it is preferably about 0.1 to 1 w / v%, more preferably about 0.25 to 0.5 w / v% in terms of the dry weight of the peptide. In the case of a supplement (food preparation), it is preferably about 20 to 80% by weight, more preferably about 30 to 60% by weight in terms of the dry weight of the peptide.

  Since the peptide mixture containing an ACE inhibitory component as a food is derived from rice, when it is ingested, it has a good flavor, and if it is ingested too much, there is no harm to the human body, and there is no concern about side effects due to long-term continuous ingestion.

  The intake amount of the peptide mixture having an ACE inhibitory effect or a blood pressure lowering effect as a food varies depending on the age, body weight, symptom, etc. of the subject, but the total dry weight of the peptide per day for an adult is about 0.3 to 2 g Is preferable, and an amount of about 0.5 to 1 g is more preferable. If it is the said range, the ACE inhibitory effect or the blood pressure lowering effect is fully acquired.

When the pharmaceutical ACE inhibitor or antihypertensive agent is a pharmaceutical, the dosage form is not particularly limited. For example, oral preparations such as tablets, pills, capsules, granules, fine granules, powders, syrups, infusions and injections Parenteral preparations such as a preparation. Since the peptides in the first to third peptide mixtures of the present invention are absorbed from the digestive tract, oral agents that are easy to use are preferred.
These preparations may contain excipients, binders, lubricants, disintegrants and the like that are usually used in the pharmaceutical field in addition to the peptide mixture. The kind of these additives is not particularly limited, and those used for ordinary tablets, capsules, granules, powders, injections and the like can be used. Specifically, examples of the excipient include sugars such as crystalline cellulose, sugar alcohols such as mannitol, starches, and anhydrous calcium phosphate. Examples of the binder include starches and hydroxypropyl methylcellulose. Examples of the lubricant include stearic acid and salts thereof, talc, and wax. Examples of the disintegrant include carboxymethylcellulose and potassium salts thereof.
In the case of a solid or gel oral preparation, the content of the first to third peptides or peptide mixture in the pharmaceutical preparation is preferably about 20 to 80% by weight, preferably 30 to 60% in terms of the dry weight of the peptide. % Is more preferable. In the case of a liquid or fluid oral preparation, it is preferably about 1 to 10 w / v%, more preferably about 2.5 to 5 w / v% in terms of the dry weight of the peptide. In the case of an injection, it is preferably about 20 to 50 w / v%, more preferably about 30 to 40 w / v% in terms of the dry weight of the peptide.

The dosage of an ACE inhibitor or antihypertensive agent as a pharmaceutical agent varies depending on the patient's symptoms, age, body weight, etc., but in the case of oral intake, the total dry weight of the peptide is about 0.3 to 2 g per day for an adult. An amount of about 0.5 to 1 g is more preferable. In the case of an injection, the amount that the total dry weight of the peptide is about 0.2 to 1.5 g per day for an adult is preferable, and the amount that is about 0.3 to 0.7 g is more preferable. If it is in the said range, sufficient ACE inhibitory effect or blood pressure lowering effect will be obtained.
Since the peptide mixture containing an ACE inhibitory component as a pharmaceutical is also derived from rice, as in the case of food, there is no harm to the human body even if it is consumed excessively, and there is no concern about side effects due to long-term continuous intake.

Subjects ACE inhibitors or blood pressure lowering agents of the present invention are humans with normal high values (systolic blood pressure 130-139 mmHg, diastolic blood pressure 85-89 mmHg), mild hypertension (systolic blood pressure 140-159 mmHg, diastolic blood pressure 90-99 mmHg). Is a suitable target. Mammals exhibiting blood pressure corresponding to this are also suitable subjects.

EXAMPLES Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to these examples.

(A) Production method of peptide mixture <Removal of sugar from sake lees (first and second steps)>
Carbohydrates contained in sake lees were removed according to the flowchart shown in FIG. Specifically, the procedure was as follows.
(1) 120 L of water was added to 60 kg of sake lees (wet weight, solid content 58.4%) and stirred sufficiently. While maintaining this mixture at 50 ° C., 30 g of cellulase (Cellleucine T2; manufactured by HIBI) and 30 g of β-amylase (Biozyme M; manufactured by Amano Enzyme) were added and decomposed at 50 ° C. for 16 hours (first step). ).
(2) After the decomposition, the reaction was stopped by heating at 90 ° C. for 10 minutes.
(3) Squeeze at 5 kg / cm ² for 3 hours with a press (NSK Engineering Co., Ltd. ONS automatic press filter press 500 type), 41.9 kg of sake lees with high protein content (wet weight, solid content 56.8%) Was obtained (second step).
<Decomposition of sake-rich sake lees (steps 3 to 4)>
According to the flowchart shown in FIG. 2, the protein-rich sake lees obtained in the process shown in FIG. 1 were decomposed. Specifically, the procedure was as follows.
(1) 180 L of water was added to 41.9 kg (wet weight, 56.8%) of protein-rich sake lees obtained in the step shown in FIG. 1, and the pH was adjusted to 6.8 with KOH, followed by thorough stirring.
(2) While maintaining the temperature at 50 ° C., 210 g of neutral protease (derived from Bacillus genus, protease N Amano G; manufactured by Amano Enzyme) was added and decomposed at 50 ° C. for 16 hours. Furthermore, after adjusting to pH 4.2 with HCl and stirring well, 250 g of acidic protease (derived from Aspergillus sp., Sumiteam AP; manufactured by Shin Nippon Chemical Industry Co., Ltd.) was added and further decomposed at 60 ° C. for 22 hours (No. 3 Process).
(3) After the decomposition, the reaction was stopped by heating at 90 ° C. for 10 minutes.
(4) The mixture was squeezed at 5 kg / cm ² for 3 hours with a pressing machine (NSK Engineering Co., Ltd. ONS automatic filter press 500 type) to obtain 200 L of a peptide mixture having an ACE inhibitory action (fourth step).
(6) The obtained peptide mixture was concentrated by a vacuum concentration method and further powdered by a freeze-dry method. Concentration under reduced pressure was performed using a rotary evaporator (RE121 Rotavapor manufactured by Nihon Büch Co., Ltd .: hot water bath temperature 50 ° C., 100 rpm) and an aspirator (Neocool Aspirator BP-51 manufactured by Yamato Scientific Co., Ltd .: cooling temperature 5 ° C.). Freeze-drying was performed by dehumidifying and drying to a powder at −80 ° C. and a vacuum degree of 0.4 Pa using a freeze dryer (EYELA Tokyo Rika Kikai Co., Ltd. FDU-2100).

表１から、セルラーゼ単独でも実用的な高いＡＣＥ阻害活性が得られるが、さらに、βアミラーゼ、α−アミラーゼ、又はα−グルコシダーゼを併用する場合はさらに高いＡＣＥ阻害活性が得られることが分かる。特に、β−アミラーゼを併用した場合に最も高いＡＣＥ阻害活性が見られた。 (B) Examination of types of saccharide-degrading enzymes The types of enzymes used to remove saccharides from sake lees were compared. In the above procedure (A), a peptide mixture was obtained in the same manner as in (A) except that cellulase and β-amylase were used as carbohydrases and the enzymes shown in Table 1 below were used. It was. The results are shown in Table 1. In Table 1, the total activity is a relative value obtained by multiplying the solid content ratio by the value of the ACE inhibitory activity.

Table 1 shows that practical high ACE inhibitory activity can be obtained with cellulase alone, but further higher ACE inhibitory activity can be obtained when β-amylase, α-amylase, or α-glucosidase is used in combination. In particular, the highest ACE inhibitory activity was observed when β-amylase was used in combination.

表２から、中性プロテアーゼと酸性プロテアーゼとを併用することにより、ＡＣＥ阻害ペプチドの製造効率に相乗効果が得られたことが分かる。 (C) Examination of type of protease The type of protease was compared and examined. In the above procedure (A), a peptide mixture was obtained by using the enzymes shown in Table 2 below instead of using a neutral protease and an acidic protease in combination. The results are shown in Table 2.

From Table 2, it can be seen that a synergistic effect was obtained in the production efficiency of the ACE-inhibiting peptide by using a neutral protease and an acidic protease in combination.

In the examples of the present invention, the ACE inhibitory activity was measured by the following method.
Method for measuring ACE inhibitory activity ACE inhibitory activity was measured according to the method described in JOURNAL of Chromatography, 233 (1982) 123-130. Specifically, it measured by the following procedures.
<Reagent>
(1) Substrate solution 6.5 mM Hip-His-Leu (manufactured by Peptide Institute), 520 mM NaCl, dissolved in 0.2 M borate buffer (pH 8.3) was used.
(2) Enzyme solution Rabbit lung ACE enzyme (manufactured by SIGMA) 1 U / ml dissolved in 0.2 M borate buffer (pH 8.3) (final concentration 0.1 U / ml) was used.
(3) Reaction stop solution A 3% w / v metaphosphoric acid solution was used.
<Measurement procedure>
(1) 60 μl of a test sample (distilled water in the case of control) prepared at 250 to 1000 ppm and 30 μl of enzyme solution were kept at 37 ° C.
(2) Three minutes later, 300 μl of the substrate solution was added and reacted at 37 ° C. for 30 minutes.
(3) After 30 minutes, 500 μl of a reaction stop solution was added to stop the reaction.
(4) Hippuric acid concentration was measured by HPLC. The measurement conditions are shown below.
Model used SHIMADZU LC10AVP
Column Waters SunFire C18 4.6mm × 150mm
Moving bed A solution: 20 mM phosphate buffer pH 3.0, B solution: methanol A: B = 55: 45
Injection volume 20μl Flow rate 1ml / min Column temperature 40 ℃
(5) ACE inhibitory activity was calculated according to the following formula.
ACE inhibitory activity = [(hippuric acid concentration of control−hippuric acid concentration of test sample) / hippuric acid concentration of control] × 100 (%)
(6) The sample concentration (IC ₅₀ ) when the ACE inhibitory activity was 50% was calculated.

＜ペプチド混合物の測定＞
（１）上記（Ａ）により得られたペプチド混合物１０ｍｇを純水1ｍＬに溶解し、ペプチド混合物水溶液とした。
（２）ペプチド混合物水溶液と内部標準原液と混合し、ペプチド混合物と内部標準物質が各0.002ｇ／ｍＬ、0.1ｍｇ／ｍＬになるよう試料溶液を調製し、標準溶液の場合と同様に各ペプチドのMSピーク面積を測定した。
（３）得られたペプチドのMSピーク面積より、以下の式に従って各々のペプチド含有量を算出した。
試料のペプチド含有量（ｍｇ/g）＝標準溶液のペプチド濃度（mｇ/ml）×{（試料溶液のペプチドピーク面積/試料のＩＳピーク面積）/（標準溶液のペプチドピーク面積/標準溶液のＩＳピーク面積）}/試料のペプチド濃度（ｇ/ml）
＜結果＞
結果を表４に示す。

表中の従来法は、特許文献１に記載された方法であり、酒粕からのＡＣＥ阻害ペプチド混合物の製造方法である。表４より、本発明方法により得られるペプチド混合物には、４種類のＡＣＥ阻害ジペプチドが含まれており、それらの濃度は従来法により酒粕から得られる混合物より高いことが分かる。
なお、上記（Ａ）により得られたペプチド混合物のＡＣＥ阻害活性を、従来の製法により得られるＡＣＥ阻害ペプチド混合物のＡＣＥ阻害活性と比較したものを以下の表５に示す。従来製法によるペプチド混合物のＡＣＥ阻害活性は表中に記載した公報から引用した値である。

本発明方法により、従来の食品由来のＡＣＥ阻害ペプチド混合物に比べて格段に活性の高いＡＣＥ阻害ペプチド混合物が得られることが分かる。 (D) Quantification of ACE-inhibiting peptide The ACE-inhibiting peptide contained in the peptide mixture obtained in (A) above was quantified by LC / MS analysis. The analysis conditions are shown below.
<Measurement of peptide standard sample>
(1) Preparation of peptide standard solution Weigh 10 mg of various peptides (Phe-Trp, Arg-Trp, Ser-Trp, Gln-Trp; manufactured by Kokusan Kagaku Co., Ltd.), add water or hydrochloric acid aqueous solution to completely dissolve, and add to 1 mL. The volume was constant.
(2) Preparation of internal standard substance solution 10 mg of N-carbamyl-L-Arg (manufactured by Sigma) was weighed and completely dissolved in 1 mL of 0.06N aqueous hydrochloric acid solution to obtain an internal standard solution stock solution.
(3) Preparation of standard solution Peptide standard solution and internal standard stock solution were mixed, and standard solutions were prepared so that various peptides and internal standard substances would be 0.01 mg / ml and 0.1 mg / ml, respectively. The MS peak area of each peptide was measured under the following LC / MS analysis conditions.
Equipment: Waters Alliance 2695-2996-ZQ4000 (LC-photodiode array detector-MS detector)
Column: Imtakt UK-C18 75 × 4.6mm
Mobile phase: Liquid A: 0.05% HCOOH / water, liquid B: Acetonitrile (0-25% / 0-7.5 minutes, 100% / 7.5-8.5 minutes)
Injection volume: 10 μL
Flow rate: 1 mL / min Column temperature: 35 ° C
Detection condition: UV 190-300 nm
MS electrospray ion (ESI) method positive SIR mode

<Measurement of peptide mixture>
(1) 10 mg of the peptide mixture obtained by the above (A) was dissolved in 1 mL of pure water to obtain a peptide mixture aqueous solution.
(2) Mix the aqueous peptide mixture solution with the internal standard stock solution, and prepare the sample solution so that the peptide mixture and the internal standard substance are 0.002 g / mL and 0.1 mg / mL, respectively. MS peak area was measured.
(3) Each peptide content was computed from the MS peak area of the obtained peptide according to the following formula | equation.
Sample peptide content (mg / g) = standard solution peptide concentration (mg / ml) × {(sample solution peptide peak area / sample IS peak area) / (standard solution peptide peak area / standard solution IS Peak area)} / peptide concentration of sample (g / ml)
<Result>
The results are shown in Table 4.

The conventional method in the table is a method described in Patent Document 1 and a method for producing an ACE-inhibiting peptide mixture from sake lees. From Table 4, it can be seen that the peptide mixture obtained by the method of the present invention contains four kinds of ACE-inhibiting dipeptides, and their concentrations are higher than those obtained from sake lees by the conventional method.
Table 5 below shows a comparison of the ACE inhibitory activity of the peptide mixture obtained by the above (A) with the ACE inhibitory activity of the ACE inhibitory peptide mixture obtained by the conventional production method. The ACE inhibitory activity of the peptide mixture by the conventional production method is a value quoted from the publications described in the table.

It can be seen that the method of the present invention provides an ACE inhibitory peptide mixture that is significantly more active than conventional food-derived ACE inhibitory peptide mixtures.

  According to the method for producing an ACE-inhibiting peptide mixture of the present invention, a peptide mixture having an ACE inhibitory action can be obtained from a natural product at a high concentration without requiring a special purification step. This peptide mixture contains many peptides with blood pressure lowering effects, and can be widely used in the pharmaceutical and functional food fields as a safe antihypertensive agent derived from natural products that does not have adverse side effects on the human body even if taken continuously for a long time. .

It is a flowchart which shows the carbohydrate removal process of sake lees. It is a flowchart which shows the process of decomposing | disassembling the protein high content sake lees obtained in FIG. 1 to an effective peptide.

Claims (5)

A first step in which cellulase and β-amylase are allowed to act on sake lees and / or rice shochu , a second step in which the liquid fraction is removed from the mixture obtained in the first step, and a solid content obtained in the second step And a third step in which a neutral protease and / or an acidic protease is allowed to act on, and a method for producing an angiotensin converting enzyme-inhibiting peptide mixture. The method according to claim 1 , wherein the neutral protease is a neutral protease derived from Bacillus bacteria. The method according to claim 1 or 2, wherein the acidic protease is an acidic protease derived from Aspergillus. Furthermore, the method in any one of Claims 1-3 including the 4th process of removing solid content from the mixture obtained by a 3rd process. 2. The angiotensin converting enzyme-inhibiting peptide mixture contains Phe-Trp and / or a salt thereof, Arg-Trp and / or a salt thereof, Ser-Trp and / or a salt thereof, and Gln-Trp and / or a salt thereof. The method in any one of -4.

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