KR20030003462A - Antihypertensive casein protein hydrolysate and manufacturing method thereof - Google Patents

Abstract

PURPOSE: Provided is a process of preparing casein protein hydrolysate which is not decomposed by angiotensin converting enzyme while having excellent inhibitory activity on angiotensin converting enzyme, by using an enzyme which acts in neutrality, thereby increasing productivity and reducing the cost of production. The product is convenient to eat and can be used as a protein supply source. CONSTITUTION: A protease is added to an aqueous casein protein solution in the amount of 0.1 to 3% and reacts at 30 to 50deg.C and a pH of 6 to 9 for 4 to 48hr. The protease is one or more selected from the group consisting of MP(Aspergillus melleus), Promod 192(derived from Aspergillus), Pescalase(Bacillus licheniformis), Protamex(derived from Bacillus) to produce hydrolyzed casein vegetable protein which lowers a blood pressure and has a molecular weight of 773 daltons.

Description

Casein protein degradant having a blood pressure lowering effect and a method for manufacturing the same {Antihypertensive casein protein hydrolysate and manufacturing method}

The present invention relates to a casein protein lysate and a method for preparing the same, and more particularly, to a casein protein lysate exhibiting a blood pressure lowering effect by inhibiting angiotensin converting enzyme that produces angiotensin II, which is a blood pressure increasing substance, and a method for producing the same. It is about.

Essential hypertension, which accounts for most of hypertension, a representative disease of modern adult diseases, is not precisely identified. However, the renin-angiotensin system is known to be associated with high blood pressure as a mechanism for raising blood pressure in vivo.

In this system, angiotensin-I converting ensyme (ACE) in pulmonary capillaries decomposes two terminal amino acids of angiotensin-I in an inactive state. It is known to cause hypertension by deactivating and inactivating bradykinin, which is activated by (angiotensin-II) as well as lowering blood pressure.

ACE inhibitors that are effective in patients with hypertension include chemically synthesized captoprile, enalapril, etc., but when taken, they cause rapid breathing, weakness in the whole body, vomiting, and coughing. In order to solve this problem, angiotensin I converting enzyme inhibitors have been developed, which are separated from various foods, have no side effects, are stable to heating conditions, and are easily absorbed by the body.

ACE activity inhibitors isolated from the various food products include artificial synthetic peptides (peptides), phenol (phenol) components of tea, fig milk and hydrolyzate of by-products, sardines of fish meat protein, and hydrolysates of mackerel muscle protein. In addition, many angiotensin converting enzyme inhibitory peptides, gelatin, and casein degrading peptides produced in fermented milk by culturing lactic acid bacteria or other microorganisms have been reported (Japanese Patent Application No. H3-267198, So 62-270533). , So 64-5497, So 64-83096).

However, most of the peptides had a disadvantage of having a strong bitter taste, and the angiotensin converting enzyme inhibitory peptides produced in fermented milk by cultivation of the lactic acid bacteria or other microorganisms should be cultured for a long time, and mass production is impossible as well as other foods. There were problems that were not easy to use.

In addition, angiotensin converting enzyme inhibitory peptides derived from gelatin and casein degrading products have high angiotensin converting enzyme inhibitory activity, but α -casein, β -casein and κ -casein, which are used as raw materials, are more expensive than normal casein. Since it is very expensive and sold only as a reagent, there is a problem that it is difficult to put into practical use as food.

In general, in the case of a protein degradation product (peptide mixture), the effect of inhibiting angiotensin converting enzyme does not necessarily have a blood pressure lowering effect.

This is because the angiotensin converting enzyme itself is an enzyme that cuts a specific amino acid present at the end of the peptide, and thus, in most cases, the angiotensin converting enzyme inhibiting peptide present in the protein hydrolyzate is decomposed in vivo and does not exert its effect. And development, 1995, Vol. 31, No. 8, p 50-52).

Therefore, whether the blood pressure lowering effect as an ACE inhibitor may be determined by measuring the blood pressure lowering effect in vivo. However, in the past, the protein lowering effect having angiotensin converting enzyme inhibitory activity was directly administered in vivo to confirm the blood pressure lowering effect. There were few cases.

It is an object of the present invention to provide a casein protein degradation product which is excellent in angiotensin converting enzyme inhibitory action as well as substantially lowering blood pressure in vivo.

It is another object of the present invention to provide a casein protein degradation product having an improved blood pressure lowering taste and a good blood pressure lowering effect.

The above object of the present invention is achieved by providing a casein protein degradation product prepared by hydrolyzing a casein protein corresponding to about 80% of the milk protein in neutral and hydrolyzing with an enzyme having appropriate endopeptidase and exopeptidase activity. Casein protein degradation products are easy to take because the bitter taste is improved, and in biochemical experiments, it inhibits angiotensin converting enzyme and also inhibits angiotensin converting enzyme in vivo.

1 is a graph showing the blood pressure lowering effect of the casein protein degradation product of the present invention coated with cured oil.

The present invention comprises the step of adding a protease in an amount of 0.1 to 3% to the aqueous solution of casein protein and reacting for 4 to 48 hours at a temperature of 30 to 50 ℃, pH 6-9, And a casein protein degradation product.

The protease is MP {(Aspergillus melleus)}, Promod 192 {Promod 192; derived from Aspergillus}, Pescalase {Pescalase (Bacillus licheniformis) And at least one selected from the group consisting of protamex (protamex; derived from Bacillus), and among them, it is most preferable to use promod 192 alone.

Hereinafter, a casein protein degradation product having a blood pressure lowering effect of the present invention and a preparation method thereof will be described in detail by way of examples, but the present invention is not limited thereto.

Example 1

Protamex

1. Casein was dissolved in warm water at 55-60 ° C. to prepare a 5% aqueous casein protein solution.

2. The casein protein solution was cooled to about 35 ° C. to adjust the pH to 7, and then 1% of protamex having endopeptidase activity was added.

3. The solution was allowed to react with stirring for about 5 minutes and then for 40 hours in a 47 ° C. water bath.

4. After the reaction was completed, the enzyme was inactivated by maintaining at 90 ° C. for about 15 minutes.

5. The solution was cooled and then centrifuged, and only the supernatant was collected and dried in a dryer to prepare casein protein digest.

Example 2

Promod 192

1. Casein was dissolved in warm water at 55-60 ° C. to prepare a 5% aqueous casein protein solution.

2. The casein protein solution was cooled to about 40 ° C. to adjust the pH to 6, and then 1% of promod 192 having endopeptidase and exopeptidase activity was added. .

3. The solution was allowed to react with stirring for about 5 minutes and then reacted in a 45 ° C. water bath for 24 hours.

4. After the reaction was completed, the enzyme was inactivated by maintaining at 90 ° C. for about 15 minutes.

5. The solution was cooled and then centrifuged, and only the supernatant was collected and dried in a dryer to prepare casein protein digest.

Example 3

Pescalase

1. Casein was dissolved in warm water at 55-60 ° C. to prepare a 5% aqueous casein protein solution.

2. The casein protein solution was cooled to about 45 ° C. to adjust the pH to 9, and then 2% of pescalase having endopeptidase activity was added.

3. The solution was allowed to react with stirring for about 5 minutes, followed by 30 hours in a 47 ° C. water bath.

4. After the reaction was completed, the enzyme was inactivated by maintaining at 90 ° C. for about 15 minutes.

5. The solution was cooled and then centrifuged, and only the supernatant was collected and dried in a dryer to prepare casein protein digest.

Example 4

MP

1. Casein was dissolved in warm water at 55-60 ° C. to prepare a 5% aqueous casein protein solution.

2. The casein protein solution was cooled to about 50 ° C. to adjust the pH to 8, and 2.5% of MP having endopeptidase and exopeptidase activity was added thereto.

3. The solution was allowed to react with stirring for about 5 minutes, followed by 10 hours in a 47 ° C. water bath.

4. After the reaction was completed, the enzyme was inactivated by maintaining at 90 ° C. for about 15 minutes.

5. The solution was cooled and then centrifuged, and only the supernatant was collected and dried in a dryer to prepare casein protein digest.

Test Example 1

Angiotensin converting enzyme inhibition rate measurement

end. Measurement by conventional method (Cushman and Cheung's method)

1. 50 μl of substrate (hippuric-His-Leu) was added to 50 μl of each casein protein lysate prepared in Examples 1 to 4, followed by mixing and preincubation for 5 minutes.

2. 150 μl of ACE enzyme solution was added to the solution and reacted at about 37 ° C. for about 1 hour.

3. After the reaction was completed by adding 250 µl of 0.5N HCl to the reaction solution, 1.5 ml of ethyl acetate was added and mixed.

4. The mixture was centrifuged at 2800 rpm for 10 minutes, 1 ml was taken, dried, and dissolved in 3 ml of 1 M NaCl, and the absorbance was measured at 228 nm.

I. Pretreatment

1. 150 μl of ACE enzyme solution was added to 50 μl of each casein protein digest prepared in Examples 1 to 4 and reacted at about 37 ° C. for about 2 hours.

2. 100 μl of substrate (hippuric-His-Leu) was added to the solution, mixed, and reacted at 37 ° C. for about 1 hour.

3. After the reaction was completed by adding 250 µl of 0.5N HCl, 1.5 ml of ethyl acetate was added and mixed.

4. The mixture was centrifuged at 2800 rpm for 10 minutes, 1 ml was taken, dried, and dissolved in 3 ml of 1 M NaCl, and the absorbance was measured at 228 nm.

ACE inhibition rate of the casein protein degradation product was calculated from the absorbance measured in Test Example 1 and is shown in Table 1 below.

E _c : absorbance when distilled water is added instead of the sample

E _s : absorbance at sample addition

E _b : absorbance of the sample added after stopping the reaction

Angiotensin Converting Enzyme Inhibitory Activity of Casein Protein Degradation with Single Enzyme Example enzyme Conventional IC _50- value (㎍ / ㎖) Pretreatment IC _50- value (㎍ / ㎖) Example 1 protamex 221.98 406.58 Example 2 Promod 192 254.45 255.70 Example 3 Pescalase 326.75 423.93 Example 4 MP 302.67 529.35

IC ₅₀ value is the concentration of the sample that can inhibit the activity of angiotensin converting enzyme 50%, the smaller the value is excellent in the activity inhibitory effect of the angiotensin converting enzyme and can exhibit a strong blood pressure lowering effect.

In general, experiments to find casein hydrolysates (peptides) that exhibit ACE inhibitory activity are used in the conventional method, and at the same time, when measured by pretreatment, the fake ACE inhibitory peptide (angiotensin converting enzyme inhibitory activity is high in in vitro experiment, but membrane in In vivo experiments can distinguish hydrolysates that do not have hypotensive effects. The pretreatment method only reacts with the hydrolyzate (peptide) and the angiotensin converting enzyme first, and thus, the fake angiotensin converting enzyme inhibiting peptide is degraded by the angiotensin converting enzyme, thereby lowering the ability to inhibit the angiotensin converting enzyme. Therefore, the hydrolyzate, which exhibits a low IC ₅₀ value in both the conventional method and the pretreatment method, is likely to have a high blood pressure lowering effect in vivo.

The angiotensin converting enzyme inhibitory activity of the casein protein degradation products prepared in Examples 1 to 4 using a single enzyme was measured by the conventional method and the pretreatment method. As shown in Table 1, MP, promod 192 (Promod 192), Fes Both Pescalase and Protamex enzymes showed good angiotensin converting enzyme inhibitory activity. Especially, IC ₅₀ value of Example 2 treated with promod 192 enzyme was good in both pretreatment and conventional methods. The results show that the most preferred protease in the present invention is promod 192.

Examples 5-9

Promodal 192 (Promod 192) by adding 1% in the same manner as in Example 2, but the reaction time was changed to 4 hours, 8 hours, 12 hours, 24 hours, 48 hours respectively to prepare a casein protein degradation product .

Test Example 2

The casein protein degradation products prepared in Examples 5 to 9 were measured by angiotensin converting enzyme inhibitory activity in the same manner as in Test Example 1, and the results are shown in Table 2 below.

Angiotensin Converting Enzyme Inhibitory Activity According to Reaction Time Example Response time (hours) Conventional IC _50- value (㎍ / ㎖) Pretreatment IC _50- value (㎍ / ㎖) Example 5 4 274.02 601.99 Example 6 8 251.38 368.84 Example 7 12 248.71 313.48 Example 8 24 239.45 248.11 Example 9 48 244.93 303.32

As a result of the test, even if the enzyme reaction time was increased from 4 hours to 48 hours, the IC ₅₀ value in the conventional method tended to decrease slightly, and the IC ₅₀ value in the pretreatment method gradually decreased with increasing the reaction time. And it was found. As a result, the enzyme reaction time for preparing casein-derived ACE inhibitory hydrolyzate was 24 hours.

Examples 10-17

The same procedure as in Example 2, except that the casein protein degradation product by varying the amount of Promod 192 (Promod 192) to 0.001%, 0.1%, 0.3%, 0.5%, 0.7%, 1.0%, 2.0%, 3.0%, respectively Was prepared.

Test Example 3

The casein protein degradation products prepared in Examples 10 to 17 were measured by angiotensin converting enzyme inhibitory activity in the same manner as in Test Example 1, and the results are shown in Table 3 below.

Angiotensin Converting Enzyme Inhibitory Activity According to Enzyme Content of Promod 192 Example Enzyme amount (%) Conventional IC _50- value (㎍ / ㎖) Pretreatment IC _50- value (㎍ / ㎖) Example 10 0.001 1905.49 1368.38 Example 11 0.1 341.10 538.10 Example 12 0.3 278.53 460.66 Example 13 0.5 284.79 386.36 Example 14 0.7 279.61 303.82 Example 15 1.0 256.63 266.42 Example 16 2.0 261.03 252.13 Example 17 3.0 252.58 260.66

The test results in the enzyme but by adding 0.3% a reduction in IC ₅₀ values significantly with an increase in the enzyme added amount was less than 0.3%, the change in IC ₅₀ values for a conventional method. On the other hand, in the pretreatment method, the IC ₅₀ value decreased as the amount of enzyme added increased.

As a result of this, when preparing casein protein-derived ACE inhibitory hydrolyzate, the addition amount of Promod 192 enzyme was 1%, and the reaction time at 45 ° C. was suitable for 24 hours.

As can be seen from the results of Test Examples 1 to 3, the present invention was excellent in inhibiting the activity of angiotensin converting enzyme, but the hypertensive rat was used to find out whether the angiotensin converting enzyme showed a blood pressure lowering effect without being degraded by angiotensin converting enzyme in vivo. The degree of blood pressure drop according to the present invention was measured.

Test Example 4

Measurement of Blood Pressure Drop in Hypertensive Rats

1. Seven-week-old hypertensive rat (SHR) male was fed from Samyuk Experimental Animal Research Institute Co., Ltd. Casein protein lysate was forced orally administered to zonde.

2. The hypertension-induced rats were warmed in a box of 38-40 ° C. for 5-15 minutes, and then fixed in a holder, followed by tail cuff method using a non-vascular blood pressure measuring device (PowerLab 800 / ADInstruments). The tail arterial pressure was measured accordingly.

The measurement was performed by measuring the SBP (systolic blood pressure) more than five times when the rat is stationary, the average value was used.

Table 4 below shows the blood pressure was 7%, 13% lower as a result of oral administration of 300, 600 mg / kg of the casein protein degradation product prepared in Example 2 to hypertensive mice, respectively, and 3 hours later.

Blood Pressure Reduction Effect of Casein Protein Degradation division Lowering blood pressure after oral administration (3 hours later) 300 mg / kg -7% 600 mg / kg -13%

Test Example 5

Blood Pressure Reducing Effect of Egg Yolk Treated Casein Protein Degradation

Test in the same manner as in Test Example 4, but the casein protein degradation product prepared in Example 2 was dissolved in water to add egg yolk (egg yolk) to 30% and emulsified for 5 minutes using an ultrasonic cleaner (ultrasonic cleaner) And then administered to hypertensive mice.

As a result of oral administration of the yolk-treated casein protein degradation product to hypertensive rats, as shown in Table 5 below, when 300 mg / kg was orally administered, systolic blood pressure decreased by 8.8% after 3 hours, and 600 mg / kg was orally administered. In one case, 15.9% was reduced.

Blood pressure lowering effect of egg yolk treated casein protein hydrolysate division 3 hours after oral administration 300 mg / kg -8.8% 600 mg / kg -15.9%

In the results of Test Examples 4 and 5, the blood pressure lowering effect of oral administration of egg yolk treated casein decomposed product was more excellent than that of egg yolk treated casein so that the phospholipids contained in egg yolk wrapped the hydrolyzate (coating) This is because when the hydrolyzate is taken, it can move to the lower intestine and be absorbed into the body (blood vessels) while receiving less action of proteolytic enzymes such as pepsin, the digestive enzymes above, or trypsin and chymotrypsin secreted by the small intestine. .

Test Example 6

Blood Pressure Reduction Effect of Casein Protein Degradation Coated with Curing Oil

The test was conducted in the same manner as in Test Example 4, but the casein protein lysate prepared in Example 2 was coated with hardened oil and then administered to the hypertensive rat.

The microencapsulation of casein protein digest was used as a coating material, and cured oil was used as a coating material, and 3% of span 80 was added as an emulsifier.

After dissolving the hydrolyzate in distilled water to contain 0.2% tween 80, the ratio of the peptide dissolved in hardened oil and water is 7: 3, and this is emulsified at 8,000 rpm for 10 minutes with an airless spray gun ( An airless spray gun was used to disperse in a cold room at 4 ° C. through an orifice of 0.6 mm at 355 watts to form microcapsules.

1.5g / kg of hydrolyzate, microencapsulated with hydrogenated milk, was mixed with feed daily for 20 days to give hypertension rats the effect of lowering blood pressure. As shown in FIG. A lowering effect was seen.

As can be seen from the results of Test Examples 4 to 6, the present invention showed a blood pressure lowering effect of up to 16% when administered to a rat inducing hypertension, whereby the casein protein degradation product of the present invention was induced by angiotensin converting enzyme. It was confirmed that it inhibits the activity of angiotensin converting enzyme in vivo without degradation.

Test Example 7

Molecular weight determination of casein protein digests

The molecular weight of the casein protein digests prepared in Examples 1 to 4 was measured using HPLC in which two TSKgel G3000PWXL (7.8 mm × 30 cm) columns were connected.

The standard materials used for the measurement of surface molecular weights were β- lactogloblin (molecular weight: 18,400), aprotinin (6500), insulin B fragment (3,450), bradykinin (1,060), glutathione (307), tyrosine (181), and eluent 0.1. % TFA: acetonitril (50:50) was used, the flow rate was 0.3 ml / min, detection was measured by ultraviolet absorption of 220nm.

Table 6 shows the molecular weight distribution and average molecular weight of the casein protein degradation product of the present invention. In Example 2 using Promod 192, about 80% of the casein protein degradation product had a molecular weight of 1,000 or less, and the average molecular weight was 773. It was Dalton.

Molecular weight distribution and average molecular weight of casein degradate Example enzyme Molecular weight distribution (dalton) Average molecular weight (dalton) ≥1000 1000-500 ≤500 Example 1 protamex 50.3244 27.3614 22.3142 1,528 Example 2 Promod 192 21.0034 34.9080 44.0886 773 Example 3 Pescalase 37.5555 35.7904 26.6541 1,189 Example 4 MP 39.1098 31.6121 29.2781 1,149

Test Example 8

Determination of the Freeness of Bitter Taste-Related Amino Acids in Casein Protein Degradates

1. In 8 ml of distilled water, 1 ml of the casein protein degradation product (0.1 g / ml) prepared in Example 2 and 1 ml of 20% sulfur salicylic acid (sulfosalicylic acid) were added and stirred, followed by centrifugation at 3000 rpm for 15 minutes.

2. After centrifugation, the supernatant was filtered with a 0.45 μl microfilter, and then analyzed by an amino acid autoanalyzer (Hitachi L-8500A) to quantify free amino acids.

Hydrophobic amino acids such as valine, leucine, and isoleucine or lysine at the ends of the peptides have a strong bitter taste and when they are free, they tend to reduce bitter taste.

Test Example 8 measured the free amino acid content of the casein protein hydrolyzate of the present invention to investigate the degree of free amino acid associated with bitter taste, the results are shown in Table 7 below.

Free Amino Acid Content of Casein Protein Degradants amino acid Casein protein digests Cys 0.159 Met 0.590 Asp 0.293 Thr 1.525 Ser 0.621 Glu 0.704 Gly 0.086 Ala 0.397 Val 1.094 Ile 1.174 Leu 3.802 Tyr 2.013 Phe 3.788 Lys 2.673 His 1.269 Arg 0.168 Pro 0.572 Total (g / 100g) 20.932

The casein protein hydrolyzate of the present invention has a high content of free valine, leucine, isoleucine, and the like as 1.094 g / 100g, 3.802 g / 100g, and 1.174 g / 100g, respectively, as shown in Table 5 above. The glass was 0.704 g / 100 g. Therefore, it can be seen that the present invention reduces the bitter taste of hydrolyzate and specifically appropriate release of glutamic acid, thereby improving flavor by using enzymes having appropriate endopeptidase and exopeptidase activity.

The casein protein degradation product of the present invention is not only excellent in the inhibitory activity of angiotensin converting enzyme, but also is not degraded by angiotensin converting enzyme, and thus also has an excellent blood pressure lowering effect in vivo.

In addition, the casein protein degradation product of the present invention is prepared using an enzyme that acts in neutral, so that it can be prepared through a simple process without addition of an acid or a base, and thus has high productivity and low manufacturing cost.

In addition, the present invention, unlike blood pressure lowering agents prepared by using a casein protein present in natural milk and manufactured by chemical synthesis, has no side effects such as cough and vomiting, and has high safety, is well soluble in water, and absorbed into the body by low molecular weight substances. After digestion, it is not easily decomposed by other enzymes, so digestion absorption is good.

Therefore, the present invention can be easily ingested as food by adding milk or other milk processed products, egg processed products, etc., and can be used in various sports drinks, fatigue recovery and medical protein sources, elderly nutrition products, and the like.

Claims (7)

A casein protein having a blood pressure lowering effect, which is prepared by adding a protease in an amount of 0.1 to 3% to an aqueous casein protein solution and reacting for 4 to 48 hours at a temperature of 30 to 50 ° C. and a pH of 6 to 9. resolvent. The method according to claim 1, wherein the protease is MP {(Aspergillus melleus)}, Promod 192 {Promod 192; derived from Aspergillus}, Pescalase {Pescalase (Bacillus richen) A casein protein degradation product having a blood pressure lowering effect, characterized in that it is at least one selected from the group consisting of Bacillus licheniformis and Protamex (from Bacillus). The casein protein degradation product according to claim 2, wherein the protease is promod 192 (Promod 192). The casein protein degradation product according to claim 3, wherein the mean molecular weight is 773 daltons. A casein protein having a blood pressure lowering effect, comprising the step of adding a protease in an amount of 0.1 to 3% to a casein protein aqueous solution and reacting for 4 to 48 hours at a temperature of 30 to 50 ° C. and a pH of 6 to 9 Method of Preparation of Decomposition The method according to claim 5, wherein the protease is MP {(Aspergillus melleus), Promod 192 {Promod 192 derived from Aspergillus}, Pescalase {Pescalase (Bacillus richen) Method for producing a casein protein degradation product having a blood pressure lowering effect, characterized in that it is at least one selected from the group consisting of Bacillus licheniformis, Protamex (from Bacillus). The method of claim 6, wherein the protease is Promod 192 (Promod 192), characterized in that the casein protein degradation product having a blood pressure lowering effect.