JPH04341193A - Production of peptide or its salt - Google Patents

Abstract

PURPOSE:To easily and quickly obtain peptides having inhibitory action on angiotensin converting enzymes and suitable as a food material by treating an enzymic decomposition product of milk-originated casein with an ion exchange resin and a hydrophobic resin, thereby separating and purifying the peptides. CONSTITUTION:The objective peptide or its salt capable of inhibiting angiotensin converting enzymes and having the amino acid sequence of formula I or formula II can be produced by suspending casein originated from cow milk in water in a jar fermenter, adjusting the pH of the suspension to 7.5 with 10N KOH, adding trypsin while keeping the suspension at 37 deg.C, digesting for 4 hr under agitation, subjecting to the heat treatment under high temperature and pressure condition at 121 deg.C for 10min, removing the precipitate by continuous centrifugal separation, collecting the supernatant to obtain an enzymic decomposition product of casein, passing the supernatant through an anion exchange resin column to effect the adsorption of the component, eluting the adsorbed component with a buffer solution of pH 5.0, contacting the eluate with a hydrophobic resin, washing with water and eluting the adsorbed component with ethanol.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention purifies an enzymatic decomposition product of casein using an ion-exchange resin and a hydrophobic resin to produce angiotensin convertase, which is particularly suitable as a material for foods for specified health uses.
ting enzyme, hereinafter abbreviated as ACE. )
The present invention relates to a method for obtaining an inhibitory peptide or a salt thereof.

0002

[Prior Art] It is well known that the renin-angiotensin system is deeply involved in the onset of hypertension. The renin-angiotensin system contains ACE, which is involved in blood pressure regulation, and this enzyme causes an increase in blood pressure by converting angiotensin-I to angiotensin-II, which has a strong vascular wall smooth muscle contraction effect. Therefore, by suppressing this enzyme activity, it is possible to prevent an increase in blood pressure (lower blood pressure).

Various substances have already been found as angiotensin converting enzyme inhibitors, for example, D-2-methyl-3-mercaptopropanoyl-L-
Proline (generic name: captopril) and the like are currently in practical use as oral antihypertensive agents due to their high inhibitory activity.

[0004] Also known as inhibitors derived from natural products are snake venom peptides, and peptides obtained by enzymatically decomposing casein derived from milk and zein derived from corn.

Among these, natural products or inhibitors derived from natural products have the potential to become safer antihypertensive agents, whereas synthetic products still have problems in terms of toxicity and side effects. In particular, casein-derived ACE inhibitory peptides are expected to be advantageous in terms of cost as well as safety, so their practical application as antihypertensive materials is being considered (Unexamined Japanese Patent Publication No. Publication No. 58-109425, Japanese Unexamined Patent Publication No. 2-23885, etc.).

[0006] Casein-derived ACE-inhibiting peptides include, for example, those having the amino acid sequences shown below.

CEI12: Phe-Phe-Val-A
la-Pro-Phe-Pro-Glu-Val-Ph
e-Gly-Lys

[Formula 1] CEI6: Thr-Thr-Met-Pro-Leu
-Trp

[0008] These ACE-inhibiting peptides have been purified using a gel filtration column or the like on a laboratory scale (Agricultural and Biological Chemistry; Agric. Biol. Ch.
em. , 46, 1393, 1982/Agric. Bi
ol. Chem. , 49, 1405, 1985/Agr.
ic. Biol. Chem. ,51,2557,198
7).

[0009] However, purification by gel filtration cannot increase the throughput of the sample very much and requires purification at least 3 to 4 times in combination with an ion exchange resin, so it is not very suitable for industrial use. In addition, gel filtration carriers are generally expensive, and peptides purified using them cannot be added to foods.

0010

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a simple, rapid, and large-scale method for obtaining ACE-inhibiting peptides that can be used as food materials.

[0011]

[Means for Solving the Problems] The present invention provides an angiotensin converting enzyme inhibiting peptide or a salt thereof, which is characterized in that an enzymatic decomposition product of milk-derived casein is purified using an ion exchange resin and a hydrophobic resin. It's a method.

The present invention will be explained in detail below. The casein used in the present invention, which is a raw material for the enzymatically decomposed product of milk-derived casein, is preferably milk-derived acid casein and caseinate, but unpurified ones such as skim milk may also be used. The enzyme is not particularly limited, and any enzyme that specifically cleaves the peptide bond at the carboxyl terminal of a basic amino acid may be used. Although lysyl endopeptidase or arginyl endopeptidase may be used in combination, trypsin is industrially more suitable. However, in the case of a crude enzyme such as pancreatin, the improvement in yield of the target peptide may be small and is not very desirable.

[0013] Any method known in the art may be used for enzymatically degrading casein. For example, trypsin is
A casein decomposition solution is obtained by adding 0.1 to 5% by weight based on milk-derived casein substrate under conditions of 5.0 to 10.0 and reacting at 15 to 60°C for 30 minutes to 18 hours. I can do things. The reaction is stopped by inactivating the enzyme by adding acid or heating.

The casein decomposition product obtained in this way (hereinafter referred to as crude peptide-containing product) may be subjected to the next step as a solution, but for preservation, it may be spray-dried or freeze-dried. It may also be made into a powder.

[0015] Next, the ionic strength and pH of this crude peptide-containing material are adjusted using a conventional buffer solution and, if necessary, an acid or an alkali so that it can be adsorbed onto an ion exchange resin. When using a cation exchange resin, the pH
If the pH is adjusted to 5 or less, and in the case of an anion exchange resin to 8 or more, it is possible to adsorb the target peptide.

[0016] The adsorption method may be either a batch method or a column method, but the column method is preferable in view of the degree of purification.

[0017] After adsorption, elution by changing the pH is necessary because normal changes in ionic strength alone are not sufficient for elution. The pH for elution is preferably in the range of 6 to 8 for cation exchange resins and 4 to 6 for anion exchange resins.

The ion exchange resin used may be any commercially available ion exchange resin, but as a result of examining the degree of crosslinking and exchange capacity, we found that it has a large network structure and is porous, such as styrene resin or acrylic resin. A resin that is rich in properties and has a weakly acidic carboxyl group or a weakly basic dimethylamino group as an exchange group is more desirable in terms of adsorption amount and recovery rate. The resin used is, for example, Amberlite IR-118, IRC-50, IRA-94S, XT-
5028 (manufactured by Organo), etc.

Next, the fraction containing a large amount of the desired ACE-inhibiting peptide obtained by fractionation using an ion exchange resin was
When applied to a porous hydrophobic resin such as polystyrene beads, the target peptide is adsorbed.

The hydrophobic resin to be used may be a commercially available styrene resin or acrylic resin having a large porous network structure, such as Amberlite XAD-2, XAD-2, etc.
7 (manufactured by Organo), Revachit OC-1064 (manufactured by Bayer), etc. are suitable.

[0021] The adsorption method may be either a batch method or a column method, but the column method is preferable in terms of the degree of purification.

[0022] After washing the resin with water, it is eluted by changing the concentration of an organic solvent such as ethanol, thereby performing purification at the same time as desalting. Ethanol concentration for elution is 20-60
A range of % is preferred.

[0023] After removing ethanol from the eluate under reduced pressure, it is dried using a method such as freeze drying or spray drying to obtain the ACE inhibitory peptide as a white powder.

As in the above process, by using a combination of ion exchange resin and hydrophobic resin, the desired ACE can be achieved.
We were able to obtain inhibitory peptides at a much higher content than expected.

Either the ion exchange resin or the hydrophobic resin may be used first, but if the hydrophobic resin is used later, purification and desalting can be carried out at the same time, which is very effective.

The ACE-inhibiting peptide obtained by the present invention is mainly composed of the effective peptide CEI12 or CEI6, and its content is extremely high. This ACE inhibitory peptide was confirmed to have an enhanced blood pressure lowering effect in an evaluation system using spontaneously hypertensive rats (SHR). Here, we have discovered that it is possible to provide materials such as foods for specified health uses for preventing and improving hypertension.

As described above, the casein-derived ACE inhibitory peptide is usually isolated and obtained in the form of a powder, and then it can be prepared as it is or, more preferably, with an appropriate carrier for oral ingestion, from an appropriate shape or form. Provides an oral diet consisting of: As methods for producing these foods, conventional methods for formulations or food production applicable to proteins and peptides can be used.

[0028]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto in any way. In addition, the amount of CEI12, the amount of CEI6, the amount of protein, and the blood pressure lowering effect were measured by the following methods.

(Quantification of CEI12: Quantification by HPLC) The sample was dissolved in 30% acetonitrile containing 0.1% trifluoroacetic acid so that the CEI12 content was 0.1 to 1 mg, and the sample was dissolved in 30% acetonitrile containing 0.1% trifluoroacetic acid. ODS column A
-312). Elution was performed using 30% acetonitrile containing 0.1% trifluoroacetic acid (flow rate 2 m).
l/min), and its CEI12 content is determined from the absorbance at 260 nm or 220 nm using a calibration curve using pure CEI12.

(Quantification of CEI6: Quantification by HPLC) CEI6 is quantified in the same manner as for the quantification of CEI12, using 23% acetonitrile containing 0.1% trifluoroacetic acid as the eluent.

(Measurement of protein amount) The sample is diluted with the solvent used for elution if necessary, and the absorbance at 280 nm is measured to determine the protein amount.

(Measurement of blood pressure lowering effect) We treated SHR (Japanese Charles River, male, 15-16 weeks old)
The method of eks et al. (Proceedings of the Society for Experimental Biology and Medicine; Proc. Soc. Exp.
Biol. Med. 104, 646, 1960), a cannula is inserted from the artery of the greater cuboid toward the abdominal aorta. After recovering physical strength over a day and fasting overnight, a pressure transducer (manufactured by Nihon Kohden Co., Ltd., TP-
400T). For administration of the sample, 1 g/kg of the rat body weight was dissolved in water using a gastric tube, and the sample was forcibly administered orally to a volume of 10 ml/kg of the rat body weight. Changes in blood pressure were observed over time after administration.

Example 1 (Purification of CEI12) 2 kg of milk-derived casein (manufactured by Beignet, food grade acid casein) was
Suspend in 20 l of water in a 0 l jar fermenter (manufactured by Mitsuwa) and adjust the pH to 7.5 with 10 N KOH. Incubate at 37°C and add trypsin (manufactured by Novo Industries, PTN3.0S, EC.33.4.21.4)
, derived from pig pancreas) was added, and digestion was performed for 4 hours while stirring. This was heated under high temperature and pressure in a jar fermenter (121°C, 10 minutes), and the resulting precipitate was
The supernatant was obtained by continuous centrifugation at 00 rpm and 4°C. This supernatant was dried to obtain 1.4 kg of crude peptide-containing material.

Amberlite I, an anion exchange resin
RA-94S (10 l) was added to the column (φ15 x 57 cm
) and pre-filled with Mcllvaine's buffer (0
．． It was equilibrated with 1M citric acid/0.2M phosphate buffer) pH 8. 100 g of the above crude peptide-containing material was dissolved in 2 liters of the same buffer pH 8, and after adsorption, 50 liters of H2
Wash with water and add 100 liters of Mcllvaine's buffer (0.2M citric acid, 0.4M phosphate buffer, pH 5).
Elution was performed using

[0035] The eluate was separated into 2 liter portions, and the protein amount and CEI12 amount thereof were measured according to the method described above. The results are shown in Figure 1.

[0036] The elution pattern shows that the unadsorbed protein (
By elution at pH 5, CEI12 was first observed to be eluted, and then other adsorbed proteins were eluted.

Next, among the above elution fractions, 1 kg of hydrophobic resin Amberlite XAD-2 was added to the fraction containing a large amount of CEI12 (underlined part in FIG. 1), mixed well for 1 hour, and washed with water. Elution was performed twice with 2 liters of 40% and 60% ethanol. For each fraction, CEI1
2 amount and protein amount were measured.

CEI12 was mainly eluted in the 60% ethanol fraction, and the ethanol was removed from this fraction under reduced pressure, and a dry powder was obtained by freeze-drying. The CEI12 content in this powder was 23.1% by weight (CEI12
recovery rate of 34.7%).

A summary of the purification process is shown in Table 1. These purification methods increased the content of CEI12 by approximately 23 times relative to the crude peptide content.

[0040]

[Table 1]

[0041] Even when the order of using the anion exchange resin and the hydrophobic resin was changed, similar results were obtained.

Example 2 (Purification of CEI6) Under the same column conditions as in Example 1, CEI6 was purified using an anion exchange resin. Figure 2 shows the results. C
EI6 was eluted later than CEI12. Next, the fraction containing a large amount of CEI6 (underlined part in Figure 2) was
As a result of purification using a hydrophobic resin under the same column conditions as in Example 1, the CEI6 content increased significantly as in Example 1.

Comparative Example 1 (C using only anion exchange resin)
Purification of EI12) The crude peptide-containing material obtained in Example 1 was dissolved in a pH 8 Tris/HCl buffer and applied to an anion exchange resin Amberlite IRA-94S equilibrated with the same buffer in advance. A batch method was used as the adsorption method, and elution was performed using 1N HCl. The yield of CEI12 is 60%, CEI1
The content of 2 increased only about twice that of the crude peptide-containing material.

Comparative Example 2 (C using only cation exchange resin)
Purification of EI12) The crude peptide-containing material obtained in Example 1 was dissolved in an acetate buffer of pH 4, and the cation exchange resin Amberlite IRC-50 or IR-11 equilibrated with the same buffer was used.
Served on 8th. Using a batch method as an adsorption method, 1N N
Elution was performed using aOH. The yields of CEI12 were 35% and 32%, respectively. The content of CEI12 increased only about twice that of the crude peptide-containing material.

Comparative Example 3 (CEI1 with only hydrophobic resin)
Purification of 2) The crude peptide-containing material obtained in Example 1 was dissolved in water, washed with ethanol in advance, and then equilibrated with water. 1064 (manufactured by Bayer). Adsorption was performed by a batch method, and after washing with water, elution was performed using 95% ethanol. The yield of CEI12 was 75, respectively.
%, 50%. The content of CEI12 increased only about 3 times and about 2 times, respectively, compared to the crude peptide-containing material.

Test Example 1 Using the freeze-dried powder obtained in Example 1, the blood pressure lowering effect on animals was investigated according to the method described above. The antihypertensive effect was enhanced compared to the crude peptide-containing product before purification, which was administered as a control (FIG. 3).

[0046] Hereinafter, an example of oral food will be shown as an application example of the ACE-inhibiting peptide obtained by the present invention.

Application example 1 Yogurt composition
weight% milk
70.0 whole milk
4.0 skimmed milk powder
5.0
Granulated sugar
7.0 water
12.0 ACE inhibitory peptide of Example 1 2.0 Prepared using the above-mentioned formulation according to a conventional manufacturing method (100 g/cup).

Application example 2 Soft drink composition
weight% milk
90.0 liquid sugar
7.0 A of Example 1
CE inhibitory peptide 0.5 cocoa powder
2.5 Produced using the above formulation using a normal manufacturing method (200 g/can).

[0049]

[Effects of the Invention] Since the present invention is constructed as described above, it produces the effects as described below. By the method for obtaining peptides of the present invention, ■ simple, rapid, and large quantities of peptides can be obtained;
■With no contaminating salts, it is possible to obtain an ACE-inhibiting peptide with increased target peptide content and enhanced blood pressure lowering effect. Therefore, it is of great significance that this method has made it possible to put into practical use an oral food that is derived from natural products, is highly safe, and contains a large amount of effective peptides and has a strong blood pressure lowering effect.

[Brief explanation of the drawing]

FIG. 1 is an elution curve of protein and CEI12 when the crude peptide-containing material obtained in Example 1 was purified using an anion exchange resin.

FIG. 2 is an elution curve of protein and CEI6 when the crude peptide-containing material obtained in Example 1 was purified using an anion exchange resin.

FIG. 3 is a diagram showing the temporal variation pattern of blood pressure when the ACE inhibitory peptide obtained in Example 1 was administered to animals.

Claims (2)

[Claims] Claim 1: When obtaining an angiotensin converting enzyme inhibiting peptide or a salt thereof from an enzymatic decomposition product of milk-derived casein, the enzymatic decomposition product of casein is separated and purified using an ion exchange resin and a hydrophobic resin. A method for obtaining a peptide or a salt thereof. 2. The method for obtaining a peptide or a salt thereof according to claim 1, wherein the angiotensin converting enzyme inhibiting peptide is represented by the following structural formula (I) or (II). CEI12: Phe-Phe-Val-Ala-Pro
-Phe-Pro-Glu-Val-Phe-Gly-
Lys...(I) CEI6 :Thr-Thr-Met-Pro-Leu
-Trp...(II)