JP5000273B2 - Method for producing peptide having ACE inhibitory activity - Google Patents

Description

  The present invention relates to a method for producing a peptide, and more particularly, to a method for producing a peptide having ACE (angiotensin I converting enzyme) inhibitory activity.

Waste liquids containing protein discharged from potato starch manufacturing plants, etc. are discharged into rivers after water treatment by various methods. Recently, by using proteins contained in this waste liquid, Attempts have been made to reduce the cost of waste liquid treatment as well as to effectively use the protein.
For example, Non-Patent Document 1 proposes a method for producing a peptide having ACE inhibitory activity from a protein contained in decanter juice, which is a waste liquid discharged from a potato starch production factory. According to the method proposed by this non-patent document 1, first, the residue deposited by concentrating the waste liquid is filtered and dried with hot air. Then, distilled water is added to the dried residue, and after stirring, the pH is adjusted to 6.5 using NaOH, after heat sterilization (95 ° C. reaching temperature), cooled to 70 ° C., and Amano AD (manufactured by Amano Pharmaceutical) is added. Stir for 1 hour. Subsequently, the temperature was adjusted to 55 ° C., 0.6 L of Alcalase (registered trademark) was added and stirred for 4 hours, the temperature was further adjusted to 50 ° C., flavorzyme L (manufactured by Novo Nordisk) was added, and the mixture was stirred for 20 hours. Hydrolysis. After completion of the reaction, the enzyme is inactivated by heating at 98 ° C. for 30 minutes, allowed to cool and then purified using an ion exchange resin, further filtered, desalted by electrodialysis, and lyophilized to purify the peptide. And the peptide which has ACE inhibitory activity is manufactured by isolate | separating the peptide which has ACE inhibitory activity from this refine | purified peptide.
“Development of new functional ingredients from potato starch mill effluent (2000)” R & D Division Toshie Minomiya Kiyoshi Oba, Cosmo Foods Co., Ltd. Haruo Miyasaka, Obihiro University of Agriculture and Veterinary Sciences Area Research Center

  However, in the method proposed in Non-Patent Document 1, a peptide is generated through many stages of enzyme addition and purification processes as described above. For this reason, the manufacturing process is complicated, and quality control at each stage is difficult.

  The present invention has been made in view of the above-described problems, and an object thereof is to facilitate the production of a peptide having ACE inhibitory activity.

  In order to achieve the above object, the present invention provides a hydrothermal reaction treatment step in which a hydrothermal reaction treatment is performed on a raw material solution containing protein or a dehydrated cake obtained by a predetermined treatment on the raw material solution, and the hydrothermal reaction. And a separation step of separating a peptide having ACE (angiotensin I converting enzyme) inhibitory activity from the hydrothermal reaction treatment liquid obtained in the treatment step.

  According to the present invention having such characteristics, a hydrothermal reaction treatment liquid is generated by performing a hydrothermal reaction treatment on a raw material liquid or a dehydrated cake obtained by a predetermined treatment on the raw material liquid. A peptide having ACE inhibitory activity is separated from the treatment solution.

  Further, in the present invention, the dehydrated cake is obtained by a heat treatment step of performing a heat treatment on the raw material liquid and a solid-liquid separation step of taking out a solid phase containing a solid content obtained by the heat treatment step. Can be adopted.

  Moreover, in this invention, the said heat processing process WHEREIN: The structure of heating the said raw material liquid to 100-110 degreeC is employable.

  Moreover, in this invention, the structure of adjusting the pH of the said raw material liquid to 5.0-5.5 in the said heat processing process is employable.

  Moreover, in this invention, the said hydrothermal reaction process WHEREIN: The structure that the process temperature is 160 to 230 degreeC, and the said hydrothermal reaction process can be employ | adopted for 10 to 15 minutes of process time is employable.

  Further, in the present invention, a drying step for obtaining a dried cake by performing a drying treatment on the dehydrated cake, and a hydration step for returning to the dehydrated cake by adding water to the dried cake obtained in the drying step. It is possible to employ a configuration in which the hydrothermal reaction treatment is performed on the dehydrated cake that has been returned by the hydration step.

  Moreover, in this invention, the structure that the said raw material liquid is a decanter soup of potato is employable.

According to the present invention, the raw material liquid or the dehydrated cake is subjected to a hydrothermal reaction treatment by a hydrothermal reaction treatment using subcritical or supercritical water. Since the raw material liquid or dehydrated cake contains protein, the protein is reduced in molecular weight by hydrothermal reaction treatment, and as a result, a peptide is produced. That is, the peptide is contained in the hydrothermal reaction treatment liquid obtained by the hydrothermal reaction treatment. And the peptide which has ACE inhibitory activity is isolate | separated from a hydrothermal reaction process liquid.
Thus, according to the present invention, in order to produce a peptide from a raw material solution or a dehydrated cake, it is not necessary to go through many stages of enzyme addition and purification processes, and only a hydrothermal reaction process may be performed. Therefore, it is possible to facilitate the production of a peptide having ACE inhibitory activity.

  Hereinafter, an embodiment of a method for producing a peptide according to the present invention will be described with reference to the drawings.

(First embodiment)
FIG. 1 is a flow diagram showing a schematic configuration of a peptide production apparatus S1 for producing a peptide having ACE inhibitory activity using the peptide production method of the present embodiment. In this embodiment, potato decanter juice (potato juice), which is a waste liquid discharged from a potato starch manufacturing plant, is used as a raw material liquid.
As shown in this figure, the peptide production apparatus S1 includes a deproteinization apparatus 1, a hydrothermal apparatus 2, a centrifuge 3, a separation / purification apparatus 4, an IC reactor 5, and an activated sludge treatment facility 6.

  The deproteinizer 1 heats the supplied decanter soup X1 to coagulate the coagulable protein contained in the decanter soup X1. The deproteinization apparatus 1 has a separator that separates the solid phase and the liquid phase, and separates into a separation liquid X2 that is a liquid phase and a dehydrated cake X3 that is a solid phase containing a coagulated protein. Are discharged separately.

  The hydrothermal apparatus 2 performs a hydrothermal reaction treatment on the dehydrated cake X3 discharged from the deproteinizer 1, and is liquid by reducing the molecular weight of the protein contained in the dehydrated cake X3 with subcritical water or supercritical water. The peptide is produced. Then, the hydrothermal apparatus 2 discharges the hydrothermal reaction treatment liquid X4 containing the peptide obtained by hydrothermally treating the supplied dehydrated cake X3.

  The centrifuge 3 removes the residual dehydrated cake X5 contained in the hydrothermal reaction treatment liquid X4 discharged from the hydrothermal apparatus 2. Then, the centrifuge 3 discharges the removed residue dehydrated cake X5 and the hydrothermal reaction treatment liquid X4 separately.

  The separation / purification device 4 separates and purifies peptides having ACE inhibitory activity from the hydrothermal reaction treatment liquid X4 discharged from the centrifuge 3. And the peptide P which has ACE inhibitory activity is discharged | emitted from the separation / purification apparatus 4. FIG.

  The IC reactor 5 is a secondary decanter juice X6 (liquid phase component obtained by solidifying and then separating the starch after adding the starch, after the separation liquid X2 is supplied from the deproteinizer 1 and is produced in the production process at the potato starch production plant. ) And potato starch production plant, the production process water X7 generated in the production process in the potato starch production plant is supplied. The IC reactor 5 performs methane fermentation of the supplied separation liquid X2, secondary decanter juice X6, and production treated water X7. The IC reactor converts the methane gas G obtained by methane fermentation of the supplied separation liquid X2, secondary decanter juice X6, and production treated water X7 into steam and returns it to the deproteinizer 1, and the treated liquid X8 The activated sludge treatment facility 6 is supplied.

  The activated sludge treatment facility 6 generates a discharge water X9 that can be discharged into a river or the like by performing an aerobic process on the treatment liquid X8 supplied from the IC reactor 5, and discharges the generated discharge water X9 to the outside. To do.

  Next, a method for producing a peptide according to this embodiment using the thus configured peptide production apparatus S1 will be described.

First, the decanter juice X1 discharged from the potato starch manufacturing factory is supplied to the deproteinization apparatus 1. In the deproteinization apparatus 1, the decanter soup X1 is subjected to heat treatment (heat treatment step). Further, in the deproteinization apparatus 1, the decanter juice X1 after the heat treatment is separated from the separation liquid X2 which is a liquid phase and the dehydrated cake X3 which is a solid phase component containing the protein coagulated by the heat treatment (solid liquid Separation step). Then, the separation liquid X2 and the dehydrated cake X3 are separately discharged from the deproteinization apparatus 1.
That is, the dehydrated cake X3 is obtained by heat treatment on the decanter juice X1 and solid-liquid separation that takes out a solid phase containing a coagulated protein that is a solid content obtained by the heat treatment.
In addition, in the heat processing of the decanter soup X1 in the deproteinization apparatus 1, it is preferable to heat the decanter soup X1 to 100-110 degreeC. By heating the decanter juice X1 in this temperature range, the coagulation of the coagulable protein contained in the decanter juice X1 can be promoted. In addition, the degradation of the coagulated protein progresses, and it becomes possible to improve the yield of the peptide having ACE inhibitory activity after the hydrothermal reaction treatment which is the subsequent step.
In addition, in the heat treatment of the decanter juice X1 in the deproteinizer 1, it is preferable to adjust the pH of the decanter juice X1 to 5.0 to 5.5 using hydrochloric acid or the like. By adjusting the pH of the decanter soup X1 within this range, it becomes possible to further promote the coagulation of the coagulable protein contained in the decanter soup X1.
Further, by adjusting the pH of the decanter soup X1 within the above range, the separation efficiency of the separation liquid X2 and the dehydrated cake X3 can be improved, the amount of solid content remaining in the separation liquid X2 can be reduced, and the dehydrated cake It becomes possible to reduce the moisture content of X3.
Moreover, in the heat processing of the decanter soup X1 in the deproteinization apparatus 1, the processing time is preferably 10 to 15 minutes.

  The separation liquid X2 is supplied to the IC reactor 5 and subjected to methane fermentation together with the secondary decanter juice X6 and the production treated water X7 supplied to the IC reactor 5 from the potato starch manufacturing factory. The methane gas G generated as a result is converted into steam and supplied to the deproteinization apparatus 1, and is used for the heat treatment of the decanter soup X1 in the deproteinization apparatus 1. On the other hand, the treatment liquid X8 generated as a result of the methane fermentation is subjected to an aerobic treatment in the activated sludge treatment facility 6 and then discharged to an external river or the like as discharge water X9.

The dehydrated cake X3 discharged from the deproteinization apparatus 1 is supplied to the hydrothermal apparatus 2 after being moderately hydrated. In the hydrothermal apparatus 2, the protein contained in the dehydrated cake X3 is reduced in molecular weight and liquefied. As a result, a hydrothermal reaction treatment liquid X4 containing a liquid peptide is generated. Then, the hydrothermal reaction treatment liquid X4 is discharged from the hydrothermal device 2.
In addition, in the hydrothermal reaction process in the hydrothermal apparatus 2, it is preferable that process temperature is 160 to 230 degreeC, and process time is 10 to 15 minutes. Further, the treatment pressure is set to a pressure at which water is maintained in a subcritical state or a supercritical state at the treatment temperature (for example, around 3 MPa). By using such treatment temperature and treatment time, the yield of the peptide having ACE inhibitory activity can be improved.

Subsequently, the hydrothermal reaction treatment liquid X4 is supplied to the centrifuge 3, and after the residue dehydrated cake X5 contained therein is removed, the hydrothermal reaction treatment liquid X4 is supplied to the separation / purification device 4.
In addition, the residue dewatering cake X5 is supplied to the compost 10 after drying, for example, composted in the compost 10 and reused.

When the hydrothermal reaction treatment liquid X4 is supplied to the separation / purification apparatus 4, the separation / purification apparatus 4 separates / purifies the peptide P having ACE inhibitory activity from the hydrothermal reaction treatment liquid X4 (separation step).
Specifically, the extract extracted from the hydrothermal reaction treatment solution X4 with 70% ethanol is concentrated under reduced pressure at a hot water temperature of about 40 ° C. using a rotary evaporator. In this concentrated extract, amino acids and peptides are separated from other components in order to separate them from strong acid ion-exchange resin at room temperature, and after sufficient washing, the peptides are eluted with 2N ammonia water. It was. Thereafter, in order to purify a peptide having ACE inhibitory activity, only peptide P having ACE inhibitory activity is separated and purified by repeated cellulose column chromatography.

According to such a method for producing a peptide of the present embodiment, the dehydrated cake X3 that has been moderately hydrated by the hydrothermal reaction process using subcritical or supercritical water is subjected to a hydrothermal reaction process. Since the dehydrated cake X3 contains a protein, the protein is reduced in molecular weight by a hydrothermal reaction treatment, and as a result, a peptide is generated. That is, the hydrothermal reaction treatment liquid X4 obtained by the hydrothermal reaction treatment contains a peptide. And the peptide which has ACE inhibitory activity is isolate | separated from the hydrothermal reaction process liquid X4.
As described above, according to the method for producing a peptide of this embodiment, in order to produce a peptide from the dehydrated cake X3, it is not necessary to go through many stages of enzyme addition and purification processes, and only a hydrothermal reaction process may be performed. . Therefore, it is possible to facilitate the production of a peptide having ACE inhibitory activity, thereby simplifying the production process and reducing the equipment cost.

  Moreover, according to the method for producing a peptide of the present embodiment, the decanter juice X1 is heat-treated in the deproteinization apparatus 1. By performing such a heat treatment, the degradation of the coagulated protein proceeds, and it becomes possible to improve the yield of the peptide having ACE inhibitory activity after the hydrothermal reaction treatment, which is a subsequent step.

FIG. 2 is a graph showing experimental data for verifying the method for producing the peptide of the present embodiment, and the vertical axis shows the ACE inhibitory activity rate. In this experiment, the hydrothermal reaction liquid obtained by hydrothermal reaction treatment of the dehydrated cake X3 with the sample A and the dehydrated cake X3 at 220 ° C. is the sample B, and the dehydrated cake X3 is hydrothermally reacted at 270 ° C. The hydrothermal reaction treatment liquid obtained by the treatment is used as Sample C. Then, a crude extract obtained by extracting each sample with about 70% ethanol at room temperature for about one month was dissolved in methanol so as to have a final concentration of 1000 ppm as a measurement sample. Then, each measurement sample was measured by a measurement method utilizing that ACE recognizes and cleaves a His-Lue residue, and the ACE inhibitory activity rate of each sample was calculated from the measurement result. Specifically, in this measurement method, Hip (hippuric acid) -His-Lue is used as a substrate, and the amount of hippuric acid released within a unit time is measured to determine the degree of ACE inhibition by the measurement sample. Used. In addition, the amount of hippuric acid decreases with respect to control, so that the ACE inhibitory activity of a measurement sample is high.
In the actual experimental operation, 20 μl of the measurement sample was added to 250 μl of the substrate solution, and 100 μl of ACE that was also kept warm was added to the sample kept at 37 ° C. for 5 minutes, and the enzyme reaction was performed at 37 ° C. for 30 minutes. The reaction was stopped by adding 750 μl of 3% phosphoric acid. The amount of hippuric acid released in this solution was quantified by high performance liquid chromatography (HPLC), and the ACE inhibitory activity rate of the sample relative to the control was calculated. For control, methanol was used instead of the measurement sample. The HPLC conditions were H ₂ 0:10 mM KH ₂ PO ₄ (pH 3.0) = 1: 1 (v / v), Flow rate = 0.5 ml / min, ODS column (φ4.6 mm × 250 mm). is there.

  As shown in FIG. 2, in this experiment, sample A (dehydrated cake X3) had an ACE inhibitory activity rate of 72.6%, and sample B (dehydrated cake X3 obtained by hydrothermal reaction at 220 ° C. was obtained. The ACE inhibitory activity rate of the thermal reaction treatment solution) is 96.4%, and the ACE inhibitory activity rate of the sample C (hydrothermal reaction treatment solution obtained by hydrothermal reaction treatment of the dehydrated cake X3 at 270 ° C.) is 64. 1%.

  From this experiment, the dehydrated cake X3 obtained by heat-treating the decanter juice X1 in the deproteinizer 1 is further subjected to a hydrothermal reaction treatment at 220 ° C., whereby the yield of the peptide having ACE inhibitory activity is increased. It turns out that it improves. Moreover, it turned out that it is preferable that the process temperature in the hydrothermal reaction process with respect to the dehydration cake X3 is 220 degreeC rather than 270 degreeC.

FIG. 3 is a pie chart showing the results of analyzing the components contained in the decanter juice X1 and the separation liquid X2, wherein (a) shows the components of the decanter juice X1, and (b) shows the separation liquid X2. Ingredients are shown.
As shown in this figure, the decanter juice X1 contains 23% peptide-derived nitrogen and 53% protein-derived nitrogen, and the separation solution X2 contains 44% peptide-derived nitrogen and protein-derived nitrogen. Contains 13% nitrogen.
For this reason, it turns out that the protein is decomposed | disassembled and changed into the peptide before and behind the heat processing in the deproteinization apparatus 1. FIG. Therefore, it can be seen that the yield of the peptide containing the peptide having AEC inhibitory activity is improved by performing the heat treatment on the decanter juice X1.

(Second Embodiment)
Next, a second embodiment of the present invention will be described. In the description of the second embodiment, the description of the same parts as in the first embodiment will be omitted or simplified.

FIG. 4 is a flowchart showing a schematic configuration of a peptide production apparatus S2 for producing a peptide having ACE inhibitory activity using the peptide production method of the present embodiment.
As shown in this figure, in the peptide production apparatus S2, a drying apparatus 7 is installed between the deproteinization apparatus 1 and the hydrothermal apparatus 2. The drying device 7 discharges the dehydrated cake X3 discharged from the deproteinization device 1 as a dry cake X10 by drying.
In such a peptide production method in the peptide production apparatus S2, the dehydration cake X3 is dried by the drying apparatus 7 (drying process), and the resulting dried cake X10 is hydrated (hydration process), thereby dehydrating cake. What has been returned to is supplied to the hydrothermal apparatus 2.

By drying the dehydrated cake X3 once to obtain the dried cake X10, it can be stored for a long time and can be easily transported by volume reduction. For this reason, according to the method for producing a peptide of this embodiment, the same effect as the method for producing a peptide of the first embodiment can be obtained, and the dried cake X10 can be stored or transported for a long period of time and dehydrated by addition as necessary. The hydrothermal reaction treatment can be performed after returning to the cake X3.
For this reason, for example, even if decanter juice X1 cannot be obtained throughout the year and can only be obtained for a period of time, the peptide having ACE inhibitory activity throughout the year can be stored as a dry cake X10. Can be manufactured.
Further, even when the hydrothermal apparatus 2 is far away from the place where the decanter soup X1 can be obtained, the hydrothermal apparatus can be manufactured at low cost by transporting the dried cake X10 and returning it to the dehydrated cake X3. 2 can be supplied with dehydrated cake X3.

  As shown in FIG. 4, in the peptide production apparatus S2, methane gas G obtained by the IC reactor 5 is converted into steam and supplied to the drying apparatus 7, and is used for the drying process of the dehydrated cake X3 in the drying apparatus 7. ing.

  As mentioned above, although preferred embodiment of the manufacturing method of the peptide which concerns on this invention was described referring drawings, it cannot be overemphasized that this invention is not limited to the said embodiment. Various shapes, combinations, and the like of the constituent members shown in the above-described embodiments are examples, and various modifications can be made based on design requirements and the like without departing from the gist of the present invention.

  For example, in the said embodiment, in order to improve the yield of the peptide which has ACE inhibitory activity, the structure which carries out the hydrothermal reaction process of the spin-drying | dehydration cake X3 obtained by heat-processing decanter juice X1 was demonstrated. However, this invention is not limited to this, You may perform a hydrothermal reaction process directly with respect to decanter soup X1.

  Moreover, in the said embodiment, the structure which uses decanter soup X1 as a raw material liquid was demonstrated. However, this invention is not limited to this, For example, the whole raw material liquid containing the protein derived from foodstuffs other than a potato can be used as a raw material liquid.

  Moreover, in the said 1st Embodiment, the structure which heat-processes the decanter soup X1 using this hydrothermal apparatus can also be employ | adopted for the deproteinization apparatus 1 with a hydrothermal apparatus.

It is the flowchart which showed schematic structure of the peptide manufacturing apparatus for manufacturing the peptide which has ACE inhibitory activity using the manufacturing method of the peptide of 1st Embodiment of this invention. It is a graph which shows the experimental data for verifying the manufacturing method of the peptide of 1st Embodiment of this invention. It is a pie chart which shows the result of having analyzed the ingredient contained in decanter juice and a separation liquid in the manufacturing method of the peptide of a 1st embodiment of the present invention. It is the flowchart which showed schematic structure of the peptide manufacturing apparatus for manufacturing the peptide which has ACE inhibitory activity using the manufacturing method of the peptide of 2nd Embodiment of this invention.

Explanation of symbols

  S1, S2 ... Peptide production equipment, 1 ... Deproteinization equipment, 2 ... Hydrothermal equipment, 3 ... Centrifugal separator, 4 ... Separation / purification equipment, 5 ... IC reactor, 6 ... Activated sludge treatment Equipment, 7 ... Drying device, P ... Peptide having ACE inhibitory activity, X1 ... Decanter juice (raw material), X2 ... Separation solution, X3 ... Dehydrated cake, X4 ... Hydrothermal reaction treatment solution, X5 ...... Remaining dehydrated cake, X6 ... Secondary decanter juice, X7 ... Manufacturing treated water, X8 ... Processed liquid, X9 ... Discharged water, X10 ... Dry cake

Claims (2)

A heating step of adjusting the pH of the decanter soup of potato to 5.0 to 5.5 and heating the decanter soup of potato to 100 to 110 ° C;
A solid-liquid separation step of taking out the solid phase containing the solid content obtained by the heat treatment step as a dehydrated cake;
A hydrothermal reaction treatment step for performing a hydrothermal reaction treatment at a treatment temperature of 160 ° C. to 230 ° C. and a treatment time of 10 to 15 minutes for the dehydrated cake ;
A separation step of separating a peptide having an ACE (angiotensin I converting enzyme) inhibitory activity from the hydrothermal reaction treatment solution obtained in the hydrothermal reaction treatment step;
I have a,
In the separation step, the extract extracted with 70% ethanol from the hydrothermal reaction treatment liquid is concentrated under reduced pressure, the concentrated extract is held in a strong acid ion exchange resin, and the peptides are extracted with 2N aqueous ammonia. A method for producing a peptide having ACE inhibitory activity, wherein the peptide having ACE inhibitory activity is obtained by extraction and cellulose column chromatography . A drying step for obtaining a dried cake by performing a drying treatment on the dehydrated cake; and a hydration step for returning to the dehydrated cake by adding water to the dried cake obtained in the drying step. method for producing a peptide having ACE inhibitory activity according to claim 1, wherein the relative returned the dehydrated cake and performing the hydrothermal reaction treatment.

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