JP5561850B2 - Method for producing protein degradation product - Google Patents

Description

  The present invention relates to a method for producing a protein degradation product obtained from a protein derived from sesame seeds, and more specifically, a protein having an angiotensin converting enzyme inhibitory activity obtained by hydrolyzing a protein derived from sesame seeds with subcritical water. The present invention relates to a method for producing a decomposition product.

  Conventionally, hypertension is a typical lifestyle-related disease and is known to cause various complications such as cerebral hemorrhage, subarachnoid hemorrhage, cerebral infarction, myocardial infarction, angina pectoris, nephrosclerosis and the like. It is known that angiotensin converting enzyme (hereinafter referred to as ACE) is involved in the cause of hypertension, and those having ACE inhibitory activity are attracting attention as preventing hypertension. A peptide that is an enzyme degradation product of food material protein has been proposed as one of such ACE inhibitory activities. As a method for producing such a peptide, a peptide having an ACE inhibitory activity is prepared by allowing the enzyme collagenase to act on gelatin. A method for obtaining (for example, see Patent Document 1), a method for enzymatically degrading a protein derived from sesame seed (for example, see Patent Document 2), a κ casein glycomacropeptide from a milk raw material, etc., and this κ casein glycomacropeptide A protein is hydrolyzed with a proteolytic enzyme (see, for example, Patent Document 3), and a protein is hydrolyzed with supercritical or subcritical water under a high temperature and high pressure of 221 to 1000 atmospheres at a temperature of 300 to 400 ° C. A method (for example, refer to Patent Document 4) has been proposed.

  However, the production method using an enzyme is complicated and the production efficiency is low, and the method of hydrolysis with supercritical or subcritical water under high temperature and high pressure requires appropriate equipment and skill. However, there is still a problem that the risk of work is high.

Japanese Patent Laid-Open No. 52-148631 JP 7-69922 A JP-A-8-269088 Japanese Patent Laid-Open No. 9-268166

  The problem to be solved by the present invention is to provide a production method capable of safely mass-producing a protein degradation product having ACE inhibitory activity.

  As a result of researches to solve the above-mentioned problems, the present inventor has found that a method of hydrolyzing a protein derived from sesame seeds with subcritical water under specific conditions is correctly suitable.

That is, the present invention relates to a method for producing a protein degradation product (hereinafter referred to as the production method of the present invention), and is characterized by passing through the following steps including the first step, the second step and the third step.
First step: 10 to 50 parts by mass of water is mixed with 1 part by mass of protein derived from sesame seeds, the mixture is sealed in a pressure vessel, and the atmosphere gas in the pressure vessel is N ₂ gas. After the replacement and pressurization, the mixture in the pressure vessel is brought to an ultimate temperature of 160 to 280 ° C. in 10 to 90 minutes, and the pressure in the pressure vessel is 4.0 to 8.0 MPa at the ultimate temperature. The process of heating to.
2nd process: The process hold | maintained at the same temperature of 160-280 degreeC for 0-20 minutes.
3rd process: The process which cools the produced | generated hydrolyzate in a pressure vessel, takes out from a pressure vessel, isolate | separates a liquid part, and dries the isolate | separated liquid part.

  In the production method of the present invention, a protein derived from sesame seeds is hydrolyzed under specific conditions with subcritical water. Examples of such sesame seeds include various sesame seeds such as raw sesame seeds, roasted sesame seeds, kneaded sesame seeds, cut sesame seeds, and defatted sesame seeds. preferable. In the production method of the present invention, a protein derived from sesame seeds extracted from such sesame seeds is used in order to efficiently obtain a protein degradation product.

  A protein derived from sesame seeds can be obtained by a method known per se, for example, see Journal of Food Science, Vol. 47p. 457-460 (1982). According to this method, the raw sesame seeds are defatted with hexane, the protein is extracted from the defatted sesame seed treated product with the alkaline solution, and further obtained as a precipitate by a treatment such as an isoelectric precipitation method.

In the first step, first, a protein derived from sesame seeds (hereinafter referred to as protein) and water are mixed to obtain a mixture. The amount of water is sufficient if it is greater than or equal to the stoichiometric amount necessary to hydrolyze the protein, but water is mixed at a ratio of 10 to 50 parts by mass with respect to 1 part by mass of the protein. The temperature of the mixture at this stage is preferably 10 to 80 ° C. The mixture is sealed in a pressure vessel and heated so that the ultimate temperature of the mixture in the pressure vessel is 160 to 280 ° C, preferably 200 to 240 ° C. The heating time to reach the ultimate temperature is 10 to 90 minutes, preferably 20 to 60 minutes, and the rate of temperature increase at this time is preferably 3 to 20 ° C./minute. The atmospheric gas in the pressure vessel is replaced with N ₂ gas in advance and pressurized.

  The second step is a step of further proceeding the hydrolysis while maintaining the temperature in the pressure vessel after completion of the first step. In the second step, the same temperature reached in the first step is held for 0 to 20 minutes.

  The third step is a step of cooling the hydrolyzate produced in the pressure vessel, taking it out of the pressure vessel, separating the liquid part, and drying the separated liquid part.

  A hydrolyzate corresponding to the reaction product is generated in the pressure vessel, and this hydrolyzate is usually in a state where the liquid part and the solid part are mixed. Therefore, the hydrolyzate is liquid by decantation, filtration, centrifugation, etc. The separated liquid part can be dried by a method such as spray drying, drum drying, freeze drying, etc. to obtain a protein degradation product.

  As the water in the subcritical state, clean water, distilled water, ion exchange water, ultrapure water, or the like can be used. As the pressure vessel used for the reaction, any one that can withstand the use temperature and the use pressure can be used, and among them, a pressure vessel made of stainless steel is preferable. The reaction system can be either continuous or batch.

  As will be described in detail later, the protein degradation product obtained by the production method of the present invention exhibits excellent ACE inhibitory activity and is useful as a blood pressure lowering agent.

  The present invention described above has an effect that a protein degradation product having an ACE inhibitory activity can be safely mass-produced from a protein derived from sesame seeds.

The graph which illustrates the change of systolic blood pressure when the protein degradation product obtained by the manufacturing method of this invention is orally administered. The graph which illustrates the change of the diastolic blood pressure when the protein degradation product obtained by the manufacturing method of this invention is orally administered. The graph which illustrates the change of the average blood pressure when the protein degradation product obtained by the manufacturing method of this invention is orally administered. The graph which illustrates the change of the heart rate when the protein degradation product obtained by the manufacturing method of this invention is orally administered.

  Hereinafter, in order to make the configuration and effects of the present invention more specific, examples and comparative examples will be described. However, the present invention is not limited to these examples. In the following Examples and Comparative Examples, “part” means “part by mass” and “%” means “% by mass”.

Test category 1 (Manufacture of protein degradation products-1)
Example 1
The raw sesame seeds were defatted with hexane, immersed in a 0.05N aqueous sodium hydroxide solution, heated, and stirred for 45 minutes while maintaining the liquid temperature at 55 ° C. to extract proteins. The residue is removed from the extract by centrifugation (8000 × g, 15 minutes), the pH of the liquid part from which the residue has been removed is adjusted to 4.5 with 2N hydrochloric acid, and the water-containing product containing protein is removed by isoelectric point precipitation. Obtained.

30 g of the water-containing product containing the protein obtained above was put in a stainless heat-resistant container, and 200 g of distilled water was added to obtain a mixture (protein 8.3 g / water 221.7 g). The temperature of the mixture was 25 ° C. After the lid was closed and the gas in the pressure vessel was replaced with N ₂ gas, the exhaust valve was closed and N ₂ gas was injected and sealed until the pressure in the pressure vessel reached 3.0 MPa. The mixture was heated at a heating rate of 7 ° C./min until the temperature of the mixture in the sealed pressure vessel reached 180 ° C. The pressure in the pressure resistant container when the temperature of the mixture reached 180 ° C. was 4.0 MPa. When the temperature of the mixture reached 180 ° C., it was immediately cooled to 50 ° C., the exhaust valve was opened and the pressure was restored, and then the lid was opened and the hydrolyzate was taken out. The aqueous phase was separated from the extracted hydrolyzate by centrifugation, and this aqueous phase was freeze-dried to obtain a protein degradation product.

Examples 2-5 and Comparative Examples 1-2
The protein degradation products of Examples 2 to 5 and Comparative Examples 1 to 2 were obtained in the same manner as in Example 1, but under the conditions shown in Table 1.

Test Category 2 (Measurement of ACE inhibitory activity-1)
Rabbit lung angiotensin converting enzyme (manufactured by Sigma) of 6.0 mU (1 U is an enzyme titer that produces 1 μmol of hyprilic acid per minute), 5 mmol of hippuriu L-histidiru L-leucine (manufactured by Sigma), 400 mmol of 100 μg of an aqueous solution prepared by dissolving sodium chloride and the protein degradation product obtained in Test Category 1 in pure water so as to have a concentration of 1 mg / ml was stirred in 350 μl of borate buffer (pH 8.3), and the liquid temperature was adjusted. The reaction was allowed to proceed for 30 minutes while maintaining the temperature at 37 ° C. After stopping the reaction by adding 250 μl of 1N hydrochloric acid, 1 ml of ethyl acetate was added and vigorously stirred for 15 seconds. The contents were separated by processing with a centrifuge (3000 rpm × 10 minutes), and 0.5 ml of an ethyl acetate layer was collected. The collected sample was dried at a temperature of 120 ° C. to remove the solvent, and redissolved with 1 ml of distilled water to obtain a measurement sample. With respect to this measurement sample, the absorbance (B) at a wavelength of 228 nm, which is the absorption wavelength of hyperylic acid, was measured. The same test was carried out without containing a protein degradation product, the absorbance (A) was measured, and the ACE inhibitory activity (%) was determined from the following formula 1.

Test Category 3 (Production of protein degradation product-2)
Example 6
30 g of the protein obtained in the same manner as in Example 1 of Test Category 1 was placed in a stainless steel pressure vessel, and 200 ml of distilled water was added to obtain a mixture (protein 4.9 g / water 225.1 g). The temperature of the mixture was 25 ° C. After the lid was closed and the gas in the pressure vessel was replaced with N ₂ gas, the exhaust valve was closed and N ₂ gas was injected and sealed until the pressure in the pressure vessel reached 3.0 MPa. The mixture was heated at a heating rate of 7 ° C./min until the temperature of the mixture in the sealed pressure vessel reached 200 ° C. The pressure in the pressure vessel when the temperature of the mixture reached 200 ° C. was 4.5 MPa. After the temperature of the mixture reached 200 ° C., it was immediately cooled to 50 ° C., the exhaust valve was opened and the pressure was restored, and then the lid was opened and the hydrolyzate was taken out. The aqueous phase was separated from the extracted hydrolyzate by centrifugation, and this aqueous phase was freeze-dried to obtain a protein degradation product.

Example 7
30 g of the protein obtained in the same manner as in Example 1 of Test Category 1 was placed in a stainless steel pressure vessel, and 200 ml of distilled water was added to obtain a mixture (protein 4.9 g / water 225.1 g). The temperature of the mixture was 25 ° C. After the lid was closed and the gas in the pressure vessel was replaced with N ₂ gas, the exhaust valve was closed and N ₂ gas was injected and sealed until the pressure in the pressure vessel reached 3.0 MPa. The mixture was heated at a heating rate of 7 ° C./min until the temperature of the mixture in the sealed pressure vessel reached 200 ° C. After the temperature of the mixture reached 200 ° C., the same temperature was further maintained for 10 minutes. The pressure in the pressure vessel during the holding was 4.5 MPa. Thereafter, the mixture was immediately cooled to 50 ° C., the exhaust valve was opened and the pressure was restored, and then the lid was opened and the hydrolyzate was taken out. The aqueous phase was separated from the extracted hydrolyzate by centrifugation, and this aqueous phase was freeze-dried to obtain a protein degradation product.

Examples or Reference Examples 8 to 16
In the same manner as in Example 6 or 7, but under the conditions shown in Table 2, the protein degradation products of Examples or Reference Examples 8 to 16 were obtained.

Test category 4 (Measurement of ACE inhibitory activity-2)
Similar to the measurement in Test Category 2, except that several samples of aqueous solutions with different concentrations of proteolysates were prepared, and ACE inhibitory activity (%) was obtained for these samples. The ACE inhibitory activity was determined from the line connecting the ACE inhibitory activities. The concentration of the proteolysate at 50% was calculated, and the inhibitory proteolysate concentration (IC ₅₀ ) was determined. The results are summarized in Table 2.

Test category 5 (evaluation as antihypertensive agent)
Eleven weeks old spontaneously hypertensive rats (Nippon Charles River Co., Ltd.) temperature 22 ± 3 ° C., humidity 60 ± 15%, ventilation rate 12-15 times / hour and lighting 12 hours / day (7: 00-19: 00) In the breeding room set to 1, water and feed (manufactured by Oriental Yeast Co., Ltd.) were freely ingested and bred. Rats with a body weight of 270 to 300 g and a systolic blood pressure of 160 mmHg or more were divided into 5 rats per group, and the protein degradation product of Example 8 of Test Category 3 dispersed in water was orally administered at a rate of 1000 mg / kg body weight. . After administration, systolic blood pressure, diastolic blood pressure, average blood pressure, heart rate, and body weight were measured over time by a tail-cuff method using a non-invasive blood pressure measuring device (BP-98A, manufactured by Softron). . As a control, the same measurement was performed for the group not administered with the protein degradation product. The average value of the measured values is shown in FIGS.

  As is clear from FIGS. 1 to 4, the proteolysate having ACE inhibitory activity of the present invention exhibited a blood pressure lowering effect after oral administration. Moreover, after oral administration, there were no trends such as an increase in heart rate or an increase in body weight.

11-14 Polygonal line when the protein degradation product of the present invention is used 21-24 Contrast line

Claims (4)

A method for producing a protein degradation product, comprising a step including the following first step, second step and third step.
First step: 10 to 50 parts by mass of water is mixed with 1 part by mass of protein derived from sesame seeds, the mixture is sealed in a pressure vessel, and the atmosphere gas in the pressure vessel is N ₂ gas. substituted, after pressurization, the pressure of the mixture 160 to 280 in the pressure vessel at by the Hare and reaching temperature becomes temperature reached ℃ in 10-90 minutes the pressure vessel becomes 4.0~8.0MPa heating to.
2nd process : The process hold | maintained for 0 to 20 minutes at the same temperature of 160-280 degreeC.
3rd process: The process which cools the produced | generated hydrolyzate in a pressure vessel, takes out from a pressure vessel, isolate | separates a liquid part, and dries the isolate | separated liquid part.   The method for producing a protein degradation product according to claim 1, wherein the sesame seed is one or more selected from raw sesame seed, roasted sesame seed and defatted sesame seed. The first step is a step of heating the mixture in the pressure vessel so that the temperature reaches 200 to 240 ° C. in 20 to 60 minutes, and the second step sets the same temperature of 200 to 240 ° C. to 0 to The method for producing a protein degradation product according to claim 1 or 2, which is a step of maintaining for 20 minutes. Producing how the 蛋 white matter degradation product having ACE inhibitory activity of any one of claims of claims 1-3.

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